Epigenetic and Post-Transcriptional Control

Epigenetic processes such as DNA methylation, histone modifica-tions, generation of small RNAs (sRNA) molecules and transposable element activity, play essential roles in modulating gene activity in response to environmental stimuli (Elenderson and Jacobsen et al, 2007; Feng et al., 2010). While most mechanisms involved in epigenetic and its heritance have not yet indentified, they play a major role in gene silencing on one hand and as a target for manipulation on the other. Abiotic stress can induce changes in gene expression through hypomethylation or hypennethylation of DNA which are related with stress tolerance. The stress-induced-specific CpHpG-hypennethylation in the halophyte Mesembryanthemum crys-tallinum L. induced the switch in photosynthesis mode from C₃ to CAM, contributing to the adaptation to salt stress (Dyachenko et al, 2006). In wheat, the use of the methylation inhibitor 5-azacytidine resulted in the increased tolerance to salt stress at the seedling stage. Decrease levels of histone acetylation levels (antisense) in tomato resulted in higher photosynthetic rates under water-stress (Scippa et al, 2004). The control of methylation and histone patterns is emerging as a potential tool for improving tolerance to abiotic stress in crops, however, little is known about how to control the effect of post transcriptional manipulation.

The small RNAs (sRNAs) are the bioregulators of plant stress response, regulates by transcriptional gene silencing (TGS), controlling mRNA stability and translation, or targeting epigenetic modifications (Khraiwesh et al, 2011). Abiotic stress can induce both the over- or under-expression of specific sRNAs that are involved hi pathways that contribute to re-program complex processes of metabolism and physiology. Several reports have recently indicated the possible use of these sRNAs as targets for the genetic manipulation of crops. The over expression of miR398 in Arabidopsis, which targets two closely related Cu/Zn superoxide dismutases (cytosolic CSD1 and chloroplastic CSD2) resulted in increased tolerance to oxidative stress (Sunkar et al, 2006).

Transgenic tomatoes expressing Sly-miR169c displayed decreased stomata opening, a decrease in leaf water loss and enhanced drought tolerance (Zhang et al., 2011). Several miRNAs regulating drought and salinity stress have been discovered till the date (Table 5). Transgenic rice constitutively expressing osa- MIR396c showed increased sensitivity to salt stress (Gao et al, 2010). The identification and characterization of the role(s) of sRNAs in the regulation of gene expression, together with the development of artificial miRNA methodologies open new avenues for the generation of transgenic stress tolerant crops (Schwab et al, 2005).

Choice of Promoters: When and How Much to Express

An important aspect of transgenic technology is the regulated expression of transgenes. Tissue specificity of transgene expression is also an important consideration while deciding on the choice of the promoter so as to increase the level of expression of the gene. Tims, the strength of the promoter and the possibility of using stress inducible, developmental stage, or tissue-specific promoters have also proved to be critical for tailoring plant response to these stresses (Bajaj et al, 1999). Some gene products are needed in large amounts, such as LEA3, thereby necessitating the need for a veiy strong promoter. With other gene products, such as enzymes for polyamine biosynthesis, it may be better to use an inducible promoter of moderate strength. The promoters that have been most commonly used in the production of abiotic stress tolerant plants so far, include the CaMY 35S, ubiquitin 1 and actin promoters.

These promoters being constitutive in nature, by and large express the downstream transgenes in all organs and at all the stages. However, constitutive over production of molecules, such as trehalose (Romero et al, 1997) or polyamines (Capell et al, 1998) causes abnormalities in plants grown under normal conditions. Also, the production of the above-described molecules can be metabolically expensive. In these cases, the use of astress inducible promoter may be more

Type of Stress	miRNA biomarkers	Up/Down regulated	References
Drought Stress	miR393, miR319, miR397 miR169g, miR171a miR156, miR159, miR168, miR170, miR171, miR172, miR319, miR396, miR397, miR408, miR529, miR896, miR1030, miR159, miR169, miR171, miR319, miR395, miR474, miR845, miR851, miR854, miR896, miR901, miR903, miR1026, miR1125 miR1035, miR1050, miR1088, miR1126, miR159.2, miR393, miR2118, miR398 a, b, miR408		Sunkar and Zhu 2004 Zhao et al., 2007, Jian et al., 2010 Zhou et al., 2010 Arenas-Huertero et al., 2009 Trindade et al., 2010
Salinity Stress	miR169g, miR169n, miR169o, miR393 miR156, miR158, miR159, miR165, miR167, miR168, miR169, miR171, miR319, miR393, miR394, miR396, miR397 miR398		Zhao et al., 2009, Gao et al., 2011 Liu et al., 2008

Table 6: Various promoters used for targeted expression of transgenes in crop plants to develop tolerance against drought and salinity stress

Fold increases of PAs in transgenic plants are from the basal level unless otherwise stated (NA, not available) desirable. In plants, various types of abiotic stresses induce a large number of well characterized and useful promoters. An ideal inducible promoter should not only be devoid of any basal level of gene expression in the absence of inducing agents, but the expression should be reversible and dose-dependent.

The transcriptional regulatory regions of the drought-induced and cold-induced genes have been analyzed to identify several cis-acting and trans-acting elements involved in the gene expression that is induced by abiotic stress (Shinwari, 1999). Most of the stress promoters contain an arr ay of stress-specific cis-acting elements that are recognized by the requisite transcription factors; for example, the transcriptional regulation of lisp genes is mediated by the core “heat shock element” (HSE) located in the promoter region of these genes, 5’ of the TATA box. All the plant hsp genes sequenced so far have been shown to contain partly overlapping multiple HSEs proximal to TATA motif. Apart from these hsp promoters, rd29 and adh gene promoters induced by osmotic stress and anaerobic stress, respectively, have also been studied. The Arabidopsis rd29A and rd29B are stress responsive genes, but are differentially induced under abiotic stress conditions. The rd29A promoter includes both DRE and ABRE elements, where dehydration, high salinity and low temperatures induce the gene, while the rd29B promoter includes only ABREs and the induction is ABA-dependent. Over expression of DREB1A transcription factors under the control of stress inducible promoter from rd29A showed a better phenotypic growth of the transgenic plants than the ones obtained using the constitutive CaMV 35S promoter (Kasuga et ah, 1999). A stress inducible expression of Arabidopsis CBF1 in transgenic tomato was achieved using the ABRC1 promoter from barley HAV22 (Lee et ah, 2003). Gene expression is induced by the binding of DREB1A, which in itself is induced by cold and water stress, to a cisacting DRE element in the promoters of genes such as rd29A, rdl7, cor 6.6, cor 15A, erd 10, and kinl, thereby, initiating synthesis of gene products imparting tolerance to low temperatures and water stress in plants. The regions of respiratory alcohol dehydrogenase adhl gene promoter in maize and rice that are required for anaerobic induction include a string of bases called anoxia response element (ARE) with the consensus sequence of its core element as TGGTTT. Besides, other stress-responsive cis-acting promoter sequences like low temperature responsive elements (LTRD) with a consensus sequence of A/GCCGAC have been identified in genes such as Cor 6.6, Cor 15 and Cor 78 These basic findings on stress promoters have led to a major shift in the paradigm for genetically engineering stress tolerant crops (Katiyar-Aggarwal et ah, 1999).