Simultaneous expression of regulatory genes associated with specific drought-adaptive traits improves drought adaptation in peanut.

Adaptation of crops to drought-prone rain-fed conditions can be achieved by improving plant traits such as efficient water mining (by superior root characters) and cellular-level tolerance mechanisms. Pyramiding these drought-adaptive traits by simultaneous expression of genes regulating drought-adaptive mechanisms has phenomenal relevance in improving stress tolerance. In this study, we provide evidence that peanut transgenic plants expressing Alfalfa zinc finger 1 (Alfin1), a root growth-associated transcription factor gene, Pennisetum glaucum heat-shock factor (PgHSF4) and Pea DNA helicase (PDH45) involved in protein turnover and protection showed improved tolerance, higher growth and productivity under drought stress conditions. Stable integration of all the transgenes was noticed in transgenic lines. The transgenic lines showed higher root growth, cooler crop canopy air temperature difference (less CCATD) and higher relative water content (RWC) under drought stress. Low proline levels in transgenic lines substantiate the maintenance of higher water status. The survival and recovery of transgenic lines was significantly higher under gradual moisture stress conditions with higher biomass. Transgenic lines also showed significant tolerance to ethrel-induced senescence and methyl viologen-induced oxidative stress. Several stress-responsive genes such as heat-shock proteins (HSPs), RING box protein-1 (RBX1), Aldose reductase, late embryogenesis abundant-5 (LEA5) and proline-rich protein-2 (PRP2), a gene involved in root growth, showed enhanced expression under stress in transgenic lines. Thus, the simultaneous expression of regulatory genes contributing for drought-adaptive traits can improve crop adaptation and productivity under water-limited conditions.

A high-throughput virus-induced gene silencing protocol identifies genes involved in multi-stress tolerance.

BACKGROUND: Understanding the function of a particular gene under various stresses is important for engineering plants for broad-spectrum stress tolerance. Although virus-induced gene silencing (VIGS) has been used to characterize genes involved in abiotic stress tolerance, currently available gene silencing and stress imposition methodology at the whole plant level is not suitable for high-throughput functional analyses of genes. This demands a robust and reliable methodology for characterizing genes involved in abiotic and multi-stress tolerance. RESULTS: Our methodology employs VIGS-based gene silencing in leaf disks combined with simple stress imposition and effect quantification methodologies for easy and faster characterization of genes involved in abiotic and multi-stress tolerance. By subjecting leaf disks from gene-silenced plants to various abiotic stresses and inoculating silenced plants with various pathogens, we show the involvement of several genes for multi-stress tolerance. In addition, we demonstrate that VIGS can be used to characterize genes involved in thermotolerance. Our results also showed the functional relevance of NtEDS1 in abiotic stress, NbRBX1 and NbCTR1 in oxidative stress; NtRAR1 and NtNPR1 in salinity stress; NbSOS1 and NbHSP101 in biotic stress; and NtEDS1, NbETR1, NbWRKY2 and NbMYC2 in thermotolerance. CONCLUSIONS: In addition to widening the application of VIGS, we developed a robust, easy and high-throughput methodology for functional characterization of genes involved in multi-stress tolerance.

Drought stress acclimation imparts tolerance to Sclerotinia sclerotiorum and Pseudomonas syringae in Nicotiana benthamiana.

Acclimation of plants with an abiotic stress can impart tolerance to some biotic stresses. Such a priming response has not been widely studied. In particular, little is known about enhanced defense capacity of drought stress acclimated plants to fungal and bacterial pathogens. Here we show that prior drought acclimation in Nicotiana benthamiana plants imparts tolerance to necrotrophic fungus, Sclerotinia sclerotiorum, and also to hemi-biotrophic bacterial pathogen, Pseudomonas syringae pv. tabaci. S. sclerotiorum inoculation on N. benthamiana plants acclimated with drought stress lead to less disease-induced cell death compared to non-acclimated plants. Furthermore, inoculation of P. syringae pv. tabaci on N. benthamiana plants acclimated to moderate drought stress showed reduced disease symptoms. The levels of reactive oxygen species (ROS) in drought acclimated plants were highly correlated with disease resistance. Further, in planta growth of GFPuv expressing P. syringae pv. tabaci on plants pre-treated with methyl viologen showed complete inhibition of bacterial growth. Taken together, these experimental results suggested a role for ROS generated during drought acclimation in imparting tolerance against S. sclerotiorum and P. syringae pv. tabaci. We speculate that the generation of ROS during drought acclimation primed a defense response in plants that subsequently caused the tolerance against the pathogens tested.

Identification and functional validation of a unique set of drought induced genes preferentially expressed in response to gradual water stress in peanut.

Peanut, found to be relatively drought tolerant crop, has been the choice of study to characterize the genes expressed under gradual water deficit stress. Nearly 700 genes were identified to be enriched in subtractive cDNA library from gradual process of drought stress adaptation. Further, expression of the drought inducible genes related to various signaling components and gene sets involved in protecting cellular function has been described based on dot blot experiments. Fifty genes (25 regulators and 25 functional related genes) selected based on dot blot experiments were tested for their stress responsiveness using northern blot analysis and confirmed their nature of differential regulation under different field capacity of drought stress treatments. ESTs generated from this subtracted cDNA library offered a rich source of stress-related genes including signaling components. Additional 50% uncharacterized sequences are noteworthy. Insights gained from this study would provide the foundation for further studies to understand the question of how peanut plants are able to adapt to naturally occurring harsh drought conditions. At present functional validation cannot be deemed in peanut, hence as a proof of concept seven orthologues of drought induced genes of peanut have been silenced in heterologous N. benthamiana system, using virus induced gene silencing method. These results point out the functional importance for HSP70 gene and key regulators such as Jumonji in drought stress response.

Glycinebetaine-induced water-stress tolerance in codA-expressing transgenic indica rice is associated with up-regulation of several stress responsive genes.

Rice (Oryza sativa L.), a non-accumulator of glycinebetaine (GB), is highly susceptible to abiotic stress. Transgenic rice with chloroplast-targeted choline oxidase encoded by the codA gene from Arthrobacter globiformis has been evaluated for inheritance of transgene up to R5 generation and water-stress tolerance. During seedling, vegetative and reproductive stages, transgenic plants could maintain higher activity of photosystem II and they show better physiological performance, for example, enhanced detoxification of reactive oxygen species compared to wild-type plants under water-stress. Survival rate and agronomic performance of transgenic plants is also better than wild-type following prolonged water-stress. Choline oxidase converts choline into GB and H2O2 in a single step. It is possible that H2O2/GB might activate stress response pathways and prepare transgenic plants to mitigate stress. To check this possibility, microarray-based transcriptome analysis of transgenic rice has been done. It unravelled altered expression of many genes involved in stress responses, signal transduction, gene regulation, hormone signalling and cellular metabolism. Overall, 165 genes show more than two-fold up-regulation at P-value < 0.01 in transgenic rice. Out of these, at least 50 genes are known to be involved in plant stress response. Exogenous application of H2O2 or GB to wild-type plants also induces such genes. Our data show that metabolic engineering for GB is a promising strategy for introducing stress tolerance in crop plants and which could be imparted, in part, by H2O2- and/or GB-induced stress response genes.

Introgression of Root and Water Use Efficiency Traits Enhances Water Productivity: An Evidence for Physiological Breeding in Rice (Oryza sativa L.).

BACKGROUND: Semi-irrigated aerobic cultivation of rice has been suggested as a potential water saving agronomy. However, suitable cultivars are needed in order to sustain yield levels. An introgression of water mining and water use efficiency (WUE) traits is the most appropriate strategy for a comprehensive genetic enhancement to develop such rice cultivars. RESULTS: We report a novel strategy of phenotyping and marker-assisted backcross breeding to introgress water mining (root) and water use efficiency (WUE) traits into a popular high yielding cultivar, IR-64. Trait donor genotypes for root (AC-39020) and WUE (IET-16348) were crossed separately and the resultant F1s were inter-mated to generate double cross F1s (DCF1). Progenies of three generations of backcross followed by selfing were charatcerised for target phenotype and genome integration. A set of 260 trait introgressed lines were identified. Root weight and root length of TILs were 53% and 23.5% higher, while Δ13C was 2.85‰ lower indicating a significant increase in WUE over IR-64. Five best TILs selected from BC3F3 generation showed 52% and 63% increase in yield over IR-64 under 100% and 60% FC, respectively. The trait introgressed lines resembled IR64 with more than 97% of genome recovered with a significant yield advantage under semi-irrigated aerobic conditions The study validated markers identified earlier by association mapping. CONCLUSION: Introgression of root and WUE into IR64, resulted in an excellent yield advantage even when cultivated under semi-irrigated aerobic condition. The study provided a proof-of-concept that maintaining leaf turgor and carbon metabolism results in improved adaptation to water limited conditions and sustains productivity. A marker based multi-parent backcross breeding is an appropriate approach for trait introgression. The trait introgressed lines developed can be effectively used in future crop improvement programs as donor lines for both root and WUE.

Assessment of variability in acquired thermotolerance: potential option to study genotypic response and the relevance of stress genes.

High-temperature stress affects all growth stages of crops and ultimately yields. This is further aggravated by other environmental stresses like intermittent drought and high light. Management options are few and hence developing intrinsically tolerant plants is essential to combat the situation. As thermotolerance is a multigenic trait, emphasis needs to be on relevant approaches to assess genetic variability in basal and acquired tolerance. This is in fact the major aspect in crop improvement programmes. The relevance of temperature induction (acclimation) response (TIR), a high throughput approach to identify thermotolerant individuals and its utility as potential screening method is described here. This is based on the concept that stress-responsive genes are expressed only during initial stages of stress (acclimation stress) and bring about requisite changes in cell metabolism for adaptation. The fact that acclimation response is ubiquitous has been demonstrated in different crop plants in our studies and by others. Significance of acclimation in acquired tolerance and thus in assessing genetic variability in thermotolerance is discussed. The limitations of present approaches to validate the relevance of specific stress genes either in transgenics or in mutants or knock downs have been analyzed and the need to characterize transformants under conditions that trigger acquired tolerance is also highlighted. This review also focuses on the potential of exploiting acclimation response approach to improve the thermotolerance of crop plants by suitable breeding strategies.

GBF3 transcription factor imparts drought tolerance in Arabidopsis thaliana.

Drought transcriptome analysis of finger millet (Eleusine coracana) by cDNA subtraction identified drought responsive genes that have a potential role in drought tolerance. Through virus-induced gene silencing (VIGS) in a related crop species, maize (Zea mays), several genes, including a G-BOX BINDING FACTOR 3 (GBF3) were identified as candidate drought stress response genes and the role of GBF3 in drought tolerance was studied in Arabidopsis thaliana. Overexpression of both EcGBF3 and AtGBF3 in A. thaliana resulted in improved tolerance to osmotic stress, salinity and drought stress in addition to conferring insensitivity to ABA. Conversely, loss of function of this gene increased the sensitivity of A. thaliana plants to drought stress. EcGBF3 transgenic A. thaliana results also suggest that drought tolerance of sensitive plants can be improved by transferring genes from far related crops like finger millet. Our results demonstrate the role of GBF3 in imparting drought tolerance in A. thaliana and indicate the conserved role of this gene in drought and other abiotic stress tolerance in several plant species.

Transcriptome Analysis of Sunflower Genotypes with Contrasting Oxidative Stress Tolerance Reveals Individual- and Combined- Biotic and Abiotic Stress Tolerance Mechanisms.

In nature plants are often simultaneously challenged by different biotic and abiotic stresses. Although the mechanisms underlying plant responses against single stress have been studied considerably, plant tolerance mechanisms under combined stress is not understood. Also, the mechanism used to combat independently and sequentially occurring many number of biotic and abiotic stresses has also not systematically studied. From this context, in this study, we attempted to explore the shared response of sunflower plants to many independent stresses by using meta-analysis of publically available transcriptome data and transcript profiling by quantitative PCR. Further, we have also analyzed the possible role of the genes so identified in contributing to combined stress tolerance. Meta-analysis of transcriptomic data from many abiotic and biotic stresses indicated the common representation of oxidative stress responsive genes. Further, menadione-mediated oxidative stress in sunflower seedlings showed similar pattern of changes in the oxidative stress related genes. Based on this a large scale screening of 55 sunflower genotypes was performed under menadione stress and those contrasting in oxidative stress tolerance were identified. Further to confirm the role of genes identified in individual and combined stress tolerance the contrasting genotypes were individually and simultaneously challenged with few abiotic and biotic stresses. The tolerant hybrid showed reduced levels of stress damage both under combined stress and few independent stresses. Transcript profiling of the genes identified from meta-analysis in the tolerant hybrid also indicated that the selected genes were up-regulated under individual and combined stresses. Our results indicate that menadione-based screening can identify genotypes not only tolerant to multiple number of individual biotic and abiotic stresses, but also the combined stresses.

Root transcriptome of two contrasting indica rice cultivars uncovers regulators of root development and physiological responses.

The huge variation in root system architecture (RSA) among different rice (Oryza sativa) cultivars is conferred by their genetic makeup and different growth or climatic conditions. Unlike model plant Arabidopsis, the molecular basis of such variation in RSA is very poorly understood in rice. Cultivars with stable variation are valuable resources for identification of genes involved in RSA and related physiological traits. We have screened for RSA and identified two such indica rice cultivars, IR-64 (OsAS83) and IET-16348 (OsAS84), with stable contrasting RSA. OsAS84 produces robust RSA with more crown roots, lateral roots and root hairs than OsAS83. Using comparative root transcriptome analysis of these cultivars, we identified genes related to root development and different physiological responses like abiotic stress responses, hormone signaling, and nutrient acquisition or transport. The two cultivars differ in their response to salinity/dehydration stresses, phosphate/nitrogen deficiency, and different phytohormones. Differential expression of genes involved in salinity or dehydration response, nitrogen (N) transport, phosphate (Pi) starvation signaling, hormone signaling and root development underlies more resistance of OsAS84 towards abiotic stresses, Pi or N deficiency and its robust RSA. Thus our study uncovers gene-network involved in root development and abiotic stress responses in rice.

Cloning, functional expression in Pichia pastoris, and purification of potato cystatin and multicystatin.

In the tubers and leaves of potato, Solanum tuberosum, cysteine protease inhibitors are thought to play roles in the defence against herbivores and in regulating physiological processes like senescence and cell death. The cDNAs for two such inhibitors, potato multicystatin (PMC) with 8 cystatin domains and potato cystatin (PC) with a single domain, were cloned and expressed in the yeast Pichia pastoris. PC yielded on average 100 mg of purified active protein from 1l of culture supernatant. Purification to homogeneity was done in one step by cation exchange. The apparent equilibrium dissociation constant (K(i)) for papain was 0.1 nM. Cloning of the PMC cDNA was successful despite apparent toxicity for Escherichia coli and a high frequency of recombination events in RecA- strains of E. coli. In yeast, the expression of the cloned full length PMC gene was poor compared to that of the single domain.

Stable expression of mtlD gene imparts multiple stress tolerance in finger millet.

Finger millet is susceptible to abiotic stresses, especially drought and salinity stress, in the field during seed germination and early stages of seedling development. Therefore developing stress tolerant finger millet plants combating drought, salinity and associated oxidative stress in these two growth stages is important. Cellular protection through osmotic adjustment and efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms in plants. Mannitol, an osmolyte, is known to scavenge hydroxyl radicals generated during various abiotic stresses and thereby minimize stress damage in several plant species. In this study transgenic finger millet plants expressing the mannitol biosynthetic pathway gene from bacteria, mannitol-1-phosphate dehydrogenase (mtlD), were developed through Agrobacterium tumefaciens-mediated genetic transformation. mtlD gene integration in the putative transgenic plants was confirmed by Southern blot. Further, performance of transgenic finger millet under drought, salinity and oxidative stress was studied at plant level in T1 generation and in T1 and T2 generation seedlings. Results from these experiments showed that transgenic finger millet had better growth under drought and salinity stress compared to wild-type. At plant level, transgenic plants showed better osmotic adjustment and chlorophyll retention under drought stress compared to the wild-type. However, the overall increase in stress tolerance of transgenics for the three stresses, especially for oxidative stress, was only marginal compared to other mtlD gene expressing plant species reported in the literature. Moreover, the Agrobacterium-mediated genetic transformation protocol developed for finger millet in this study can be used to introduce diverse traits of agronomic importance in finger millet.

Root traits and cellular level tolerance hold the key in maintaining higher spikelet fertility of rice under water limited conditions.

Reduced spikelet fertility appears to be one of the major factors responsible for the decreased rice grain yield when cultivated under semi irrigated aerobic condition. We demonstrate that genotypes with better root systems coupled with higher cellular level tolerance (CLT) can significantly improve spikelet fertility under semi-irrigated aerobic condition in the field. A set of 20 contrasting rice accessions differing in root traits and CLT with significant molecular diversity were subjected to specific soil moisture regimes during a period between five days before and 10 days after anthesis. Lowest spikelet fertility was observed among the plants grown under water limited (WL) conditions followed by the plants grown aerobically in field conditions (AF). Deep rooted genotypes generally maintained higher spikelet fertility under both WL and AF conditions. Furthermore, genotypes that had high roots biomass as well as high CLT recorded the lowest reduction in spikelet fertility under WL and AF compared with the low root and low CLT genotype. This study emphasised the relevance of combining water acquisition and CLT for improving field level tolerance of rice to water limitation. Such genotypes recorded significantly higher grain yield under stress as well as well watered conditions. The study led to the identification of promising trait donor genotypes which can be exploited in breeding to develop superior trait pyramided cultivars suitable for semi irrigated aerobic cultivation.

Expression of a finger millet transcription factor, EcNAC1, in tobacco confers abiotic stress-tolerance.

NAC (NAM, ATAF1-2, and CUC2) proteins constitute one of the largest families of plant-specific transcription factors and have been shown to be involved in diverse plant processes including plant growth, development, and stress-tolerance. In this study, a stress-responsive NAC gene, EcNAC1, was isolated from the subtracted stress cDNA library generated from a drought adapted crop, finger millet, and characterized for its role in stress-tolerance. The expression analysis showed that EcNAC1 was highly induced during water-deficit and salt stress. EcNAC1 shares high amino acid similarity with rice genes that have been phylogenetically classified into stress-related NAC genes. Our results demonstrated that tobacco transgenic plants expressing EcNAC1 exhibit tolerance to various abiotic stresses like simulated osmotic stress, by polyethylene glycol (PEG) and mannitol, and salinity stress. The transgenic plants also showed enhanced tolerance to methyl-viologen (MV) induced oxidative stress. Reduced levels of reactive oxygen species (ROS) and ROS-induced damage were noticed in pot grown transgenic lines under water-deficit and natural high light conditions. Root growth under stress and recovery growth after stress alleviation was more in transgenic plants. Many stress-responsive genes were found to be up-regulated in transgenic lines expressing EcNAC1. Our results suggest that EcNAC1 overexpression confers tolerance against abiotic stress in susceptible species, tobacco.

Functional characterization of water-deficit stress responsive genes using RNAi.

In response to water-deficit stress, plants alter expression of thousands of genes and as a result, cellular, physiological, and biochemical processes are modified. Understanding the functional role of water-deficit stress-responsive genes is important in order to develop stress-tolerant plants. RNA interference (RNAi) technology is one of the potential reverse genetics tool for assessing the functional significance of these genes. We describe here the protocols for developing stable gene knockdown lines for stress-induced genes using RNAi. In addition, stress imposition method that allows plants to experience gradual acclimation stress is enumerated. Further, precautions that should be taken while developing RNAi lines and during stress imposition are discussed.

Relationship between 18O enrichment in leaf biomass and stomatal conductance.

Models that explain the oxygen isotope enrichment in leaf water (and biomass) treat the relationship between the kinetic fractionation that occurs during evapotranspiration and the stomatal conductance in an empirical way. Consequently, the isotopic enrichment is always predicted to decrease with increasing stomatal conductance, regardless of the experimental evidence to the contrary. We explain why and suggest an alternative method to reconcile theory and experiment. We support this with our experimental data on rice and groundnut plants.

Functional characterization of three water deficit stress-induced genes in tobacco and Arabidopsis: an approach based on gene down regulation.

Functional characterization of water deficit stress responsive genes is important to understand their role in stress tolerance. RNAi-based silencing of gene of interest and studying the stress response of knockdown plants under stress can be one of the potential options for assessing functional significance of these genes. Several genes showing higher transcript expression under water deficit stress were cloned earlier from a stress adapted crop species, groundnut. In this study, a few selected gene homologs have been characterized in Nicotiana tabacum and Arabidopsis. Using post transcriptional gene silencing (PTGS) based RNAi approach we developed N. tabacum knockdown lines for three of the genes namely alcohol dehydrogenase (ADH), trans caffeoyl coA-3-O-methyl transferase (CcoAOMT) and flavonol-3-O-glucosyl transferase (F3OGT). By quantitative RT-PCR we demonstrated that the RNAi lines showed significant reduction in target gene transcripts. We followed a stress imposition protocol that allows the plants to experience initial gradual acclimation stress and subsequently severe stress for a definite period. The RNAi knockdown lines generated against ADH and F3OGT, when subjected to water deficit stress showed susceptible symptoms signifying the relevance of these genes under stress. Knockdown of CcoAOMT showed higher chlorophyll degradation and less cell viability upon stress compared to control plants. Further, the Arabidopsis mutant lines clearly showed susceptibility to salinity and water deficit stresses validating relevance of these three genes under abiotic stresses.

STIF: Identification of stress-upregulated transcription factor binding sites in Arabidopsis thaliana.

UNLABELLED: The expressions of proteins in the cell are carefully regulated by transcription factors that interact with their downstream targets in specific signal transduction cascades. Our understanding of the regulation of functional genes responsive to stress signals is still nascent. Plants like Arabidopsis thaliana, are convenient model systems to study fundamental questions related to regulation of the stress transcriptome in response to stress challenges. Microarray results of the Arabidopsis transcriptome indicate that several genes could be upregulated during multiple stresses, such as cold, salinity, drought etc. Experimental biochemical validations have proved the involvement of several transcription factors could be involved in the upregulation of these stress responsive genes. In order to follow the intricate and complicated networks of transcription factors and genes that respond to stress situations in plants, we have developed a computer algorithm that can identify key transcription factor binding sites upstream of a gene of interest. Hidden Markov models of the transcription factor binding sites enable the identification of predicted sites upstream of plant stress genes. The search algorithm, STIF, performs very well, with more than 90% sensitivity, when tested on experimentally validated positions of transcription factor binding sites on a dataset of 60 stress upregulated genes. AVAILABILITY: Supplementary data is available at http://caps.ncbs.res.in/download/stif.

Virus-induced gene silencing and its application in characterizing genes involved in water-deficit-stress tolerance.

Understanding post-transcriptional gene silencing (PTGS) phenomena in plants has provided breakthroughs in advancing plant functional genomics. A recently developed approach based on one of the strategies adopted by plants to defend against viruses, called virus-induced gene silencing (VIGS), is being widely used to enumerate the function of plant genes. Since its discovery, VIGS has been widely used to characterize plant genes involved in metabolic pathways, homeostasis, basic cellular functions, plant-microbe, plant-nematode and plant-herbivore interaction. Recently, the application of this technique has been extended to characterize the genes and cellular processes involved in abiotic-stress tolerance, and in particular drought and oxidative stress. Because abiotic-stress tolerance is multigenic, identification and characterization of genes involved in this process is challenging. VIGS could become one among the several potential tools in understanding the relevance of these stress-responsive genes. Development of VIGS protocols for the use of heterologous gene sequences as VIGS-inducers has extended its applicability to analyze genes of VIGS recalcitrant plant species. This article describes the methodology of VIGS for characterizing the water-deficit-stress-responsive genes, precautions to be taken during the experimentation, and future application of this technology as a fast forwarded as well as a reverse genetics tool to identify and characterize plant genes involved in drought tolerance. We also describe the importance of accurate water-deficit-stress imposition and quantification of stress-induced changes in the silenced plants during the process of screening to identify genes responsible for tolerance. Further, limitations of VIGS in characterizing the abiotic-stress-responsive genes are noted, with suggestions to overcome these limitations.

Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene.

Freshwater is a limited and dwindling global resource; therefore, efficient water use is required for food crops that have high water demands, such as rice, or for the production of sustainable energy biomass. We show here that expression of the Arabidopsis HARDY (HRD) gene in rice improves water use efficiency, the ratio of biomass produced to the water used, by enhancing photosynthetic assimilation and reducing transpiration. These drought-tolerant, low-water-consuming rice plants exhibit increased shoot biomass under well irrigated conditions and an adaptive increase in root biomass under drought stress. The HRD gene, an AP2/ERF-like transcription factor, identified by a gain-of-function Arabidopsis mutant hrd-D having roots with enhanced strength, branching, and cortical cells, exhibits drought resistance and salt tolerance, accompanied by an enhancement in the expression of abiotic stress associated genes. HRD overexpression in Arabidopsis produces thicker leaves with more chloroplast-bearing mesophyll cells, and in rice, there is an increase in leaf biomass and bundle sheath cells that probably contributes to the enhanced photosynthesis assimilation and efficiency. The results exemplify application of a gene identified from the model plant Arabidopsis for the improvement of water use efficiency coincident with drought resistance in the crop plant rice.