Abstract: Changes in the level of total and Free Amino Acid (FAA) and analysis of protein profiles on SDS-PAGE were investigated and compared in root and shoot tissues of two green gram (Phaseolus aureus) cultivars (T-44 and SML-32) differing in salt tolerance during plant growth in the absence and presence of NaCl salinity. Total as well as individual FAA were found to increase progressively with growth in both the tissue and in both the cultivars under absence and the presence of salinity. The level of total and individual FAA was more in shoot than in root during plant growth from day 1 to 5. Salt tolerant cultivar T-44 had significant higher (p<0.05) level of total as well as individual FAA than that of salt sensitive cultivar SML-32 in both the tissues in absence as well as in the presence of salt. In salt tolerant cultivar the increased accumulation of amino acid (AA) under salt stress was more pronounced in case of alanine, arginine, aspartic acid, glutamic acid, glutamine, phenylalanine and serine. Analysis of the protein profiles of root and shoot tissues of both the cultivars in absence and presence of salinity on SDS-PAGE revealed that in both tissues of salt tolerant cultivar T-44, 3 to 4 polypeptides (PPs) were specially synthesized while synthesis of some PPs increased under salt stress. Salt induced changes in protein profile in root and shoot of salt tolerant cultivar T-44 were mostly tissue specific. On the other hand no such salt specific proteins were synthesized under salt stress in salt sensitive cultivar SML-32. Correlation between the degree of salt tolerance of the cultivar and levels of AAs and those of stress protein synthesis has been discussed.
Introduction
Amino acids, being the primary products of inorganic assimilation and precursors of proteins, play essential role in plant metabolism. Accumulation of high concentrations of nitrogenous organic solutes in the cytoplasm has been reported to occur in various plants subjected to salinity stress (Hasegawa et al., 2000; Meloni et al., 2001, 2003; Greenway and Munns, 1980). Solutes, which accumulate during salinity stress in many species, are glycinebetaine (Ghoulam et al., 2002), proline (Ghoulam et al., 2002; Girija et al., 2002; Balibera et al., 1997; Bajji et al., 1998; Alian et al., 2000) and various other AAs like aspartic acid, glycine, asparagine, glutamine and serine, etc. These soluble nitrogenous compounds accumulating under salt stress have been regarded as components of salt tolerance mechanism (Stewart and Larher, 1980) as they contribute for osmotic balance in the cytoplasm when electrolytes are higher in vacuoles and also impart a protective role on enzymes in the presence of high electrolytes in the cytoplasm (Hasegawa et al., 2000; Meloni et al., 2001, 2003; Greenway and Munns, 1980). Many salt tolerant plants have been reported to possess in built higher levels of certain AAs. In general, plants when grown without NaCl have low levels of such solutes but show increased accumulation under salinization (Stewart and Larher, 1980).
Stress induced changes in protein synthesis have been reported in response to a variety of environmental stresses such as cold (Kawata and Yoshida, 1988), dehydration (Jung and Shin, 1992; Ingram and Bartels, 1996; Ramanjulu and Sudhakar, 1997; Loderio et al., 2000), salinity (Ben-Hayyim et al., 1993; Lopez et al., 1994; Bajji et al., 1998), anaerobiosis (Sachs and Ho, 1986) and heat shock (Vierling , 1991; Wu et al., 1993). In case of anaerobic stress certain induced proteins have been assigned specific functions in known metabolic pathways (Sachs and Freeling, 1978).
Increased synthesis of some of the existing proteins as well as synthesis of entirely new proteins under salt stress has been reported. Thus salt induced ten fold increases in a 26 KD PP is reported in tobacco cells (Singh et al., 1985), maize callus (Ramgopal, 1986) and in barley root meristems (Ramgopal, 1988). A variety of salt induced proteins have been reported from a number of other sources. Thus, 23, 25 and 27 KD protein from citrus (Ben-Hayyim et al., 1989; 1993), 26 and 27 KD from rice (Shirata and Takagishi, 1990) and barley roots (Hurkman et al., 1989; Robinson et al., 1990), 18, 27 and 49 KD PPs from Jojoba calli (Chretein et al., 1992), 22 KD PP from leaves of Raphanus sativus (Lopez et al., 1994). Many salt induced changes in proteins are species specific and that no obvious similarity has existed (Ben-Hayyim et al., 1989). Studies have also been carried out in a number of tissues for the identification of the genes responsible for salt tolerance (King et al., 1988; Singh et al., 1989; Winicov et al., 1989). Among these, the 26 KD proteins of salt adapted cells of tobacco (called osmotin) and tomato has been examined in detail and a gene sequence has been cloned (Singh et al., 1989; Lopez et al., 1994).
We have been pursuing the biochemical studies on the two green gram cultivars (namely T-44 and SML-32, differing in their relative salt tolerance) for identification of biochemical markers for salinity tolerance (Misra and Dwivedi, 1990, 1995). In the present paper we report the metabolic levels of total FAAs and those of individual FAAs and also analysis of the soluble protein profile of root and shoot on SDS-PAGE in the absence as well as presence of NaCl salinity during seed germination.
Materials and Methods
Plant Material and Stress Treatment
Green gram (Phaseolus aureus L. Roxb Family: Leguminosae)
seeds were screened for their salinity during germination in our laboratory
and thus cultivar T-44 was found to be fairy tolerant while cultivar SML-32
was found to be sensitive to saline stress (Misra and Dwivedi, 1990). Seeds
were surface sterilized and germinated as described (Misra and Dwivedi, 1995).
Two concentrations of NaCl viz. 200 mM and 50 mM were used for cultivar T-44
and SML-32, respectively. Starting with the 4 hrs soaked seeds (zero hrs. of
germination), the germinated seeds were taken out at 24 h intervals up to 5
days, roots and shoots (along with cotyledons) were separated from the seeds
and proceeded further.
Estimation of Total FAAs
Total FAAs were measured by ninhydrin assay (Yemm and Cocking, 1955) with
some modifications. Five hundred mg tissue (root/shoot) were extracted in 5
mL of 80% ethanol and centrifuged at 18,000-x g for 30 min. The test extract
was taken (0.1 mL) and total FAA was estimated using (7.6 mL) ninhydrin reagents
containing 1% ninhydrin in 0.5 M citrate buffer, pH 5.5, glycerol (87%) and
0.5 M citrate buffer pH 5.5 in ratio of 5:12:2. After vigorous shaking contents
were heated in boiling water bath for 10 min and after cooling, absorbance was
measured at 570 nm in a Gilford Spectrophotometer. 0.1 mL 80% of ethanol served
as blank in place of test extract. Absorbance readings were converted to mg
amino acid g-1 fresh weight tissue using a glycine standard curve.
Estimation of Individual FAAs
For separation and quantitative estimation of individual FAAs, a known volume
of test extract of shoot and root tissues were applied on reversed phase HPLC
using the method of Brierley et al. (1996).
Protein AnalysisProteins were extracted from root and shoot (along with cotyledons) at 48 h of seed germination. Two grams of tissues were homogenized in 5 mL Tris-HCl buffer (0.1 M, pH = 7.5) containing 1 mM phenyl methyl Sulfonyl Fluoride (PMSF), at 4°C. The homogenate was centrifuged at 18,000-x g for 30 min. Protein in the supernatant was measured by the some modification of the Bradford (1976) using crystalline bovine albumin as a reference. This protein extract was concentrated before loading over the gel. For concentration, the extract was kept in a dialysis bag and the ends were tied with thread tightly and the dialysis bag containing extract was kept in powdered sucrose for the appropriate time (3-5 h) at 4°C during which period the extract was concentrated more than 3 times. Thereafter, the contents were taken out from dialysis bag, the volume of the extract was measured and protein was again determined in a suitable aliquot Bradford (1976).
Sample preparation and SDS-PAGE at pH 8.3 was done as described (Laemmili, 1970). The gels were photographed and scanned using 2202 ULTROSCAN Laser Densitometer (LKB).
Statistical Analysis
Each treatment was analyzed with at least three replicates and Standard
Deviation (SD) was calculated. Statistical analysis was performed using the
students t-test; p<0.05 was considered statistically significant.
Results
Effect of Salinity on Free Amino Acid
With the progress of germination the FAA level increased throughout in root
and shoot of both the cultivars, in the absence as well as in the presence of
salinity (Table 1). However, in the presence of salinity the
AA content of both root and shoot increased significantly (p<0.05) in the
cultivar T-44 while those of SML-32 exhibited only a marginal increase. Moreover,
shoot maintained higher level of AA as compared to root in both the cultivars.
Thus, on day 5 of plant growth in cultivar T-44, root exhibited approximately
3.9 fold (p<0.05) increased in the presence of 200 mM NaCl as compared to
non saline control while only 1.0 fold increase was observed in root of cultivar
SML-32 in the presence of 50 mM NaCl as compared to respective non saline control
(Table 1).
| Table 1: | Effect of salinity on total free amino acid (mg/g FW) in root and shoot of green gram cultivar T-44 (a and b) and SML-32 (c & d) during plant growth. Each value represents mean of three independent observations and SD determined. Data are statistically significant at p<0.05 |
| Table 2: | Effect of salt stress on individual amino acid (mg/g FW) in root and shoot on day 5 plant growth of green gram cultivars T-44 and SML-32. Each value represents mean of three independent observations and SD determined. Data are statistically significant at p<0.05 |
Similarly, in shoot of cultivar T-44 the AA content was observed aprox 5.9 fold (p<0.05) increase in the presence of 200 mM NaCl as compared to non saline control while only 1.0 fold increase was observed in root of cultivar SML-32 in the presence of 50 mM NaCl as compared to respective non saline control.
Effect of Salinity on Individual Free Amino Acid
Levels of all 20 AAs measured were found to be increased in presence of
salinity in both root and shoot, with shoot maintaining a high level of all
these AAs as compared to that of root under the conditions of both absence and
presence of salinity in both the cultivars (Table 2). However,
the increase in the levels of individual AA under salinity stress was significant
in T-44 whereas it was only marginal in SML-32 in both roots as well as in shoot.
Thus, in cultivar T-44, AAs whose concentration increased more in both root
and in shoot under salinity stress were alanine, arginine, glutamic acid, glutamine,
proline, cystein, isoleucine, asparagine and serine (a total of nine Aas).
| Fig. 1: | SDS- PAGE of soluble proteins from shoot (A and B) and root (C and D) of salt tolerant T-44 cultivar in absence (a) and in the presence (b) of 200 mM NaCl. The proteins were extracted, concentrated and approximately 200 μg protein was loaded in each case on a 12% polyacrylamide gel containing SDS. Numbers in KD indicated the position of standard marker. Arrows indicated the position of specific proteins synthesized only during salt stress. B and D show scan of the gels A and C, respectively. Arrows indicate the specific proteins synthesized during salt stress |
The corresponding increase in the levels of these AAs under salinity stress in root and shoot of cultivar SML-32 was only between 2-7%.
We also investigated the developmental profile of all the nine AAs that showed marked increase under salt stress (data not shown). Out of all 20 AAs measured, the most remarkable was the proline, which was increased significantly in the presence of 200 mM NaCl in root as well as in shoot of T-44 cultivar. However in the presence of salinity stress (200 mM NaCl) proline was increased ≈ 5.0 fold (p<0.05) in shoot and 2.1 fold in root of T-44 cultivar. Developmental profiles of these AAs were characterized by a gradual increase in their content both in absence as well as in the presence of salinity in both root and shoot of both the cultivars. However, the increase in the amount of these AAs in presence of salinity stress was more pronounced in case of cultivar T-44 while it was only marginal in case of cultivar SML-32. Shoot always maintained a higher level as compared to root in both the cultivars in the absence as well as in the presence of salinity (data not shown).
Influence of Salinity on the Soluble Protein Profiles of Shoot
Soluble protein profile of shoot tissue from germinating green gram seeds
of salt tolerant cultivar T-44 was analyzed by SDS-PAGE in absence and in the
presence of salinity (Fig. 1A). In presence of salinity few
PPs were found to be newly synthesized during salt stress while synthesis of
some existing proteins increased. Thus, three PPs with approximate mw of 25
KD (IV), 27 KD (III) and 49 KD (II) were specifically synthesized in shoot under
salt stress. In addition, nearly four PPs were specifically synthesized in the
range of mw 150-170 KD as indicated by zone I on Fig. 1A.
The gels were scanned using a laser densitometer as shown in Fig.
1B. The 25, 27 and 49 KD PPs in 150-170 KD synthesized specifically in presence
of salt are also evident from the scan. Furthermore, the existing PPs whose
synthesis increased during salt stress were those in the mw range of 36 and
20 KD as evident from scan by an increase in the peak(s) height as well as total
area under the peak Fig. 1B. Soluble protein profile from
the shoot of germinating green gram seeds of cultivar SML-32 (sensitive) was
also analysed by SDS-PAGE. It was observed that no salt stress PPs similar to
those of salt tolerant cultivar was specifically synthesized in responce to
salt stress. However, few PPs showed increased synthesis under salt stress in
comparison to nonsaline control, in the range of 36 and 20 KD similar to those
of tolerant cultivar (Data not shown).
Influence of Salinity on the Soluble Protein Profile of Root
SDS-PAGE profile of soluble proteins in root of germinating seed of salt
tolerant green gram seed cultivar T-44 was investigated and compared in the
absence and in the presence of salinity (Fig. . 1C). In presence
of salinity few PPs were specifically synthesized in root also. They had mw
in the range of 27(I), 20(II) and 13(III) KD. In addition, synthesis of few
existing PPs increased under salt stress. The more predominant PPs belonging
to this group has mw in the range of 36, 23, 18 and 16 KD. It is noteworthy
that in root most of the PP whose synthesis increased under salinity was small
mw PPs in the range 23-16 KD. In contrast some high mw PPs in the range of 100
KD showed decreased synthesis under salt stress. No such decrease was found
in case of shoot tissue on the contrary, they exhibited specific synthesis of
some high mw PPs in this range (150-170 KD). The conclusions drawn from the
gel profile were consistent with the data of scan (Fig. 1D).
We also investigated the SDS-PAGE profile of soluble proteins from root of germinating
seed of cultivar SML-32 (salt sensitive). It was observed that no PPs in the
mw range of 27, 20 and 13 KD were specifically synthesized under salt stress,
as observed in the case of salt tolerant cultivar T-44 though the PPs which
exhibited increased synthesis in T-44 were also observed in SML-32 (36, 23,
18 and 16 KD) (data not shown).
Discussion
Quantitative and qualitative changes in AAs revealed increased accumulation of total FAAs as well as a group of nine individual FAAs in salt tolerant cultivar (T-44) under salt stress during plant growth. The corresponding increase in salt sensitive cultivar was negligible. The individual AAs, alanine, arginine, glutamic acid, glutamine, proline, cystein, isoleucine, asparagine and serine (a total of nine AAs) that showed significant increase during salt stress in tolerant cultivar. Proline was unique in all that it was significant under salinity stress in salt tolerant cultivar. Similar reports of increased accumulation of total as well as a group of six individual AAs namely alanine, arginine, valine, leucine, glutamine and proline have been reported for rice plants differing in salt sensitivity under salinity stress (Dubey and Rani, 1990). Increased accumulation of proline (Misra and Gupta, 2005; Ghoulam et al., 2002), glycine, aspartic acid, serine, asparagine and glutamine (Ahmed et al., 1978), glycinebetaine (Girija et al., 2002) under salinity stress has been reported. Increased accumulation of asparagine, aspartic acid, glycine, serine, glutamic acid, alanine, proline under salinity stress was also observed in pigeon pea (Rao and Rao, 1981). Similar observations of high in built levels of certain AAs or combination of 3-4 AAs were markedly increased accumulation in salt tolerant cultivars have also been reported under salinity stress (Stewart et al., 1978). A combination of asparagine, glutamine, serine and proline is reported to be specific for salt tolerance in many higher plants (Ahmed et al., 1978). Increased level of arginine has been correlated with polyamine synthesis, which are involved in osmotic regulation during salinity stress (Bray et al., 1991). It has been suggested that some of them AAs functions as compatible cytoplasmic solutes and their increased accumulation serves as means of intracellular osmotic adjustment in order to maintain the osmotic potentials of cytoplasm in adverse conditions of salinity (Bray et al., 1991).
Proline accumulation is also related with salt tolerance in many higher plants (Bray et al., 1991; Perez-Alfocea and Larher, 1995; Lopez et al., 1994; Ghoulam et al., 2002; Girija et al., 2002). Higher osmolyte accumulation, especially proline seem to be related to salt tolerance in salt tolerant genotype T-44 not to be a consequence of tissue dehydration or tissue reaction to stress damage (Misra and Gupta 2005). Proline accumulation also appeared to be a reaction to salt stress damage and not a plant response associated with salt tolerance (Lacerda et al., 2003). Proline has been shown to protect the PEG induced precipitation of some enzymes and protein complex in vitro (Paleg et al., 1985). Such protection of proteins has been shown to be increased further in presence of a combination of 3-4 AAs along with proline. These observations suggested that the protective effect of some AAs are at least additive and are consistent with the conclusion that the compatible solutes protect proteinBcontaining systems against the unfavorable consequences of dehydration (salt stress also has dehydration component) by increasing the tendency of the system to maintain the status quo. A similar suggestion can be made from our findings.
Analysis of the protein profiles of the shoot of cultivar T-44 on SDS-PAGE revealed that some PPs are specifically synthesized in response to salt stress (25, 27 and 49 and 150-170 KD) while synthesis of few already existing PPs (20 and 36 KD) were increased under salt stress. In root of the cultivar T-44 the PPs specifically synthesized under salt stress were 13, 20 and 27 KD while PPs whose synthesis increased during salt stress were 36, 23, 18 and 16 KD. Similar reports of specific synthesis of proteins under salt stress have been reported. Thus, a 25 KD protein was found to be specially synthesized in salt adapted cells of Citrus sinensis, which was found to be associated with salt tolerance (Ben-Hayyim et al., 1989, 1993). Increased synthesis of some 10 PPs in cells adapted to salinity has been also reported (Singh et al., 1985). Synthesis of a 26 KD protein under salt stress was unique in such tobacco cells which they latter called as osmotin (Singh et al., 1987a,b), because it was synthesized and accumulated by cell undergoing gradual osmotic adjustment to either salt or desiccation stress (Chretein et al., 1992; Perez-Alfocea and Larher, 1995). Chretein et al., (1992) reported that PPs of mw of 18, 27 and 49, synthesized in salt adapted calli of Jojoba, which were suggested as marker proteins for salt adaptation. Ericson and Alfinito (1984) reported increased synthesis of a 20 and 32 KD PPs in salt adapted tobacco cells. Ramgopal and Carr (1991) reported that in sugarcane suspension cells 15 proteins were induced or enhanced and other three proteins were repressed or abolished. The most obvious change concerned a 22 KD, PI 7.5 PP, which accumulated after exposure of the Raphanus sativus to NaCl. A cDNA clone corresponding to the radish 22 KD PP was obtained and sequenced (Lopez et al., 1994).
From our study it is noteworthy, that the PPs specifically synthesized under salt stress were not common in root and shoot except for 27 KD PPs. Similarly, PPs synthesized at increased rates under salt stress were also not common in root and shoot except for 36 KD PP. In contrast to shoot, where none of the PPs exhibited decreased synthesis, root exhibited a PP in the range of 100 KD whose synthesis decreased under salt stress as reported for some PPs in tobacco cells (Singh et al., 1985). Polypeptides synthesized specifically under salt stress in root and shoot tissues of salt tolerant cultivar T-44 were found to be missing from those of salt sensitive cultivar SML-32 under salinity. Similar results have been reported from barley roots (Hurkman et al., 1989) in which 26 and 27 KD PPs synthesized in salt tolerant cultivar under salinity stress was missing from the salt sensitive cultivar in presence of salinity. Since the proteins which are specifically synthesized (in shoot as well as in root tissue) under salt stress only in cultivar T-44 and was absent in the cultivar SML-32 under the conditions of absence as well as presence of salinity, these proteins may be considered as markers for salt tolerance in salt tolerant cultivar T-44. These proteins that are synthesized either specifically or at increased rate under salt stress are suggested to have adaptive role in plants in osmotic adjustments (osmotic as well as ionic components of salt stress) (Shirata and Takagishi, 1990; Singh et al., 1987a,b) protecting the key cytoplasmic enzymes and protein synthesizing apparatus against adverse effects of high salt concentrations. Salt induced changes in protein in our case are predominantly, tissue specific and that no obvious similarity exist, which is consistent with the differences in the physiological functions played by the two types of tissue (root and shoot) and also with the environment around these tissues. However, the possibility that a variety of environmental stresses may lead to the production of one or more common proteins suggesting a general stress tolerance mechanism and the possibility that there may be a keyset of proteins (Harrington and Alm, 1988) is not ruled out from our findings. A number of proteins synthesized in our case are, at least, in close range, on the basis of mw, to those of proteins specifically synthesized under heat shock in tobacco cell lines, namely 15 to 20 KD (Harrington and Alm, 1988). Thus, conclusions drawn from the studies of Ben-Hayyim et al., (1993) that salt induced proteins are species specific and that no obvious similarities exists among them is not consistent with our findings, as a number of proteins synthesized in response to salt stress in our case are common (on the basis of their molecular weight) with those reported from other sources under salt stress cf. PPs 23,25,27, 20 KD, etc. (Ben-Hayyin et al., 1989) 49 KD (Chretein et al., 1992).
In conclusion, green gram cultivars differing in salt tolerance have in built varying levels of AAs, alanine, arginine, glutamic acid, glutamine, proline, cystein, isoleucine, asparagine and serine (a total of nine AAs) and higher levels of these AAs in germinating seed parts (root and shoot) of tolerant cultivar T-44. Increased accumulation of these AAs in tolerant cultivars T-44 under salinization can be correlated with salt tolerance ability of this green gram cultivar. These all AAs along with proteins synthesized/accumulated under salt stress should be considered together in combination as indices for salt tolerance in green gram as suggested by other workers (Dubey and Rani, 1989).
Acknowledgements
Financial assistance of the Council of Science and Technology, U.P., is gratefully acknowledged. The study was partially supported from the Department of Biotechnology and University Grants Commission, New Delhi. The authors are also thankful to Dr. Aseen Chauhan, Chairman and Director General K.K. Ohari, Amity University, Lucknow Campus for their support and encouragement.