Lectures

Guru Gobind Singh Indraprastha University School of Biotechnology

The Eighth Dr Yellapragada SubbaRow Memorial Lectures 2009

12 January 2009

"Linking Genes To Plant Stress Tolerance" by Sudhir K. Sopory Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnolgy, Aruna Asaf Ali Road, New Delhi 110067

temperature extremes etc. Based on trends recorded on changes in the global climate it is predicted that there will be an increase in average temperatures, higher sea levels and changes in rainfall patterns. These changes can lead to depletion of fresh water and increase in saline areas which will have a negative impact on crop productivity. Salinisation is one of the most serious environmental problems facing many countries. According to FAO over 30% land loss due to salinity may take place by 2020. Depletion of freshwater is another worrying aspect. There is thus a need to produce more food for growing population on less land with less water, or bring other areas like saline areas under cultivation. This is a challenge for science and technology

Presently conventional breeding has yielded limited success towards development of resistant varieties for abiotic stress tolerance in crop plants. It is therefore essential to complement the present plant breeding methods by more genomic level approaches and bring in genetic engineering technologies. This should enable one to tailor-make specific new crop types that will be highly productive in more harsh environments. To achieve these objectives a better understanding of the molecular basis of stress tolerance will be a prerequisite for undertaking genetic manipulation of crop plants for developing stress tolerance.

Abiotic stress leads to an enhanced expression of a number of genes and results in the modulation of various physiological and metabolic factors. The aim of our work is to identify genes and processes that are regulated under stress and move these genes into stress –sensitive plants. We have cloned a large number of stress up-regulated EST’s from plants like Pennisetum, and saline tolerant pokkali rice and have obtained from these a number of full length cDNAs . The expression of these genes in response to various abiotic stresses has been checked by northern and RT- PCR. Gene expression data analyzed using macroarrays and Northern blots suggest that salinity tolerance of pakkali may be due to constitutive expression of a number of genes. A detailed work on some of the genes encoding components in the signal transduction, transcription factors and those involved in glutathione and ion homeostasis and trafficking has been undertaken. In addition upstream elements of some of these genes have been cloned and analyzed using promoter:: reporter assays. Our studies also indicate that in addition to regulation through transcription factors, epigenetic changes like histone modifications may also be playing an important role in stress mediated up- or down regulation of genes. We have also cloned some of the identified genes in plant transformation vectors and transgenic rice plants have been raised for their functional validation. A detailed analysis has been done for genes encoding glyoxalase I and II and vacuolar sodium/proton antiporter. Glyoxalase pathway enzymes (Glyoxalase I and II) are required for glutathione based detoxification of methylglyoxal which is potent toxic compound. We have found that methylglyoxal levels do increase in response to stress and hence for the survival of the plants, its detoxification is required. We have developed transgenic tobacco and also rice that overexpress both glyoxalase I and II. These transgenic plans were found to tolerate higher levels of NaCl and were able to grow, flower and set seeds under stress conditions. The transgenic plants also showed tolerance to drought stress. Our detailed biochemical studies on these transgenic plants have shown that enhanced detoxification of methylglyoxal and maintenance of glutathione homeostasis is one of the possible mechanisms behind this tolerance. However, despite higher expression of both the enzymes, methylglyoxal was never totally detoxified. Our preliminary studies have indicated that methylglyoxal could be a signal molecule also. In the transgenic plants it was also found that under salinity stress there was in fact an enhanced uptake of Na. The tolerance to higher Na+ could be due to its sequestering into vacuoles via vacuolar sodium proton antiporter (NHX). We have cloned gene encoding for NHX and have developed NHX overexpressing transgenic rice. These plants also showed higher levels of tolerance which was further enhanced following exogenous application of calcium. A brief account on how calcium helps in increasing the tolerance limits will also be presented. Our studies with glyoxalase pathway engineering and also with some other genes show that it may be possible in future to grow crop plants under unfavorable conditions like saline and arid regions.

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References:

Kumari, S., Sabharwal- Panjabi, V., Khushwa, H.R., Sopory, S.K., Singla-Pareek, S.L. and Pareek, A.P. 2008. Transcriptome map for seedling stage specific salinity Stress response indicates a specific set of genes as candidates for saline tolerance in Oryza sativa L. Funct. Integ. Genomics (in press).

Singla-Pareek, S.L, Yadav, S.K., Ashwani Pareek, M.K.Reddy and Sopory, S.K.2008. Enhancing salt tolerance in a crop plant by overexpression of glyoxalase II. Transgenic Research 17:171-180

Mishra, R.N., Reddy, P.S., Nair, S., Markandeya, G., Reddy, A.R., Sopory, S.K. and Reddy, M.K. 2007. Isolation and characterization of expressed sequence tags (ESTs) from subtracted cDNA libraries of Pennisetum glaucum seedlings. Pl. Mol. Biol 64:713-732.

Shikha Misra , Yuliang Wu , Gayathri Venkatraman , Sudhir Sopory , Narendra Tuteja 2007 Heterotrimeric G-proteins complex and GPCR from a legume (Pisum sativum): role in salinity and heat stress and cross talk with PLC . Plant Journal 51:656-66

Sreenivasulu, N., Sopory, S.K. and Kavi Kishor, P.B. 2007. Deciphering the regulatory mechanisms of abiotic stress tolerance in plants by genomic approaches. Gene 388:1-13

Yadav, S.K., Singal-Pareek, S.L., Kumar, M., Pareek, A. Saxena, M., Sarin, N.B., and Sopory, S.K. 2007. Characterization and functional validation of glyoxalase II from rice. Protein Expr. Purification 51: 126-132

Agarwal, P, Agarwal, P.K. Nair, S., Reddy, M.K. and Sopory, S.K. 2007 Stress-inducible DREB2A transcription factor from Pennisetum galucum is a phosphoprotein and its phosphorylation negatively regulates its DNA –binding activity. Mol Gen. Genomics 277:189-198

Rajgopal, D, Agarwal, P. , Tyagi, W., Singla-Pareek, S.L., Reddy, M.K., Sopory, S.K. 2007 Characterization of an isoform of Na/H antiporter from Pennisetum galucum that confers high level of salinity tolerance in transgenic Brassica juncea. Molecular Breeding 19:137-151.

Singla-Pareek, S.L, Yadav, S.K., Pareek, A., M.K.Reddy, and S.K.Sopory 2006. Transgenic tobacco overexpressing glyoxalase pathway enzymes grow and set viable seeds in zinc spiked soils. Plant Physiology 140:613-623.

Yadav, S.K., Singla- Pareek, S.L., Ray, M., Reddy, M.K. and Sopory, S.K. 2005 Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase 1 and glutathione. Biochem. Biophys. Res. Commun. 337: 61-67.

Yadav. S.K., M.K. Reddy, S.K.Sopory, Singla –Pareek, S.L. 2005. Transgenic tobacco plants over expressing glyoxalase enzymes resist an increase in methylglyoxal and maintain higher reduced glutathione levels under salinity stress. FEBS Letts. 579: 6265 - 6271

Tyagi , W., Rajgopal, D., Singla-Pareek, S., Reddy, M.K., and Sopory, S.K. 2005. Cloning and regulation of a stress regulated Pennisetum glaucum vacuolar ATPase c gene and characterization of its promoter that expresses in shoot hairs and floral organs. Plant Cell Physiol. 46: 1411-1422.

Sannan-Mishra, N., Pham, X-H., Sopory, S.K. and Tuteja, N. 2005. Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield. Proc. Natn. Acad. Sci. USA 102 : 509-514

Singla-Pareek, S.L., Reddy, M.K. and Sopory, S.K. 2003. Genetic engineering of glyoxalase pathway in tobacco leads to enhanced salinity tolerance. Proc. Natn. Acad. Sci. USA 100: 14672-14677

Sahi, C., Agarwal, M , Reddy, M.K., Sopory, S.K. and Grover, A. 2003. Isolation and expression analysis of salt stress associated expressed sequence tags from contrasting rice cultivars using PCR-based subtraction method. Theor. Appl. Genet.106: 620-628

Veena, Reddy, V.S. and Sopory, S.K. (1999) Glyoxalase I from Brassica juncea: molecular cloning, regulation and its over expression confer tolerance in transgenic tobacco under stress. Plant Journal 17:385-396.