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The many strategies undertaken for improving abiotic stress tolerance in a particular genetic background have included screening of diverse genetic resources, wide crossing and subsequent recurrent backcrossing; identification and selection of the major conditioning genes through linkage mapping and quantitative trait loci (QTL) analysis; the production and screening of mutant populations and the transgenic introduction of novel genes (Figure 1). Although some success has been achieved in introducing tolerance traits into crop varieties from wild relatives (i.e. barley; Forster et al. 2000 and tomato; Foolad, Zhang and Lin 2001), in general there has been very little success reported in achieving high abiotic tolerance into elite germplasm (Flowers 2004).
As previously mentioned, breeding for, or induction of, abiotic stress tolerance traits is almost always limited by the genetic complexity of the underpinning mechanisms as well as potential interaction among genetic determinants. Also, differential selection of a particular stress may be affected by additional environmental factors, plant development stage, poor or irreproducible selection techniques, and the logistical constraints of physiological screening of large breeding populations on a field scale (Flowers et al. 2000). In this regard, the identification of discrete chromosomal regions that have a major effect on the specific tolerance trait through quantitative trait loci (QTL) mapping and marker-assisted selection remains a valuable option for many breeding programs (Cushman 2009, Cuartero et al. 2010). This is particularly so when whole genome knowledge is lacking and no candidate tolerance genes are known.
For accurate selection of the related phenotype, reliable and realistic screening techniques are required. However, uniformity and reliability of field based screening may suffer from heterogeneity in the stress across the site (i.e. boron or salinity level) as well as the potential compounding environmental factors (i.e. disease, rainfall, temperature). Also, when the starting material is genetically wide, heterogeneity among the genetic backgrounds may also impact on the ability to accurately select the most superior or different tolerances. As an alternative, cellular based mutant induction and subsequent selection initially under controlled in vitro conditions offers a method to quickly screen large populations with homogeneous backgrounds for novel fortuitous changes related to tolerance. Subsequent field screening then ensures adequate performance of the tolerance trait under the external potentially mitigating factors previously mentioned. Unsurprisingly, this method has generated great interest in selecting for abiotic stress tolerances in several crop species (Suprasanna, Sidha and Bapat 2008).
Transgenic approaches for engineering tolerance
Many genes linked to different pathways and processes such as stress perception and signaling, contributing to molecular, biochemical, cellular, physiological and morphological adaptations are differentially regulated in response to plant stress (Munns and Tester 2008). Stress responsive genes include those that alleviate the effect of the stress and lead to adjustment of the cellular environment and plant tolerance. The gene products are classified into three major groups: those that encode products that directly protect plant cells against stresses, those that are involved in signaling cascades and in transcriptional control and those that are involved in water and ion uptake and transport.
Engineering metabolic and stress-signaling pathways to produce stress-tolerant crops is one of the major interests of agricultural research. Genetic transformation with stress-inducible genes has been employed to gain an understanding of their functional role in the tolerance response and ultimately to improve the tolerance trait in the target genotype (Zhang, Creelman and Zhu 2004, Cuartero et al. 2010). To date, by far the majority of these studies have been limited to single-gene transfers within known multigenic pathways and mostly those involved in signaling and regulatory pathways, or effector genes that encode enzymes present in pathways leading to the synthesis of functional and structural protectants (Wang, Vinocur and Altman 2003, Chinnusamy, Jagendorf and Zhu 2005; Jewell et al. 2010). When selecting for success of the transformation experiment, a common prime considerations is whether the transgenic plants expresses a higher level of the transgene (i.e. an osmoprotectant or a protein) only under the stress conditions (Zhu 2001). In general, specific inducible promoters are used rather than constitutive promoters since the tolerance/stress induced mechanisms may be energy and nucleic acid greedy and divert essential resources away from normal growth processes (Su et al. 1998).
As examples, transgenic rice plants have been developed with choline oxidase (codA), D-pyrroline-5-corboxylate synthase (P5CS), LEA protein group 3 (HVA1), alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) genes exhibited drought tolerance (Datta 2002, Soren et al. 2010). Potato and rice (Yeo et al. 2000 and Garg et al. 2002, respectively) transformed with trehalose synthesis genes displayed tolerance to drought (in case of potato), and salt, drought, and low-temperature stress (in case of rice). Tobacco plants transformed with ectoine biosynthesis genes from the halophilic bacterium Halomonas elongate showed enhanced salt tolerance. Also transformation with genes for sorbitol (Sheveleva et al. 1997) or mannitol (Shen, Jensen and Bohnert 1997) resulted in an increased accumulation of these osmolytes and tolerance to high salinity (Table 1). Overexpression of genes encoding the enzymes pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) has resulted in proline over production and enhanced abiotic stress tolerance (Szabados and Savoure 2010). P5CS over expression in transgenic tobacco dramatically elevated free proline (Kishor et al. 1995) with improved germination and growth of seedlings under salt stress. Transgenic petunia plants transformed with Arabidopsis P5CS gene showed resistance to drought conditions for longer duration than control plants (Yamada et al. 2005).
The enhancement of glycine betaine (GB) synthesis in transgenic plants using genes that encode for enzymes (choline monooxygenase, betaine aldehyde dehydrogenase and choline oxidase) in GB biosynthesis is another strategy to achieve enhanced tolerance to drought, salt and chilling stress (Rontein, Basset and Hanson 2002; Chen and Murata 2008). Transgenic rice plants expressing the codA (choline oxidase) gene recovered from an initial growth inhibition under salt and low-temperature stress and grew normally than the wild type (Sakamoto, Murata and Murata 1998). Several other plants that have been genetically engineered for obtaining salt, drought, freezing and heat tolerance through GBS accumulation include; Arabidopsis thaliana, Brassica napus, Brassica juncea,Gossypium hirsutum, Lycopersicon esculentum, Nicotiana tabacum, Solanum tuberosum and Zea mays (Chen and Murata 2008).
Trehalose is a nonreducing disaccharide and is an effective osmoprotectant (Goddijn and van Dunn 1999) and transgenic plants over expressing trehalose biosynthetic genes showed increased tolerance to different abiotic stress conditions (Penna 2003; Almeida et al. 2007). Using a stress-inducible promoter to drive the overexpression of Escherichia coli trehalose biosynthetic genes (otsA and otsB) as a fusion gene (TPSP), tolerance to different abiotic stresses has been demonstrated in rice (Garg et al. 2002). The TPSP fusion gene has the dual advantages of needing only a single transformation event to introduce both genes simultaneously into the rice genome, while at the same time increasing the catalytic efficiency for trehalose formation by the bifunctional enzyme (Jang et al. 2003; Almeida et al. 2007).
Research on genetic engineering efforts with other osmolytes such as mannitol, fructans, ononitol, proline, glycinebetaine and ectoine has also shown promise for generating tolerant genotypes (Suprasanna, da Silva and Bapat 2005). To avoid over-production of compatible solutes burdening the plantââ‚¬â„¢s metabolic machinery and potentially diminishing pleiotropic effects, engineering for over-production should be done under stress-inducible and/or tissue specific regulation. In addition, production of the osmolytes should be targeted to the chloroplast by placing a signal sequence in front of the engineered enzymes (Shen, Jensen and Bohnert 1997).
As previously stated, abiotic stress generates an increase in reactive oxygen species that may be deleterious to normal cellular functions. Therefore, several oxidative-stress-related genes have been employed in developing transgenic plants tolerant to various stresses (Hussain et al. 2008). For example, transgenic tobacco plants overexpressing chloroplastic Cu/Zn-SOD showed increased resistance to oxidative stress caused by salt exposure (Tanaka et al. 1999; Bartel 2001). Transgenic alfalfa (Medicago sativa) plants expressing Mn-SOD had reduced injury from water-deficit stress, as determined by chlorophyll fluorescence, electrolyte leakage and re-growth (McKersie et al. 1996). Simultaneous expression of genes encoding three antioxidant enzymes; copper zinc superoxide dismutase, ascorbate peroxidase and dehydroascorbate reductase in the chloroplasts of tobacco plants conferred enhanced tolerance to oxidative stresses caused by paraquat and salt (Lee, Kim and Bang 2007). Similarly, overexpression of AtNDPK2 efficiently modulated oxidative stress caused by various environmental stresses in sweet potato through enhanced antioxidant enzyme activities such as peroxidase, ascorbate peroxidase and catalase (Kim et al. 2010). Thus it seems promising to target detoxification pathways as an approach for obtaining plants with multiple stress-tolerance traits.
Transgenic manipulation of detoxification pathways through overexpressing genes involved in oxidative protection, such as glutathione peroxidase, superoxide dismutase, ascorbate peroxidases and glutathione reductases is an area of current interest. Constitutive expression of the Nicotiana PK1 gene (regulatory protein NPK1) enhanced freezing, heat and salinity tolerance in transgenic maize plants (Shou, Bordallo and Wang 2004). In a further study, Shou et al. (2004) expressed a tobacco MAPKKK (NPK1) constitutively in maize and showed enhanced drought tolerance. The transgenic maize plants maintained significantly higher photosynthesis rates, suggesting that NPK1 induced a mechanism that protected photosynthesis machinery from dehydration damage.
When under salt stress, tolerant plant cells must maintain high K+ (100-200mM) and lower Na+ (less than 1mM) levels to maintain normal metabolic function. An important strategy for achieving greater tolerance to salinity stress is to help plants to re-establish homeostasis under stressful environments, restoring both ionic and osmotic homeostasis. This strategy continues to be a major approach to improve salt tolerance in plants through genetic engineering, where the target is to achieve Na+ excretion, or vacuolar storage. A number of abiotic stress tolerant transgenic plants have been produced by increasing the cellular levels of proteins (such as vacuolar antiporter proteins) that control the transport functions. For example, AtSOS from Arabidopsis has been shown to encode a plasma membrane Na+/H+ antiporter (NHX) with significant sequence similarity to the respective antiporter from bacteria and fungi (Shi et al. 2000). Constitutive expression of vacuolar Na+/H+ antiporter (NHX1) or AVP1 (Arabidopsis thaliana vacuolar H+- translocating pyrophosphatase) gene energized the pumping of Na+ into the vacuole, and increased both accumulation and Na+ tolerance in Arabidopsis (Gaxiola et al. 2001). Thus more efficient sequestration of these ions to the vacuole could improve tissue tolerance to salinity by reducing the cytosolic Na+ concentrations. The importance of Na+ sequestration in salt tolerance has been further demonstrated in transgenic tomato plants over expressing the AtNHX1 gene (Zhang and Blumwald 2001). Also, a vacuolar chloride channel gene, AtCLCd, involved in cation detoxification has been cloned and over expressed in Arabidopsis and shown to confer salt tolerance. Up-regulation of the Salt Overly Sensitive 1 (SOS1) gene in Arabidopsis resulted in a greater proton motive force necessary for elevated Na+/H+ antiporter activities (Shi et al. 2000).
Apart from the single gene approach, tolerance towards multiple stresses may be achieved by targeting a stress inducible signal transduction molecule and/or transcription factor (Chinnusamy, Jagendorf and Zhu 2005). The transcription factors play an important role in the acquisition of stress tolerance, which may ultimately contribute to agricultural and environmental practices (Century, Reuber and Ratcliffe 2008). A large numbers of transcription factors are involved in the plant response to abiotic stress (Vincour and Altman, 2005). Most of these falls into several large transcription factor families, such as AP2/ERF, bZIP, NAC, MYB, MYC, Cys2His2 zinc-finger and WRKY. Accordingly, over expression of the functionally conserved At-DBF2 gene resulted in wide and high levels of multiple stress tolerances in Arabidopsis (Lee, van Montagu and Verbruggen 1999). Salt stress-tolerant tobacco plants were produced by over expressing the calcineurin, a Ca2+/calmodulin-dependent protein phosphatase gene, formally identified as being involved in salt-stress signal transduction in yeast (Pardo, Reddy and Yang 1998; Grover et al. 1999).
Some stress responsive genes may share the same transcription factors, as indicated by the significant overlap of the gene expression profiles that are induced in response to drought and cold stress (Seki et al. 2001; Chen and Murata 2002; Mantri et al. 2007). The activation of stress-induced genes has been possible in transgenic plants by over-expressing one or more transcription factors that recognize regulatory elements of these genes. In Arabidopsis, the transcription factor DREB1A specifically interacts with the DRE and induces expression of stress tolerance genes (Shinozaki and Yamaguchi-Shinozaki 1997). CaMV 35S promoter-driven over-expression of DREB1A cDNA in transgenic Arabidopsis plants provided tolerance to salt, freezing and drought stress through strong constitutive expression of the stress inducible genes (Liu et al. 1998).
The transcription factors involved in the ABA-dependent (such as NAC, AREB/ABF, and MYB) and ââ‚¬"independent (AP2/ERF gene) stress response pathways regulate cascade of downstream genes and events that enhance tolerance to drought stress. Transforming crops with such transcription factor genes should be more meaningful in the development of drought tolerance (Zhang, Creelman and Zhu 2004; Ashraf 2010). Over-expressing Arabidopsis CBF1 (CRT/DRE) cDNA in tomato improved tolerance to salt, chilling and drought stress, however, the plants exhibited a dwarf phenotype as well as reduced fruit set and seed number (Hsieh et al. 2002). Over-expression of Alfin1 (transcriptional regulator) in alfalfa plants exhibited salinity tolerance through regulated endogenous MsPRP2 (NaCl-inducible gene) mRNA levels (Winicov and Bastola 1999).
The future of transgenic approaches
The current scenario in plant genetic engineering for developing salt stress tolerant transgenic plants has included approaches of altering the expression levels of native genes or by incorporating alien genes for osmolytes, ion transporters, transcription factors and other signaling genes. The advent of global transcription profiling has demonstrated that large numbers of other genes are also up and down regulated simultaneously in response to salt stress. These second category of genes, encode proteins related to the regulation of transcriptional and translational machineries with distinct roles in mediating the salt stress response. Particularly, coordinated induction and action of the transcript of several RNA binding proteins, ribosomal genes, helicases, cyclophilins, F-box proteins, dynamin-like proteins, translation initiation and elongation factors seems to be important in salt stress tolerance. The functionality of these genes at the cellular level should be investigated also to assess suitability for targeted transgenic approaches (Sahi et al. 2006).
The evaluation of genetically engineered salt tolerant transgenic lines needs critical, careful, and thorough experimentation (Flowers 2004). The fourth or fifth generation genotypes should be evaluated along with parents (wild-types) line under controlled saline and non-saline treatment conditions. Validation should not stop at the laboratory or green house level, since quantitative measures of growth are required including at emergence and through the vegetative developmental stages.
Conclusions and perspective
In the last decade, significant progress has been made in our understanding of the complex mechanisms governing abiotic stress tolerance in crop plants. However, we are still far from pinning the exact battery of gene activation responsible for tolerance to a particular abiotic stress condition. This situation is complicated when one considers plants have to simultaneously cope with numerous biotic stresses along with various abiotic stresses. Our struggle to understand these complex mechanisms is ongoing and recent development of new tools for high-throughput genotyping and phenotyping gives us a new ray of hope. A complete understanding on physiological and molecular mechanisms especially signalling cascades in response to abiotic stresses in tolerant plants will help to manipulate susceptible crop plants and increase agricultural productivity in the near future.