Improvement of water use efficiency in rice and tomato using Arabidopsis wax biosynthetic genes and transcription factors

Drought is a common occurrence in rainfed agriculture which is mainly dependent on the seasonal rainfall of the region. Many agricultural regions, especially in tropical-subtropical countries, have consistently low rainfall and unlike other abiotic stresses, the water availability directly affects productivity. Therefore, efforts to increase water uptake and/or conservation by the plants under water limiting conditions would lead to increased biomass, which contributes to fruit/grain yield. The threat of global warming and demands of an increasing world population will increase water scarcity, resulting in a growing need for water use efficient and drought tolerant crop plants.Water use efficiency (WUE), the ratio of biomass produced to the water used, is an important crop trait under water limiting and drought affected environments. Variation in WUE could arise either from variation in biomass (determined by the canopy carbon gain via photosynthesis, which is regulated by net carbon assimilation rate and functional leaf area) or through variation in the amount of water lost by transpiration (determined by stomatal conductance, cuticular properties and leaf area).Regulatory genes or transcription factors (TF), more specifically AP2/ERF family TF's have been shown to improve stress tolerance by regulating the coordinated expression of several stress related genes in heterologous transgenic plants. Subsequently, in this study an overexpression approach has been employed for the introduction of TFs and a few target drought adaptive genes from Arabidopsis, in rice and tomato to improve WUE.Oryza sativa ) cultivation requires two to three times more water when compared to other major food crops such as wheat or maize. Therefore, water deficit is a major constraint for rice production in rainfed areas. Plants develop adaptive strategies towards drought avoidance, such as an extensiveroot system in response to soil drying where the roots of resistant genotypes continue elongation at water potentials that are low enough to inhibit shoot growth completely.A previously identified Arabidopsis gene HARDY ( HRD), an AP2/ERF-like transcription factor, on overexpression shows increased leaf mesophyll layers, extended root branching and thickness and confers drought resistance in Arabidopsis (Dixit et al., unpublished). As the gene displayed various features useful for drought/abiotic stress resistance, the gene was transformed into the crop model rice to characterize the drought resistance related phenotype.HRDoverexpressors in rice exhibit improved WUE characterized by a higher biomass both with and without drought stress. This improvement in WUE is mostly due to an increase in root biomass under stress, and increases in leaf and shoot biomass without stress. HRD overexpression shows significantly higher net photosynthetic rate, which is exemplified under drought stress conditions. The major contributing factor for higher biomass and WUE, therefore, seems to be higher net carbon assimilation rate due to better mesophyll efficiency in the HRD lines. Higher net photosynthesis and relative quantum yield of PSII at steady state photosynthesis reflect the higher mesophyll capacity in HRD lines. Though the drought stress reduces the efficiency of PSII reaction centre similar to other studies in rice, HRD overexpression maintains higher efficiency of open PSII reactions compared to wild-type, both under well-irrigated and drought conditions, directly indicating the higher photosynthetic efficiency. The increased root biomass due to water deficit is an adaptive response specific to the HRD genotypes, indicating that HRD expression primes the plant to adapt to water deficit by inducing roots to harvest the scarce water.Plants lose a large amount of water in form of transpiration during assimilate/photosynthate production. Under water deficit conditions, most of the transpirational loss is due to non-stomatal/cuticular water loss since drought induces decreased stomatal conductance and stomatal closure. Therefore,decreasing transpiration by changing plant cuticular properties has potential applications. In overexpression studies with wax biosynthetic genes, that alter the cuticular properties either by increasing the cuticular waxes or the cutin monomers (resulting in changed characteristics of the cutin matrix), we show reduction in water lost through the cuticle and thus an increase in WUE.The variation for cuticular wax in rice has been suggested to be related to drought resistance, indicating that increase in wax content might be used to improve water use and drought resistance. Therefore, overexpression of genes and TFs involved in wax/cuticle biosynthesis holds great promise in reducing unrestricted cuticular water loss. In this study, rice plants overexpressing an ERF/AP2 TF SHN2 gene and a drought adaptive wax biosynthesis gene CER6, a key elongase enzyme in the wax biosynthesis pathway, show increase in WUE and drought resistance.Increased membrane stability under drought stress in theSHN2and CER6 transformants exhibits their increasedcapacity to avoid membrane damage andhelp stabilize the underlying photosynthetic apparatus.SHN2overexpressors show a reduction in transpiration at whole plant level, in both well watered and water deficit conditions. However, CER6 overexpressors did not show any change in transpiration at the whole plant level, probably because the increase in stomatal conductance is compensated by a decrease in cuticle permeability.Constitutive expression of stress resistance genes such as TFs has been shown to result in abnormal phenotypes and reduced growth. Therefore, identification of promoters with tissue specificity and stress inducibility is essential. We characterized the CER1 promoter, showing expression in epidermis and young tissue, as well as stress inducibility, and used it in tomato for overexpression studies of CER1 ,an aldehyde decarbonylase. Since the reactions of wax biosynthesis are localized in epidermal cells, the epidermal specific CER6 promoter was used in tomato to drive the expression of SHN1 , an AP2/ERF TF and a few other genes involved in the wax biosynthetic pathway, such as CER6 ,amajor condensing enzymeand WAX2 ,a gene involved inboth cutin and cuticular wax deposition.Cuticle and epidermal development are highly integrated and the nature of their interaction is complex, coregulated changes in the epidermis and cuticular permeability are observed in all the tomato transgenics.Overexpressors of the epidermal/cuticle genes changed epidermal cell shape, with SHN1 overexpressors also showing an increase in cell size. All the overexpressors showed a decrease in cuticular permeability of the leaves and fruit. Stomatal index of these overexpressors is reduced compared to wild-type plants under drought stress, which might be due to the stress inducible nature of the promoters. Decrease in transpiration and a resultant increase in WUE is observed in all the overexpressing tomato lines.As crop water loss is driven by the gradient in water vapor concentration between the crop canopy and the atmosphere, increasing leaf surface reflectance as observed for the tomato SHN overexpressors is expected to reduce the surface temperature of the photosynthetic tissue and increase WUE. This mechanism is supported by theSHN1overexpressors that showbetter photosynthetic mesophyll efficiency and higher efficiency of PSII reaction centre in light adapted conditionsunder both well watered and drought conditions.WAX2overexpression results in a 2.6 fold increase in the amount of cutin monomers compared to wild-type, probably responsible for the increased resistance to cuticular water loss from the plant surface. Consequently these lines display better WUE and reduced transpiration, and interestingly better mesophyll efficiency and efficiency of the PSII reaction center compared with wild-type only under drought stress.In nature, plants also have to cope with the interaction of multiple stresses that often arise concomitantly with drought, and ultimately involve oxidative stress. Protective responses at the leaf level must then be triggered in response to stress to prevent irreversible damage to the photosynthetic machinery. The molecular understanding of mechanisms involving stress perception, signal transduction, and transcriptional regulation of stress tolerance, may help engineer tolerance to multiple stresses in crop plants. Application of TFs may provide a more adaptive function in improving tolerance, either through protection or repair mechanisms. Advances in the molecular biology of stress responses in tolerant organisms are introducing the potentials of stress tolerance genes in agricultural programmes, not only to ensure survival but also to ensure productivity under drought.