Effect of some stresses on free proline content during pigeonpea (Cajanas cajan) seed germination

Abiotic stresses influences survival, biomass production and crop yield in negative manner. Proline is an electro cyclic low molecular weight protein amino acid of glutamate family has attracted considerable attraction of many ecophysiologists in last two decades. It is an organic osmoprotectant accumulates in a large number of plant species exposed to environmental stresses such as salinity, drought, extreme temperature, UV radiations and heavy metals (Hare and Cress 1997; Zhu et al.,

1998; Rhodes et al., 1999; Taylor 1996; Yamada et al., 2005; Eraslan et al., 2007). Proline is involved in the regulation of osmotic homeostasis, scavenging of reactive oxygen species and protection of cell structures (Srinivas and Balasubramanian 1995; lyer and Caplan 1998). Proline is a typical adaptive response in plants and it may be a part of stress signal (Maggio et al., 2002 Yang et al., 2009). Accumulation of proline has been reported from increased proline biosynthesis, lower proline utilization, decreased degradation of proline and proteins (Delauney and Verma 1993). The level of proline accumulation varies from species to species and also among the plant organs, its highest levels found in flowers and seeds and lowest levels in roots. (Verbruggen et al., 1993; Nathalie and Christian 2008). Proline ameliorates the stress tolerance by preventing membrane and protein damage (Santoro et al., 1992; Santarius 1992). Catabolism of proline in stress free conditions generates reducing equivalents especially ATP to repair stress-induced damage (Hare and Cress 1997; Hare et al., 1998). Ashraf and Foolad (2007) suggested that the application of proline successfully improved stress tolerance in plants. Hence it is worthwhile to study the fate of free proline under the influence of NaCl, Boron and aluminium treatment at the time of seed germination.

Thus the present work was carried out to investigate the effect of salinity and Boron and Aluminium toxicities on proline accumulation so as to assess the significance of proline in stress tolerance in germinating seeds of pigeonpea.

MATERIAL AND METHOD

Healthy seeds of Pigeonpea (var. ICPL-87) were sorted out and surface sterilized with 1% sodium hypochloride for two mins. Petri-plates were sterilized with absolute alcohol and lined with filter paper at bottom. Twenty seeds of Pigeonpea were allowed to germinate on Whatman filter paper No- 1 in sterilized petridishes at 30⁰ C in BOD under the influence of various concentrations of NaCl (0.4, 0.8 and 1.2%), Boron (10, 50 and 100 ppm) and Aluminium (5, 10 and 50 ppm) and investigations were covered at different stages of germination from 24 to 120 hrs. The free proline content was determined by employing the method of Bates et al . (1973).

• Fig.1    Effect of salt stress on the free proline content

  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  

  during pigeonpea seed germination.

  
  
  
  
  

  • □    control

  • ■    0.4 NaCl%

  • □    0.8 NaCl%

  • ■ 1.2 NaCl%

  
  

Germination hrs.

RESULTS AND DISCUSSION

The results revealed highest proline content at 120 hrs of seed germination by lower doses of NaCl as compared to 24 whereas decrease in proline content was found at 24 and 96 hrs. (Fig. 1). The role of proline as osmolyte in salt stressed tissues is supported by many physiologists. The increase in proline content due to NaCl treatment has been reported by some workers (Durgaprasad et al., 1996 Kuzhetsov and Shevyakova 1997). Increased proline contents and reduction in proline dehydrogenase enzyme activity has been reported in Brassica juncea (Madan et al., 1995) and in Solanum tuberosum (Rahnama and Ebrahimzadeh 2004). Similarly Wang et al. (2011) found higher proline contents, increased activity of orn-δ aminotransferase and reduced proline dehydrogenase in the leaves of Saussurea amara under salinity stress. In contrast to it decrease in proline content under salinity noticed by Singh and Jain (1982). Murumkar (1986) speculated that decrease in proline content under salt stress reflects salt sensitive nature of chickpea seeds at germination stage. It is evident from our observations that accumulation of proline at 120 hrs may be playing a significant role against the salt stress.

• Fig.2    Effect of various concentrations on Boron on free proline content during pigeonpea seed germination

  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  

  • □    control

  • ■    10ppm B

  • □    50ppm B

  • ■    100ppm B

  
  

24      48      72      96     120

Germination hrs.

Excess boron can limit plant growth on soils of arid and semiarid environments throughout the world. There was no attention has been paid so far to the influence of boron in the medium on proline level. Induction in proline accumulation was found at different stages of pigeonpea seed germination more particularly by 10 and 50 ppm of boron. However higher concentrations of boron (100 ppm) in the medium adversely affect the proline concentration (Fig. 2). Boron toxicity results into osmotic imbalances, photooxidative damage, memberane leakiness, lipid peroxidation and proline accumulation (Reid 2007; Eraslan 2007; Herrera-

Rodriguez et al ., 2007, 2010). Papadakis et al . (2003) has been emphasized increased proline contents in the leaves of Cleementine mandarin plant grafted on sour orange and Swingle citrumelo under boron toxic condition. If a level of proline is related to stress tolerance potential it can be concluded that boron concentrations above 50 ppm are not desirable for the pigeonpea seed germination.

The problem of soil acidity is becoming acute in the world. In acidic soil major problem is aluminium toxicity involved in limiting growth in plants (Tang et al., 2001; Kochiana et al., 2005; Valle et al., 2009). Soil acidity is reported to cause accumulation of proline in pea, soybean, wheat, barley, maize, sorghum and Polygonum (Klimashevskii 1984 Guo et al., 2004). The proline accumulation was found to be increased by aluminium treatments at 48, 72 and 120 hrs, while opposite trend was noticed at 24 and 96 hrs of pigeonpea seed germination (Fig. 3). The initial decrease in proline content is in agreement with the earlier report of Satokopan et al. (1990). They observed that increasing concentrations of Al2(SO4)3 from 1µM to 100 µM reduced proline content during chickpea seed germination. Guo et al. (2004) stated that Al and Cd toxicity caused increase in proline levels in barley seedlings. Aluminium decreased proline accumulation in leaves of Sorghum bicolor plant (Rodrigues da Cruz et al., 2011). It is difficult to pinpoint the exact role of proline under the aluminum toxicity.

• Fig. 3    Effect of various concentrations of Aluminium onfree Proline content during pigeonpea seed germination.

• □    control

• ■    5 ppm Al

• □    10ppmAl

• ■    50ppmAl

24     48     72     96     120

Germination hrs

From present work it has been concluded that the stimulation in proline accumulation may reflect a common strategy to overcome the stresses due to NaCl, boron and aluminium in pigeonpea .

ACKNOWLEDGEMENTS

One of the authors Mr. S.B. Bhamburdekar is thankful to UGC Western Region Pune for award of Teacher fellowship and Principal, Krishna Mahavidyalaya, Rethare Bk.

REFRENCES

Ashraf, M. and Foolad, M.R. (2007) Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environmental and Experimental Botany, 59(2): 206-216.

Bates, L.S.; Waldern, R.P. and Teare, T.D. (1973) Rapid determination of proline for water stress studies. Plant Soil , 39 :205-207.

Delauney A.J, Verma D.P.S (1993) Proline biosynthesis and degradation in plants. Plant J ., 4 : 215-223.

Eraslan, F., Inal, A., Gunes, A., Alpaslan, M. (2007) Boron toxicity alters nitrate reductase activity, proline accumulation, memberane permeability and mineral constituents of tomato and Pepper plants. Journal of Plant Nutrition , 30 : 981-994.

Guo, T., Zhang, G., Zhou, M., Wu, F. and Chen, J. (2004) Effects of aluminium and cadmium toxicity on growth and antioxidant enzyme activities of two barley genotypes with different Al resistance. Plant and Soil , 258 (1): 241-248.

Hare, P.D. and Cress, W.A. (1997) Metabolic implications of stress-induced proline accumulation in plants. Plant Growth Regulation , 21 : 79-102.

Hare, P.D., Cress, W.A., Van Staden, J. (1998) Dissecting the roles of osmolyte accumulation during stress. Plant Cell Envir . , 21 : 535-553.

Herrera-Rodriguez, M.B., Gonzalez-Fontes, A., Rexach, J., Camacho-Cristobal, J.J., Maldonado, J.M. and Navarro-Gochicoa, M.T. (2010) Role of boron in vascular plants and response mechanism to boron stresses. Plant Stress , 4 (2): 115-122.

Herrera-Rodriguez, M.B., Perez-Vicente, R. and Maldonado, J.M. (2007). Expression of asparagines synthetase genes in sunflower ( Helianthus annus ) under various environmental stresses. Plant Physiology and Biochemistry , 45 : 33-38.

Iyer, S. and Caplan, A. (1998) Products of proline catabolism can induce osmotically regulated genes in rice. Plant Physiol ., 116 : 203-211.

Klimashevskii, E.L. (1984) Akad.S-Kh Nauk. Im VI Lenina, 10 : 3-5.

Kochiana, L.V., Pineros, M.A. and Hoekenga, O.A. (2005). The physiology, genetics and molecular biology of plant aluminium resistance and toxicity. Plant Soil, 274: 175195.

Kuznetsov, V.V. and Shevyakova, N.I. (1997) Stress responses of tobacco cells to high temperature and salinity. Proline accumulation and phosphorilation of polypeptide. Physiol. Plant , 100: 320-326.

Madan, S., Nainawatee, H. S., Jain, R. K. and Chowdhury, J. B. (1995) Proline and proline metabolizing enzymes in in-vitro selected NaCl-tolerant Brassica juncea L. under salt stress. Ann. Bot ., 76 : 51-57.

Maggio, A., Miyazaki, S., Veronese, P., Fujita, T., Ibeas, J.I., Damsz, B., Narasimhan, M.L., Hasegawa, P.M., Joly, R.J. and Bressan, R.A. (2002) Does proline accumulation play an active role in stress-induced growth reduction? Plant J ., 31 : 699-712.

Murumkar, C.V. (1986) Physiological studies in chickpea ( Cicer arietinum L) Ph.D Thesis, Shivaji University, Kolhapur (MH), India.

Nathalie, V. and Christian, H. (2008) Proline accumulation in plants: a review. Amino Acids , 35 : 753-759.

Papadakis, I.E., Dimassi, K.N. Bosabalidis, A.M., Therios, I.N., Patakas, A. and Giannakoula, A. (2003) Boron toxicity in ‘Cleementine’ mandaris plants grafted on two rootstocks . Plant Science , 166 (2): 539-547.

Rahnama, H. and Ebrahimzadeh, H. (2004) The effect of NaCl on proline accumulation in potato seedlings and calli. Acta. Physiologiae Plantarum , 26 (3): 263-270.

Reid, R. (2007) Update on boron toxicity and tolerance in plants. In: Xu, F., Goldbach, H.E., Brown, P.H., Bell, R.W., Fujiwara, T., Hunt, C.D., Goldberg, S., Shi, L. (Eds).

Advances in Plant and Animal Boron Nutrition, Springer, Dordrecht , 83-90.

Rhodes, D., Verslues, P.E. and Sharp, R.E. (1999) Role of amino acids in abiotic stress resistance. In (B.K. Singh ed.) "Plant Amino Acids: Biochemistry and Biotechnology", Marcel Dekker, NY, pp. 319-356.

Rodrigues da Cruz, F.J., Silva Lobato, A.K., Lobo da Casta, R.C., Santos Lopez, M.J., Borges Neves, H.K., Oliveira Neto, C.F., Silva, M.H.L, Santos Filho, B.G., Lima Junior, J.A. and Okumura, R.S. (2011) Aluminium negative impact on nitrate reductase activity, nitrogen compounds and morphological parameters in Sorghum plants. Australian Journal of Crop Science, 5(6): 641-645.

Santarius, K.A. (1992) Freezing of isolated thylakoid membranes in complex media. VIII. Differential cryoprotection by sucrose, proline and glycerol . Physiol. Plant , 84 : 8793.

Santoro, M.M., Liu, Y., Khan, S.M.A., Hou, L.X. and Bolen, D.W. (1992) Increased thermal stability of proteins in the presence of naturally occurring osmolytes. Biochemistry 31 : 5278-5283.

Satokopan, V.N., Sankar, M. and Baskaran (1990) Determination of toxicity levels of Al³⁺ in chickpea ( Cicer arietinum L) seedlings. Indian Jr. Plant Physiol., 35 :90-94.

Singh, G., and Jain, S. (1982) Effect of some growth regulators on certain biochemical parameters during seed development in chickpea under salinity. Indian Jr. Plant Physiol ., 25 :167179.

Srinivas, V. and Balasubramanian, D. (1995) Proline is a protein-compatible hydrotrope. Langmuir, 11 : 2830-2833.

Tang, C., Diatloff, E., Rangel, Z. and McGann, B. (2001) Growth response to surface soil acidity of wheat genotypes differing in aluminium tolerance. Plant Soil , 236 : 1-10.

Taylor, C.B. (1996) Proline and water deficit: ups and downs. Plant Cell , 8 : 1221-1224.

Valley, S.R., Carrasco, J., Pinochet, D. and Calderini, D.F. (2009) Grain yield above ground and root biomass of Al-tolerant and Al-sensitive wheat cultivars under different soil aluminium concentrations at field conditions. Plant Soil , 318 : 299-310.

Verbruggen, N., Villarroel, R. and Van Montagu M. (1993) Osmoregulation of a pyrroline-5-carboxylate reductase gene in Arabidopsis thaliana . Plant Physiol ., 103 : 771-781.

Wang, K., Liu, Y., Dong, K., Dong, J., Kang, J. Yang, Q., He, Z. and Sun, Y. (2011) The effect of NaCl on proline metabolism in Saussurea amara seedlings. African Journal of Biotechnology, 10 (15): 2886-2893.

Yang, S.L., Lan, S.S. and Gong, M. (2009) Hydrogen peroxide-induced proline and metabolic pathway of its accumulation in maize seedlings. J. Plant Physiol ., 166 : 1694 1699.

Zhu, B.C., Su, J., Chan, M.C., Verma, D.P.S., Fan, Y.L. and Wu, R. (1998) Overexpression of a delta (1)-pyrroline-5-carboxylase synthase gene and analysis of tolerance to water and salt-stress in transgenic rice. Plant Science , 139 : 41-48.