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Crops are faced with a number of external factors that affect growth and development. These factors, biotic and abiotic, causes stress which can lead to the production of free radicals and reactive oxygen which can be toxic and cause damage to the plant. Abiotic stress include environmental factors like heat, salinity, drought, freezing, etc., which plays a huge role in the reduction of average crop yield. In the wake of global warming and unpredictable weather conditions, stress-tolerant plants are needed to tolerate the normal fluctuations of abiotic stress to maintain growth and development to improve crop yield. Furthermore, with the increase in human population and demand for food, stress-tolerant crops are needed to be grown in extreme environmental conditions like in deserts and high-salt soils (Yang et al, 2010). In agriculture, about 75% of the total global water is used up in many developing countries in irrigation (UNEP, 2009).
As opposed to animals, plants are sessile in nature and therefore depend largely on internal mechanisms to maintain homeostasis in normal fluctuations in the external environment (Yang et al, 2010). In other words, plants maintain growth and development by regulating internal metabolic processes in response to environmental stress. Many abiotic stress including drought cause similar damage to plants usually by water deficit. The flow of water from root to leaves is determined by the difference in water potential. Droughts is a water deficit stress which reduces soil water potential or increases the leaf water potential due to hot and dry windy conditions. Drought is a major environmental factor affecting crop productivity worldwide (Rivero et al, 2007) and water shortages are expected to become frequent as climate change alters rainfall patterns and increases the proportion of arid land in some key agricultural regions (Salinger et al, 2005). Plants use different strategies to cope with drought stress, for example, closing of the stomata to avoid water loss by transpiration, increase in root growth, reduced growth and wilting/shedding of old leaves (Fischer and Turner, 1978). Leaf senescence, a type of cell death, is believed to be a key survival strategy during drought but not economical to farmers as it reduces crop yield. Therefore, engineering drought tolerant crop plants to reduce leaf senescence during a drought episode is of economic importance. Recent studies in gene expression, transcriptional regulation and signal transduction in plants has led to practical approaches for the engineering of plant tolerance to drought (Umezawa et al, 2006).
Drought triggers a whole range of plant responses and its adaptation is one of the most complex biological processes involving reduction in growth, transcriptional activation/inactivation of specific genes, increases in abscisic acid (ABA) levels, accumulation of compatible solutes and protective enzymes, increased levels of antioxidants and suppression of energy-consuming pathways (Muhammad et al, 2011). Although conventional methods of plant breeding are important, they are labour-intensive and time-consuming during water deficit for a stable yield. The basic strategy for genetically engineering drought tolerance is the introduction of functional and/or regulatory genes that govern stress-related genes during drought, identified by the advancement in DNA microarray technology (Umezawa et al, 2006).
As mentioned above, plants respond to drought stress by accumulating non-toxic compounds called, compatible solutes or osmoprotectants to reduce the osmotic potential. These compounds can accumulate in high concentrations without disrupting the hydration shell around proteins and membranes during drought stress (Slater et al, 2008). The compounds are of 2 classes, sugar and sugar alcohols e.g. mannitol, sorbitol, pinitol e.t.c. and zwitterionic compounds (amino acids, proline and quaternary ammonium compounds, glycine betaine). Different plant species produce different compatible solutes to combat drought stress. However, some important crop plants (rice and tobacco) lack significant amount of these compatible solutes, hence a target for genetic engineering. Introduction of functional genes that encode enzymes associated with the synthesis of compatible solutes has been an area of focus.
Glycine betaine is one of such major osmoprotectant which is synthesized and accumulated in response to drought and able to restore and maintain osmotic balance in living cells (Nyyssölä et al, 2000). According to Waditee et al. (2005), the expression of N-methyltransferase genes in Arabidopsis via a novel betaine synthetic pathway accumulated betaine in higher levels than those produced by choline-oxidizing enzymes; and under stress, an improvement in seed yield was observed. Although the increased accumulation of glycine betaine level in the transgenic plant was slightly higher than the normal plants under stress, the transgenic plant showed more resistance to drought and other abiotic stress (Waditee et al, 2005). The increased tolerance is believed to be ascribed by the protective effect of glycine betaine on macromolecules (Slater et al, 2008) and its ability as an osmolyte, to increase cell-water retention while still maintaining normal cellular functions (Yancey et al, 1982).
Another of such important metabolite produced by plant during drought stress is abscisic acid (ABA). ABA is a stress hormone, produced in the leaves and induces the closure of the stomata to reduce water loss by transpiration (Ikegami et al, 2009). In plants, the 9-cis-epoxycarotenoid dioxygenase (NCED) gene was identified to participate in ABA synthesis (Schwartz et al, 1997). The AtNCED3 gene in Arabidopsis was found to be induced by drought stress and is responsible for the production of ABA under drought conditions. Overexpression of the AtNCED3 gene enhanced the expression of drought-inducible genes and also decreased leaf transpiration with accumulation of ABA. This overexpression of the AtNCED3 gene in the transgenic Arabidopsis plants improved drought tolerance (Iuchi et al, 2001).
The hormonal action of ABA in plants is also controlled by its catabolism. In addition to the NCED gene, the ABA catabolic pathway is triggered by ABA 8′-hydroxylase enzyme catalysed by the cytochrome P450 CYP707A(1-4) family in Arabidopsis, which plays a key role in the regulation of ABA levels in seed imbibition and drought stress (Saito et al, 2004). During a drought stress condition, all the CYP707A genes were upregulated with CYP707A2 responsible for the rapid decrease in ABA level during seed imbibition (Kushiro et al, 2004). In another expression analysis according to Umezawa et al (2006), the CYP707A3 gene of Arabidopsis was found most highly induced during dehydration and rehydration. A T-DNA insertional mutant, cyp707a3-1, contained higher ABA levels in turgid plants and showed a reduced transpiration rate. Conversely, expression of the CYP707A3 gene showed reduced growth retardation by ABA and increased transpiration in drought.
As stated above, plants respond to drought stress by reducing canopy size through leaf senescence and leaf abscission amongst others which in contrast reduces crop yield. Enhancing plant tolerance to drought-induced leaf senescence is a good target for genetically engineering drought resistance with reduced yield losses. Leaf senescence, the final stage of leaf development, is a type of programmed cell death in which many plants respond to developmental and stress processes regulated by nuclear gene expression. It is characterised by the transport and storage of assimilated nutrients from the senescing leaves to the developing leaves and seeds (Hayati et al, 1995). Leaf abscission, which follows senescence, reduces water loss through transpiration, thus contributing to the water retention of the whole plant but as an effect, also reduces yield (Munné-Bosch and Alegre, 2004). A number of plant hormones inhibit leaf senescence including Cytokinins. A number of different genes have also been identified to be either down-regulated or up-regulated during leaf senescence making regulation complex with a number of different pathways (Gan and Amasino, 1997). Blocking a particular pathway may not give the desired effect in the control of leaf senescence. Therefore a physiological approach for manipulating leaf senescence is preferred. This involves enhancing the overproduction of the plant hormone cytokinins in crop plants by expressing the isopentenyl transferase (IPT) responsible for its production (Ori et al, 1999). Production of cytokinins by the expression of the IPT gene in plants will delay senescence in leaves triggered by drought stress. The mechanism of engineering involves the introduction of the IPT gene with a senescence-specific promoter (SAG12) to direct its expression. At the onset of drought, the SAG12 promoter drives the expression of the IPT gene which in turn increases the production of cytokinins inhibiting leaf senescence. This method has been used to enhance drought tolerance in the crop plant Manihot esculenta (Cassava). According to the report by Zhang et al. (2010), the expression construct SAG12-IPT was introduce into the cassava plant which catalysed the production of cytokinins during drought stress. It was shown that the SAG12-IPT transgenic plants were resistant to drought-stress by limiting leaf abscission, producing stay-green leaves and a quick stress-recovery. It was also shown that the basal leaves of the transgenic plant had a higher rate of photosynthesis compared to the wild type. This was associated with the increase in cytokinin production in the transgenic cassava plant. This method has also been used in other crop plants like tobacco (Ori et al, 1999) and rice (Peleg et al, 2011) to confer resistance to drought stress by delaying leaf senescence.
Late embryogenesis abundant (LEA) proteins are highly hydrophilic proteins expressed at high levels in plant seeds and drought stressed tissues which confers the ability to withstand dehydration during embryo development (Tunnacliffe and Wise, 2007) . They were first identified in cotton plant (Gossypium hirsutum) (Dure and Chlan, 1981) and later in anhydrobiotic plants, animals and microorganisms (Wise and Tunnacliffe, 2004) where it is said act as chaperone proteins to prevent damage to cellular structures during drought stress. The LEA proteins are classified into at least 6 different groups with Group 1, 2 and 3 being the most important. It is noted also that some LEA proteins in plants and yeast can induce tolerance to water deficit (Xu et al, 1996). This has been reported by Chandra-Babu et al. (2004) when a LEA-encoded gene (HVA1) from barley was engineered into rice which conferred resistance to drought stress. Analysis revealed that the transgenic rice plant retained more leaf water and delayed wilting by 2 weeks relative to the wild rice type. It also showed the transgenic plant to have a better cell membrane protection after stress. This result showed how engineering LEA proteins encoded genes into crop plants can help improve tolerance to drought stress. Other crop plants like Mulberry (Morus Indica) (Lal et al, 2008), Chinese cabbage (Brassica campestris) (Park et al, 2005), tobacco (Wang et al, 2006) etc. have been engineered with LEA proteins which conferred resistance to drought stress.
The use of micro array technology and other molecular approaches have revealed a number of transcription factors associated with drought stress response (Tran et al, 2007). Their expression regulates the expression of target downstream genes responsible for drought stress tolerance (Umezawa et al, 2006). This makes transcription factors a target for engineering drought stress resistance. Some of the genes encoding transcription factors identified in the model Arabidopsis plant include AP2/ERF family, the basic leucine-zipper protein (bZIP), MYB/MYC, NAC, Zinc finger proteins, etc. have been implicated in the role of regulating drought stress response (Yang et al, 2010). However, overexpression of these Transcription factors induced by stress promoters in transgenic plants exhibit growth retardation and metabolic alterations (Vinocur and Altman, 2005). In Arabidopsis, overexpression of the ABA-responsive elements and ABRE binding factors ABF3, AREB2/ABF4, AREB1/ABF2 (types of bZIP transcription factor) resulted in reduced transcription and enhanced drought tolerance (Kang et al, 2002). They are called ABA-dependent transcription factors. In 2 transgenic rice plants expressed with CBF3/DREB1A and ABF3, both transgenic lines were shown to be resistant to drought stress. The transgenic plants exhibited normal growth and phenotype which are not usually associated with the Arabidopsis plant (Oh et al, 2005).
Another type of transcription factor belonging to the AP2/ERF family, DREB/CBFs, are also expressed in response to dehydration. They are called ABA-independent dehydration-responsive transcription factors (Nakano et al, 2006) with the DREB1/CBF signalling pathway being the most widely explored for engineering crop plants against drought stress. The DREB1A transcription factor from Arabidopsis expressed by a stress inducible promoter from the rd29A gene was introduced to peanut (Arachis hypogaea, Bhatnagar-Mathur et al, 2007) and wheat (Triticum aestivum, Pellegrineschi et al, 2004). During a drought stress episode, both transgenic plants maintained their transpiration rate and did not show growth retardation and phenotypic alterations. Overexpressing the transcription factors in both plants conferred resistance to drought.
The Nuclear Factor (NF-Y) transcription factor has been identified in Arabidopsis (AtNF-YB1) which when expressed improved performance under drought conditions. Its ortholog, ZmNF-YB2, was found in Zea mays and shown to have similar results in drought conditions (Nelson et al, 2007). In water deficit, the transgenic maize plants were greener and showed increased recovery compared to wild type. They also exhibited less wilting, delayed senescence, higher photosynthetic rate, higher chlorophyll index and less leaf rolling.
Aside from transcription factors, several genes encoding signalling factors have been identified to be involved in drought stress response (Chinnusamy et al, 2004). One of such method has been seen in the β-subunit of farnesyltransferase (ERA1) which regulates ABA signalling and drought tolerance (Wang et al, 2005). Down-regulation of the β-subunit in Arabidopsis results in increased ABA and in turn regulation of stomata closure (Cutler et al, 1996). This method was used to confer drought resistance in canola (Brassica napus). The target protein farnesylation, an antisense ERA1 construct, expressed by a drought-inducible promoter rd29A was introduced into the canola plant (Wang et al, 2005). The transgenic canola plant compared with the wild type showed increased ABA levels as well as reduced transpiration rate during drought stress. It was also shown to have an increased seed yield during mild drought conditions, however in normal conditions, seed yield was equal. Therefore targeting protein farnesyltransferase in crop plant is important in conferring drought resistance.
Another target in this group of signalling factors are the protein kinases. They have been linked to be involved in the phosphorylation of transcription factors in response to environmental stress (Boudsocq and Laurière, 2005). These protein kinases include MAPKKK, SnRK2, SnRK3, CDPK, CBL, and CIPK. Overexpression of an MAPKKK gene, Nicotiana protein kinase (NPK1), activated stress signals involved in abiotic tolerance in transgenic tobacco plants. This was also shown in maize where a tobacco MAPKKK (NPK1) was expressed which conferred drought tolerance in the transgenic maize (Shou et al, 2004). As well as being tolerant, the transgenic maize showed a significant increase in photosynthetic rate and also increased kernel weight compared to the wild type. This suggests that NPK1 induces protection to photosynthetic machinery during drought stress. A similar NPK1 gene has been found in rice, OsNPKL, which shows similar results to that seen in maize (Ning et al, 2008). Calcium-dependent protein kinases (CDPK) have also been implicated in plants to respond to abiotic stress (Ludwig et al, 2004). They are encoded by a number of genes and are involved in signal transduction during stress. The signalling pathway of different abiotic stress resistance is characterised by specific CDPK isoforms (Ludwig et al, 2004). A CDPK encoding gene in rice, OsCDPK7, was over expressed which enhanced stress-responsive genes in response to salinity and drought (Saijo et al, 2001). The transgenic rice showed increase in tolerance to drought and salinity. Another group of calcium protein kinases are the CIPKs activated by interacting with CBL proteins (Hirayama and Shinozaki, 2007). This pathway has been elucidated in rice and potato (Nookaraju et al, 2012) and their overexpression has conferred enhanced drought tolerance.
Genetically engineering drought tolerance in crop plants cannot be overemphasized. With unpredictable weather conditions due to climate change increasing the amount of arid land, crop plants need to be able to survive and produce yield enough to sustain the ever increasing world population. To combat this growing problem, molecular approaches have been devised to identify target genes, proteins and transcription factors which can confer drought resistance to crop plant. Although not a crop plant, Arabidopsis has played an important role in identifying basic processes underlying stress tolerance, and the knowledge obtained transferred to a number of important crop plants (Zhang et al., 2004). It also should be noted that these biological stress cross-talk and the engineering of one stress can affect another positively.