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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.