- Email: [email protected]
Salt Stress Responsive Polypeptides in Germinating Seeds and Young Seedlings of Indica Rice (Oryza sativa L.)
]. Plant Pbysiol. VoL 143. pp. 250-253 (1994)
Short Communication
Salt Stress Responsive Polypeptides in Germinating Seeds and Young Seedlings of Indica Rice (Oryza sativa L.) U. RADHA RANI and A. R. REDDY Plant Molecular Genetics Laboratory, School of Life Sciences, University of Hyderabad, Hyd.erabad - 500 134, India Received May 26, 1993 · Accepted August 11, 1993
Summary
Changes in protein profiles induced by salt stress were investigated both during germination and early seedling growth in rice. Two predominant polypeptides with apparent mol. wts. of 70 and 23 kDa, respectively, were induced under salt stress in germinating seeds of which the 23 kDa is the most abundant. Two other polypeptides with apparent mol. wts. of 15 and 26 kDa were found to be specifically induced in shoots ofNaCl treated seedlings. These two salt responsive polypeptides (SRPs) were partially purified and their properties investigated. It has been clearly demonstrated that the 15 and 26 kDa SRPs exhibit a unique property of boiling stability.
Key words: Salt stress, salt responsive polypeptides, boiling stability. Abbreviations: SRP
=
salt responsive polypeptide; SDS = Sodium dodecylsulphate.
Introduction
The most prominent class of events of salt-stress response include transient adjustment of intracellular fluctuations in ionic balance (Watad et al., 1986; Binzel et al., 1988; Garbarino and DuPont, 1989) and pronounced changes in metabolism of small molecular weight compounds like proline, betaine, sugars and ABA (Binzel et al., 1987; La Rosa et al., 1987). Another important consequence is the alteration in polypeptide profiles (Bruggeman and Janiesch, 1988; Singh et al., 1985; Hurkman and Tanaka, 1987; Ramagopal and Carr, 1991). These events reflect mainly transient changes in polypeptide synthesis and/or turnover as a function of osmotic adjustment. In rice, not much is known about the biochemical and molecular basis of the salt stress response. Recent studies with salt-treated rice seedlings revealed significant changes in polypeptide profiles and also in gene expression. One such salt-induced gene is sal T, which encodes a 15 kDa polypeptide (Claes et al., 1990). In addition, there is another class of genes in rice that are induced by a number of stresses like dehydration by PEG or desiccation and osmotic stress caused by ABA and also salt, or both (Bostock and Quatrano, 1992, Mundy and Chua, 1988; Skriver and Mundy, 1990). The rab © 1994 by Gustav Fischer Verlag, Stuttgart
16 gene, for instance, belongs to this class of genes (Skriver and Mundy, 1990). The physiological function of these induced proteins and their precise role in salt stress response of rice plant is not yet known. Elucidation of the functional association of these salt responsive polypeptides (SRPs) with salt tolerance of the plant requires characterization of the induced proteins. The present report deals with the induction of specific polypeptides by salt in germinating seeds and growing seedlings of rice. Two of the induced polypeptides were partially purified; they exhibit the property of boiling stability in aqueous solutions.
Materials and Methods Seeds of indica rice (Oryza sativa L. cv. Hamsa) were imbibed in water, surface sterilized with 5% sodium hypochlorite (v/v) for 5 min, thoroughly washed with sterile water and germinated in either water or NaCl solutions upon moistened filter papers in the dark. The average temperature during seedling culture ranged from 28 °C ± 2 °C. In one set of experiments, seeds were germinated in different concentrations of NaCl (50 to 600 mM) for 3 days and the whole seedlings were used for protein studies. In the second set,
Salt responsive polypeptides in rice
66
45 36
29 24
14
.......-
-
-
Fig.1: SDS-PAGE (15%) proftles of shoot proteins of control and NaCl treated 10-day-old Hamsa seedlings. All lanes carry 50 J.Lg protein. Lane A, control; Lane B, 0.25 M NaCl; Lane C, 0.5 M NaCl; Lane D, 0.75M NaCl; Lane E, l.OM NaCl; Lane F, 1.25M NaCl; Lane G, 1.5 M NaCl. Arrows indicate the NaCl induced polypeptides. seeds were germinated in water and allowed to grow for 8 days. The 8-day-old seedlings were treated with 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5M NaCl for 2days. Control plants received only water. Shoots were harvested on the first and second day after treatment and quick frozen in liquid nitrogen and stored at - 80 °C until further analysis. Total proteins were extracted as per Goday et al. (1988). Protein content was determined by the method of Lowry et al. (1951) and the protein extracts were subjected to one dimensional SDS-PAGE according to a modified Laemmli procedure (Laemmli, 1970). The SRP 15 and SRP 26 were purified by electroelution. Both the control and treated protein extracts were subjected to preparative SDS-PAGE and the gels were stained with Coomassie Brilliant Blue. The induced 15 and 26 kDa polypeptide bands were identified, cut out and electroeluted. The eluted proteins were precipitated in cold acetone at -80 °C overnight. The pellets were collected by centrifugation and resuspended in 10 mM sodium phosphate buffer (pH 7.4). The purity of the two SRPs was tested by SDS-PAGE. Boiling stability of SRP 15 and SRP 26 was tested both in crude extracts and purified samples. In both cases the protein extracts were boiled for 10, 30 or 60 min in a water bath, and centrifuged at 12,000 rpm for 10 min in a microfuge. The soluble polypeptides were precipitated with seven volumes of chilled acetone overnight and collected by centrifugation. The pellets were resuspended in the SDS-PAGE loading sample buffer and electrophoresed.
251
the induction of two major polypeptides with apparent mol. wts. of 15 and 26 kDa (Fig. 1). Interestingly, the SRP 15 is induced only at the highest concentration of NaCl used (1.5M), while the SRP 26 was detectable from 0.5M NaCl onwards. Besides, there was a clear decrease in the relative abundance of several major polypeptides, including the large subunit of RUBP-carboxylase (55 kDa), while the 16 and 29 kDa practically disappeared under high salt. Such high concentrations of NaCl were deliberately used in order to induce new polypeptides that are different from those reported earlier. From the protein profiles of germinating seeds (Fig. 2) it can be seen that though the SRP 26 was induced from 200 mM NaCl onwards, there was no induction of the SRP 15 in the shoot extracts. Similarly, a protein with an apparent mol. wt. of 41 kDa was induced in germinating seeds but not in shoots of 10-day-old seedlings. The most prominent one is the 23 kDa polypeptide whose induction starts at 100 mM NaCl, and its intensity increases with increasing concentrations of NaCl. This polypeptide was absent in the shoot extracts of the seedlings. In addition, a polypeptide with an apparent mol. wt. of 70 kDa also increases with increasing concentrations of NaCL There was a corresponding decrease in another polypeptide with an apparent molecular weight of 72 kDa. The absence of the SRP 23 in the shoots of 10-day-old seedlings of rice subjected to salt stress is probably because it is an embryo specific protein. However, 23 kDa polypeptide has been reported to be induced in 10-day-old rice seedlings subjected to various stresses other than NaCl, like ABA, PEG-mediated water stress and air drying (Rao et al., 1993) and in cell suspension cultures grown in ABA and PEG (Reddy et al., 1993).
NaCI(mM) kD CON 50 100 200 300 400 500 600
36 24.
Results and Discussion
The SDS-PAGE profiles of control and NaCl treated Hamsa shoot protein extracts consistently revealed detectable differences from the second day after treatment only (shoots after 1 day of salt treatment do not show any changes in their profiles). The most prominent effect of salt stress is
14 Fig.2: SDS-PAGE (15%) profiles of proteins extracted from 3-dayold Hamsa seedlings germinated in NaCI. All lanes carry 75 J.Lg protein.
252
U. RADHA RANI and A. R. REDDY
45.· 38' ..
2~.
Fig.3: SDS-PAGE of purified 15 and 26kDa polypeptides before and after boiling. Lane (a) 15kDa (25J.&g), Lane (b) 15kDa (40J.&g) (boiled), Lane (c) 26kDa (20 Jlg), Lane (d) 26kDa (40 Jlg) (boiled).
kD
NaCI
Boiled
·""' - o;----: -- ~ c:P "' ,a ~a ....,a ~
66·
clearly demonstrate that both SRP 15 and SRP 26 are boiling stable in aqueous buffer solutions. From Fig. 4, it can be seen that while the boiling step resulted in the coagulation of a majority of proteins, the 15 and 26 kDa SRPs remained soluble. However, 60 min of boiling seems to remove the SRP 26. The purified SRPs were also found to be heat stable (lanes b and d in Fig. 3). Two additional faint bands in the high molecular weight region, produced upon boiling, may not be contaminants since they do not occur in the purified nonboiled samples (lanes a and c in Fig. 3). These are probably a result of oxidation occurring in the sample due to the destruction of 13-mercaptoethanol in the boiling process. A variety of stress induced proteins were found to be heat stable. These include the cold regulated (cor) proteins of Arabidopsis and wheat (Lin et al., 1990), ABA responsive rab family proteins of rice (Mundy and Chua, 1988), the dehydrin proteins of barley, corn Gacobson and Shaw, 1989; Close et al., 1989) and mosses (Werner and Bopp, 1993) and late embryogenesis abundant (lea) proteins of cotton (Baker et al., 1988). All these stress related proteins are hydrophilic in nature and therefore remain soluble at elevated temperatures. The functional role of these stress responsive proteins is not yet unequivocally established. It has been suggested that the boiling resistant lea and rab proteins (Baker et al., 1988; Mundy and Chua, 1988) play a role in drought and desiccation tolerance, and the cor polypeptides (Lin et al., 1990) may be involved in freeze tolerance. Our data suggest that the 15 kDa polypeptide, which also appears under a variety of other stresses like PEG, ABA and air drying, may play a role in salt stress response of rice plants. In summary, our data reveal the tissue specific induction of different polypeptides by salt stress. Presently, we are screening a number of rice lines, both salt-sensitive and tolerant types, for the appearance of the polypeptides SRP 23 (rab) and SRP 15 (seedling) under salt stress. Acknowledgements
We wish to thank the European Economic Commission for funding this project in collaboration with the Max-Planck-Institute, Koln, Germany (Dr. Dorothea Bartels). The fellowship from CSIR to URR is gratefully acknowledged. References
20 . . . ,
14. Fig. 4: SDS-PAGE of boiled crude protein extracts from the shoots of control and NaCl treated Hamsa seedlings. Each lane carries 50 Jlg of protein.
The shoot specific SRP 15 and SRP 26 that were purified by SDS-PAGE and electroelution are shown as single bands on SDS-PAGE (lanes a and c in Fig. 3). Further, our data
BAKER, J., C. STEELE, and L. DURE: Plant Molecular Biology 11, 277-291 (1988). BINZEL, M. L., P.M. HAsEGAwA, D. RHoDES, S. HANDA, H. K. HANDA, and R. A. BRESSAN: Plant Physiol. 84, 1408 -1415 (1987). BINZEL, M. L., F. D. HESs, R. A. BllESSAN, and P.M. HAsEGAwA: Plant Physiol. 86, 607-614 (1988). BoSTOCK, M. R. and S. QuARTANO: Plant Physiol. 98, 1356-1363 (1992). BRUGGEMAN, W. and P. JANIEScH: J. Plant Physiol. 134, 20-35 (1987). CLAES, B., R. DEKEYSl!ll, R. VILLARROEL, M. vAN DEN BULCKE, G. BAuw, V. M. MoNTAGU, and A. CAPLAN: The Plant Cell2, 19-27 (1990). CLOSE, T. J., A. A. KoRTr, and P.M. CHANDI.Ell: Plant Molecular Biology 13, 95-108 (1989). GARBARINo, J. and F. M. Du PoNT: Plant Physiol. 89, 1-4 (1989).
Salt responsive polypeptides in rice GoDAY, A., M. ToRRENT, M. D. LUDEVID, and P. PumELOMENECH: Electrophoresis 9, 738-741 (1988). HuRKMAN, W. J. and C. K. TANAKA: Plant Physiol. 83, 517-524
253
MUNDY,}. and N.H. CHuA: EMBO J. 7, 2779-2786 (1988}. RAMAGOPAL, S. and J. B. CARR: Plant, Cell and Environment 14, 47-56(1991).
(1987).
RAo, A. H. K., B. KARUNASREE, and A. R. REDDY: J. Plant Physiol.
(1989).
REDDY, K. R. K., A. H. K. RAo, B. KARUNASREE, and A. R. REDDY: J. Plant Physiol. 141, 373-375 (1993). SINGH, N. K., A. K. HANDA, P.M. HAsEGAWA, and R. A. BREsSAN: Plant Physiol. 79, 126-137 {1985). SKJUVER, K. and J. MUNDY: The Plant Cell2, 503-512 (1990). WATAD, A. E. A., P. A. PESCI, L. REINHOLD, and L. LERNER: Plant Physiol. 81, 503-512 (1986). WERNER, 0. and M. BoPP: J. Plant Physiol. 141, 93-97 (1993).
JACOBSEN, J. V. and C. D. SHAw: Plant Physiol. 91, 1520-1526 LAEMMu, U.K.: Nature 227, 680-684 (1970). LA RosA, P. C., P. H. HAsEGAWA, D. RHoDES, J. M. CuTHERO, A. E. A. WATAD, and R. A. BREsSAN: Plant Physiol. 85, 174-181 (1987).
LIN, C., W. W. Guo, E. EvERSON, and F. M. THOMASHow: Plant Physiol. 94, 1078-1083 (1990). LoWRY, 0. H., N.J. RosEBROUGH, A. L. FARR, and R. J. RANDALL: J. Biol. Chern. 193, 265-275 (1951).
142, 88-93 (1993).
Short Communication
Salt Stress Responsive Polypeptides in Germinating Seeds and Young Seedlings of Indica Rice (Oryza sativa L.) U. RADHA RANI and A. R. REDDY Plant Molecular Genetics Laboratory, School of Life Sciences, University of Hyderabad, Hyd.erabad - 500 134, India Received May 26, 1993 · Accepted August 11, 1993
Summary
Changes in protein profiles induced by salt stress were investigated both during germination and early seedling growth in rice. Two predominant polypeptides with apparent mol. wts. of 70 and 23 kDa, respectively, were induced under salt stress in germinating seeds of which the 23 kDa is the most abundant. Two other polypeptides with apparent mol. wts. of 15 and 26 kDa were found to be specifically induced in shoots ofNaCl treated seedlings. These two salt responsive polypeptides (SRPs) were partially purified and their properties investigated. It has been clearly demonstrated that the 15 and 26 kDa SRPs exhibit a unique property of boiling stability.
Key words: Salt stress, salt responsive polypeptides, boiling stability. Abbreviations: SRP
=
salt responsive polypeptide; SDS = Sodium dodecylsulphate.
Introduction
The most prominent class of events of salt-stress response include transient adjustment of intracellular fluctuations in ionic balance (Watad et al., 1986; Binzel et al., 1988; Garbarino and DuPont, 1989) and pronounced changes in metabolism of small molecular weight compounds like proline, betaine, sugars and ABA (Binzel et al., 1987; La Rosa et al., 1987). Another important consequence is the alteration in polypeptide profiles (Bruggeman and Janiesch, 1988; Singh et al., 1985; Hurkman and Tanaka, 1987; Ramagopal and Carr, 1991). These events reflect mainly transient changes in polypeptide synthesis and/or turnover as a function of osmotic adjustment. In rice, not much is known about the biochemical and molecular basis of the salt stress response. Recent studies with salt-treated rice seedlings revealed significant changes in polypeptide profiles and also in gene expression. One such salt-induced gene is sal T, which encodes a 15 kDa polypeptide (Claes et al., 1990). In addition, there is another class of genes in rice that are induced by a number of stresses like dehydration by PEG or desiccation and osmotic stress caused by ABA and also salt, or both (Bostock and Quatrano, 1992, Mundy and Chua, 1988; Skriver and Mundy, 1990). The rab © 1994 by Gustav Fischer Verlag, Stuttgart
16 gene, for instance, belongs to this class of genes (Skriver and Mundy, 1990). The physiological function of these induced proteins and their precise role in salt stress response of rice plant is not yet known. Elucidation of the functional association of these salt responsive polypeptides (SRPs) with salt tolerance of the plant requires characterization of the induced proteins. The present report deals with the induction of specific polypeptides by salt in germinating seeds and growing seedlings of rice. Two of the induced polypeptides were partially purified; they exhibit the property of boiling stability in aqueous solutions.
Materials and Methods Seeds of indica rice (Oryza sativa L. cv. Hamsa) were imbibed in water, surface sterilized with 5% sodium hypochlorite (v/v) for 5 min, thoroughly washed with sterile water and germinated in either water or NaCl solutions upon moistened filter papers in the dark. The average temperature during seedling culture ranged from 28 °C ± 2 °C. In one set of experiments, seeds were germinated in different concentrations of NaCl (50 to 600 mM) for 3 days and the whole seedlings were used for protein studies. In the second set,
Salt responsive polypeptides in rice
66
45 36
29 24
14
.......-
-
-
Fig.1: SDS-PAGE (15%) proftles of shoot proteins of control and NaCl treated 10-day-old Hamsa seedlings. All lanes carry 50 J.Lg protein. Lane A, control; Lane B, 0.25 M NaCl; Lane C, 0.5 M NaCl; Lane D, 0.75M NaCl; Lane E, l.OM NaCl; Lane F, 1.25M NaCl; Lane G, 1.5 M NaCl. Arrows indicate the NaCl induced polypeptides. seeds were germinated in water and allowed to grow for 8 days. The 8-day-old seedlings were treated with 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5M NaCl for 2days. Control plants received only water. Shoots were harvested on the first and second day after treatment and quick frozen in liquid nitrogen and stored at - 80 °C until further analysis. Total proteins were extracted as per Goday et al. (1988). Protein content was determined by the method of Lowry et al. (1951) and the protein extracts were subjected to one dimensional SDS-PAGE according to a modified Laemmli procedure (Laemmli, 1970). The SRP 15 and SRP 26 were purified by electroelution. Both the control and treated protein extracts were subjected to preparative SDS-PAGE and the gels were stained with Coomassie Brilliant Blue. The induced 15 and 26 kDa polypeptide bands were identified, cut out and electroeluted. The eluted proteins were precipitated in cold acetone at -80 °C overnight. The pellets were collected by centrifugation and resuspended in 10 mM sodium phosphate buffer (pH 7.4). The purity of the two SRPs was tested by SDS-PAGE. Boiling stability of SRP 15 and SRP 26 was tested both in crude extracts and purified samples. In both cases the protein extracts were boiled for 10, 30 or 60 min in a water bath, and centrifuged at 12,000 rpm for 10 min in a microfuge. The soluble polypeptides were precipitated with seven volumes of chilled acetone overnight and collected by centrifugation. The pellets were resuspended in the SDS-PAGE loading sample buffer and electrophoresed.
251
the induction of two major polypeptides with apparent mol. wts. of 15 and 26 kDa (Fig. 1). Interestingly, the SRP 15 is induced only at the highest concentration of NaCl used (1.5M), while the SRP 26 was detectable from 0.5M NaCl onwards. Besides, there was a clear decrease in the relative abundance of several major polypeptides, including the large subunit of RUBP-carboxylase (55 kDa), while the 16 and 29 kDa practically disappeared under high salt. Such high concentrations of NaCl were deliberately used in order to induce new polypeptides that are different from those reported earlier. From the protein profiles of germinating seeds (Fig. 2) it can be seen that though the SRP 26 was induced from 200 mM NaCl onwards, there was no induction of the SRP 15 in the shoot extracts. Similarly, a protein with an apparent mol. wt. of 41 kDa was induced in germinating seeds but not in shoots of 10-day-old seedlings. The most prominent one is the 23 kDa polypeptide whose induction starts at 100 mM NaCl, and its intensity increases with increasing concentrations of NaCl. This polypeptide was absent in the shoot extracts of the seedlings. In addition, a polypeptide with an apparent mol. wt. of 70 kDa also increases with increasing concentrations of NaCL There was a corresponding decrease in another polypeptide with an apparent molecular weight of 72 kDa. The absence of the SRP 23 in the shoots of 10-day-old seedlings of rice subjected to salt stress is probably because it is an embryo specific protein. However, 23 kDa polypeptide has been reported to be induced in 10-day-old rice seedlings subjected to various stresses other than NaCl, like ABA, PEG-mediated water stress and air drying (Rao et al., 1993) and in cell suspension cultures grown in ABA and PEG (Reddy et al., 1993).
NaCI(mM) kD CON 50 100 200 300 400 500 600
36 24.
Results and Discussion
The SDS-PAGE profiles of control and NaCl treated Hamsa shoot protein extracts consistently revealed detectable differences from the second day after treatment only (shoots after 1 day of salt treatment do not show any changes in their profiles). The most prominent effect of salt stress is
14 Fig.2: SDS-PAGE (15%) profiles of proteins extracted from 3-dayold Hamsa seedlings germinated in NaCI. All lanes carry 75 J.Lg protein.
252
U. RADHA RANI and A. R. REDDY
45.· 38' ..
2~.
Fig.3: SDS-PAGE of purified 15 and 26kDa polypeptides before and after boiling. Lane (a) 15kDa (25J.&g), Lane (b) 15kDa (40J.&g) (boiled), Lane (c) 26kDa (20 Jlg), Lane (d) 26kDa (40 Jlg) (boiled).
kD
NaCI
Boiled
·""' - o;----: -- ~ c:P "' ,a ~a ....,a ~
66·
clearly demonstrate that both SRP 15 and SRP 26 are boiling stable in aqueous buffer solutions. From Fig. 4, it can be seen that while the boiling step resulted in the coagulation of a majority of proteins, the 15 and 26 kDa SRPs remained soluble. However, 60 min of boiling seems to remove the SRP 26. The purified SRPs were also found to be heat stable (lanes b and d in Fig. 3). Two additional faint bands in the high molecular weight region, produced upon boiling, may not be contaminants since they do not occur in the purified nonboiled samples (lanes a and c in Fig. 3). These are probably a result of oxidation occurring in the sample due to the destruction of 13-mercaptoethanol in the boiling process. A variety of stress induced proteins were found to be heat stable. These include the cold regulated (cor) proteins of Arabidopsis and wheat (Lin et al., 1990), ABA responsive rab family proteins of rice (Mundy and Chua, 1988), the dehydrin proteins of barley, corn Gacobson and Shaw, 1989; Close et al., 1989) and mosses (Werner and Bopp, 1993) and late embryogenesis abundant (lea) proteins of cotton (Baker et al., 1988). All these stress related proteins are hydrophilic in nature and therefore remain soluble at elevated temperatures. The functional role of these stress responsive proteins is not yet unequivocally established. It has been suggested that the boiling resistant lea and rab proteins (Baker et al., 1988; Mundy and Chua, 1988) play a role in drought and desiccation tolerance, and the cor polypeptides (Lin et al., 1990) may be involved in freeze tolerance. Our data suggest that the 15 kDa polypeptide, which also appears under a variety of other stresses like PEG, ABA and air drying, may play a role in salt stress response of rice plants. In summary, our data reveal the tissue specific induction of different polypeptides by salt stress. Presently, we are screening a number of rice lines, both salt-sensitive and tolerant types, for the appearance of the polypeptides SRP 23 (rab) and SRP 15 (seedling) under salt stress. Acknowledgements
We wish to thank the European Economic Commission for funding this project in collaboration with the Max-Planck-Institute, Koln, Germany (Dr. Dorothea Bartels). The fellowship from CSIR to URR is gratefully acknowledged. References
20 . . . ,
14. Fig. 4: SDS-PAGE of boiled crude protein extracts from the shoots of control and NaCl treated Hamsa seedlings. Each lane carries 50 Jlg of protein.
The shoot specific SRP 15 and SRP 26 that were purified by SDS-PAGE and electroelution are shown as single bands on SDS-PAGE (lanes a and c in Fig. 3). Further, our data
BAKER, J., C. STEELE, and L. DURE: Plant Molecular Biology 11, 277-291 (1988). BINZEL, M. L., P.M. HAsEGAwA, D. RHoDES, S. HANDA, H. K. HANDA, and R. A. BRESSAN: Plant Physiol. 84, 1408 -1415 (1987). BINZEL, M. L., F. D. HESs, R. A. BllESSAN, and P.M. HAsEGAwA: Plant Physiol. 86, 607-614 (1988). BoSTOCK, M. R. and S. QuARTANO: Plant Physiol. 98, 1356-1363 (1992). BRUGGEMAN, W. and P. JANIEScH: J. Plant Physiol. 134, 20-35 (1987). CLAES, B., R. DEKEYSl!ll, R. VILLARROEL, M. vAN DEN BULCKE, G. BAuw, V. M. MoNTAGU, and A. CAPLAN: The Plant Cell2, 19-27 (1990). CLOSE, T. J., A. A. KoRTr, and P.M. CHANDI.Ell: Plant Molecular Biology 13, 95-108 (1989). GARBARINo, J. and F. M. Du PoNT: Plant Physiol. 89, 1-4 (1989).
Salt responsive polypeptides in rice GoDAY, A., M. ToRRENT, M. D. LUDEVID, and P. PumELOMENECH: Electrophoresis 9, 738-741 (1988). HuRKMAN, W. J. and C. K. TANAKA: Plant Physiol. 83, 517-524
253
MUNDY,}. and N.H. CHuA: EMBO J. 7, 2779-2786 (1988}. RAMAGOPAL, S. and J. B. CARR: Plant, Cell and Environment 14, 47-56(1991).
(1987).
RAo, A. H. K., B. KARUNASREE, and A. R. REDDY: J. Plant Physiol.
(1989).
REDDY, K. R. K., A. H. K. RAo, B. KARUNASREE, and A. R. REDDY: J. Plant Physiol. 141, 373-375 (1993). SINGH, N. K., A. K. HANDA, P.M. HAsEGAWA, and R. A. BREsSAN: Plant Physiol. 79, 126-137 {1985). SKJUVER, K. and J. MUNDY: The Plant Cell2, 503-512 (1990). WATAD, A. E. A., P. A. PESCI, L. REINHOLD, and L. LERNER: Plant Physiol. 81, 503-512 (1986). WERNER, 0. and M. BoPP: J. Plant Physiol. 141, 93-97 (1993).
JACOBSEN, J. V. and C. D. SHAw: Plant Physiol. 91, 1520-1526 LAEMMu, U.K.: Nature 227, 680-684 (1970). LA RosA, P. C., P. H. HAsEGAWA, D. RHoDES, J. M. CuTHERO, A. E. A. WATAD, and R. A. BREsSAN: Plant Physiol. 85, 174-181 (1987).
LIN, C., W. W. Guo, E. EvERSON, and F. M. THOMASHow: Plant Physiol. 94, 1078-1083 (1990). LoWRY, 0. H., N.J. RosEBROUGH, A. L. FARR, and R. J. RANDALL: J. Biol. Chern. 193, 265-275 (1951).
142, 88-93 (1993).