Many genes linked to different pathways and processes such as stress perception and signalling, 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 encoding products that directly protect plant cells against stress, those that are involved in signalling cascades and in transcrip-tional control and those that are involved in water and ion uptake and transport.
Engineering metabolic and stress-signalling 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 et al. 2004, Cuartero et al. 2010). To date, by far, majority of these studies have been limited to single-gene transfers within known multigenic pathways and mostly those involved in signalling and regulatory pathways, or effector genes that code for enzymes catalysing the synthesis of structural and functional defendants (Wang et al. 2003; Chinnusamy et al. 2005; Jewell et al. 2010). When selecting for success of the transformation experiment, a common prime consideration is whether the transgenic plants express 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 developed with choline oxidase (codA), d-pyrroline-5-cor-boxylate 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 halo-philic bacterium Halomonas elongate showed enhanced salt tolerance. Also transformation with genes for sorbitol (Sheveleva et al. 1997) or man-nitol (Shen et al. 1997) resulted in an increased accumulation of these osmolytes and tolerance to high salinity (Table 1.1). Overexpression of genes encoding the enzymes pyrroline-5-carboxylate
(P5C) synthetase (P5CS) and P5C reductase (P5CR) resulted in proline overproduction and enhanced abiotic stress tolerance (Szabados and Savoure 2010). P5CS overexpression in trans-genic 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 et al. 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 et al. 1998). Several other plants that have been genetically engineered for obtaining salt, drought, freezing and heat tolerance through GBS accumulation include; A. thaliana, Brassica napus, Brassicajuncea, Gossypium hirsutum, Lycopersicon esculentum, Nicotiana tabacum, Solanum tuberosum and Zea mays (Chen and Murata 2008) .
Trehalose is a non-reducing disaccharide and an effective osmoprotectant (Goddijn and van Dunn 1999) . Transgenic plants overexpressing trehalose biosynthetic genes showed increased tolerance to different abiotic stress conditions (Penna 2003; Almeida et al. 2007). A stress-induc-ible promoter has been utilised to overexpress Escherichia coli trehalose biosynthesis genes (otsA and otsB) as a fusion gene (TPSP) in rice, to confer tolerance to different abiotic stresses (Garg et al. 2002). The TPSP fusion gene is dually advantageous as both the genes can be simultaneously introduced into the rice genome leading to increased catalytic efficiency for trehalose synthesis (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 have also shown promise for generating tolerant genotypes (Suprasanna et al. 2005). To avoid overproduction of compatible solutes burdening the plant's metabolic machinery and potentially diminishing pleiotropic effects, engineering for overproduction 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 et al. 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 regrowth (McKersie et al. 1996). Simultaneous expression of genes encoding three antioxidant enzymes: copper zinc superoxide dismutase, ascorbate per-oxidase and dehydroascorbate reductase in the chloroplasts of tobacco plants conferred enhanced tolerance to oxidative stresses caused by paraquat and salt (Lee et al. 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 per-oxidase, superoxide dismutase, ascorbate peroxi-dases 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 et al. 2004b). In a further study, Shou et al. (2004a) expressed a tobacco MAPKKK (NPK1) constitutively in maize resulting in enhanced drought tolerance. The transgenic maize plants maintained significantly higher photosynthesis rates, suggesting, NPK1 induced a mechanism that protected photosynthesis machinery from dehydration damage.
Under salt stress, tolerant plant cells must maintain high K+ (100-200 mM) and lower Na+ (less than 1 mM) levels for 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 (A. 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 overex-pressing the AtNHX1 gene (Zhang and Blumwald 2001) ; Also, a vacuolar chloride channel gene, AtCLCd, involved in cation detoxification has been cloned as well as overexpressed 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 et al. 2005). The transcription factors play an important role in the acquisition of stress tolerance, which ultimately contribute to agricultural and environmental practices (Century et al. 2008). A large number 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, overexpression of the functionally conserved At-DBF2 gene resulted in wide and high levels of multiple stress tolerances in Arabidopsis (Lee et al. 1999) ; Salt stress-tolerant tobacco plants were produced by overexpressing the calcineu-rin, a Ca)+/calmodulin-dependent protein phos-phatase gene, formally identified as being involved in salt-stress signal transduction in yeast (Pardo et al. 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 overexpressing 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 overexpression 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 -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 et al. 2004; Ashraf 2010). Overexpressing 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). Overexpression of Alfin1 (transcrip-tional regulator) in alfalfa plants exhibited salinity tolerance through regulated endogenous MsPRP2 (NaCl-inducible gene) mRNA levels (Winicov and Bastola 1999).
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