Journal of Experimental Botany, Vol. 52, No. 360, pp. 1455-1463,
July 1, 2001
© 2001 Oxford University Press
Original Papers |
Genotype-dependent proteolytic response of spring wheat to water deficiency
niewski1
ska1,2,3
1 Plant Physiology and Biochemistry Department, Plant Breeding and Acclimatization Institute, Radzików, POB 1019, 00-950 Warszawa, Poland
2 Biochemistry Department, Warsaw Agricultural University, Rakowiecka 26/30, 02-528 Warszawa, Poland
Received 2 May 2000; Accepted 13 March 2001
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Changes in proteolytic activities in response to water deficiency have been investigated in ten genotypes of spring wheat (Triticum aestivum L.) differing in response to water deficit stress and ability to acclimate. To determine subcellular localization and the type of proteases, mesophyll protoplasts isolated from wheat leaves were purified. Proteolytic activities were assayed using azocasein in the case of vacuolar proteinases at pH 5.0 and 125I-lysozyme in the case of extravacuolar ATP-dependent proteinases at pH 8.2. ATP-dependent proteolytic activity was found to be confined to the extravacuolar fraction while the azocaseinolytic activity to vacuoles. Dehydration increased vacuolar azocaseinolytic activity at both stages of plant development (shooting and heading), but the increase was significantly lower in more tolerant genotypes. The extravacuolar energy-dependent 125I-lysozyme degradation was low at the shooting stage but it was higher in the genotypes with a greater critical water saturation deficit. At the heading phase in the non-acclimated flag leaves ATP-dependent 125I-lysozyme degradation decreased in a genotype-dependent manner, but was enhanced upon acclimation to the same extent irrespective to the genotype ability to acquire dehydration tolerance during acclimation. The results presented indicate that both pathways of protein degradation are interlinked upon dehydration and are genotype dependent.
Key words: Wheat, proteolytic activity, dehydration tolerance, acclimation.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The ability of higher plants to tolerate water deficit varies considerably among different species in which lethal leaf water potential ranges from -1.2 MPa to -500 MPa (Ludlow, 1993
). Also large differences in water loss may occur within single species and in individual organs of the same species (Levitt, 1980; Bray, 1997). Among higher plants, only a small group of angiosperms (resurrection plants) can survive dehydration and can recover from complete dryness within several hours of rehydration (Bartels and Nelson, 1994). Despite significant progress in molecular biology of the drought response, the genes and/or gene products required for dehydration tolerance remain unknown (Ingram and Bartels, 1996; Bray, 1997).
Plants have the ability to improve their dehydration resistance by acclimation. A transient dehydration induces phenotypic alterations without inheritable genetic alterations, and involves important modifications of gene expression, which enables adjustment to water deficit. For reorganization of plant metabolism under water deficiency, proteolysis may be an important mechanism of regulation of cellular activity. Amino acids may be released for synthesis of new proteins, aberrant proteins formed under water deficit may be degraded and certain proteins may be activated (Guerrero et al., 1990; Jones and Mullet, 1995).
Plant proteolysis is mediated by two different proteolytic pathways: ATP-independent and ATP-dependent (Callis, 1995; von Kampen et al., 1996). Different compartments within the cell contain distinct proteinases: major endo- and carboxypeptidases are localized in vacuoles, whereas aminopeptidases are found mainly in chloroplasts and in the cytoplasm (Huffaker, 1990). The ubiquitin-proteasome system, responsible for the majority of selective protein degradation is located in the nucleus and cytosol (Vierstra, 1993). Clp-type proteinases probably account for at least part of the ATP-dependent proteolysis in chloroplasts (Clarke et al., 1994; Shanklin et al., 1995; Anderson and Arö, 1997).
Proteolytic activities in response to senescence are well documented (Noodén et al., 1997; Fischer et al., 1998) but present knowledge of proteolytic activities under dehydration remains fragmentary. Previous studies have indicated that, in response to dehydration, the activities of ATP-independent proteinases degrading azocasein in wheat leaf extracts increased about 7-fold at the acid pH optimum (Zagdaska and Winiewski, 1996), while ATP-dependent 125I-lysozyme hydrolysis decreased (Zagdaska and Winiewski, 1998). Acclimation had no effect on the quantitative induction of azocaseinolytic proteolysis, but the contribution of cysteine proteinases (Zagdaska and Winiewski, 1996) and ATP-dependent 125I-lysozyme degradation was significantly increased in total proteolytic activity (Zagdaska and Winiewski, 1998).
To understand the role of proteolysis in the response to water-deficit stress, ten spring wheat genotypes differing in critical water saturation deficit (WSD) and in the ability to acclimate to water deficiency were examined. Differences between the genotypes were determined in a simple laboratory test; detached leaves were dehydrated under vacuum and were tested for injury by electrolyte leakage. WSD was also determined (Zagdaska, 1995). The WSD at which 50% injury occurred was defined as critical WSD. ATP-dependent (125I-lysozyme degradation) and ATP-independent (azocaseinolytic) proteolytic activities were determined in leaf extracts and in their vacuolar and extravacuolar fractions at two phases of plant development (shooting and heading). Therefore, changes in proteolytic activities have been examined in wheat leaves under direct influence of soil water deficit, when water deficiency affected the expanded blade of the fifth leaf (shooting phase) or flag leaf (heading phase). This experiment also included the leaves acclimated to water deficit, when they were transiently dehydrated during tissue differentiation. These comparative studies revealed genotype-dependent differences in the plant proteolytic responses to water deficit.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material
The experiments were carried out on spring wheat (Triticum aestivum L.) plants (eight cultivars and two strains). Plants were grown in soil in a growth chamber and watered daily. The day/night temperature was 10/5 °C, relative humidity 70/80% and photoperiod 12 h, until the second leaf was fully expanded. The seedlings were then subjected to a day/night temperature of 20/15 °C and a 16 h photoperiod for about 14 d. After this period the temperature was raised to 25/21 °C with relative humidity of 60/80% and a 16 h photoperiod. Irradiance was 260 µmol m-2 s-1 PFFD provided by metal halide lamps (VEB NARVA, Oschatz, Germany). Control (non-stressed) plants were grown under these conditions throughout the whole experimental period. When the fifth leaf was fully expanded (shooting phase), plants were drought pretreated (drought acclimated) by cessation of watering during a sufficiently long period to evoke 50% tissue injury measured by electrolyte leakage. As described in detail previously, the WSD of these leaves were determined and defined as critical WSD (Zagda
ska, 1995). After this dry period, the plants were rewatered and then at the heading phase (fully expanded flag leaf ) subjected to soil water-deficit conditions together with non-pretreated (non-acclimated) plants. Developmental stages were determined as described previously (Zadoks et al., 1974). To eliminate the indirect effect of drought on plant development, all measurements were carried out with the leaves at the same developmental stage (developmental control). Additionally, some control plants were grown for the same period as water-deficit-treated plants (age control). Also, to eliminate the possible effect of leaf age, control leaves (non-acclimated and acclimated) were analysed at the beginning and end of the soil-water-deficit treatment. An average WSD of the leaves from both non-acclimated and acclimated plants after the stress period was 50%. Water saturation deficit (WSD) in the studied leaves was calculated according to Stocker as follows: WSD=(weight of fully saturated leaf-actual fresh weight)/(fully saturated leaf weight-dry weight) (Stocker, 1929). Five leaves were analysed from each experiment run in triplicate. The experimental error in WSD determinations did not exceed 0.5%.
Enzyme extraction
About 1 g of leaf tissue was homogenized in liquid nitrogen and extracted with 5 ml of cold extraction buffer (50 mM TRIS/HCl pH 7.2) containing 0.2 g insoluble PVP and 5 mM mercaptoethanol. The extract was filtered and centrifuged at 15 000 g for 10 min at 4 °C and the supernatant was used for the enzyme assay.
Isolation of protoplasts
Protoplasts were isolated from leaves (control and stressed) of cv. Eta by the modified method of Blom-Zandstra et al. (Blom-Zandstra et al., 1990). The leaves were cut into 2–3 mm wide fragments and digested in incubation medium containing 25 mM MES-TRIS ( pH 5.6), 0.6 M sucrose, 1.0% (w/v) Cellulase Onozuka R-10, 0.3% (w/v) Macerozyme R-10 (final osmolarity about 700 mOsmol kg-1). After digestion, the enzyme medium was removed and protoplasts were washed with 25 mM MES-TRIS (pH 5.6) and 0.6 M sucrose. Protoplasts were isolated by gentle shaking in hypotonic solution (0.1 M K2HPO4, pH 8.0, 1 mM DTT, 5 mM EDTA; osmolarity about 275 mOsmol kg-1). Protoplasts were collected by centrifugation at 500 rpm for 2 min. The equilibration medium containing 1 M sorbitol, 12% (w/v) Ficoll, 30 mM HEPES-imidazol (pH 8.2), 2 mM EDTA, 1 mM MgSO4, 1 mM CaSO4 and 0.2% (w/v) BSA was added to raise the osmolarity up to about 500 mOsmol kg-1. After purifying the suspension through membrane filters (pore diameter 200–500 µm) about 1 ml of suspension was overlaid on 3 ml of the iso-osmolal step-density gradient containing 2 ml of 0.4 M sorbitol, 30 mM HEPES-imidazol, 2 mM EDTA, 1 mM MgSO4, 1 mM CaSO4, 0.2% (w/v) BSA, adjusted to pH 8.0 and centrifuged at 800 rpm for 5 min to separate vacuoles. Protoplasts and vacuoles were counted using a haemocytometer. The following marker enzymes were used to monitor purity of the fractions: PEP carboxylase (EC 4.1.1.31) for the extravacuolar fraction and 
-mannosidase (EC 3.2.1.24) for vacuoles. In preliminary experiments the protoplasts were checked for the possible presence of contaminating proteinases from the Cellulase and Macerozyme preparations by comparing specific proteolytic activity of protoplasts and leaf tissues including (i) their response to specific inhibitors of cysteine (10 mM iodoacetate), aspartic (5 µg ml-1 pepstatin) and serine (2.5 mM phenylmethanesulphonylfluoride) proteinases (Lin and Wittenbach, 1981) and (ii) by monitoring the fate of 125I-isolation enzymes during protoplast purification (Wagner et al., 1981).
Measurement of azocaseinolytic activity
It has been shown previously that the maximum azocaseinolytic activities in crude wheat leaf extract was at pH 5.0 (Zagdaska and Winiewski, 1996) and therefore changes in total azocaseinolytic activities in either fifth or flag leaves were monitored at this acid pH optimum. The reaction mixture contained in 1 ml: 0.1 ml of the enzyme extract, 0.3 ml of 0.5% azocasein and 0.6 ml of 0.25 M citrate/phosphate buffer pH 5.0. After 2 h at 37 °C the reaction was stopped by adding 2 ml of 12% trichloroacetic acid and acid-soluble products were determined spectrophotometrically at 340 nm. One unit of azocaseinolytic activity was defined as the amount of the enzyme causing a 0.01 increase in A340. To determine the activity of the cysteine endoproteinases, 1.0 mM iodoacetate was added to the enzyme extracts and the mixtures were preincubated for 1 h prior to the addition of azocasein. The protein content in leaf extracts was determined using BSA as a standard (Bradford, 1976).
Assay of ATP-dependent 125I-lysozyme breakdown
Lysozyme was radio-iodinated by the chloramine-T procedure (Parker, 1990). The specific activity was between 1.6–3.1x104 cpm pmol-1. The breakdown of 125I-lysozyme to acid-soluble material was determined as described previously (Herschko et al., 1983). The reaction mixture contained 10 µl of 0.25 M TRIS/MES/KOH (pH 8.2), 40 µl of plant extract and 15 µl of 125I-lysozyme. For ATP-dependent activity, ATP and ATP-regenerating system, which consisted of 3 µl of 2 mM ATP, 5 µl of 0.005 M MgCl2, 0.001 M DTT, 0.01 M creatine phosphate in 0.05 M HEPES/KOH at pH 8.2, and 2 µl of phosphocreatine kinase (1 unit µl-1) was added. For ATP-independent activity, the ATP-depleting system, which consisted of 5 µl of 0.005 M MgCl2, 0.001 M DTT, 0.01 M deoxyglucose in 0.05 M HEPES/KOH at pH 8.2, and 5 µl of hexokinase (1 unit µl-1) was added. The reaction mixture was preincubated under ATP-depleting conditions for 1 h at 37 °C, whereafter the radio-iodinated lysozyme was added. Following incubation at 37 °C for 2 h, the reaction was terminated by the addition of 300 µl of 24% trichloroacetic acid and the samples were centrifuged at 15 000 rpm for 15 min. Degradation of 125I-lysozyme into acid soluble 125I was measured by scintillation counting. The difference between degradation of 125I-lysozyme under ATP-regenerating and ATP-depleting systems was a measure of ATP-dependent proteolysis. Acid-soluble radioactivity in the sample at zero time was subtracted. Participation of ATP-dependent proteolytic activity in total proteolytic activity (rate of 125I-lysozyme degradation in the ATP-regenerating system) was expressed in per cent. This relative method of expression of ATP-dependent proteolytic activity is widely used (Rivett, 1994).
Statistical analysis
All data were calculated according to Statistica 5. 97 edition program.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Critical WSD of spring wheat cultivars
Spring wheat genotypes showed considerable variability in critical WSD at shooting (fifth leaf ) and heading (flag leaf ) phases of plant development. As shown in Table 1
WSD in seven out of ten genotypes was lower during heading (flag leaves) than at the shooting phase (fifth leaves) indicating that 50% leaf injury occurred at a higher leaf relative water content. In one genotype (Jota) no difference in WSD was found and in two (Eta and Henika) the critical WSD was higher at the heading phase. The WSD increased in half of the genotypes acclimated to water deficit (Fig. 1). In two cases (cvs Broma and KOH 193) this increase was up to 23% WSD. As can be seen from Table 1 and Fig. 1 the response to water deprivation and acclimation of individual cultivars is equivocal. Wheat genotypes responded differently to water deficiency during shooting and heading and the genotypes resistant at shooting phase (e.g. Broma, Sigma, KOH 193, and 393) were susceptible during heading or showed the same highest tolerance (KOH 393) although somewhat lower than at shooting phase. These genotypic differences during plant development were not related to the critical WSD at heading phase.
|
|
Protein content in dehydrated leaves
At the shooting phase, in dehydrated fifth leaves, the content of soluble proteins (expressed as % of control) in the wheat genotypes with the highest WSD was reduced to 10% of the control (Fig. 2A). The protein content of dehydrated fifth leaves of the genotypes with the lowest WSD, was reduced to 25% of control, fully-turgid leaves. In contrast, protein content in dehydrated non-acclimated flag leaves was higher in the genotypes with a higher WSD. The protein content was still higher in the acclimated flag leaves (Fig. 2B). The correlation coefficients between soluble protein content in dehydrated leaves and critical WSD of spring wheat genotypes were high at both stages of plant development (Table 2 ).
|
|
Vacuolar and extravacuolar proteolytic activities
To assess the intracellular localization of proteolytic activities vacuolar and extravacuolar fractions were separated from protoplasts of wheat leaf cells. The activities of -mannosidase, a marker of vacuolar fraction, and PEP carboxylase, designating the extravacuolar fraction, illustrate the relative purity of the obtained fractions (Table 3). The vacuolar fraction contained about 90% of the total activity of -mannosidase and about 16% of the PEP carboxylase, the cytosolic marker enzyme. Distribution of azocaseinolytic activity closely corresponds to that of -mannosidase. ATP-dependent degradation of radio-iodinated lysozyme in extravacuolar fractions from protoplasts was comparable to that obtained in total extracts from the leaves of the same cultivar (Zagdaska and Winiewski, 1998). This activity in the vacuolar fraction was 8% of total ATP-dependent 125I-lysozyme degradation (Table 3). Thus taking into account contamination of this fraction with extravacuolar fraction it can be assumed that ATP-dependent radio-iodinated lysozyme degradation is confined to the extravacuolar fraction. The results obtained indicate that 125I-lysozyme degradation in the extracts under the described conditions can be used for comparative characteristics of wheat genotypes.
|
Azocaseinolytic activities
Total azocaseinolytic activities in the control, fully-turgid wheat leaves were found to be slightly but insignificantly higher in flag leaves (2.97–5.76 units mg-1 protein h-1) than in the fifth leaves (2.20–3.78 units mg-1 protein h-1) of non-acclimated plants (Fig. 3B and A, respectively). Critical WSD at both developmental stages was not correlated with azocaseinolytic activity (Table 2
). At the shooting phase (fifth leaves), azocaseinolytic activity increased under water deprivation from 7–20-fold (Fig. 3A). At heading phase (flag leaves) the enhancement of azocaseinolytic activity was slightly lower from 4–7-fold (Fig. 3B). In acclimated flag leaves (Fig. 3C), azocaseinolytic activity increased to a still lesser extent about 2-fold from 4.11 to 9.72 units mg-1 protein h-1 in control leaves to 8.17–2.4 units mg-1 protein h-1 in dehydrated flag leaves (Fig. 3C).
|
Independent of the stage of wheat development, shooting (Fig. 3A
) or heading (Fig. 3B), critical WSD of the genotypes was highly negatively correlated with vacuolar proteolysis (Table 2), although at the shooting phase (Fig. 3A) azocaseinolytic activities were twice as high as at the heading phase (Fig. 3B). The vacuolar proteolytic activity in the acclimated flag leaves (Fig. 3C) decreased with the increasing genotypic critical WSD in a similar way as that in non-acclimated leaves (Fig. 3B).
The cysteine proteinases are preferentially enhanced under water deficiency when assessment is based on the inhibitory effect of iodoacetate on azocasein hydrolysis. Their activity is low in fully turgid control leaves and ranged between 0.18–0.96 in the fifth leaves and between 0.45–1.6 units mg-1 protein h-1 in non-acclimated flag leaves and in acclimated leaves between 0.19–1.9 units mg-1 protein h-1. However, the net increase in the cysteine endoproteinase activities (differences between the enzyme activity in dehydrated and control, fully turgid leaves) induced by water deficit (Fig. 4) was highly positively correlated with the genotypic critical WSD (Table 2). Based on the azocaseinolytic (Fig. 3) and cysteine endoproteinase activities (Fig. 4) it was possible to calculate the activity of cysteine endoproteinase in total proteolytic activities. Upon water deficit, the activity of cysteine endoproteinases increased up to 77% in fifth leaves with a 2-fold increase in flag leaves. The pattern of enhancement of this specific ATP-independent protein breakdown followed the same course as the total azocaseinolytic activity, i.e. the enhancement being lower in tolerant genotypes with a higher critical WSD (Fig. 4).
|
ATP-dependent 125I-lysozyme breakdown
In contrast to azocaseinolytic activity, the ATP-dependent 125I-lysozyme degradation (% of total degradation of labelled lysozyme, Fig. 5) was low in dehydrated fifth leaves of genotypes with a critical WSD of 55–67%, but it was increased in genotypes with higher critical WSD (Fig. 5A). In genotypes with critical WSD above 75% the level of ATP-dependent radiolabelled lysozyme degradation is higher than in the control well-watered fifth leaves. As a result, critical WSD is highly correlated with the ATP-dependent proteolytic activity (Table 2). Generally in flag leaves (Fig. 5B, C), 125I-lysozyme degradation is about three times higher than at the shooting phase (Fig. 5A). In the non-acclimated leaves, water deficit resulted in decreased ATP-dependent 125I-lysozyme degradation which negatively correlated with critical WSD. Acclimation of the flag leaves prevented a decrease in ATP-dependent proteolytic activity in all genotypes. Under water deficit, ATP-dependent 125I-lysozyme degradation was enhanced (Fig. 5C) compared to the dehydrated non-acclimated leaves (Fig. 5B). This increase was not related to the critical WSD (Table 2) and the activity of ATP-dependent 125I-lysozyme degradation was the same in all genotypes.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The increase of ATP-independent and reduction of ATP-dependent proteolytic activity under water deficit previously found in the leaves of spring wheat (Zagda
ska and Winiewski, 1996, 1998) has been confirmed in the present studies with wheat genotypes which differ in critical WSD. Critical WSD, used in our work to characterize plant response to water deficiency, seems to be an important determinant of enzyme mediated processes and thus, plant metabolic activity. Also a preferential enhancement of cysteine proteinases activities and the increased contribution of these proteinases in total proteolytic activity is consistent with our previous results (Zagdaska and Winiewski, 1996). It has now been demonstrated that both of these responses are genotype-dependent at each developmental stage.
At the same time both critical WSD and proteolytic response depended on the phase of plant development. Generally, wheat genotypes were more tolerant in the shooting than in the heading phase. Although determinations of critical WSD (leaf dehydration tolerance) may allow the prediction of the plant growth and yield responses to water deficiency in grain sorghum (Sullivan, 1972), spring wheat (Zagdaska and Pacanowska, 1979) and other plants (Levitt, 1980; Blum, 1988) at present it is not clear whether the observed variability in critical WSD during plant ontogeny would be of some benefit for plants experiencing water deficit. Despite the significant progress that has been achieved in analyses of drought-inducible gene expression and cis- and trans-acting elements involved in stress-responsive gene expression, present understanding of dehydration tolerance mechanisms remains far from complete (Shinozaki et al., 1999). Nevertheless at each developmental stage significant correlation existed between critical WSD and two types of proteolytic activities.
Significant correlation was found between the content of soluble protein and critical WSD, negative in dehydrated fifth leaves and positive in dehydrated flag leaves. Higher ATP-dependent 125I-lysozyme degradation and lower azocaseinolytic activities referred to genotypes set in order of increasing critical WSD were associated with a distinct reduction of soluble protein content in the fifth leaves. In non-acclimated flag leaves, a negative correlation between protein content and either type of proteolytic activity was found. These findings indicate that the reduction in soluble protein content of leaves upon exposure to water deficit is associated with the phase of plant development rather than critical WSD. This is consistent with the finding by Brouquisse et al. that the decrease in protein content differed according to the maize tissue (Brouquisse et al., 1998). Thus, it may be proposed that at the shooting phase protein hydrolysates are exported to sinks such as the youngest leaves. In contrast, at the heading phase amino acids for the synthesis of new, stress-induced proteins more suited for survival under water deficiency are needed (Callis, 1995; Ingram and Bartels, 1996; Bray, 1997). The above suggestion raises a question concerning the role of endoproteinases in the response to soil water deficit.
Increase in cysteine endoproteinase activities, negatively correlated with critical WSD of the studied leaves under water deficiency, suggests that specific vacuolar enzymes are potentially involved in the plant response to dehydration. This increase may be due either to the activation of pre-existing enzymes or to de novo synthesis, but the results presented do not make it possible to conclude whether these endoproteinases are new or pre-existing or both. However, it has been shown recently that PsCyp15a cDNA, which previously was recognized as an up-regulated transcript in wilted pea shoots (Guerrero et al., 1990; Jones and Mullet, 1995), is a cysteine proteinase associated with the vacuole and cytoplasmic vesicles (Vincent and Brewin, 2000). Since the proposed function of this enzyme is in maintenance of cell turgor (Vincent and Brewin, 2000), higher activity of cysteine endoproteinase in genotypes susceptible to water deficiency is logical.
At the fifth leaf stage (shooting), the lower azocaseinolytic activity in dehydrated leaves was compensated in genotypes with a higher critical WSD by the extravacuolar energy-dependent 125I-lysozyme degradation. In these genotypes, this type of proteolysis exceeded even that in the non-dehydrated leaves. A similar mutual compensation of the proteolytic activities was observed in the dehydrated non-acclimated flag leaves at the heading phase in genotypes with a lower critical WSD. At this phase of development when plants were less tolerant than at the fifth leaf stage the ATP-independent vacuolar proteolytic activity was about 50% lower and the ATP-dependent proteolytic activity about 5-fold higher than in the leaves at the shooting phase. However, in genotypes with a higher critical WSD such compensation did not take place. Generally, in the non-acclimated flag leaves lower proteolytic activity of both types was associated with a higher critical WSD.
It is of great interest that, under water deprivation conditions, the ATP-dependent extravacuolar proteolytic activity in the acclimated flag leaves, as compared to the non-acclimated ones, increased irrespective of the genotype. This is consistent with previous findings that high-energy expenditure for proteolytic activity during dehydration of acclimated plants was not related to the critical WSD (Zagdaska, 1995). However, a 2-fold increase in ATP-dependent protein degradation in sensitive genotypes and an 8–10-fold increase in tolerant genotypes, but to the same level for all examined genotypes, indicates a profound shift in energy-dependent proteolytic activity of genotypes with a higher critical WSD upon acclimation. Also it may be noted that in control non-stressed fifth leaves and in the non-acclimated flag leaves azocaseinolytic activity did not depend on the response of the genotype to water stress. In contrast to ATP-independent proteolytic activity the ATP-dependent extravacuolar degradation of 125I-lysozyme was highly negatively correlated with the critical WSD. These current findings demonstrate that the molecular and metabolic basis for the correlation between the genotypic values of critical WSD and either type of proteolytic activity, presented in Table 2, is highly complex. Moreover, until recently the mechanisms by which multiple pathways may be involved in degradation of the same protein remain unknown (Tanaka, 1998). This not only suggests that the metabolic cross-talk between ATP-dependent and independent degradation of proteins is associated with the precise regulation of energy input but also may imply differences in energy metabolism between the tolerant and the sensitive genotypes.
When considering the energy requirement in the remodelling of cellular proteins in dehydrated leaves, it has to be remembered that this is a multi-step process. Energy is needed not only for the phosphorylation of many proteins prior to ubiquitination (Tanaka, 1998), and ubiquitination of proteins targeted for degradation (Varschavsky, 1997) but also to final degradation by the proteasome (Baumeister et al., 1998). Moreover, in some cases ATP-dependent ubiquitination of proteins is needed for their further energy-independent degradation in vacuoles (Hicke and Riezman, 1996; Tanaka, 1998). In this case energy-dependent proteolysis supplemented vacuolar proteolysis. Moreover, ATP is probably needed for the transport of substrates to vacuoles (Vierstra, 1993). Thus, availability of ATP is needed not only for protein degradation via the Ub-proteasome pathway but also for supporting the ATP-independent pathway. Moreover, ubiquitination and also phosphorylation precede ubiquitination of proteins destinated for degradation involve specific proteins (von Kampen et al., 1996; Tanaka, 1998). For global energy-dependent proteolysis in dehydrated wheat leaves availability of ATP should be of decisive importance. Earlier observations indicated that the ATP pool declined upon dehydration although its molar concentrations remained unchanged (Zagdaska, 1991) and at the same time the energy requirement for ATP-dependent proteolysis is increased (Zagdaska, 1995).
|
Acknowledgments |
|---|
This work was supported by a grant from the State Committee for Scientific Research (5 PO6A 013 10). The authors would like to thank Professor Konstancja Raczy
ska-Bojanowska for her valuable discussion and critically reading the manuscript. |
Notes |
|---|
3 To whom correspondence should be addressed. Fax: +48 22 725 4714. E-mail: zagdanska{at}delta.sggw.waw.pl
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Andersson B, Aro E-M. 1997. Proteolytic activities and proteases of plant chloroplasts. Physiologia Plantarum 100, 780–793.
Bartels D, Nelson D. 1994. Approaches to improve stress tolerance using molecular genetics. Plant, Cell and Environment 17, 659–667.
Baumeister W, Cejka Z, Kania M, Seemuller E. 1998. The proteasome: a macromolecular assembly designed to confirm proteolysis to a nanocompartment. Biological Chemistry 378, 121–130.
Blom-Zandstra M, Koot HTM, van Hattum J. 1990. Direct isolation of vacuoles from leaf tissue of lettuce (Lactuca sativa) retaining protoplast within the leaves. Physiologia Plantarum 79, 693–699.
Blum A. 1988. Plant breeding for stress environments. Boca Raton, Florida: CRC Press.
Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254.[Web of Science][Medline]
Bray EA. 1997. Plant responses to water deficit. Trends in Plant Science 2, 48–54.
Brouquisse R, Gaudillière J-P, Raymond P. 1998. Induction of a carbon-starvation-related proteolysis in whole maize plants submitted to light/dark cycles and to extended darkness. Plant Physiology 117, 1281–1291.
Callis J. 1995. Regulation of protein degradation. The Plant Cell 7, 845–857.[Web of Science][Medline]
Clarke AK, Gustafsson P, Lidholm JA. 1994. Identification and expression of the chloroplast clpP gene from the conifer Pinus contorta. Plant Molecular Biology 26, 851–862.[Web of Science][Medline]
Fischer A, Brouquisse R, Raymond P. 1998. Influence of senescence and of carbohydrate levels on the pattern of leaf proteases in purple nutsedge (Cyperus rotundus). Physiologia Plantarum 102, 385–395.
Guerrero FD, Jones JT, Mullet JE. 1990. Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Molecular Biology 15, 11–26.[Web of Science][Medline]
Herschko A, Heller H, Elias S, Ciechanover A. 1983. Components of ubiquitin-protein ligase system. Resolution, affinity, purification and role in protein breakdown. Journal of Biological Chemistry 258, 8206–8214.
Hicke L, Riezman H. 1996. Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell 84, 277–287.[Web of Science][Medline]
Huffaker RC. 1990. Proteolytic activity during senescence of plants. New Phytologist 116, 199–231.[Web of Science][Medline]
Ingram J, Bartels D. 1996. The molecular basis of dehydration tolerance in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 377–403.[Web of Science][Medline]
Jones JT, Mullet JE. 1995. A salt- and dehydration-inducible pea gene, Cyp15a, encodes a cell-wall protein with sequence similarity to cysteine proteases. Plant Molecular Biology 28, 1055–1065.[Web of Science][Medline]
Levitt J. 1980. Responses of plants to environmental stresses. New York: Academic Press.
Lin W, Wittenbach VA. 1981. Subcellular localization of proteases in wheat and corn mesophyll protoplats. Plant Physiology 67, 969–972.
Ludlow MM. 1993. Physiological mechanisms of drought resistance. In: Mabry TJ, Nguyen HT, Dixon RA, Bonnes MB, eds. Biotechnology of arid plants. The University of Texas, Austin IC2 Institute, 11–34.
Noodén LD, Guiamét JJ, John J. 1997. Senescence mechanisms. Physiologia Plantarum 101, 746–753.
Parker CW. 1990. Radiolabelling of proteins. In: Deutscher MP, ed. Methods in enzymology, Vol. 162. Academic Press Inc., 723–726.
Rivett AJ. 1994. Multicatalytic endopeptidase complex: proteasome. In: Barret AJ, ed. Methods in enzymology, Vol. 244. Academic Press Inc., 331–350.
Shanklin J, DeWitt ND, Flanagan JM. 1995. The stroma of higher plant plastids contain ClpP and ClpC, functional homologs of Escherichia coli ClpP and ClpA: an archetypal two-component ATP-dependent protease. The Plant Cell 7, 1713–1722.[Abstract]
Shinozaki K, Yamaguchi-Shinozaki K, Liu Q, Kasuga M, Ichimura K, Mizoguchi T, Urao T, Miyata S, Nakashima K, Shinwari ZK, Abe H, Sakuma Y, Ito T, Seki M. 1999. Molecular responses to drought stress in plants: regulation of gene expression and signal transduction. In: Smallwood MF, Calvert CM, Bowles DJ, eds. Plant responses to environmental stress. Bios Scientific Publishers Ltd., 133–143.
Stocker O. 1929. Das Wasserdefizit von Gefasspflanzen in verschiedenen Klimazonen. Planta 7, 382–387.
Sullivan CY. 1972. Mechanisms of heat and drought resistance in grain sorghum and methods of measurements. In: Rao NPG, House LR, eds. Sorghum in the seventies. New Delhi: Oxford & IBH Publishing Co., 247–264.
Tanaka K. 1998. Proteasomes: structure and biology. Journal of Biochemistry 123, 195–204.
Varschavsky A. 1997. The N-end rule pathway of protein degradation. Genes and Cells 2, 13–28.
Vierstra RD. 1993. Protein degradation in plants. Annual Review of Plant Physiology and Plant Molecular Biology 44, 385–410.[Web of Science]
Vincent JL, Brewin NJ. 2000. Immunolocalization of a cysteine protease in vacuoles, vesicles and symbiosomes of pea nodule cells. Plant Physiology 123, 521–530.
von Kampen J, Wettern M, Schulz M. 1996. The ubiqutin system in plants. Physiologia Plantarum 97, 618–624.
Wagner GJ, Mulready P, Cutt J. 1981. Vacuole/extravacuole distribution of soluble protease in Hippeastrum petal and Triticum leaf protoplasts. Plant Physiology 68, 1081–1089.
Zadoks JC, Chang TT, Konzak CF. 1974. A decimal code for the growth stages of cereals. Weed Research 14, 415–421.
Zagdaska B, Pacanowska A. 1979. Dehydration tolerance of spring wheat and its relation to plant growth and productivity under soil drought conditions. Biologia Plantarum 21, 452–461.
Zagdaska B. 1991. Water stress-induced changes in adenine nucleotide level in wheat leaves. Plant Physiology and Biochemistry 29, 409–413.
Zagdaska B. 1995. Respiratory energy demand for protein turnover and ion transport in wheat leaves upon water deficit. Physiologia Plantarum 95, 428–436.
Zagdaska B, Winiewski K. 1996. Endoproteinase activities in wheat leaves upon water deficits. Acta Biochimica Polonica 43, 512–520.
Zagdaska B, Winiewski K. 1998. ATP-dependent proteolysis contributes to the acclimation-induced drought resistance in spring wheat. Acta Physiologiae Plantarum 20, 55–58.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||