Search

Plants in the natural conditions are often challenged by a combination of two or more stressors. A combination of drought and pathogen is one of the most pressing threats to the plant’s growth and survival in the field, and thus warrants a mechanistic understanding. Susceptible plant-pathogen interaction, owing to effector-mediated suppression of plant defense responses, limits its scope for combined stress studies. In the present study, we have investigated the morpho-physiological responses of Arabidopsis thaliana to simultaneous drought and nonhost bacterial pathogen Pseudomonas syringae pv. tabaci. Combined stress treatment provoked an early and more pronounced hypersensitive response in the plant as compared to the non-host pathogen treatment. We have further deciphered the molecular basis for the robust defense response observed under combined stress by transcriptomic profiling carried out using whole-genome microarray. We found that the enhanced resistance to the combined stress is accompanied by a massive transcriptional reprogramming involving several transcripts specifically responding to the stress combination. A prominent over-representation of genes involved in basal defense-related machinery was observed under the combined stress. Genes involved in various defense signaling cascades, accumulation of secondary metabolites and those encoding for receptor-like kinases were highly up-regulated under the combined stress. Up-regulated genes related to redox homeostasis and hypersensitive response (HR)-mediated cell death were also found to be markedly enriched under combined stress. We also compared the global gene expression profile of A. thaliana subjected to combined drought-nonhost bacteria to those treated with a combination of drought-host bacteria Pseudomonas syringae pv. tomato DC3000. A significant induction of genes responding to drought as well as bacteria was observed during both the interactions. However, the amplitude of induction was more pronounced under the combination of drought and nonhost bacteria. Our results also indicate that plant activates multiple defense pathways upon exposure to combined stress which strengthens the overall basal immunity of the plant, characterized by a stronger HR response.

Plants under combined stresses exhibit a prominent shift in molecular responses compared with plants exposed to the same stresses independently. Profiling responses to individual and combined stressors at the gene expression level have identified several genes with intersecting responses to these stressors. However, the upstream regulators at the intersection of plant responses to individual and combined stresses are not known. Here, using the transcriptome of Arabidopsis thaliana under individual and combined drought and Pseudomonas syringae infection, we identified several genes whose expression overlaps between individual and combined stresses. To study the key regulator of such an overlapping gene, we predicted that the expression of 1-Pyrroline-5-carboxylate synthase 1 (AtP5CS1) is regulated by ath-miR164c at post-transcriptional level. Our results from the stem-loop RT-PCR based expression analysis revealed significant downregulation of ath-miR164c in response to P. syringae infection under both well-irrigated (pathogen only) and drought stress (combined stress) conditions. Furthermore, an Arabidopsis loss-of-function mutant of the miRNA ath-miR164c exhibited resistance to pathogen infection under combined stress, unlike the wild-type plants, implicating the role of ath-miR164c in regulating plant immunity. AtP5CS1 gene expression and proline accumulation were enhanced in the ath-miR164c mutant plants relative to the wild-type plants, demonstrating that ath-miR164c regulates AtP5CS1 of the proline biosynthesis pathway, which was also validated by 5’RLM-RACE results. This miRNA-mediated modulation of AtP5CS1 gene expression under combined stress fills crucial gaps in identifying the key convergent players in the current understanding of plant stress responses.

In field conditions, plants are often exposed to a combination of abiotic and biotic stresses, for instance, drought and pathogen infection. Transcriptome studies on Arabidopsis thaliana and other plants under individual and combined drought and pathogen stresses have unveiled the activation of shared molecular defense mechanisms. These shared plant responses are characterized by commonly regulated genes under both individual as well as combined stresses. Therefore, the identification of commonly regulated genes during individual and combined stress conditions can reveal plant responses towards combined stress. Available transcriptome studies on combined-stressed plants have hinted at G-Box Binding Factor 3 (GBF3) as one of the regulatory components of the shared response. However, the mechanistic understanding of the role of AtGBF3 under combined drought and pathogen stress is not yet decoded. In the current study, we used genetic approaches to identify the role of AtGBF3 in conferring tolerance to individual and combined drought and pathogen stress. Atgbf3 mutant plants showed increased susceptibility, while AtGBF3-overexpressing plants were tolerant under individual and combined drought and Pseudomonas syringae pv. tomato infection stresses as compared to wild-type plants. We further analyzed the global transcriptome of Atgbf3 mutant plants under combined stress to identify its downstream targets. We also established a high-throughput method to apply combined polyethylene glycol and pathogen stress on Murashige and Skoog medium-grown plants to further validate the role of AtGBF3 in combined stress.

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.

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.

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.

The authors regret that in the above published article, the figure captions of Figs. 1e5 were incomplete, which have been corrected to read as follows: Fig. 1. Antibiotic selection and confirmation of transgenic RNAi lines. Seeds from T0 generation RNAi lines were germinated on MS medium supplemented with kanamycin. A representative plates for wild-type and each transgenic tobacco (Nicotiana tabacum) lines are shown here (A). PCR amplificationwas performed in the leaf samples collected from T1 generation RNAi lines using attB1-attB2 primers and amplification data for two lines are shown here (B). Arrow marks in the panel A show representative white (dead) seedlings. Fig. 2. Real time PCR (qRT-PCR) analysis showing the down regulation of ADH or CcoAOMTor F3OGTgene transcripts in respective tobacco RNAi lines. The transcript levels were studied by qRT-PCR in non-stressed and water deficit stressed plants of RNAi lines of ADH (A), CcoAOMT (B) and F3OGT (C) at T1 generation. Plants from two independent lines were analyzed and relative expression ratio values over their corresponding wild-type are represented in the graph. In all the experiments vector control plants were also analyzed and they did not show silencing. Relative amounts of transcripts were calculated using method described in Pfaffl (2001). The data was normalized to the expression of elongation factor 1-a (EF1a) as the internal housekeeping gene and the wild-type (WT) plants of stress or non-stress mRNA levels of respective genes as an expression reference (WT stress1⁄4 1; WT non-stress1⁄4 1). Silenced lines are shown by filled bars and theWT control(s) is shown by empty bar. Bars represent standard deviation values. Fig. 3. Phenotype of the RNAi transgenic tobacco plants down regulated with ADH or CcoAOMT or F3OGT under water deficit stress. Fortyday-old wild-type and RNAi plants (RNAi line 1) were subjected to water deficit stress by gradually with-holding water for 7 d and later maintained at 40% FC for 7 d. The photographs of representative plants from each gene silenced lines were taken at the end of stress. Values indicate leaf relative water content (%) at the end of 7 d severe stress. Fig. 4. Performance of the transgenic RNAi plants down regulated for ADH or CcoAOMT or F3OGT under water deficit stress. RNAi transgenic lines were initially exposed to acclimation treatment by gradually with-holding the irrigation and later exposed to severe stress (40% FC-7 d). Data from a selected representative line (RNAi line 1) is presented here. Cellular level tolerance was assessed by estimating chlorophyll reduction (A), cell viability by TTC assay (B) and cell membrane stability (C). Percent reduction values under stress in (A) and (B) are calculated over corresponding well watered (100% FC) plants. Bars represent standard error values. The same letters are not significantly different at 5% level by Duncan’s Multiple Range Test. WT, wild-type; FC, field capacity. Fig. 5. Response of Arabidopsis mutant plants to salinity and water deficit stress. The f3ogt, ccoaomt and adh mutants seedlings were initially grown in MS medium and transferred to 100 mM NaCl medium for root bending assay. Root growth was studied at the end of 7 d (A, B). To assess the desiccation response, seedling rosettes were detached and left on the lab bench for drying. Fresh weight loss was measured at the indicated time intervals and water lose was assessed (C). Water deficit stress was imposed by gradually with-holding water from pottingmix (acclimation) and exposing to severe stress. The cell membrane stability was assessed (D) in the stressed seedlings at 14.5% soil water content as measured by soil moisture monitor (Decagon devices Pullman WA USA; probe EC-5). The non-stress plants were maintained at 46% soil water content. Bars represent standard deviation values.