Plant Cell Biology for Masters

http://plantcellbiology.masters.grkraj.org/Plant_Cell_Biology_Table_Of_Contents.html
i. ii. iii. iv. v. vi. vii.

Plant Cell Structure Plant Cell Division Plant Cell Apoptosis Plant Cell Genetics Plant Cell Physiology Plant Cell Biochemistry and Metabolism Plant Growth and Development
i. ii. iii. iv. v. vi. vii. viii. ix. x. xi. xii. xiii. xiv. xv.

Plant Development Introduction Plant Hormones introduction Auxins Gibberellins Cytokinins Abscissins Plant Cell Vacuoles Ethylene Morphactins and Others Application of Plant Hormones Physiology Of Flowering Physiology Of Vernalization Physiology Of Dormancy Physiology Of Plant Movements Circadian Rhythm and Biological Clock

viii. ix.

Plant Cell Gene Engineering Plant Molecular Biology

Plant Development-Introduction
Concepts of Growth and Differentiation

In living organisms production of a zygote to produce a zygote are two events separated by space and time. The zygote in an embryo sac divides and redivides to produce a mass of cells. Concomitant with the cell divisions polarity of future shoot apex and root apex is also determined and established which is accompanied with physiological and structural changes. After a period of active growth, the embryo suspends its active state and becomes dormant. At this stage, the embryo which is enclosed in a seed coat is shed from the mother plant. Then after a period of rest, which may be short or long, if conditions are favorable, the seed germinates and develops into a full fledged plant. Such plants inturn, after going through a period of vegetative development produce reproductive structures in which once again a zygote develops due to the act of fertilization. And the cycle is repeated. Between these two stages the time required for the development varies from as short as 24 hours, to several years. This is however determined by the genetic potentiality of the given plant,

but environmental factors have a strong and stimulating influence on the plant genome for its expression of manifestation. A careful analysis of various events in the development of higher plants reveals that there are two important processes that go hand in hand. One is growth and the other is differentiation; together it is called development. Growth is generally defined as an irreversible increase in size/volume accompanied with an increase in the protoplasmic mass. Differentiation, on the other hand, is a process in which new structures and new functions generate from the preexisting cellular components. For that matter, these two events are not mutually exclusive.

Increase in the number of cells accompanied with the increase in volume has an apparent effect on the increase in the size of the plant body. The same can be determined by either counting the number of cells or measuring the volume or both. But differentiation involves the molecular changes in undifferentiated cells, leading to the formation of new structures, like tissues, leaves, buds, branches, flowers and fruits. So the development can be considered as the summation of all physiological activities.

Measurement of growth: Normally growth is measured in terms of increase in the number of cells, increase in height or weight with time. These can be determined by known physical methods. Even the number of leaves, branches, roots, flowers and fruits produced can be measured by simple methods. The developments of later said structures are the products differentiation which can be further subjected to quantitative or qualitative studies. The growth of a seedling, in terms of increase in length or weight, can be plotted as the function of time which may be in hours or days. The graphical expression, which results in a curve called growth curve. Different plants, for those matter different organs of the same plant, exhibit different kinds of curves. For example, a tree plant increases in height and weight with time. Similarly, the number of leaves, flowers and fruits produced show different pattern, which is quite distinct from that of growth pattern of a single leaf, flower or fruit. The plants exhibit indefinite growth and determinate growth pattern. But in both one can observe the growth pattern which passes through three different phases one leading to the other. They are lag phase, log phase and steady or constant phase.

It is possible to utilize the above said growth curves in determining overall growth rate or the growth rate of individual phases. This can be done by taking a tangent angle (90) degree derived from two points on the curve, which provides the information about the increase in length or weight in a given period of time. By integrating them together, one can obtain the growth rate. The same value i.e. growth rate (increase in length or wt.xTime) can be plotted against a larger time scale. This plot gives a general pattern of growth rate at different phases of growth of the whole plant. The finer analysis of these growth patterns can be employed to determine the efficiency of plants in terms of its increased or deceased dry matter, photosynthetic efficiency, flowering, fruit production, etc. In fact, using the above methods, it is also possible to determine relative growth rate, not assimilation rate and the ratio of leaf area to dry weight or leaf area ratio. The above methods provide an invaluable data in determining the genetic potentiality of plants in response to different environmental conditions. The efficiency of the plants in terms of input and output can also be determined. Growth Rate and Phases of Growth: Different plants exhibit different growth rate; this is the law of nature. Generally, most of the plants initially show a period of slow growth. This is called lag phase. At this stage most of 5he available reserve food material is metabolized to generate energy rich compounds and various organic compounds necessary for building the structural components of cells. In this period, most of the reserve food material is drawn into metabolic pool. Further development depends upon the ability of the plants to synthesize its own food material, which is achieved by the development of leaves. Once sufficient number of leaves and roots are produced, the plant becomes self sufficient and then the plant grows at a faster pace. In fact, the increment of growth doubles with time. This phase of growth is called log period or exponential period of growth. The term exponential is derived from the fact, the increase in the number of cells or increase in height or weight, when measured at equal periods of time always doubles, etc., 1, 2, 4, and 8, 16, 32, 64 and so on. Such a relationship between the number of cells produced in a given time is expressed n the form of an equation i.e. N=4t, where t is the time interval and N is the number of cells or weight or length. If the growth pattern obeys the said equation, then it is called exponential growth. If fact, it is at this period of time, the plant reaches the maximum differentiation and development, in terms of production of leaves, buds, branches, etc., and the plant gains maximum height and weight. The magnitude of exponential growth in this period however, varies from plant to plant. For example, Calamus gigantica (bamboo) grows at the rate of 2 ft./day, Asparagus at 1 ft/day, inflorescence axis in Agave 6 inches/day, Basella bacella 2 inches a day. On the contrary Cycas, a gymnosperm, grows at

the rate of 1.2 to 2 inches/year. The above examples clearly indicate that the ability of growth of each plant is ultimately determined by its genetic potentiality. Once plants reach their maximum growth point, they start producing flowers. This of course varies from species to species. With this, the growth rate of plants falls sharply, but the other non flowering branches continue to grow but slowly. Thus a steady rate of growth is maintained. Such a phase is called steady phase. But I may annuals, with flowering and fruiting, the growth comes to stand still and ultimately the plant dies. Nevertheless, the perennial plants (herbs, shrubs or trees) exhibit further seasonal vegetative growth and seasonal flowering/fruiting periods, where the growth curve is different from that of annuals. Such a behavioral pattern in exhibited by evergreen plants as well ad deciduous plants. While the above observations are true for the entire plant, the growth rate of leaves, flowers and fruits exhibit a different pattern. They too have a lag phase, a log phase and constant phase where the growth of the said organs is virtually stopped. All structures or organs which have determinate growth show this pattern. Furthermore, the magnitude of growth of different organs varies from species to species. Mechanism of Growth and Differentiation: It is very important to understand the regions of growth that are involved in the development of the plant body. This can be taken as the basis for understanding the mechanism of growth and differentiation. The apices of shoots and roots provide valuable information about the structures involved in growth and differentiation of cells during development of plant structures. Refer to the topic cell differentiation as the function gene expression to understand the molecular basis of differentiation. Both root apex and shoot apexes are made up of three important regions i.e. the region of meristems, the region of elongation and the region of differentiation. The meristematic region is restricted to the apexes, where the cell will be in an active state of cell division. In the region of elongation, cells are in the process of physical elongation, thus less region exhibits an apparent growth. Just behind this region, the elongated cells or the cells that are still in the process of elongation show marked differentiation in their cellular structures and functions. In this region, one can see different types of cells and tissues like pith, xylem, phloem, endodermis, cortex etc. The analysis of each of these structures and their functions will give a comprehensive picture of how cells grow, differentiate and produce specific organs.

Meristems:

The higher plants, in their embryonic state itself, acquire polarity for their future

development. The embryo possesses plumule at one end and radicle at the other end of the plant axis. The plumule develops into shoot system and radicle into root system. But shoot apex and root apexes contain a single or a group of meristematic cells. Each cell in this region is endowed with a potentiality of active cell division and this potentiality is retained throughout the lifetime of the plant. Such meristematic cells are rich in cytoplasm, compactly arranged with a number of intercellular protoplasmic strands as interconnecting structures. Vacuoles in these cells are nearly absent. Nucleus is active. Cells exhibit maximum respiratory activity to generate energy rich compounds. Other metabolic pathways are very active in generating various compounds to build up various cellular components and structures. The nuclear DNA undergoes periodic replication and various sets of genes are expressed in producing required TRNAs, RRNAs and RNAs. Once these RNAs reach the cytoplasm, they are utilized in synthesizing a host of proteins of which some are used in the organization of different cellular structures like membranes of ER, plasma lemma, mitochondria, plastids, nucleus, lysosomes, golgi bodies, cytoskeletal structures, microtrabaculae, chromatin, ribosomes, etc. and other proteins act as enzymes and regulatory factors for various metabolic processes. These activities in meristatic cells are maintained as well as sustained till

the death f the plant. Except under dormant conditions where all the cellular activities are temporarily suspended, they are fully active through out the life of the plant. Cell Division and Plane of orientation: Once the cellular components are made and the protoplasmic mass reaches a particular proportion in terms of mass, the cells initiate mitotic divisions. This results in the production of daughter cells. Some of the daughter cells which are away from the mother meristem cell act as derivatives. T he most crucial part of mitotic divisions is the plane of division. This is however determined by the plane of orientation of mitotic apparatus in the dividing cells. As shown in the fig upper cells undergo another period of preparation for another cycle of cell division to produce another set of derivative cells and the process goes on and on thus the terminal cells fund in the apical dome continue to divide and redivides ceaselessly to generate new cell derivates. The regulatory factors that control cell divisions continue to be produced in mitotically active meristematic cells there by the said cells keep on their mitotic activity unhindered. On he contrary, the cell derivatives produced by the upper meristematic cells, because of their positional effect, nutritional and hormonal influences produce certain regulatory factors some of which block gene expression required for continuous mitotic activity and others stimulate the cells to undergo certain transformation leading to differentiation. Mutation studies in yeast cells clearly suggest that mitotic cycle is controlled by at least 36-38 mutational sites an each one of them are distinct and specific in their activity. Some of the genes, if

activated, may block the continuous mitotic activity. On the other hand, if some specific regulatory genes undergo mutation, it induces uncontrolled mitotic activity which may lead to cancerous growth. However in higher plants to keep pace with the increase in the number of meristematic cells, the tunica layer of cells which cover the apical dome divide and redivides radially. It is interesting to note that the outer tunica layer of cells or epidermal cells are also meristematic but their plane of cell division is always radial. The intrinsic factors that are responsible for such a behavior of these cells are perplexing and fascinating. Nothing is known about this phenomenon. Transitional Zone: The basal cells though derived from the terminal meristematic cell(s) exhibit different pattern of growth. This is however, determined by the underlying mature or partially matures cells which supply nutrients in the form of mineral elements, organic compounds and plant hormones. The gradient and magnitude of various chemical components supplied from the lower cells have profound influence on the cells derived from meristematic cells. The positional effect of transitional zone cells, compounded by the hormonal and nutrient gradients the cells found in the transitional may undergo one or more cell divisions or they may directly undergo transformation leading to differentiation. Onset of cell elongation and cell differentiation: The cell derivatives thus produce, utilizing nutritional ingredients and phytohormones, undergo dramatic transformations. Not all the cells behave in the same way. Some elongate considerably and undergo transformation to produce different cell types, such as xylem elements, phloem elements, and endodermal cells and so on. Others without much elongation develop into cortical parenchyma, pith parenchyma and pericyclic cells. In stem apex, just below the meristematic zone cells, just below the tunica layer, some of the cells organize into leaf primordia, which actually cover the entire meristematic dome.

In roots, epidermal cells in the region differentiation elongate and develop into root hairs. However, among the vascular elements the first to be differentiated is phloem and then xylem elements develop. Though most of the cells are derived from the same group of meristems because of intrinsic factors, and programming the derivatives undergo transformation and develop into various cell types perform different functions.

Phloem as a Model for Understanding the process of differentiation: Whether it is stem apex or root apex the first cell type to be differentiated are sieve tubes. The cells that are destined to become sieve elements are in line with the sieve cells of vascular bundles found below. The destined cell receives auxins from the cells found above and nutritional factors and sucrose from the lower vascular elements. Here sucrose appears to play a significant role in the formation of sieve cell. In response to hormones and nutritional factors, the said cell first undergoes an unequal cell division in the vertical plane. The smaller cell produced undergoes one or two divisions and develop into companion cells. But the larger cell undergoes remarkable transformation. First the cell elongates and expands. Concomitantly the nucleus disintegrates into small chromatin bits. It s at this juncture, the chromatin becomes active and produces substantial amount of mRNAs required for the formation of P-proteins and other factors. The mRNAs thus produced retain their translational activity for a period of 4-5 months. Once the p-proteins and its associated elements organize into longitudinally oriented tubular structures the tonoplast membrane disappears rendering the peripheral protoplasm and vacuolar materials, indistinguishable. functional activity of such sieve tubes and companion cells are sustained for quite a long time. Differentiation of xylem elements: Similar to the development of sieve elements, some of the cells found by the side and just below the newly formed sieve tubes undergo differentiation and development into xylem structu5es. Experiments involving the induction of vascular elements in callus clearly show that the application of sucrose favor the development of sieve tubes and indole acetic acid on the other hand, stimulates the formation of xylem elements. So supply of IAA to these cells is crucial in the development of the xylem elements. Responding to IAA and other nutritional factors, the cells that are destined to become xylem elements enlarge in length as well as in breadth. At the same time, the entire cellular machinery gears up to produce cell wall materials in massive amounts. Golgi bodies become very active, the vesicle loaded with cell wall components and enzymes are transported across the cell and the same are unloaded outside the plasma membrane. Synthesis and deposition of various components of secondary wall takes place on a large scale. Meanwhile endoplasmic reticulum starts associating with the plasma membrane at different sites. These regions become free from cell wall deposition and the same develop into pit canals. But in other regions, cell wall is deposited in a characteristic pattern in different cells of different xylem elements. During the development of xylem the protoplasm is completely used and finally it disappears; it is an apoptotic phenomenon, a programmed cell death. At the molecular level, it is clear that differential expression of genes is mainly responsible for the production of massive amounts of MRNAs required for the formation of enzymes needed for cell wall components. Even the deposition of cell wall material is regulated. Thus undifferentiated cells undergo differentiation into xylem structures to perform their specific functions. Molecular Paradox in Morphogenesis The

During morphogenesis, the derivatives of meristems are subjected to nutritional and hormonal pressures. As a result cellular programs of these cells get modified due to the activity of regulatory factors. A set of genes which were active during early stages of mitotic cycle, at least, some of them get repressed, thus they switch off all the programmes for cycle events. At the same time, a new set of genes are activated which inturn exhibit cascade effect. In the sense, synthesis of a new group of mRNAs and their translation products activate another set of genes and such sequential gene inactivation and activation results in the transformation of undifferentiated cells into new cell types which by virtue of possessing a characteristic structure and functional potential perform specific functions; thus the division of labour sets into the system. There must be a master switch (s) in the form of regulatory factors that act at every stage of differentiation and growth. This can be similar to that of Homeobox or Hox gene clusters of animal system. Existence of such gene clusters have been discerned but not fully evaluated. The regulation of gene expression may operate at the transcriptional level, translational level or at post translational level, but each of these events have their own feed back mechanisms. Besides nutritional factors and environmental factors phytohormones have a profound influence on gene regulation. Unlike animal systems plants have just a set of hormones like auxins, gibberellins, cytokinins, abscissins and ethylene. Among them the first three act as growth promoters and the others act as growth inhibitors. The site of synthesis, the time of synthesis, the concentration of each of these hormones at any given site and time, nflu8ence the molecular expression according to their specific effects. Though individual hormones elicit certain specific responses, their activity is greatly influenced by the presence of other hormone. One hormone in one tissue type may promote or inhibit the activity of the other hormone. Thus by modulating the levels of growth hormones, they induce different morphological structures. This is possibly achieved through a very complex gene regulation mechanism about which we know nothing. Molecular aspects of IBA induced new root formation: Adventitious root initiation in response to auxins like IBA has been used in understanding the molecular events that lead to redifferentiation in plants. In response to IBA, pericyclic cells found in the hypocotyl tissues of phaseolus vulgaris undergo transformation and reorganize into root primordial cells. The entire process takes place within 24-36 hours after the treatment with the hormone.

Molecular events: Within 15-30 minutes of auxin treatment, even in the presence of actinomycin the rate of protein synthesis in the hypocotyls increases without any concomitant increase in mRNA synthesis, which suggests that early effect, is on IBA translational machinery. This view has been experimentally borne out by invitro translational studies. After another 60-90 minutes transcription and translational process further increase by 4-5 fold over control tissues. Among many changes observed in the pattern of proteins synthesized, the increased synthesis of 55-58 K Dalton proteins and another high molecular weight protein is very significant. Using SDS page, invitro translational system and immunoprecipitation methods the 55-58K protein has been identified as Tubulin.

Tubulin is known to be an important component of microtubule needed for mitotic apparatus and cytoskeleton structures. Furthermore tubulin polymerization into microtubules also increases in the presence of IBA and cytoplasmic factors. The above observations suggest that the IBA induced microtubules formation has some role in dedifferentiating the cells. In order to ascertain this view when the IBA pretreated hypocotyls are exposed to colchicine or cytochalasin B for a period of 36 hours, IBA mediated new root formation is completely inhibited, but after IBA mediated new root formation is completely inhibited, but after 36 hours of IBA pretreatment colchicine has no effect. The above results indicate that auxin mediated increase in tubulin synthesis, tubulin polymerization into microtubules, the organization and orientation of mitotic apparatus ultimately determines the plane of division. Besides another important observation is the transformation of parenchymatous cells into mitotically active cells. Thus IBA induces cell transformation and also determines the plane of division in parenchymatous tissue which ultimately determines the organization and development of root primordia.

Interestingly, the IBA mediated root initiation can be inhibited by higher concentration of cytokinins. Anatomical studies show that in the hypocotyls, treated with both auxin and cytokinins, the pericyclic cells show greater mitotic activity but root primordial organization is totally absent. But in the segments treated with cytokinin alone pericyclic cells do not show any changes. In order to find out the interesting interaction between IBA and cytokinins and their effect on molecular events studies on the rate f protein synthesis and RNA synthesis show that the rate of protein synthesis increases as early as 15-30 minutes as cytokinin treated segments but the increase in RNA synthesis is delayed by 4-6 hours. Similarly in the hypocotyl which are exposed to both IBA and cytokinins, the rate of protein synthesis increases at 15-30 minutes, but the IBA mediated RNA synthesis is inhibited by cytokinin for a period of 4-6 hours. Later RNA synthesis and protein synthesis increase substantially in both the cases. IBA+cytokinin, the increase is highest. Analysis of in vivo protein pattern and invitro protein pattern reveals that though cytokinin increases the level of tubulin synthesis as in the case of IBA. It inhibits the synthesis of high mol.wt. Protein induced by IBA. The above features suggest that cytokinins inhibit IBA mediated root initiation by modulating the microtubular organization by way of inhibiting high moo. wt. proteins that are needed for root primordial organization. Probably this is the only example in plant system which explains the molecular events that lead to the initiation of root formation and the inhibition of it in response to combination of phytohormonal treatment. A model has been given to follow the events for new root formation in response to IBA and BAP is given.
Fucus as a Model to Understand Structural Changes during Morphogenesis:

Particularly in the stems treated with

From the earlier discussions it is clear that the temporal and quantitative regulation of gene activity through transcription and translation ultimately determines structural changes during differentiation. Though not much is known about the molecular events that lead to ultra structural changes during development, it is well known that the fertilized egg in the embryo sac always undergoes a polar division as if it is predetermined and produces cells which develop into radicle and plumule. Such behavior of cells during the early part of its development is known to be a function of intracellular compartmentalization regulated by certain intrinsic interactions of nucleocytoplasmic components. Development of umbrella shaped reproductive gametangial structures in Acetabularia, development of hold fasts in cladophora and rhizoidal formation in the zygotic development of Fucus, are some of the examples which have been used in understanding ultra structural changes during organogenesis. The developing zygote is Fucus first produces a rhizoidal process a rhizoidal process at the region of contact with the substratum. If such spherical zygotes are subjected to unilateral illumination from one side, rhizoidal structures develop at the opposite pole. If the direction of illumination on the zygote is changed at short intervals of time, the end at which the rhizoid develops is determined by

the last treatment. The time required for such polarity fixation is about 10-16 hours of treatment, beyond that the polarity of rhizoidal development remains unchanged.

If such programmed cells are studied under electron microscope, it is revealing to find a large number of osmophilic bodies some unknown granular materials, dense fibrillar structures, ribosomes

and mitochondria congregated at the region of rhizoidal formation. Interestingly, the perinuclear region facing the rhizoidal pole shows polarized finger shaped projections indicating the flow of informational materials from the nucleus in the direction of rhizoidal pole. The above observations suggest that the duration between the time of induction by light and the time at which rhizoids develop is the most crucial period at which various molecular and structural events fix the polarity. In fact, these events are the basis for differentiation. Furthermore, if zygotes, which re subjected photo inductive treatment, are treated with inhibitors of protein synthesis like CHI for above 9-10 hours, rhizoidal formation is not inhibited. This clearly suggests that the denovo protein synthesis is not required for polarity fixation. The polarity fixation is achieved with whatever structures or components available in the cell. Instead if such zygote is provided with cytochalasin B or colchicine or both for the same period of time, i.e. 9-10 hours, the polarity of rhizoidal formation is totally inhibited and it remains labile. Cytochalasin B and colchicine are known for disrupting the formation of microfilaments and microtubules respectively. Transmission electron microscopic studies of polarized zygotes of Pelvetia fastigata reveal cortical clearing. in the region of rhizoidal formation. the polarity fixation is due to the organization and Bundles of microfilaments and of microtubules and microtubules are found oriented in line with rhizoidal pole. The above studies clearly suggest that orientation microfilaments. The intrinsic components that lead to the organization of such cytoskeleton are not yet clear. Furthermore, the plane of cell division is also controlled by the orientation of mitotic apparatus. It is very important to know more about microtubules and micro filaments and their role in organogenesis (refer chapter proteins). The assembly of tubulin units into microtubules and their orientation within the cell is determined by the position of nucleating centers and the availability of polymerization factors like Tau, Maps, and GTP & ATP. In a developing system, the assembly and orientation of microtubules and microfilaments are under close spatial and temporal regulation. In recent years, the role of microtubules and microfilaments in hormone induced growth and development is attracting greater attention.

Morphogenesis involves growth and differentiation. Growth generally refers to the enlargement of the cells or the production of a group of cells of similar kind. In contrast to growth, differentiation involves the development of a cell or an organ which is structurally and functionally different from the cell it originates. In the development of a plant both growth and differentiation, go hand in hand. Plants like animals start their development from an unicellular structure, which by a series of pre-determined cell divisions produces a variety of cell structures and organs. Except for a few cell types such s sieve tubes among phloem elements, almost all living cells of all kinds are totipotent and if proper nutritional conditions are given they are capable of giving rise to a complete plant. This developmental potential is inherent in their genetic make-up. In recent years, however, it is becoming clear that the differential expression of the genetic potential is controlled by plant growth substances that are produced in the system itself. It is their interaction with the genetic apparatus which results in the full expression of the phenotype of the plant.

Supplement material from my thesis

PLANT GROWTH REGULATING SUBSTANCES: HISTORICAL ACCOUNT: The existence of a plant growth substance was first perceived by Charles Darwin [1], who in his own elegant way, described how plants perceive the stimulus of light and respond to phototropic growth. Since then, botanists have made great strides in unraveling the mystery of the diffusible substance, which is responsible for growth. Kogl and Haagen Smit isolated and identified the growth promoting substance and coined the name hetero auxin or as it is known today, indole-3-ylacetic acid (IAA) [2,3]. This gave a great impetus to the plant scientists all over the world, and the search culminated in a series of discoveries of plant growth substances, such as Gibberellin B (GAB) by Yabuta and Sumuki (1938), Cytokinin by Miller and Skoog (1955), and Abscisic acid (ABA) by Robinson and Wareing (1964). Simultaneously a host of synthetic growth promoting as well as growth inhibiting substances were also made available to plant scientists. This led to intensive investigations on the effect of the various plant hormones on plants. Various hormones, elicited a wide array of responses in different kinds of tissues. It was soon realized that the physiological response to any given growth substance depends, in the first instance, on the type of cell receiving the stimulus. For example, a developing leaf cell, in a barley plant, will respond to GA3 by elongating, whereas an aleurone cell, from the same plant, will respond by producing a-amylase. The specificity of response is built into the cell by its previous developmental programme. It is curious to note that the highly specific animal hormones affecting specific cells have no counterpart in plants. In general, it can be stated that the auxin is known as growth promoting substance, but the same substance is also capable of eliciting responses such as, apical dominance, root initiation, prevention of leaf abscission, fruit setting and so on. While Gibberellic acid is known to be effective in promoting growth, induction of a-amylase, bolting and flowering, Cytokinin is considered as the hormone that controls cytokinesis, a part of cell division. Abscisic acid is known to act as a growth inhibitor, controlling processes such as bud dormancy and seed dormancy. Ethylene, recognized as a hormone recently, shows bizarre effects. In the development of the plant body, it is the interaction between the various hormones that controls and regulates the process of morphogenesis.

HORMONAL INTERPLAY

Although Haberlandt (1913) considered the tissue and organ culture in sterile conditions as a theoretical possibility, it was realized as a practical proposition for research by two independent workers, Kottle (1992) in Germany and Robbins (1922) in America. The advent of tissue culture techniques opened up a new vista in the studies of hormonal interactions in morphogenesis. When plant growth substances are supplied to plant tissues exogenously, each of them elicits unique responses. However, many responses are overlapping. But if two or more different hormones are supplied in known concentrations, in many cases synergistic or antagonistic responses are exhibited and this effect is referred to as hormonal interaction.

Studies on tobacco pith callus tissue have revealed that the relative concentrations of hormones control the expression of callus either into roots or shoots. Low, high and intermediate ratios between auxin and cytokinin induce roots, shoots and callus respectively [4]. Auxin-cytokinininduced shot formation in the callus is inhibited by GA3; however GA3 enhances the growth of the callus. The inhibitory effect of GA3 can be alleviated by the addition of ABA, which by itself is a weak inhibitor of callus growth [ 5,6 ]. The form of shoots developed from callus is dependent on GA3-cytokinin ratio. The higher ratios induce the formation of tall, spindly shoots with narrow leaves, whereas lower ratios produce dwarf shoots with rounded leaves [7].
Specific hormones are found to influence the relative levels of other hormones and this is very well substantiated in the culture system, where auxin-cytokinin induced callus growth is reduced by certain culture conditions, which also inhibit the biosynthesis of GA [6]. Thus, the interactions of hormones appear to be very complex.

This complexity is further compounded by the recent observations, in which, it has been shown that each hormone can influence the biosynthesis, degradation, conjugation or transport of one or the other hormones. The end ogenous level of IAA in a plant tissue is enhanced by the exogenous application of GA or Kinetin. But, ethylene, which is known to be induced by excess auxin, reduces the level of auxin itself. Again, auxin levels can be increased by the addition of cytokinin. This complex relationship appears to be very fascinating, because of the findings in which cytokinins are known to bring down the levels of ABA, through the increased biosynthesis of GA. But ethylene promotes the synthesis of ABA, which in turn enhances the levels of ethylene. The close relationship between ethylene and auxin provides an excellent feed back control mechanism. Letham et al. (1978) have illustrated the complex interaction between plant growth substances as indicated in the diagram (Fig. 1) [8]. Further investigations into hormonal interactions at molecular level, will have far reaching impact in understanding morphogenesis at the molecular and sub cellular levels. ULTRASTRUCTURAL CHANGES DURING DIFFERENTATION: MICROTUBULES, MICROFILAMENTS IN CELL POLARITY AND ORGANOGENESIS: The temporal and quantitative changes in the regulation of gene activity forms the basis for the ultra structural changes determining the process of cell development and

Fig. 1: Hormonal Interactions. Fig.1: Hormonal interactions which affect hormonal levels in plant tissues. Open arrow from A B indicates hormone A causes increase in the levels of B. Closed arrow from A B indicates that hormone A decreases the levels of B. differentiation. The molecular events leading to such ultra structural organization are not very clear. For example, it is not clear how and why a fertilized egg in the embryo sac, always undergoes a polarized division in such a way, that one of the two cells, equal or unequal, develops into radicle and the other into plumule. This polar behavior of the cells at the earliest part of their development is the function of intracellular compartmentalization, regulated and governed by certain intrinsic interactions of nucleo cytoplasmic components, whose expression in turn is controlled by the inherent genome. Polar growth like cap (reproductive organ) formation in Acetabularia, rhizoidal development in Fucus, hold-fast development in Cladophora are some of the best studied systems in the plant kingdom. In the case of zygote development in Fucus, if the spherical zygote is subjected to unidirectional light treatment, the rhizoid develops at the shaded side of the cell. If such a programmed zygote is subjected to electron microscopic investigation, it is observed that in the region of rhizoid formation, large number of osmophilic bodies and undefined extracellular materials accumulate. The peri nuclear region showed highly polarized finger shaped projections towards the site of rhizoid initiation. Along with these charges, heavy concentration of mitochondria, ribosomes and dense fibrillar vesicles are also found. These structural changes represent an expression of a fixed axis for rhizoidal growth [9]. The time taken for such polarity fixation has been determined to be 10-16 hrs. During this period, if the cells are treated with cycloheximide (CHI) for 9-15 hrs after fertilization, they still show a fixed polar axis. However, if cytochalasin-B, which is known to disrupt the microfilaments, is present during polarity fixation, the polar axis remains labile and axis establishment is prevented [10].

The role of cytochalasin-B and colchicines, in lactin Con-A induced cap formation in animal cells, has been reviewed by Edelman (1976) and Nicolson (1976) [11,12]. The authors have explained how the microtubules and microfilaments control the mobility, stabilization and topographical distribution of cell membrane and surface components during lectin induced cap formation. The cross linking of membrane receptors embedded in the plasma membrane, to a variety of fluorescent labeled polyvalent ligands such as antibodies or lectins lead to form patches and clusters which ultimately culminates in the cap formation at one end of the cell. Similar to cap formation in animal cells, cortical clearing in the region of rhizoidal emergence is observed in zygotes of the plant Pelvetia fastigata [13]. In the same system, Peng etal. (1976) have shown the deposition of new membrane patches (30 um) covering the rhizoidal region at the time of cortical clearing [14]. These studies clearly suggest that microtubules and microfilaments play a significant role in cell polarity, which is a prerequisite for organogenesis. Ever since, the discovery of microtubular components in few plant cells by Lederbetter and Porter (1963) [15] the presence of such structures, in most of the plants, has been established. Basic components of the microtubules are not known to be tubulin monomers, which exist in the form of heterodimers, consisting of and subunits, of molecular weight 110,000- 120,000 daltons [16]. Several proteins are associated with microtubules, of which the proteins known as TAU and microtubule associated proteins (MAPS) appear to be important, for they take part in assembly and disassembly of microtubules [17,18,19]. In addition to the above components, the presence of nucleating centers or organizing centers within the cells, account for an ordered or controlled assembly of microtubules [20,21]. In a developing system, microtubule assembly is under close spatial and temporal regulation. The nucleating centers function in controlling initiation, orientation, directionality and patternization of microtubular polymerization. Recently attempts have been made to isolate microtubules and study their organization in plants [22,23]. The cross reaction of antibodies, raised against porcine brain tubulin, with the plant protein, strongly suggests the similarity of plant tubulin with animal tubulin, at least at the antigenic level. Considerable attention has been given to relate the orientation of microtubules during plant hormone induced growth. Hormone induced growth in Vigna angularis, hypocotyl of Lactuca sativa and coleoptile segments of Triticum vulgare, is inhibited by colchicines, urea and vineblastin sulfate [24-26] and this inhibitory effect is attributed to microtubular disruption [27]. Similarly, the inhibition of protoplasmic streaming in Avena, and growth inhibition in Zea mays root by cytochalasin-B are again attributed to the role of microfilaments and microtubules [28,29]. Electron microscopic studies on the deposition of cellulose in cell walls during secondary growth and movement of secretory vesicles are shown to be directed by microtubules [30]. However, the available information on plant hormone induced changes in the microtubular assembly, synthesis and their role in morphogenesis is scanty.

PLANT HORMONES AND NUCLEIC ACID METABOLISM

Plant growth regulating substances, very often, exhibit dramatic effects on growth and development in plants. The molecular mechanisms which control such processes involve changes in nucleic acid and protein metabolism. DNA replication, transcription and translation are the three

major molecular events involved in growth and differentiation. An understanding, as to how plant hormones control such molecular mechanisms, as to how plant hormones control such molecular mechanisms, is very pertinent to interpret morphogenesis at the molecular level. In spite of the enormity and complexity of the problem involved, considerable Progress has been made in this field.

DNA METABOLISM

A majority of investigations reveal that hormone induced cell elongation does not require DNA synthesis [31,32]. However, Wardell (1975) has reported that auxin, in flowering tobacco plants, enhances DNA synthesis within an hour of treatment. On the contrary, in pea root cortical cell cultures [33], Libbenga and Torrey (1973) have shown that enhanced DNA synthesis is possible, only in the presence of auxin and kinetin together, but not with auxin alone [33a].

In long term experiments, most of the growth promoting substances enhances DNA synthesis which is always accompanied by cell division [34]. In cucumber hypocotyls, GA3 promoted growth is fluoro-deoxyuridine (FUDR) sensitive. This increase in DNA synthesis, in response to GA3 is due to the increased chloroplast proliferation, which is prevented by chloramphenicol. The above experiments suggest that GA3 has some preferential effect on chloroplast biogenesis [35, 36]. On the other hand, hormones such as ethylene and Abscissic acid inhibit DNA synthesis. This correlates well with their growth inhibitory effects in vivo [37-39]. However, it is not clear whether this effect is direct or indirect. Although, cytokinin is known to induce cell divisions, it does not affect DNA synthesis by itself as a prelude to cytokinesis. However, it promotes DNA synthesis in the presence of auxin [40]. Hence, it is suggested that auxin has a permissive role in DNA synthesis, while kinetin stimulates it. This is further exemplified in an experiment, where hormone depleted and aged tobacco pith explants, could be induced to undergo mitosis without the accompanying DNA replication by the addition of cytokinin. This suggests that cytokinin perhaps regulates cell division, by controlling the synthesis of specific proteins for mitosis rather than acting directly on DNA replication [41,42].

DNA SEQUENCE COMPLEXITY

In higher plants, it is estimated that the amount of DNA present per cell ranges from 1pg 100 pg and this amount of DNA codes for more that 10 6 different proteins. The rough estimates indicate that there are 50,000 or more structural genes which are expressed at one time or the other during the life of the plant [43]. The number of structural genes operating in different plant structures like root, stem, leaves and flowers may vary. On the basis of hybridization studies between polysomal RNA and 3H-labelled single copy DNA, it has been estimated that there are about

2700 diverse structural gene transcripts in the tobacco leaf cells [44]. DNA, in higher plants, consists of three base sequence classes i.e., highly repeated, moderately repeated and unique. Unique sequences are believed to code for mRNA sequences [45]. These sequences can be detected, characterized and quantified by studying the reassociation kinetics of denaturated and sheared DAN. Highly repeated sequences reassociate rapidly at low cot values ( cot = concentration of DNA in mole nucleotide per liter X time in seconds ) and unique sequences reassociate slowly at high cot values [45]. Studies on changes in DNA sequence complexities in response to phytohormones in plants are few. Nevertheless, reannealing studies of DNA isolated from the control and auxin treated artichoke tubers, suggest that auxin causes an apparent loss in the rapidly reannealing fraction of DNA, but leads to some dramatic changes in the cot values for other fractions [46]. On the contrary, GA3 has no effect on the DNA isolated from the roots of Cucumber but DNA from the treated shoots and leaves of the same plants, exhibit a nine-fold increase in the intermediate reassociation fractions. According to Britten and Davidson (1969) such sequences may have a regulatory role [47]. In cymbidium protocorm tissue, while auxin causes an increase in AT-rich DNA, GA3 promotes an increase in GC-rich DNA [48]. Similar studies have been carried out in various plants such as wheat embryos, pea epicotyls etc. [49] However, the above studies do not indicate whether, phytohormone induced changes in DNA population is due to primary or secondary effects of the hormones.

PHYTOHORMONES AND TRANSCRIPTION

Protein synthesis is a crucial event in growth and differentiation, and therefore the effect of phytohormones on transcription and processing of different kinds of RNA would determine the process of morphogenesis.

AUXIN-MEDIATOR/ACCEPTOR PROTEINS AND RNA SYNTHESIS

The mechanistic aspects of auxin function appear to be very complex. Previous investigations have suggested that there may be one or two cellular sites at which auxin may act and elicit its responses on growth and differentiation. One such site is found to be located in the plasmamembrane and the other may be a non-plasma membrane site located either in the cytoplasm or nucleus. Auxin-mediated early cell enlargement is known to function through the activation of membrane bound factors [50-52]. The early growth is insensitive to both cycloheximide and actinomycin D; hence neither RNA nor protein synthesis is required for this function. However, the second phase of growth requires both RNA and protein synthesis. The auxin-induced mechanism of early growth is speculated as due to the cell-wall loosening effect caused by the secreted protons, which are pumped out by the auxin-activated membrane bound factors [53]. The existence of soluble auxin-binding proteins of molecular weight 3,15,000 and 10,000 daltons have been reported in dwarf

bean seedlings and soybean cotyledons respectively [54,55]. It is not clear, whether these auxin binding proteins have any role in transcription. Hardin et al. (1972) [56-58] have demonstrated that in soybean hypocotyls and onion stems, auxin treatment results in the release of a plasma membrane factor into the cytosol, which stimulates -Amanitin sensitive RNA polymerase activity. However Mathyse and Philips (1969) have shown the existence of auxin-receptor complex in the nuclei and this factor is responsible for the enhanced RNA synthesis. Auxin-receptor protein has been isolated from coconut nuclei and shown to have a molecular weight of 10,000 daltons. It binds to the auxin non-covalently at a molar ratio of 2:1. In the presence of transcribing system, consisting of amanitin sensitive RNA polymerase (presumably RNA polymerase II), native DNA, and an initiation factor, the auxin binding factors stimulate RNA synthesis 2-3 fold, only in the presence of auxin. Although, these factors are not well characterized, this constitutes a good evidence for the existence of auxin-receptor proteins. Using changes in melting point profiles and bathochromic shifts in DNA and chromatin, Fellenberg (1971) and Bamberger (1971) have claimed that auxin interacts with DNA and chromatin directly, there by facilitating transcription [62,63]. However, these observations turned out to be due to changes in the PH of the medium, rather than to the direct effects [64]. While the studies on auxin-histone interactions have not yielded any significant information, nonhistone proteins are now considered to be the most likely candidates influencing specific gene transcription. Non-histone proteins are heterogenous and include a number of enzymes and structural proteins. They are species-specific and tissue-specific. Their phosphorylation pattern changes with the different states of gene activity [65,66]. Such an involvement is very well exemplified in the studies involving progesterone and estrogen receptor complexes with the chromatin of target cells in different animal systems.

In plants, the existence of zone-specific non-histone proteins is known in soybean hypocotyls [68]. Recently, 2,4-dichlorophenoxy acetic acid (2,4-D) induced changes in the phosphorylation of nuclear proteins has been shown in soybean, but the correlation between auxin induced RNA synthesis and the phosphorylation pattern ahs not been established unequivocally [69]. Tissere et al. (1975) have isolated factors from the acidic fraction of the chromatin proteins from the lentil roots, referred to as , , and factors. On auxin treatment, the , and factors remain unchanged but the factor increases two-fold. While the factor regulates RNA polymerase I , affects polymerase I and II. Hence these factors are known to regulate the activity of RNA polymerases without enhancing the synthesis of the enzyme [70]. Generally, auxins induce RNA synthesis. Silber and Skoog (1953) for the first time correlated auxin-induced cell enlargement with enhanced RNA synthesis [71]. Since then, considerable work has been carried out on the auxin induced changes in the levels of various RNA species.

Auxin has been reported to preferentially increase rRNA content in pea epicotyls and soybean hypocotyls [72]. But, it has also been shown that the enhancement in RNA synthesis occurs well after auxin has effected growth. It has been found that 5-fluorouracil [5-FU] does not prevent cell enlargement in soybean hypocotyls and artichoke tuber, but auxin induced growth in oat coleoptile and soybean hypocotyls is inhibited by actinomycin D. The above observations suggest that auxin induced growth in the earlier stages does not require RNA synthesis, but requires the synthesis of messenger RNA later [73,74]. Investigations, using polysome content as a measure of quantification of mRNA synthesis have shown that auxin induced polysome levels are actinomycin D sensitive, but 5-FU insensitive. This indicates that the synthesis of new ribosomes is not necessary for the increase in the levels of polysome [75-78]. Verma et al. (1975) using in vitro cell free protein synthesizing system and cellulase specific antibody, have shown that the auxin induces the synthesis of cellulase-specific RNA [79]. However, increase in other m RNA populations is not ruled out. It is not clear, whether, the auxin induced messenger RNA is synthesized as a precursor heterogenous RNA (hnRNA) in plants. In short-term labeling experiments (45 minutes), auxin enhances the labeled precursor incorporation, preferentially into the nuclear fraction. Long term labeling experiments, however indicate that incorporation into both nuclear and cytosolic fractions is enhanced [80]. Recent studies with Petroselium sativum (parsley), have demonstrated that most of the poly (A)-RNA is synthesized as hnRNA. This is established by DNA-RNA hybridization-kinetic studies [81]. Attempts have also been made to use Escherichia coli RNA polymerase core enzyme, to measure transcription of DNA/chromatin, as an index of differential gene expression chromatin, isolated from auxin treated soybean seedlings has been found to code for synthesis of many fractions of RNA species, of which the synthesis of TB-RNA, several fractions of hetero disperse RNA AND 4S-5S RNA are much more than the control. This chromatin preparation enhances labeled amino acid incorporation into proteins by 2-3 fold in coupled transcription-translation assays [82,83]. Similar studies involving the plant tissues have clearly indicated that auxin can influence polymerase activity, as well as template activity.

GA-BINDING/MEDIATOR PROTEINS AND RNA SYNTHESIS

The presence of GA3 binding protein in pea stems, epicotyls and other plants has been reported. The approximate molecular weight of the GA 3 binding proteins, extracted from dwarf epicotyls of pea plant, has been determined to be 5, 00,000 and 60,000. Both the fractions bind GA3 noncovalently and easily exchange with non-labeled GA3. Not much is known about the effects of GA3 binding proteins on transcriptional events. Isolated pea nuclei do not respond to exogenously added GA3 but, if the nuclei are isolated after GA3 treatment, transcription is enhanced [86]. Attempts to localize GA3 binding sites, in cells by

autoradiography or by cell fractionation, have yielded limited results [87]. GA7 has been shown to interact with DNA containing higher AT than GC content. It is also reported that GA7 interacts With DNA isolated from a number of plant sources, as well as some phages, but it does not interact with either animal or bacterial DNA. This specificity of GA7 interaction has not been explained. In the presence of DNA Ligase, GA7 causes the formation of covalently linked loops in cucumber DNA, but cytokinins and auxins do not have any effect. Recently, it has been reported that at low concentrations of ethidium bromide, GA4, GA7, or GA3, show synergistic effects on elongation in cucumber hypocotyls, perhaps by exposing binding sites in DNA and thus making gibberellic acid more effective. Studies on the effect of GA3 on transcription of either isolated chromatin or isolated nuclei from pear or cucumber show, that the increase in RAN synthesis is due to increased RNA polymerase, as well as due to the increased template availability [90]. A wide variety of tissues such as barley leaf segments, clover seedlings, lentil epicotyls, potato buds, cucumber hypocotyls and sugar beet have been used to study GA3s effect on RNA synthesis. A significant amount of information on the effect of GA3 on RNA synthesis comes from the studies with barley aleurone cells. GA3 causes a selective increase in m RNA for -amylase, which on release to the endosperm facilitates the mobilization of carbohydrate source for the growing embryo. If actinomycin-D is added along with GA3, the increase in -amylase synthesis is inhibited [91]. The labeling of poly (A)RNA, specific for -amylase, shows an increase after GA3 treatment with a lag period of 3-4 hrs. This correlates well with the lag period of -amylase synthesis observed in vivo. However, Higgins et al. (1976) and Carlson (1977) have suggested that GA3 activates the preexisting -amylase m RNA by post transcriptional process into an active translatable form of m RNA but, Rodaway et al. (1978) have considered this possibility as very unlikely similar effects of GA 3 on poly (A)-RNA synthesis have been established in etiolated maize seedlings, but their translational products have not been characterized.

CYTOKININ-RECEPTOR PROTEIN AND RNA SYNTHESIS

The presence of cytokinin-receptor protein has been indicated by the work of Mathyse et al. (1969) [97]. does not stimulate RNA The cytokinin-protein complex enhances RNA synthesis in vitro in synthesis. Similar studies using nuclei-rich preparations presence of pea bud chromatin or DNA as template and e. coli or calf thymus, kinetin-receptor from soybean, tobacco callus and pea buds, have shown that the cytokinins enhance [ 14 C] ATP incorporation into TCA precipitable and KOH digestible RNA fractions by 40-100% within 5-10 minutes after treatment. To the phytohormone-starved tobacco pith callus, the addition of cytokinin alone does not cause any major charges in RNA metabolism, but cytokinin with auxin treatment enhances [32 p] orthophosphate incorporation into polysomal RNA within 3 hrs of treatment [98,99]. On the contrary, in the hypocotyls of soybean, even at early stages after treatment, the auxin induced incorporation of [14 C] uridine into RNA is inhibited by kinetin. After 6 hrs of

incubation with cytokinin and auxin together, the inhibition of labeled precursor incorporation into rRNA and tRNA is 90% and TB-RNA 30-50%. However, the labeling into D-RNA is not affected [ 100-103 ].
Cytokinin-mediated increase in the specific isoacceptor leucyl-tRNA species has been detected in cotyledons and hypocotyls of beans [ 104, 105 ]. This could be the result of and effect of direct synthesis or on posttranscriptional modifications. In general, it seems that the occurrence of cytokinins in tRNA is not longer viewed as necessary for eliciting the cytokinin response [106-108].

ETHYLENE-RECEPTOR AND RNA METABOLISM

Although no coherent information is available regarding ethylene binding to receptor proteins, transcriptional studies with isolated chromatin from ethylene treated tissue, show qualitative changes in the RNA species synthesized. There is a change in the nearest neighbor frequency of the RNA synthesized, thus indicating transcription of new template in response to ethylene [37]. Generally ethylene is known to induce abscission and fruit ripening in plants. During abscission, ethylene enhances the synthesis of cellulase and pectinase, which are responsible for the breakdown of the cell wall and pectin [109, 110]. In cotton, Coleus and bean plants, ethylene induced abscission can be prevented by actinomycin D. Furthermore, using 5-FU as an inhibitor of rRNA synthesis, investigations have demonstrated that the ethylene mediated abscission involves induction of specific mRNA, but the products have not yet characterized. Similar results have been obtained in the ethylene induced ripening of Pyrus mallus fruits [111]. In ripening tomato fruits, ethylene brings about changes in the relative amounts of isoaccepting species of leucyl-, lysyl-, methionyl- and tyrosinyl-tRNAs.

ABA-RECEPTOR AND RNA SYNTHESIS

ABA affects a number of physiological processes, such as dormancy, leaf senescence, abscission, seed germination and flowering, and also antagonizes the effects of other plant hormones, in a number of plant tissues. It is not clear, whether such physiological responses to ABA are brought about through mediator molecules. However, various reports indicate that the effect of ABA is on RNA turnover and translational process.

ABA has been shown to inhabit the transcriptional capacity of isolated chromatin [112]. This inhibitory effect is ascribed to the effect of ABA on polymerase activity [113,114 ]. In Fraxinus embryos [115], ABA

inhibits labeled precursor incorporation into RNA, and reduces polysome formation, but protein synthesis is not affected. The above results have been interpreted to mean that ABA prevents the synthesis of mRNA and other RNA species, thereby affecting growth.

ABA, in pear embryo and lentil roots, causes a preferential increase in UMP rich RNA and a substantial decrease in GMP, but there is not change in AMP, CMP contents of RNA (115 a). This observation is significant in view of the recent observation, that translation control RNA (tcRNA), which controls translation of certain mRNAs has very high (50%) content of UMP nucleotides [116]. Additional support to the above observation comes from the findings that ABA treated germinating seedlings contain UMP rich (40-50%) RNA species [117]. It is not clear whether this high level of UMP rich RNA is due to new synthesis or preferential degradation of the other RNA species.

ABA also inhibits GA3 - promoted

-amylase synthesis, in barley aleurone cells. Some investigators

have shown that ABA inhibits [14 C] uridine incorporation into polydisperse RNA [92, 118]. There are also reports that ABA does not inhibit the synthesis of any species of RNA [119]. In this context, the observation about certain short chain fatty acids (of C5 and C9 length) at a concentration of 10-4 M, inhibiting GA3 induced amylolysis in barley endosperm is interesting. Such short chain nonionic fatty acids have been detected in many germinating seeds such as oat and fenugreek [120].

PHYTOHORMONES AND POST TRANSCRIPTIONAL EVENTS

Perichromatin and Precursor RNAs:

Most of the RNA species, transcribed within the nucleus, are in the form of larger molecules and these are processed and transported to the cytoplasm across the nuclear membrane pore complex [121]. The existence of extra chromosomal and extra nucleolar ribonucleoprotein structures called perichromatin granules (presumably precursors of mRNAs) in rat liver nuclei and plant cells, has been reported as early as 1969 [122]. The transport of such particles across the membrane pore complex has been speculated. Active movement of such chromatoid body (containing RNA) across the pore complex, during rat spermatogenesis is every well documented [123]. In plants, synthesis of large precursor RNAs (hnRNA) and their processing into the mature forms have been reported [43,124,125].

POLY (A) AND CAP

During the processing of hnRNA, additional structures such as the cap at the 5 en d poly (A) chain at the 3 end are tagged to the mRNA. Addition of poly (A) segments of 50-200 nucleotides, at the 3 end of the hnRNA or mRNA is catalyzed by poly (A) polymerase [126]. The existence of such polyadenylated mRNA has been demonstrated in the nuclei and cytoplasm of plant cells [93,127,125]. However, not all mRNAs of RuDP carboxylase in chloroplasts lack such mRNA (?), it is one such example [128].

Capping at the 5 end of the mRNA involves the addition of 7 -methylguanosine, which is linked by a 5opopopo5 triphosphate bridge to a 2-0-methylated Adenine [129,130]. The cap structure is implicated in influencing the processing of pre-mRNA, initiation of translation , transport of mRNA and stability of the same [131, 132]. However, cap is not required for the translation of all mRNAs [133]. Recent reports suggest that the prokaryotic mRNAs, which are capped in vitro, translate very efficiently in cell-free eukaryotic protein synthesizing system [134]. Sonenberg et al. (1979) have isolated a cap binding protein whose molecular weight is 24,000. This protein stimulates translation of capped mRNAs in an in vitro system derived from HeLa cell extracts, under conditions that do not increase translation of non-capped mRNAs from satellite necrosis virus [134a]. In plants, there are not many reports about cap structures in mRNAs. However,

addition of 7-methyl guanosine 5-monophosphate (m 7G5P) to the French bean seed mRNA programmed cell free protein synthesizing system derived from wheat germ, inhibits [35 S] - methionine incorporation into

proteins by 90%, thereby suggesting that the mRNA isolated from French be an cotyledons are capped [135]. The role of plant growth substances in the capping process is not clear.

The non-coding sequences found between the first AUG codon and the cap at the 5 end and the last terminator codon and the first adenine nucleotide of the poly(A) segment at the 3 end are suggested to play an important role in initiation and termination of protein synthesis [136]. Such information is not available with plant mRNAs.

INFORMOSOMES

The messenger RNA is transported from the nucleus to cytoplasm as m RNA protein complex (m RNP). These m RNP particles may release the m RNA for polysome formation or the m RNPs may remain in an inactive form. The latter particles are referred to as informosomes. Such informosomes are found in a number of embryonic tissues [93,137,138] and other non-embryonic structures such as stems, tubers, etiolated leaves, dry mosses, spores of ferns, Acetabularia and Dictyostelium . Such inactive m RNPs have also been reported in animal cells [145-147].

The m RNPs that are found in most of the organisms have poly(A) tail at 3 end [148]. The presence of such m RNPs ensure the preservation of m RNAs during periods of dormancy and certain adverse environmental conditions such as drought, lack of optimal photoperiodic stimulus and lack of light of actinic wavelength.

ACTIVATION AND INACTIVATION OF m RNAs

The activation of preexisting dormant m RNPs to external stimuli has been reported in a variety of systems such as germinating seeds [138,159] rejuvenating moss[141], etiolated leaves exposed to light [140], ferritin synthesis in rat liver [146] and fertilization in sea urchin eggs [160]. Specific effects of plant hormones on this process have also been documented. These examples include GA3 effect in barley aleurone

cells and spores of Anemia phyllitidis [93,149], IAA effect in pea epicotyls [79] and cytokinin effect in Glycine max [102,103]. In all the above mentioned cases, when external stimuli are provided, the dormant m RNPs get activated and the rate of protein synthesis is enhanced, even in the presence of actinomycin-D, or 5-FU, thereby indicating that the enhanced protein synthesis is independent of new RNA synthesis. It is not clear, whether these external stimuli or added phytohormones act directly on m RNPs or function through other mediating-factors, which may either activate ribosomal machinery or bring about the conformational changes in the secondary structure of m RNPs.

TRANSFER RNA:

Transfer RNA (tRNA) is also synthesized as a large precursor and then processed into smaller products [131]. Transport, slicing and secondary modifications such as methylation, thiolation of pre-tRNAs are some of the important controlling processes, which make available, the functional tRNAs for protein synthesis. Addition of CCA at 3 terminal of t RNA and activation of amino-acyl tRNAs and activation of amino-acyl t RAN synthetases are other crucial steps that regulate the translation processes in Pisum sativumseedlings, GA3 is known to enhance aminoacyl t RNA synthetases by 5-8 fold [149]. In early 1960s, auxin was suspected of form a complex with t RNA thus regulating t RAN function [150]. However, ti is now considered that this may not be the case [151,152].

As the genetic code is degenerate, it is thought, that multiple iso-accepting species of tRNAs may exert a regulatory role in the translation of mRNAs [153] and the relative changes in the population of such isoaccepting species of t RANs [154] and the relative changes in the population of such iso-accepting species of

tRNA during different stages of development, has been recorded in plants [155,156]. The presence of cytokinins-effect may be mediated through tRNAs [106]. The occurrence of the highly active cytokinin6 de (6-3 methyl-2-butenyl amino purine) adjacent to the A base of the anticodon triplet has been demonstrated in yeast serine- tRNA I, II and E. coli- tRNA [157]. Such cytokinin containing tRNAs have been identified in various plants and it is felt, that cytokinins may exert their effect on growth through regulating the translational process. However, recent studies [158] show that there is no correlation between the effect of hormone-induced growth and the presence of cytokinin in tRNA species. Nevertheless, there are evidences to show that cytokinins can affect the tRNA activities either by regulating the levels of tRNAs or by secondary modification of tRNA by methylation or by changing amino acyl-synthetase activity [108].

CONTROL OF PROTEIN SYNTHESIS BY PHYTOHORMONES

In radish cotyledons, cytokinin-enhanced protein synthesis does not require mRNA synthesis de novo [161]. Similarly cytokinin enhances protein synthesis in soybean callus, as early as 30 minutes after treatment [102]. Klambt (1976) [99] has demonstrated that N5 (2- isopentyl)adenine stimulates cell-free protein synthesis slightly in systems derived from tobacco pith and corn extracts. Such stimulation is suggested as due to selective binding of cytokines to a specific protein of the ribosome. Fox et al. (1975) [162] have identified one low and one high affinity cytokinin binding site on wheat germ ribosomes. The binding affinity is lost on boiling the 0.5M KCL wash or on treatment with trypsin, suggesting that this high affinity receptor is a ribosomal protein. Such cytokinin-binding protein from tobacco callus is associated with 4OS ribosomes [163]. In animal systems [164] increased or reduced phosphorylation of ribosomal protein controls the rate of protein synthesis. Cytokinin is also thought to enhance protein synthesis through dephosphorylation of ribosomal proteins. Tapfer et al. (1975) [165] have found that, when cytokinin requiring soybean callus is transferred to a cytokinin deficient medium, the decreased rate of protein synthesis is accompanied by increased phosphorylation of some ribosomal proteins.

In soybean hypocotyls, a parallel situation is found where Auxin causes increased protein synthesis in a cell-free protein synthesizing system [166]. Travis et al. (1976), have proposed, that the polysome formation is dependent on ribosome activation as well as mRNA supply. In the same system they have also observed the incorporation of labeled amino acids into 3 ribosomal proteins, one of which shows several fold higher incorporation that that of the control. However, ribosomal dissociation studies, clearly indicate that these auxin induced proteins are associated with 60S ribosomal sub-units. Evidence for hormone-induced protein synthesis, independent of mRNA synthesis, is also found in Pea epicotyls for [99]. Here, auxin has been found to activate the mRNA specific for cellulase at very early periods, when there is no increased mRNA synthesis. The poly (A) RNA isolated from treated segments, on translation in vitro, shows enhanced synthesis of

cellulase. The poly(A) RNA from control plants hardly codes for any cellulase. The above results suggest that the auxin perhaps activates the preexisting, dormant mRNAs into active translatable form of mRNA. Thus, the evidences presented above indicate that most of the growth promoting hormones increases protein synthesis through increased mRNA synthesis as well as through activation of dormant mRNAs or the ribosomal system. Such dual action of phytohormones is well documented (165 and 166) ABA, in general, appears to exert an inhibitory effect on the synthetic processes. Although, ABA inhibits light-promoted unrolling and greening of etiolated leaves in wheat, it does not prevent light induced polysome formation [167]. In etiolated leaves of barley seedlings, ABA not only reduces the amount of polysome formation in the dark, but also inhibits light-induced increase in polysome size [168]. In barley aleurone cells, GA3 induced -amylase synthesis is inhibited by ABA and this inhibition is prevented by the addition of cordycepin. This observation indicates that ABA has both transcriptional as well as translational control [169]. In this context, increase in UMP-rich RNA component, in response to ABA treatment, is highly significant. Heywood et al. (116) have isolated a translational control RNA of mol.wt. 10,000 daltons, which is rich in UMP. This translational control RNA is believed to play a significant role in translational control where it inhibits translation of mRNAs, probably by pairing with poly-AMP segments of mRNAs [116 &170]. This observation correlates well with the effects of ABA on growth inhibition in plants.

PHYTOHORMONES AND RNA PROTEIN TURNOVER

The regulation of RNA and protein turnover is another important facet of cellular differentiation. Phytohormones are known to regulate such events. ABA, when applied to excised barley leaves, enhances chromatin bound nuclease and deoxy-ribonuclease (DNase) activities [180]. In excised Avena leaves, it affects one out of the four nuclease isoenzymes [171]. Similarly in lentils (Lens culnaris), ABA reduces the levels of RNA and also inhibits growth [172]. However, in maize coleoptiles, ABA suppresses the incorporation of radioactive precursors into RNA without affecting the levels of RNase for 8 hours [173]. Auxin is also implicated in suppressing the RNase activity in number of cases [174,175]. However, no direct relationship between nuclease levels and RNA stabilization has been established. In pea epicotyls, IAA elicits a large increase in membrane-bound RNase, which in turn is suppressed by kinetin [176]. Similarly, a correlation has been established between nucleases and auxin treatment in wheat root callus cultures [177]. In senescing leaf tissues, increased activities of RNase and DNase are correlated well with the decreased content of RNA and yellowing of the leaf. Cytokinins suppress such activities and delay senescence, which is popularly known as Richmond and Land effect [178-180]. In the absence of a

water stress on leaves RNase activity is suppressed by kinetin but the same hormone under water stress condition enhances RNase activity [181]. In mustard and corn leaves, during sene scence kinetin and 6-benzylaminopurine retard the loss of radioactive label from prelabelled proteins [182, 183]. In wheat leaves, cytokinin suppresses proteolysis of RuDP-carboxylase and this is prevented by CHI, suggesting that the proteolytic enzymes are being synthesized de novo during senescence [184]. During differentiation in Dictyostelium (from plasmodial stage to sporangium) [144], bluegreen algae (vegetative to heterocyst) [185], and soybean callus cells (non-dividing to dividing state) [103], appearance and disappearance of some specific proteins have been noticed. This selective appearance and disappearance of certain proteins during developmental process may involve differential expression of genes. Such transitory proteins may play a very important role in morphogenesis.

ADVENTITIOUS ROOT INITIATION AS A MORPHOGENETIC SYSTEM

Introduction: In the present investigation, hormone induced root initiation in the hypocotyls tissue of Phaseolus vulgaris Linn. Is used as a model system, to study the molecular events involved in the differentiation process. A brief review of the literature on adventitious root formation is therefore presented here. The root that develops from any part of the plant body other than the radicle is called an adventitious root. Duhmel du Monceau (1758) for the first time explained adventitious root formation in stem as due to the downward movement of sap. Sachs (1880) postulated the existence of a highly potent root forming substance originating from leaves [186]. Van Tieghen and Douliot (1888) observed adventitious root formation in 67 dicotyledonous and 21 monocotyledonous families [187]. In 1933, Bouilleune and went propounded a hypothesis that Rhizocauline, a specific factor, is responsible for root initiation [188]. Went and Thimann (1937) observed for the first time that auxin, in addition to its growth-promoting effect, also induces root initiation in stem outings. Thus, a practical use was also found for the auxin [189]. Anatomical studies:
Previous anatomical studies reveal that during root initiation, meristematic activity is found in the region of endodermis, pericycle, especially over the vascular bundles [190-192]. However, there are many examples of adventitious roots originating from phloem parenchyma or phloem ray cells. It is also not uncommon to find roots arising from tissues such as primary xylem parenchyma, callus etc. [193]. The common observation of

adventitious roots arising near vascular bundles, prompted Priestly and Swingle (1929) to suggest that nutrient supply may be a factor determining the site of root formation (194).

In recent years, pea epicotyls (Pisum sativum) and hypocotyls of Glycine max (soybean) have been used as systems for studying the effect of plant growth substances in the regulation of cambial activity and organization of root primordial in plants. Excised or intact hypocotyls, epicotyls segment and callus in response to auxin treatment, undergo dedifferentiation leading to the formation of root primordial. Preliminary anatomical studies have shown that parenchymatous cells, in the cortex of 24 hr auxin treated soybean hypocotyls and pea epicotyls, appear to be swollen and exhibit few cell divisions [72,195,196]. After 48 hr the number of cell divisions increase and disintegration of many cortical cells sets in, leaving lacunae. Root primordials originate from the cambial cells near vascular tissues and become recognizable after 3 days of hormone treatment. The primordial cells continue to divide and occupy the lacunae. In 4-5 days the new roots break through the epidermal layers of the segments.

Factors other than phytohormones:

Many environmental factors such as water supply, temperature, sunlight, acidity of the soil, etc. are known to affect root initiation. Young leaves promote root initiation but old leaves are found to inhibit them. Roots themselves inhibit new root formation, perhaps as a result of the production of some inhibitors [197].

The role of organic micronutrients is new root formation is significant. Adenine, arginine, aspargine and uracil promote root initiation [197] but guanine inhibits root formation in Phaseolus mungo [198]. Among other organic micronutrients, nicotinamide, niacin, thiamin (vitamin B1), vitamin K an vitamin H and pyridoxyl enhance root formation only in the presence of auxin [197]. It is also interesting to note, that some cortisols stimulate adventitious root formation in the intact hypocotyls segments of mung beans [199]. The cortisol sensitive phase during root initiation is between 2nd and 4th day of treatment. However, cortisols in the presence of auxins, stimulate root initiation, but inhibit growth of the roots [200]. Recently it has been reported that vitamin D and its analogues by themselves promote root formation slightly, but in the presence of IBA, IAA or NAA, root production is greatly enhanced [201]. Gentics of root formation:
Zobel (1975) has described mutants in tomato with respect to root formation. These mutants are referred to as rosette (ro), lateral-les (dgt) and dwarf (drf), of which r has been established as adventitious rootless mutant [202]. The diploid chromosome number of these plants is 24 (2n = 24). Breeding experiments

and genetic analysis have revealed that ro gene is located on the second chromosome and follows simple Mendelian inheritance. Hence, it is suggested that a single pair of alleles controls adventitious root formation.

Plant hormone interactions: The most effective adventitious root forming substances known are IBA and NAA [188]. Staurt (1938) demonstrated that excised stem cuttings of Phaseolus mobilize carbohydrate reserves as soluble sugars within 24 hr of auxin treatment [203]. Altman and Wareing (1975) obtained similar results using [14 C]-CO2 in Phaseolus vulgaris cuttings [204]. However, the application of exogenous sugars could not replace the requirement of auxins for new root formation [205]. Ethylene stimulates root initiation in many leafy stem cuttings [206], but there are also cases where ethylene has no promotive effects [207,208]. Batten et al. (1978) have claimed that there is no correlation between the effectiveness in rooting and induction of ethylene production by a variety of auxins. Application of ethylene has no effect on root initiation and it proceeds normally even in the presence of 7% carbon dioxide or in hypobaric conditions [209]. In this context, it is pertinent to note, that in the hypocotyls of mung bean, IAA induces a specific protein, which in turn inhibits IAA induced ethylene production [210]. While ABA exhibits both stimulatory and inhibitory effects on new root formation [211,212], cytokinins and GA3 in general are inhibitory [213-215]. However, it has been suggested that the early stages of root formation are most sensitive to cytokinin and GA 3inhibition, but under certain conditions GA3 promotes root formation in Citrus embryoids [216]. Verga et al. (1974) have found that pretreatment of leaves with GA3, before excision, enhances adventitious root initiation. This effect is believed to be due to GA3induced enhancement of auxin synthesis [217].
In callus cultures, in general, root formation is dependent on the ratio between auxin and cytokinin concentration. While higher ratios promote root formation, a lower ratio inhibits this process but induces shoot primordial. However, at intermediate ratios, both roots and shoots are produced. Hence, it is deduced that the relative concentration of auxin to cytokinin is very critical in the differentiation and development of organs such as roots and shoots [4] Fig.2.

BIOCHEMICAL EVENTS: During adventitious root initiation in the epicotyls of pea and hypocotyls of soybean, increase in DNA, RNA and protein contents has been observed. After 3 days of auxin treatment, cellulase activity increases 30-40 fold, pectinase 7-8 fold, pectin esterase and 1 -> 3 Gluconase by 3-fold [195]. Similarly, during rhizogenesis, in detached cotyledons of mustard (Sinapis alba L.) increase in RNA and protein contents have been noticed [218]. Some evidence has been presented to show that the increase in cellulose activity is totally abolished, if the segments are treated with actinomycin D or azaguanine or puromycin along with the auxin. Hence, it is discerned that the continued synthesis of RNA and protein is essential for the increase in the activity of cellulase [196].

Trewavas (1976) has documented some interesting observations on the changes in nucleic acid metabolism and protein synthesis, specifically cellulose, during adventitious root initiation in pea epicotyls and soybean hypocotyls [196] (Fig. 3) In epicotyls of pea plant, over a 3 day period, auxin causes an increase in DNA and protein contents by 2.5 fold and RNA content 4.0 fold. In soybean hypocotyls, a 4-5 fold increase in DNA and protein contents and a 10-foldincrease in RNA content have been observed. Under similar conditions, actinomycin D and puromycin inhibit the auxin induced increase in DNA, RNA and protein contents. In the presence of auxin, the ratio of microsomal RNA to soluble RNA is 6.9 compared to the control value of 4.8. These changes are attributed to auxin induced differential turnover of RNA. Furthermore, labeling studies have indicated that initial lag period for auxin mediated increase in RAN synthesis is l hr in pea segments and 3 Hr in soybean. After several hours of treatment with auxin, increased labeling has been found in rRNA and tRNA [72,151]. Using in vitro transcriptional assay system and DNA-RNA hybridization techniques, O Brien et al. (1968) and Verma et al. (1975) have demonstrated qualitative and quantitative changes in RNA [79,83]. This implies selective unmasking of the genome, which correlates well with the time point at which cell divisions start. In a time-course study in pea segments, Davies et al. (1963) have detected a further shift in the labeling pattern of the polysomal populations. At the end of the third hour the proportion of polysomes containing 10 ribosomes or more has increased 3 fold. Ribosomal populations double within 6-10hours. After 10 hr of treatment, the ratio of polysomes to monosomes reaches a value of 9, compared to 0.5 in control tissue [219]. In soybean hypocotyls a shift from monosomes to polysomes is noted after 3 hr of auxin treatment and this is sensitive to actinomycin D, but not to 5-FU, 6-methylpurine in polysome size is independent of ribosomal RNA synthesis, but dependent upon the continued synthesis of tightly bound (TB) and DNA-like RNA (D-FNA). It has been further demonstrated that the increase in protein synthesis by individual polysomes in vitro is due to the increased activation of chain initiation, probably through the synthesis of 3 specific 60s ribosomal proteins [77,166]. The present investigation has been undertaken to study (1) the early molecular events leading to dedifferentiation of pericyclic cells into root primordial; (2) the identification of specific gene products essential for adventitious root initiation; (3) a delineation of sequence of events at molecular level leading to the emergence of root initials. Adventitious root initiation in hypocotyls segments of Phaseolus vulgaris Linn. In response to the hormone IBA treatment has been used as the model system.

Fig.2: Interaction

between

Auxins

and

Cytokinins

during

Organ

differentiation

in

callus

cultures. Open arrows indicate the promotion of organs and closed arrows indicate inhibition.

Fig.3: Time of initiation of metabolic changes induced by IAA after addition to Soybean hypocotyls or pea epicotyls.

PLANT HORMONES-Introduction

By convention hormone are said to be a substances whose site of synthesis and site of action are different; the two events are separated by space and time. Hormones are known to elicit specific responses. Charles Darwin first demonstrated the existence of such substances in plants. Who in his own inimitable way explained growth of plant tips, respond to light and exhibits photo induced curvature movements. Since then botanists all over the world made studies in unraveling the mysteries of diffusible substances called hormones which control growth and development of the plant body.
DISCOVERY OF PLANT HORMONES

Darwin used canary grass coleoptile tips to demonstrate the sensitiveness of the stem apex to light mediated curvature movements. Later Boysen Jensen used Avena coleoptile tips to demonstrate the presence of plant hormones. A gelatin block was placed on the decapitated coleoptile tip, then the tip

was replaced over the gelatin block and the tip was illuminated from one direction. In response to light, the stem tip bent towards the light source. This was explained as due to the downward movement of some substance from the tip through gelatin block down wards. And the substance was considered as the cause for growth curvature. Paal.A, on he other hand, placed the cut coleoptile tip asymmetrically over the decapitated coleoptile segments and placed the seedlings in tip dark. After few hours, he observed the curvature of the stem tip away from the side at which apical tip was placed. Pals experiment further demonstrated the presence of some kind of growth promoting substances in coleoptile tip, from which the substance was able to diffuse and bring about growth on one side hence the curvature. What ones people called diffusible substances have be restated as transportable substances for there are carriers in the cell membranes which do the role. These are carriers specific. F.W. Went collected growth promoting substances by placing the coleoptile tips on the square agar blocks. By placing such loaded agar blocks asymmetrically on the decapitated coleoptile tips in dark, showed the growth curvature movements. Persuing the above methods he established quantitative bioassays. The bioassays explain and correlate quantitative relationship between the amount of hormone applied and the magnitude curvature as a response.

It is at this juncture of time biologists and chemists started identifying the chemical component that is responsible for growth promoting activity. To their surprise, they found the human urine as a rich source for the said hormone. Kogl and Haagen Smit starting with 33 gallons of urine, extracted 40 mg of the active principle in the form of crystalline powder which showed 50,000 fold grater activity. First they called this substance as Auxin A. Using the same extraction procedures they isolated another active substance from corn germ oil and called it as Auxin B. Not satisfied with their purification methods, they used charcoal adsorption column chromatographic procedures

for isolating a pure form of growth substance. The substances obtained from this method were called Heteroauxin. Later heteroauxin was identified as Indole Acetic Acid. But this substance was known as a chemical to them for it was already identified by E & H Salkowaski. However, Salkowskis did not know about the property of IAA as growth hormone. The discovery of Indole acetic acid as the plant growth hormone gave impetus to plant physiologists. As a result, new hormones were discovered from different plant sources. Their site of synthesis, chemical structure, site of action and their physiological and morphological effects have been studied in detail. So far, five phytohormones have been identified from different parts of the plant body; most importantly all the hormones can be detected in the same plants. They are Indole

Acetic Acid (IAA), Gibberellic acid or Gibberellins (GA), cytokinin, Abscisic acid (Abscissin or ABA) and Ethylene. Simultaneously a host of synthetic compounds have been developed which stimulate plant hormones in many respects. The figure below shows each hormone synthesis pathway.

PHYTOHEROMONES and PLANTS RESPONSES

Investigation on the effects of hormones on plants has revealed that the hormones elicit a wide array of responses in different types of tissues in the same plant body. Physiologists have realized that the responses to the hormonal treatments depend upon the kind of tissue and the physiological state of the tissue. For example, in a developing stem segment, in response to GA3, the internodes elongate considerably but the same hormone in maize grains elicit the synthesis of alfa amylase enzymes is aleurone cells. Thus the specific response to a particular hormone depends upon the inbuilt potentiality of the said tissues; this behavior is because of its previous developmental programmes. It should also be remembered that the specialized hormones found in animal systems have no counterparts in plants with respect to the target cells and specific functions. The auxin which induces the growth in one part of the plant body, fails to bring about the same effect in the other part, but it may have different effects like apical dominance, new root formation or parthenocarpy, etc, at different site. While GA is known to bring about the gene activation in aleurone cells leading to the synthesis of alfa amylase, the same hormone acts on rosette shaped Hyoscyamus plant and induces bolting and flowering. But in pea plants, it overcomes genetic dwarfism. The above observations suggest that each and every phytohormone elicit more than one response in the same plant body but at different sites. Furthermore as all hormones are synthesized at different sites within the same plant body the said hormones interact with each other and control the growth and development.
HORMONAL INTERPLAY

Although Haberlandt considered the tissue and organ culture under sterile conditions as a theoretical possibility, Kattle in Germany and Robbin in USA have succeeded in developing methods to culture

tissues in a defined media. With the advent of tissue culture techniques, studies on experimental morphogenesis progressed in leaps and bounds in the past 45 years or so. Using tissue culture methods, it is possible to treat the tissue with one or the other hormone at will. Use of phytohormones in tissue cultures, have revealed that though a single hormones has a specific effect, two or more hormones together at different concentrations, elicit different responses in tissue from. In the presence of two different hormones, the effects may be promotive, synergistic or antagonistic where one may modify the activity of the other. Such reactions are referred to as Hormonal interplay.

Callus tissue can be grown from tobacco pith cells and the same can be maintained in a defined nutrient medium in the presence of both IAA and cytokinins at particular concentrations. The same callus can be induced for organogenesis by changing the relative concentrations of IAA to that of cytokinins. In the presence of higher amounts of auxin to cytokinins, callus generates roots. On the contrary if the concentration of cytokinin is higher in relation to auxins, the callus cells produce shoots only. If the concentration of the two said hormones is balanced, the callus induces the formation of both roots and shoots. The above mentioned observations suggest that the hormonal interplay has a significant role in organogenesis. Similarly, GA3 promotes callus growth but the same hormone inhibits auxin-cytokinin induced shoot formation. The above said hormonal effects are not just direct effects, but they also influence the levels of other hormones either by activating the synthesis of a particular hormone or by inhibiting the synthesis of it. Various studies on hormonal effects show that the endogenous levels of auxin in plant tissues is elevated by the applications of GA, Cytokinins or both. While auxin induces the synthesis of ethylene which in turn induces the formation of ABA, which on the contrary enhances the levels of

ethylene? Thus, they show both cooperative and promotive effects on each other. It is also known that ethylene and ABA together bring down the levels of the auxin. This effect can be overcome by the addition of cytokinins, for cytokinins are capable of bringing down the levels of ABA through the increased levels of GA biosynthesis. The close relationship and interplay between GA, auxins, cytokinins, ABA and ethylene, exhibits an excellent feedback control mechanism. important in interpreting the hormonal interplay and effects. However, understanding of hormonal interaction at the level of gene expression and their product is very

Plant Hormones-AUXINS
Distribution Though auxin is synthesized in the plant apices of shoots and roots, it is transported towards their respective basal parts. Quantitative estimation of auxins found in the segments of seedlings, by spectrophotometric analysis; show that stem apex possesses 1.5 to 2.0 fold higher amounts than found in the root apices. The lowest concentration is found in the region of the stem where cotyledons are attached. Even leaves contain some amount of Auxins.

Looking at the structure, functional responses, plants are no different from animals system neuronal network. Holistically one can imagine plants having a system are no different from animal systems.

Auxins exist either in bound form or in a free state. The non diffusible form is considered as bound form and it is active, but the freely diffusible form is referred to as an inactive form. The bound

state of the auxin has been explained as due to the binding of receptor proteins or binding proteins to auxins. Such proteins are found in the plasma membranes, cytoplasm and chromosomes. The mol. wt. of auxin binding proteins has been determined as 10,000 and 31, 5000 Daltons. Such proteins have been isolated from the free nuclei of coconut liquid endosperm. These proteins in the presence of IAA are found to be active in inducing gene expression. In living cells, the

relative concentrations of bound form and free forms of auxin is not constant but depends upon the environmental conditions or the functional state of cells.

TRANSPORTATION

Auxin is transported from the site of synthesis to the site of action, which is not far away. Nevertheless auxin is also translocated to other regions of the plant body. The transportation of auxin is polar, i.e., from apex to the base, which is called basipetal movement, but acropetal movement i.e. from the base to apex has also been observed but the amount transported is almost negligible. The ratio between basipetal and acropetal movement is approximately 3:1.

Furthermore, the transportation is more or less an active process. The long distance transportation appears to take place through sieve tubes and it is a facilitated mechanism. The rate of auxin movement is about 6.4-20 mm/hr, which is many times faster than the rate of diffusion. It is now clear that the transportation is through carriers. Auxin exists in ionized form called A (-) and uncharged form called AH. Transportation across the membranes is through carriers and in ioized form. But transportation in free space is diffusion and it is in AH form. One of the proteins that is responsible for efflux transport of auxins is PIN; there are several forms of these proteins. SITE OF SYNTHESIS: The natural auxin found in plants is called Indole Acetic Acid (IAA) and it is the first of the phytohormones to be discovered. Indole acetic acid is mostly synthesized in the stem and root apexes. Using radioactive 14C as the tracer, it has been demonstrated that the meristematic cells, just above differentiating into vascular tissues, are found to be active in synthesizing IAA. Shoot apexes synthesize more auxin than the root species.

BIOSYNTHESIS OF INDOLE ACETIC ACID

Tryptophan has been found to be the precursor for IAA synthesis. In some cases Indole acetonitril has also been found to be used in the synthesis of IAA. The enzymes responsible for the

biosynthesis of IAA have been identified. There are two pathways which converge in the production of IAA. To start with, tryptophan is converted to Indole pyruvate by deamination reaction catalyzed by specific deaminase enzymes. The indole 3D pyruvate is then subjected to decarboxylation to produce indole acetaldehyde, which is then oxidized by alde-hydrases to indole 3- acetic acid. On the other hand, tryptophan may be first subjected to decarboxylation step. The tryptamine synthesized in this reaction is deaminated to indole 3- acetaldehyde then it is oxidized to IAA.

Indole aceto nitrile is also found in plants and the same is used in the synthesis of IAA by converting IAN to Indole acetamide then to IAA or IAN to IAA directly.

SYNTHETIC AUXINS

Once the native auxin has been identified as indole acetic acid, plant biochemists started looking for similar compounds in nature as well as in the laboratory. As a result, a host of synthetic auxins have been discovered and their effects as growth promoting hormones have been characterized, ex. Indole propionic acid Indole butyric acid Naphthalene acetic acid, phenyl acetic acid, 2,4 Dichlorophenoxy acetic acid are just few of the known synthetic auxins.

The hormonal activity of IAA and the other synthetic compounds has been attributed to the presence of a ring system as the nucleus, a side chain possessing a carboxyl group and the presence of at least one carbon atom between the ring and the carboxyl end. The length of the side chain, the number of substituents in the nucleus and the side chain and the basic structure of the nuclear ring have been found to exert profound influence in eliciting physiological responses.
EFFECT ON GENERAL METABOLISM

Isolated tissues, hypocotyls segments, epicotyl segments, leaves, excised roots and even whole plants have been used to monitor various biochemical and physiological responses to hormone treatment. Auxin has been found to accelerate cellular metabolism in treated tissues. Particularly respiratory rate increases by at least by 20%.

The general activation of metabolic processes in terms of turnover is all pervading.

Even

mitochondria and plastids show increased activity. Many of the increased metabolic activities in response to auxin treatment have been attributed to the changes in membrane permeability and activation of some membrane factors. As a consequence of increased respiratory activities,

photosynthetic activity, amino acid metabolism, nucleic acid synthesis and protein synthesis and others, cells build up the required materials for their growth.

Auxin binding protein

Plant growth involves interaction between metabolites such as sugars, phytohormones and their action on gene expression. Auxin as a signaling molecule has various effects depending upon the tissue where it acts.

EFFECT ON NUCLEIC ACID SYNTHESIS

Auxins show some dramatic effects on growth and development of plants. Though the immediate effect of auxin is known to be at the plasma membrane and cytosolic level, with time its effect on gene activation and protein synthesis is over bearing. The effectiveness of Auxins activity is due to

the presence of auxin binding proteins. They act as receptors and after complexing with the auxins, they are rendered highly active. The effect of auxins induced cell elongation does not require DNA synthesis, but in long term effect, DNA synthesis is always accompanied with cell division. The interaction between auxin and cytokinin has been interpreted as at the auxin plays permissive role in DNA synthesis and cytokinin stimulates it.

Auxins do not induce transcription immediately after application of the hormone. But auxin induced transcription sustained for 49-50 minutes. But auxin after effect on increased translational activity has been explained as due to the activation of translational machinery through the activation of translational factors. However, transcriptional activity in response to auxin at later stages results in the synthesis of all species of RNAs, such as mRNAs, tRNAs and rRNAs. Among the population of mRNAs induced by auxins, some specific mRNAs for cellulase, tubulin and others, are found is higher concentrations.
EFFECT ON PROTEIN SYNTHESIS

As mentioned earlier, auxin, in many systems enhances the rate of protein synthesis very early without the concomitant increase in RNA synthesis; which suggests that auxins earlier effect is on translational activity. This may be achieved either through the activation of inactive mRNPs, or through the activation of translational machinery and translational factors. Increase in polysome content in response to auxin treatment in many plant cells is good evidence in support of auxins promotive effect on protein synthesis. However, the increased level of protein synthesis at later stages is actually due to the auxin induced transcriptional activity, which really sustains the translational activity for a longer period of time. Though auxin enhances the synthesis of almost all house keeping enzymes, it also induces the synthesis of specific proteins like cellulase, cellulose synthase, tubulins and other specific factors required for cell elongation. It should be remembered that the specific effect of auxin on the expression of a particular protein product depends upon kind of tissue involved. Auxin induced activation of polymerization of tubulins into microtubules is another interesting feature of auxins effect.
EFFECT ON CELL ELONGATION

Among all the hormonal effects the effect of auxin on cell elongation has been studied in detail. For a long time, the exact mechanism of auxin induced cell elongation has remained unsolved. For historical interests it is essential to understand a few theories which where proposed in the past to explain the mechanism. In this text recent views have also been provided.
TURGIDITY THEORY

This is probably the oldest of the theories that have been proposed so far. This theory is based on the assumption, that auxin stimulates respiratory and other metabolic activities.

As a result, the osmotically inactive components of cells are degraded to osmotically active molecules. This in turn affects the diffusion pressure deficit of the cell and water potential gradient is created between the growing cells. So water from neighboring other cells, particularly xylem diffuses into the activated cell passively. As a consequence of this turgour pressure that

builds up within the cell, the cell is stretched from within, thus the cell elongates. According to this concept the cell elongation is a passive phenomenon.

It is true that auxins enhances the respiratory activity, but for the cell to elongate, due to the turgour pressure developed inside, the cell also requires loosening of the cell wall, without which whatever turgour pressure that develops, cannot force the cell to elongate or expand. In recent years, with the use of refined techniques, it has been demonstrated that in many plant systems the auxin induced cell elongation takes place under negative turgour pressure. Furthermore, respiratory inhibitors like DNP, cyanide inhibit auxin mediated cell elongation, which suggests that cell elongation is an active process. Because of these the turgour theory is not favored. One of the proteins involved in cell expansion is expansin

GENE ACTIVATION THEORY

With new discoveries in the field of molecular biology, plant scientists also thought, that auxin brings about the cell elongation through gene activation. Bonneret.al. suggested that as auxins are acidic in nature, they can easily bind to basic proteins found associated with the chromatin material. The binding of auxins to histones, certain segments of DNAs are freed from the surrounding histone proteins for transcriptional activity. As a result, the required mRNAs for cellulase are produced. As cellulase is required for degradation and loosening of the cell wall, they proposed gene activation. The combined effect of gene activity and auxin induced turgour pressure has believed to be mechanism of cell elongation. Further elongation stops at later stages and the synthesis of cell wall materials continues which again is due to the activation of gene expression.

This theory however falls short of its expectations because transcriptional activity in response to auxin increases only after 15-30 minutes later, but the apparent growth of cell starts as early as 1015 minutes after auxin treatment. So this theory also fails to explain the early phase of growth, moreover, the binding of auxin to the histone in bringing about gene expression is no more tenable.
ACID THEORY

Cleland and others have demonstrated that stem segments on exposure to auxin, secrete protons into external medium and render it acidic. Sharp fall in pH of the medium due to auxin treatment has been demonstrated by many workers. Proton secretion has been attributed to the activation of H/ATPase found in the plasma membrane. As the protons diffuse through the cell wall, the pH in that region falls and acidic form cellulase will be activated. As a result, the cell wall fibrils are cut and loosened which greatly facilitates the elongation of cells.

Acid theory explains the early effect of auxin on cell elongation even before the auxin induced transcription starts. This theory also assumes that the internal pressure responsible for the elongation as turgour pressure. It is now known that not in all cases turgour pressure has been demonstrated as the cause for cell elongation. However, this theory explains the early effect of auxin on cell elongation.

Auxin also induces cellulase enzymes which act on cellulose fibers and loosen the cell wall and facilitate the expansion due to increase in turgid pressure.

CYTOSKELETON THEORY

This theory is the most recent theory and it is based on many experimental evidences. It is very important to know that auxin induced cell elongation or growth exhibits two phases. The first phase is initiated as early as 5-15 minutes after treatment. It is rapid and lasts for 30-45 minutes. This phase is insensitive to the inhibitors of transcription and translation. But it is highly sensitive to colchicine treatment. The above features suggest that the early rapid growth does not require transcription or translation products. But it requires microtubule formation because colchicine inhibits polymerization of tubulin monomers into microtubules. On the other hand, the second phase of growth is slow but steady. It is sensitive to Actinomycin-D, CHI and also colchicine. It means the second phase requires transcriptional products, translational products and polymerization of tubulins into microtubules which act as cytoskeletons.

In the first phase, auxin first enhances the rate of respiration, thereby ATP production increases. At the same time, it also activates nucleating centers and H/ATPase pump located in the plasma membrane. Nucleating centers are a group of aggregated tubulin monomers which on activation start assembling available tubulin monomers from the cytosolic pool. Thereby, a large number of microtubules grow and elongate from the nucleating centers. Polymerization of tubules requires GTP and ATPs as energy source. The hydrolysis of them generates significant amount of H+. Meanwhile, the activated H/ATPase pumps excrete H+ into the exterior of the plasma membrane by active process. As the pH in the cell was becomes acidic, the acidic form of cellulase enzyme which are already present become active glucanase activity they hydrolyze the cellulose fibrils, thus the cell walls fibrils loosened. In this process, certain amount of Ca2+ ions are also removed from the middle wall, which renders the middle wall more labile and plastic. As more and more microtubules grow and elongate and build up, they build up a kind of mechanical force within the cell and the cell is stretched. MTs contribute to the deposition of cell wall materials to be transported and deposited out side the plasma membranes. The elongation is further facilitated by the loosened cell wall. Thus the cells grow in length. The second phase of cell elongation however required more and more of tubulin pool because most of it is depleted in the first phase. So it also requires fresh synthesis of tubulins, cellulase enzymes and other required factors for the sustained cell elongation as per the demand transcriptional activity increases and the RNAs produced are used by the translational machinery and more of proteins are synthesized. Though there is an increase in the synthesis of most of the house keeping proteins, the increased synthesis of tubulin, cellulase and cellulose synthesizing enzymes is significant. Utilizing tubulin monomers continue to polymerize and more of microtubules generate at the same time loosening of cell wall continues. There by the second phase of cell elongation proceeds slowly but steadily. That is why the second phase is so sensitive to the inhibitory actions of actinomycin D, CHI and colchicine.

This theory appears to explain most of the observed properties of cell elongation. Furthermore, it also explains how cells elongate even in the absence of sufficient turgour pressure within the cell.
EFFECT ON NEW ROOT FORMATIONS

Roots developing from any part of the plant body other than the radicle are called adventitious roots. It is not an uncommon phenomenon to see the plant parts are propagated by inducing new root formation. But the exogenous supply of auxins to stem and leaf cuttings readily induces the new root formation, which ensures vegetative propagation. The synthetic hormones like IBA and NAA are more effective in new root formation than the native IAA.

If defoliated stem cuttings are continuously washed in water to leach out the endogenous auxins, they do not produce any adventitious roots in a nutrient media. But if such washed segments are treated Indole Butyric Acid for about 10-20 minutes and placed in a nutrient medium, stem cuttings

produce a large number of roots. These roots normally develop from the terminally differentiated pericyclic tissue found around phloem tissue. Within 36-72 hours after treatment some of the founder cells in the said pericyclic region undergo transformation and organize into root primordia, which later grow through the cortex and emerge out of the stem.

The transformation and organization of pericyclic cells into root primordia is a phenomenon of dedifferentiation. Studies on molecular aspects of IBA induced new root formation in the hypocotyls of phaseolus vulgaris reveal that the new root formation is due to the differential gene expression. Using techniques like translation of isolated mRNAs from treated and untreated segments at different time periods in a cell free system and the analysis of invitro and in vivo protein products by polyacrylamide SDS gel electrophoresis and autoradiography and immunoprecipitation of labeled proteins by using specific antibodies, show that among the many proteins synthesized, the synthesis of 55-58 KD proteins and 105 kd proteins in the IBA treated segments increases significantly. The 55-58% proteins have been identified as and tubulin, which are the precursor for microtubules. Involvement of microtubular assembly and orientation during the differentiation and organization of pericyclic cells into root primordia is confirmed by the use of colchicine and cytochalasin B. The said drugs prevents the root initiation in the IBA treated hypocotyl segments within 36

hours after hormonal treatment, but the drugs have no effect if the hypocotyls that have already passed through 36 hours after IBA treatment. The above results indicate how auxins can bring about new root formation by inducing differential gene expression. Some of the concepts of auxin action on gene expression has been shown below in the form consolidated figures which are self explaining.

Lateral root develop from preexisting roots and new roots develop from stems. The root initials start from cambial founder cells. It is important to remember that even plant tissue, as in the case of animal systems, contain stem cells, called them as founder cells. Proper signals can induce organ formation from such stem cells. Auxin activates founder cells in pericycle to enter into G1-S transition. It is now clear that auxin induces certain cyclins and CDKs (B) types. Auxin induced transcriptional mechanism perhaps go through activators, Auxin binds to auxin response factors TIR1, which in turn translocates into the nucleus and bind to auxin response elements of genes. However the degradation ARF/IAA/Auxin protein complex by SCF TIR is important for the ARFs to bind to auxin response elements properly. It is also possible several other factors are involved in induction of lateral roots; probably auxin binding protein (ABP) is one among them.

Once auxin is transported into cells, it binds to its receptor protein TIR, which inturn binds ARFs /IAA/Auxin protein complex which are acting as repressors. IAA-TIR binding leads to activation of SCF mediated degradation of IAA/Aux binding protein which frees ARFs and now ARFs bind to ARE promoter elements. Aux/IAA binding proteins were acting as repressors. The auxin-TIR1 now binds to Aux/IAA proteins and using SCF complex ubiquitinate AUX/IIA proteins and feed to proteosomes for degradation. Thus the ARFs become active and activate specific genes to which they are bound. This is general mechanism for auxin induced gene activation.

Auxin binding protein (ABP)

TIR1 protein bound to auxin activates Auxin response factors and thus gene expression.

The above figure proposed by Thomas Guiylfoyle provides the input how auxin mediated specific genes are activated. This proposal is considered to be the most famous among plant molecular biologists.

EFFECT ON APICAL DOMINANCE Plants like pines and other conifers exhibit a growth pattern, which is quite distinct from a banyan tree or a tamarind tree. The conical growth of the pine plant is due to the predominant growth activity of the apical meristems where the growths of lateral buds are more or less suppressed. On the other hand, the growth pattern in banyan tree is diffused, where lateral branches grow as vigorously as the apical branches. The suppressive effect of apical buds on the growth of lateral buds is often called apical dominance. Such a phenomenon is not just restricted to only conifers, but it is also found in other plants.

Apical dominance has been explained as due to the action of auxin present in the main apex. This view is amply supported by an experiment, where if the stem tip is cut off, the axillary buds found below sprout immediately. Instead, if an agar block containing auxin placed over the decapitated stem, the axillary buds remain suppressed. It is clear from the above experiment, that auxin present in main apex some how inhibits the growth of the axillary bud. The severity of apical dominance is greater on the axillary buds present nearer to the main apex. Nevertheless, apical dominance exerted by the auxins can be overcome by the application of cytokinins to axillary buds. This is because cytokinin induces cytokinesis. Probably, the mitotic block that is operating in axillary buds may be due to the inadequate supply of cytokinins. But how the supply of cytokinin to axillary buds is made inadequate by the apical bud is not clear.

The idea of nutritional inadequacy to the axillary buds due to the stronger influence of apical meristems has been considered by many scientists. It is assumed that because of apical dominance, most of the nutrition is drawn towards the apex than to the axillary bud. This explanation for apical

dominance has no conclusive evidences to prove the claim. On the other hand, studies have shown that higher concentrative of IAA induces the synthesis of ethylene. As apical buds contain more of auxins, it may induce the synthesis more of ethylene which in turn may inhibit the normal growth of the axillary bud. But by removing the apical bud, the concentration of auxin drops, so also ethylene hence axillary buds sprout.

Effect on Phototropism

The growth curvature in stem apex in response to light is called phototropic movement. Such growth curvature in shoot tips can be induced even in the absence of light by placing auxin containing agar blocks asymmetrically on decapitated stem tips. This indicates that unequal concentration of auxin is responsible for unequal growth there by the curvature. But in light induced curvatures, how does light brings about unequal concentration of auxin and which part of the light spectrum is responsible for the curvature are the few questions that needs explanation. Using monochromatic light, it has been determined that the most effective spectrum is 445 mm. But the answer to the first question is still elusive and various theories have been proposed from time to time to explain this phenomenon.

Lateral Transport Theory

This theory was proposed by Cholodney & F.W. Went. Accordingly, the concentration of auxin in the stem apex is uniform all-round. Once the light rays fall on the stem from one side, it induces the movement of auxin from the illuminated region towards the darker region. By such movement, auxins accumulate in greater amounts in the darker region. The motive force for the movement of the auxin in response to light is attributed to the differential electrical charges on the said surfaces. Then he tips were illuminated from one side and the basipetally diffused auxin was collected and the amount of auxin found in the agar blocks was determined. According to the authors in the control tips they detected more of auxin in the far side the surface that was in the dark than from the surface that was illuminated. But in the tip with a mica plate the amount of auxin found in both the blocks was same. So it was deduced that the difference in the amount of the auxin detected in the blocks on the darker side is actually was due to lateral movement. dependent auxin concentration. Accordingly resultant differential concentration is responsible for differential growth, hence the phototropic curvature. The growth is

Recombinant GUS gene expression of a gene that is responsive to auxin shows the distribution of Auxin in response to light Their claim was disputed by other workers, who used radioactive C-labeled auxin for exact quantitative determination of the transported auxin from one region to another. They did not find any substantial lateral transportation. So the lateral movement theory has remained unconvinced. Inhibition of basipetal Transport Theory: Gordon and others proposed that the unequal distribution of auxins in response to light treatment is not due to lateral movement, but due to the inhibition of basipetal movement from the illuminated side, which causes unequal concentration of auxins and growth curvature is due to it. Slow growth of the stems in day times and steady growth of the plants in intense sun light has been attributed to this effect. However this theory has not been tested with covincing experiments. Photo inactivation Theory: When blue light at 445 nm has been detected as the action spectrum for the induction of phototropic curvature, plant physiologists started looking for pigments that absorb blue light at 445 nm. This logic was based on the fact that known photo responses like photosynthesis and photoperiodic responses are due to specific pigments. The search for such pigments revealed the presence of B carotenes and riboflavin in the stem tips. As some plants, which respond to light induced phototropic movement, did not possess B carotenes it was deduced that riboflavin as the causative pigment. The photo inactivation was explained on the assumption that riboflavin after

absorbing light gets activated and the same inactivates the auxin directly or destroys the auxin through certain IAA oxidase. This result in unequal concentration of auxin in the stem tip which in turn is believed to be responsible for the phototropic curvature again this theory has never been proved unequivocally.
Present concept:

In the past, many experimental results were based on very crude extraction methods. Such experimental results are not very convincing today. Use of radioactive isotopes i.e. (14C) IAA, solvent extraction methods combined with GLC analysis for qualitative and quantitative estimations of auxins have cast grave doubts about previous theories. In the light of recent work, it has been suggested that light has profound effect on inducing the synthesis or releasing the growth inhibitors like ABA. The release of ABA in the region where it is illuminated causes inhibition of growth in the said region. The effect of ABA on osmotic changes by creating effluxes of ions is well known. Moreover, it is now known that auxins could be made inactive or active by auxin binding proteins. If binding protein complexes with IAA, it is rendered physiologically active, if it is free, it remains inactive. So light induced changes in the concentration of ABA and other auxin binding factors are believed to bring about variations in the endogenous levels of active auxins. Such changes are ultimately responsible for phototropic growth curvatures, For a long time the above concepts were accepted as facts, however, finding of phototropin, a blue light absorbing protein, has role in phototropic curvature is also an accepted fact. Phototropin exists in two forms; both are flavin binding proteins of mol.wt 110 Kda and 124Kda. Interestingly these proteins contain at least 11 kinase (PAS) domains; these domains are celled LOVE domains (Relation to Light, Oxygen and Voltage). They also contain different domains such as BTB and D1-to D15 domains. Phototropins act as light receptors and absorption leads to autophosphorylation at serine/threonine sites and they are involved in phosphorylation of many other membrane bound proteins. Membrane bound proteins have many roles in transportation of ions, transportation of Auxin/auxin receptor.

However the exact mechanism by which they bring about photrophic curvature movement is yet to be discerned.

Conformational changes of Love domain proteins called Phototropin in the presence of light and in the absence of light. Plants regulate their growth directions in response to light direction, and its response is called phototropism. Phototropism is a model research to understand the regulatory mechanisms of auxin metabolisms in response to environmental stimuli, and we are studying on this topic by a molecular genetic approach using Arabidopsis mutants. We identified a signal transducer RPT2 and a novel blue-light photoreceptor phot2, both of which are required for the phototropic responses of Arabidopsis. We indicated that phot1 and phot2 show partially overlapping functions in two different responses, phototropic response and chloroplast relocation, in a fluence rate-dependent manner, as blue light receptors. To reveal the phototropin-signaling pathways, We studied on the regulation of cytosolic Ca2+ concentrations by phototropins, involvements of RPT2 and another phototropin-signaling factor NPH3 in chloroplast relocation and stomatal opening, and functional sharing between phototropins and other blue-light receptors cryptochromes. Now, We are studying on a dephosphorylation mechanism of NPH3 and transcriptional and post-transcriptional regulations of RPT2 in response to blue light irradiation.

In addition, we revealed that photoreceptors phytochromes and cryptochromes regulate auxin biosynthesis, metabolism, and transport in response to light irradiation and indicated that a suppression mechanism of expression of auxin transporter PGP19 by phytochromes and cryptochromes is one of mechanisms to enhance the phototropic response by phytochrome and cryptochromes. Other analyses on the light signaling and auxin mechanisms were also published by collaborations with other research groups.

Light activates three kinds of photoreceptor families, phototropins, phytochromes, and cryptochromes, and affects the plant growth directions through changes of auxin biosynthesis/metabolism and transport. Effect on Growth Curvatures: Growth movement of any part or the organ of the plant body in response to gravitational pull of the earth is referred to as geotropic movements. The most remarkable feature of the plant body is that, though both shoot and root system are derived from the same embryonic cells, they respond differently to the same gravitational stimulus. This property may be due to unique embryonic developmental programmes dictated by the inbuilt genetic factors, which probably enforce the respective organism to behave differently to different environmental factors. Growth curvature away from the earths gravity is called negative geotropism and the growth of the roots towards earth is called positive geotropism. Most of the roots, with the exception of pneumatophores and coralloid roots which are negatively geotropic, exhibit positive geotropism. However, not all stem show absolute negative geotropism. It is a common observation that some of the underground stems like rhizomes, suckers, etc, grow obliquely in the soil and such growth movements are called dia-geotropic movements. Mechanism of geotropic for with Curvatures: Again, Cholodney and went are the pioneers in explaining geotropic movements. They proposed that stem apexes and root apexes require different concentration of auxins to bring about the maximal growth. In the sense, it is said, that the concentration of auxin that is favorable for the growth of shoot apex is inhibitory to the growth of the root apex. On the contrary, the concentration, that is optimal for the growth of the root apexes, is not adequate for the growth of the stem tips. It implies that stem cells require greater amount of auxins for the in maximal growth and root cells require very low concentration of auxins for their optimal growth. Based on these premises the mechanisms of geotropic responses have been explained.

If a straight seedling is placed horizontally on the soil, shoots curl upwards and roots bend towards the soil. This behavior has been attributed to the gravitational force that acts upon the respective organs for they are endowed with different programmes, though both have the same genetic properties. Cholodney and went assumed that the gravitation once acts on both root and shoot. So auxins having certain mass of their own move downwards and accumulate in greater amounts in the lower cells which are in contact with the soil. As higher concentration of auxin is promotive in stem tips the cells grow faster than the others. So the stem curves and grows upwards. On the contrary, as the higher concentration of auxin is inhibitory for the root cells, the growth of root cells is inhibited, but the cells containing less amount of auxins show maximal growth activity. Hence root tips curve towards soil and grow forwards in to it. Thus stems exhibit negative geotropic growth movements and roots show positive geotropic movements. The above said theory enjoyed the general acceptance for a long period of time. But people realized that gravitational force, by its mass action, has greater effect on amyloplasts than on auxins found in the plant cells. Because of their greater mass, amyloplasts settle down on the plasma membranes of the cells as shown in the figure. The contact of the amyloplasts with plasma membranes acts as on irritant as a result growth of cells on that side of the membrane is inhibited but the growth on he

other side is favored. But this explanation has never been favored because some plants which are lacking in amyloplasts also exhibit geotropic responses. In recent years, research work on geotropic movements has revealed that the site of geotropic perception is not the root apex, and it requires root cap for its response. If the root cap is cut off, the decapitated root dies not show any geotropic responses. When the cap is replaced he roots respond normally for geotropic stimulus. This observation has been explained as due to the synthesis of ABA in the root caps. The ABA is transported basipetally, and then it also moves downwards due to the gravitational force. As the lower cells receive ABA their growth is inhibited. But the upper cells grow normally and bring about geotropic curvature. Effect on Parthenocarpy: Development of fruits without fertilization is known as parthenogenesis and the fruit is said to be parthenocarpic fruit. Auxins have been found to be effective in inducing parthenocarpic fruits in some plants. It has been demonstrated that the extracts of pollen grains also induce the development of parthenocarpic fruits, discharge of pollen tube contents into embryo sac is believed to be cause for the increase in he content of auxin in embryo sac. Some botanists suspect that pollen tubes carry some enzymes which

produce more auxins. It is important to note the synthesis of more auxins not lead to fertilization. Whether the act of fertilization has any stimulatory effect on the synthesis of auxin is not clear nonetheless many plants respond to auxin treatment produce parthenocarpic fruits.

Induction of parthenocarpic fruits by auxins has greater application value in cultivating fruit yielding plants. The auxin induced fruits, besides seedless, they are larger in size and sweeter. Commercial production of such fruits brings more income to farmers.
Effect on Abscission

It is common to observe that older leaves, debladed petioles, abortive flowers, and often fruits, fall off from the plants. In the above said cases a distinct and characteristic layer of cells develop at the base of the petiole of the leaf or the pedicel of fruit, which acts as a weak point, hence the said structures fall down. The layer that is responsible for this process is called abscission layer or abscission zone.
Structure of Abscission Layers

It consists of a number of layers of thin walled cells which are rich in cytoplasm and actively dividing. Disappearance of middle lamella from the cells of this layer is a characteristic feature. This is due to the activity of pectinase enzyme. As a result, this part of the stalk becomes weak and the leaf or the fruit falls down by its own weight. In many plants, just below the abscission zone towards the stem, a layer of actively dividing cells develops. This layer is called protective layer. This layer develops after the fall of leaf at the free surface, but in some cases the protective layers develop even before the fall of the leaves. These cells by repeated cell divisions produce many layers of cells, which get suberized and protect the inner layer of cells from injury or infection. Thus, this layer acts as wound healing tissue. Development of Abscission Layer:

Leaves fall with the age and fruits fall down after ripening, but in deciduous plants, onset of winter acts as the signal for the plants to shed their leaves. Sometimes, unseasoned cold waves induce premature falling of fruits. In all these cases, the falling of leaves or falling of fruits is due to the formation of abscission layer at the base of their stalks. The factors that induce the formation of abscission layers vary. In some cases, ageing acts as an important causative factor, in others environmental conditions like winter may act as the factor. In the case of aging the accumulation of senescing factors induce abscission layer formation. One of the senescing factors is believed to be ABA. Reduction in the quantity of auxin n the distal parts of the leaf is also known to be another causative factor. In the initial stages of abscission layer formation, if IAA is applied to distal part of the leaf, the development of the abscission layer is inhibited. Instead, if auxin is applied at a later stage of abscission the process I shortened and the leaf falls quickly. Once, it was thought that ABA is mainly responsible for leaf abscission, but recent investigations indicate that ethylene is highly effective in inducing abscission layer formation, but the role of ABA is not totally ruled out. Incorporation of radioactive label during the abscission layer formation suggests, differential gene expression in the abscission zone results in the production of pectinases and cellulases, which by their activity breakdown the middle wall and also some cellulose. At the same time the cells in this zone become meristematic. Uses: The application of auxin to leaves and fruits is now known to prevent the development of abscission layer. Many synthetic hormones like NAA, IBA, 2,4-D have been found to very effective in preventing the premature fall of fruits, particularly commercial crops like citrus, apple, oranges, mangoes, grapes. The use of such hormones not only prevents the premature falling of fruits, but also increases the quantum of fruit setting. And fruits thus produced are larger and sweeter. Thus auxins can be used for commercial gains in the field of pomiculture. Auxin and Cytokinins interactions: In plants each of the phytohormones, though have independent effects, often they affect the other or they may have synergistic effect. For example Auxin prepares the cell but cytokinins execute cell division. When auxin induces new root formation at a particular concentration, cytokinins inhibit auxin induced new root formation.

Plant Hormones-Gibberellins Gibberillic acid was first discovered in Japan under unusual circumstances. Farmers in Japan, before the World War, found that their paddy crop plants were afflicted with a strange disease called Bakane. loss. The diseased plants showed unusual growth where plants were very tall, weak and sterile. This disease affected 20-30% of the crop plants and the farmers were subjected to great

Investigation into the cause, Hori a Japanese plant pathologist discovered that this disease was due to a fungus called Gibberella fujikoroi, but it is now identified as Fusarium moniliforme. Later Sawada and Kurosowa found that the extracts of this fungus simulated what the fungus could cause on infection. Then a group of Japanese workers led by Yabuta and Sumuki obtained an active principle from the extracts of the causative fungus, and later Yabuta and Hayashi identified the active principle as Gibberellic acid, which is new popularly called as Gibberellins (GA). Western scientists did not know anything about these discoveries before the First World War. After the war, they came to know about GA. Then they searched for this substance in higher plants and found this substance in all known higher plants. In fact they succeeded in obtaining GA in pure crystalline form and also they established its chemical structure.

Maryland mammoth
Distribution

Almost every kind of plant, including algae, ferns, fungi, bacteria, is known to contain GA. Quantitative analysis of various parts of the higher plants shows that young leaves, which are just unfurling, are rich in GA. Even germinating seedlings contain significant amount of GA. Further studies revealed that proplastids found in young and developing leaves act as the site of GA synthesis and it is of great interest to know that their synthesis and release from plastids is under the subtle control of phytochromes. Though GAs is synthesized in plastids, they are translocated to different regions of the plant body through sieve tubes by active translocation mechanism.

The diagram shows the concentrations and spatial distribution of different phyhormones in a plant body under normal conditions.

Structure and Classification of GA

Gibberellins are diterpenoid acids derived from tetra cyclic diterpenoid known as Kaurene. The basic carbon skeleton of GA is known as Gibbane ring. But today the term Gibbane is in use. However, the systematic nomenclature of GAs is based on Kaurene and Gibbane structures.

Modified solvent extraction methods and the use of sophisticated analytical tools like HPLC etc. have greatly helped plant biochemists in identifying different forms of GAs. So far, 52 to 57 different kinds of Gas have been identified. Some of the gibberellins are known to be conjugated with glucose as glucose esters. Chemical analysis of different parts of the plant body for GAs shows that different kinds of GAS are located in different parts of the plant body; their content and kinds vary at different stages of development of the plant body. Two or more GAs are found in the same plant but in different organs. In addition, Gibberellins exist in a dynamic state, in the sense; they undergo rapid interconversions from one kind to another, which however depends upon the intrinsic or extrinsic

factors. It is also known that some of the gibberellins which are active in one organ are found to be inactive in another organ and vice versa. The dynamic equilibrium between different kinds of GAs, their synthesis and their multifaceted effects in plants is really fascinating. Biosynthesis: Biosynthesis of GA takes place in young plastids. Acetate is the precursor for the synthesis of all kinds of gibberellins.

In a series of multi step reactions acetate is used to produce a diterpene compound called Kaurene. Specific enzymes responsible for each creates have been more or less identified. The site of synthesis is proplastids or plastids or both. Plastids are also the site of fatty acid synthesis in plants.

Kaurene thus synthesized is converted to Gibberellic acid, which is then subjected to hydroxylation, methylation or glycosylation to produce different forms of Gibberellins. The remarkable feature of the biosynthetic pathway of GAs is that the same pathway is also used by plastids to synthesize certain plant pigments, Abscisic acid and some sterols. The needed enzymes for the biosynthesis of the above said compounds are regulated by phytochromes and other factors. The plastogenome and nuclear genome play a significant role in regulating the biosynthesis of Gibberellins and other mentioned compounds, how plastogenome contributes to the synthesis and its regulation is not known.
Effect of GA on of Plants

1. Effect on General Metabolism:

Effect of various factors on cellular activities.

Gibberellins, as growth promoting hormones, accelerate the rate of cellular metabolic pathways such as respiration, protein synthesis etc. During the germination of cereal grains, GA is known to promote mobilizing the food materials for the growing embryo. It plays a significant role in metabolizing lipids to glucose and then to sucrose through gluconeogenesis process. The promotive effect of GA on nitrogen metabolism and HMP pathway is very significant. GA is also known for its effect on membrane transformation through rapid turnover of lipids. Interestingly, GA also favor C3 pathway in C3 plants, but the same hormone adversely affects Hatch and Slack pathway in C4 plants. Thus GA exerts a wider influence on general metabolic pathways.

2. Effect on Transcription and Translation: The metabolic effects exerted by Gibberellins show variations and it depends upon the structure, species and developmental stage of the plant body that receives it. The presence GA binding proteins have been identified from various sources like pisum epicotyls, phase his etc. Mol. Wt. of such proteins has been determined as 6.0 x 104 to 5.0 x 105 Daltons. GAs are also known to elicit specific responses through such receptor proteins. In cucumber hypocotyls, GA3 promotes the replication of DNA in plastids; thereby it exhibits its specific effect on biogenesis of chloroplasts. Gibberellins are also known to stimulate transcriptional activity of DNA templates through the binding of GA to AMP rich regions of DNA. In some cases, GA effect on transcriptional activity has been attributed to the activation of RNA polymerase through their receptor proteins, where the synthesis of all species of RNAs is enhanced. The most specific effect of GA on transcription has been observed in the germinating barley grains. GA3 induces the mRNA synthesis for alfa amylase enzymes in aleurone cells and the endospermal do not produce mRNA for alfa amylase. Probably this effect of GA is one of the best exemplified actions of phytohormones on differential gone expression. In spite of having voluminous data about GAs effect on different plant species, nothing is clear how different GAs bring about different transcriptional activities leading to morphological expressions. Gibberellins are also known to activate dormant mRNPs and the translating machinery. This view is supported by the fact that GA enhances polysome formation even in the absence of concomitant synthesis of mRNA. Synthesis of alfa amylase enzyme in barley aleurone layers, appearance of some new proteins during bolting and flowering are few examples to demonstrate the effect of Gibberellins on protein synthesis. Effect on Genetic Dwarfism: Genetic dwarfism, in many plants including garden peas and some species of French beans, is controlled by single genes. Such genetic dwarfs respond very favorably to GA treatment and in response to the hormone they grow as tall as normal tall varieties.

However the phenotypic transformation of genetic dwarfs into tall plants is not heritable. Quantitative analysis of tall and dwarf peas and bean plants reveal the presence of high concentrations of GA in tall plants than is dwarf varieties, which suggests that dwarfism is due to a single gene mutation affecting one of the steps in GA biosynthesis. Cross breeding experiment do support this view. The fascinating aspect of Gibberellins effect on genetic dwarfs in its specific action is on internodal growth. The internodes respond favorably than any other parts. The elongation of internodes is possible by two simultaneous processes i.e. one by meristematic divisions of intercalary meristems and the second is by the elongation of meristematic cells. The exact mechanism by which cell elongation is achieved is not clear, nonetheless, GA induced early growth of internodes can be inhibited by colchicine, but not by the inhibitors of transcription and translation. This indicates that the cytoskeleton structures like microtubules are involved in GA mediated growth similar to that of auxin induced cell elongation. Dwarf rice plants also respond very well to GA treatment. GA induced internode elongation in rice plants is due to the activation of inter-calary meristems. After few cell divisions, the cell derivatives elongated 80-1000 times the original size. Elongation, in this case, is due to the excretion of protons and labializing the cell wall to be plastic. Meanwhile, intracellular turgidity also builds up due to the action of GA on plasma membranes. The combined effect of loosening the cell wall, the increased turgour pressure results in the cell elongation.

Effect on Aleurone Layers in Cereal Grains: Cereal grains of Zea mays, sorghum, Hordium, Oryza etc have a distinct layer around the endosperm called aleurone layer, the cells of which are rich in protein granules. During germination, aleurone

cells become active and with time, they secrete enzymes into endospermous tissue, where the reserve starch gets degraded to glucose and the same is utilized by the growing embryos.

There is a definite relationship between the activity of aleurone cells and developing embryo. If the embryo is separated from the rest of the grain and incubated separately, aleurone cells fail to secrete any enzymes. On the other hand if the embryo is also incubated with the rest of the grain, after a period of time, aleurone cells start secreting alfa amylase. Instead of incubating embryo with the rest of the grain, if GA3 is added to the incubating medium, aleurone cells produce alfa amylase enzymes. Thus it is clear that during the early part of germination the young embryo produces Gibberellins which on reaching the aleurone layer activates the cells to produce the required enzymes like alfa amylase, protease, phosphoryl choline glyceride transferase and phosphoryl choline citidyl transferase. The last two mentioned enzymes are required in lecithin biosynthesis which activates membranes. Recently it has been reported that GA besides inducing the expression of alfa amylase inducing enzyme, it also triggers the expression of ribosomal RNA synthesis to a greater extent.

The synthesis of GA induced enzymes can be inhibited by actinomycin D or cycloheximides, which are the inhibitors of transcription and translation respectively. This suggests that GA preferentially activates the gene expression for the above said enzymes.

GA binding protein

Studies on GA induced molecular events clearly suggest than GA at very early stages activates preexisting mRNPs for alfa amylase but later the hormone activates specific genes in aleurone cells. However, the increase in the levels of transcription for amylase and protease reaches maximum at 6-7 hours after hormone treatment. The above results further supported by the fact, that when GA induced mRNAs from aleurone layer are translated in an vitro system, among the many proteins produced, the levels of alfa amylase and protease is found to be high. In actuality, the newly synthesized enzymes are loaded into membrane vesicles and the same is secreted out of the aleurone cells into endospermous tissue, where they bring about hydrolytic activity and thus mobilize the food materials for the developing embryos.

Endosperms cells are dead cells. In recent investigations it has been shown that GA not only induces the synthesis of mRNAs for alfa amylase but also induces the synthesis of RNA. But ABA suppresses the GA induced mRNA for alfa amylase. However ABA does not prevent the synthesis of RNAs.

GA signaling takes through trimeric G receptor protein. This leads to pathways, one-Ca+ dependent pathway and the other Ca+ independent pathway; not much is known about how GA induces signal transduction. The Ca+ independent pathway activates phosphorylation of DELLA proteins, which are repressors of GAMB gene ( GA induced Amylase-beta). Phosphorylation of F-box proteins (SLN1 and SLR1) leads to ubiquitinated protein degradation. This facilitates the GA-GA receptor complex bind to GAMYB gene promoter and activates the transcription of the genes. Translated product GAMY beta protein, a transcriptional regulator protein, enter the nucleus and binds Amylase gene regulatory elements called GARE (GA response elements) and induce amylase gene expression. Transcripts on translation produce amylase proteins which are transferred into ER and loaded into vesicle and stored in cytoplasm. They are released in response to GA through what is called Ca+ dependent pathway. Note MYB is a family of genes there cans any where 80-108 in number; they are the most abundant transcription factors.

Effect on Vernalization: Not all seeds released from the fruits germinate immediately. Some pass through a period of dormancy. And those seeds which exhibit temporary suspension of growth activity are called dormant seeds. In order to make dormant seeds germinate they have to be subjected to scarification or stratification or some of them have to be subjected cold treatment. Plants or seeds acquiring the ability to accelerate flowering in response to cold treatment is called vernalization.

GA is known to overcome cold treatment for plants requiring vernalization, but it can also break the seed dormancy. Quantitative estimation of GA in vernalized seeds do not show any increase in GA content in response to cold treatment, however, during germination at normal temperature, the concentration of GA increases significantly; these studies suggest that cold treatment per se does not increase GA content, but provides the ability to synthesize more GA during germination and growth. In some instances, where dormant seeds requiring far-red treatment to break the dormancy GA acts as the substituent. Effect on Parthenocarpy: Where auxins fail, GAs are found to be very effective in inducing Parthenocarpy. In addition to it, GA induced parthenocarpic fruits are larger in size and sweeter in content. GA is used to increase the total yield of grape fruits. It is also highly effective in increasing the yield and sugar content of

sugar cane. However, the application of GAs should not be more than what is required in grape cultivation; otherwise fruits will be damaged .

Gibberellins and Auxin Interactions: Gibberellins and auxins are found to induce cell elongation, parthenocarpy and metabolic activities including RNA and protein synthesis. But GA and auxins have their own specific effects on different tissues of the plant body. While GA promotes internode elongation, overcomes genetic dwarfism, induces amylase synthesis in aleurone cells and in some cases it can substitute cold treatment or far red treatment. IAA does not elicit any of these effects. On the other hand, auxins impose apical dominance, induce adventitious roots and induce cellulase synthesis. On the other hand GAs dont elicit any of these responses. However, in the case of cell elongation though GA and IAA have independent actions in promoting the growth of etiolated normal pea stem sections, if both the hormones are provided together, their total effect is just additive but not synergistic. On the contrary, if internodes of dwarf pea stems are treated with either GA or IAA, the promotive effect on stem segments is very little, but if both are provided together the stimulation in terms of growth is highly pronounced and the total effect is synergistic. The above observation indicates that gibberellins need auxins for synergistic activity. This particular conclusion is further substantiated by the fact that a decapitated internodal segment does not respond to GA treatment alone but if the apical meristem or an agar block containing auxin is placed on the decapitated segment, elongation of the internode is stimulated in the presence of GA. This is because apical meristems do synthesize auxins, which interact with gibberellins to bring about the combined effect. Recent studies in in vivo and invitro system strongly suggest that GA has an important role in promoting the biosynthetic pathway of auxin, thus in the presence of GA, the levels of auxins increase. Probably GAs enhances the rate of IAA synthesis.

Microarray of GA induced gene expression

Effects Stem Growth Parthenocarpy Root initiation Callus formation Induction of amylase Bolting and flowering

IAA + + + + -

GA + + + +

GA and ABA:
Plant hormonal interactions are fascinating for the simple reason that many of them act antagonistically and some cooperatively and few synergistically. Some of the interactions are concentration dependent. GA and Auxin induce parthenocarpy; Cytokinins and auxins act antagonistically in Auxin induced new root formation. Induction of dormancy and breaking dormancy are two opposite development pathways in seeds. ABA can induce dormancy and GA can break the dormancy. Cold treatment induces vernalization in some plants in ABA dependent manner, but vernalization can be broken by GA.

The diagram illustrates how ABA and GA act in opposite ways in response to environmental inputs.

GA and Flowering:
Plants which require longer photoperiodic conditions for flowering are called long day plants and those plants requiring shorter photoperiodic are called short day plants. Long day plants do not flower under short day conditions and vice versa. Correct photoperiod treatments stimulate the synthesis of flowering hormone called florigin which inturn induces flowering by differential gene expression. However, there are a large number of plants which do not require such photoperiodic conditions for flowering.

FCA and the control of Arabidopsis flowering time. (A and B) Schematic representation of the principal genetic pathways controlling flowering time in winter annual and rapid cycling accessions of Arabidopsis. Promotive activities are denoted by arrowheads, repressive activities are denoted by T-bars. The photoperiod (PP) and gibberellin signal transduction (GA) pathways are shown activating genes with a floral meristem identity function (FMI), while FLC is shown to repress this. FRI, the FCA-containing autonomous pathway (AUT) and vernalization pathway (VRN) regulate FLC in an antagonistic manner. The most frequently found difference between winter annual (A) and rapid cycling (B)Arabidopsis accessions is allelic variation at FRI, with most rapid cycling accessions carrying inactive, loss-of-function fri alleles. (C) A comparison of the phenotype of wild-type Ler plants and late flowering fca1 mutant plants. Although grown for the same time and under identical conditions, wild-type plants have already flowered while the fca-1 plants have remained in the vegetative state and continued to produce more leaves as opposed to floral organs. ( D) Schematic representation of the alternative processing ofArabidopsis FCA pre-mRNA. Exons are represented as filled boxes and intron by lines.

Some of the long day plants under non inductive short day conditions exhibit rosette leaves because of extreme condensation of internodes. If such plants under non inductive conditions are sprayed with Gibberellins, they get stimulated and the internodes elongate dramatically; such GA induced elongated stems also initiate flowering, such a phenomenon is known as Bolting and Flowering. Though bolting and flowering are two different events, GA has been found to effect the elongation of internodes first during which process flowering substances are also produced as a result floral buds are initiated.

Furthermore, quantification of GA in untreated plants and plants which are in flowering show very low amount of GA in the former and higher content in the later. It amounts to the fact that during proper photoconductive conditions synthesis of Gibberellins increases. However, GA has no effect on majority of short day plants. Strangely particular variety of short day rice plants responds very well to GA treatment and produce flowers. From the above discussions, it is clear that Gibberellins actually over come long day treatments are photo period requiring plants. The interrelationship between long day photoperiodic treatments and GA effect has been explained on the basis of the following facts. Plastids synthesize and store the same under long day photo periodic condition. At the same time, the phytochrome pigments which are also present within plastids, absorb red light and transform into far red light absorbing pigment. This form of pigments labelises the plastid membranes and thus Gibberellins are released from the plastids. Thus the concentration of GA increases in photo period induced long day plants. In spite of it, the exact mechanism by which GA brings about differential gene expression during flowering is not clear. Probably, it may also require other factors for floral induction. Effect on inflorescence and floral structure: Though GA is known to induce bolting and flowering in long day plants, the effect of GA on a short day plants like sorghum bicolor is very interesting. In these plants, GA3 stimulates flowering even under non inductive conditions. But in combination with far red light treatment GAs effect is synergistic. The hormone also brings about marked increase in the number of spikelets and glumes. Along with these promotive responses, GA also increases the number of floral structures like ovaries and promotes fertilization, but inhibits stamen development. Effects of GAs on Flowering and Flowering Genes
By Valerie Sponsel, Biology Department, University of Texas, San Antonio, TX, USA, with acknowdgment

Flowering in Arabidopsis, a plant for which more tools exist to study the molecular genetics of the process than in any other species, is regulated by four separate pathways: (a) the photoperiodic (long day) pathway, which operates in the leaves; (b) the convergentautonomous (leaf number)/vernalization (low temperature) pathway; (c) the carbohydrate (sucrose) pathway; and (d) the gibberellinpathway. The latter three pathways all operate in the shoot apical meristem. The four pathways converge on a number of floral pathway integrators that together regulate floral initiation. The recent identification of FLOWERING LOCUS T (FT) as the gene expressed in leaves that encodes a phloem-mobile mRNA or protein that fits the description of a universal flowering stimulus, florigen, has been an exciting development after the decades-long search for this elusive flowering signal (Huang et al. 2005; and see Zeevaart 2006, for a review). Additional work is seeking to further define the integrative networks that exist between the different pathways. Web Essay 25.2, from which Figure 1 is taken, provides a complete consideration of this subject. This topic briefly addresses the GA pathway, when it operates, and what is known about how it is integrated with the other pathways.

Figure 1 Multiple developmental pathways for flowering in Arabidopsis: photoperiodism, the autonomous (leaf number) and vernalization (low temperature) pathways, the energy (sucrose) pathway, and the gibberellin pathway. The photoperiodic pathway is located in the leaves and involves the production of a transmissible floral stimulus, FT protein. In LDPs such as Arabidopsis, FT protein is produced in the phloem in response to CO protein accumulation under long days. It is then translocated via sieve tubes to the apical meristem. In SDPs such as rice, the transmissible floral

stimulus Hd3a protein accumulates when the repressor protein, Hd1, is not produced under short days, and the Hd3a protein is translocated via the phloem to the apical meristem. In Arabidopsis, FT binds to FD, and the FT/FD protein complex activates the AP1 and SOC1 genes, which trigger LFY gene expression. LFY and AP1 then trigger the expression of the floral homeotic genes. The autonomous (leaf number) and vernalization (low temperature) pathways act in the apical meristem to negatively regulate FLC, a negative regulator of SOC1. The sucrose and gibberellin pathways, also localized to the meristem, promoteSOC1 expression. (After Blzquez 2005.) (Click image to enlarge.)

In Arabidopsis, flowering occurs in long days (LD) even in the GA-deficient ga1-3 mutant, although it is somewhat delayed compared to wild-type plants. However, this mutant will not flower in short days (SD) unless treated with GA. Double mutants of ga1-3 and co, (the wild-type CONSTANS is normally expressed in leaves and is upstream of FT in the LD pathway) will not normally flower, either in SD or LD. From this and other information it is proposed that the LD and GA pathways are independent, with the GA pathway being essential for flowering in noninductive conditions. Input from the LD pathway (transduced through FT) and from the GA pathway converge on SOC 1 (SUPPRESSOR OF OVEREXPRESSION OF CO 1), that is expressed in the apical meristem. Thus, GA treatment toga1-

3 mutants in SD causes both enhanced expression of SOC in the apical meristem, and flowering. Over
expression of SOC1 in ga1-3mutants allows them to flower in SD without GA treatment (Moon et al. 2003). An additional floral pathway integrator is LFY, the product of a floral meristem identity gene that is expressed in apical meristems.LFY expression, which is high in LD-grown plants, is only observed in SD-grown ga1-3 mutants that have been treated with GA. Constitutive LFY expression will also allow SD-grown ga1-3 to flower without GA treatment (Blazquez et al. 1998). Using deletion analysis, Blazquez and Weigel (2000) identified an 8 bp motif in the LFY promoter that is necessary for GA responsiveness, but not for response to day length. This sequence is a potential target for MYB transcription factors, and candidate MYBs whose expression increases in the shoot apex in response to exogenous GA4, or to elevated levels of native GA4, have been identified in both Lolium

temulentum and Arabidopsis (Gocal et al. 1999, 2001b). Interestingly, LDsbut not GA treatment
cause a rapid increase in expression of the L. temulentum ortholog of FT (LtFT), indicating in this plant too that the LD and GA pathways converge downstream of FT (King et al. 2006).

Plant Hormones-CYTOKININS
Skoog and his students, while working on the callus cultures under in vitro conditions, Cytokinins were discovered. They found the callus that develops from the stem explants, containing both pith and vascular elements, develops well. But the explants containing just pith cells produces callus, but further growth of its stops, even in the presence of optimal concentration of auxins. This is because the cell in the callus somehow rendered incapable of cell division. If such callus is supplanted vascular tissues, extract of vascular tissues, coconut milk or malt extracts, the growth of the callus will be restored and the cells exhibit mitotic activity. This effect has been attributed to the presence of some active principle in the supplanted coconut milk. Cytokinins have various functions in association with other hormones or its accessories.

The active principle responsible for inducing cell division was isolated first from the extracts of yeasts. Such a substance was called kinetin, later the name was changed and called as cytokinin; kinetin terminology was misleading for another class of compounds called kinins which were already known to be found is animal systems. However, the term cytokinin has been given to all those compounds that are capable of inducing cell division in the presence of optimal concentration of auxin. Now it is known that a good source for cytokinin is coconut liquid endosperm and milky endosperm of sweet corns.

Some of the naturally occurring cytokinins are 6-furfuryl-aminopurine, Ribosyl zeatin, Zeatin, Isopentinyladenine and Dihydrozeatin. Interestingly, the above compounds are also found in denatured products of nucleic acids. Many synthetic cytokinins are also available in the market, ex., 6 Benzyl aminopurine, 6 Phenyl aminopurine.

Distribution: Cytokinins have been detected in a wide variety of plants; from unicellular yeasts, algae to multi cellular higher plants. Particularly in higher plants, cytokinins are found in root tips, xylem, young leaves; endosperms of developing fruits, germinating seeds and tumour tissues. Site of Synthesis: Most of the cytokinins required for the plant body are synthesized in the root tips, and then they are translocated to different regions particularly to meristematic and expanding tissues; transportation is through xylem stream. This observation has been supported by many studies.

Site of CK synthesis For example, the amount of cytokinin found in the excised petiole is less than the petiole that has rooted. If the root tips are cut, the growth of the stem apex is more or less inhibited till adequate supply of cytokinin is restored by new root formation. Significant amount of cytokinin is synthesized in required endosperm of palm fruits, kernel of cereal grains and others. Almost all naturally occurring cytokinins are the products of purine nucleotide derivatives. The presence of iospentenyl adenine in some tRNAs has misled people to believe that the source of cytokinins is tRNAs but it is not the case. Nevertheless the site of synthesis of cytokinins in young and developing plants is restricted to meristematic regions of the root tips; where certain enzymes utilize purine and convert them to cytokinins. The presence of isopentenyl adenine in many tRNA is not due to the incorporation of cytokinins into tRNAs. biosynthetic pathway of cytokinins is yet to be elucidated. Effects: However, the exact mechanism and

General Metabolism: Cytokinins effect on respiration is very interesting. It enhances the rate of respiration in the callus, but the same hormone when applied to senescing leaves, brings down the rate of respiration, retards the degradation of chlorophyll and enhances the rate of chlorophyll synthesis. In addition it also increases the rate of metabolic activities involved in C3 pathway. In certain systems cytokinin induces nitrate reductase activity, but in callus it does not. The root nodule development in legumes and its metabolic activity is subtly regulated by the interaction between auxins and cytokinins. Nucleic acid Synthesis: Although cytokinin is known to stimulate cell division, it does not induce DNA synthesis as a prelude to cell division. But in the presence of auxin, it promotes DNA synthesis. So it is suggested that cytokinin stimulates and auxin promotes DNA synthesis. For example, an aged and non proliferating callus cells can be stimulated to undergo mitotic divisions by the application of cytokinin. Cytokinin binding protein i.e. Receptor proteins have been identified in many plant systems. The cytokinin protein complex is known to induce transcription activity a pea bud chromatin in a cell free system. In Soy bean and French been hypocotyls, it transitorily inhibits IBA induced RNA synthesis for a period of 6-7 hours, but later it stimulates RNA synthesis. Specific species of tRNAs, containing cytokinin activity, is seen in the cotyledons and hypocotyl segments of bean plants. But this has been identified as due to post transcriptional modification of tRNA. Recent investigations have clearly elucidated that the presence of cytokinin moiety in some tRNA structures is not responsible for eliciting any cytokinin mediated responses, however it is to be noted that many tRNAs contain isopentenyl adenine (IPA) in the anticodon loop region PROTEIN SYNTHESIS:

Quite a number of experiments, involvement in vivo systems or cell free invitro systems, have demonstrated that cytokinins first stimulate translational activity without any concomitant increase in the synthesis of mRNAs. This particular effect has been attributed to cytokinins ability to activate pre existing mRNPs and ribosomal proteins needed for chain initiation. Fox et al, have demonstrated that cytokinins bind to ribosomal surface through a receptor protein. This protein activates ribosomes by dephosphorylating a specific ribosomal protein. Furthermore, the increased activity of protein synthesis in response to cytokinin has been found to be concentration dependent. It is also interesting to note that cytokinin mediated protein synthesis; either as short term or long term effects, shows the synthesis of new proteins in treated tissues. At least in the hypocotyls of French bean, cytokinin inhibits some proteins that are induced by IBA, but cytokinin by itself enhances the rate of tubulin synthesis similar to that of IBA. Cell Division: The name cytokinin is derived from its ability to induce cytokinesis during cell divisions. Though cytokinins stimulate the process, the permissive effect of it is controlled by auxins. When cytokinin is provided to a liquid culture medium containing plant cells, the protein synthetic activity of the cells is greatly stimulated. Moreover, some of the proteins thus synthesized are new ones. These observations suggest that cytokinins control cytokinesis by regulating the synthesis of some specific protein factors that are required for cytokinesis. Cell Enlargement: Isolated cotyledons of Cucumis, radish and other plants expand dramatically when they are treated with cytokinin. The expansion of cotyledons is rather more due to cell enlargement than due to cell divisions. During cytokinin induced cell enlargement, respiratory activity increases significantly and greater amounts of K+ ions are accumulated in the cells. At the same time, cells in response to the hormones induce the synthesis of few minor species of RNAs and some proteins. But the inhibitors of respiration, transcription and translation cotyledons completely also respond inhibit to cytokinin light mediated treatment cell and enlargement. Interestingly such red

enlarge. However the red light induced enlargement cannot be reversed by far red light treatment which further suggests that cytokinins bring about permeability changes within the membranes.

Richmond & Langs effect: Senescense of leaves leads to yellowing and finally leads to the fall from the plant. If a young excised leaf is kept in water, it slowly changes its color to yellow and dies. If such leaves are

provided with cytokinin, the yellowing is significantly delayed and such an effect is called Richmond and Langs effect; named after the discoverers. Senescense is a common feature exhibited by parts of the plant which show definite growth pattern. With age, the structures like leaves, flowers, etc., senesce and die. Among various factors, decrease n the content of auxin acts as a very important factor in inducing senescence. In matured leaves, still attached to the plant body with time, senescence sets in and leads to the degradation of chlorophyll. Catabolic activity increases and the formation of abscission layer begin. In detached leaves also one finds similar processes. But cytokinins prevent and prolong the initiation of senescence for a quite a period of time. This effect of cytokinin has been explained as due to the prevention of degradative catabolic processes by the way of repression activity of few hydrolysing enzymes like protease, RNAse, DNAse etc. Furthermore, cytokinin facilitates the chlorophyll synthesis. It also sustains the activity of carbon fixation, RNA synthesis and protein synthesis. Still the exact mechanism by which cytokinins prevent aging and senescence is not known. Effect on Dormancy: Dormant buds that develop due to certain adverse environmental factors remain inactive for a long time. If such buds are treated with cytokinins they come out of dormant state and sprout. This is due to the effect of cytokinin in activating cell division which was prevented by mitotic blocks present in the dormant besides. Interestingly, cytokinins also overcome auxin imposed apical dominance and stimulate the growth of the axillary buds, probably by overcoming the factors such as mitotic blocks. Interaction of cytokinins with auxins in morphogenesis: Plant development is a series of biochemical events which ultimately leads to morphogenic changes. This process is regulated by a number of phytohormones which bring about differential gene activity leading to the development or specific phenotype. The interaction between various hormones is very complex and they operate at different levels like differentials transcription, protein synthesis, enzyme activity, permeability etc. Here the interaction between cytokinin and auxin id discussed briefly.

The culturing of plant explants with various combinations of cytokinins and Auxins show the inhibitory effect of cytokinin on auxin induced new root formation. In tissue culture experiments, the plant explants are supplemented with optimal nutrients including carbohydrate as the energy source and hormones as growth regulators. Plant explants, which may be leaf segments stem segments, roots or embryos develop and produce an undifferentiated tissue called callus is a defined culture medium. This behavior is due to the presence of balanced concentration of auxin and cytokinin in the nutrient medium. The callus is like a cancerous tumour, where the cells are undifferentiated and they are under the spell of uncontrolled mitotic activity. If the concentration of auxin with respect to that of cytokinin ratio is changed, from the ratio that is favorable for the growth of only callus, the cells of the callus undergo transformation to produce organs like roots or shoots. If the ratio between auxin and cytokinin is high, the callus cells undergo transformation and produce roots. On the other hand, if the ratio between auxin and cytokinin is low the callus cells initiate shoots. As all cells in the callus are totipotent, depending upon the hormonal concentration, cellular genetic material is differentially expressed and their products induce specific morphogenetic events leading to organ formation. In this system it is the concentration of each of the hormones provide the signal for differentiation of undifferentiated cells into new organs.

In spite of having sufficient knowledge about the hormonal effects in organogenesis, the molecular basis of morphogenesis is not very clear; however, one example can be used to illustrate how auxins and cytokinins interact and modulate molecular events during morphogenesis. Adventitious root formation in the hypocotyls of phaseolus vulgaris in response to auxin treatment is a process of redifferentiation and reorganization of pericyclic cells into root primordia. While IBA induces new root formation, cytokinins inhibit IBA mediated root initiation. At molecular level both auxin and cytokinins increase the rate of protein synthesis without increase in transcriptional activity at 30 minutes of treatment. In the case of auxin treated hypocotyls increase in transcription is detected after 45-60 minutes, but not in cytokinins treated. In the segment treated with both auxin and cytokinins one does not observe such changes at short time but observed at longer periods. This observation comes from quantitating in vivo and in vitro protein synthesis using 14C leucine. Also purified poly-A is used for quantitation using in vitro translation. The results show increase in translation at later stages is due to transcriptional activation. General Protein analysis on SDS-PAGE at 24hrs, 48hr and 72 hrs, shows a remarkable increase in two bands at 55Kda and 58Kda and few other bands of high molecular weight. However increase in the said proteins was not detected in cytokinin treated segments. The bands were identified as Tubulin alpha and beta proteins. More over in Auxin treated segment discerned from in vitro translation of Pol(A) RNA shows one more 115Kds band showed up only in auxin treated but not in any others. This increase is only transitory from 24hrs to 36hrs. Then the protein band disappears. The role of this protein is not known. At molecular level Auxin acts through Auxin response factor for activation of genes. But the activation of transcription at 36 hrs or more, by cytokinins is yet to be found. However cytokinins action of activation of genes has been discerned to some extent in Arabidopsis. Cytokinin acts as signal molecule and it binds to dimeric receptors anchored in plasma membrane. The receptors are believed to be similar to receptor tyrosine kinases (RTKs), but their kinase activity is restricted to histidine residues (HPK), so they are called Arabidopsis histidine protein kinases (HKs). This kinase activates histidine phospho transfer proteins (AHPs) transducers. These are response regulators which can activate or repress gene expression. AHK-ps enter the nucleus and interact with ARRs (nuclear response regulators) and activate transcription. Such components were also observed in maize.

Outline of cytokinins pathway There are four steps in cytokinins signaling pathway as shown in the obove diagram. AHK sensing and signaling, AHP nuclear translocation and localization and activation of ARR genes and a negative feedback loop through Cytokinin inducible ARR gene products. Arabidopsis encode three cytokinins receptors; cytokine response gene1 (CRE1 also called AHK4) ,Woodenleg (Wol) and AHK2/3. There are other histidine kinases such as CK11 and CK12 (AHK5) which are independent of cytokinins but they do respond to cytokinins. Mutants of CRE1 and Wol show defects in cytokinins mediated shoot formation and defects in root vasculature.

The ARR (A response regulator proteins) ( such asARR1, ARR2,and ARR10, which are transcriptional activators carry MYB like DNA binding domains, and also contain glutamine Q rich activating domains. ARR p-lation activates transcription of A-type ARRs. Expression of cyclin D and ARR5 show they are the major sites of cytokinins action in root and shoot meristems. There are 54 gene encode AHKs, AHPs and ARRs.

Cytokinins requirement for nodulation

In procambial cells (PCs), the coordinated signaling by cytokinin and auxin induces the expression of genes that are involved in the maintenance and growth of procambial activities into xylem elements. The auxin-signaling pathway might be involved in gene expression of auxin-response factors, such as MONOPTEROS (MP), that also function as transcriptional activators, and their repressors, the AUX/IAA proteins. Cytokinin might be perceived by the WOL/CRE1/AtHK4 cytokinin receptor, which, in turn, transmits an intracellular signal that is mediated by a His Asp phosphorelay mechanism to PC-related histidine-containing phosphotransfer factors (AHPs) and then to PC-related type-B response regulators (ARRs). The type-B ARRs might function as transcriptional activators of PCrelated genes including the genes of their repressors, the type-A ARRs. The presence of repressors in auxin- and cytokinin-signaling pathways might allow cytokinin and auxin signaling to be temporal. Brassinosteroids (BRs in the figure) are biosynthesized actively in PCs and secreted, but brassinosteroids do not work as a signal for the maintenance of procambial activities. Instead, brassinosteroids, in the presence of auxin, might initiate differentiation of procambial cells to precursors of xylem cells (pXCs) after recognition by a receptor, which might be a heterodimers, composed of either brassinosteroid-insensitive-1 (BRI1) or one of the BRI1-like proteins (BRL1 BRL3), plus BRI1-associated receptor kinase-1 (BAK1). The brassinosteroid signal inactivates the negative regulator BIN2 (brassinosteroid-insensitive-2), which allows the unphosphorylated form of bri1-EMS-suppressor-1 (BES1) and brassinazole-resistant-1 (BZR1) to translocate to the nucleus and to promote pXC-related gene expression. Among the most important pXC-related genes that are induced by brassinosteroids might be the HD-ZIP-III-homeobox gene family, which might function in further xylem cell differentiation. KANADI and the microRNAs MIR165 and MIR166 might suppress differentiation of PCs to pXCs. The suppression by the microRNAs might be caused by the rapid degradation of the HD-ZIP-III gene mRNA through RNAi machinery.

The above diagram shows how cytokinins play a role in localization of CRF6 in cytosol.

RFs in Arabidopsis
We have taken a genetic approach identifying knockout mutants in each the CRF genes in order to better understand their function in plant growth and development. One research area we on is determining the b

of

U C
Cytokinins induce CRF genes. There are six such CRFs; they are cytokinin response/regulatory factors and transcriptional activators. They play important role in plant development. Mutants in CRF genes produce malfunctions in leaf and cotyledon development shown in the diagrams above.

as

Arabidopsis, each of which into pairs of related genes that arose as ancient genome duplication in plants. Each pair of genes has been maintained over time and may have become specified in function. -.
This figure provides general information about cytokinin signal pathways.

While only three have taken a genetic approach identifying knockout mutants in each of the CRF genes in order to better understand their function in

plant growth and d

Cytokinin activated gene expression is shown in the form of microarray. CRFs act in parallel with type-B ARRs to mediate cytokinin regulated gene expression. (A) Wildtype, arr1,12, crf1,2,5, andcrf2,3,6 seedlings were treated with either 10 M BA or a DMSO control for 1 h and gene expression analyzed by using a microarray. Genes that displayed a 2-fold change in response to cytokinin in the wild type are shown. (B) Venn diagram of the 135 cytokinin-regulated genes affected by the arr1,12, crf1,2,5, and/or crf2,3,6 mutations. (C) Model of cytokinin signaling. Both AHPs and CRFs move into the nucleus in response to cytokinin. Once there, the AHPs phosphorylate the type-B ARRs, which, together with CRFs, mediate cytokinin-regulated gene expression. Another diagram shows few more Cytokinin signaling features.

Plant hormones-ABSCISINS
Plant physiologists knew presence of growth inhibiting compounds in plants for sometime. But the active substance responsible for it was discovered by Ohkuma and Cornforth, later Ohkuma established its structure in 1965; Cornforth further confirmed this in the same year by synthesizing the compound in the laboratory. Today, we know that growth inhibiting substance as Abscisic acid or Abscissin II, which was once called as Dormin. Besides ABA, plants also contain other natural growth inhibitors such as Coumerin, Ferulic acid, Para ascorbic acid, phaseic acid, violoxanthin, etc. In addition, plant chemists have identified some synthetic growth inhibitors. Ex. 2,3,5 tri iodo-benzoic acid, morphactins, caproic acid, phenyl propionic acid, Malic hydrazide, etc.

Distribution: Abscissin has been isolated from a wide variety of plants. The amount of ABA found in plants varies from species to species and from organ to organ. For example, in the pulp of avocado fruits the concentration of ABA is 10 mg/kg. And in the dormant buds of cocklebur it is 20 mg/kg. Again depending upon the environmental conditions the concentration of ABA varies in the same part or the organ of the plant body. In the leaves of phaseolus vulgaris, if the plant is subjected to water stress within 90 minutes the amount of ABA raises from 15 mg/kg to 175 mg/kg. Intense light and other dormancy inducing factors are highly effective in increasing the concentration of ABA is the plant body.

ABA like other phytohormones exists in free state or in bound form. The free state of ABA is supposed to be an active form. The bound forms of ABA are conjugates of glucose and its derivatives. Depending upon the influencing factors, they undergo rapid changes from one form to another to elicit their respective functions. ABA is mostly transported through sieve tubes in the plant body. But the rate of transportation ranges from 25-45 cm/hr, which is far below the rate of sucrose transport that takes place in sieve tubes, which suggests that ABAs transport is independent of sucrose.

Structure and Biosynthesis: Abscissin I is a sesquiterpene consisting of 3 isoprenoid units. It has a 6-carbon ring and a 5-carbon chain ending with a carboxyl group. The ring has a double bonded oxygen group. However, naturally accuring ABA exists in two stereo isomeric forms like (+) ABA and (-) ABA but the active form is ABA (-). The biosynthetic pathway of ABA is almost similar to that of Gibberellins. In fact, mevolonate acts as the precursor. But some research workers consider that ABA is a break down product of violoxanthin, however, the detailed studies, using radioactive isotopes as labels, have revealed that it is derived from simple isoprenoid compounds and not from violoxanthin or such products.

The site of synthesis of ABA has been identified as plastids, which includes proplastids, etiolated and also mature chloroplasts. The initial biosynthetic reactions are more or less the same as that of GA up to Farnesyl pyrophosphate FPP and GGMP. From GGMPP onwards, the synthesis of GA and ABA go through two different pathways; which is controlled by phytochrome, water stress and environmental factors. Depending upon the factors, one pathway is favored over the other. Adverse environmental factors like water stress, salt stress and severe cold favor the synthesis of ABA. But red light favorable conditions enhance the synthesis of GAs.

Thus plastids play an important role in regulating the synthesis of anyone of these two hormones at any given time depending upon phytochrome state or water potential of the cell.

Effects of ABA: Dormancy: Abscisic acid is a stress induced hormone. During winter season in temperate and cold regions, most of the axillary and terminal buds undergo a period of suspended physiological activities called a period of dormancy. Even seeds belonging to various species exhibit dormancy; which has been attributed to ABAs effect on physiological activities of the cells. Studies on ABAs effect on transcription and translation indicate, the hormone inhibits RNA polymerase activity. Its effect on translation has been attributed to its inductive ability to synthesize a RNA species, which is poly (A) rich a (mol. Wt. of 10,000 Daltons). This poly(A) RNA is capable of inhibiting the translation of mRNA by hybridizing with 3 region of mRNA. Thus it affects both transcription and translation. During the seed development or during induction of dormancy transcriptional activity has been found to be normal but the mRNA synthesized is made inactive by ABA by the above said mechanism. Most of the mRNAs remain as inactive mRNPs or informosomes. Thus ABA imposes dormancy in the said structures like buds and seeds. ABA is also known to promote dehydration in seeds and render them dormant. How ABA brings about this effect is not clear, but it is known that ABA has profound effect on Ca+, K+ and H+ fluxes across the membranes. ABA inhibits GA mediated Alfa amylase synthesis: It is very interesting to know that one hormone interacts with another hormone in regulating the gene expression. Interestingly, both GA and ABA are synthesized in the plastids and they follow the same pathway in the initial steps. Curiously enough, both the said hormones inhibit each others physiological effects.

While Gibberellins activate aleurone cells to produce alfa amylase and proteases through differential gene expression, ABA completely inhibits the GA mediated synthesis of amylase and protease by inhibiting the translation of mRNA through ploy-A rich RNA. By another mechanism ABA also activates the release of specific short chain fatty acids that inhibits GA3 induced amylosis in barley endosperm by inhibiting transcription and translation of alfa amylase mRNAs.

Role of GA in countering ABA action during seed gemrination

Effect on Cell Expansion: Normal plants treated with GA or IAA exhibit pronounced growth by way of activating cell elongation, but ABA inhibits the cell growth considerably. Considering this, some botanists think that the genetic dwarfism is probably due to the presence of excess of ABA in their plant body. But quantitative studies reveal that there is no such correlation between the genetic dwarfism and ABA content. And genetic dwarfism is rather due to the deficiency of Gibberellins than to the presence of excess ABA. But the inhibitory effect of ABA has been attributed to its effects on membrane

permeability and ion fluxes. It is well known that ABA brings about the extrusion of K+ ions from guard cells and causes closing of the stomata. Thus ABA may act on other cells as in the case of guard cells and inhibit GA or IAA mediated cell elongation. Senescence: Abscisic acid is known to initiate aging in leaves in which process the detached leaves loose their green color and becomes yellow. During ABA induced senescence, degreening takes place by the synthesis of an enzyme called chlorophyllase, whose synthesis can be blocked by transcriptional or translational inhibitors. ABA also induces high rate of respiration for only a short period of time. The labalizing effect of ABA on lysosomes leads to the release of enzymes, which start degrading cellular macromolecules. Photosynthesis and other anabolic processes like protein synthesis, etc., are greatly reduced. The induction of ethylene synthesis is believed to be activated by ABA in certain cases, where the auxin concentration is very low. The ABA induced ethylene in turn causes the formation of abscission layers in the stalks of leaves and fruits. On the other hand, cytokinins are known to overcome ABAs effect, by decreasing degradative processes. ABA influences the formation of abscission layer. This process has been exploited commercially in harvesting fruits and cotton balls. By spraying ABA on cotton plants, most of the stalks of cotton balls develop abscission layers simultaneously and it will greatly help in mechanical harvesting of cotton balls. Similarly, in fruit orchards, to obtain uniform harvesting, controlled application of ABA facilitates the harvesting of most of the fruits at one time. Effect on Turgour Movement: In recent years, ABAs effect on membranes, in changing ion fluxes, is drawing the attention of all plant physiologists. ABA has a positive effect on guard cells, where the cells loose most of the K+ ions to subsidiary cells and collapse to induce the closing of the stomata. In fact, guard cells also contain chloroplasts and it is suspected that ABAs are released from them under certain conditions like water stress or high temperatures, which in fact induce the closing of the stomata.

Stomatal movement in closing and opening of guard cell is an excellent system in understanding interaction of cellar and external factors. Co2 depletion in the atmosphere, water stress leads to activation of ABA, that is released from chloroplast found in guard cells. They acxtivate Potassium and H-channels for expulsion og ions and another class of channels opens for inward movement of Ca+ ions. This leads to the closing of the stomata; this process is reverse of opening, where K+ ions are translocated in to build up of ion concentration so as to facilitate the movement of water into guard cells, so the cells turgour pressure increase and stomata opens.

The figure is not very explanative, though looks good.

Similarly, the turgour movements of motor or bulliform cells in grass leaves, turgour movements of parenchymatous cells in the pulvinous of Mimosa pudica and other plants which exhibit nastic movements are believed to be due to the action of ABA. In recent years, the role of ABA in photo induced growth curvatures has been gaining importance. When stem tips are unilaterally illuminated from one side, the cells that receive light release some quantity of ABA, which causes the efflux of K+ ions, which results in the collapsing of cells. At the same time, they also inhibit cell elongation. But on the other side i.e. darker side cells grow normally and bring about growth curvatures. It look like ABA by their controlled synthesis and release bring about innumerable physiological is plants effects.

ABA and Gene Expression: ABA is also involved in gene expression of ABA response factor (TFs), whose upstream binding sequences are given in the table. Activation leads to the production of factors that provide stress tolerance. ABA is involved in a variety of stress tolerance activities such osmotic stress, cold stress, superoxide (ROS) stress, salt stress and others. Only diagrams have been provided they are self explanatory in nature.

Ros response involves dsRNA mediated mi RNA

Plant Cell Vacuoles

Mature plant cells contain a single large central vacuole and it is the most conspicuous compartment of the cell which occupies nearly 50-70% of the total cell volume. On the contrary, meristematic cells are lacking in such vacuoles. Plant cell vacuoles are distinct and characteristic in having a single unit membrane called tonoplast, which separates the vacuolar content from the rest of the cytoplasmic fluid. The liquid present within the vacuole in called cell sap this contains a host of inorganic and organic compounds. Cell derivatives of meristems; first undergo expansion, and then differentiation. During this stage many smaller vacuoles arise from cytoplasmic membranes of both RER and SER and fuse with one another to form a large central vacuole. Tonoplast membranes at their cytoplasmic surfaces are associated with polysomal complexes engaged in protein synthesis. In the course of time the central vacuole gets loaded with wide variety chemical

components. The most perplexing situation is that during transformation of mature parenchyma cells into meristematic cells the central vacuole disappears. What happens to the vacuolar components is not known. Plant vacuoles can be identified from other membranous vesicles by vital staining techniques. The neutral red stain being basic in its properties binds components of cell vacuole. These are highly inducible enzymes. ER is involved in the intracellular transportation of various components like proteins, lipids, carbohydrates and others via golgi. Some of ER membranes, containing specific proteins within their lumen, are pinched off into vesicles which later fuse with each other or get processed via Golgi complex and get integrated with specific cell organelles. Even cellular vacuoles of various kinds and dimensions show metabolic activities like amino acid metabolism, fatty acid oxidation. The loading of variety of components in to cell vacuoles bring about changes in the osmotic potential of the cell sap. As a consequence of this, water may freely enter into protoplast; this has been taken as evidence to argue that tugour changes within the cell sap is mainly responsible for the growth of the negative turgour pressure. But now it is known that the growth of the cell takes place without development of turgour pressure. Vacuolar expansion and concentration is another dynamic feature, where vacuoles play a predominated role in the development of young buds. The swelling of vacuole forces the cell wall to bulge into a bud. Similarly in the case of stomata, guard cells contain a number of small contracted vacuoles, but at the time of opening, the same vacuoles fuse and enlarge into a large central vacuole in the guard cells, thus they bring about the movement of guard cells. Nonetheless, it is speculated that hormones like Abscisic acid and cytokinin play important roles in closing and opening of stomata

Vacuolar Contents:

Inorganic substances found in the vacuole show variation from cell type to cell type. For example, more than 90% of the total cellular Mg2+ ions are found within the vacuole. On the contrary, the total concentration of calcium ions and copper ions is just 6% and 2% respectively. But the most common ions like K+ ions are equally distributed between cytoplasm and vacuoles. In certain cases more than 40% of the total phosphates are found in vacuoles and most of it is in the form of polyphosphates called volutin threads. Enzymatic components: It is really interesting to observe the presence of a wide variety of hydrolysing enzymes within the vacuoles. The common enzymes found are carboxypeptidase, RNase, DNase, phosphotases, bglycosidase, alfa and betaamylase etc. The concentration and composition of such enzymes vary from cell to cell and species to species, strangely leaf cells of Solanaceae members like tomato, potato, contain significant amount of protease inhibitors within their vacuoles. How such variety of enzymes and other components are retained and released is a mystery. Other organic compounds:

Plant cell vacuoles also contain a good number of organic carboxylic acids, amino acids, amides, mucilage, anthocyanins, flavones, gums, alkaloids, anthocyanin and other pigments and even tannins. In certain plants like citrus, the vacuoles are filled with highly acidic citrate compounds whose pH is about 2.5 and curiously enough such acidic pH is prevented from inactivating cytosolic components by tonoplast membranes and provides a distinct compartmentalization. In some cases more than 25 per of the total amino acid pool is found in vacuoles.
Specialized Vacuoles:

Aleurone vacuoles: Certain vacuoles in seeds or grains of various pulses and cereals store a variety of proteins; such granular components are referred as aleurone grains. Inulin, legume, vanillin, glycerin, etc. are some of the common proteins found as storage products in Aleurone vesicles. Such vesicles are derived from RER-SER membrane transitions. Most of the stored or aggregated nonliving structures are called ergastic substances. Spherosomes and starch vacuoles: Similar to Aleurone vesicles, lipids and starch are also stored in special vacuoles called spherosomes and amylosomes. Spherosomes are derived from RER SER transitional endomembranes. Such membranous vesicles are endowed with enzymatic system from synthesis interconversion. On the other hand, starch is synthesized in chloroplasts but transported to be stored in amyloplasts. Such starch containing membranous organs are found in large numbers in storage tissues like root tubers, stem tubers, cotyledons and endosperms of seeds and grains. The role of pyrenoids found in lower algal cells and their role in storing starch is very interesting. Functions: Vacuoles are highly dynamic. They store many inorganic and organic compounds including a host of enzymes. At the same time they are engaged in protein synthesis at the cytosolic surfaces of tonoplast where they of found as polysomal complex. Some of the ions are transported across tonoplast membranes against concentration gradient. In crassulacian plants carboxylic acids are released into cytoplasm at nights. The diurnal behavior of vacuoles is a unique feature for succulents. The synthesis and aggregation of various substances like calcium oxalate crystals, raphides and other ergastic is very common for plant central vacuoles.

Plant Hormones-ETHYLENE
In olden days, villagers, even now, used to accelerate the ripening process of banana, mango and other fruits, just before they were taken to market places. The method employed by them was simple. They used to keep the raw and unripened fruits in tightly closed earthern pots and fill the pot with smoke generated by burning cow dung at the base and then seal it. After 12 to 24 hours of this treatment, the fruits would appear yellowish and just started for full ripening and they were ready for marketing. Even today, villagers use this method without knowing why and how ripening is accelerated by the smoke generated by burning the cow dung. But plant physiologists discovered ethylene as the gas that induces and augments fruit ripening. This phenomenon and technology of fruit ripening was known to our village farmers many many centuries back. Even now renowned plant physiologists don't know this. All of us know cow dung produces atmospheric polluting gases such as methane and ethylene. For that matter animal feca and fruit release more pullutants than all the cars (put together) that emit pullutants. Though scientific studies were initiated as early as 1900s, understanding the process of fruit ripening and identification of the causative factor was possible only in 1924. Since then, detailed studies have been made on ethylene and its effect on plants.

Many plant physiologists call ethylene as a plant hormone in gaseous state. But some do not agree with this view instead, they consider ethylene as a byproduct of reactions induced by other phytohormones and not an hormone perse. Two critical enzymes involved are SAM and ACC synthase. Both gene can be used for antisense technology for prolonging commercial fruit ripening, which is help full for farmers for their products will have longer shelf life and this protectiveness ha sno deleterious effect on eaters.

Ethylene is produced in response to exogenous stimuli as shown in the above diagram. In any of the plant developmental processes plant hormones interact with one another.

Distribution: Ethylene is found in almost all parts of the plant body. But it is found in greater amounts particularly in old and yellowing leaves and ripening fruits. This compound being a gaseous substance diffuses through the intercellular spaces easily and rapidly reaches different regions of the plant body. Biosynthesis: Ethylene consists of two CH2 groups held by a common double bond H2C=CH2. The synthesis of ethylene is greatly enhanced by higher concentrations of auxin. Even Gibberellins and cytokinins induce the synthesis of ethylene indirectly. The precursor for ethylene was once believed to be methionine.

But recent investigations, using radioactive isotopes have shown that the precursor for ethylene is 1amino cyclopropane carboxylic acid and not methionine. ACC acts as the direct and immediate precursor. In fact, higher concentration of auxin induces the synthesis of a group of ethylene synthetase enzymes. These enzymes require FMN, H2O and Cu2+ as the cofactors for their activity. The auxin induced enzymatic activity can be inhibited by actinomycin D and CHI, which suggests that ethylene synthases are inducible enzymes. The site of synthesis of ethylene has been suspected to be chloroplasts and its release is believed to be regulated by phytochromes.

Apart from Auxins, factors like wounding, aging, irritation, light, cold temperature and drought can also induce ethylene synthesis. Most of the above mentioned are stress factors even; ABA is known to induce ethylene production.

Effects: Abscission: Onset of winter, cold treatment, drought and such conditions induce the formation of abscission layer in the stalks of leaves, flowers and fruits. Eventually the said structure separates by death of cells from the plant body and withers, it is also called in Greek language as Apoptosis. The structure and the development of abscission layer has been explained in the chapter Auxin. To put it in a nut shell, ethylene induces differential gene expression in the region, where the abscission zone develops. As a result, pectinase and cellulase enzymes produced and the same act upon the cell wall and degrade the same. Thus the abscission layer becomes the weak point and the leaves, fruits, etc., fall down by their shear weight.

Fruit Ripening: Once the fruit reaches a particular stage of development, the raw, hard, green colored fruits undergo transformation to produce matured, soft, sweeter and yellow/red/orange/coloured fruits. The repining process requires period of time ranging from 24 hours to a week or so. And ethylene is known to initiate this phenomenon. Once the ripening is on, there is way to stop it. With the maturity of fruits, the synthesis of ethylene is induced and ethylene induces more ethylene production. During early part of ripening process, ethylene initiates a cascade of events, which follow one after another and end up in a crescendo of biochemical reactions or what is called climacteric state at which all the biochemical reactions are at their maximum efficiency. Ethylene to be effective in its action requires a copper containing metallo protein. Strangely, CO2 is known to bind to the same site of the protein at which ethylene binds and thus it competitively inhibits ethylene action. The active ethylene in its complex form first activates respiratory process by which reserve food materials and organic acids if found, are subjected to oxidative and decarboxylation reactions. The increase in respiratory activity is unusually cyanide insensitive, which means that the electron transport chain used in this process appears to be different from usual mitochondrial electron transport chain. Studies in this regard have revealed that the electron transport chain in this process branches of from Cyt.C and bypasses the cyt.a3 oxidase enzyme which is actually the site of cyanide inhibition. Most of the respiratory and other metabolic pathways that are stimulated by ethylene lead to the formation of more and more of sucrose and organic acids.

As the respiratory activity is reaching its climax ethylene simultaneously affects the membrane permeability and also activates a set of genes resulting in the synthesis of specific mRNAs. On translation of these mRNAs, specific proteins such as pectinase and cellulases are produced. The enzymes then act on the middle wall and primary walls to loosen up the cells, thus render the

hard

fruit into soft fruit.

Ethylene induced fruit ripening can be effectively inhibited by

actinomycin and CHI, which suggests that ethylene induces differential gene expression.

Ethylene also affects the membrane stability and permeability. As a result, the pigments found in the tonoplast leak out and most of the membrane structures get disturbed. Furthermore, ethylene induces the degradation of chlorophyll by chlorophyllase which is again a product of gene expression. Simultaneously some anthocyanins are also synthesized which develop attractive coloration to the skin and the flesh of the fruit. Thus ethylene ultimately makes the fruit into a softer, sweeter and colorful commodity. Effect on apical dominance;

Plants which show conical growth, ex., conifers, is known to have a strong apical dominance effect on the lateral buds. This has been attributed to strong influence of IAA present in the apical meristems found in the main axis. Recent investigations, it has been found that IAA induced apical dominance is more due to ethylene production than to auxin itself. Apical meristems of the main axis synthesize auxin and the same is translocated downwards. At the same time, some amount of IAA produced in very young leaves is also translocated towards the stem and more of auxin gets accumulated in the nodal regions. As higher concentration of IAA stimulates the synthesis of ethylene, which on synthesis, diffuses into lateral buds inhibits the growth. So the apical dominance is actually enforced by IAA through its second messenger i.e. ethylene. But the apical dominance can be overcome by the application of cytokinin, which removes the mitotic block imposed by ethylene and activates cell division, so the lateral buds grow into branches. Effect on Geotropic Movements: Geotropic responses are explained as due to the sensitivity of stem tip and root tip to different concentrations of auxins. But ethylene, a product induced by higher concentration of auxin brings about a reverse of geotropic curvatures called ageotropic effects, where roots instead of growing downwards into the soil curl upwards. In fact, ethylene treated roots loose their sense of directional growth. Sometimes, the effect of ethylene will be similar to the effects of morphactins. In addition, the other effects induced by ethylene, such as stunting, stem enlargement and prostrate habit by an impaired response to gravity are called Triple response to ethylene. Ethereal: In recent years, ethylene derivatives are sold in the market as ethereal or ethephon, a patented product. This compound is nothing but 2 chloro ethyl phosphonic acid. When these compounds are applied to plants in solution form, they release ethylene which in turn brings about its effects. Application value of this compound in agriculture, pomiculture is very well exploited during harvesting cotton balls and other fruit products. Application of ethereal induces not only ripening in most of the fruits irrespective of the age and degree of ripening, it also induces the abscission layer formation uniformly in stalks, which greatly facilitates harvesting either by mechanical means or manually at any given time. Ethylene signal transduction pathways: Ethylene though exists in gaseous form, it diffuses across cells, but when it enters a cell it binds to specific receptor. The signal transduction process are more like RTK but for they have histidine kinase activity. Very often in some cases it looks like an RTK pathway. The ultimate effects are different dpending upon the organ on which it works and the time at which it works. Belwo only the self explanatory diagrams are give for your reference and looking for more information.

Plant hormones-MORPHACTINS And Others

Morphactins are a group of substances which act on morphogenesis and modulate the expression of plants. Chemically, they are the derivates of fluorene compounds. Fluorene by itself is inactive, but the addition of COOH group in the 9th position makes it active. Majority of morphactins, as synthetic compounds, have very diverse effects on plant growth and development. Ex., chloroflurenol, flurenol, methyl benzilate, methyl chloroflurenol, methyl dichloroflurenol.

Most of these components have transitory effect and they are degraded rapidly in plants. The half life varies from compound to compound and plant to plant. Moreover, cellular and environmental factors have greater influence on the stability of them. Sometimes, the applied morphactins may be modified by glycosylation or demethylation. They are translocated in plants basipetally as well as acropetally through both sieve tubes and xylem elements.

The effect of morphactins is very interesting. Particularly in the presence of other natural hormones, they exhibit both synergistic and antagonistic effects, which is however depends upon the relative concentrations. Generally, morphactins have adverse effects on plant morphogenesis. They inhibit seed germination, but sprouting, growth of seedling, internode elongation etc. In many cases, they depolarize cell division which probably leads to distorted morphogenesis. Morphactins are very effective in inducing lateral bud development so tillering will be profuse. morphactins stimulate flowering in certain short day plants. Strangely, some

In many respects, morphactins resemble ABA in inducing seed dormancy, bud dormancy, suppressing stem elongation, etc. Most of their effects can be reversed by GA3 treatment. The overall effect of morphactins appears to be polyvalent antiregulators. Other known hormones Other identified plant growth regulators include:

Brassinolides - plant steroids that are chemically similar to animal steroid hormones. First isolated from pollen of the mustard family and extensively studied inArabidopsis. They promote cell elongation and cell division, differentiation of xylem tissues, and inhibit leaf [17] abscission. Plants that are deficient in brassinolides suffer from dwarfism. Salicylic acid - activates genes in some plants that produce chemicals that aid in the defense against pathogenic invaders. Jasmonates - are produced from fatty acids and seem to promote the production of defense proteins that are used to fend off invading organisms. They are believed to also have a role in seed germination, and affect the storage of protein in seeds, and seem to affect root growth. Plant peptide hormones - encompass all small secreted peptides that are involved in cell-to-cell signaling. These small peptide hormones play crucial roles in plant growth and development, including defense mechanisms, the control of cell division and [18] expansion, and pollen self-incompatibility . Polyamines - are strongly basic molecules with low molecular weight that have been found in all organisms studied thus far. They are essential for plant growth and development and affect the process of mitosis and meiosis. Nitric oxide (NO) - serves as signal in hormonal and defense responses.

Strigolactones, implicated in the inhibition of shoot branching.

[19]

Jasmonate
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Jasmonic acid

Methyl jasmonate The jasmonates (JAs) are a group of plant hormones which help regulate plant growth and development. Jasmonates include jasmonic acid and its esters, such asmethyl jasmonate (MeJa). Like the related prostaglandin hormones found in mammals, the jasmonates are cyclopentanone derivatives which are derived biosynthetically from fatty acids. They are biosynthesized from linolenic acid by the octadecanoid pathway. The level of JA in plants varies as a function of tissue and cell type, developmental stage, and in response to several different environmental stimuli.[1] High levels of JA are also found in flowers and pericarp tissues of developing reproductive structures and in the chloroplasts of illuminated plants;[1] JA levels also increase rapidly in response to mechanical perturbations such as tendril coiling and when plants suffer wounding.[2][3] Demonstrated roles of JA in planta include:

JA and MeJA inhibit the germination of nondormant seeds and stimulate the germination of dormant seeds High levels of JA encourage the accumulation of storage proteins; genes encoding vegetative storage proteins are JA responsive and [4][5] tuberonic acid (a JA derivative) has been proposed to play a role in the formation of tubers JA application can induce chlorosis and inhibition of genes encoding proteins involved in photosynthesis, although the purpose of this response is unknown it is proposed that this response to JA could help reduce the plant's capacity for carbon assimilation under [1] conditions of excess light or carbon The role of JA accumulation in flowers and fruit is unknown; however, it may be related to fruit ripening (via ethylene), [1] fruit carotenoid composition, and expression of genes encoding seed and vegetative storage proteins JA plays a role in insect and disease resistance. Many genes during plant defense are induced by JA; JA and ethylene may act [6] together in defense response
[1]

The perception of jasmonate is via the ubiquitin system, like auxins. After the conjugation of jasmonate and an amino acid isoleucine, it led to the SCFCOI1 complex degrade the ubiquitin markerd JAZ protein, and then releasing the transcription of other transcription factors

Yi Zhang and John Turner at the University of East Anglia found that when leaves of the model plant Arabidopsis are wounded, cell division in the apical meristem is reduced, growth of the plant is arrested within days, and the new leaves grow to only one-half of their normal size although the size of leaf cells is unaffected. Unexpectedly, the suppression of cell division in the apical meristem occurs through a signal pathway initiated by the wound hormone, jasmonate, which is synthesised in the damaged mature leaves. Mutant Arabidopsis lines unable to synthesise or to respond to jasmonate are not only larger than normal plants, but their growth is not reduced by the wound stress.

The jasmonates are a group of plant stress hormones that naturally occur in plants following exposure to certain types of stresses, including pathogen and herbivore attacks. (+/-)-Methyl jasmonate is a mixture of trans (3R,7R and 3S,7S) isomers. Methyl jasmonate induces the synthesis of proteinase inhibitors in plant leaves. 1 In cancer cells, it suppresses proliferation and induces apoptosis.2 More specifically, methyl jasmonate inhibits hexokinase that is bound to mitochondria.3 As hexokinase is overexpressed in cancer cells and contributes to cancer cell growth and survival, methyl jasmonates disruption of mitochondrial hexokinase activity selectively targets, and kills, cancer cells. Methyl jasmonate derivatives also have potential as anti-inflammatory agents.4
Brassinosteroids There are approximately 60 steroidal compounds known as brassinosteroids named after the first one identified, brassinolide, which was found in mustard pollen. Effects include: 1. Stimulation of stem elongation. 2. Inhibition of root growth and development. 3. Promotion of ethylene biosynthesis and epinasty. Salicylates Salicylates have been known to be present in willow bark for quite some time. Salicylic acid is synthesized from the amino acid phenylalanine. Effects include 1. Thermogenisis in Arum flowers. 2. Plant pathogen resistance-stimulates plant pathogenesis protein production. 3. Reported to enhance longevity of flower ( practice of adding a bit to cut flowers). 4. Reported to inhibit ethylene biosynthesis. 5. Reported to inhibit seed germination. 6. Blocks the wound response. 7. Reverses the effects of ABA. Jasmonates Jasmonates are represented by Jasmonate and its methyl ester. They were first isolated from the jasmine plant in which the methyl ester is an important product in the perfume industry. Jasmonic acid is synthesized from linolenic acid which is an important fatty acid. Jasmonates have a number of effects such as: 1. Inhibition of many processes such as growth and germination. 2. Promotion of senescence, abscission, tuber formation, fruit ripening,

pigment formation, and tendril coiling. 3. They appear to have important roles in plant defense by inducing proteinase synthesis.:defence mechanism against fungi

Practical applications of Plant Hormones:

Agriculturists all over the world have developed certain unusual methods by which they successfully cultivate the crop plants. It is only in recent years plant physiologists discovered how plant hormones can be effectives used in agriculture, horticultures, pomiculture and other related fields. As described earlier, plant hormones have a wide variety of effects and most of these responses are concentration dependent. Fortunately phyto chemists have also identified many synthetic hormones, some of which are more potent than natural hormones. Experimentation and experience have shown that the judicial use of hormones or combination of hormones can e employed in agriculture and related industries to get the maximum benefit. Quantity and quality of agricultural products are very important factors in the agricultural economics. How best the phytohormones can be utilized in this direction requires imagination and training.

There are many areas in agriculture, horticulture, pomiculture, moriculture, etc., where phytohormones can be used in successful cultivation to obtain greater yield. The high percentage of germination of sown seeds in the field has a bearing on the output. Pretreatment of seeds with IAA, NAA, GA, etc. has been found to be very effective not only in increased the percentage of germination but also in the total yield of the crop plants. Suitable concentration and combination have to be determined for each and every crop plants.

The overall growth of plants, number of tillers and branches that produce from every plant in the field contribute to the total yield. Use of GA or IAA greatly enhances the growth of plants and total area of leaf surfaces. Some morphactins can also be used to produce more tillers. In the case of sugar cane, use of GA has been found to increase the length of the internodes and also the sugar content.

Plants can be multiplied by vegetative propagation. Many horticultural plants are propagated by this way. The success of this method depends upon rooting. Hormones like NAA and IBA are very effective in inducing roots in stem cuttings. These hormones can also be used fro in grafting propagation. This way most of the plants can be propagated in large numbers and is quick time.

Use of hormones like IAA, NAA, IBA and Gibberellins ensures fruit setting and many of the fruits which develop from such hormone treatments are seedless, larger in size and sweeter in content. In many cases the total yield will be very high. Quantitatively and qualitatively such products yield more income to the farmer. Grapes, apples, oranges, mangoes and other fruit yielding crop plants can be treated with some of these phytohormones for the better yield. Furthermore, these hormones prevent premature falling of fruits, otherwise nearly 50-70% of the set fruits fail to mature and most of them fall off because of the formation of abscission layer in their stalks.

Preservation of agricultural products before marketing is another area in which hormones can be used effectively. Tubers, rhizomes, bulbs and such products sprout while they are stored. This will affect the farmer in terms of financial gain. Some of the synthetic hormones like NAA, maleic hydrazide can be used to preserve the said products for quite a period of time, thus one can improve the keeping quality of agricultural products.

Another judicial use which can be commercially exploited is the use of GA and other hormones in inducing flowering in unseasonal periods. But one should known which hormone is effective on which plant; otherwise phytohormones fail to produce the desired results.

Today farming is labour oriented and economically it is becoming impossible. Particularly removing weeds in the field is a nuisance, costly and time consuming. However specific synthetic hormones are available in the market to destroy the weeds selectively. 2, 4-D, 2, 4, 5-T can be used in paddy fields to destroy weeds. Similarly, monocot grasses can be destroyed by using specific weedicide hormones. Sometimes, water hyacinth and such water plants grow and multiply so fast they spread and establish their population in large tracts of tanks and streams. This causes considerable damage to water storing capacity of tanks. For example, 2 methyl 4 chloro 5 isopropyl phenoxyacetic acids can be effectively used to destroy water hyacinth and save the tanks. Thus one can use different hormones for different purposes, it can be for good or for bad, and it is like a knife with two cutting edges. It is left to the man who uses it.

Application of Plant Hormones

Agriculturists all over the world have developed certain unusual methods by which they successfully cultivate the crop plants. It is only in recent years plant physiologists discovered how plant hormones can be effectives used in agriculture, horticultures, pomiculture and other related fields. As described earlier, plant hormones have a wide variety of effects and most of these

responses are concentration dependent. Fortunately phyto chemists have also identified many synthetic hormones, some of which are more potent than natural hormones. Experimentation and experience have shown that the judicial use of hormones or combination of hormones can be employed in agriculture and related industries to get the maximum benefit. Quantity and quality of agricultural products are very important factors in the agricultural economics. How best the phytohormones can be utilized in this direction requires imagination and training.

There are many areas in agriculture, horticulture, pomiculture, moriculture, etc., where phytohormones can be used in successful cultivation to obtain greater yield. The high percentage of germination of sown seeds in the field has a bearing on the output. Pretreatment of seeds with IAA, NAA, GA, etc. has been found to be very effective not only in increased the percentage of germination but also in the total yield of the crop plants. Suitable concentration and combination have to be determined for each and every crop plants.

The overall growth of plants, number of tillers and branches that produce from every plant in the field contribute to the total yield. Use of GA or IAA greatly enhances the growth of plants and total area of leaf surfaces. Some morphactins can also be used to produce more tillers. In the case of sugar cane, use of GA has been found to increase the length of the internodes and also the sugar content.

Plants can be multiplied by vegetative propagation. Many horticultural plants are propagated by this way. The success of this method depends upon rooting. Hormones like NAA and IBA are very effective in inducing roots in stem cuttings. These hormones can also be used fro in grafting propagation. This way most of the plants can be propagated in large numbers and is quick time.

Use of hormones like IAA, NAA, IBA and Gibberellins ensures fruit setting and many of the fruits which develop from such hormone treatments are seedless, larger in size and sweeter in content. In many cases the total yield will be very high. Quantitatively and qualitatively such products yield more income to the farmer. Grapes, apples, oranges, mangoes and other fruit yielding crop plants can be treated with some of these phytohormones for the better yield. Furthermore, these hormones prevent premature falling of fruits, otherwise nearly 50-70% of the set fruits fail to mature and most of them fall off because of the formation of abscission layer in their stalks.

Preservation of agricultural products before marketing is another area in which hormones can be used effectively. Tubers, rhizomes, bulbs and such products sprout while they are stored. This will affect the farmer in terms of financial gain. Some of the synthetic hormones like NAA, maleic

hydrazide can be used to preserve the said products for quite a period of time, thus one can improve the keeping quality of agricultural products.

Another judicial use which can be commercially exploited is the use of GA and other hormones in inducing flowering in unseasonal periods. But one should known which hormone is effective on which plant; otherwise phytohormones fail to produce the desired results.

Today farming is labor oriented and economically it is becoming impossible. Particularly removing weeds in the field is a nuisance, costly and time consuming. However specific synthetic hormones are available in the market to destroy the weeds selectively. 2, 4-D, 2, 4, 5-T can be sued in paddy fields to destroy weeds. Similarly, monocot grasses can be destroyed by using specific weedicide hormones. Sometimes, water hyacinth and such water plants grow and multiply so fast they spread and establish their population in large tracts of tanks and streams. This causes considerable damage to water storing capacity of tanks. For example, 2 methyl 4 chloro 5 isopropyl phenoxyacetic acids can be effectively used to destroy water hyacinth and save the tanks. Thus one can use different hormones for different purposes; it can be far good or far bad. It is left to the man who uses it.

Application of plant hormones: Experimental morphogenesis

Tissue Culture:

Since the days of Haberlandt attempts to grow plant cells, tissues and organs in an artificial but defined nutrient medium have met with great success. Various methods have been established to raise plantlets starting from single cells, pith, leaf, roots, etc. By modulating the nutrients and hormonal concentration, it is possible to regenerate the entire plant body from any living cell from any part of the plant body, which suggests that all cells are totipotent. Hormonal concentrations play a significant role in culturing explants into undifferentiated callus and callus to differentiate into roots, shoots or the entire plant from eh callus.

Plantlets from Callus:

Tissue culture techniques have been very well exploited in understanding the role of different hormones and their interaction in organogenesis. Such systems offer excellent experimental

materials for biochemical or molecular studies. Tissue explants in the presence of a particular concentration of auxin, proliferate and produce an undifferentiated mass of cells called callus. However, further growth of the callus depends upon the availability of cytokinin, for the callus by itself cannot synthesize cytokinins.

The callus cells can be further induced to develop into shoots, roots or both by providing auxins and cytokinins in a defined ratio. As shown in the figure, at high ratio of auxin to cytokinin callus produces only shoots, at lower ratio the callus induces only roots, but at an intermediate ratio both shoots and roots develop. In certain tissues, by manipulating the concentration of auxin, cytokinins and gibberellins one can induce flowering directly from the callus.

Tissue culture methods can be employed in propagating somoclonal variations from the same callus. By inducing numerous plantlets from the single callus, the plantlets can be transplanted to field conditions and then desirable varieties can be elected in a shortest possible time. This is because cells in the callus show cot of variation in the chromosomal number. Tissue culture systems also provide an opportunity to understand the effects of each hormone, nutrients, interactions between the hormones and their effects on plant systems. Using the same methods, it is also possible to study molecular events that lead to organogenesis. The potentiality of this technique in the applied fields like horticulture, agriculture and other related fields is great; actually thee is not limit. These methods can be employed to create haploid plants from pollen grains, which is very useful in hybridization techniques.

Somatic Cell Hybridization

Protoplasts: Plant cells from leaves, stem and roots, for that matter from any source, can be separated from one another by subjecting the tissues to certain cell wall digesting enzymes like pectinases. The enzyme pectinase digests the middle wall, thereby the cells get separated. Once the cells are freed from one another, cellulase can be used to remove the surrounding cell walls. The resultant cells lacking is cell walls are called protoplasts which contain only plasma lemma as the outer membrane and such cells assume spherical shape.

Cell Hybridization:

Insolated protoplasts can be used for single cell cultures in a defined medium and the calluses can be obtained from such cells. Further, embryoids or organs can be raised from such callus tissue. For

every plant species the defined nutrient medium has to be determined for obtaining callus and plantlets.

Protoplasts obtained from two different species can be forced or induced to fuse with one another by various methods. It can be done by treating the protoplasts with polyethylene glycol (PEG) or (X) viruses. These agents make the cell membrane labile and favor the binding of the membranes between two protoplasts. Once the cells bind to each other, the membranes fuse with one another leading to the fusion of cytoplasm between the cells. If the protoplasts thus fused belong to two different species variety or genera, the product is called as somatic cell hybrid.

It is also very important to note that just fusion of two cells by their cytoplasm is not an end. The fusion of nuclei is also very important. Once the nuclei fuse, the hybrid cell is ready for propagation. Such cells can be plated onto a defined medium which is suitable for the development of both the species. In such a medium the hybrid cell divides and redivides to produce the callus; again by manipulating hormonal combinations and nutrient media, it is possible to induce plantlets from such callus. The success of these methods requires a large number of trial and error experimentations, where one has to determine the suitable media for each species and then one has to obtain another proper medium to make the hybrid cells to respond. The success of these techniques required efforts and imagination and lastly the luck.

Introduction of recombinant DNA into protoplasts:

Protoplast culturing techniques can be very well exploited in incorporating exogenously supplied DNA into protoplasts. When protoplasts are incubated in a known medium containing DNA, the cells take up DNA slowly. The amount of DNA that is incorporated into the cells has to be determined. More than that, the entry of DNA into the cell is not enough; the DNA has to reach the nucleus and it has to be incorporated into the host chromatin. The DNA that is supplied may belong to different species or it may be recombinant DNA of a known gene or gene(s). The DNA can also be injected into the nucleus directly by simple injecting transgenic techniques. If the protoplasts incorporate such supplied DNA into their nuclei, then the protoplasts can be cultured. One can develop a new species from the said techniques. However, one has to find out suitable factors for the expression of incorporated genome. These methods have been successfully employed in many laboratories.

In recent years the technique of cloning of known or desired gene has been perfected. A cloned gene can be transferred to a bacteria or a phage easily and the same can be made to express. The same technique can be used to transfer known genes into animal cells by directly injecting the known genetic material into zygotes and by transplant back into uterus it possible obtain the offspring

with the exogenously supplied DNA incorporated into the chromatin material unfortunately plant cells are not amenable for such techniques. However, the plasmids from Agrobacteriam tumificians (which causes can be like tumors are plants) have been isolated and characterized. Methods have been developed to inactivate the plasmid into harmless structure, but such plasmids are still active when transferred as exogenously supplied genetic material. By transferring known genes with using such plasmids it can be used as an effective vector to transfer genes into protoplasts or to the living plant itself either by rubbing the plants into wounds or by using a gene gun. By this was many desirable games can be introduced into different plants.

Application of somatic cell hybridization techniques:

Somatic cell hybridization is a reality, but this is a technology of the twenty first century. The success of this technology has raised new hopes in mankind. Already plant technologists have succeeded in fusing different species of tobacco protoplasts and also they have obtained a complete plant put of such somatic cell hybrids. The fusion of potato and tomato protoplasts has produced pomato. But this hybrid has yielded plantlets which are capable producing a tuber like structures in the terminal region than at the base of the plant. What one wishes is to obtain hybrids which can yield two crops by a single plant.

The most ambitious project that man is thinking of is to develop plant and animal cell hybrid. In fact, the hybrids between HeLa cell (Human cell line) and an yeast cell has been achieved. What futurist plant genetic engineers expect is to fuse a plant cell with an animal cell and make the plants to produce animal tissue. Ex. Plants producing pork or animal cell proteins can be used as vegetables. Though the wishes are wild the success of such dreams are becoming a reality.

Similarly introducing of recombinant DNA into protoplasts has generated great expectations in the field of agriculture. For example, if nitrogen fixing genes are introduced into cereal crop plant cells, the plants obtained from such experiments do not need nitrogen fertilizers. It saves the farmer from providing nitrogen fertilizers and saves him lot of money. Similarly, introduction of some protein genes, which yield proteins of good nutritional value. It is of immense help in saving man from malnutrition. So the application value of this technology has no limit. Many industries have been established to develop new varieties of plants and plant products which have a greater commercial value.

PHYSIOLOGY OF FLOWERING

Plants, to begin with go through a period of vegetative growth. The extent of vegetative growth is endowed with its genetic potentiality. Accordingly, they may grow into herbs or shrubs and some may develop into trees or climbers. Generally, every plant after going through a period of vegetative growth, responding to environmental clues, start producing floral structures, which may be in the form of characteristic single flowers or inflorescences.

Many plants for that matter, a large number of plant species (higher plants), after a period of vegetative growth, start flowering irrespective of the season. But some plants flower only in a particular season of the year. Based on the duration required for the plants to produce flowers, they have been classified into annuals, biennials and perennials. All plants have to acquire ripeness to flowering. Annuals complete their vegetative growth and flowering in one season and then they die. Biennials produce vegetative growth in one season and flower in the next season. But perennials remain for many years and flower seasonally.

In fact, some trees do not flower till they reach a certain age. For example, coconut and areca nut plants start producing flowers only when they reach an age of 6-8 years. On the other hand in the case of bamboo plants, they grow for a number of years, and flower only once in their life span. As soon as they flower, produce seeds and plants die (monocarpic plants). Interestingly there are many plants which flower throughout the year, ex., Catharathus roseus.

Parts of a flower from outer to the central region

Aestivation of some flowers- refers to the organization of each floral part with respect to each other, especially holds good for sepals and petals.

Plants growing in different regions of the globe are exposed to different climatic conditions and different day lengths. In fact they are adapted to such environs in such a way, they exhibit alternate vegetative and flowering cycles. It means that plants with their inherent genetic potentiality interact with environmental conditions, accordingly, they respond and behave. Humans, homo sapiens present day species, just about 25000yrs to 40000yrs old, copulated with Homo eructus, mostly in Asia and made them extinct; when they evolved and colonized sites of their own, after observing for many centuries the above said natural phenomenon, they have devised different methods to cultivate crop plants in different seasons of the year, so as to get the harvest at the right time of the year. They also domesticated animals for their use. The common knowledge of the farmer has been extended and explained by plant physiologists ; why and how the said plants behave in response to different environmental conditions. Plants have all the needed signal transduction pathways to respond to environmental signals. Such signal pathways has been worked out in Arabidopsis.

Developmental pathway of plants and its structures start from the zygote and end in all the structures.

Discovery of flowering response:

Though it is a common knowledge that different kinds of plants respond to different seasons of the year in producing the flower, it was left to G.Gassner & W.W. Garner to explain the phenomenon by their pioneering scientific studies. Gassner observed that winter variety of petkus rye plants called Secale cereal, responded favorably to cold treatments. Almost at the same period of time, Garner and Allard demonstrated how plants produce flower in response to different lengths of the day and night in a 24 hours day cycle. The above two phenomenon are popularly called as Vernalization and Photoperiodism respectively. The above studies have lead to the discovery of how plants rhythmically respond and behave to day and night duration or to temperature fluctuation in different seasons of the year and they also observed rhythmical behavior of the plants which is referred to as biological rhythm. And the operational time measuring system found with in the plant structures is called Biological Clock.

PHOTOPERIODISM (PP):

Earth, because of its revolution on its own axis and rotation around the sun, exhibits a period of day and night and seasonal changes. The duration of the day and night again shows variations because of the angle and distance between the earth and the sun at any given time of the year. Thus plants and animals living on different parts of latitudes or longitudes are subjected to different periods of photo periods and different temperatures at different seasons of the year.

If we use three points or places on the globe, located at different positions as the reference point, to measure the day and night periods, it will be apparent how different are the day periods and temperatures of such places. Brazil in South America and Congo in Africa exhibit almost 12 hours of day and 12 hours of night in all the months of a year. But a city like Philadelphia located in the east coast of USA at latitude of 40 degree N, in the month of December; it experiences 9 hours of day and 15 hours of night. On the other hand, in the month of June, the day period is 14 hours and night is 9 hours long. Similarly, cities of Norway, during December, experience 6 hours of day and 18 hours of night, but in June, it enjoys 18 hours of day and 6 hours of night. Such day periods also accompany with changes in extreme temperatures. The above observations suggest that organisms living in these regions are subjected to seasonal variations of day and night and also to changes in seasonal temperature fluctuations. Garner and Allard , while working the department of Agricultural Station, Beltsville, Maryland, USA, demonstrated remarkable relationship between the effect of the day period and flowering in a mutant tobacco plant called Maryland Mammoth. They observed that the mutant failed to produce flowers but tall, so they are called Maryland Mammoth. They also observed that the same plant started flowering in summer under field conditions. But the same plant started flowering when transferred to green house where it was subjected to short day and long night conditions. So the plant was called short day plant. Since then, a large number of plants have been subjected to various cycles of photoperiod i.e. treatment and according to their responses, plants have been classified into different groups. The flowering response in plants to photoperiodic treatment is now called photoperiodism. Light induced responses in photo morphogenesis are many and intricate, and this can be only represented in the form of network.

Classification:

Based on the responses to different photoperiods, most of the plants are grouped into 3 major classes, viz. Short day plants, long day plants and day neutral plants. However, detailed studies on each of these groups resulted in further classification of them into sub groups like long short day plants, short long day plants etc. Each of these groups has been further grouped into qualitative and quantitative varieties based on the specificity of the appropriate light periods.
Critical Day Period:

It is the duration of the photoperiod or the dark period that ultimately determines whether the plant has to go through vegetative growth or to produce flowers. Different plants require different periods of light or dark for 100% flowering. If that period falls short then plants do not produce 100% flowering. Such requirement of a minimum of photoperiod or dark period for effective flowering is called critical day period. The length of light and dark period for different long day and short day plants varies. For example Xanthium requires a critical length of 15 hours of dark period for its effective flowering. If the dark period is less than 15 hours plants do not induce any flowering, but longer dark periods d not inhibit flowering. On the other hand, the long day plant, ex. Hyoscyamus niger requires a critical 11 hours of exposure to light. Anything less than that, plants fail to produce flowers. If the length of the day period for this plant is more than 11 hours, it does not affect the flowering. Similarly, different plants have different critical day periods and the correct photoperiod has to be determined individually by subjecting them to photoperiodic treatment.
Ripeness to flowering and site of perception:

Not all photoperiodic plants respond to the light treatment until and unless the plant has grown to certain vegetative maturity. For example, Wulfia requires at least one leaf: Xanthium responds well if it has few partially mature leaves. In Zea mays, at least there should be 5-6 leaves to respond for photo periodic treatment.

Stem apex is the site for development of flower; a dramatic change in the structural and functional features of the floral structures; they are nothing but modified leaves. In spite of it is to be noted, the floral structures are same as the vegetative structure but modified; sepals, petals, stamens and carpels are all derived from leaves. The most intriguing question that has puzzled scientists for such a long time, how the genes hitherto remain silent, start expression and develop these structure. The molecular aspects of gene expression that changes leaves into floral organs are just opening up, yet only in bits.

Looking at the start of the plant body from the zygote a fertilized egg, in course of time divides and redivides and differentiates and develops tissues and organs. Developmental process in plants, at molecular level is more or less the same pattern as in animal systems.

Look at the Stem Apex Meristem (SAM) has undifferentiated central dome of progenitor cells (pleuripotent in nature) covered by a layer of epidermal cells. Signals have to come from different modes and methods to convert such potent cells to go through developmental programmes. It is possible one such cell is enough for the development, just like stem cells in animal systems. Signals are varied such as sunlight, sunlight duration, temperature, and organic chemicals such as Gibberellins and sucrose are internal signals. Cellular transduction, in response different signals, is more complex, for the signals arrive as environmental factors Light (Photoperiodic pathway) temperature (Vernalization pathway) or inbuilt factor (autonomous pathway). Most of the environmental signals impinge on leaves and resultant downstream products have to be translocated to the SAM; this can be long distance for the site of perception and the site of response are separated in time and space (Einstein).

Plants without leaves do not respond to any photoperiodic treatment, which suggests that the vegetative buds are incapable of perceiving the stimulus. Similarly, plants with only old and mature leaves not only fail to respond but also they inhibit or nullify the photoperiod effect. However, partially mature leaf or leaves that are just unfolding, are highly sensitive. A remarkable feature is that even one such sensitive leaf is enough to respond to proper photoperiodic induction. It means whatever reactions or a product produced in one leaf is enough to induce flowering in the entire plant. Let us start with light mediated induction.
Light and Duration mediated: Photo inductive Cycle:

A plant, if subjected to certain length of day period and night period in 24-36 hours duration, then it is called one photoperiodic cycle. Such periodic cycle responsible for inducing flower formation, is referred to as photo inductive cycle. The required number of photo inductive cycles varies from species to species; this is because of inherent genetic makeup of the said plant. For example, Cockleburr needs just one photo inductive cycle but plants like Plantago lanceolata or Salvia accidentales require at least 17-25 cycles for 100% flowering. If the provided inductive cycles are less than what is required, the number of flowers produced in such plants will be less than 100%. This suggests that during the inductive cycles some flower inducing material gets accumulated and if such a substance produced reaches a threshold value the flower production is maximal. It is important to know that the photoperiodic response is all or none phenomenon. Once the provided stimulus produces a proper impact on the genome, the flowering is initiated and once it is initiated it cannot be reverted to vegetative condition under normal circumstances.

Importance of Dark Period:

Systematic studies, using different lengths of light and dark period, it is noticed that the dark period is important for short day plants for flowering. Shortening of photoperiod has profound influence on the quantitative yield of flowers. The importance of photoperiod, i.e. Day period on short day plants is very interesting. For example Xanthium requires 15 of continuous dark period and 8 hours of day period for 100% flowering. If such a plant is maintained with 15 long dark period and the day period is shorted by 2 or 3 hours, the total number of flowers produced will be significantly lower than the plant that is exposed to 8 hours of day period. This suggests that the photoperiod affects flowering ability in quantitative terms, it means that proper dark period is essential for flowering but one cannot expect the plant to grow and produce flowers in continuous dark. This is because light is required for photosynthesis for it provides energy rich and components for the development of floral primordia. If the photosynthate supply is not adequate the total growth of the floral axis is affected. In fact, in one of the experiments, a short day plant which is kept in continuous dark conditions is induced to produce flowers by providing sugar solution to the leaves. Similarly if the CO2 supply is cut off during photosynthetic period, the short day plants in spite of receiving proper dark and light periods, they do not produce flowers to their maximum ability. The above observations suggest that photosynthate provides the necessary raw materials for floral primordia for full expression.

Another important factor that affects the floral induction is the intensity of light. If the light provided is of low intensity i.e., less than 100 ft candles, flower production is totally inhibited, though the meristems are organized into floral primordia. But if the intensity of light is increased above a critical level, the number of flowers produced also increases up to certain level. This is because the light intensity affects the total yield of photosynthate, so flower production is also affected by the said factor.
Action Spectrum of Light:

Finer analysis of the active part of white light that is effective is photoperiodic inductions reveals that the red light at 660 mm and far-red light at 730 mm are the most effective wavelengths in inducing or inhibiting the flower initiation. It has been established that continuous far-red

irradiation inhibits flowering in long day plants, on the contrary, continuous red light treatment blocks flowering in short day plants. The dramatic effects of red light and far red light can be demonstrated on a short day plant like xanthium. It is known that a short break in continuous dark period with white light in a short day plant brings about the total inhibition of flowering. If the short break is due to red light, the inhibition is 100%, but if the break is due to far red light flowering is not inhibited. Interestingly, if the red light and far red treatment is repeated alternately but ending in Far Red as short breaks results in the reduction of total number of flowers produced. If the number of cycles is extended the flowering will be totally inhibited though the last light treatment is far red light. This is possibly due to the breakdown or exhaustion of some intrinsic factors generated during short day treatments.
Phytochrome as the Photoreceptor:

The effectiveness of red light and far red light inducing or inhibiting the induction of flowers strongly suggests the presence of some substance or substances that could absorb light in the said wavelengths. By absorbing the Light at particular wavelength, the said substances probably undergo excitation of chromophore and the protein bound undergoes conformational change.

This protein complex induces signal transducing activities leading to flower induction. Search for such a compound in plants resulted in the discovery of a blue / yellow colored pigment called phytochrome.

This complex when get excited with the absorption of light at red wave length, its protein gets activated and acts as a aspartate kinase and it autophosphorylates itself. When phytochromes were discovered there was great excitement among plant physiologists. This led to intensive research work on various aspects of the structure and functions of phytochromes in plant morphogenesis. Now scientist have extracted and identified five phytochromes; Phy A to PhyE. Yet in recent years another pigment was discovered, called Cryptochrome whose role is not well discerned. Distribution:

In recent years, the techniques for extraction and quantification of this subtle pigment have been standardized. The pigment has been found to be located in all possible organs of the plant body. At the cellular level, it is mainly associated with plasma membrane, cytoplasm and the membranes of plastids. The presence of this pigment in plastid membranes is significant, because plastids are also known to perform many other photo biological processes.
Chemical composition and structure:

The pure form of phytochrome appears blue / yellow colored pigment in solution form. Phytochrome, in fact, is made up of two moieties; one is a protein and the other is a chromophoric component. The phytochrome-associated protein has been isolated from maize seedlings and other sources. The Mol.wt. of phytochrome is 123-125 KD and it is a tetramer. But chromophore part is made up of linear chain of four pyrole rings. The protein subunits are firmly bound to the A-pyrole ring via Sbond of the chromophore unit (chromophorobilin). With the absorption of light at 660 mm or 730 mm, the double bonds found within the chromophore get disturbed and shifted. These changes inturn bring about conformational changes in 3-D structure of the pigment and also in protein chains either in in trans form (activated) or cis form (inactive form). Probably, the above said changes due to absorption of light transform them into excited form of molecules and they inturn elicit certain physiological functions in the cells.

The two leaf like structures are dimeric phytochrome apoproteins

Dual forms of phytochromes:

In the earlier days of its discovery, people suspected the presence of two kinds of phytochrome pigments because they showed different absorption spectrum at 660 mm and 730 mm. But Norris and others, using dual wavelength spectrophotometer, demonstrated that the same phytochrome pigment exists in two alternate forms. They are called red light absorbing pigment and far red light absorbing pigment. The Pr form after absorbing red light gets transformed into Pfr form which by absorbing Far red light gets converted back to PR form. The Pfr form naturally undergoes decay back to Pr form. Thus phytochrome exhibit dual forms. The concentration of each form is dependent upon quality of the light source and the physiological state of the cell. The PFR form of the pigment formed due to the absorption of red light is not very stable. It decays back to PR form or it is destroyed by some enzymes in dark

condition, but this process is slow. On the other hand, if the PFR form absorbs far red light, it gets converted to PR quickly. But the decay of PfR pigment to PR form on its own takes place in dark or it is converted by certain enzymes and it is temperature dependent. In the presence of oxygen, the pigment undergoes irreversible destruction. In spite of their labileness and sensitivity, they remain quite stable at pH 6 and pH 8. Furthermore, the stability of these forms of pigments is controlled by the firm binding of protein moiety to the chromophore part of the pigment.

Activated phytochrome undergoes autophosphorylation at serine/threonine residue, which then binds to its receptor protein and moves into the nucleus, where it associates with transcriptional factors and activate their target genes whose products inturn activate other genes and promote flowering.

Phytochrome in response light undergoes conformational change and moves into the nucleus where it binds to PIF3. The Phy-PIF3 dimers bind to G-box of the response gene and activate the gene expression. Light also triggers phytochrome mediated G-trimeric membrane protein activation, that leads to the production of cGMP and also the release of Ca2+ ions. One of the early gene expressed in response to Phytochrome activation is CCA1.

PHY-Pfr (), not only moves into the nucleus but also interact with membrane trimeric alpha/beta/gamma G protein, that activates cGMP and release of Ca^2+.
The effect of which is not clear (at least to me). The PKS is serine protein kinase and NDPK2 is Nucleotide biphosphate kinase.

Involvement of Pfr-Phy-A and other in chromatin remodeling is speculated but not discerned.

The effects activated Phy-A is many where it activates many genes and their effect is seen or felt in cytoplasm, this includes flowering time locus FT synthesis.

Functions:

Probably phytochrome is the only pigment that is known to have multifarious activity and elicit diverse responses. Among the 35 to 45 functions attributed to this pigment-protein complex, bud dormancy, seed dormancy, flower induction is the well known phenotypic effects. At the metabolic level, the phytochrome is known to act upon cell respiration, permeability of membranes, transcription, translation and enzyme activity. Some of these activities have been very well demonstrated in different plant systems. In this text the discussion is restricted to its role in flower inductions and dormancy.
Role of phytochromes in flower induction:

Phytochromes being omnipresent in the plant body, they are always subjected to both red and far red radiations in the day conditions. Accumulation of pR forms and pFR forms of phytochrome in sufficient amounts in plants is critical and important. The effective concentration of any of these forms over a threshold values in the perceptive organs like leaves is absolutely essential to bring about certain biochemical functions which may ultimately lead to the induction of flowers. In long day plants, the Pr form of the pigment by absorbing red light throughout the day transforms the substance to Pfr form and it accumulates in greater amounts. Such Pfr pigments, when preset in higher concentration above the threshold value, activate the cell machinery and ultimately induces flowering. However, recent studies indicate that the PfR

form alone is not active, but it also requires another substance called X whose properties are not well characterized. The PER-X complex is believed to be highly effective in inducing flower formation in long day plants. The X factor is known to be Phytochrome interacting factor (PIF3). PR > PfR > PRR.X Induction of

flowering. Light activated Phy-A binds to the receptor FHY1-FHL (FHY means elongated hypocotyls) that enters the nucleus where it activates light response genes including flowering genes. Accumulation PhyA represses the production of FHY3-FAR1 for they bind to gene loci of the same. With the dissociation of Phy-A from FHY1-FHL the genes for FHY3 and PAR1 get activated to produce FHY1FHL that is found in cytosol after transcription and translation, which are used for the binding of Activated Phy-A. On the contrary in short day plants, because of long dark periods, whatever PfR pigments formed in the day conditions are subjected to decay back to PR form. However, higher levels of PR pigments are effective in inducing flower formation in short day plants. Conversely, higher amounts of PR forms inhibit flower initiation in long day plants and PfR forms prevent initiation of flowers in short day plants. So the kind of pigment or the form of pigment that has a promotive effect on one kind of plant acts as an inhibitor to the other kind. The dual form of pigments performing dual role is really intriguing.

The PHY PIF3 complex binds to LHY/CCA (long hypocotyls and Chlorophyll a/b binding proteins) region of the said genes and activate its transcription. The CCA has its own target genes such as TOC which in turn interacts with PHY-PIFr dimer complex. The ultimate effect is on the synthesis of FT in conjunction with CO. This FT moves into phloem vessels that translocates to the base of SAM.

Light activated signal transduction pathway (general): This actually explains why gibberellins are effective on long day plants but not so on short day plants. Extrapolating this view it is possible to visualize that there are two or more different genetically regulated regulatory factors acting at two regulatory sites. Added to this, plants requiring vernalization can be short cut the flowering by GA treatment. But one thing is certain that one of the factors is GA and other factor, i.e. Anthesins may be anything, possibly it may be a highly labile protein or it may be one of the mRNPs, RNPs or a protein or signal factor or a kind of ligand that can induce signal transduction just binding to the receptors on plasma membranes or cytoplasmic receptor found in of the receiver tissues. The most enigmatic situation was GA actually synthesized in the apex of the future stem tip or inflorescence meristems, precursors for GA synthesis are found in proplastids and fully developed plastids. The photoperiodic stimulation takes place in young leaves; if so translocation of the phytochrome induced signal has to be transported a long distance to the non determinant stem meristem the future floral meristem. What is the structural element that can transport such substances? Is it Xylem or phloem? Xylem elements participation can be ruled out for the simple reason that the xylem elements transport is mostly from root to all other regions. But phloem transportation takes place in both directions; starting form vienlets to veins and to the midrib of the leaf and from there the transportation is bidirectional. Whether the bidirectional movement takes lace in the same sieve tube-companion dimers or separate sieve tube elements are involved is little ambiguous.

In addition to photoperiodic and GA pathway, plants use autonomous pathway and vernalization pathways also, where GA pathway provides a kind of interlink between photoperiodic and vernalization pathways.
Concept of Florigin:

The PR and PfR forms of pigments are the products of photoperiodic stimulus and they in turn are responsible for inducing flower formation. So the respective pigments elicit certain responses in the plants which probably produce some kind of a substance (s) which is/are responsible for transforming vegetative shoot into reproductive shoot. Production of such substances by plants has been suspected by many plant physiologists long ago. But so far, no one has succeeded in isolating or identifying such compounds. In spite of it, the presence of substance (s) responsible for flower induction has been proved by different methods and by different investigators. The grafting experiments conclusively prove the presence of flowering substance(s). If a short day plant, kept under proper photo inductive conditions, is grafted to another plant or plants (by serial grafting) which are maintained under non photo inductive conditions, flowering is induced in all plants. Whether the plants receiving the graft are short day plants or long day plants, it does not make any difference. This experiment clearly indicates the production and existence of a flower inducing substance in a photo induced plant made product in response to stimulus, which is capable of diffusing through the graft to the receiver plant. What is this substance is it a chemical signal such as cAMPs like or is it a mRNA or a protein. Any substance that is induced and produced in leaf cells has to be transported to long distances and should be stable. It is possible that the substance produced should cross through the cell wall of mesophyll cells into sieve tube cells, and then it has to be transported to stem apex meristems (SAMs). There again it has to cross cell wall barriers to reach a whole mass of cells. So this substance should be a small molecule that can be easily transportable and easily induce signal transduction that is capable of spreading. Chailkhyan, a noted Russian botanist named such flower inducing elusive hormone as Florigin. Attempts to isolate such a substance have failed. In fact, people have made attempts to collect the substance from the donor plant to a receiver plant through a water jacket, but failed to obtain any stable compound which could induce flowering in other plants. Probably, the suspected florigin may be an extremely unstable, labile and sensitive compound, which could not withstand the most simple extraction methods. However recent experiments involving solvent extraction methods indicate that florigin might be a compound similar to sterols or mRNA-mRNP complex or a labile protein. But there are may plant physiologists who suspect the very existence of such a compound because they feel that some of the known growth promoting hormones by themselves may bring about flower induction by some complex interactions. Most of the known hormones are small molecules, easily transportable and bind to receptors and induce signal transduction. Either such hormones and their binding proteins or the combined complexes may be involved. However, the transport of such substances has been found to be through sieve tubes, but the rate of translocation in short day plants and long day plants varies. In short day plants, the rate of transportation is about 45-50 cm/hr. but in long day plants, it is about 2-2.5 cm/hr. The rate of translocation of the said flowering substance is found to be 40-100 times slower than the rate of

transportation of sucrose, though the components involved in transportation are the same. The different rates of transportation observed in short day plants and long day plants are suspected to be due to the presence of two different substances. It is also known that sieve tubes translocate different substances at different rates because of specific carriers involved in the translocation process. The puzzling feature that is not known is that whether the so called florigin is one compound or a complex of compounds. If it is one compound, then the flowering substance produced by both the short day plants and the long day plants should be the same. If there are two compounds, the rates of translocation may differ. But why should they differ? The probable explanation is that one of the compounds is constitutively synthesized and such substances reach their destination earlier and the other compound that is synthesized when it is subjected to photo inductive conditions reaches the destination later. However, for the inductive action, both compounds are required, but it is difficult to visualize whether these compounds elicit their action in complexed form or independently at one or two different sites. But recent studies reveal that plants produce the elusive florigin, which is synthesized in leaves and translocated through sieve cell and reach the base of SAM. The complex of substance now called the actual Florigin, is a Molecule of the century has been identified as FT (flowering time protein correctly Flowering locus T), not the FT mRNA suspected earlier. Actually FT is synthesized in response to constans (CO). It is now believed that it combines with another protein called FD, together activate AP1, SOC1 in the apical Meristem, they inturn activate the expression of LFY. Then AP1-LFY triggers the expression of floral homeotic genes. This just explains light induced components, but the flower initiation is also due to GA, sucrose, vernalization and in a large number of plants it is autonomous. There is a kind of confluence of the products of these effects ultimately responsible for triggering the floral homeotic genes.

Red light activated Phy-A induces certain genes, not very well documented, the ultimate flowering product is FT; so also GA has an effect on inducing FT.

Effect of GA on Flowering:

Fascinating aspect of flower inducing substance (s) is that when the extracts, obtained from photo induced leaves of Xanthium is applied to lemma plant kept under non inductive conditions, the extracts induce flowering in lemma plants. However, the induction of flowering by the extracts should be supplemented with gibberellins, without which the extracts alone or gibberellins alone has no effect. This experiment suggests that gibberellins are probably one of the components of elusive florigin (like elusive Himalayan snow man), but the nature of the other component is still a mystery. It is very well known that gibberellins induces bolting and flowering in rosette leaved long day plants, but not in short day plants (with some exceptions). In long day plants, GA not only stimulates the elongation the condensed internodes, but at the same time, it also promotes the formation of factors needed for flowering. Thus GA treatment substitutes photoperiodic treatment in long day plants. Added to this complexity, application of high concentrations of gibberellins and cytokinins to the callus, obtained from Arachis hypogea (peanut plant), results in the induction of flowers directly from the callus. Paradoxically ABA, a growth inhibitor, is very effective in inducing flowers in some short day plants like Fragilis, Pharbatis, etc. But ABA does not induced flowers in xanthium, another short day plant. This particular case is very interesting, at the same time, it is also intriguing and it further raises doubts about the existence of florigin per se. But such experimental results are very few and conditions used for such materials have to be carefully analyzed. Much more perplexing that is observed in some cases is that the application of cytokinins to the whole plant induces flowering, when the plant is at a particular stage of development. Based on gibberellins promotive effect on long day plants and its failure on short day plants, Brain and is colleagues (1958-59) came out with a working model to explain the action of photoperiods on flower inducing substances. According to this model, during photo inductive red light treatment a precursor gets converted to Gibberellin or Gibberellin like substance. The same substance is believed to undergo decaying back to the precursor either in dark or under far-red light treatment. According to their concept it is assumed that Gibberellin like substances have to be maintained at high concentrations in long day plants to be effective in producing the elusive compound called florigin. But in short day plants, according to Brain, et.al. GA like substances are effective only when their concentration is very low for higher concentration of GA is inhibitory. That is why when GA is applied to short day plant there is no effect in terms of flower induction. But the said scheme of events fails to explain how low levels of GA like substances can produce sufficient quantities of florigin in short day plant to bring about the induction of flowers and it is expected that the florigin that is produced in short plants or long day plants should be the same. Even today, with all the knowledge of molecular biology of the exact processes involved in inducing flowers are not known. Still, it is very important to understand the model proposed by Chailkhyan, a great Russian plant scientist. He toiled his entire life time to understand this phenomenon and his proposed model is very worth understanding. Cajlachjan, another way to pronounce his name, has

assumed that the florigin formation takes place at two levels but in two steps. Further, florigin is not one substance but it is a complex of two substances, i.e., gibberellins and Anthesins. It is also assumed that long day plants synthesize anthesins irrespective of photoperiods, which means anthesins are produced constitutively, which indicates that the genes responsible for the synthesis of the above said compound are constitutively expressed all the time in long day plants. But the synthesis of GA or GA like compounds is under the control of long day photoperiodic conditions. In the sense, the pFR produced in long day plants has an important role in activating the pathway of GA synthetic. On the other hand, short day plants are believed to synthesize GA constitutively and the synthesis of anthesins is under the control of short day photoperiods. It means the pR form of the pigments is effective in inducing the synthesis of anthesin. Gibberellin pathway and Light quality pathway. Each of them has certain products and they get integrated at the base of the meristem and determine and differentiate the floral meristem. Many of the components that are synthesized in floral meristem or axis, very young leaf primordials ultimately interact and integrate in activating the apical Meristem into floral meristem.

Light has another discerning, but subtle activity is inducing the synthesis of GA1 and its derivatives that activates LFY, and hypocotyl elongation and shade avoidance processes.

In spite of numerous experiments and detailed studies well over 50 years or so, the explanation given to describe the entire mechanism of flower induction is confusing and often contradictory in nature. Particularly investigations at the molecular level are few and they are not convincing. Skoog and his students working on a cultivar variety of Nicotiana tabbacum plant demonstrated that when the total DNA extracted from the leaves of photo induced plants is applied to the stem tips of tobacco plants kept under non photo inductive conditions, the receiver plant flowered. It is not clear from their experiments whether on not the DNA extracted is really pure and free from proteins and other stimulatory small molecules that can easily diffuse into stem apex.. Yet it indicates that pure DNA perse cannot express until and unless they are provided with regulatory factors which are mostly proteins. If proteins are associated with such extracted DNA, what are those proteins? Unfortunately, nothing is known about them. But it is clear from their experiments that the applied DNA is not free from proteins and such proteins are highly stable when they are associated with DNA. It would be interesting to isolate chromosomal proteins from induced plant and provide the same to non induced plants and see whether the receiver plants produce flowers or not. A group of Japanese workers demonstrated an increase in the levels of different species of RNAs and different species of proteins is buds during floral initiation. Unfortunately it is not clear whether there are any qualitative difference among the mRNAs and proteins produced during floral initiation. It is very important to understand the molecular events that lead to the transformation of vegetative buds into floral buds. It beats any ones imagination why such work has not been successfully elucidated so far; even though the technology is available at hand. Nevertheless, it is now clear that the induction of flowering due to photoperiodic effects is regulated at two sites separated by space and time. In the first event the site of response to photoperiodic induction is young leaves. The second site is the terminal or axillary shoot buds. Which on receiving the flower inducing substance(s) are stimulated to produce floral axis or floral buds? Shoot buds per se do not respond to photo inductive treatments, but they respond when leaves are subjected to such treatment. Which means whatever substances produced in leaves reach the vegetative buds and induct flowering in them? At the level of leaves, phytochromes able to receive signals form correct photoperiodic treatments, undergo excitation and induce gene expression in the cells leading to the synthesis of flower inducing substances. According to the prevailing views, the pFR pigments produced in long day plants induce the synthesis of GA or Gibberellin like compounds in proplastids/plastids and the same are released into cytoplasm. In the same long day plants, whatever small amounts of pR pigment found in the leaf cells might be able to induce gene expression to produce anthesins all the time irrespective of photoperiodic treatment. On the contrary in short day plants, the pR form of pigments that accumulated during long dark periods are suppose to induce anthesins production and the little amount of pFR present might be involved in producing GA like compounds constitutively all the time. The anthesins appears to be proteinaceous or protein like compounds. That is why the rate of translocation of flowering compounds varies. Anthesins appears to be translocated faster than gibberellins. Whether the genes that respond to two different photoperiodic treatments are located in the nucleus or plastids or both, is difficult to visualize, until and unless one obtains nuclear or cytoplasmic mutants for flowering.

Once such components are produced in leaves they are translocated to different site of cell vegetative buds. It is conceivable that as long day plants produce anthesins and short day plants produce GA like compounds constitutively; they reach the meristems of the vegetative buds earlier. On the contrary as GA like substances in long day plants and in short day plants produced under photo inductive conditions; reach the vegetative mounds later. If one of the components reaches the vegetative meristems earlier, it cannot bring about the total gene expression without the presence of the other factor. So both the factors are required in the vegetative buds for total and comprehensive effect to be effective. Once the vegetative buds receive both factors (i.e. GA and Anthesins) in required quantities, the florigin complex activates a battery of genes requiredfor the transformation of vegetative buds into floral buds. Whether the florigin components activate one gene or a group of genes, the activation leads to a cascade effects. It means a specific gene or gene products may activate group or group of genes, which inturn may activate another set of genes. The products of these genes ultimately bring about the transformation of vegetative meristems into floral meristems and commit them to develop into floral structures. It is important to remember that the production of stalk, bracts, sepals, petals, stamens and pistils is not a single event nor is it regulated by a single gene. They are a series of sequential events which require a sequential expressions and gene products. The totality of this highly regulated gene expression results in the transformation of vegetative buds into highly organized floral buds of a particular type. As many plants belonging to different species produce flower where all are sepals, some lacking petals, stamens or pistils or the combination of any of them. It is predictable that the development of each of these structures is under the control of specific genes or gene clusters. It is to be remembered that the molecular biology of flower induction and development is in progress and we have to wait the D-day for understanding the whole process in Toto. Phytochrome Pfr activated gets autophosphorylated. It can act on membrane G protein receptor that can lead to cGMP and calcium modulated Cam, both can enter into the nucleus. What is their function is not clear. The activated PhyA-Pfr act as serine-threonine protein kinase. Once it enters in to the nucleus it interacts and binds to PIF3 dimeric (phytochrome interacting factor) proteins and also binds to HFR1-PIF3 dimers (HFR1 = long hypocotyl far-red protein1 is a TF); then PhyA-p and receptor proteins bind to their respective gene regulatory elements called G-box receptor and recruit TFs and RNAP II and activate genes like CCA (TF), LHY (TF) and HY5 (TF). The fate of these gene products perhaps activate transcription of FT in association with constans in leaf cells. Probably they activate FT gene in the vascular bundles, especially companion cells, where the role of PhyB plays antagonistically in the production of FT.

Vernalization pathway:

Many plants have to go through wintering to flower in the next season. The famous plant is petkus rye, which is essential for bread makers in Russia. It is not the only plant that requires cold treatment for the plant to flower. These plants, particularly seeds contain specific proteins and its associated component which bind to loci that are involved flower induction and silence the chromatin by heterochromatization. Heterochromatization is achieved by histone methylation and histone deacetylation at specific loci. One such protein is known as flowering locus C (FLC). FLC acts as a repressor. Cold treatment in fact induce few FLC antogonizing genes and FLC dissociates from the loci and get degraded or remain free. There are several genes that are involved are VRN1, VRN2, VRN3 and VIN3. Even Frigida (FRI) proteins are involved. Frigida promotes FLC and FRI antagonize FLC. Once the chromatin is free from FLA and its associated protein, they interact with floral integrators such as SOC, CO, FT and LFY, which inturn activate genes for floral parts. In certain cases GA can overcome vernalization.

Cold has positive effect on the activation of genes such as BMS genes that leads to floral transition.

Major pathways: Pathways are shown in the following diagrams, copied from different sources and acknowledged too- the pathways are Photoperiodic pathway, Autonomous pathway, Vernalization pathway,

Vernalization pathway:
Many plants have to go through wintering to flower in the next season. The famous plant is petkus rye, which is essential for bread makers in Russia. It is not the only plant that requires cold

treatment for the plant to flower. These plants, particularly seeds contain specific proteins and its associated component which bind to loci that are involved flower induction and silence the chromatin by heterochromatization. Heterochromatization is achieved by histone methylation and histone deacetylation at specific loci. One such protein is known as flowering locus C (FLC). FLC acts as a repressor. Cold treatment in fact induce few FLC antogonizing genes and FLC dissociates from the loci and get degraded or remain free. There are several genes that are involved are VRN1, VRN2, VRN3 and VIN3. Even Frigida (FRI) proteins are involved. Frigida promotes FLC and FRI antagonize FLC. Once the chromatin is free from FLA and its associated protein, they interact with floral integrators such as SOC, CO, FT and LFY, which inturn activate genes for floral parts. In certain cases GA can overcome vernalization.

Pathways in general- modalities of integration of signals generated by each of the inducing stimuli:

light

Diiferent pathways integrating activate floral meristem and organ identity gene.

Genetics of Flowering:
Gregor John Mendel showed that the color of the flower id controlled by the action of a pair of heritable factor, now called Genes. They are independent discrete units of heredity. Inductive stimulus should transform the entire shoot apical meristem (SAM) into flower inducing meristem; there again from the same meristem different organs like sepals, petals; stamens and ovary have to develop. The flower is indeed is a modified shoot with leaves representing different parts of the flower. Stamen and ovary differentiation is more complex than sepals and petals for the simple reason they have highly specialized structures and they are gamete producing super-specialized reproductive organs. Whether it is light induced pathway or vernalization pathway or GA induced pathway or autonomous pathway, signals are produced and translocated to phloem and the base of SAM. The important component that induce FT is CO which activates the expression of FT which is a 20Kda protein. The FT is association with FD (TF) activates AP1 and LFY; this leads to the activation floral organ genes.

The above figure is self illustrative and shows how LD and SD effect the activation of gene, and the products move into the sieve cells, from there they reach the SAM. Note Hd3a is a homolog of FT in rice plants Once FT and FD together act on AP1 and LFY floral Meristem evocates and the floral organs start developing. Each of the structures is controlled by single or a combination of genes. The famous called ABC model has been used to explain the genes involved. Mutants in said gene exhibit their

phenotypic characters. The gene-A produces sepals and combination of AB generates petals and the combination of B and C generate stamens and C alone produces Carpels. As stamens and carpels contain different set of structures, to explain them the ABC model has be extended as ABCD and E model.

What is interesting thing about these genes is the said gene products are a group of MADS genes and they are homeotic genes. Each of these have been identified and studied.

MADS box proteins in combination induce specific floral organs. Look at A, AB, CD and D alone and the pervasive E overlaps ABCD. MADS are mostly transcription factors and they act in combination with one another. They have many domains from N to C end.

Ap3 expression with Gus as a reporter gene in apical meristems destined to Become floral organs.

MADS=MCM, Agamous, Dificiens and SRF Ap3 expression with Gus as a reporter gene in apical meristems destined to Become floral organs.

Ap3 expression with Gus as a reporter gene in apical meristems destined to Become floral organs.

Ap3 expression with Gus as a reporter gene in apical meristems destined to Become floral organs.

This figure is an ultimate starting and finishing of flowering, starts at leaves and ends in flowers. I have taken few authors written articles and put them together so student can understand experts views on flowering. I duly acknowdge the authors for their great insight into the flowering process.

FT Protein, not mRNA, is the Phloem-Mobile Signal for Flowering

Jan A. D. Zeevaart, MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI

September, 2007
Introduction Florigen as a Physiological Concept: Specific flower-inducing substances were first postulated by Julius Sachs (1865), but more convincing evidence had to await the discovery of photoperiodism (Garner and Allard 1920). The seminal finding was that in photo periodically sensitive plants the day length is perceived by the leaves, whereas flower formation takes place in the shoot apical meristem (Knott 1934). This finding demonstrates that a long-distance signal, called the floral stimulus or florigin (Chailkhyan 1936), moves from an induced leaf to the shoot apex. The floral stimulus can be transmitted from a flowering partner (donor) via a graft union to a non-flowering partner (receptor), as illustrated in Figure 25.29 of the textbook. Furthermore, it was shown that florigen is exchangeable between related species and genera, as well as among different photoperiodic response types (Lang 1965; Zeevaart 1976; Zeevaart 2006). These observations led to the hypothesis that florigen is widespread, if not universal, in flowering plants, and that only the conditions that regulate its production vary among the different response types. Although certain physiological characteristics of florigen, such as its movement in the phloem (e.g., King and Zeevaart 1973), could be investigated, its identity remained unknown. Thus, florigen remained a physiological concept rather than a chemical entity. With the isolation of auxin as the first-identified plant growth hormone in the 1930s and the discoveries of cytokinins and gibberellins in the 1950s, many attempts were made to extract florigin. In the early approaches, it was assumed that like the classical plant hormones, florigen would be a small organic molecule. Extracts prepared from flowering material were tested for flower-promoting activity in vegetative plants. Positive results were reported occasionally, but none of them was reproducible (reviewed in Zeevaart 1976). As a result, skeptics challenged the adequacy of florigin as a single substance causing flowering. Instead, it was proposed that florigen consists of multiple factors and that flowering is induced by a specific ratio of known hormones and metabolites (Bernier 1988; Bernier et al. 1993). Molecular-Genetic Research on Flowering With the advent of Arabidopsis as a model plant for molecular-genetic studies, genetic and molecular analyses became popular approaches in studies on flowering. Through mutagenesis, many mutants

were isolated in the quantitative long-day plant (LDP) Arabidopsis thaliana. Of interest here are those mutants that exhibit changes in flowering time in comparison with wild-type (WT) plants. Mutants flowering later than WT plants represent a loss-of-function that must involve positive regulators of flowering. Conversely, early-flowering mutants have lost an inhibitor of flowering. These molecular-genetic studies have led to identification of four pathways that regulate flowering in Arabidopsis: the photoperiod, vernalization, autonomous, and GA pathways (e.g., Komeda 2004; Corbesier and Coupland 2005, 2006; Imaizumi and Kay 2006). In the present essay, we will deal mainly with the photoperiod pathway. The genes called CONSTANS (CO) and FLOWERING LOCUS T (FT) are principlal to long day induced flowering in Arabidopsis. CO encodes a nuclear zinc-finger protein, which in response to LD induces transcription of FT in the phloem of leaves (?).Does it happens in phloem or in leaf tissues, but where in the cells of leafs; is it mesophyll cells, bundle sheath cells or in cells in contact with phloem progenitors. Visualize phloem food conducting vessels are like human lymphocyte system. Neither CO nor FT is expressed in the shoot apex. Expression of CO from a meristemspecific promoter does not enhance flowering, but early flowering is induced in short days (SD) when FT is overexpressed in the shoot apex. Expression of CO from a phloem-specific promoter is sufficient to generate a phloem-mobile stimulus that induces flowering, as shown by grafting experiments between Arabidopsis donor plants over expressing CO and co mutant shoots as receptor (An et al. 2004; Ayre and Turgeon 2004). Because FT must act in the shoot apex in order to elicit flowering, this result gives a strong indication that FT or its product is the signal that moves from an induced leaf to the shoot apex and induces flowering. FT acts in the shoot apex by forming a complex with the bZIP transcription factor FD. The essential role of FD in flowering is demonstrated by the finding that fd mutants flower late and that FT over expression is partially suppressed by fd (Abe et al. 2005; Wigge et al. 2005). The FT/FD complex activates the downstream genesAPETALA1 (AP1) and SUPPRESSOR OF OVEREXPRESSION OF

CO1 (SOC1); the latter, in turn, activates LEAFY(LFY). So, although FT and FD are produced at
different sites, they act together in the shoot apex (Figure 1). This finding suggests that the FT gene product has to move from the leaf to the shoot apex and strongly implicates the FT gene product as part of florigin, if not florigin itself. The concept of Ft as florigin, a protein of 20Kds how does it induce the SAM. Is it through the binding to FT receptors found in the basement cell membranes of SAM or what? Is the Phloem-Mobile Floral Stimulus FT mRNA or FT Protein? A substance functioning as florigin must fulfill at least the following criteria: 1. It must be produced in the leaf, presumably only under inductive conditions for flowering. At least, it would be expected to be more abundant in an induced than in a non-induced leaf. 2. It must be phloem-mobile, moving from an induced leaf to the shoot apex. 3. It must be required for flowering in all plants.

From the hierarchy of regulatory proteins that controls flowering, it follows that FT is the terminal gene expressed in the leaf, whereas its effect is in the shoot apex (see above; Corbesier and Coupland 2006). Consequently, three possibilities can be envisaged: 1. FT mRNA is phloem-mobile and moves from an induced leaf to the shoot apex. 2. FT protein moves from an induced leaf to the shoot apex. 3. FT in the leaf controls the synthesis of a small compound in the leaf that then moves to the shoot apex, where it induces FT expression to produce FT protein, which complexes with FD. Results relevant to all three possibilities will be discussed below. Huang et al. (2005) investigated the possibility that FT mRNA is the mobile stimulus. It was shown in Arabidopsisthat FT under control of a heat shock promoter was transiently expressed in a single heated leaf and FT mRNA was detected in the shoot apex by RT-PCR 6 hours later. In addition, the heat-treated leaf did induce flowering. Thus, in this work FT mRNA would seem to fulfill some of the criteria of florigen. However, it was later reported that the real-time RT-PCR data were analyzed incorrectly and that in new experiments the movement of FT mRNA from leaf to shoot apex was not detected. Consequently, the conclusion that FT mRNA is part of the floral inductive signal moving from leaf to shoot apex was retracted (Bhlenius et al. 2007). Other authors also failed to obtain evidence favoring FT mRNA as a mobile floral stimulus. In the case of Arabidopsis and rice,FT mRNA as measured by real-time RT-PCR was lower in shoot apices than in leaves (Corbesier et al. 2007; Tamaki et al. 2007). Also, no FT mRNA could be detected in the shoot apex of Arabidopsis by in

situhybridization (Jaeger and Wigge 2007). In Cucurbita, FT mRNA was not detected either in the
phloem sap or in the shoot apex (Lin et al. 2007). Finally, in grafting experiments with tomato SFT transcript of the donors did not move across the graft union to the receptor shoots (Lifschitz et al. 2006; Lin et al. 2007).Thus, a rather extensive body of evidence argues against FT mRNA functioning as florigen, although considering the presence of mRNAs in phloem as components of a long-distance signaling network, the possibility cannot be completely ruled out (Lough and Lucas 2006). Simultaneously with the retraction of the publication on FT mRNA as a phloem-mobile signal, two new publications reported results that FT protein rather than the mRNA is the mobile signal in the phloem that induces floral initiation in Arabidopsis as well as in the SDP rice (Figure 1). To determine the distribution and movement of FT protein, George Coup lands team at the Max Planck Institute for Plant Breeding Research, fused FT with the gene encoding GREEN FLUORESCENT

PROTEIN (GFP) and expressed this construct in ftmutant plants. Expression from phloem-specific
promoters demonstrated the presence of FT:GFP not only in leaf phloem, but also in the shoot apex. FT:GFP transgenic plants also flowered earlier than ft plants. Assuming that the promoters employed in these experiments are specific for expression in phloem, these results demonstrate that the FT protein expressed in the leaf phloem moves to the shoot apex to induce floral initiation (Corbesier et al. 2007). FT and FT:GFP were also expressed from the GALACTINOL

SYNTHASE (GAS1) promoter, which acts specifically in the phloem companion cells of the minor veins
of leaves. In transgenic plants expressing GAS1:FT:GFP, GFP fluorescence was observed only in the

minor veins of leaves. Transgenic GAS1:FTplants flowered early, but GAS1:FT:GFP plants flowered as late as ft plants, although the transgene was active in the leaves, as shown by activation of FRUITFULL (FUL). The authors speculated that the fusion protein was not mobile in the minor veins and as a result did not move to the shoot apex and induce flowering. This result confirms that FT protein is the floral stimulus in Arabidopsis and that no secondary product of FT is involved (Corbesier et al. 2007). In the SDP rice, Heading date 3a (Hd3a) is the ortholog of FT in Arabidopsis. Under SD conditions, Hd3a shows highest expression in leaf blades of rice. Ko Shimamotos group at the Nara Institute of Science and Technology, transformed rice with the Hd3a: GFP construct under control of phloem-specific promoters. The transgenic plants flowered early and GFP fluorescence was observed in the vascular tissues of the leaf blade, stem, and shoot apex. Because the Hd3a:GFP construct was expressed only in the phloem of leaf blades, whereas the protein was detected in the shoot apex, it must be concluded that Hd3a protein is moving in the phloem and that Hd3a functions as florigin in rice (Tamaki et al. 2007). Additional evidence obtained with Arabidopsis further supports the notion that FT protein moves from an induced leaf to the shoot apex. When expressed from a phloem-specific promoter, an epitope-tagged version of FT induced early flowering; the protein was detected by immuno localization in the shoot apex. By contrast, when MycFT was targeted to the nucleus (immobilized), it had no effect on flowering and remained localized in the phloem of the leaf, whereas the constitutive 35S promoter did induce early flowering (Jaeger and Wigge 2007). Furthermore, FT expressed ectopically as a large fusion protein promoted flowering. But in the case of a phloemspecific promoter, flowering was promoted only if the FT protein was released from the complex by a specific protease (Mathieu et al. 2007). Thus, export of FT from companion cells was required and correlated with flowering. In the experimental approaches used in Arabidopsis to demonstrate the movement of FT protein, FT was expressed as a fusion protein in transgenic plants for ready detection by either confocal microscopy or immunolocalization. It should be realized that expression of FT transgenes is usually much higher than that of the native FT gene. Although all the results are consistent with movement of FT protein from induced leaves to the shoot apex, the presence of native FT protein in the phloem translocation stream was not demonstrated in these experiments. In a close relative of Arabidopsis, Brassica napus, FT protein has been identified in phloem exudate of inflorescence stems (Giavalisco et al. 2006), so that it is reasonable to assume that native FT protein is also present in the phloem sap of Arabidopsis. Because of its small size, Arabidopsis is not a suitable plant for phloem translocation studies. For this purpose,Cucurbita has been a favorite, especially because phloem exudate can be readily collected from cut stems. However, cucurbits are day-neutral and had not been used for flowering studies until Bill Lucass laboratory at the University of California, Davis, surveyed 97 cucurbit accessions. They found one, C. moschata (Cmo), that flowered only under SD conditions. The group tested this species, along with day-neutral C. maxima (Cm), to determine whether long-distance movement of FT is required for flowering. They used the Zucchini yellow mosaic virus (ZYMV) as vector for introducing

the FT gene of Arabidopsis (AtFT) into Cmo. Infection with this vector caused flowering of Cmo in LD. The virus was present in developing leaves, but not in apical tissues (Lin et al. 2007). This result demonstrates that the function of AtFT as an inducer of flowering is conserved when expressed in Cmo, and further, that flowering was most likely induced by movement of FT protein from virusinfected leaves to apical regions. To investigate the role of FT-like (FTL) genes in Cucurbita, two orthologs of FT were isolated from both Cm(Cm-FTL1 and Cm-FTL2) and Cmo (Cmo-FTL1 and Cmo-FTL2). Transcripts of FTL genes were restricted to phloem of stems and leaves, but were not detected in phloem sap. By contrast, the FTL proteins were detected in phloem sap by a combination of liquid chromatography-tandem mass spectrometry. Cmo-FTL2 was approximately 10 times more abundant in phloem exudate than was Cmo-FTL1. Both proteins were present only in phloem sap obtained from SD-grown plants. Although transcript and protein levels of Cmo-FTL2 were much up-regulated in the phloem of stems in SD, an important regulatory mechanism by the photoperiod appeared to be entry of FTL proteins into the phloem translocation stream, in addition to transcriptional control (Lin et al. 2007). Finally, Cmo(receptor) was grafted onto Cm (donor) in long days. All receptor shoots were induced to flower and the CmFTL2 protein was identified in phloem sap collected from Cmo scions. These results show convincingly that FTL proteins are present in the phloem translocation stream of Cucurbita and that they can move across a graft union in a heterograft to induce flowering in a vegetative receptor shoot (Lin et al. 2007). This experiment elegantly demonstrates that transmission of florigen from a donor to a receptor plant is associated with transfer of FT protein from donor to receptor. FT Protein Is the Universal Signal for Flowering The basic tenet of the florigin hypothesis is that florigin is common to all flowering plants. Physiological evidence for the universality of florigin is based on results of grafting experiments between closely related species in which one response type (e.g., a LDP) induces flowering in a related species of another response type (e.g., a SDP). However, this approach has been limited by graftincompatibility between unrelated species. With the advent of molecular genetics and plant transformation, this barrier can now be readily overcome. Instead of grafting, a specific gene from one species can be transplanted into an unrelated species and its role in flowering demonstrated. For example, the SFT gene of day-neutral tomato can substitute for the LD requirement in Arabidopsis (Lifschitz et al. 2006). Likewise, FT expressed in the SDP C. moschata induces flowering under LD conditions (Lin et al. 2007). One of the criticisms of the universality of florigin was that there were many examples of grafting experiments in which receptor shoots failed to flower (reviewed in Zeevaart 1976). Does this mean non-identity of florigin in the two grafting partners? Work with tomato provides an answer to this question. Transgenic tomato plants over expressing SINGLE-FLOWER TRUSS (SFT), an ortholog of FT, under control of the 35S promoter, were excellent donors, but wild-type tomato could not complement sft mutant plants in grafting experiments (Lifschitz et al. 2006). This result suggests that the failure to induce flowering in receptors is not due to non-identity of florigin, but most likely due to a low level of florigin in the donor and/or rapid decay of florigin in the receptor.

The functions of FT orthologs appear highly conserved in flowering plants regardless of response type, and in monocots as well as in dicots. Although the control of expression of FT varies across response types, the end product, FT protein, appears to be always the same. The evidence obtained so far provides strong support for the universality of FT protein as florigin not only in herbaceous plants, but also in trees (Bhlenius et al. 2006; Hsu et al. 2006). In Lolium temulentum, GAs, specifically GA5 and GA6, have been assigned a role as florigen (Web Essay 25.1). But it is of interest that also in this species LtFT was strongly up-regulated in the leaves after plants had been shifted from SD to LD (King et al. 2006). The question is often asked: Why did it take so long to elucidate the molecular nature of florigin? There are several aspects to a complete answer. For many years it was assumed that florigin, like the classical plant hormones, would be a small molecule that could be extracted and re-introduced into test plants. Some physiological evidence indicated that in some species florigen has virus-like properties, but techniques to extract nucleic acid or proteins and apply them to assay plants were not available. So, further progress had to await the application of molecular-genetic techniques to studies on physiology of flowering. It then took considerable time before the various flowering pathways had been worked out in Arabidopsis and the pivotal role of FT in flowering became apparent (e.g., An et al. 2004; Ayre and Turgeon 2004; Abe et al. 2005; Wigge et al. 2005; Imaizumi and Kay 2006). Rather than applying extracts to test plants, transgenes could now be expressed and their products, mRNA and protein, could be visualized by techniques of cell biology, or identified by mass spectrometry. It took 70 years, but finally florigin has been identified as FT, a mobile protein of approximately 20 Kda. Perspective With the identification of FT protein as florigin, questions regarding its production, transport, and persistence can be studied at the molecular level. For example, is FT permanently activated in plants with localized induction? Does FT induce its own production via a positive feedback loop in species that exhibit the phenomenon of indirect induction of flowering? (see textbook pp. 661662). These intriguing phenomena, as well as other classical observations on the physiology of flowering, can now be studied from a molecular-genetic perspective. It is expected that results of further studies will provide a solid underpinning for the florigen theory. References Abe, M., Kobayashi, Y., Yamamoto, S., Daimon, Y, Yamaguchi, A., Ikeda, Y., Ichinoki, H., Notaguchi, M., Goto, K., and Araki, T. (2005) FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 309: 10521056. An, H.L., Roussot, C., Surez-Lpez, P., Corbesier, L., Vincent, C., Pieiro, M., Hepworth, S., Mouradov, A., Justin, S., Turnbull, C., and Coupland, G. (2004) CONSTANS acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of Arabidopsis. Development 131: 36153626

The Role of Gibberellins in Floral Evocation of the Grass Lolium temulentum

Rod W. King and Lloyd T. Evans, CSIRO, Plant Industry, GPO Box 1600 Canberra, ACT 2601, Australia

September, 2006
Introduction Almost 70 years ago it was recognized that the leaf is the site of the initial response in daylengthregulated flowering. However, the nature of the factors transported from the leaf to the shoot apex where the flowers form has remained elusive. Here we summarize the evidence from studies with the grass Lolium temulentum, which shows that specific gibberellins (GAs) act as its "floral stimulus." This evidence satisfies five requirements, namely: The production of florigenic GAs in the leaf when exposed to a photo inductive long day Their transport from the leaf to the shoot apex Their sufficient increase in the shoot apex when floral evocation occurs Morphological and molecular changes at the apex, especially involving floral organ identity genes Replacement of the long-day requirement by specific GAs Our studies over the last 40 years, reviewed by King and Evans (2003), have established a continuous trail of evidence for the gibberellin (GA) class of plant hormone as a floral stimulus in the long-day regulated flowering of the grass, Lolium temulentum. Its long day (LD) photoresponse in the leaf blade involves far-red rich phytochrome-active light, which enhances the expression of a GA biosynthetic enzyme and alters GA precursor and product levels. Most significantly, the florigenic GAs increase in the shoot apex not long after they rise in the leaf, and at the time of the earliest molecular and morphological changes at the shoot apex. Finding the final pieces of this scientific jigsaw has involved GA application studies. Not only was it necessary to show that applied GAs could replace the need for a LD, but by utilizing a range of structurally related synthetic and natural GAs as well as inhibitors of GA synthesis, we have established that some GAs promote inflorescence initiation while others are more active for stem elongation or may participate in the later stages of flowering of L. temulentum. Gibberellins for Flowering In 1956, Anton Lang reported for the first time that applied gibberellic acid (GA3), like vernalization or exposure to long days (LDs), could cause plants of Hyoscyamus niger first to bolt, and subsequently, to flower. His finding was soon confirmed both for other dicot species, for grasses and for GAs other than GA3 (Lang 1957). Flowering of many temperate grasses is induced by LD or by treatment with GA 3. For example, the grass Lolium temulentum requires a single LD for inflorescence initiation, and this requirement can be

met in noninductive short days (SD) by a single application of some GAs, either to the leaf blade or to the shoot apex (Evans 1964a, 1969; Evans et al. 1990). However, there are differences between some GAs and LD in this response. Over at least the first three weeks of inflorescence development, there is little or no stem elongation/bolting associated with the LD-induced flowering of L. temulentum and perhaps other grassesa response duplicated by application of some GAsbut many GAs that induce flowering of grasses also cause early and excessive stem elongation. The responses to GAs may be similar across dicot and monocot species even though the effects on stem elongation and flowering may occur in a different order, presumably reflecting a different order of gene activation associated with different GAs. For example, GAs that stimulate stem elongation may not be involved in the process of floral evocation in grasses, although they could be involved in subsequent inflorescence development. Conversely, other GAs may be florally effective because they cause inflorescence initiation with little or no effect on stem elongation. The Search for GAs that are Florigenic but Ineffective for Stem Elongation Beginning in the mid 1980s, and in collaboration with Dick Pharis and Lew Mander, we compared the effects of many different gibberellins on floral evocation and stem elongation in L. temulentum. Some, like GA8 and GA9, influence neither process. 16,17-dihydro GA5 promotes flowering while inhibiting stem growth (Evans et al. 1994b), while GA1 and GA3 enhance both and are therefore unlikely to be the floral stimulus in L. temulentum(Evans et al. 1990). However, exogenous GA5 and GA6 are both able to cause floral evocation and induce the early stages of inflorescence development in SD at doses that have no effect on stem elongation (King et al. 1993, 2003). Two questions arise from such findings. Firstly, are either or both GA5 and GA6 agents of floral evocation by one LD in L.

temulentum? Secondly, are the more growth-effective GAs such as GA1 and GA4involved in the
subsequent processes of inflorescence development in this grass? To answer these questions, in the following sections we consider evidence based on endogenous gibberellin contents of the leaf and apex of L. temulentum along with information on metabolic pathways involved in GA synthesis and degradation. Critical Steps in GA Metabolism The relevant steps in GA metabolism as summarized by Hedden and Phillips (2000) are shown below:

Figure 1 (Click image to enlarge.) The key to biological activity among many gibberellins is the closure of the lactone ring with the conversion of GA19 to GA20 and GA24 to GA9. This step involves a 20-oxidase whose activity increases in LD, as shown for spinach and Arabidopsis (Wu et al. 1996; Xu et al. 1997). A multifunctional 3oxidase completes the conversion of the biologically inactive GA20 or GA9 to bioactive GAsincluding GA5 and GA6and to the 3-hydroxylated GA1, GA3, and GA4. A key inactivation step involves the 2oxidase responsible for adding a hydroxyl at C-2 to give GA8, GA29, or GA34. GAs for Floral Evocation: Production in the LD Leaf and Transport to the Shoot Apex In L. temulentum, when the leaf is exposed to a single photo inductive LD, expression of the messenger RNA for a 20-oxidase increases dramatically after 16 hours of light and peaks 4 hours later (Figure 2). Most significantly, the timing of the increase in the 20-oxidase in LD matches the minimum duration of light required for floral induction of L. temulentum by one LD. In SD, the expression level of mRNA for this key enzyme is much weaker and peaks 12 hours later during the next 8-hour-daylight period.

Figure 2 Expression of a L. temulentum 20-oxidase GA biosynthesis gene. Compared with an 8h short day, LD exposure of the leaf (8h SD extended by 9.5h using incandescent lamps) led to a large increase in gene expression. (Semi-quantitative RT PCR assays from Blundell, MacMillan, and King; unpublished). The consequence of increased 20-oxidase activity in L. temulentum leaves is a fall in GA19 levels when the critical day length is reached around midnight, and an equivalent rise in GA20 (GA19 falls from a peak of 35 ng g-1 DW to a minimum of 12 ng g-1 around midnight, while GA20 rises from 3 ng g-1 to 23 ng g-1; King et al. in preparation). GA5, an immediate product of GA20, also increases in LD but not in SD leaves so that the difference by midnight (i.e., after 16 hours of light) is fourfold. GA 1 could also be expected to rise in the LD leaves, but this is not apparent until the following day. The next step, the export of GA5 from the leaf blades and down the leaf sheath has not been directly documented. The difficulty is to isolate specific vascular tissue and to sense potentially small changes in GAs. As an alternative, we have assessed movement from the leaf to the apex of labeled GA5. In LD, 2H4-GA5 applied to the leaf blade was transported intact and quantitatively to the shoot apex (King et al. 2001). Earlier experiments in which the blade and sheath of the single LD leaf were cut off at various positions and times suggest that the LD stimulus is translocated to the shoot apex at a speed of 12.4 cm h-1, compared with the simultaneous transport of sugars at about 80100 cm h1

(Evans and Wardlaw 1966). Furthermore, evocation can be maximal in shoot apices, which throughout

the low intensity daylength extension, show no increase in their sucrose content over the 2% found in the vegetative plants exposed to SD (King and Evans 1991). The LD florigenic stimulus in L.

temulentum is clearly not sucrose, and an increase in the content of sucrose in the shoot apex is
neither necessary nor sufficient for its floral evocation. Based on its apparent speed of translocation, the LD floral stimulus should begin to arrive at the shoot apex and floral evocation begins on the morning after the LD. Such timing of floral evocation was confirmed by excising shoot apices at various times after the LD, onto media supporting apex development (McDaniel et al. 1991). King et al. (1993) confirmed these results but also found with plants of various ages that shoot apices excised from vegetative plants in SD were induced to flower by supplying GA3 in the medium and could reach a similar stage of flowering to those from plants given one LD but with no GA3 in the medium. Floral evocation in plants of L. temulentum is associated with an increase on the day after the LD in
32

P incorporation into RNA, and of

35

S into protein in shoot apices (Rijven and Evans 1967; Evans

and Rijven 1967). These increases occur in the dome of the apex and in the sites of future spike lets, down both sides (Knox and Evans 1968). Thus, the timing of these changes fits with the estimated time of arrival of the LD photoperiodic stimulus in the apex (Evans and Wardlaw 1966; McDaniel et al. 1991; King et al. 1993). However, to complete the evidence relating GAs to floral evocation in L.

temulentum, it was essential to measure their content in the shoot apex.

The minute size of the shoot apex (<3 g dry weight) had made it difficult to measure its GA content, but this became feasible through a recent collaboration with Thomas Moritz in Sweden. Using highly sensitive GCMS techniques, he analyzed GAs at subpicogram levels in the shoot apex of L.

temulentum. As shown in Table 1, by the end of the daylight period following the overnight LD, the
content of the highly florigenic GA5 and GA6 at least doubles in the shoot apex (King et al. 2001, 2003). Moreover, at this time the maximum GA5 concentration reached in the shoot apices is 3 107

M which is close to that needed in an agar medium if shoot apices excised from plants in SD are to

flower (about 5 10-7M GA5; King et al. 1993). It is highly probable, therefore, that translocation of these two GAs from leaf blades in LD causes floral evocation of L. temulentum.

Table 1 Changing the Guard: Inflorescence Development and Growth-Active GAs A number of bioactive GAs and precursors including GA1, GA4, and GA9 are notably absent from the shoot apex or minor in SD and on the day after the inductive LD, and remain so for 610 days. After exposure of the leaf to 2 or 3 LD, they all increase in content at the apex (King et al. 2001), whereas the content of GA5, GA19, and GA24 falls more rapidly with additional LD. Such additional LDs accelerate inflorescence development (Evans 1958, 1960; Evans and Blundell 1996) and, as well, increase the content of GA1, GA4, and GA9 in the LD leaf (Gocal et al. 1999). Thus, as for floral

evocation, for inflorescence development there are increases in GAs in LD but now for a new group of GAs that are highly active for stem elongation. During inflorescence development, the many fold increase in the shoot apex of the "new guard" GAs GA1 and GA4along with evidence that their application can now promote flowering (King et al. 2001), shows their importance for inflorescence development. That a GA input is effective during floral development is also evident from studies with apices induced to flower by one LD, and then excised. Progress to flowering is weak unless GAs are supplied by about six days after the end of the LD (King et al. 1993). Which GAs are important at this time is indicated by the response to application of two growth retardants, Trinexapac Ethyl and LAB 198 999. These retardants block synthesis of GA 1 and GA4 by 3-oxidases and inhibit inflorescence development but show no inhibition when applied earlier during floral evocation (Evans et al. 1994a). Thus, whereas GA5 and GA6 are the gibberellins most active in floral evocation of L. temulentum, it is the elongation-active, C-3-hydroxylated GAs such as GA1 and GA4 that play a role in subsequent inflorescence development. Since GA1 and GA4 can be detected in leaves of L. temulentumwhether from vegetative or floral plantsthere must be some mechanism first to exclude them from the shoot apex and then allow them to access the shoot apex late in inflorescence development. As discussed below, this exclusion mechanism may involve: (i) degradation of specific GAs by a 2-oxidase; (ii) localization of a 2-oxidase just below the apex of vegetative plants and; (iii) disappearance of this 2-oxidase during inflorescence development. The implication that some GAs are more readily degraded than others (e.g., GA1 or GA4 vs GA5 or GA6) also provides a focus in the following section for understanding how GA structure affects response. Structural Considerations: The Lord of the Rings The functional groups contributing to GA activity are indicated in Figure 3, and from our comparisons of many GAs (Evans et al. 1990, 1994 a,b; King et al. 2003; Mander et al. 1998 a,b), it is clear that the structural requirements for florigenicity are quite different from those for stem elongation. As an example, for four GAs2,2-dimethyl GA4, GA32, GA1, and GA4although not greatly different in their effectiveness in promoting stem elongation, there is up to a ten thousandfold range in their florigenic activity.

Figure 3 Gibberellin structure and carbon numbering.

Differences in activity of an applied GA are likely to be determined not only by their ability to resist degradation (see above) but also by structural features altering uptake and transport, their inherent bioactivity, their potential as a biosynthetic substrate, and their capacity to interfere with endogenous GA synthesis and degradation. GA uptake and transport is unlikely to be critical to florigenicity, as GAs could variously promote either or both stem elongation and flowering. Those structural elements favoring florigenicity in particular include: (i) Formation of the lactone bridge between carbons 4 and 10, so adding a fifth ring to the 4-ringed structure of the biologically inactive GAs. This requirement seems to apply to all the LD grasses whose flowering is induced by gibberellins. For example, GA19 (a 20-carbon precursor GA) is wholly inactive, whereas GA3, GA5, GA7, and several other 19-carbon GAs have reported florigenic activity in other species besides L. temulentum. However, formation of the lactone ring itself is insufficient, and a further step involving activity of a 3-oxidase enzyme is also essential (see iii). (ii) A free carbon-7 carboxy group is an absolute requirement for all plant GA responses and may reflect ability to bind to a GA receptor. (iii) Hydroxylation at carbons 3, 12, 13, and 15 and/or the presence of a C-1, 2 double (C-C) bond (as in GA3), or a 2,3 double bond (as in GA5), or a 2,3 epoxide (as in GA6). (iv) Absence of a C-2 hydroxyl. While features listed under (iii) may enhance the stability of a GA against inactivation by 2hydroxylation, structural elements at C-2 are the most significant in this regard. A good comparison involves the responses to applications of three closely related GAs: the highly florigenic and growth active 2,2-dimethyl GA4, the modestly florigenic but reasonably growth-active 2-methyl GA4, and the nonflorigenic, growth-active GA4 (Evans et al. 1990). Because it lacks any C-2 structural elements, 2-hydroxylation of GA4 is highly likely and this apparently rings the death knell for any florigenic activity. GA4 is taken up byand transported inthe plant as it is highly active for stem elongation but quite clearly it is not transported intact into the shoot apex. The most likely explanation, for this observation would involve the presence of a concentrated zone of 2-oxidase just below the vegetative apex, and while this has yet to be demonstrated for L. temulentum, such a highly localized zone of 2-oxidase RNA expression has been reported recently for rice (Sakamoto et al. 2001). The concept for rice of the exclusion of 2-hydroxylation-sensitive GAs from the shoot apex fits well with the structures of the GAs that are naturally found in the shoot apex of L. temulentum. Neither of the readily 2-hydroxylated GAs we examined, GA1 and GA4, was detectable in the shoot apex of vegetative plants or for about the first week after floral evocation (King et al. 2001) although their content increased in LD leaves (Gocal et al. 1999). By contrast, we detected highly florigenic but weakly growth-active GAssuch as GA5 and GA6these GAs probably being protected from C-2 hydroxylation by the C-2,3 double bond in GA5 and the C-2,3 epoxide in GA6. High florigenicity with limited growth activity for GA32 also fits with its having a C-1,2 double bond. Later, as floral development proceeds GA5 and GA6 decrease in the apex while there is a dramatic increase in GA1 and GA4 (King et al. 2001) and it is at this stage of flower development that the localized band of 2-oxidase disappears from just below the rice inflorescence (Sakamoto et al. 2001).

Apparently, the localized band of 2-oxidase activity protects the shoot apex from "growth-active" GAs, so guaranteeing the integrity of the apex during vegetative growth, floral evocation, and early floral differentiation. Later, during inflorescence development, disappearance of the 2-oxidase barrier allows the influx of highly growth-active GAs. Explaining a Paradox: Flowering Promotion by GA Biosynthesis Inhibitors Classic studies by Baldev and Lang (1965) with the rosette LDP Samolus parviflorus showed that flowering in LD is inhibited when GA synthesis is blocked using either of the GA biosynthesis inhibitors, Amo-1618 and Cycocel (CCC). Such results supported a role for GAs in the LD response. Paradoxically, however, with L. temulentum, several "anti-gibberellins"which might be expected to inhibit floral inductionactually promote it, and act synergistically with some GAs. For example, CCC alone did not change the flowering response to one LD, but greatly enhanced the promotive effect of GA3, although other "anti-gibberellins" such as B9 and Amo 1618 gave the expected inhibiting effect on flowering (Evans 1969). With the acylcyclohexanedione inhibitors, LAB 198 999 and Trinexapac Ethyl, promotion of flowering could reflect an interference with the 2-oxidase. Whether or not this enzyme is localized below the vegetative shoot apex, endogenous or applied bioactive GAs would be spared from inactivation. Another explanation involves either or both inhibition of the 3-oxidase(s) responsible for conversion of GA20 to GA1 and inhibition of the 2-oxidase involved in converting GA20 to inactive GA29. By inhibiting the conversion of GA20 to either GA1 or GA29, LAB 198 999 and Trinexapac Ethyl treatments could promote GA20 conversion to GA5, thereby accounting for their strongly promotive effects on floral evocation inL. temulentum. Their subsequently inhibitory effects on inflorescence development (Evans et al 1994a) when synthesis of GA 1 and GA4 becomes important, fits with their known action as 3-oxidase competitors. The greater inhibition of the 2-oxidase by the derivative 16,17-dihydro GA5 than by LAB 198 999 (Junttila et al. 1997), indicates further interesting effects of "ant gibberellins," which preclude drawing too simple an interpretation of how they may alter GA metabolism. The Genes Involved at the Shoot Apex Of the various genes known to be involved in floral determination and differentiation, the earliest to be expressed in the shoot apex of L. temulentum is LtCDC2, which greatly increases in activity in the future spikelet sites by the afternoon after the long day, just when floral evocation is completed (Gocal 1997). By the next day, the API-like gene LtMADS2 and a related gene LtMADS1 have also increased their expression at the spikelet sites and subsequently at the floret sites (Gocal et al. 2001). Whereas the LEAFY (LFY) gene is expressed early in the floral apices of Arabidopsis, Gocal et al. (2001) found it not to be expressed until about 12 days after LD induction in L. temulentumpossibly associated with the great difference between the two species in the timing of stem elongation vis-vis floral evocation, and, as well, in the GAs involved. Conclusion

For no other species is there such a complete and consistent trail of evidence on the identity of the LD floral stimulus as in L. temulentum, from the daylength-sensitive, physiologically mobile GA5, and probably GA6, in the leaf blades, translocated intact and at appropriate velocity to reach the shoot apex in sufficient concentration to effect floral evocation at the clearly identified time. Moreover, several previously puzzling features of this investigation, especially those relating to the great range in florigenicity among the gibberellins and to the synergistic effects of several inhibitors of GA synthesis, can now be resolved. If, as in rice, the vegetative shoot apex in L. temulentum is protected by a ring of 2-oxidase, a very coherent picture of the control of flowering in L.

temulentum by gibberellins emerges. In addition, such localized action of the 2-oxidase explains much
of the variation among bioactive GAs in their relative promotion of floral evocation vis--vis stem elongation. Our findings also highlight a feature of the growth habit of temperate grasses, which may have been crucial to their evolutionary success when close-grazed by ungulates. Except for the relatively brief stage when the inflorescences must grow upwards for wind pollination and seed dispersal, the terminal meristems are kept close to the ground by the absence of stem elongation until the later stages of inflorescence development. The initial involvement at floral evocation of GAs such as GA5 and GA6, which do not cause stem elongation at concentrations that are florigenic, and the initial exclusion from the apex of GAs such as GA1 and GA4, which do cause stem elongation, presumably aids survival under both close-grazing and adverse environmental conditions. Such an evolutionary explanation is required, because stem elongation per se is not antagonistic to flowering. Applied 2,2dimethyl GA4, for example, not only induces flowering but also causes massive stem elongation. For a number of reasons, our findings that specific GAs are florigens in grasses, cannot be generalized to all species. Based on their evolutionary relatedness (see Kellogg 2001), we believe our findings are applicable to other temperate grasses and cereals, which retain a LD responseas most do (Evans 1964b; Heide 1994). However, the warm climate grasses and cerealsincluding rice, corn, and sugarcaneoften have no photoperiodic response and if they are sensitive, they often respond to SD, which may lead to decreases, not increases, in GA content. Where a species is insensitive to photoperiod, a role for GAs appears unlikely and one of many other florigenic factors must be limiting. In temperate, LD-responsive dicots, GA increases have often been documented but it is also clear that their primary action may be on stem elongation rather than on flowering. This latter observation is especially interesting as the GAs detected in dicots have always been the growthactive ones, and the florigenic but weakly growth-active GAs have yet to be examined in such dicots. Nevertheless, there is no reason at present to suggest that the growth-inactive GAs that are florigens in grasses play any role in flowering of dicots. References Baldev, B., and Lang, A. (1965) Control of flower formation by growth retardants and gibberellins in Samolus parviflora, a long-day plant. Amer. J. Bot. 52: 408417. Evans, L. T. (1958) Lolium temulentum L., a long-day plant requiring only one inductive photo cycle. Nature 182: 197198.

Evans, L. T. (1960) The influence of environmental conditions on inflorescence development in some long-day grasses. New Phytol. 59: 163174. Evans, L. T. (1964a) Inflorescence initiation in Lolium temulentum L. V. The role of auxins and gibberellins. Aust. J. Biol. Sci. l7: l023 Signals produced in leaves are transported to the shoot apex where they cause flowering. Protein of the geneFLOWERING LOCUS T (FT) is probably a long day (LD) signal in Arabidopsis. In the companion paper, rapid LD increases in FT expression associated with flowering driven photosynthetically in red light were documented. In a far red (FR)-rich LD, along with FT there was a potential role for gibberellin (GA). Here, with the GA biosynthesis dwarf mutant ga1-3, GA4-treated plants flowered after 26 d in short days (SD) but untreated plants were still vegetative after 6 months. Not only was FT expression low in SD but applied GA bypassed some of the block to flowering in ft-1. On transfer to LD, ga1-3 only flowered when treated simultaneously with GA, and FT expression increased rapidly (<19.5 h) and dramatically (15-fold). In contrast, in the wild type in LD there was little requirement for GA for FT increase and flowering so its endogenous GA content was near to saturating. Despite this permissive role for endogenous GA in Columbia, RNA interference (RNAi) silencing of the GA biosynthesis gene, GA 20-OXIDASE2, revealed an additional, direct role for GA in LD. Flowering took twice as long after silencing the LD-regulated gene, GA 20-OXIDASE2. Such independent LD input by FT and GA reflects their non-sympatric expression (FT in the leaf blade and GA 20-OXIDASE2 in the petiole). Overall, FTacts as the main LD floral signal in Columbia and GA acts on flowering both via and independently of FT

Fig. 7. Summary of findings here and in the companion paper of positive effects (arrows) on flowering andCO/FT for two commonly used LD photoresponses. This schematic incorporates effects on FT and

flowering of: mutants; gene silencing; change in light intensity; and a block to photosynthesis. Predominantly, in LD, photosynthetic sucrose amplifies CO/FT expression (see companion paper) while phytochrome acts directly and also via GA, which plays a permissive and, often, non-limiting role. There is also a direct but lesser LD-mediated increase in GA supply via the petiole response to FRrich light. A dashed arrow indicates a potential step of regulation, and weaker responses are indicated by thinner arrows. The electronics symbol for a speaker is used to show sucrose amplification of CO/FT expression

GA4 applied to ga1-3 shows an FT-independent effect on flowering in SD and a permissive effect involving FTexpression in LD. A 10 l drop of GA4 [1 mM in 20% ethanol: water (v:v)] was applied to each of three leaves on consecutive days either in SD or at the start of a far-red-rich LD (LD-FR). Plants of ga1-3 flowered, bolted, and leaves grew (A). Its FT expression increased most after GA treatment in LD (B), and (C) shows the effect of GA 4 on FT expression in Columbia. Prior to treatment, the plants of ga1-3 had been grown in SD for 12 weeks and those of Columbia for 5 weeks. The low intensity FR-rich LD exposure was for 2 d. GA4 was applied 8 h after starting the day, and leaf blades were harvested 19.5 h later for assays of FT expression (leaves harvested at 16 h showed similar increases; not shown). There was no effect of solvent application on flowering or gene expression (not shown). All FT expression was normalized to the value in SD without GA application. The means and SE were based on three replicates for FT assays and 10 replicates for flowering time.

Intergrative pathway

In Arabidopsis, the four floral pathways converge through genes of the integrative pathway [7]. The activation of floral integrators, such as AtFLOWERING LOCUS T (AtFT) and AtSUPPRESSOR OF

CONSTANS 1(AtSOC1), in turn, lead to the activation of floral meristem identity genes such
as LEAFY (LFY) and APETALA1(AP1). We did not observe differential expression of TaFT (see above discussion of TaVRN3). On the other hand, transcripts for wheat genes that share sequence similarity with AtSOC1 did show differential expression. Zhaoet al. [38] identified seven MADS-box genes (TaAGL1, TaAGL7, TaAGL18, TaAGL20, TaAGL21, TaAGL23 andTaAGL38) that, upon phylogenetic analysis, were placed in the SOC1-like clade of MADS-box genes. Only three probe-sets on the wheat array corresponded with these seven genes (see Additional file 5). The gene TaAGL7corresponds with the probe set Ta.25343. The

genes TaAGL1, TaAGL18 and TaAGL23 (the most similar toAtSOC1) all correspond to one probe-set (Ta.21250), and TaAGL20, TaAGL21 and TaAGL38 (the least similar toAtSOC1) to another (TaAffx.19661) given the high sequence similarity between the genes in the respective groups, they are probably homoeologues and so we cannot report on their individual behavior. However, the three probe-sets that correspond to the genes of the SOC1 clade of MADS-box genes all evidenced essentially the same profile of abundance (data not shown). That is, in both leaf and crown tissue of all three varieties, transcript increased slightly (Figure 5).

Photoperiod pathway The principal components of the photoperiod pathway are conserved in the monocots and, more pertinent to this discussion, in the cereals On the Wheat Genome Array, there are probe-sets that correspond to many of the genes belonging to the photoperiod pathway. In Arabidopsis, AtCONSTANS (AtCO) encodes a transcription factor that activates genes required for floral initiation. It integrates circadian clock and day-length signals and, under long-days, activates the floral promoters AtFT, AtSOC1 and AtLFY [8]. The two circadian clock genesAtLHY and AtTOC1 influence the expression of AtCO. They form part of a feedback mechanism, each directly affecting the expression of the other: AtLhy.p is a repressor of AtTOC1, and AtToc1.p is required for the expression of AtLHY [8]. The cyclic expression of these two genes, which occurs over a 24 hour period, entrains that of AtGIGANTEA (AtGI). This latter activates AtCONSTANS. In this study, the profiles of abundance for TaLHY and TaTOC1 were complementary to each other, as one might expect from their relationship to each other in circadian cycling. In crown tissue, transcript of TaLHYincreased in abundance and then declined; conversely, that of TaTOC1 declined and later increased. The profile for TaGI was very similar to that of TaTOC1 (see Additional file 4). Given that in both rice and Arabidopsis, GI is a promoter of CO expression [8], one might have expected the transcript for HEADING DATE 1 (TaHD1), the supposed wheat orthologue of AtCONSTANS, to follow the profile of TaGI. However, it did not show differential expression in this study. Interestingly, other transcripts that appear to be members of theCONSTANS-like family of genes exhibited profiles that did reflect those of TaLHY, and TaTOC1, and TaGI. In particular, a sequence highly similar to barley CONSTANS-like 9 (the most divergent of the barley CONSTANS-

like genes which has no counterpart in Arabidopsis [35]) had a profile of abundance very similar to
that ofTaLHY (see Additional file 4). A transcript with similarity to CONSTANS-like 1 in Lolium

perenne, a gene which has been reported to increase after extended periods of exposure to
cold [36], had a profile of transcript abundance that echoed that of TaTOC1 and TaGI. Ciannamea et

al., using a similar experimental approach to that used in this study, suggested that the profile of
transcript abundance for LpCOL1 was suggestive of the gene being involved in the vernalisation response [36]. We observed a very similar profile of responses in both the cold treated plants and the controls. This would suggest that this gene in wheat is responding to shortening day length.

GA pathway genes The Affymetrix array doesn't include probe-sets for AtGA1 (codes for ent-copalyl diphosphate synthase) orAtGA INSENSITIVE (the wheat orthologue is REDUCED HEIGHT B1 [RHT B1]). There is a probe-set forAtRGA1 (the wheat orthologue of RHT D1), but we did not observe differential accumulation of this gene. However, there was a clear genotype-dependent, cold response of some components of the gibberellin pathway: transcripts for ent-kaurene synthase and ent-kaurene oxidase (correspond to AtGA2 and AtGA3, respectively), showed leaf specific accumulation (> 20-fold increase after 12 weeks) in the two winter varieties and no response at all in Paragon (Figure 4). This result was confirmed by qRT-PCR (Pearson correlation = 0.99). Ent-kaurene synthase and ent-kaurene oxidase are two of the principal enzymes of the gibberellin biosynthetic pathway [30,31]. Thus, given the profiles of abundance that we observed, one might assume that, in the two winter varieties, there was an increase in gibberellins. In Arabidopsis, gibberellic acid (GA) activates the expression of AtSOC1(SUPPRESSOR OF OVER EXPRESSION OF CO 1) [32], an important integrator of several flowering pathways (discussed below), which in turn promotes flowering through its action on floral meristem identity genes or their products (Komeda 2004). What's more, Moon et al. [32] report that the gibberellin pathway is the only pathway to promote flowering under short days. Thus, it would appear that we have evidence to show that in wheat the gibberellin pathway functions in a similar manner to that in Arabidopsis, and that as a consequence of vernalization under short-days it tends to promote flowering. However, the complete lack of response in the spring variety, Paragon, is intriguing: does the gibberellin pathway not function to promote flowering in spring varieties of wheat.

Vernalization pathway:
Prolonged exposure to low temperatures (vernalization) accelerates the transition to reproductive growth in many plant species, including the model plant Arabidopsis thaliana and the economically important cereal crops, wheat and barley. Vernalization-induced flowering is an epigenetic phenomenon. In Arabidopsis, stable down-regulation of FLOWERING LOCUS C (FLC) by vernalization is associated with changes in histone modifications atFLC chromatin. In cereals, the vernalization response is mediated by stable induction of the floral promoter VERNALIZATION1 (VRN1), which initiates reproductive development at the shoot apex. We show that in barley (Hordeum vulgare), repression of HvVRN1 before vernalization is associated with high levels of histone 3 lysine 27 trimethylation (H3K27me3) at HvVRN1 chromatin. Vernalization caused increased levels of histone 3 lysine 4 trimethylation (H3K4me3) and a loss of H3K27me3 at HvVRN1, suggesting that vernalization promotes an active chromatin state at VRN1. Levels of these histone modifications at 2 other flowering-time genes,VERNALIZATION2 and FLOWERING LOCUS T, were not altered by vernalization. Our study suggests that maintenance of an active chromatin state at VRN1 is likely to be the basis for epigenetic memory of vernalization in cereals. Thus, regulation of chromatin state is a feature of epigenetic memory of vernalization in Arabidopsis and the cereals; however, whereas vernalization-induced flowering in Arabidopsis is mediated by epigenetic regulation of the floral

repressor FLC, this phenomenon in cereals is mediated by epigenetic regulation of the floral activator, VRN1. Vernalization-induced flowering in cereals is associated with changes in histone methylation at theVERNALIZATION1 gene. 1. Contributed by W. James Peacock, April 1, 2009 (received for review March 17, 2009)

epigenetic
MADS

intron barley

chromatin

Plants respond to seasonal cues, such as temperature and day-length, to ensure that flowering coincides with favorable conditions. Prolonged exposure to low winter temperatures (vernalization) accelerates the progression from vegetative to reproductive growth in many plant species, including the temperate cereals (such as wheat and barley) and dicot species (such as Arabidopsis) (13). In both these lineages, plants retain a memory of the prolonged cold of winter, which stimulates flowering when days lengthen during spring (13). The memory of cold is then reset in the next sexual generation to ensure progeny are competent to respond to vernalization (13). In Arabidopsis, the vernalization response is mediated by epigenetic regulation of the floral repressor,FLOWERING LOCUS C (FLC), which encodes a MADS-box transcription factor that represses genes involved in floral initiation, including SUPPRESSOR

OF

CONSTANS

1 and FLOWERING LOCUS T (FT) (1, 46). FLC is expressed before vernalization and delays
flowering, but its expression is repressed by vernalization (1, 4). FLCremains repressed when plants are subsequently exposed to warm temperatures, allowing activation of FT, which promotes flowering (1, 4). The stable down-regulation of FLC by vernalization is associated with an increase in the levels of repressive histone modifications at FLC chromatin, such as histone H3 lysine 27 di- and trimethylation (H3K27me2, H3K27me3), histone H3 lysine 9 dimethylation, and histone H4 arginine 3 symmetrical dimethylation, as well as the loss of histone modifications associated with active transcription, such as histone H3 acetylation and histone H3 lysine 4 di- and trimethylation (H3K4me2, H3K4me3) (713). Repression of FLC by vernalization involves the vernalization-dependent association of Polycomb-Group (PcG) complexes to FLC chromatin, which are required for addition and maintenance of H3K27me3 at FLC (14, 15). Taken together, these studies indicate that vernalization induces an alteration of FLC chromatin state from actively transcribed to stably repressed (715). The cellular memory of transcriptional repression of FLC is maintained during successive cell divisions by mitotic inheritance of repressive histone modifications at the gene (11), but

active FLC transcription is restored in progeny, ensuring that the next generation is competent to respond to vernalization (1, 4, 16). In temperate cereals, the vernalization response is mediated by the stable induction of a floral promoter,VERNALIZATION1 (VRN1) (3, 1719). VRN1 encodes a FRUITFULL-like MADS-box transcription factor required for the initiation of reproductive development at the shoot apex (20 22). In vernalization-requiring cereal plants, VRN1 is expressed at low levels and is induced by vernalization, with the level of expression being dependent on the length of cold exposure (1719, 23 25). VRN1 expression remains high when plants are exposed to warm temperatures following vernalization, and promotes the transition to reproductive development (1719,2325). VRN1 downregulates the floral repressor VERNALIZATION2 (VRN2), and allows long-day induction of the floral activator FT to accelerate subsequent stages of floral development (3, 2426). The vernalization response of VRN1 shows characteristics of epigenetic regulation, in that VRN1 is induced by vernalization, expression is maintained following vernalization, and the prevernalization level of VRN1 expression is reset in the next generation (1719, 2325). In this article we analyze the effect of vernalization on the levels of histone modifications at the barley (Hordeum

vulgare) VRN1 gene (HvVRN1). Our study indicates that vernalization-induced flowering in cereals is
mediated by epigenetic regulation of VRN1 chromatin state. Our results suggest that regulation of the histone methylation status of VRN1 chromatin is important for repression of VRN1 before vernalization, for activation of VRN1 by vernalization, and for maintaining a memory of vernalization following cold exposure

Flowering time genes in Arab

Co induced FT with FD and soc1 activate genes like LFY and AP1 in SAM; they take their own course of development of structures.

The monocot Rice or dicot Arabidopsis have their homologs that operate in inducing flowering.

Environmental signals that favor GA synthesis leads to the bonding of GA to its receptor and interact with their binding proteins, where the regulator of GA called RGA gets degraded through proteosomes. Now the GA signals SOC1/PFP/GAMYB like components that lead to the production FT and FT with FD acts on apical meristem

This figure shows how the GA synthesis leads activation of different response factors that leads to the synthesis of SOC1 and FT, where FT acts on AP1 (?) and SOC1 acts on LFY (?) which ultimately act on flower identity genes.

Below some general and specific flowering pathways have been given and they are self illustrative.

Integration of different pathways

The role of GA in stem elongation seed germination and flower development.

The effect of combinatorial expression of gene in the presence and absence of VRN on phenotype is wells illustrated. The figure below shows even Rice palnts produce a FT homolog called Hd3a and it has the same effect as FT of dicots.

Ft homolog Hd3a operates in rice plants, they move from leaves to stem apex, Hd3a in combination with FD (?) induce flowering

Integration of different pathways on meristem identity genes

MADS box proteins action on floral development

MADS MADS protein domains

Hour glass model showing the effects of PhyB and Phy A on CO synthesis and degradation. Co is transcription factor for the expression of FT.

PHYSIOLOGY OF PLANT MOVEMENTS Higher plants, being fixed to soil cannot move from place to place. But within the plant body various protoplasmic components are in constant motion, ex., movement of water, minerals, food etc. But certain parts of the plant body in response to external stimuli, exhibit physical displacement called movement. Lower unicellular plants also show movement from place to place. Such movements may be autonomic or induced.

The movement of plant structures in response to stimuli is very interesting. The stimuli may be in the form of light, touch, chemicals, temperature, gravity or water. Thus the agency or factor that causes movement is called stimulus. Not all parts of the plant act as the site of perception to stimuli. Only certain regions or structures are capable of receiving the stimuli and such structures or organs are called perception site. The site of perception need not be the structure that responds to movement. In fact, in many cases the site of response and the site of perception are different. Nonetheless, the stimulus has to cross through the plasma membrane of the cell or cells found in such structures that receives the stimulus. Plasma membrane has all the inbuilt components and the potentiality to receive stimulus and transmit it into intracellular mileu for proper response. There is a time lag between the time at which the stimulus is applied and the time at which the response begins. This time is called reaction time. It may vary and depends upon the intensity of stimulus and the kind of response. If the stimulus is weak, there may not be any response at all, but if the stimulus is adequate or in right quantity the response is positive. The time required to cause the proper stimulus is called presentation time. Once the plant body responds to the stimulus say sleeping movement (change in the position direction), structures involves always come back to their original position. This process is called recovery and the time required is referred to as relaxation time, which again varies from species to species. If the stimulus is provided repeatedly the receiver structures do not respond with the same intensity as the first instance, but it slows down. This effect is due to fatigue. Such behavior is probably due to the loss of same material components required for the response. If the stimulation is continued at increased frequency, the plant organs do not respond and behave as if they are dead structures. Such a state is called Tetanus or extreme fatigue. Now it is believed that stimulation (quantity) causes irritation at the site of perception and the product of irritation are then transported to the site of response, where the structures respond and perform movement. All these events initiate with signal, and the receptor that receives the signal becomes active and induces signal transduction pathway in the cell cells. There will be a cascade of events that finally leads to the response. Plants endowed with different structural adaptation and different potentialities exhibit different types of movement either voluntarily or involuntarily. Based on the behavior as pattern, movements stimulus, the plant movements have been grouped into physical movements and vital movements.

Physical Movements: The structures involved in this type of movement are mostly dead. Xerochasy: Structures like the wall of fruits, sporangia, capsules, have differential wall thickenings. During dry weather conditions, they loose water to atmosphere. Because of this, the thick walls contract as a result they break open along with the line of dehiscence, where the cell walls are thin and susceptible. Hydrochasy:Certain structures, made up of hydrophilic substances, are capable of imbibing water as well. Due to imbibition of water they swell. Due to imbibition of water peristomial teeth in Moss capsules, elators in equisetum, etc. show movements. In fact such movements help in the dispersal of spores. Vital Movements: Plant movements due to the activity of living structures are called vital movements. They are further classified into different kinds. In some plants, particularly unicellular algae, the entire cell moves from place to place or from one position to the other or the protoplasm by itself shows continuous flux by physical displacement. In others, where the plant body is fixed in the soil, certain structures show bending or curvature movements. Furthermore, some of the movements are auto regulated and propelled by innate mechanisms but other movements are induced by stimuli. In this case the entire living cell is involved either in the movement of protoplasm or the entire body of the plant cell from one place to another. These movements may be autonomous and induced. Autonomous: Protoplasmic Streaming: Protoplasm is not a static fluid. With all its complicated structures the entire protoplasmic fluid is in constant sweeping motions. These streaming movements can be observed under high resolution light microscopes. For example, in the cells of staminal hairs of tradescantia, the streaming is localized and in each of these areas the direction is clockwise or anticlockwise. Tiny particulates are seen swept along with the stream in a particular direction and particular directed path. Such compartmentalized movements are known as Cyclosis. But in Elodea and other plants, the protoplasm shows uniform movement but in one direction. One can observe chloroplasts movement along the cytoplasmic streaming. Such movements are called Rotational Movements. Protoplasmic streaming is due to the activity of contractile proteins found associated with other microtubule network found within the cytoplasm. It is an active process microtubules play an important role in such intra cellular movements. These structural elements contain motor proteins and they perform the movements. The energy required for this process is derived from ATP molecules. These movements help in the even distribution of chemical components. And the protoplasmic movement across the plasmodesmata brings about the transportation of materials from one cell to another. Auxin has been found to accelerate the rate of protoplasmic

movements. Addition of colchicine and cytochalasin B totally inhibits the movement, thereby indicating the involvement of microtubules and microfilaments. Respiratory poisons like DPN, KCN, also inhibit the movement, thus suggesting that these are active movements. Even amoebae show such movements. For that matter all living cells exhibit autonomous movements. Paratonic movements: These movements are stimulated by external agents like light, chemical heat, etc. Hence they are called taxis or tactic

movements. Phototactic Movement: Unicellular algae suspended in a test tube moves towards the light source to obtain solar energy for photosynthesis. If the light is very intense, they move away from light; this may be due to the raise in temperature. The movement in these cases is due to locomotor structures like flagella or cilia. The beating of these structures propels the cells towards light. The flagellar activity utilizes ATP. Hence these movements are active.

Chemotactic Movement; Certain flagellated or non-flagellated bacteria move towards the source of food by lashing their flagella or by tumbling. In these cases, the stimulus is chemicals. Similarly, the movement of spermatozoids in lower organisms like Bryophytes is directional, because the chemicals released by mature archegonia provide the chemo stimulus. Sensing the chemicals, the flagellated spermatozoids swim towards archegonia and enter the neck canal and bring about fertilization. Thermotactic: Again lower organisms sense the temperature and move towards the compatible temperature or move away if the temperature is incompatible. Sensing the change in the temperature by the plants is autonomic but the cells always show directional movements. All the above mentioned movements involve signal transduction pathway. Elucidation of these pathways at molecular level is necessary and exciting. Movements of Curvature: Plants with their fixed plant body cannot move from place to place, but certain structures show bending movements which may be directional or non-directional or they may be autonomous or induced. However, some of the plant movements are due to the growth of the cells or due to change in the turgidity of the cells. Based on the above features movement of curvature has been further classified such as:

Autonomic Growth Movements: Natatory; Plant structures like tendrils and stem tips exhibit differential growth alternately. This results either in sideward or circular movements. Certain sub cylindrical stems (flat or angular) due to differential growth at the sides show zig zag movement. On the other hand, cylindrical tendrils in search of getting a hold on to a substratum, a waves of growth activity takes place around the tendril. That is why it appears as if it is moving in circles. Such type of movements is called circumnutatory movement. The growth activity of the above said structures is in-built and they show a rhythmic pattern. Whether the hormonal fluxes are responsible for this type of growth movement or any other innate mechanism involved is not known. But the treatment of these stems with ABA inhibits the movements, indicating that these are phytohormone mediated. Ephemeral Movements: During the development of leaves and floral organs, the growth pattern determines the growth direction of the structures. For example, leaves expand laterally by continuous radial divisions and expansions. Similarly sepals and petals because of continuous growth of cells at the base on the inner surface make the flower open. The hypocotyl hook of the bean seed straightens up because of one sided growth due to expansion cells on that side. Such growth movements are called Ephemeral. Once the movement reaches certain stage, the bending movement stops.

Autonomic Turgour Movement: Desmodium gyrans (Indian telegraphic plant) and Eleiotis sorria have trifoliate compound leaves. In the former case, central leaflet is larger and straight. But the lateral two leaflets are smaller which show regular upward and downward movements. Such movements are observed only during daytimes but not at night. Such rhythmic gyratory movements require about 5-8 minutes for the completion of one cycle. This is due to autonomic turgour changes in the cells found in the swollen pulvinus of the leaflets. How light brings about turgour changes or is it due to phytochrome mediated responses or hormonal responses is not clear.

Paratonic Nastic Movements: Paratonic movements are induced by external stimuli. Whatever may be the point or direction of stimulus applied the movement of the plant structures is already predetermined and they exhibit movement only in one direction. Most of the movements are turgour movements, but growth movements are not uncommon. For example, the opening of hypocotyl hook of germinating bean seedling, opening of circinately coiled leaves of ferns and cycas are the examples of growth mediated nastic movements. Differential distribution of growth hormone in the adaxial (dorsal) surfaces of the leaves; it is this that is mainly responsible for such growth movements. These movements are permanent and they do not show temporary day and night fluctuations. Photonasty: Many leguminous plants, with their pulvinous bases, show characteristic sleeping

movements and they show a rhythmic pattern of opening of leaf-lets in the day and closing in the evening with a precision of a clock. In fact, such movements are attributed to circadian rhythm operated by an inbuilt biological clock.

The photonastic mechanism has been explained on the basis if hormonal distribution in the cells of the pulvinus. During daytimes, auxin is found in greater amount at the upper region of the pulvinus, because of this the cells become more turgid and leaf lets open. The same process is reversed during night because of the redistribution of auxin to lower side which causes folding by the way of turgour changes. Investigations into such movements have shown that these are phytochrome mediated, because red and far red lights are very effective in opening and folding of leaflets. Phytochrome being an excitable molecule in response to light it is known to bring about changes in the permeability of membranes. Thus the turgour movements bring about so called sleeping movements. Added to this the change in the permeability also involves the efflux and influx of K+ ions. The loading or unloading of potassium ions causes increased or decreased DPD which acts as the driving force for the entry or the exit of water which results is turgour movements. Chemonasty: Drosera, Venus fly trap and such insectivorous plants have devised mechanisms to trap insects to obtain nitrogenous compounds. These plants are found growing in boggy areas and they are incapable of utilizing soil nitrogen.

The plant Drosera consists of leaves arranged in a rosette pattern. The leaf blades are flat and racket shaped. The upper surface is made up of colorful glands which secrete sticky juice, where you very high activity of golgi complex. There are a large number of sensitive tentacles spread out at the margins. The tentacles have a sensitive broad base and a terminal glandular globosely head which also secretes juice. In sunlight, these structures glisten like dew drops; hence the name sundew plant.

Insects, mistaking the glistening juice for honey, settle down on these surfaces where their legs get stuck because of the sticky juice and they cannot escape. As insects have nitrogenous compounds in their body, they diffuse and sensitize the tentacles, because nitrogen compounds act as signals, in response, they immediately fold upon the insect and the same gets trapped. In this case nitrogenous compounds found in insects provide the stimulus. On the other hand, if a metal piece is placed, tentacles do not fold, instead if a piece of meat is dropped, the tentacles close immediately. However, after digestions, tentacles open slowly and this process takes few hours. Such chemonastic movements are found in utricularia, Venus fly trap, etc.

Thermonasty: Flowers in Tulips and Crocus are very sensitive to temperature variations. Accordingly the accessory whorls close or open. These movements are known to be due to turgour changes rather than growth movements. Siesmonasty: Mimosa pudica (Touch me not plant), Biophytum, Neptunia oleracea, Desmanthus are very sensitive to touch, rather shock generated by the touch. Mimosa pudica has pinnately compound leafless with a swollen pulvinus at the base every leaflet. Touching the leaves is believed to cause a seismic shock to the leaves and this stimulus is transmitted all along the rachis downwards and reaches the basal pulvinus of the leaf. The irritability caused, due to touch is actually transmitted through sieve tubes, because these are the only structures which are capable of transmitting the stimulus as fast as 1.5-20 cm/sec. The material basis for the transpiration stimulus has been found to be ABA and ABA mediated ions. As the ABA ions diffuse along the rachis, it also trans-diffuses into pulvinus of the leaflets.

The mechanism of upward movement of pinnules and downward bending of the entire leaf at the base is now considered as due to the activity of the motor cells found in the pulvinus. Anatomical studies indicate that at the lower region the pulvinus consist of thin walled parenchymatous cells, which are loosely arranged with lot of intercellular spaces. Cells also contain many contractile vacuoles. The

cell membranes and vacuolar structures have contractile proteins. These vacuoles are loaded with K+ ions when the leaflets are open. These motor cells are highly sensitive and active. Such cellular organization is also found at the upper region of the pulvinus found at the base of the leaflets. When the stimulatory hormone ABA ,which is also called as stress hormone (released due to irritability) reaches these specialized cells, they are stimulated to contract, collapse and extrude water and K+ ions into intercellular spaces. This action brings about the collapsing of cells on this side and the leaf downwards. Similarly mechanism is involved in the folding of leaflets upwards. The recovery takes place within 5-10 minutes. The opening process takes place by active pumping in of K+ ions back into cells. The K/ATPase pumps are supposed to be found in the cell membranes. This active process requires energy. If the production of ATP is inhibited by respiratory inhibitors like DNP or KCN recovery does not take place. If plants are kept is continuous dark, they fail to produce any siesmonastic responses, which suggest the siesmonastic movements are ATP dependent active processes. Paratonic Tropic Movements: These movements are due to growth activity. The curvature movement is always directional, either towards the stimulus or away from the stimulus. Most of these are phytohormone mediated movements. Phototropism: Phototropic movement is mostly exhibited by stem tips; for they always bend towards the light source. The action spectrum of the light has been found to be at blue light. The blue light is now known to bring about unequal distribution of active auxins than the total auxin content. The pigment for absorption of blue light has been found to be Cryptochrome. The older view of destruction of auxin and the lateral movements of auxins in response to light has been more or less ruled out. The difference in the quantity of active auxin brings about differential growth and also curvature. Details of this process have been explained elsewhere.

Photropic movement in in one of the fungi called Phycomycs blacesleeanus The apoprotein cry in association with flavins bring about photoptropism, it gets activated by absorbing blue light and autophosphorylation of ser/thr residues, that leads to transport of ions to one side and brings about bending movement.

Geotropism: Growth of roots towards soil and the stem away from the soil in response to gravitational force is an inbuilt mechanism endowed in stem tip and root tip of the plant. Curvature movement of roots towards gravitational is termed as positive geotropism and the movement of the stem away or against gravitational force is called negative geotropism. However in some plants, rhizomes and runners grow parallel to the surface of the soil. Auxin). The differential response of stem tip and root tip to the same gravitational force is due to their different innate structural and functional potentials. Though both structures are derived from the same embryonic cell and possess the same genetic potential they behave differently probably this is Such a response is called digeotropism. The obliquely growing stems show plagio geotropism (For details refer the Chapter

because of the programming of process driving the development of in such structures. The mechanism of geotropism has been explained on the assumption that differential responses are due to different concentrations of auxins. The concentration of auxin that promotes the growth of the stem apex inhibits the growth of the root tip. On the other hand, the concentration of auxin that is effective in the growth of the root tip is not adequate for the growth of the stem tip. This concept suggests that stem apex requires higher concentration of auxins for its growth and roots require low levels of auxins as optimal concentration for the normal growth. If a seedling is placed on the soil horizontally, due to mass action of gravitational force, auxins move downwards in both stem apex and root apex. In the stem apex, as more and more of auxin accumulates at the lower surfaces the cells found in this region grow faster than the upper cells, thus the stem curls upwards. But in roots, as more of auxin accumulates at the lower region, the higher concentration inhibits the growth of cells found at the lower surface, but the cells at upper surface grow faster because of lower concentration of auxin. Furthermore, the root cap being the preceptor of gravitaropism produces ABA, which on translocation basipetally, reaches the lower cells by sheer gravity. With the accumulation of more and more of ABA at lower cells, the growth of these cells is further inhibited, but the cells at the upper region grow normally and thus bring about the downward growth curvature for roots. Chemotropism: Pollen tubes and certain fungal hyphae exhibit chemotropic movements, because they grow towards organic nutrient rich media. In the case of pollen tubes, the direction of growth is dictated by the chemical gradient generated by the embryo sac. This chemical gradient greatly facilitates the growth movement of pollen tube towards the embryo sac, irrespective of the position of ovules in the ovary. Hydrotropism: Growth of roots towards water is called hydrotropism. Whatever may be the positions of seedlings, as shown in the figure, the roots curl towards water. This positive growth curvature might be due to greater water potential, probably acts as the motive force for the growth of root tip. Stem and other structures are insensitive to water and they do not show any hydrotropic curvature movements. Thigmotropism: Plants with weaker stem spread around and require support for their growth. Many of the climbers have developed hooks, pads and tendrils for getting a firm foot hold onto the substrate; which may be a rock or a wooden stick or any such hard structure. For example, in the case of tendrils of cucurbita and other species the terminal regions are soft, tender. The terminal region of the tendrils consists of a number of fine pits, which are highly sensitive to touch. When the tendril comes in contact with any supporting structures, the pits get stimulated and the same is transmitted a few millimeters basally. This results in the differential growth of the tendril on one side. This causes the curvature in the tendrils and finally they coil around the stem the supporting structure. The time required for such coiling after the stimulus is just 3 to 5 minutes. Once the initial reaction is set in at the basal region, the tendrils coil round the support and thus draw the climber nearer to the support. It is suspected that auxins and ABA are involved in thigmotropic

curvatures. ABA inhibits the growth of the cells in the region of the contact and auxins stimulate the growth of the cells on the opposite side and bring about eh growth curvature called coiling.