Lecture notes for Plant Biology and Physiology (AGRO 101 / NRM 102) Prepared by Chandiposha M.

PHOTOSYNTHESIS
Primary source in crop production (organic compounds) from light energy and CO2. The products of photosynthesis are Carbohydrates, Lipids etc which makes up the total biomass yield. Leaves are functional units for photosynthesis; there facilitate the movement or diffusion of CO2 to sites of fixation. The process of diffusion of CO2 from outside to fixation sites is the limiting factor of photosynthesis (CO2 Assimilation rates). Photosynthesis is measured in 2 ways: a) Net CO2 intake / CER using Infra- red gas analyser. b) Dry weight increment per unit time per unit leaf area. Use growth analysis techniques. Photosynthesis is expressed as mg CO2 per leaf area per time or biomass per area per time. Canopy photosynthesis is affected a lot by plant geometry (Plant spacing, architecture) which influences radiation interception, extinction coefficient and proportion of incident radiation intercepted by a canopy. Full ground cover intercept 95% radiation Agronomic practices influence the amount of radiation intercepted

Leaf Photosynthesis
There are 2 processes 1) Light reaction Energy input comes from terrestial radiation. This energy input provides excitation energy which allows electrons to move against REDOX potential gradient. The electron movement in the light reactions according to ChemiOsmostic theory creates Hydrogen ion gradient. When this Hydrogen ion gradient is dissipated or equalizes it leads to ADP +P ATP, this is called photo-phosphorylation.

There is a protein called coupling factor (CF) on the thylakoid protein through which the Hydrogen ion gradient is dissipated to result in ATP formation. The terminal step of the electron transfer system is NADP + eH+

NADPH (Reduction process)

Thus light reaction result in ATP and reductant NADPH

2) Dark Reaction The ATP from light reaction provides the bond energy to connect C-C, C-O, C-H. NADPH2 is the reductant, provide the H and electrons in the manufacture of organic compounds. Other reduction reactions in the plant such as S reduction and Nitrate reduction requires electrons which come from photosynthesis e.g. NO 3- + eNO2NH4+ During excitation, the electrons produced are not all used photosynthesis but some electrons are released as inflorescence. by

Calvin Cycle
1) CO2 react with D-Ribulose 1,5 diphosphate to form 2 molecules of D-3-Phosphoglyceric acid (3PGA) under the influence of the enzyme 1,5 Biphosphate Carboylase Oxygenase (RUDP carboxylase). This is a complex reaction in which CO2 is added to a 5 C sugar to form a 6 Carbon intermediate which then splits into two 3C sugars. Note that CO2 is the substrate for carboxylation reaction and not HCO3-. The enzyme RUDP carboxylase requires Mg2+ in order to catalyse the carboxylation reaction. There evidence that the initial step of the mechanism is the formation of an enzyme-CO2-Mg2+ complex. Illumination increases Mg2+ concentration and raises pH (movement of H+ into cell).

RUDP carboxylase has another catalytic activity. It can behave as RUDP oxygenase catalysing the cleavage of RUDP in the presence of O2 into 3PGA and Phosphoglycolate in Photo-respiration. The carboxylase activity of RUDP carboxylase is competitively inhibited by O2. Similarly the oxygenase activity is competitively inhibited by CO2. Thus the relative rate of the 2 reactions in vivo will be dependent upon concentration of CO2 and O2 in the chloroplast stroma.

2) The 3 PGA is phosphorylated to give 1,3 diphosphoglyceric acid and ATP generated in the light reaction is the phosphorylating agent. Phosphoglycerate kinase catalyses this reaction that is freely reversible. During daylight, the continuous regeneration of ATP drives the reaction in the direction of 1,3 diphosphoglyceric acid.

3) The 1,3 diPGA is now reduced to 3-Phosphoglyceraldehyde or 3PGAld by NADPH produced in the light phase of photosynthesis. 4) The 3PGAld is used to make 5C, 6C and 7C sugars NB: Pi is required to move TPs out of the cell. Pi deficiency cause starch accumulation in the cells The 3PGAld can be used in the Calvin cycle in 4 different ways.

a) It is isomerized to Dihydroxyacetone phosphate (DiHOAcP) or Triose phosphate catalysed by triose phosphate isomerase. b) The 3PGAld condenses with Dihydroxyacetone phosphate to form Dfructose I,6 diphosphate (F 1,6 diP). The reaction is catalysed by fructose diphosphate aldose. The F 1,6 diP is dephosphorylated when added to water to produce Dfructose-6-phosphate and the reaction requires Mg2+. Also the enzyme responsible for this is fructose biphosphatase.

c) 3PGAld can react with equivalent amount of F6P previously formed to produce equal amounts of D-Xylulose-5-phosphate (Xu-5-P) and DErythrose-4-phosphate (E-4-P). The reaction is catalysed by fructose transkictolose and requires thiamine pyrophosphate and Mg for activity. The E-4-P formed then reacts with an equal amount of Dihydroxyacetone phosphate to form D-Sedoheptulose 1,7 iphosphate (Su 1,7 diP). The reaction is catalysed by fructose diphosphatase.

The Su 1,7 diP is then dephosphorylated to Su-7-P using the enzyme sedoheptulose biphosphatase.

d) The Su-7-P then react with equal amount of 3PGAld to form equimolar amounts of Xu-5-phosphate and D-ribose-5-phosphate catalysed by transketolase.

5) Regeneration of RUDP The D xylulose-5-phoshate generated in the last reaction undergo epimerization in C3 catalysed by ribulose phosphate 3 epimerase to form D ribulose-5-Phosphate (Ru-5-P). The Ru-5-P formed is phosphorylated at the expense of ATP producing Ru1-5-diP.

Summary of Calvin Cycle (Dark reactions)

CO2

Ribulose 1,5 Biphosphate Phosphoglyceric acid

RUBISCO

NADPH + ATP

ATP

Fixed Carbon Product Triose Phosphate

5C 6C 7C Sugar Phosphates

To produce one molecule of a hexose (6C) sugar, 6 molecules of CO 2 are incorporated into the Calvin cycle by ribulose biphosphate carboxylase at the expense of 12 molecules of NADPH and 18 molecules of ATP.

qqTherefore for every molecule of CO2 utilized, 2 molecules of NADPH and 3 Molecules of ATP are required. The stoichiometry does not take into account the effect of photorespiration which causes loss of fixed C in C3 spp, loss is between 20 40% and net numbers of NADPH and ATP required to fix one CO2 molecule are much higher than 2 and 3 respectively.

C4 Photosynthesis Leaf morphology / compartmentalization of functions C3 and C4 plants have a wide range and overlapping stomatal concentrations 5000 30000 stomata / cm2 depending on species. Stomata of C4 have higher resistance to CO2 diffusion than C3, ecologically it is thought that they developed a system of concentrating CO2 around RUBISCO to overcome this problem. The higher stomatal resistance to gaseous movement in C4 plants reduce water loss from the plant through transpiration, saving water and allowing C4 plants to grow in drier, hotter environments than C3 plants. Leaves of C4 species characterized by an extensive networks of air spaces which allow air to bath in a high proportion of photosynthetic cells; few photosynthetic cells are more than 2 3cell layers away from air.

Summary of the path of C in C4 Photosynthesis

Mesophyll cells sheath cells

Bundle

CO2 RUBP

C 4 (OAA)

C4

RUBISCO

CO2

Calvin Cycle

C3 (PEP) 3 PGA

C3 F6P

PEP Phosphoenol pyruvate (C3) OAA Oxaloacetate (C4)

NB: C4 substance decaboxylated can be i) Malate-the enzyme used in decarboxylation is NADP malic enzyme ii) OAA the enzyme used in decarboxylation is PEP carboxykinase iii) NAD the enzyme used in decarboxylation is malic enzyme.

RUBISCO is confined in the bundle sheath cells of C4 and absent in mesophyll cells while in C3 species it is found in mesophyll cells. PEP carboxylase is absent in the C3 species. C4 species have a Krantz anatomy meaning they have two types of cells, Mesophyll cells which surround bundle sheath cells. Mesophyll cells contain PEP carboxylase that capture CO 2 and combine it with a 3 C compound to form a 4 C compound hence C4. Bundle shealth cells contain RUBISCO that intiates the normal Calvin Cycle of C3 photosynthesis on receiving CO2 from the decarboxylated C4 compound.

Differences in C4 species arise in the way the C4 dicarboxylic acid is decaboxylated in the bundle sheath cells. 1) Zea mays, Saccharum officinarum, Sorghum sudanense use NADP malic enzyme. 2) Panicum maximum, Chloris Guyana and Sporobolus fimbriatus use PEP carboxykinase (Very efficient). 3) Atriplex spongiosa, Portulaca oleracea, Amaranthus edulis use NAD malic enzyme.

Chloroplasts are present in both mesophlly and bundle sheath cells but markedly different in appearance. The difference is most marked in advanced C4 species (Panicoid grasses) where bundle sheath chloroplasts have no grana or grane that are reduce in size.

Reasons why C4 species are more efficient that C3 species are: 1) Source to sink distance is small in C4 plant species. 2) Due to concentration of CO2 to RUBISCO in C4 species this causes elimination of O2. 3) Also the bundle sheath cells are found in deep tissues of C4 species where O2 is difficult to move.

PEP Carboxylase / CO2 Compensation point Has a high affinity for CO2. KmCO2 for RuBP carboxylase is 11 18 um KmCO2 while for PEP carboxylase is 0.02 um. The carboxylation for PEP form a C4 compound (OAA) is therefore very rapid and can occur at very low CO2 concentration. This accounts for the low CO2 compensation point for C4 species versus C3 species. The CO2 compensation point is the CO2 concentration at which respiration and photosynthesis are equal, therefore net photosynthesis is zero. Any increase in CO2 concentration above its CO2 compensation point results in

an increase on net photosynthesis or an increase in dry matter in the plant. C4 plant species begin net dry matter production at very low CO2 concentration because of high affinity for CO2 by PEP carboxylase, they concentrate CO2 around RUBISCO in the bundle sheath cells.

Stoichemistry of C4 Photosynthesis The operation of the Calvin cycle is similar for both C3 and C4 species. Thus the stoichemistry is the same and 3 ATP and 2 NADPH are required for the fixation of each CO2 molecule. In the C4 species, an additional 2 ATPs are required for the conversion of pyruvic acid to PEP in the mesophyll cells. Therefore the C4 mode of photosynthesis requires 5 ATP and 2 NADPH for the fixation of each CO2. From this regard, there is therefore an extra cost in C4 species expended in the mesophyll to regenerate the CO2 acceptor, PEP. C4 plants can be said to be less efficient in C fixation. This conclusion is wrong because no account has been taken of photorespiration. Photorespiration is non-existent or very low in C4 plants, making them more efficient despite having extra energy requirements for CO2 capture.

CAM (Crassulacean Acid Metabolism) Plants that exhibit CAM are succulents. Many are dicotyledons, Aizoaceae, Cactaceae, Portulaceae, Chenopodiaceae, Euphorbiaceae and monocotyledons, Liliaceae and Orchidaceae.

CAM plants exhibit the following characteristics: i) Normally their stomata open during the night (i.e. during the dark and closed during the day). ii) CO2 is fixed during the hours of darkness in chloroplast containing cells of photosynthetic

tissue of the leaf or stems and considerable quantities of L-malic acid synthesized. iii) The malic acid is stored in the vacuole, CAM plants have characteristically large vacuoles in their photosynthetic cells. iv) During the following daylight this malic acid is decarboxylated by NADP malic enzyme and the resulting CO2 enters the Calvin cycle which is light driven. It results in the manufacture of sucrose, amylopectin and storage glucan. v) During the next period of darkness, some of the stored glucan present in the cell is catabolized to provide an acceptor molecule for the dark CO2 fixation reaction (PEP).

The CAM plants use an initial C4 cycle to capture CO2

PEP

CO2

PEP Carboxylase

OAA

CAM plants also benefit from the high affinity of PEP carboxylase for CO2. The decarboxylation of malic acid is achieved by NADP malic enzyme. CAM is an adaptation that allows plants to survive and grow in extremely arid environments. Not only does the stomatal control rely on illumination (Light Darkness) but on water supply to the plant, day and night temperature, thermoperiod, and photoperiod. There is also evidence that are 2 types of CAM plants:

1) Obiligate or Constitutive CAM plants e.g. Cacti. 2) Faculatative or crystallinium. Inducible CAM plants e.g Mesembryathemum

Photorespiration It is CO2 efflux from the plant on exposure to light. CO2 efflux over and above that which was attributable to normal respiration. It is associated with C3 species, it is negligible or non-existent in C4 species. It increases with the concentration of O2. The higher the O2 concentration, the greater the photo respiratory loss of CO2 in plants (Warbug effect). Photorespiration is responsible for reducing net photosynthesis by between 20 45% in C3 species at normal atmospheric O2 condition (21% O2) C3 plants will grow fast under pollution conditions. 1 O2 is captured for every 4CO2 by RUBISCO

O2 + RuBP (5C)

3PGA (3C)

Phosphoglycolate (2C)

CO2

+ RuBP (5C)

2 x 3PGA (3C)

Photoperiodism and Vernalisation

Flowering is an important phase of lifecycle because the transition from vegetative growth to reproductive phase involves several changes in physiology of a plant. The physiological mechanism responsible for flowering has been found to be controlled by 1) Periodicity of light (photoperiodism) 2) Temperature (vernalisation)

Photoperiodism The phenomenon in which the influence of day length on plants is studied is called photoperiodism. It may also be defined as the response of plants to the relative length of day and night. On the basis of the length of photoperiod requirements, they have classified plants into: 1) Short-day plants 2) Long-day plants 3) Day-neutral plants

1) Short day plants For short-day plants to flower, the day length must not exceed a certain critical value In other words, the day length is less than a certain critical length. Short-day plants may be more correctly called long night plants as a certain minimum of uninterrupted dark period in 24 hours is necessary for their flowering. If the dark period is less than the critical length, flowering will not occur. Short day plants will not flower even if a flash of light or weak light is provided during continuous dark period. However, the light interruption is nor very effective if it is nearing the beginning or end of the dark period. These plants will not flower if short dark and short light are provided alternatively.

Research has shown photosynthesis.

that

these

plants

require

light

only

for

Some examples of short day plants are soybean, potato, sugarcane, chrysanthemum, tobacco (Maryland mammoth), rice, datura, onion, upland cotton, hemp and strawberry e.t.c.

2) Long-day plants Long day plants require photoperiod of more than a critical length which may vary from 14 to 18 hours. The best flowering of long day plants usually occurs in continuous light. Long day plants require either no dark period or very short dark period. Research has shown that a flash of light given to long day plants can induce flowering in them even during short day periods. The long day plants can flower even in short day periods if these short day periods are accompanied with still shorter dark periods. The flowering in long day plants is inhibited not because of short light periods but because of the too long dark periods and because of this, long day plants can also be called short-night plants. Examples of long day plants are spinach, lettuce, radish, alfalfa, sugar beet, oats, wheat etc.

3) Day neutral plants Examples of day neutral plants are tomato, cucumber, cotton, sunflower, maize. Pea, dandelion. Their flowering is not affected by the length of the day. They can flower even if the light period provided is from few hours to continuous illumination..

Photoperiodic induction Research has shown that continued favourable photoperiod till flowering is not essential but a short and appropriate photoperiod exposure is required for the production of flowers.

This kind of photoperiod persists even when the plant is subjected to unfavourable photoperiods or conditions. This phenomenon of producing photoperiodic influence is known as photoperiodic induction or photo-induction. Dark interruption of the day had little or no effect but night interruption by light inhibited flowering in short day plants and promoted flowering in long day plants.

Site of Photo-inductive perception Researchers confirmed that photo-induction is perceived by leaves. The sensitivity of perceiving influence increases with the growth of the leaf till full expansion and decreases in the older leaves.

Theories on plant flowering 1) Theory of endogenous rhythms According to this theory flowering of plants depends upon the length of photoperiods. These photoperiods affect the daily endogenous rhythms (everyday internal activities) which consist of two phases: a) Photophilous phase- It occurs in light and is characterized by intensive photosynthesis and weak respiration. In this phase the synthetic processes are dominant. b) Skotophilous phase- It occurs in the dark and is characterized by intensive respiration. In this phase, hydrolytic activities are increased and decomposition of starch into sugar takes place.

2) Phytochrome theory This theory believes that pigment (chromoprotein) is present in two interconvertible forms:

Pr (P660)

Pfr (P730)

One form of phytochrome is P730 which absorbs light at 730nm and the other form of phytochrome is P660 which absorbs light at 660nm. Under long-day photoperiods, P730 is retained for a longer time and stimulates flowering of long day plants and suppresses flowering of short day plants. Under short photoperiods, P660 is retained for longer time which stimulates flowering of short day plants and inhibits flowering of long day plants.

3) Theory on the relation of rates of light and dark reaction The rate of dark reaction in long day plants is relatively higher and the rate of light reaction in short day plants relatively lower.

4) The theory of two phase flowering This theory proposes that during flowering certain metabolic changes occur . For long day plants under long day conditions, day length affects plant metabolism by increasing carbohydrate contents and metal containing oxidases in leaves, auxins in stem apices and gibberallins in leaves. There is considerable decrease in contents of nitrogenous compounds. For short day plants under short day conditions, increase nitrogenous compounds, residual respiration, oxidases in leaves, metabolites of nucleic acid, metabolism in stem apices and anthesins in leaves.

Vernalisation Vernalisation is defined as the method of inducing early flowering in plants by pretreatment of their seeds at very low temperatures. Other scientists, has defined vernalisation as the acceleration of the ability to flower by chilling treatment. acquisition or

Importance of Vernalisation Crops can be produced earlier, that is a crop can be harvested much earlier than the control crop.

Crops can be grown in the regions where they do not naturally reproduce and Plant breeding work can be accelerated.

Vernalisation process The seeds are allowed to germinate for some time and then are given cold treatments by keeping them at 0 50C. The period of cold treatment varies from few days to many weeks from species to species. After the cold treatment seedlings are allowed to dry for some time and then sown. They should not be sown immediately after the cold treatment. The drying period also should not be very long as this decreases response of vernalisation and may cause seedlings to completely devernalised.. Devernalisation can also be affected if the vernalised seeds are subjected to heat treatment. It is also possible to revernalise the devernalised seedlings. It has been observed that mainly stem apex is the region which perceives the effect of vernalisation although leaves and roots can be used in vernalisation. The response to vernalisation depends on the duration for which and the temperature at which the seed is subjected to vernalisation.