GENES AND ENZYMES OF CAROTENOID BIOSYNTHESIS IN PLANTS

INTRODUCTION Indispensable and Other Roles of Carotenoids in Plants The carotenoid

pigments are essential components of the photosynthetic membranes in all plants, algae, and
cyanobacteria and serve an extraordinary variety of functions in plants (18, 28, 32, 38, 111). A
defining characteristic of these organisms is the use of readily available water as the reductant for
photosynthesis. This ability has been of central importance to the evolutionary success of plants
and algae. However, the production of molecular oxygen that accompanies the oxidation of water
near the reaction centers of photosystem II is problematic. Excited triplet state chlorophyll,
formed in the reaction centers and light-harvesting antenna upon illumination, can interact with
oxygen to form the reactive and damaging singlet oxygen. Carotenoids associated with the
reaction centers and antenna complexes react with and efficiently quench triplet chlorophyll,
singlet oxygen, and also superoxide anion radicals (18, 28, 32). These protective functions are so
critical that an inability to form cyclic carotenoids is eventually lethal in oxygen-evolving
photosynthetic organisms (84). Carotenoids in plants also dissipate excess light energy absorbed
by the antenna pigments (28, 32, 111), harvest light for photosynthesis (18, 28, 32, 111), serve as
precursors for biosynthesis of the plant growth regulator abscisic acid (108, 112), and are
exploited as coloring agents in flowers and fruits to attract pollinators and agents of seed
dispersal (38). The yellow, orange, and red colors provided by these pigments are of important
agronomic value in many crop and ornamental plants. Carotenoids are precursors of vitamin A in
human and animal diets (52), play other roles in human nutrition (74), and are of interest as
potential anticancer agents (64). A number of carotenoids are valuable poultry and fish feed
additives (61), or important colorants in the cosmetics and food industries (49). There is
currently considerable interest in the manipulation of carotenoid content and composition in
plants to improve the agronomic and nutritional value for human and animal consumption.
Carotenoids in the Context of Plant Metabolism The carotenoid pigments are synthesized in the
plastids of plants. In chloroplasts they accumulate primarily in the photosynthetic membranes in
association with the light-harvesting and reaction center complexes. In the chromoplasts of
ripening fruits and flower petals and in the chloroplasts of senescing leaves the carotenoids may
be found in membranes or in oil bodies or other structures within the stroma. The lipid-soluble
carotenoid pigments are but one example of the plethora of chemical compounds that are
produced by what are collectively known as the pathways of isoprenoid biosynthesis. The one
feature in common to the many isoprenoids (more than 20,000 in plants; 6, 24, 65) is their
biosynthesis from the central metabolite and building block for all isoprenoid compounds: the 5-
carbon compound isopentenyl pyrophosphate (IPP; Figure 2). A modular assembly process that
produces compounds of 5, 10, 15, 20, or more carbons (in multiples of 5) allows the biosynthesis
of the basic skeletons for the many and various isoprenoids with a relatively small number of
basic reaction steps (65). The C40 skeleton of plant carotenoid pigments, for instance, is
assembled from two molecules of a C20 compound, geranylgeranyl pyrophosphate (GGPP), that
is itself assembled from four units of IPP and that also serves as a precursor for many other
branches of the isoprenoid pathway in plants (Figure 2).
Roles of glycine betaine and proline in improving
plant abiotic stress resistance

3. Proline
3.1. Natural production and accumulation
Amino acid proline is known to occur widely in higher plants
and normally accumulates in large quantities in response to
environmental stresses (Rains, 1989; Ashraf, 1994; Ali et al.,
1999; Rhodes et al., 1999; Ozturk and Demir, 2002; Hsu et
al., 2003; Kavi Kishore et al., 2005). In addition to its role as
an osmolyte for osmotic adjustment, proline contributes to stabilizing sub-cellular structures (e.g.
membranes and proteins),
scavenging free radicals, and buffering cellular redox potential under stress conditions. It may
also function as a proteincompatible hydrotrope (Srinivas and Balasubramanian, 1995),
alleviating cytoplasmic acidosis, and maintaining appropriate
NADP+/NADPH ratios compatible with metabolism (Hare and
Cress, 1997). Also, rapid breakdown of proline upon relief of
stress may provide sufficient reducing agents that support mitochondrial oxidative
phosphorylation and generation of ATP for
recovery from stress and repairing of stress-induced damages
(Hare and Cress, 1997; Hare et al., 1998). Furthermore, proline is known to induce expression of
salt stress responsive
genes, which possess proline responsive elements (e.g. PRE,
ACTCAT) in their promoters (Satoh et al., 2002; Oono et al.,
2003; Chinnusamy et al., 2005).
In response to drought or salinity stress in plants, proline
accumulation normally occurs in the cytosol where it contributes
substantially to the cytoplasmic osmotic adjustment (Leigh et
al., 1981; Binzel et al., 1987; Ketchum et al., 1991). For example, in cells of Distichlis spicata
treated with 200 mM NaCl,
the cytosolic proline concentration was estimated to be more
than 230 mM (Ketchum et al., 1991). In apical region of maize
roots growing at a water potential of -1.6 MPa, proline concentration reached approximately 120
mM and accounted for up
to 50% of the osmotic adjustment (Voetberg and Sharp, 1991).
Furthermore, it was determined that, in response to water deficit,
increased concentration of proline in maize root apical meristem was paralleled with increased
concentration of abscisic acid
(Ober and Sharp, 1994; Sharp et al., 1994).
In plants, the precursor for proline biosynthesis is l-glutamic
acid. Two enzymes, pyrroline-5-carboxylate synthetase (P5CS)
and pyrroline-5-carboxylate reductase (P5CR), play major roles
in proline biosynthetic pathway (Delauney and Verma, 1993)
(Fig. 2). Transgenic tobacco plants over-expressing P5CS have shown increased concentration of
proline and resistance to both
drought and salinity stresses (Kishor et al., 1995). However,
whether proline accumulation in these transgenic plants resulted
in increased stress tolerance through osmotic adjustment or other
mechanisms is unknown (Sharp et al., 1996).
Accumulation of proline under stress in many plant species
has been correlated with stress tolerance, and its concentration
has been shown to be generally higher in stress-tolerant than in
stress-sensitive plants. For example, while in salt-tolerant alfalfa
plants proline concentration in the root rapidly doubled under
salt stress, in salt-sensitive plants the response was slow (Fougere
et al., 1991; Petrusa and Winicov, 1997). Similarly, salt-tolerant
ecotypes of Agrostis stolonifera accumulated more proline in
response to salinity than salt-sensitive ecotypes (Ahmad et al.,
1981). Besides positive effects of proline on improving plant
salt tolerance at the organismal level, considerable improvement
in salt tolerance has also been observed at the cellular level.
For example, in vitro studies with brown mustard (Brassica
juncea) indicated that salt-adapted calli had higher accumulation of free proline compared with
non-stressed calli (Madan et
al., 1995; Gangopadhyay et al., 1997). Similarly, salt-tolerant
calli of C. limon had significantly higher proline concentration
compared with salt-sensitive calli (Piqueras et al., 1996). Proline accumulation also occurs in
plants subjected to drought
stress. For example, in rice plants subjected to water deficit,
the concentration of proline was increased in the leaves (Hsu
et al., 2003). This drought-induced accumulation of proline
was related to increased contents of the precursors for proline
biosynthesis, including glutamic acid, ornithine, and arginine.
In wheat, an assessment of the effects of drought stress on proline accumulation in a drought-
tolerant and a drought-sensitive
cultivar revealed that the rate of proline accumulation and utilization was significantly higher in
the drought-tolerant cultivar
(Nayyar and Walia, 2003). Furthermore, in B. juncea plants
grown under stress conditions, activities of proline biosynthetic enzymes P5CR and ornithine
-aminotransferase (OAT)
increased mainly in tolerant lines though the activity of proline degrading enzyme proline
oxidase decreased in all lines
(Madan et al., 1995).
Although enhanced accumulation of proline in plants under
abiotic stress is well documented, information on signaling
mechanism(s) that regulates proline biosynthesis is scarce. In
light of the available information, however, it seems that proline
accumulation in plants is mediated by both ABA-dependent and
ABA-independent signaling pathways (Chiang and Dandekar,
1995; Shinozaki and Yamaguchi-Shinozaki, 1997; Hare et al.,
1999; Zhu, 2001b, 2002). ABA is known to mediate signals in
plant cells subjected to environmental stresses. These signals
can bring about expression of stress-related genes followed by
synthesis of compatible osmolytes such as proline (Thomashow,
1999; Kavi Kishore et al., 2005). Furthermore, ABA accumulation in plants in response to
osmotic stress has been determined
to regulate expression of P5CS gene (Fig. 2), which is involved
in proline biosynthesis (Xiong et al., 2001). However, it has been
reported that ABA is not solely sufficient for the induction of
P5CS transcript. The role of calcium in ABA-mediated induction of P5CS gene during drought-
and salt-stress has also been reported (Knight et al., 1997). Furthermore, a more recent study
suggested the role of phospholipase D as a signaling component along with calcium and ABA in
the regulation of proline
biosynthesis (Thiery et al., 2004). Whether MAP kinases have
any role in the regulation of proline biosynthesis is not fully
known (Kavi Kishore et al., 2005). However, it is imperative to
elucidate the role of stress-related signaling pathways in modulation of proline biosynthesis, so
as to help devise effective
strategies to improve plant stress tolerance.
Despite the presence of a strong correlation between stress
tolerance and accumulation of proline in higher plants, this relationship may not be universal.
For example, in rice plants grown
under salt stress, accumulation of proline in the leaf was deemed
to be a symptom of salt injury rather than an indication of salt
tolerance (Lutts et al., 1999). Similarly, assessment of proline
accumulation and distribution during shoot and leaf development in two sorghum genotypes
contrasting in salt tolerance
suggested that proline accumulation was a reaction to salt stress
and not a plant response associated with tolerance (de-Lacerda
et al., 2003). And yet in another study, under salt stress, sensitive
rice cultivars accumulated greater amounts of proline than did
the tolerant genotypes (Lutts et al., 1999). However, further studies are needed to determine
whether the relationship between
stress tolerance and accumulation of proline is species-specific
or if it can be altered by experimental conditions. Currently,
there is more evidence supporting the presence of a positive
relationship.
Transgenic approach to improve plant stress tolerance via
over-producing proline has had some success. For example,
engineered tobacco plants over-producing proline significantly
reduced the level of free radicals and improved tolerance to
200 mM NaCl (Hong et al., 2000). In Arabidopsis, plants engineered with an antisense proline
dehydrogenase cDNA resulted
in an increase in accumulation of proline and a constitutive tolerance to freezing temperatures (as
low as -7 C) as well as
salinity (up to 600 mM NaCl) (Nanjo et al., 2003). The level of
stress tolerance of these transgenic plants was comparable to that
of rd29A::DREB1A transgenics. The RD29A promoter-driven
DREB1A transgenic plants exhibited tolerance to water deficit,
salt stress, and freezing temperatures (Kasuga et al., 1999). However, an alternative, and may be
quicker, approach to improving
plant stress tolerance is through exogenous application of proline, as discussed in below.
3.2. Exogenous application of proline
Exogenous application of proline can play an important role
in enhancing plant stress tolerance. This role can be in the
form of either osmoprotection (Wyn Jones and Gorham, 1983;
Handa et al., 1986) or cryoprotection (Songstad et al., 1990;
Santarius, 1992) (Table 2). For example, in various plant species
growing under saline conditions, exogenously-supplied proline
provided osmoprotection and facilitated growth (Csonka and
Hanson, 1991; Yancey, 1994). In rice, exogenous application of
30 mM proline counteracted the adverse effects of salinity on
early seedling growth, though higher concentrations of proline
resulted in reduced growth (Roy et al., 1993). Exogenous appli-cation of proline to stressed
plants of the halophyte Allenrolfea
occidentalis increased their growth and halted increased production of ethylene due to salt- or
drought-stress (Chrominski
et al., 1989). Proline can also protect cell membranes from
salt-induced oxidative stress by enhancing activities of various
antioxidants (Yan et al., 2000). For example, growth of tobacco
suspension cells under salt stress was promoted by exogenous
application of 10 mM proline, which was proposed to be due
to proline action as a protectant of enzymes and membranes
(Okuma et al., 2000). In soybean cell cultures maintained under
salt stress, exogenous application of proline increased activities
of superoxide dismutase and peroxidase, which normally contribute to increased salt tolerance
(Yan et al., 2000; Hua and Guo,
2002) (Table 2). In barley embryo cultures under saline conditions, exogenous application of
proline resulted in a decrease in
Na+ and Cl- accumulations and an increase in growth (Lone et
al., 1987). Such ameliorative effects of proline were indicated
to be due to plasma membrane stabilization (Mansour, 1998)
(Table 2).
In suspension cultures of halophilic D. spicata, metabolism
of 13C-glutamic acid to proline was determined to be greater
in cells adapted to NaCl than in cells not adapted to the salt
(Heyser et al., 1989). Such metabolism, however, was inhibited
when 5.0 mM proline was added to the growth medium. This
inhibition was not due to a reduction in glutamic acid transport
in response to proline application, but rather regulated by either
feedback-inhibition of existing enzymes or repression of transcription (or translation) of genes
encoding enzymes involved in
proline biosynthesis (Heyser et al., 1989). In general, accumulation of proline in the cytoplasm is
associated with a reduction in
the concentration of toxic ions and an increase in the cytosolic
water volume (Cayley et al., 1992).
In contrast to the above findings on beneficial effects of
exogenous application of proline, there are a few reports cautioning its use. For example, foliar
application of proline to
rice plants growing under saline conditions did not change concentrations of either Na+ or Cl- in
the leaves (Krishnamurthy
and Bhagwat, 1993) (Table 2). Moreover, in Arabidopsis plants exogenous application of proline
was suggested to cause damages to ultra-structures of chloroplast and mitochondria (Hare
et al., 2002). Such loss of organelles integrity in response to
exogenous proline can result in a significant increase in reactive oxygen intermediates in the
chloroplast and mitochondria.
Such destructive effects were attributed to feedback inhibition
of proline biosynthesis, causing considerable reduction in photosynthetic electron acceptor pools
(Hare et al., 2002). These
authors questioned the widely-accepted hypothesis that proline
is an inert compatible solute that can be accumulated to high levels with minimal effects on
cellular function. Thus, when using
exogenous proline as a tolerance-inducing agent, the effective
and beneficial concentrations must be first determined.
3.3. Effective concentrations of exogenous proline
Although exogenous application of proline to plants exposed
to abiotic stresses generally provides a stress preventing or
recovering effect, high concentrations of proline may be harmful
to plants, including inhibitory effects on growth or deleterious effects on cellular metabolisms
(Ehsanpour and Fatahian,
2003; Nanjo et al., 2003). Therefore, it is essential to determine
optimal concentrations of proline that provide beneficial effects
in different plant species. In mung bean (Vigna radiata), for
example, it was determined that while addition of 2033 mM
of proline to cell cultures mitigated the adverse effects of NaCl
stress, concentrations of 50 mM or higher were inhibitory to the
growth of both salt-stressed and non-stressed cultures (Kumar
and Sharma, 1989). In this study, while cellular contents of Na+
and Cl- decreased when proline reached optimal concentration,
they increased with further elevation in proline concentration. In
alfalfa (Medicago sativa) callus cultures, while 10 mM exogenous proline was very effective in
alleviating the effects of salt
stress, higher concentrations were not beneficial (Ehsanpour and
Fatahian, 2003). In rice, while 30 mM proline was the most
effective concentration in improving germination and seedling
growth under salt stress, higher concentrations (40 or 50 mM)
resulted in reduced seedling growth and lowered K+/Na+ ratio in the leaves (Roy et al., 1993).
Thus, in spite of its acclaimed
protective role, the toxicity effect of proline at high concentrations may be a problem. In a study
to examine toxicity effects of
proline in Arabidopsis, a mutant (pdh) was isolated with a defect
in enzyme proline dehydrogenase (AtProDH), which catalyzes
the first step of proline catabolism (Nanjo et al., 2003). While
pdh mutants showed hypersensitivity to exogenous application
of 10 mM proline, wild-type plants grew normally at this concentration. Further investigations
determined a dose-dependent
elevation in internal concentration of free proline in pdh mutants
during exogenous application of proline. The results of this study
indicated toxicity effects of proline and suggested that AtProDH
was the only enzyme acting as a functional proline dehydrogenase in Arabidopsis.
The available information from different studies suggest that
optimal concentrations of proline may be species or genotype
dependent, which need to be determined a priori before commercial application of exogenous
proline to improve crop stress
tolerance. Furthermore, because in most crop species stress
tolerance may vary with developmental stages (Ashraf, 1994;
Foolad, 2000), it is also as critical to determine the growth stage
at which exogenous application of proline may be the most effective.
Effects of Exogenous Silicon on Photosynthetic Capacity and Antioxidant
Enzyme Activities in Chloroplast of Cucumber Seedlings Under Excess
Manganese
Abstract
Effects of silicon on photosynthetic parameters and antioxidant enzymes of chloroplast in
cucumber seedlings under
excess Mn were studied. Compared with the control, excess Mn significantly inhibited net
photosynthetic rate (Pn),
stomatal conductance, as well as the maximum yield of the photosystem II photochemical
reactions (Fv/Fm) and the
quantum yield of photosysytem II electron transport ( PSII), application of Si reversed the
negative effects of excess Mn.
In the further investigation, it was obtained that application of Si significantly increased the
activities of enzymes related
with ascorbate-glutathione cycle including ascorbate peroxidase (APX), dehydroascorbate
reductase (DHAR) and
glutathione reductase (GR) in cucumber chloroplast under excess Mn, this could be responsible
for the lower accumulation
of H
2O2 and lower lipid peroxidation of chloroplast induced by Mn, and resulted in keeping higher
photosynthesis.
Silicon is the second most abundant element in soil, it is not considered to be an essential
element for higher
plants (Epstein 1999). However, there is increasing
evidence that it has a number of beneficial effects on
plant growth under biotic and abiotic stress (Kauss
et al. 2003; Liang et al. 2005, 2007; Ma 2000). In the
aspect of abiotic stresses, a function of Si on alleviating Mn toxicity has been observed in many
plants such
as cowpea, pumpkin, and cucumber (Iwasaki et al.
2002; Iwasaki and Matsumra 1999; Rogalla and
Rmheld 2002; Shi et al. 2005). In the past, it has
been obtained that excess Mn can inhibit photosynthesis of ricebean and white birch
(Subrahmanyam and
Rathore 2000; Kitao et al. 1997), while there were not
reports obstained about Si on photosynthesis under
excess Mn, previous study has shown that Si alleviated
the growth inhibition of cucumber by excess Mn (Rogalla
and Rmheld 2002; Shi et al. 2005), therefore it can be
concluded that Si must influence photosynthetic capacity of cucumber plants, based on this
hypothesis, in
the experiment, effects of Si on photosynthetic and
chlorophyll parameters as well as antioxidant enzymes
activities of chloroplast were investigated.
RESULTS
Plant growth
Fig.1 showed that excess Mn significantly inhibited the
shoots and roots growth of cucumber seedlings (P<0.05).
Under normal Mn level, application of Si had no signifi-cant effects on the growth of cucumber
seedlings.
However, under excess Mn, Si significantly promoted
the growth of both shoots and roots (P < 0.05).
Symptom and pigment contents
One common symptom caused by Mn toxicity is yellowing of the leaves which might be
attributed to Fe
deficiency induced by excess Mn. In the investigation, excess Mn induced obvious necrosis in
cucumber
leaves, and application of Si inhibited the appearance of
necrosis under excess Mn (Fig.2), the symptom was
in accordance with the change of pigments. Fig.3
showed that, under excess Mn conditions, chlorophyll
a, chlorophyll b and carotenoid concentrations greatly
decreased in cucumber leaves (P < 0.05), and addition
of Si significantly increased their level (P<0.05); while
addition of Si had no significant effects on pigment contents under normal Mn treatment.

Uptake system of silicon in different plant species

Introduction
Silicon (Si) is a beneficial element for plant growth. Silicon
helps plants to overcome multiple stresses including biotic
and abiotic stresses (for a recent review, see Ma, 2004).
For example, Si plays an important role in increasing the
resistance of plants to pathogens such as blast on rice
(Datnoff et al., 1997) and powdery mildew on cucumber
(Miyake and Takahashi, 1982a, b). Silicon is effective in
preventing lodging in rice by increasing the thickness of the
culm wall and the size of the vascular bundles (Shimoyama,
1958), thereby enhancing the strength of the stems. Silicon
also alleviates the effects of other abiotic stresses including
salt stress, metal toxicity, drought stress, radiation damage, nutrient imbalance, high temperature,
and freezing
(Epstein, 1999; Ma and Tahakashi, 2002; Ma, 2004). These
beneficial effects are mostly expressed through Si deposition in the leaves, stems, and hulls,
although other mechanisms have also been proposed (Ma, 2004). Therefore, the
Si effect is characterized by a larger effect associated with
a greater Si accumulation in the shoots.
However, Si accumulation in the shoots varies considerably among plant species, ranging from
0.1% to 10% Si
in the dry weight (Ma and Takahashi, 2002). Takahashi and
coworkers made an extensive survey on the Si concentrations
of nearly 500 plant species from Bryophyta to Angiospermae, grown under similar soil
conditions (for the summary,
see Ma and Takahashi, 2002). The results showed that there
is a characteristic distribution of Si accumulation in the plant
kingdom. In higher plants, only plants in Gramineae and
Cyperacea show high Si accumulation. Plants in Cucurbitales, Urticales, and Commelinaceae
show intermediate Si
accumulation, whereas most other plants species show low
Si accumulation. The difference in Si accumulation has been
attributed to the ability of the roots to take up Si (Takahashi
et al., 1990). Silicon is taken up in the form of an uncharged
molecule, silicic acid (Takahashi and Hino, 1978). Three
different modes of Si uptake have been proposed for plants
having different degrees of Si accumulation, that is, active,
passive, and rejective uptake (Takahashi et al., 1990). Plants
with an active mode of uptake take up Si faster than water,
resulting in a depletion of Si in the uptake solution. Plants
with a passive mode of uptake take up Si at a rate that is
similar to the uptake rate of water; thus, no significant changes
in the concentration of Si in the uptake solution are observed.
By contrast, plants with a rejective mode of uptake tend to
exclude Si, which is demonstrated by the increasing concentration of Si in the uptake solution.
However, the mechanisms
involved in the different uptake modes are not understood.
The objective of this study was to examine the uptake systems
of Si in rice, cucumber, and tomato, which represent high,
intermediate, and low Si accumulation, respectively.
Results and discussion
It was reported that rice, cucumber, and tomato contained
7.3, 2.3, and 0.2% Si in the shoot dry weight, respectively,
when they were grown under similar conditions (Takahashi
et al., 1990). To investigate whether this difference is due to
the difference in root uptake, a short-term (up to 24 h)
uptake experiment was performed with the three species.
When the plants were exposed to a solution containing
the same Si concentration under the same conditions, the
uptake of Si by rice was much higher than that by the other
two species at each time point (Fig. 1). Si uptake by
cucumber was also higher than that by tomato (Fig. 1). At
24 h, the Si uptake by rice was about 6-fold and 12-fold
higher than cucumber and tomato, respectively. The Si
concentration in the uptake solution for rice decreased markedly with time (data not shown), but
remained
constant for cucumber and increased slightly for tomato.
No significant difference was observed in the transpiration
rate between the three species (data not shown). These
results are in agreement with previous findings by Okuda
and Takahashi (1962), who compared only rice and tomato
in their study. The accumulation of Si in the shoots may be
related to a number of factors such as transpiration, growth
duration, growth rate, etc, but root uptake ability is the most
important factor for determining Si accumulation in the
shoots. These results confirmed a previous proposal that
the difference in Si accumulation results from the ability of
the roots to take up Si (Takahashi et al., 1990).
The uptake of Si involves at least two processes: radial
transport of Si from the external solution to the cortical cells
and the release of Si from the cortical cells into the xylem
(xylem loading). In rice, it has been demonstrated that
radial transport of Si is mediated by a type of transporter
with a K
m value of 0.15 mM Si (Tamai and Ma, 2003; Ma
et al., 2004). In the present study, this process was also
examined in cucumber and tomato. For comparison, rice
was also investigated under the same experimental conditions.
A time-course experiment showed that the Si concentration
in the root-cell symplast increased with time in all species
(Fig. 2). However, the Si concentration in the symplast was
much higher in rice, followed by cucumber and tomato,
although the Si concentration in the symplast in all species
was higher than that in the external solution. After exposure
to 0.5 mM Si for 12 h, the Si concentration in the root
symplast was 6.0, 2.0, and 0.9 mM for rice, cucumber, and
tomato, respectively (Fig. 2).
A kinetic study showed that the Si concentration in the
root-cell symplast increased with increasing external Si
concentrations, but saturated at a higher concentration,
although the Si concentration in the root-cell symplast
differed greatly between the three species (Fig. 3A, B, C).
Based on these curves, the Km value was estimated to be
0.16, 0.15, and 0.16 mM for rice, cucumber, and tomato,
respectively. The value of Vmax was 34.5, 26.9, and 13.3 ng
root1 8 h1, respectively, for rice, cucumber, and tomato.
These results suggest that the radial transport of Si from the
external solution to the cortical cells in the three plant
species is mediated by a transporter that shows a similar
affinity to silicic acid. However, the difference in the Vmax
value suggests that the density of the Si transporter on the
root cell membranes differs among the plant species,
following the order of rice>cucumber>tomato.
A higher Si concentration in the root-cell symplast than
in the external solution (Fig. 1) suggests that silicic acid is
transported against a concentration gradient from the external
solution to the cortical cells, which would be energydependent. To confirm this, the effect of a
metabolic inhibitor
(2,4-DNP) and low temperature on the radial transport of Si
was investigated. In the absence of the inhibitor, the Si
concentration in the apoplasmic solution in all species was almost the same as that in the external
solution, whereas the
Si concentration in the root-cell symplast differed among
the three plant species, being 5-, 2-, and 1.5-fold higher than
that in the external solution, respectively, for rice, cucumber, and tomato. In the presence of 2,4-
DNP or under a low
temperature (4 8C), the Si concentration in the root-cell
symplast was decreased to a level similar to that in the
apoplast and the external solution (Fig. 4). These results
suggest that Si uptake involves two components: a transportermediated component as described
above and a passive transport by diffusion. Different from most other minerals, Si is
present in the form of an uncharged molecule, silicic acid, at
a pH below 9. This uncharged form is probably permeable
across the plasma membranes in the roots (Raven, 2001).
Recently, it was reported that Si uptake by rice was
significantly inhibited by HgCl2, (Tamai and Ma, 2003).
Further, this inhibition is independent of water inhibition
caused by HgCl2 because the Hg-induced inhibition of Si
uptake occurred earlier than that of water uptake and
Hg-inhibited water uptake was completely recovered in the presence of 10 mM 2-
mercaptoethanol, while Hginhibited Si uptake was not. In the present study, the effect
of HgCl2 on the radical transport of Si was therefore
compared among rice, cucumber, and tomato. In the
presence of HgCl2, the Si concentration in the root-cell
symplast decreased to a similar level as that in the apoplast
in all three species (Fig. 4). It is suggested that HgCl2 blocks
water channels via the oxidation of cysteine residue(s)
proximal to the aqueous pore and the subsequent occlusion
of the aqueous pore by the large mercury ion (Maurel, 1997).
Therefore, these results suggest that the transporter responsible
for the radial transport of Si from the external solution to the
cortical cells contains a Cys residue.
The subsequent process, i.e. the release of Si from
the cortical cells to the xylem (xylem loading) was then
investigated in cucumber and tomato. For comparison, rice
was also investigated under the same experimental conditions, although the xylem loading
process has been characterized in rice (Ma et al., 2004; Mitani et al., 2005). The
average exudation rate of xylem sap was 35 ll plant1 30
min1 for rice, and 4050 ll plant1 30 min1 for cucumber
and tomato. When the roots were exposed to a nutrient
solution containing 0.5 mM Si, the concentration of Si in the
xytem sap of rice reached 6.0 mM Si in 30 min and 18 mM in
8.5 h (Fig. 5), suggesting that the release of Si to the xylem is
a very rapid process and that Si is loaded against a concentration gradient in rice as reported
previously (Ma et al.,
2004; Mitani et al., 2005). By contrast, in cucumber and
tomato, the Si concentration in the xylem sap was much
lower than that in rice (Fig. 5). The Si concentration in the
xylem sap of cucumber was 0.6 mM after 30 min, and
remained stable throughout the experiment period. In
tomato, the Si concentration in the xylem sap was lower
than that of the external solution throughout the experiment.
Xylem loading of Si has been reported to be a transporter-mediated process in rice (Ma et al.,
2004). A kinetic
study of xylem loading in the present study confirmed this conclusion in rice (Fig. 6A). By
contrast, in cucumber and
tomato, the Si concentration in the xylem sap increased
gradually with increasing Si concentration in the external
solution (Fig. 6B, C), but the concentration was lower than
that in the external solution. The Si concentration in the
xylem sap of cucumber was 23-fold higher than that of
tomato (Fig. 6B, C). These results suggest that different
from rice, xylem loading of Si in cucumber and tomato is
mediated by passive diffusion. In this experiment, xylem
sap was collected 8 h after exposure to Si. At this stage of
exposure, the Si concentration in xylem sap of cucumber
and tomato by diffusion did not reach the level of the
external solution (Fig. 6). However, with prolonged
exposure, the Si concentration eventually approaches to
that of the external solution. This has been demonstrated previously in tomato (Okuda and
Takahashi, 1962), in
which the Si concentration in xylem sap became similar to
that in the external solution by 37 h after the initiation of Si
treatment. A higher Si concentration in the xylem sap of
cucumber compared with that of tomato may be attributed
to the higher density of transporter for radial transport of Si
in cucumber (Fig. 3B, C), resulting in a higher concentration of Si in the root cells, and
subsequently more Si in the
xylem sap by diffusion.
Figure 7 presents a scheme for an Si uptake system,
modified from that of Ma et al. (2004) based on results
obtained in the present study. There are two components
in the radial transport of Si from the external solution to
the cortical cells: transporter-mediated transport (SIT1)
and passive diffusion. Further, the density of this transporter differs among plant species,
following the order of
rice>cucumber>tomato, although the affinity of this transporter to silicic acid was similar in
these three species.
Xylem loading of Si was mediated by a putative transporter
(SIT2) in rice, but by diffusion in cucumber and tomato.
Recent studies (Ma et al., 2002, 2004) comparing wild-type
rice and a mutant defective in Si uptake showed that xylem
loading is the most important step leading to a high
accumulation of Si in the shoots. Therefore, the much
lower accumulation of Si in cucumber and tomato could
be explained by a lower density of SIT1 and a defect of
SIT2.
Silicon is abundant in soil. However, most plants,
especially dicots, are unable to accumulate a large amount
of Si in the shoot from the soil. Therefore, they do not benefit from Si. Recently, a gene
controlling the xylem
loading of Si has been mapped to chromosome 2 of rice
(Ma et al., 2004). Cloning of this gene from rice may be
useful in genetically modifying the Si uptake ability of other
plant species, thereby enhancing the resistance of plants to
multiple stresses.