Proteins alterations associated with salinity, desiccation, high and low temperature stresses and abscisic acid application in seedlings of Pusa 169, a high-yielding rice (Oryza sativa L.) cultivar

Ashwani Pareek, Sneh Lata Singla and Anil Grover*

Department of Genetics, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi 110 021, India.

Abiotic stresses (such as salinity, water stress and low and high temperatures) adversely affect growth of rice. The objectives of the present study were to make a comparative assessment of various polypeptides which are altered in response to different abiotic stresses and to see the utility of stress proteins in distinguishing between the stress response of stress-sensitive and tolerant cultivars in rice. We have identified nearly 73 steady-state high and low molecular weight proteins which are up- or down-accumulated in shoot and root tissues of an indica rice cultivar (Pusa 169) when intact seedlings are subjected to salinity (NaCl), air drying and high and low temperatures. A set of five polypeptides ranging in molecular weight from 80 to 100 kDa was found to be co-regulated in response to various stresses studied here. Exogenous application of abscisic acid mimicked several, but not all, of the protein perturbations caused by the imposition of stresses. Comparison of salt-sensitive (Basmati 370 and Pusa 169) and tolerant (CSR 10 and CSR 19) rice cultivars revealed that most stress proteins appear to accumulate in a similar pattern in the contrasting types; however, differential pattern of accumulation of two specific stress proteins with molecular weights of 104 and 90 kDa was noted by Western blotting in CSR 19 as against CSR 10, Pusa 169 and Basmati 370 cultivars.

FIELD crops experience a multitude of stress conditions. In order to improve the performance of crops growing under stresses, it is important to understand how plants cope under such conditions. As the sublethal stress conditions generally provide protection against the ensuing lethal stress levels1, it is likely that metabolic alterations during sublethal stress conditions have a role to play in inducing stress tolerance. Importantly, several proteins are synthesized and accumulated in plant tissues under a range of stress conditions. Such proteins, referred to as stress proteins, have been noted to be

*For correspondence. (e-mail:pmb@dusc.ernet.in)

induced in response to high temperature, low temperature, salinity, drought and several other stress factors1–3.

Information on stress proteins is important for several reasons. Assessment of the sum total of alterations of cellular proteins/transcripts provides a measure of the complexity of stress response4–6, which in other words, reveals the possible number of genes that are altered in response to a given stress agent. Studies on activation of specific genes/proteins induced by stresses as well as abscisic acid (ABA) have been useful in elucidating the involvement of ABA as a cellular signal in stress–plant interactions7. Importantly, it has also been shown that contrasting genotypes in some instances can be distinguished based on stress proteins/genes4,5,8. Identification of protein markers for stress tolerance could provide an easy tool to plant breeders for analysing tolerance trait in the segregating population of a cross between stress-tolerant and sensitive cultivars. It is shown that response of plants to various abiotic stresses is common to an extent as plants showing tolerance to one stress type may as well show tolerance to another stress type9. It is imperative that stress proteins may have a role in providing plants the ability to cross-adapt10. Finally, it is unequivocally shown, at least in selected instances, that stress proteins play a crucial role for assisting the cells to carry out their metabolic activities during adverse conditions1.

Rice is a staple food for a large chunk of the population. Grain yield of rice is adversely affected by a host of abiotic stresses11. Several studies have been undertaken in the recent past to analyse gene/protein alterations induced in rice in response to salt, water stress, cold stress and high temperature stress10,12–18. However, most of the above studies have remained confined to specified genes/gene products rather than presenting an overall picture of the protein changes. To our knowledge, there is as yet no report in rice in which protein alterations induced by various abiotic stresses have been compared in a single study. In this study, we provide data on stress proteins which are altered in response to

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Table 1.  Summary of various rice cultivars and stress treatments employed in the present study

high temperature, cold stress, salinity, air drying and abscisic acid application in both shoot and root tissues of a high-yielding rice cultivar Pusa 169. Further, expression patterns are shown for two specific stress proteins (namely stress-associated proteins of 104 and 90 kDa; Pareek et al.18) in four rice cultivars (indicas) which showed a contrast in their salt response at the seedling stage.

Materials and methods

The cultivars and stress treatments employed in the present work are summarized in Table 1.

Effect of different stresses on seed germination and seedling growth in cultivar Pusa 169

Effects of different abiotic stresses on stress response of cultivar Pusa 169 of rice (Oryza sativa L.) were analysed at the stage of seed germination and seedling growth in this study. For germination tests, seeds were placed on wet cotton pads inside glass beakers. For salinity stress, solutions of various NaCl concentrations (i.e. 100 mM, 200 mM and 300 mM; distilled water was taken as control) were added to the beakers which were then kept at 28°C in a growth chamber (Heraeus, Germany; 100% RH). For low temperature stress, distilled water was added to the glass beakers and they were kept in water baths pre-set at requisite temperatures (i.e. 20, 15, 10 and 5°C; 28°C was taken as control). For high temperature stress, distilled water was added to the beakers which were in turn kept in water baths pre-set at the requisite temperatures (i.e. 35, 40, 45 and 50°C; 28°C was taken as control). ABA in various concentrations (10–4, 10–5 and 10–6 M; distilled water was taken as control) was added to the beakers which were kept at 28°C (100% RH). The germination percentage was determined after regular intervals of imbibition.

For seedling growth tests, seedlings were raised on moistened filter paper at 28°C (100% RH) under 12 h/12 h light and dark cycle in the growth chamber. After 4 days of growth, the uniform-sized seedlings (i.e.
1–1.5 cm in length) were transferred to pre-wet thin layer of cotton pads taken in 100 ml glass beakers. The seedlings were allowed to acclimatize for 24 h under the above conditions and then subjected to various stress conditions. For salinity stress, solutions of various NaCl concentrations (100, 200, 300 and 400 mM) were added to the glass beakers which were kept at 28°C (100% RH). For low temperature stress, the beakers containing the seedlings were kept inside pre-set water baths at various temperatures (i.e. 20, 15, 10 and 5°C). Similarly, beakers containing the seedlings were kept in water bath pre-set at the requisite temperatures (i.e. 35, 40, 45 and 50°C) for high temperature stress. ABA solutions of various concentrations (10–4, 10–5 and 10–6 M) were added to the bathing medium in place of distilled water for analysing the effects of ABA on seedling growth.

Analysis of stress-associated protein alterations in cultivar Pusa 169

The details for subjecting Pusa 169 cultivar seedlings to stress conditions were the same as described above. Extraction of soluble proteins from both shoot and root tissues was carried out as described before17–19. Essentially, proteins were extracted in a buffer consisting of 30 mM Tris-HCl (pH 8.5), 1 mM ascorbic acid, 1 mM EDTA-Na, 5 mM MgCl2, 1 mM dithiothreitol and 1 mM phenylmethylsulphonyl fluoride. Insoluble polyvinylpolypyrrolidone (50 mg/g fresh weight of tissue) was added to the extraction buffer. The crude homogenate was spun twice (12,000 g, 4° C, 15 min each time). Buffer-soluble proteins were precipitated using 8 vol of chilled acetone (containing 10 mM 2-mercaptoethanol). Precipitated proteins were solubilized as suggested by Laemmli20. Estimation of protein content was carried out following the procedure of Bradford21. An aliquot of the Laemmli buffer-solubilized proteins was precipitated using equal volume of 20% trichloroacetic acid, and the pellet was redissolved in 1 M NaOH, to minimize interference caused due to sodium dodecyl sulphate (SDS) and 2-mercaptoethanol in protein quantification. The polypeptides were subjected to one-dimensional, using either uniform (7.5% polyacrylamide) or gradient (15–22% linear polyacrylamide) SDS-gel electrophoresis. Silver staining was performed as described earlier19.

Analysis of stress-associated protein alterations in four different cultivars

Experiment on salt stress-responsive alterations in 104 and 90 kDa proteins was undertaken employing four cultivars namely Pusa 169 (salt-sensitive), Basmati 370 (salt-sensitive), CSR 10 (salt-tolerant) and CSR 19 (salt-tolerant). For assessing the salt response of the seedlings of these cultivars, seeds were germinated and grown at different levels of NaCl as described above for cultivar Pusa 169 seedlings. The seedlings were harvested after 7 days of salinity stress (50 to 200 mM NaCl solution) and the shoots of the seedlings were separated and employed for fresh weight and length measurements. Average of three sets of each treatment (with 30 seedlings in each set) was calculated for each of the cultivars.

For analysis of specific proteins induced in response to NaCl stress, seedlings of these cultivars were raised under normal conditions and 5-day-old uniform-sized seedlings were subjected to salinity stress (details of stress treatments mentioned above) for requisite durations. Controls were grown in distilled water for all the cultivars. Protein extraction and protein gel electrophoresis techniques were same as described above for Pusa 169 cultivar. Procedure of Western blotting was same as described by Pareek et al.18. The nitrocellulose filter was incubated with polyclonal antibodies raised against anti rice SAP 104 or anti rice SAP 90 antibodies18. The specific position of antigen–antibody complex was visualized using peroxidase-linked secondary antibodies.

Results

Stress response of Pusa 169 cultivar

In control (seeds germinated in distilled water), germination of seeds was seen after 36 h of inbibition and in the next 24 h nearly 100% seeds showed germination. The germination process was delayed in response to NaCl application as the initial emergence of embryonic axis was apparent after 70 h in case of 100 mM NaCl and after 90 h in case of 200 mM NaCl. No germination was seen when 300 mM NaCl concentration was used (Figure 1 a). The NaCl stress also caused a reduction in percentage of the seeds showing germination. In response to 200 mM NaCl, nearly 45% of the seeds germinated. Seedlings germinated and grown at 100 mM NaCl for 7 days showed poor extension growth of the shoot system (Figure 1 b). The roots of these plants showed characteristic arrest of branching. With further increase in NaCl concentration to 200 mM, the embryonic axis emerged from the seeds but it did not show any further growth with respect to its extension. No roots were seen in these seedlings. Further, seedlings germinated in control conditions (5-day-old in distilled water) were transferred to solutions containing different concentrations of NaCl. After 7 days, the seedlings grown in 100 mM NaCl solution showed reduced growth with respect to both the primary shoot axis and the root (Figure 1 c). With further increase in NaCl concentration, the root morphology was drastically affected as the development of fibrous root system was not seen in NaCl-treated seedlings.

With an 8° C decline in temperature (from 28° to 20° C), the initiation of germination was delayed by nearly 12 h and maximum of 85% germination was scored after 120 h of the imbibition (Figure 1 d). However, the seedlings germinated and grown at 20° C showed extremely poor growth as the embryonic axis failed to show any further growth and the root axis showed no branching even after 7 days (Figure 1 e). With further decline in temperature to 10 and 5° C, the seed germination process was seen to be completely halted. Further, 5-day-old seedlings germinated under control conditions were subjected to the low temperature stress levels. After 7 days of transfer at 20° C, seedlings showed growth comparable to those grown and kept at 28° C (Figure 1 f). There was slight reduction in growth of seedlings at 15° C as compared to 28° C-grown seedlings but with further decline in temperature, the seedlings failed to show any extension growth of the shoot and root.

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Figure caption 1

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Figure caption 2

 

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Figure caption 3

Importantly, the process of seed germination was hastened at 35° C as the initial embryonal axis emerged from the seeds kept at this temperature within first 24 h of imbibition. In both 28 and 35° C-grown seeds, nearly 100% seed germination was seen at about the same time (i.e. 48 h). Increase in germination temperature to 45° C delayed the process of germination up to 48 h of imbibition and the extent of germination in this set was less than 60% (Figure 1 g). The seeds germinated and grown at 35° C for 7 days showed higher extension growth than the 28° C maintained seedlings (Figure 1 h). On the other hand, seedling growth was completely arrested at 45° and 50° C. Further, the seedlings grown at 28° C for 5 days when transferred to 35° C showed primary leaf and root growth comparable to those germinated and grown at 28° C but these seedlings were characteristically devoid of second leaf expansion (Figure 1 i). The second leaf did not emerge with further increase in temperature.

Application of ABA (10–6 M) delayed the process of germination to 45 h of imbibition and the maximum germination was up to 80% in this set (Figure 1 j ). Importantly, 10–5 M ABA also gave maximum of 80% germination but in this case, the process of germination was apparent at 96 h of the seed germination. The seeds germinated and grown at 10–6 M ABA for 7 days showed significant reduction in the shoot extension growth (Figure 1 k). On the other hand, seedling growth was completely arrested at 10–5 and 10–4 M ABA. In further studies, seedlings germinated and maintained in water for 5 days were transferred to different levels of ABA. It was observed that after 7 days of growth, secondary leaf showed more expansion in 10–6 M ABA concentration as compared to control seedlings maintained in water (Figure 1 l). Also, roots were thicker at 10–6 M ABA as compared to 28° C-grown seedlings. Further increase to 10–5 to 10–4 M ABA concentration appeared detrimental to the shoot and root growth of the seedlings.

Stress proteins of Pusa 169 cultivar

Soluble proteins from rice seedlings (cultivar Pusa 169), which were exposed to a multitude of abiotic stresses were examined by SDS-gel electrophoresis in this study. Protein profiles from stressed samples were compared with unstressed control tissues harvested at the start of

the experiment. Considering that differences in protein profiles between stressed and unstressed samples could partly be due to growth of the seedlings (as some of the experiments continued from day 5 of the seedling age to day 9), protein changes associated with development of rice seedlings in this time period were also analysed.

Levels of 100, 76, 55, 49, 44, 40, 37, 31, 26, 16.8 and 15 kDa polypeptides for shoots (Figures 2 and 3) and 100, 78, 36 and 27.5 kDa polypeptides for roots (Figures 4 and 5) were increased during growth. The 55 kDa protein showed prominent accumulation in shoot tissues during growth from day 5 to day 9 of seedlings (Figure 2). Levels of proteins with molecular weights of 78, 65, 63, 58, 33, 22, 20 and 10.2 kDa for shoots (Figures 2 and 3) and 81, 65, 52, 25, 23, 22, 20 and 17 kDa for root tissues (Figures 4 and 5) were declined during this period.

Application of various stress conditions caused marked changes in the protein profiles of rice seedlings. Molecular weights of the prominent protein alterations are shown on the right side of each panel in Figures 2–5. These alterations ranged in molecular weight from as low as 10.2 kDa (in response to salinity and desiccation stress) to as high as 123 kDa (in response to salinity stress). On the whole, 29, 21, 13 and 35 polypeptides altered in response to salinity, desiccation, low temperature and high temperature stresses, respectively, in shoots (Figures 2 and 3). The equivalent number of polypeptides were 15, 14, 16 and 19 for salinity, desiccation, low temperature and high temperature stresses, respectively, in root samples (Figures 4 and 5).

To reveal the relationship amongst different stresses with respect to protein changes, protein alterations scored for different stresses are presented in the form of Venn diagrams, separately for the shoot and root tissues, in Figures 6 and 7. It is clear from these data that several proteins show overlapping patterns with respect to their stress-inducibility. These overlapping patterns stretch from two stresses to a whole range of stress conditions for individual proteins. For instance, patterns of alterations for polypeptides of molecular weights 76 and 23.5 kDa were common to salinity and high temperature stress in shoots (Figure 6). Stress proteins with molecular weights of 100, 91, 87, 85, 78, 60, 58, 55 and 16.2 kDa in shoots and 100, 91, 87, 85, 81, 78, 76, 63, 47, 44.5 and 40.5 kDa in roots were induced in response

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Figure 1.  Effect of different abiotic stresses on seed germination and growth of the seedlings emerging from the seeds germinated and maintained in stress conditions and seedlings germinated in control and then subjected to various stress conditions, in cultivar Pusa 169. a, Effect of salinity stress on seed germination. b, Growth pattern of seedlings germinated and maintained at various concentrations of NaCl. c, Growth pattern of seedlings germinated on to distilled water and then transferred to various levels of NaCl as mentioned. d, Effect of low temperature on seed germination. e, Growth pattern of seedlings germinated and maintained at various low temperatures. f, Growth pattern of seedlings germinated at normal temperature (28° C) and then transferred to various degrees of low temperature. g, Effect of high temperature on seed germination. h, Growth pattern of seedlings germinated and maintained at various high temperatures. i, Growth pattern of seedlings germinated at normal temperature (28° C) and then transferred to various degrees of high temperatures. j, Effect of various concentrations of abscisic acid on seed germination. k, Growth pattern of seedlings germinated and maintained at various concentrations of ABA. l, Growth pattern of seedlings germinated under normal conditions (without ABA) and then transferred to various concentrations of ABA.

 

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Figure 2.  Electrophoretic profiles of the high molecular weight proteins of the shoots of rice seedlings (cv. Pusa 169) as resolved on 7.5% uniform acrylamide concentration SDS-gel in response to various stress treatments. 20 m g crude protein was loaded in each lane and the gel was stained with silver nitrate. Proteins marked with asterisk (*) are those which decline in response to the stress treatments and those shown but are not marked with asterisk increase during the stress treatments. Numbers shown with various marks are the molecular weights (in kDa) of proteins. Duration of each treatment is shown at the top of each lane (ABA was given for 24 h). Positions of the standard molecular weight markers are shown towards the left side of the figure. C, control.

Figure 3.  Electrophoretic profiles of the low molecular weight proteins of shoots of rice seedlings (cv. Pusa 169) as resolved on 15–22% linear acrylamide gradient SDS-gel in response to various stress treatments. Other details remain the same as in Figure 2.

 

to various stresses (Figures 6 and 7). Several polypeptide alteration patterns were unique to a given stress signal only. In shoots, polypeptides of molecular weights 123, 21.5 and 13 kDa accumulated specifically in response to salinity stress and equivalent polypeptides for desiccation stress were those with molecular weights of 117, 21 and 10.2 kDa (Figure 6). Polypeptides of molecular weights 104, 39.5, 36, 31, 27.5 and 16 kDa were specifically accumulated in response to high temp-
erature stress while a 17 kDa polypeptide was found associated to low temperature stress in root tissues (Figure 7).

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Figure 4.  Electrophoretic profiles of the high molecular weight proteins of roots of rice seedlings (cv. Pusa 169) as resolved on 7.5% uniform acrylamide concentration SDS-gel in response to various stress treatments. Other details remain the same as in Figure 2.

Figure 5.  Electrophoretic profiles of the low molecular weight proteins of roots of rice seedlings (cv. Pusa 169) as resolved on 15–22% linear acrylamide gradient SDS-gel in response to various stress treatments. Other details remain the same as in Figure 2.

Application of ABA to intact rice seedlings resulted in three kinds of protein alterations: (i) The polypeptides with molecular weights of 100, 87, 85, 78, 60 and 55 kDa for shoots and 100, 91, 87, 85, 78, 63, 47 and 40.5 kDa for roots were accumulated in response to ABA application as well as in response to all stresses tested in this study (Figures 6 and 7). (ii) However, some ABA-associated protein alterations were shared by specific stress conditions. For example, a 14.5 kDa protein was accumulated in response to ABA as well as desiccation stress in shoot tissues. (iii) The ABA-associated alteration patterns of a few proteins like 81

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Figure 6.  Venn diagram showing proteins which accumulate in response to various stresses in shoots of rice seedlings (cv. Pusa 169). This analysis is based upon the alterations in steady state protein profiles as revealed by 7.5% uniform concentration and 15–22% linear acrylamide gradient SDS-gel. Molecular masses (kDa) of individual stress proteins are shown in the figure, those marked with asterisk (*) decline in response to stress while others increase in response to stress treatment.

Figure 7.  Venn diagram showing proteins which accumulate in response to various stresses in roots of rice seedlings (cv. Pusa 169). Other details remain the same as in Figure 6.

and 35.5 kDa in root tissues of rice seedlings were not co-triggered by any of the stress conditions employed here.

Analysis of four contrasting indica cultivars

Pusa 169, Basmati 370, CSR 10 and CSR 19 seedlings were analysed for their salt response by subjecting them

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Figure 8.  Analysis of contrasting genotypes of rice for levels of SAP expression. a, Histogram showing average shoot length per seedling of various rice cultivars. For this analysis, shoot length of seedlings which were germinated under control conditions (without NaCl) were taken as 100% and the values of other treatments were calculated accordingly. Standard error for each reading is indicated in the figure. b, Histogram showing average fresh weight of shoots per seedling. Other details remain the same as in a. c, Western blot showing the levels of accumulation of SAP 104 (upper panel) and SAP 90 (lower panel) in response to NaCl stress. For SAP 104, 12 m g protein and for SAP 90, 2 m g total protein was resolved on 7.5% SDS-gel and probed with rice anti SAP 104 and anti SAP 90 antibodies. Position of these polypeptides on blots is indicated by arrow. The details of treatments are indicated on the top of each lane. d, Morphology of 5-day-old seedlings of cv. Pusa 169 and CSR 10 before subjecting them to NaCl stress. e, Morphology of seedlings of cv Pusa 169 and CSR 10 after subjecting them to 200 mM NaCl stress for 7 days. Note the change in root morphology of the two cultivars.

 

to 50 to 200 mM NaCl and assessing their growth by gain in fresh weight and shoot length. Both CSR 10 and CSR 19 rice types showed more growth (at all concentrations of NaCl) as compared to Pusa 169 and Basmati 370, indicating that both CSR types are relatively more salt tolerant at seedling stage (Figure 8 a, b). Distinctive salt-associated changes in protein profiles were noted for all the cultivars and these alterations resembled those marked for Pusa 169 as described earlier (Figures 2–5). This analysis showed no difference among the four cultivars in response to salt-induced protein alterations. Levels of 104 kDa (SAP 104) and 90 kDa (SAP 90, which represents pooled fraction of 87 and 85 kDa polypeptides18) were assessed by Western blotting using specific antibodies. Anti-rice SAP 104 antibodies showed that SAP 104 protein is not seen in control tissues but is induced with response to NaCl application (Figure 8 c). Levels of induction of SAP 104 protein were comparable in Pusa 169, Basmati 370 and CSR 10. However, CSR 19 seedlings did not appear to accumulate this protein in response to NaCl application. Anti rice SAP 90 antibodies revealed that control rice cells contain low levels of SAP 90 protein. Levels of this protein were increased in response to salt application in Pusa 169, Basmati 370 and CSR 10 cultivars (Figure 8 c). Similar to SAP 104, no significant difference was noted for SAP 90 protein in CSR 19 cultivar in response to NaCl application. Morphological changes in seedlings of these cultivars before and after stress treatment are shown in Figure 8 d, e.

Discussion

This study was undertaken with two objectives: firstly, to make a comparative assessment of various polypeptides which are altered in response to different abiotic stress conditions and secondly, to see utility of such proteins in distinguishing between the stress response of stress-sensitive and tolerant cultivars. The resolution and utility of one-dimensional SDS-gel electrophoresis system was improved in this study by practising the following technical points: (i) for better resolution of proteins in the high molecular weight (HMW) range, proteins were over-run for 1–2 h on 7.5% SDS-gel, (ii) linear gradient of acrylamide in the range of 15–22% was employed for the effective separation of low molecular weight (LMW) stress proteins and (iii) proteins were stained with highly sensitive silver staining which can detect protein in nanogram range.

Pusa 169 is an ideal high-yielding rice cultivar recommended for cultivation in northern India22. In the initial course of this study, we analysed the stress response of this cultivar. From the comprehensive picture which emerged from these studies, it is inferred that application of NaCl reduced vigour of seedlings at all the concentrations employed, indicating its salt-sensitivity. This observation is in conformity with earlier reports on salt-sensitivity of rice23,24. Increase of temperature to 35° C appeared to hasten the process of seed germination, which suggested that high temperature (to the extent of 35° C) has no adverse effect on seed germination in this crop. In wheat also, high temperature (30° C) has been shown to enhance the growth of seedlings25,26. According to Howarth and Ougham27, rice is a thermotolerant crop. However, it was noted in the course of this study that while all other parameters suggested enhanced growth, emergence of the leaf in rice seedlings was arrested when the seeds were grown and maintained at a higher temperature of 35° C. Lowering of temperature by eight degrees (i.e. to 20° C) drastically affected the seedling growth in rice. In relative terms, low temperature stress appeared to affect the growth of rice seedlings more drastically as compared to high temperature stress. In general, both the processes of seed germination as well as seedling growth were found to be affected by various abiotic stresses. On comparative basis, the process of seed germination (the appearance of the embryonal axis) was found to be less affected than the process of seedling growth (extension of embryonal axis). This differential response may reflect more elaborate adaptations present/elicited in seeds for either evading or tolerating the stresses as compared to the seedlings.

Effect of stress conditions on protein profiles

From the general picture of stress proteins emerging from this study (Figures 2–5), two points are noteworthy. Firstly, numerically speaking, more protein alterations were scored in shoots than roots for all the stresses tested in this study. It is possible that this differential response in shoot and root tissues reflect their relative sensitivities to stress conditions. The differential response of shoot and root tissues to stress conditions has earlier been shown with respect to specific stress proteins in wheat4 and barley5. Secondly, maximum number of stress-associated polypeptide alterations were scored for high temperature stress in both shoot and root tissues as compared to other stresses tested in this study. For instance, 4 h of high temperature stress caused more prominent alterations in stress proteins than 96 h of cold shock (Figures 2–5). However, it must be mentioned here that comparisons of the complexity of these stress responses must also take into account that different stresses were imposed for variable durations in this study and no uniform parameter was employed for assessing the cellular level(s) of stress experienced by the tissues in response to imposed conditions.

Molecular weights of proteins which showed up- or down-accumulation in response to individual stresses applied are shown in Figures 2–5. Some of these proteins matched in molecular weights to stress proteins identified by earlier workers. For instance, a 15 kDa protein which accumulated in response to salt stress (Figure 3) appears close to osmotic stress-induced RAB 16 protein noted by Mundy and Chua13. This 15 kDa protein may also have a relation to SalT gene product which is of 15 kDa and is induced by salinity and high temperature stresses15. A 23 kDa stress protein noted in response to salt stress in our study may likewise have a homology to a similar molecular weight protein which is induced by water stress and shows cross reactivity with anti RAB 16 antibodies16. Guy et al.28 have reported that 87 and 85 kDa proteins are expressed in response to drought and cold stress in spinach and, importantly, two similar-sized proteins (87 and 85 kDa) were noted to accumulate in response to diverse stresses examined in this analysis (Figures 2 and 4). Clearly, more research is needed to establish the kinship between the proteins reported here and stress proteins noted by others.

Co-inducibility and non co-inducibility of various stress proteins

A range of stress proteins was noted to be co-triggered by diverse stress signal (Figures 6 and 7). Similar observations with respect to specified genes/gene products have been made by several other groups. For instance, in rice, gene/gene products common to salt and drought15,18,29, salt and high temperature10,18,29, salt and cold18,29, drought and high temperature10,18,29, drought and cold18,29, and high and low temperatures18,29 stresses have been shown. These common proteins may provide a molecular basis to the cross-adaptability phenomenon often noted for different abiotic stresses.

A set of five proteins in the molecular weight range of 80 to 100 kDa (viz. 100, 91, 87, 85 and 78 kDa) show prominent and parallel accumulation in response to high temperature as well as other stress conditions (Figures 2 and 4). It is possible that there might be a common inducer for these proteins. As gene expression is finely tuned with the help of specific trans-acting factors1, it is probable that common trans-acting factors mediate high-level accumulation of these proteins. It is further interesting to speculate that these proteins might be components of the same pathway and thus need to be co-regulated. Detailed characterization of these proteins/
corresponding genes may provide useful information on how various cellular signals mediate stress responses at the molecular level.

Apart from the proteins which were co-triggered, several proteins specific to a given stress type were noted in this study. The proteins unique to a given stress signal may play role(s) in governing stress-specific cellular responses30.

Involvement of ABA in induction of stress proteins

Multiplicity of ABA action in eliciting stress-specific genes is shown in several instances7. Rice stress proteins showed two types of patterns. One class of proteins showed inducibility in response to stress conditions as well as ABA. These proteins might have ABA as a signal transduction molecule. In rice, ABA involvement in activation of specified genes/gene products linked to drought stress, salt stress, cold stress and high temperature stress13,15,17,18 have been previously reported. On the other hand, another class of proteins showed inducibility with stress conditions but not ABA. There are several reports highlighting this pattern too. In rice, leaf-specific WcS 19 gene is shown to be induced by light during acclimation to low temperature but is not affected by ABA (ref. 31). Taken together, this analysis may indicate that the role(s) of ABA may be diverse and there might be multiple signal transduction pathways for stress responses which may or may not involve ABA.

Development-associated changes in protein levels

Associated with the development of rice seedlings from day 5 through day 9, a set of specific proteins were accumulated while a few other proteins showed a decline in shoot tissues. It is probable that proteins which accumulate in this period are associated with increased complexity of cellular activities (such as acquisition of enhanced photosynthetic activity, extension/growth of seedlings, etc.) during development. On the other hand, proteins which are important for supporting early growth based on endosperm reserves are expected to decline during the later phases of growth. The onset of stress conditions appear to modulate the expression of selected development-associated proteins. For instance, a 100 kDa protein was accumulated in the seedlings during the normal development to a marginal extent. This protein was elicited within 4 to 8 h of the high temperature stress in both shoot and root tissues (Figures 2 and 4). Exposure of seedlings to NaCl stress or water stress also modulated accumulation levels of this protein. Another point of interest relates to the 55 kDa protein which showed prominent increase during development. From the molecular weight point of view and considering that this protein is present in appreciably high levels in shoots (and not in root tissues), it appears that the 55 kDa polypeptide might correspond to the large subunit (LSU) of ribulose bisphosphate carboxylase (RuBisCO) enzyme. We are presently characterizing this 55 kDa protein using anti LSU-RuBisCO antibodies.

Differential accumulation of stress proteins in contrasting rice types

The criterion for a stress protein to be considered as molecular marker is that it should show differential accumulation pattern with respect to tolerant and sensitive cultivars. Recently, Moons et al.32 have shown that salt-tolerant cultivars of rice, namely Pokkali and Nonabokra can be differentiated from salt-sensitive Taichung native 1 cultivar on the basis of transcript abundance of the late embryogenesis abundant (group 3 LEA) proteins. The CSR varieties of rice are reportedly salt-tolerant with respect to the dry matter production and crop productivity33 while Basmati 370 is considered to be a salt sensitive-cultivar34. At the seedling stage, CSR 10 and CSR 19 showed more salt tolerance than Basmati 370 and Pusa 169 in this study (Figure 8). SDS-gel electrophoretic profiles of control (uninduced) and salt-stressed seedlings of all these cultivars showed protein alterations equivalent to that of Pusa 169 cultivar mentioned above. Importantly, on the basis of Western blotting data, relative levels of two SAPs (104 and 90 kDa proteins) were found to be differential in these cultivars: both SAP 104 and SAP 90 proteins were insignificantly accumulated in CSR 19 while accumulation of these proteins was significant in other cultivars. The two tolerant cultivars (CSR 10 and CSR 19) also differed with respect to the salt-induced relative levels of SAP 104 and 90 accumulation. Documentation of such specific gene expression changes should be crucial for pyramiding different genes associated with salt tolerance for raising superior salt-tolerant genotypes.

 

 

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ACKNOWLEDGEMENTS. Financial assistance to our laboratory from DST, Government of India, The Rockefeller Foundation, USA and DBT, Government of India, is gratefully acknowledged. We thank the Head, Genetics Division, Indian Agricultural Research Institute, New Delhi, Head, Genetics Section, Central Soil Salinity Research Institute, Karnal, and Dr J. K. Batra, Immunochemistry Lab, National Institute of Immunology, New Delhi for generously providing us seeds of Pusa 169 rice cultivar, seeds of CSR types and Basmati 370 and peroxidase-linked secondary antibodies, respectively. A.P. thanks CSIR, New Delhi, for the Research Fellowship Award. S.L.S. is thankful to UGC and DBT for financial support.

 

Received 18 December 1997; revised accepted 17 June 1998

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