Rational Strategies for Developing Drought and Salt Stress Tolerance in Crop Plants

The response to stress depends on the duration and severity of the event, as well as the age and developmental stage of the plant, which varies with the species and genotype level (Bray, 1997). For crop plants, tolerance to abiotic stresses is measured by yield loss rather than survival. A key to progress towards breeding better crops under stress has been to understand the changes in cellular, biochemical and molecular machinery that occur in response to stress. Modem molecular techniques involve the identification and use of molecular markers that can enhance breeding programs. However, the introgression of genomic portions (QTLs) involved in stress tolerance often brings along undesirable agronomic characteristics from the donor parents. This is because of the lack of a precise knowledge of the key genes underlying the QTLs. Therefore, the development of genetically engineered plants by the introduction and/or over expression of selected genes seems to be a viable option to hasten the breeding of “improved” plants. Intuitively, genetic engineering would be a faster way to insert beneficial genes than through conventional or molecular breeding. Also, it would be the only option when genes of interest originate from cross barrier species, distant relatives, or from non-plant sources. Indeed, there are several traits whose correlative association with resistance has been tested in transgenic plants.

Therefore, a second “wave” of transformation attempts to transform plants with the third categoiy of stress-induced genes, namely, regulatory proteins has emerged. Through these proteins, many genes involved in stress response can be simultaneously regulated by a single gene encoding stress inducible transcription factor (Kasuga et al., 1999), thus offering possibility of enhancing tolerance towards multiple stresses including drought, salinity, and freezing.

Interestingly, genetic engineering allows controlling the timing, tissue-specificity, and expression level of the introduced genes for their optimal function. This is an important consideration if the function of a given gene is preferred only at a specific time, in a specific organ, or under specific conditions of stress (Katiyar et al., 1999). The most widely used promoters in generating transgenic plants are constitutively expressed; this may have serious deleterious effects on the plant. However, in cases where the gene expression needs to be tailored to a specific organ or a specific time, such constitutive promoters may not be a suitable choice, especially for the stress-induced genes. Accordingly, the efforts to generate transgenic plants make use of gene cassettes drivenby stress-induced promoters. With an increasing number of stress genes becoming available and genetic transformation becoming a routine procedure, characterization ofstress-induced promoters has taken a firm footing (Bhatnagar et al., 2008). Unfortunately, limited amount of published reports involving the assessment of transgenic plants under abiotic stresses has shown effect of the transgene under growth conditions that are unlikely to occur in the natural environmental conditions. Therefore, it is needed to set down basic guidelines on the protocols to be followed to conduct a rigorous evaluation of the transformed plants to abiotic stress. Since, most of the reports published so far has emphasized on model plants, this has hindered our ability to readily translate the discoveries into improved yield in crop plants. This can lead to use of tr ansgenic plants as a sources of new cultivars (or their gemrplasm as new sources of variation in breeding programmes) and they could also exceedingly useful as proof-of-concept tool to dissect the function and inteiplay of gene networks for abiotic stress tolerance.

Resource Species Used for the Identification of Abiotic Stress Tolerant Genes

Various organisms have been employed to identify processes or genes associated with salt tolerance. These organisms vary from prokaryotic such as Escherichia coli, unicellular eukaryotic organisms such as Saccharomycescerevisiae, to halophytic land plants such as Mesem-bryanthemum crystallinum, and glycophytic plantssuch as rice, tomato, Puccinellia tenuiflora,A.thaliana and many more.

Saccharomycescerevisiae

Beside its genetic amenability, S.cerevisiae shares basic ion transport mechanisms with plants. Therefore, it can be used as an excellent model system for the study of salt tolerance at a cellular level. Genetic analysis has been very successful in elucidating salt stress tolerance determinants in yeast (Toone and Jones, 1998; Serrano et ah, 1999). A number of salt-sensitive yeast mutants have been identified and the cloning of the corresponding genes has shed light on the nature of many genes that are essential for salt tolerance (Brewster et ah, 1993; Mendoza et ah, 1994; Toone and Jones, 1998; Serrano et ah, 1999).

Yeast responds to NaCl by activating several signal transduction pathways. Two of these pathways are needed to sense the osmotic stress induced by NaCl by different osmosensors like SLN1, a transmembrane histidine kinase, and SSK1, the sensor and response regulator respectively, of a two-component system (Maeda et ah, 1994).The second osmosensor, SHOl, is an independent trans-membrane protein containing a SH3domain (Maeda et ah, 1995). Both osmosensors connect to a Mitogen Activated Protein Kinase (МАРК) that modulates the pathways that converges at the PBS2 kinase (a MAPKK) that phosphorilates the HOG1 kinase leading eventually to the induction of several defence genes including the glycerol biosynthetic genes and the ENA 1 (Na+efflux pump) (Serrano, 1996; Toone and Jones, 1998). Plant proteins homologous to those of the HOG pathway in yeast have been identified. A putative МАРК from Pisum sativum(PsMAPK), which is 47% identical to Hoglp, functionally complements the salt growth defect of the hogl yeast mutant (Popping et ah, 1996). The Arabidopsis gene ATHK1 was identified by its sequence homology to the yeast osmosensor SLN1 (Urao et ah, 1999). Over-expression of ATHK1 suppressed the lethality of the temperature- sensitive osmosensing-defective yeast mutant shil-ts. The third signal transduction pathway for tolerance to NaCl in yeast is specific for the ionic component of

NaCl stress which regulates ion homeostasis and involves the protein phosphatase calcineurin (Mendoza et al, 1994). However, it is speculated that it could be a vacuolar cation exchanger that releases Ca+2 in ex-change with Na+ (Serrano, 1996). Calcineurin is a Ca+2 and calmodulin-dependent protein phosphatase consisting of a catalytic A subunit (CnA) and a regulatory В subunit (CnB) possessing four high affinity EF-hand calcium-binding sites and foil activation of CnA requires calcium-CnB and calcium-calmodulin dependent complexes (Klee et al, 1988). Calcineurin regulates Na+, K~ and Ca+2 homeostasis (Nakamura et al, 1993; Mendoza et al, 1994).

Despite biochemical evidence for a calcineurin-like activity in plants (Luan et al, 1993), the identification of the specific plant oriented gene has been unsuccessful so far. Notwithstanding the identification of CnB-like proteins in plants, none can interact with the yeast CnA, suggesting that they are not functional homologues (Kudla et al, 1999). However, functional complementation of the calcineurin yeast mutant with Arabidopsis identified AtGSKl, which encodes a GSK3/shaggy like protein kinase (Piao et al, 1999).

Halophytes

A halophyte is a plant that grows in waters of high salinity, coming into contact with saline water through its roots or by salt spray, such as in saline semi- deserts, mangrove swamps, marshes and sloughs, and seashores. It is now clear that the halophytes’s tolerance to NaCl is not the result of unique adaptive mechanisms or metabolic processes that are unique to these plants (Yeo, 1998; Glenn et al, 1999). It seems that the biochemical mechanisms leading to salt tolerance in these plants are regulated in such way that allow a more successful response to salt stress than in other plants (Hasegawa et al, 2000a, b). The halophytic land plants like M.crystallinum and Puccinellia tenuiflora have been frequently used as model plant in salt tolerance studies (Bohnert et al, 2001; Ying et al, 2014). Identification of gene is the major problem in the use of most halophytes (i.e. searching for salt hypersensitive mutants). For this purpose, the study of ThellungieUa halophila might be of particular interest in the identification of genes involved in salt tolerance. This halophyte plants can survive at seawater level salinity and its DNA sequence have a similarity of more than 90% of Arabidopsis (Zhu, 2001) which can also be easily transformed allowing insertion tag mutagenesis (Bressan et al, 2001).

Glycophytes

Salinity stress significantly reduces growth and productivity of glycophytes, which are the majority of agricultural products. Of all the glycophytes, undoubtly

Arabidopsis is becoming very useful in the determination of processes involved in salt tolerance (Zhu, 2000). Another glycophyte recently employed in genetic analysis using mutagenesis is tomato (Borsani et ah, 2001a). However, in spite of its broad adaptation, production is concentrated in a few warm and rather dry areas (Cuartero and Femandez-Munoz, 1999) where salinity is a serious problem (Szabolcs, 1994). For this reason, a large number of physiological studies of salt stress have been performed using tomato as a model plant (Cuartero and Femandez-Munoz, 1999). Unlike Arabidopsis, direct studies on salinity, adaptation, and molecular changes in this plant can be assessed also for crop yield.

 
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