Chapter 1 Introduction

Transcription

1 Chapter 1 Introduction

2 1. INTRODUCTION Plants being sessile are exposed to environmental stresses mainly abiotic, caused by non-living effects of environment (temperature extremes, drought, and salinity) and biotic stress caused by living organism (viruses, bacteria, and insects). A. Abiotic: (physico chemical stress): Light: high intensity, low intensity Temperature: high, low (chilling, freezing) Water: deficit (drought), excess (flooding) Radiation: IR, Visible, UV, Ionizing (X-ray) Chemicals: salts, ions, gases, herbicides, heavy metals Mechanical factors: wind, pressure B. Biotic: Caused by the interaction and infection of living organism All these stress conditions induce potential injury on plant species. This injury can be reversible inhibiting metabolism and growth, or irreversible causing death of the cells. Among abiotic stress salinity, drought and temperature extremes are the major stresses. These stresses are responsible for crop loss worldwide, reducing average yields of major crop plants by more than 50 % (Vinocur and Altman 2005). Of the current 230 million ha of irrigated land, 45 million ha (20%) are salt affected (FAO 2008). 30 % of total land is affected by drought stress and 15% by cold stress (Blum 1986; Dai 2011). To meet the food demand for the rising world population, these adverse environmental factors are major challenges. The success through traditional breeding approaches in transferring the desirable traits from the wild relatives to cultivated varieties has been limited due to reproductive barriers and frequent failures of interspecific crosses. To cope up these stresses genetic engineering of crops is the better option to develop abiotic stress tolerance with different abiotic stress related genes. Various genes induced by salt stress are grouped under two categories, namely functional genes and regulatory genes. The first category of genes generally facilitates production of protective metabolites, which include osmolytes, transporters/channel proteins, antioxidative enzymes, lipid biosynthesis genes, polyamines. The second category of genes consists of regulatory proteins like bzip, DREB, MYC/MYB, and 1

3 NAC, which control expression of many downstream stress tolerance genes (Shinozaki and Yamaguchi-Shinozaki 2006; Agarwal and Jha 2010). These genes converge and interact in different pathways related to the abiotic stress and successfully lead to tolerance. Genetic engineering of plants to grow and produce an economic yield under the given environmental constraints remains the most viable solution to problems of environmental stress (Blum 1986; Ashraf 2009; Agarwal et al. 2012). Advances in understanding mechanisms for abiotic stress tolerance and molecular biology techniques have allowed us to tackle these problems more efficiently than in the past. Improved resistance to drought, salinity and extreme temperatures has been observed in transgenic plants that over-express genes for synthesis of osmolytes, specific proteins, antioxidants, ion homeostasis, transcription factors and transporters. Plants respond and adapt to abiotic stresses through various biochemical and physiological processes at cellular, tissue and whole plant level (Bohnert and Jensen 1996; Hasegawa et al. 2000: Wang et al. 2003). Several important pathways that are identified to be involved in the salt-stress signal transduction include salt-overlysensitive (SOS) pathway (SOS3-SOS2-SOS1) that regulates ion homeostasis under salt stress and results in Na + efflux, vacuolar compartmentation by NHX1, the calcium-dependent protein kinase (CDPK) pathway that plays an important role in osmotic stress, and the mitogen-activated protein kinase (MAPK) pathway that functions importantly in both abiotic and biotic stresses. Over-expression of these regulatory genes, such as transcription factors (DREB/CBF) and protein kinases (MAPK, CDPK), can increase plant salt tolerance (Liu and Zhu 1998; Kasuga et al. 1999; Kovtun 2000; Moon 2003; Tripathi et al. 2009; Matsukura et al. 2010). Transcription factors modulate the expression of specific groups of genes through sequence specific DNA binding and protein-protein interaction. They can act as activators or repressors of gene expressions, leading to specific effect of genes in terms of cellular response (Latchman 2003; Broun 2004). Abiotic stress related transcription factors follow ABA dependent and independent pathways. Abscisic acid (ABA) is an important plant hormone that plays a regulatory role in many physiological processes in plants, such as embryo maturation, seed development, seed dormancy, seed germination, root growth, fruit ripening, and regulation of stomatal closure. ABA is produced under abiotic stress and plays an 2

4 important role in the tolerance of plants to drought, salt, temperature stress and wounding (Ingram and Bartels 1996; Bray 1997; Riera et al. 2005; Agarwal and Jha 2010). Further, it is also proved that ABA is a major physiological signal that induces drought and high salinity responses by activation of stress responsive genes (Gomez et al. 1988; Verslues and Bray 2006; Farooq et al. 2009; Agarwal and Jha 2010; Cutler et al. 2010; Hubbard et al. 2010). The most of abiotic stress inducible genes contain potential Abscisic acid responsive element (PyACGTGGC) in their promoter region. An ABRE functions as a cis-acting DNA element involved in ABA regulated gene expression. Analyses of expression of water-stress inducible genes by ABA in ABA-deficient (aba) or ABA-insensitive (abi) Arabidopsis mutants have indicated that some of the stress-inducible genes do not require endogenous ABA accumulation under drought or cold conditions (Shinozaki and Yamaguchi-Shinozaki 1996; Bray 1997). ABA-dependent pathway include early responsive to dehydration 1 (ERD1) gene which has two different cis-acting elements such as (CATGTG) and a 14-bp rps1 site 1 like sequence. Another set of genes, such as RD29A (LTI78 and COR78), KIN1, COR6.6 (KIN2), and COR47 are also expressed under ABA-independent pathway (Liu, et al. 1998; Shinozaki and Yamaguchi-Shinozaki 2000). A 9 bp conserved sequence, TACCGACAT, named DRE, was found essential for the expression of RD29A gene under drought, low temperature, and high salt concentration conditions. Dehydration responsive element binding proteins (DREB) were isolated by using this DRE element as bait (Yamaguchi-Shinozaki and Shinozaki 1994). DREBs are important transcription factors that induce a set of abiotic stress related genes and impart stress tolerance to plants. They belong to the APETLA2/Ethylene Responsive Element Binding Protein (AP2/ERF) family of transcription factors which are specific to plants. The DREB transcription factors categorized as DREB1 and DREB2 subgroups are involved in two separate signal transduction pathways under low temperature and dehydration, respectively (Liu et al. 1998; Sakuma et al. 2006a; Matsukura et al. 2010). Major studies on abiotic stress tolerance has been conducted on glycophytes, however there are few studies carried on halophytes. DREB1 group members have been extensively studied from plants for stress tolerance against stresses like cold and salinity (Liu et al. 1998; Gilmour et al. 1998; Dubozet et al. 2003; Kasuga et al. 2004; Xu et al. 2009). DREB2 TFs has been reported for imparting tolerance against salinity, 3

5 drought and heat stress (Dubozet et al. 2003; Sakuma et al. 2006a; Agarwal et al. 2007; Almoguera et al. 2009; Mizoi et al. 2013). Till to date there is no report for characterization of DREB2 TF from a halophyte. Salicornia brachiata Roxb. (Amaranthaceae) is a leaf-less, annual, succulent, obligate halophyte growing in salt marshes in coastal areas. S. brachiata accumulates % NaCl of its dry matter. Salicornia brachiata accumulates salt in its pith and can survive at 2M salt concentration in field conditions. The vegetable salt from Salicornia known as Saloni, contains 5-7 % KCl along with NaCl, making it suitable for the high blood pressure patient. Calcium, magnesium and iodine micronutrients are naturally present in the Salicornia salt (Ghosh et al. 2005; US patent no ). This unique feature makes it a naturally adapted higher plant model to study salt and drought responsive gene(s). The molecular mechanism of salt adaptation in halophytes is far improved compared to glycophytes. Understanding of the molecular processes regulating these metabolic adaptations will facilitate engineering for abiotic stress tolerance. S. brachiata represents the potent germplasm for abiotic stresses tolerance mainly to salt and drought stresses. The present study was undertaken towards molecular characterization of the DREB2A gene from Salicornia brachiata Roxb. with following objectives. 1. Isolation of DREB2 transcription factor from Salicornia brachiata. 2. Transcript profiling of DREB2 under different stress conditions. 3. Cloning of the full length DREB2 in plant transformation vector. 4. Transformation of DREB2 in Nicotiana tabacum by Agrobacterium tumefaciens. 5. Confirmation of DREB2 transgenic plants and their functional analysis for studying its role in stress tolerance. 4