Targeted Phytohormone Profiling Identifies Potential Regulators of Spikelet Sterility in Rice under Combined Drought and Heat Stress.

Rice cultivated under rainfed or semi-irrigated ecosystems is frequently exposed to a combination of drought and heat stress. As a sensitive crop at the reproductive stage, exposure to combined drought and heat stress will have a deleterious effect on yield. In this study, two rice cultivars with contrasting spikelet sterility, AVT2-5315 (low sterility) and AC35027 (high sterility), under combined stress were selected for physiological characterization and phytohormonal profiling at anthesis. Under combined stress, both cultivars did not differ in the physiological parameters such as relative water content, photosynthetic rate, light-adapted chlorophyll fluorescence and biomass, suggesting a similar source activity under stress. However, AVT2-5315 showed better yield due to better pollen and spikelet fertility than AC35027, suggesting its intrinsic tolerance ability under combined stress. Targeted profiling of 15 phytohormones from drought, heat and combined stress-treated flag leaf and spikelet tissues using LC-MS/MS showed increased accumulation of auxins (indole 3-acetic acid and indole 3-butyric acid) in flag leaves and jasmonic acid in spikelets of AVT2-5315, while there was increased accumulation of ethylene in flag leaves and methyl-jasmonate in spikelets of AC35027. Increased accumulation of these hormones correlated with key biosynthetic pathway genes. In the flag leaves, increased accumulation of auxins was correlated with increased transcript levels of YUCCA-like gene 1 (OsYUCCA1) and fish bone (OsFIB), in AVT2-5315 under combined stress. In AC35027, increased ethylene content was correlated with expression of 1-aminocyclopropane-1-carboxylate synthase 1 (OsASC1) and aminocyclopropane-1-carboxylic acid oxidase 2 (OsACO2). Similarly, in spikelets, increased accumulation of jasmonic acid in AVT2-5315 was correlated with expression of allene oxide cyclase (OsAOC) and 12-oxophytodienoic acid reductase 1 (OsOPR1). The mechanism of regulating spikelet sterility by these hormones needs further investigation towards improving rice tolerance to combined stress.

Comparative analysis of gene expression in response to cold stress in diverse rice genotypes.

Cold stress is a major factor affecting rice (Oryza sativa) growth and productivity, limiting its distribution worldwide. Rice production is affected primarily due to its vulnerability to cold stress at seedling stage, as well as reproductive stage leading to spikelet sterility. We report here the analysis of 21 diverse rice genotypes from the USDA mini-core collection for cold tolerance and categorized their tolerance levels on the basis of reduction in growth measured by root and shoot length. The screening identified 12 cold tolerant genotypes from which six tolerant genotypes were characterized at the vegetative stage for cold tolerance and gas-exchange parameters. Two tolerant and two sensitive genotypes were used further for gene expression analysis. Lipid Transfer Protein (LTP) genes showed a clear difference in expression between cold tolerant and sensitive genotypes suggesting that they are good candidates for engineering cold tolerance in rice. Nipponbare was identified as a cold tolerant genotype with stress tolerance mechanism potentially operating via both ABA dependent and independent pathways.

Rice GROWTH UNDER DROUGHT KINASE is required for drought tolerance and grain yield under normal and drought stress conditions.

Rice (Oryza sativa) is the primary food source for more than one-half of the world's population. Because rice cultivation is dependent on water availability, drought during flowering severely affects grain yield. Here, we show that the function of a drought-inducible receptor-like cytoplasmic kinase, named GROWTH UNDER DROUGHT KINASE (GUDK), is required for grain yield under drought and well-watered conditions. Loss-of-function gudk mutant lines exhibit sensitivity to salinity, osmotic stress, and abscisic acid treatment at the seedling stage, and a reduction in photosynthesis and plant biomass under controlled drought stress at the vegetative stage. The gudk mutants interestingly showed a significant reduction in grain yield, both under normal well-watered conditions and under drought stress at the reproductive stage. Phosphoproteome profiling of the mutant followed by in vitro assays identified the transcription factor APETALA2/ETHYLENE RESPONSE FACTOR OsAP37 as a phosphorylation target of GUDK. The involvement of OsAP37 in regulating grain yield under drought through activation of several stress genes was previously shown. Our transactivation assays confirmed that GUDK is required for activation of stress genes by OsAP37. We propose that GUDK mediates drought stress signaling through phosphorylation and activation of OsAP37, resulting in transcriptional activation of stress-regulated genes, which impart tolerance and improve yield under drought. Our study reveals insights around drought stress signaling mediated by receptor-like cytoplasmic kinases, and also identifies a primary regulator of grain yield in rice that offers the opportunity to improve and stabilize rice grain yield under normal and drought stress 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.

High-Throughput Analysis of Gene Function under Multiple Abiotic Stresses Using Leaf Disks from Silenced Plants.

The high throughputness and affordability of "omics" technologies is leading to the identification of a large number of abiotic stress genes, with many of them responsive to multiple stresses. In vivo functional characterization of these genes under multiple stresses is challenging but essential to develop resilient crops for the changing climate. Here we describe a high-throughput Virus-Induced Gene Silencing-based methodology for functional analysis of genes under multiple abiotic stresses using leaf disks. Leaves with maximal silencing, which is localized to only a few leaves and to a short period, can be effectively used for multiple stress imposition and stress affect quantification.

Comparative metabolite profiling of rice contrasts reveal combined drought and heat stress signatures in flag leaf and spikelets.

Under semi-irrigated ecosystem, rice is often exposed to a combination of drought and heat stress, especially at the reproductive stage, leading to substantial yield loss. Combined stress studies are very limited in rice partly due to the difficulty in creating heat stress on a larger scale. Here, 24 cultivars with specific stress adaptive traits were phenotyped for spikelet sterility under combined stress using the natural summer temperatures and open drought phenotyping facility, simulating the field conditions. LC-MS/MS based metabolite profiling was performed in flag leaves and spikelets of three cultivars contrasting for spikelet sterility and source (leaf weight) treated to drought, heat and combined stress. Constitutively regulated metabolites, metabolic signatures common to all stresses, cultivars and tissues, metabolites common to both the tissues across the stresses and cultivars and metabolites common to each cultivar across the tissues and stresses were identified. Under combined stress, metabolites differentially accumulated between cultivars contrasting for spikelet sterility but similar for source and cultivars contrasting for both spikelet sterility and source have been identified. These metabolites would serve as markers towards improving combined stress tolerance of rice.

Combined Drought and Heat Stress Influences the Root Water Relation and Determine the Dry Root Rot Disease Development Under Field Conditions: A Study Using Contrasting Chickpea Genotypes.

Abiotic stressors such as drought and heat predispose chickpea plants to pathogens of key importance leading to significant crop loss under field conditions. In this study, we have investigated the influence of drought and high temperature on the incidence and severity of dry root rot disease (caused by Macrophomina phaseolina) in chickpea, under extensive on- and off-season field trials and greenhouse conditions. We explored the association between drought tolerance and dry root rot resistance in two chickpea genotypes, ICC 4958 and JG 62, with contrasting resistance to dry root rot. In addition, we extensively analyzed various patho-morphological and root architecture traits altered by combined stresses under field and greenhouse conditions in these genotypes. We further observed the role of edaphic factors in dry root rot incidence under field conditions. Altogether, our results suggest a strong negative correlation between the plant water relations and dry root rot severity in chickpeas, indicating an association between drought tolerance and dry root rot resistance. Additionally, the significant role of heat stress in altering the dynamics of dry root rot and the importance of combinatorial screening of chickpea germplasm for dry root rot resistance, drought, and heat stress have been revealed.

Using Network-Based Machine Learning to Predict Transcription Factors Involved in Drought Resistance.

Gene regulatory networks underpin stress response pathways in plants. However, parsing these networks to prioritize key genes underlying a particular trait is challenging. Here, we have built the Gene Regulation and Association Network (GRAiN) of rice (Oryza sativa). GRAiN is an interactive query-based web-platform that allows users to study functional relationships between transcription factors (TFs) and genetic modules underlying abiotic-stress responses. We built GRAiN by applying a combination of different network inference algorithms to publicly available gene expression data. We propose a supervised machine learning framework that complements GRAiN in prioritizing genes that regulate stress signal transduction and modulate gene expression under drought conditions. Our framework converts intricate network connectivity patterns of 2160 TFs into a single drought score. We observed that TFs with the highest drought scores define the functional, structural, and evolutionary characteristics of drought resistance in rice. Our approach accurately predicted the function of OsbHLH148 TF, which we validated using in vitro protein-DNA binding assays and mRNA sequencing loss-of-function mutants grown under control and drought stress conditions. Our network and the complementary machine learning strategy lends itself to predicting key regulatory genes underlying other agricultural traits and will assist in the genetic engineering of desirable rice varieties.

Low soil moisture predisposes field-grown chickpea plants to dry root rot disease: evidence from simulation modeling and correlation analysis.

Rhizoctonia bataticola causes dry root rot (DRR), a devastating disease in chickpea (Cicer arietinum). DRR incidence increases under water deficit stress and high temperature. However, the roles of other edaphic and environmental factors remain unclear. Here, we performed an artificial neural network (ANN)-based prediction of DRR incidence considering DRR incidence data from previous reports and weather factors. ANN-based prediction using the backpropagation algorithm showed that the combination of total rainfall from November to January of the chickpea-growing season and average maximum temperature of the months October and November is crucial in determining DRR occurrence in chickpea fields. The prediction accuracy of DRR incidence was 84.6% with the validation dataset. Field trials at seven different locations in India with combination of low soil moisture and pathogen stress treatments confirmed the impact of low soil moisture on DRR incidence under different agroclimatic zones and helped in determining the correlation of soil factors with DRR incidence. Soil phosphorus, potassium, organic carbon, and clay content were positively correlated with DRR incidence, while soil silt content was negatively correlated. Our results establish the role of edaphic and other weather factors in chickpea DRR disease incidence. Our ANN-based model will allow the location-specific prediction of DRR incidence, enabling efficient decision-making in chickpea cultivation to minimize yield loss.

Induction of Acquired Tolerance Through Gradual Progression of Drought Is the Key for Maintenance of Spikelet Fertility and Yield in Rice Under Semi-irrigated Aerobic Conditions.

Plants have evolved several adaptive mechanisms to cope with water-limited conditions. While most of them are through constitutive traits, certain "acquired tolerance" traits also provide significant improvement in drought adaptation. Most abiotic stresses, especially drought, show a gradual progression of stress and hence provide an opportunity to upregulate specific protective mechanisms collectively referred to as "acquired tolerance" traits. Here, we demonstrate a significant genetic variability in acquired tolerance traits among rice germplasm accessions after standardizing a novel gradual stress progress protocol. Two contrasting genotypes, BPT 5204 (drought susceptible) and AC 39000 (tolerant), were used to standardize methodology for capturing acquired tolerance traits at seedling phase. Seedlings exposed to gradual progression of stress showed higher recovery with low free radical accumulation in both the genotypes compared to rapid stress. Further, the gradual stress progression protocol was used to examine the role of acquired tolerance at flowering phase using a set of 17 diverse rice genotypes. Significant diversity in free radical production and scavenging was observed among these genotypes. Association of these parameters with yield attributes showed that genotypes that managed free radical levels in cells were able to maintain high spikelet fertility and hence yield under stress. This study, besides emphasizing the importance of acquired tolerance, explains a high throughput phenotyping approach that significantly overcomes methodological constraints in assessing genetic variability in this important drought adaptive mechanism.

Physiological and transcriptional responses to low-temperature stress in rice genotypes at the reproductive stage.

Rice growth and productivity is adversely affected by low-temperature stress. From a previous screen of diverse rice genotypes for cold tolerance parameters at the vegetative stage, we selected the tolerant Nipponbare and M202 genotypes and sensitive Cypress and Secano do Brazil genotypes for further analysis at the reproductive stage for physiological and yield parameters. Cold stress severely affected grain yield as estimated by the number of grain per panicle, panicle length, and 100 seed weight. Analysis of gene expression of 21 genes involved in physiological responses to low temperature tested, in the flag leaf and inflorescence tissue of these genotypes, showed an increased expression of the Lipid Transfer Protein genes LTP7 and LTP10 in flag leaf tissue of the tolerant Nipponbare and M202, along with a significant increase in the relative expression of stress-responsive transcription factors (TFs) and cold-inducible genes. In flag leaf tissue OsNAC9, OsNAC10 and OsNAP genes showed high correlation with photosynthesis, stomatal conductance, transpiration and Quantum Efficiency of PSII. In consequence of the foregoing results, we conclude that Nipponbare and M202 are cold tolerant genotypes and that LTP7, LTP10, OsNAC9, OsNAC10 and OsNAP genes can be used as markers in screening for cold tolerance at the reproductive stage. Furthermore based on the results we propose a model of low-temperature tolerance mechanism of how stress is perceived, and how the signal cascade acts to promote tolerance at below-ideal temperatures.

Cold tolerance response mechanisms revealed through comparative analysis of gene and protein expression in multiple rice genotypes.

Due to its tropical origin and adaptation, rice is significantly impacted by cold stress, and consequently sustains large losses in growth and productivity. Currently, rice is the second most consumed cereal in the world and production losses caused by extreme temperature events in the context of "major climatic changes" can have major impacts on the world economy. We report here an analysis of rice genotypes in response to low-temperature stress, studied through physiological gas-exchange parameters, biochemical changes in photosynthetic pigments and antioxidants, and at the level of gene and protein expression, towards an understanding and identification of multiple low-temperature tolerance mechanisms. The first effects of cold stress were observed on photosynthesis among all genotypes. However, the tropical japonica genotypes Secano do Brazil and Cypress had a greater reduction in gas exchange parameters like photosynthesis and water use efficiency in comparison to the temperate japonica Nipponbare and M202 genotypes. The analysis of biochemical profiles showed that despite the impacts of low temperature on tolerant plants, they quickly adjusted to maintain their cellular homeostasis by an accumulation of antioxidants and osmolytes like phenolic compounds and proline. The cold tolerant and sensitive genotypes showed a clear difference in gene expression at the transcript level for OsGH3-2, OsSRO1a, OsZFP245, and OsTPP1, as well as for expression at the protein level for LRR-RLKs, bHLH, GLYI, and LTP1 proteins. This study exemplifies the cold tolerant features of the temperate japonica Nipponbare and M202 genotypes, as observed through the analysis of physiological and biochemical responses and the associated changes in gene and protein expression patterns. The genes and proteins showing differential expression response are notable candidates towards understanding the biological pathways affected in rice and for engineering cold tolerance, to generate cultivars capable of maintaining growth, development, and reproduction under cold stress. We also propose that the mechanisms of action of the genes analyzed are associated with the tolerance response.

Virus-induced gene silencing database for phenomics and functional genomics in Nicotiana benthamiana.

Virus-induced gene silencing (VIGS) is an important forward and reverse genetics method for the study of gene function in many plant species, especially Nicotiana benthamiana. However, despite the widespread use of VIGS, a searchable database compiling the phenotypes observed with this method is lacking. Such a database would allow researchers to know the phenotype associated with the silencing of a large number of individual genes without experimentation. We have developed a VIGS phenomics and functional genomics database (VPGD) that has DNA sequence information derived from over 4,000 N. benthamiana VIGS clones along with the associated silencing phenotype for approximately 1,300 genes. The VPGD has a built-in BLAST search feature that provides silencing phenotype information of specific genes. In addition, a keyword-based search function could be used to find a specific phenotype of interest with the corresponding gene, including its Gene Ontology descriptions. Query gene sequences from other plant species that have not been used for VIGS can also be searched for their homologs and silencing phenotype in N. benthamiana. VPGD is useful for identifying gene function not only in N. benthamiana but also in related Solanaceae plants such as tomato and potato. The database is accessible at http://vigs.noble.org.

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.

Plant adaptation to drought stress.

Plants in their natural habitats adapt to drought stress in the environment through a variety of mechanisms, ranging from transient responses to low soil moisture to major survival mechanisms of escape by early flowering in absence of seasonal rainfall. However, crop plants selected by humans to yield products such as grain, vegetable, or fruit in favorable environments with high inputs of water and fertilizer are expected to yield an economic product in response to inputs. Crop plants selected for their economic yield need to survive drought stress through mechanisms that maintain crop yield. Studies on model plants for their survival under stress do not, therefore, always translate to yield of crop plants under stress, and different aspects of drought stress response need to be emphasized. The crop plant model rice ( Oryza sativa) is used here as an example to highlight mechanisms and genes for adaptation of crop plants to drought stress.

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.

The interactive effects of simultaneous biotic and abiotic stresses on plants: mechanistic understanding from drought and pathogen combination.

In nature, plants are simultaneously exposed to a combination of biotic and abiotic stresses that limit crop yields. Only recently, researchers have started understanding the molecular basis of combined biotic and abiotic stress interactions. Evidences suggest that under combined stress plants exhibit tailored physiological and molecular responses, in addition to several shared responses as part of their stress tolerance strategy. These tailored responses are suggested to occur only in plants exposed to simultaneous stresses and this information cannot be inferred from individual stress studies. In this review article, we provide update on the responses of plants to simultaneous biotic and abiotic stresses, in particular drought and pathogen. Simultaneous occurrence of drought and pathogen during plant growth provokes complex pathways controlled by different signaling events resulting in positive or negative impact of one stress over the other. Here, we summarize the effect of combined drought and pathogen infection on plants and highlight the tailored strategies adapted by plants. Besides, we enumerate the evidences from pathogen derived elicitors and ABA response studies for understanding simultaneous drought and pathogen tolerance.

Shared and unique responses of plants to multiple individual stresses and stress combinations: physiological and molecular mechanisms.

In field conditions, plants are often simultaneously exposed to multiple biotic and abiotic stresses resulting in substantial yield loss. Plants have evolved various physiological and molecular adaptations to protect themselves under stress combinations. Emerging evidences suggest that plant responses to a combination of stresses are unique from individual stress responses. In addition, plants exhibit shared responses which are common to individual stresses and stress combination. In this review, we provide an update on the current understanding of both unique and shared responses. Specific focus of this review is on heat-drought stress as a major abiotic stress combination and, drought-pathogen and heat-pathogen as examples of abiotic-biotic stress combinations. We also comprehend the current understanding of molecular mechanisms of cross talk in relation to shared and unique molecular responses for plant survival under stress combinations. Thus, the knowledge of shared responses of plants from individual stress studies and stress combinations can be utilized to develop varieties with broad spectrum stress tolerance.

Regulation of grain yield in rice under well-watered and drought stress conditions by GUDK.

Increasing the grain yield of cereals, which is stable under unfavorable environmental stress, is a major objective to sustain production and feed the growing world population. Recently, we functionally characterized a receptor-like cytoplasmic kinase, named GROWTH UNDER DROUGHT KINASE (GUDK), revealing its role in regulating grain yield under well-watered and drought stress conditions by transphosphorylating the OsAP37 transcription factor. GUDK is induced under several stresses and its loss-of-function increased the sensitivity of rice seedlings to salinity, osmotic stress, and abscisic acid treatment. In addition to reduced tolerance of gudk mutant plants to drought stress at vegetative stage, a significant reduction in grain yield was observed under well-watered and drought stress conditions at reproductive stage. Gene co-expression analysis supports the role of GUDK in regulating important biological processes both under control and stress conditions. Thus, our results suggest that GUDK has the potential to regulate grain yield both under favorable and unfavorable conditions.