CC BY
37
Journal of Stress Physiology & Biochemistry, Vol. 8 No. 3 2012, pp. 5-12 ISSN 1997-0838 Original Text Copyright © 2012 by Johnson, Renola Joy Jeba Ethal and Babu
ORIGINAL ARTICLE
Influence of Salinity Stress on proteomic profiles of Cicer arietinum L.
Johnson M.* Renola Joy Jeba Ethal T. and Babu A.
Centre for Plant Biotechnology, Department ofPlant Biology and Plant Biotechnology, St. Xavier’s College (Autonomous), Palyamkottai, Tamil Nadu, India.
Tel: + 9 1 97 86 92 43 34; Fax: + 91 46 22 5619 87
E-mail: ptcjohnson@ gmail.com
Received February 19 2012
The present study was aimed to study the influence of salt stress on the proteomic profiles of Cicer arietinum L. by using SDS-PAGE. Seedlings of Cicer arietinum were exposed to different salt concentrations (4%, 6%, 8% and 10%) of NaCl. Multiple regions of actively stained system were obtained for SDS-PAGE. On 15th day, a total of 79 bands were observed and their RM values ranged from 0.011 to 0.988. Based on the occurrence and non-occurrence of the proteins in the gel system, the protein profiles were classified in to three categories viz., salt tolerant proteins, salt inducible proteins and salt sensitive proteins.
Key words: Salinity, Protein profiles, SDS-PAGE, Cicer arietinum
ORIGINAL ARTICLE
Influence of Salinity Stress on proteomic profiles of Cicer arietinum L.
Johnson M.* Renola Joy Jeba Ethal T. and Babu A.
Centre for Plant Biotechnology, Department of Plant Biology and Plant Biotechnology, St. Xavier’s College (Autonomous), Palyamkottai, Tamil Nadu, India.
Tel: + 9 1 97 86 92 43 34; Fax: + 91 46 22 5619 87
E-mail: ptcjohnson@gmail.com
Received February 19 2012
The present study was aimed to study the influence of salt stress on the proteomic profiles of Cicer arietinum L. by using SDS-PAGE. Seedlings of Cicer arietinum were exposed to different salt concentrations (4%, 6%, 8% and 10%) of NaCl. Multiple regions of actively stained system were obtained for SDS-PAGE. On 15th day, a total of 79 bands were observed and their RM values ranged from 0.011 to 0.988. Based on the occurrence and non-occurrence of the proteins in the gel system, the protein profiles were classified in to three categories viz., salt tolerant proteins, salt inducible proteins and salt sensitive proteins.
Key words: Salinity, Protein profiles, SDS-PAGE, Cicer arietinum
Salinity induced reaction in growth is the consequence of modifications of several physiological process like, ion balance, water status, mineral nutrition, stomatal behaviour, photosynthesis, carbon allocation and utilization (Munns Termatt, 1986; Flowers et al., 1991). Salt accumulation in soils from natural processes and irrigation adversely affects seed germination, seedling growth as well as related metabolic processes of plants. The response of plants to salinity varied with species, organs and stage of development (Munns, 2002). Plant can act in response and adapt to salt stress by altering their
cellular metabolism and invoking various defence mechanisms (Bohnert 2007). However, plants subjected to salt stress showed increased levels of total free amino acids (Dubey and Pessarakli, 1995). In many plants, free proline accumulates in response to the imposition of a wide range of biotic and abiotic stresses (Hare and Cress, 1997; Johnson et al., 2011; Dooslin Mary et al., 2009; Dooslin Mary et al., 2011; Johnson et al., 2011). Carceller et al., (1999) found that the accumulating proline in maize cultivar showed a higher osmotic adjustment capacity than the non accumulating one. Sodium chloride stress negatively influenced
nitrate reductase activity of plants. It increased protease capacity (Muthukumarasamy et al., 2000. Harinasut et al., (1996) found that exposure of 28-day-old rice seedlings to 150 mM NaCl for 6 days induced drastic decreases in relative water contents, chlorophyll, and protein in leaves. The molecular identities of key ion transport systems that are fundamental to plant salt tolerance are now known (Sanders, 2000; Zhu, 2000; Hasegawa et al., 2000). With this knowledge the present study was intended to find out the salt influential protein expression of Cicer arietinum. MATERIALS AND METHODS
Seeds of Cicer arietinum were surface sterilized with 0.1% HgCl2 and 0.1% sodium lauryl sulfate solution for 3-5 min and rinsed thrice with sterile distilled water. The sterilized seeds were inoculated on to the MS medium supplemented with and without NaCl to study the influence of NaCl (0, 4%, 6%, 8% and 10%) on seedlings of Cicer arietinum. The seedlings were grown in the culture room for a period of 15 days. On 15th Day, the randomly collected whole plants were used as a source for protein isolation. 500 to 1000 mg of freshly harvested tissues were taken and homogenized with 3.5 ml of ice-cold 0.1M phosphate buffer (pH 7.0) in a pre-chilled pestle and mortar and centrifuged at 10,000 rpm for 10 min and the supernatant was collected and used for protein separation. The Poly acrylamide gel electrophoresis was performed by Anbalagan (1999) method. SDS - Poly Acrylamide Gel Electrophoresis was carried out at 25°C in the air conditioned room. Separation of protein was carried out at 50v till the tracking dye reaches the separating gel and at100 v thereafter for 3-5 hours or until the tracking dye had migrated to the bottom of the gel. After running the
electrophoresis, the gels were carefully removed from the mold and subjected to activity staining (Anbalagan, 1999).
RESULTS
Proteomic analyses have resulted in the identification of many proteins that are inducible by salt stress including the metabolism related genes. The relative positions of the protein bands as revealed by SDS- PAGE of the Cicer arietinum seedlings under different stress conditions are shown in Fig 1A. Multiple regions of actively stained system were obtained for SDS-PAGE. On 15th day, a total of 79 bands were observed and their RM values ranged from 0.011 to 0.988. The seedlings treated with 4% of NaCl showed fifteen bands in fifteen different positions. MW-Rf 0.329, 0.517 and 0.764 showed their unique presence in the seedlings treated with 4% of NaCl (Table - 1). The seedlings treated with 6% of NaCl showed their uniqueness by the presence of proteins with MW-Rf 0.094 and 0.552 (Table - 1). The seedlings treated with 8% NaCl demonstrated the expression of 18 different proteins in the protein gel system, with six different specific proteins viz., 0.058, 0.235, 0.741, 0.788, 0.905 and 0.964 .When the concentration of NaCl increases more than 8% the number of bands was decreased and they showed instability in their appearance. The bands were very light and fragile. The seedlings treated with 10 % NaCl showed only thirteen proteins, of which four proteins viz., 0.282, 0.835, 0.882 and 0.988 showed their restricted occurrence. Based on the occurrence and non-occurrence of the proteins in the gel system, the protein profiles were classified in to three categories viz., salt tolerant proteins, salt inducible proteins and salt sensitive proteins. Due to the salt stress, the seedlings of C. arietinum elicited the following salt inducible
proteins viz., MW- Rf 0.023, 0.058, 0.094, 0.200,
0.235, 0.282, 0.294, 0.329, 0.400, 0.423, 0.435,
0.447, 0.494, 0.517, 0.552, 0.705, 0.717, 0.741,
0.764, 0.776, 0.788, 0.823, 0.835, 0.882, 0.894,
0.964 and 0.988. The following proteins MW-Rf 0.105, 0.164, 0.388, 0.600, 0.635, 0.811, 0.929 and 0.952 were expressed in the control and salt
stressed seedlings, and they are called as salt tolerant proteins. The following proteins MW-Rf. 0.011, 0.047, 0.270, 0.470, 0.564 which are absent in the salt treated seedlings, and present only in control (distilled water treated seedlings) are called salt sensitive proteins.
Table 1: SDS - PAGE banding pattern of different concentration of NaCl treated Cicer arietinum seedlings on 15th Day
MW-Rf Positions Regions Concentration of NaCl in %
0 (L1) 4 (L2) 6 (Ц) 8 (L4) 10 (L5)
0.011 PP 11 1 + - - - -
0.02з PP 12 - + + + -
0.047 PP 1з + - - - -
0.058 PP 14 - - - + -
0.094 PP 15 - - + - -
0.105 PP 21 2 + - - + +
0.164 PP 22 + + + + -
0.200 PP 2з - - - + +
0.211 PP з1 з - + + - -
0.2з5 PP з2 - - - + -
0.270 PP зз + - - - -
0.282 PP з4 - - - - +
0.294 PP з5 - + + + -
0.з29 PP 41 4 - + - - -
0.з41 PP 42 - - + + -
0.з88 PP 4з + + - - -
0.400 PP 44 - - + + -
0.42з PP 51 5 - - - + +
0.4з5 PP 52 - + + - -
0.447 PP 5з - + + + +
0.470 PP 54 + - - - -
0.494 PP 55 - + + + +
0.517 PP 61 6 - + - - -
0.552 PP 62 - - + - -
0.564 PP 6з + - - - -
0.600 PP 64 + - + - -
0.6з5 PP 71 7 + + + + +
0.705 PP 81 8 - + - + -
0.717 PP 82 - - + - +
0.741 PP 8з - - - + -
0.764 PP 84 - + - - -
0.776 PP 85 - - + - +
0.788 PP 86 - - - + -
0.811 PP 91 9 + + - - -
0.82з PP 92 - - + + -
0.8з5 PP 9з - - - - +
0.882 PP 94 - - - - +
0.894 PP 95 - + + - -
0.905 PP 101 10 - - - + -
0.929 PP 102 + - - - +
0.952 PP 10з + - + - -
0.964 PP 104 - - - + -
0.988 PP 105 - - - - +
Figure 1. Zymogram of Salt Treated and Control Seedlings of Cicer arietinum
L1 - Control Seedlings; L2 - 4% of NaCl treated Seedlings C. Arietinum; L3 -6% of NaCl treated Seedlings; L4 - 8% NaCl treated Seedlings; L5 - 10% NaCl treated Seedlings.
DISCUSSION
Seeds of Cicer arietinum soaked for 24h in 1% NaCl, showed different percentage of seed germination and seedlings growth. The seeds soaked for more than 24 h showed very less percentage of germination and growth. The germinated seedlings were cultured on MS medium augmented with various percentage viz., 0, 4, 6, 8 and 10 of salt (NaCl). The seedlings showed the maximum tolerance up to 4% of NaCl. Growth of any organ is associated with an additional synthesis
of proteins which are building blocks of protoplasm and are again the resultant on inter- mediatory metabolism. Proteome analyses have resulted in the identification of many proteins that are inducible by salt stress including the metabolism related genes.
The results of the present study also revealed the expression of number of proteins which are induced by salt stress. The present observation directly coincides and supplements with the previous observations. When the seedlings were
treated with above 8 % of NaCl the number of protein bands and strength of bands were decreased.
Salt stress has been reported to cause an inhibition of growth and development, reduction in photosynthesis, respiration and protein synthesis in sensitive species (Boyer, 1982; Pal et al., 2004). Accumulation of metabolites that act as compatible solutes is one of the probable universal responses of plants to changes in the external osmotic potential. Metabolites with osmolyte function like sugar alcohols, complex sugars, proteins, isoenzymes and charged metabolites are frequently observed in plants under unfavorable conditions (Hasegawa et al., 2000; Satiropoulos, 2007).
Accumulation of organic solutes under saline conditions is an adaptive response of plants against osmotic stress (Tejera et al., 2004). This is in part due to the complexity of interactions between stress factors and various molecular, biochemical and physiological phenomena affecting plant growth and development (Zhu, 2001). One of the most common stress responses in plants over production of different types of compatible organic solutes viz., aminoacids, proteins, PGRs, soluble sugars and inorganic ions (Serraj and Sinclair, 2002). Salinity induces distinct protein changes in root and shoot tissues of barley (Ramagopal et al., 1987). The proteins and isoenzymes profiles of many plants are affected by addition of salts. In the present study also we observed the proteins expression with reference to salt concentrations. The banding positions and regions of activity are variable based on the developments of plants and concentrations of salts supplementations. Similar to the previous observation, the banding positions of C. arietinum salt treated seedlings showed the variation in the banding profile and expression in the protein gel
system. Some proteins and isoenzymes show their marked presence by the supplementation of salts in the media (Salt Induced Proteins), some are failed to express due to salt supplementations (salt sensitive proteins) and some showed their presence in the normal plants and salt supplemented seedlings also (Salt tolerant proteins). The results of the present study also revealed the expression of salt sensitive, salt induced and salt tolerant proteins in the C. arietinum salt treated seedlings. In this respect, Ebad et al., (1987) found a progressive and consistent decrease in concentration of total protein of maize plants with increasing NaCl. Moreover, Dell' Aquila and Spada (1992) detected a novel expression of the 26 kDa protein (osmotin) on germinating wheat embryos under salinity conditions. In addition, Hamada (1994) proved that low concentration of NaCl stimulated soluble protein production, but higher concentrations decreased the content of soluble proteins. Similar to Hamada's observation, in the present study also we observed the decreases in content and number of proteins in the seedlings treated above 8 % NaCl.
In conclusion, the data presented here revealed that salinity induced changes in protein profiles in the seedling of Cicer arietinum. This study reported the molecular weights of some salt responsive proteins. It is necessary to study further the structural and functional roles of these salt stress responsive polypeptides to enhance our understanding of the salt stress responses in Cicer arietinum.
REFERENCES
Anbalagan, K. (1999) An introduction to electrophoresis, Electrophoresis Institute Bohnert, H. J. (2007) Abiotic Stress: John Wiley & Sons, Ltd
Boyer, J.S. (1982) Plant productivity and environment. Sci. 218, 443-448.
Carceller, M. P. Prystupa and Lerncoff, J .H. (1999) Remobilization of proline and other Nitrogen compounds from senescing leaves of maize under water stress. J. A gr. Crop. Sci. 183, 61 -66.
Dell' Aquila, A. and Spada, P. (1992). Regulation of protein synthesis in germinating wheat embryos under polyethylene glycol and salt stress. Seed Sci. Res. 2, 75-80.
Dubey, R.S. and Pessarakli, M. (1995) Physiological mechanisms of nitrogen absorption and assimilation in plants under stressful conditions. In : M. Pessarakli, (e d.) Hand book of Plant and Crop Physiology Marcel Dekker, New York, 605625.
Dooslin Mary, D. Johnson, M & Gradin Jeyam.
(2009) Effect of UV-B radiation on Vigna radiata L. var. K -851. Biotechnology An Indian Journal, 3(4), 207-211.
Dooslin Mary, D. Johnson, M. and Geradin Jeyam.
(2010) UV-B Response of Orysa sativa var. ADT (R) -45l Seedlings. International Journal of Biotechnology and Biochemistry, 6(3), 411-418.
E bad, F.A. Khalaf, S.M. Ashoub, A. and Hand F.M. EL-Gaaly, (1987) Kinetin and cycocel effects on germination, growth and some metabolic products of soybean and maize growth under salinity conditions. Annals of Agric. Sci., Moshtohor. 25, 1337 -1351.
Flowers, T.J. Hajibagheri, M.A. and Yeo, A.R. (1991) Ion accumulation in the cell walls of rice plants growing under saline conditions: evidence for the Oertli hypothesis. Plant Cell Environ. 14, 319-325
Hamada, A.M. (1994). Alleviation of the adverse effects of NaCl on germination of maize grains by calcium. Biol Plant. 36, 623-627.
Hare, P. D. and Cress, W. A. (1997) Metabolic
implications of stress-induced proline
accumulation in plants. Plant Growth
Regulation. 21(2), 79-102.
Harinasut, P. K. Tsutsui, T. Takabe, M. Nomura, T. Takabe and Kishitani, S. (1996). Exogenous glycinebetaine accumulation and increased salt-tolerance in rice seedlings. Biosci. Biotechnol. Biochem. 60, 366-368
Hasegawa, P.M. Bressan, R.A. Zhu, J.K. and Bohnert, H.J. (2000) Plant cellular and molecular
responses to high salinity. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 463-499.
Munns, R. (2002) Comparative physiology of salt and water stress. Plant, Cell Environ. 25, 239250.
Munns, R. and Termaat, A. (1986) Whole plant responses to salinity. Aust. J. Plant Physiol. 3, 143-160.
Muthukumarasamy, M. Dutta Gupta, S. and Panneerselvam, R. (2000) Enhancement of peroxidase, polyphenol oxidase and superoxide dismutase activities by triadimefon in NaCl stressed Raphanus sativus L, Biologia Plantarum. 43(2), 317-320.
Pal, M. Singh, D.K. Rao, L.S. Singh, K.P. (2004) Photosynthetic characteristics and activity of antioxidant enzymes in salinity tolerant and sensitive rice cultivars. Indian J. Plant Physiol. 9, 407-412.
Ramagopal, S. (1987) Salinity Stress Induced Tissue-Specific Proteins in Barley Seedlings. Plant Physiol. June, 84(2), 324-331.
Sanders, D. (2000) Plant biology: The salty tale of Arabidopsis. Curr. Biol. 10, 486-488.
Serraj, R. Sinclair, T.R. (2002) Osmolyte
accumulation: can it really help increase crop
yield under drought conditions? Plant Cell Environ, 25, 333-341.
Stavropoulos, T.F. (2007) Effect of NaCl2 and CaCl2
on growth and contents of minerals,
chlorophyll, proline and sugars in the apple rootstock M4 cultured in vitro. Biol. Plantarum. 51, 177-180.
Tejera, N.A. Campos, R. Sanjuan T. and Liuch, C. (2004) Nitrogenase and antioxidant enzyme activities in Phaseolus vulgaris nodules formed by Rhizobium tropici isogenic strains with varying tolerance to salt stress. Plant Physiology, 161, 329- 338 Yercaud, Tamil Nadu, India.
Zhu, J.K. (2000) Genetic analysis of plant salt tolerance using Arabidopsis. Plant Physiol. 124, 941-948.
Zhu, J.K. (2001). Plant salt tolerance. Trends Plant Sci. 6, 66-71.
