Differential accumulation of mRNAs in drought-tolerant and susceptible common bean cultivars in response to water deficit

The physiological response to drought was measured in two common bean varieties with contrastive susceptibility to drought stress. A subtractive cDNA library was constructed from the two cultivars, Phaseolus vulgaris'Pinto Villa' (tolerant) and 'Carioca' (susceptible). 18 cDNAs displayed protein-coding genes associated with drought, cold and oxidative stress, signal transduction, plant defense, chloroplast function and unknown function. A cDNA coding for an aquaporin (AQP) was selected for further analyses. The open reading frames (ORFs) of AQPs from 'Pinto Villa' and 'Carioca' were compared and despite their similarity, accumulated differentially in the plant organs, as demonstrated by Northern blot and in situ hybridization. A phylogenetic analysis of the deduced amino acid sequence with other AQPs suggested a tonoplast-located protein. Under drought conditions, the levels of AQP mRNA from the susceptible cultivar decreased to undetectable levels; by contrast, 'Pinto Villa' mRNA was present and restricted the phloem tissue. This would allow 'Pinto Villa' to maintain vascular tissue functions under drought stress.     

Molecular cloning of glutathione reductase cDNAs and analysis of GR gene expression in cowpea and common bean leaves during recovery from moderate drought stress

Two cDNAs of the enzyme glutathione reductase (GR; EC 1.6.4.2) encoding a dual-targeted isoform (dtGR) and a cytosolic isoform (cGR), were cloned from leaves of common bean (Phaseolus vulgaris L.). Moderate drought stress (Psi w=-1.5MPa) followed by re-watering was applied to common bean cultivars, one tolerant to drought (IPA), the other susceptible (Carioca) and to cowpea (Vigna unguiculata L. Walp) cultivars, one tolerant to drought (EPACE-1), and the other susceptible (1183). mRNA levels were much higher for PvcGR than for PvdtGR in all cases. Moderate drought stress induced an up-regulation of the expression of PvcGR in the susceptible cultivars. On the contrary, PvdtGR expression decreased. In the tolerant cowpea EPACE-1, GR gene expression remained stable under drought. During recovery from drought, an up-regulation of the two GR isoforms occurred, with a peak at 6-10h after re-hydration. This suggests that moderate drought stress may lead to a hardening process and acclimation tolerance. The role of GR isoforms in plant tolerance and capacity to recover from drought stress is discussed.     

Enhanced tolerance to drought and salt stresses in transgenic faba bean (Vicia faba L.) plants by heterologous expression of the PR10a gene from potato

Key message

We report for the first time that expression of potato PR10a gene in faba bean causes enhanced tolerance to drought and salinity.

Abstract

Grain legumes such as soybean (Glycine max L. Merrill), pea (Pisum sativum L.) and faba bean (Vicia faba L.) are staple sources of protein for human and animal nutrition. Among grain legumes, faba bean is particularly sensitive to abiotic stress (in particular osmotic stress due to lack of water or enhanced soil salinity) and often suffers from severe yield losses. Many stress responsive genes have been reported with an effect on improving stress tolerance in model plants. Pathogenesis-related proteins are expressed by all plants in response to pathogen infection and, in many cases, in response to abiotic stresses as well. The PR10a gene isolated from the potato cultivar Desiree was selected for this study due to its role in enhancing salt and/or drought tolerance in potato, and transferred into faba bean cultivar Tattoo by Agrobacterium tumefaciens-mediated transformation system based upon direct shoot regeneration after transformation of meristematic cells derived from embryo axes. The transgene was under the control of the constitutive mannopine synthase promoter (p-MAS) in a dicistronic binary vector, which also contained luciferase (Luc) gene as scorable marker linked by internal ribosome entry site elements. Fertile transgenic faba bean plants were recovered. Inheritance and expression of the foreign genes were demonstrated by PCR, RT-PCR, Southern blot and monitoring of Luciferase activity. Under drought condition, after withholding water for 3 weeks, the leaves of transgenic plants were still green, while non-transgenic plants (WT) wilted and turned brown. Twenty-four hours after re-watering, the leaves of transgenic plants remained green, while WT plants did not recover. Moreover, the transgenic lines displayed higher tolerance to NaCl stress. Our results suggested that introducing a novel PR10a gene into faba bean could be a promising approach to improve its drought and salt tolerance ability, and that MAS promoter is not only constitutive, but also wound-, auxin/cytokinin- as well as stress-inducible.     

Cytokinin-mediated source/sink modifications improve drought tolerance and increase grain yield in rice under water-stress

Drought is the major environmental factor limiting crop productivity worldwide. We hypothesized that it is possible to enhance drought tolerance by delaying stress-induced senescence through the stress-induced synthesis of cytokinins in crop-plants. We generated transgenic rice (Oryza sativa) plants expressing an isopentenyltransferase (IPT) gene driven by P(SARK) , a stress- and maturation-induced promoter. Plants were tested for drought tolerance at two yield-sensitive developmental stages: pre- and post-anthesis. Under both treatments, the transgenic rice plants exhibited delayed response to stress with significantly higher grain yield (GY) when compared to wild-type plants. Gene expression analysis revealed a significant shift in expression of hormone-associated genes in the transgenic plants. During water-stress (WS), P(SARK)::IPT plants displayed increased expression of brassinosteroid-related genes and repression of jasmonate-related genes. Changes in hormone homeostasis were associated with resource(s) mobilization during stress. The transgenic plants displayed differential expression of genes encoding enzymes associated with hormone synthesis and hormone-regulated pathways. These changes and associated hormonal crosstalk resulted in the modification of source/sink relationships and a stronger sink capacity of the P(SARK)::IPT plants during WS. As a result, the transgenic plants had higher GY with improved quality (nutrients and starch content).     

CBL1, a calcium sensor that differentially regulates salt,drought, and cold responses in Arabidopsis

Although calcium is a critical component in the signal transduction pathways that lead to stress gene expression in higher plants, little is known about the molecular mechanism underlying calcium function. It is believed that cellular calcium changes are perceived by sensor molecules, including calcium binding proteins. The calcineurin B-like (CBL) protein family represents a unique group of calcium sensors in plants. A member of the family, CBL1, is highly inducible by multiple stress signals, implicating CBL1 in stress response pathways. When the CBL1 protein level was increased in transgenic Arabidopsis plants, it altered the stress response pathways in these plants. Although drought-induced gene expression was enhanced, gene induction by cold was inhibited. In addition, CBL1-overexpressing plants showed enhanced tolerance to salt and drought but reduced tolerance to freezing. By contrast, cbl1 null mutant plants showed enhanced cold induction and reduced drought induction of stress genes. The mutant plants displayed less tolerance to salt and drought but enhanced tolerance to freezing. These studies suggest that CBL1 functions as a positive regulator of salt and drought responses and a negative regulator of cold response in plants.     

Overexpression of a Harpin-encoding gene hrf1 in rice enhances drought tolerance

Harpin proteins are well known as eliciters that induce multiple responses in plants, such as systemic acquired resistance, hypersensitive response, enhancement of growth, resistance to the green peach aphid, and tolerance to drought. Overexpression of Harpin-encoding genes enhances plant resistance to diseases in tobacco, rice, rape, and cotton; however, it is not yet known whether the expression of Harpin-encoding genes in vivo improves plant tolerance to abiotic stresses. The results of this study showed that overexpression of a Harpin-encoding gene hrf1 in rice increased drought tolerance through abscisic acid (ABA) signalling. hrf1- overexpression induces an increase in ABA content and promotes stomatal closure in rice. The hrf1 transgenic rice lines exhibited a significant increase in water retention ability, levels of free proline and soluble sugars, tolerance to oxidative stress, reactive oxygen species-scavenging ability, and expression levels of four stress-related genes, OsLEA3-1, OsP5CS, Mn-SOD, and NM_001074345, under drought stress. The study confirmed that hrf1 conferred enhanced tolerance to drought stress on transgenic crops. These results suggest that Harpins may offer new opportunities for generating drought resistance in other crops.     

Differentially expressed proteins associated with drought tolerance in bananas (<Emphasis Type="Italic">Musa</Emphasis> spp.)

Bananas are one of the most important fruits in tropical and subtropical regions worldwide. Each year, banana plantations expand, but the areas available are mostly dry lands. The establishment of strategies for obtaining drought tolerant cultivars depends on understanding of biological responses at genetic, molecular and biochemical levels. Proteomics is a powerful tool for functional characterization of the response of plants to abiotic stress and little is known about drought tolerance in Musa spp. Therefore, the aim of this study was to identify proteins related to drought tolerance in two contrasting banana genotypes, Prata Anã and BRS Tropical, susceptible and tolerant to drought, respectively. Proteins were extracted from rhizomes of bananas grown under greenhouse conditions with control, irrigated and water deficit regimes. The differential protein expression pattern was established by two-dimensional (2-D) electrophoresis and spots analyzed in nano Q-Tof Micro UPLC. Twenty-three differentially expressed proteins were found in the tolerant genotype (BRS Tropical) under water deficit, with proteins involved in metabolism, defense and transport. Proteins were classified according to known function and biosynthetic pathways. Signaling proteins in response to water stress, especially for the biological function of growth and development of plants cells, were also encountered, whereas heat shock proteins played a significant role. This is the first report of proteomic analysis for drought tolerance in ‘Pome’ and ‘Silk-type’ bananas containing the ‘B’ genome. Our work provides insights into Musa spp. response to drought and data for further studies regarding molecular mechanisms, which determine how Musa spp. cells better overcome environmental perturbations.     

Comparative analysis of DNA methylation changes in two contrasting wheat genotypes under water deficit

DNA methylation is one of the epigenetic mechanisms regulating gene expression in plants in response to environmental conditions. In this study, analysis of methylation patterns was carried out in order to assess the effect of water stress in two contrasting wheat genotypes using methylation-sensitive amplified polymorphism (MSAP). The results revealed that demethylation was higher in drought-tolerant genotype (C306) as compared to drought-sensitive genotype (HUW468) after experiencing drought stress. Comparisons of different MSAP patterns showed a high percentage of polymorphic bands between tolerant and susceptible wheat genotypes (from 74.79 % at anthesis to 88.89 % at tillering). Furthermore, differential DNA methylation in roots and leaves also revealed tissue-specific methylation of genomic DNA. Interestingly, 54 developmental stage-specific bands and 23 bands that were found contrasting between these two wheat genotypes were detected. Furthermore, a few sites with stable DNA methylation differences were identified between drought-tolerant and drought-sensitive cultivars, thus providing genotype-specific epigenetic markers. These results not only provide data on differences in DNA methylation changes but also contribute to dissection of molecular mechanisms of drought response and tolerance in wheat.     

Cloning of Gossypium hirsutum Sucrose Non-Fermenting 1-Related Protein Kinase 2 Gene (GhSnRK2) and Its Overexpression in Transgenic Arabidopsis Escalates Drought and Low Temperature Tolerance

The molecular mechanisms of stress tolerance and the use of modern genetics approaches for the improvement of drought stress tolerance have been major focuses of plant molecular biologists. In the present study, we cloned the Gossypium hirsutum sucrose non-fermenting 1-related protein kinase 2 (GhSnRK2) gene and investigated its functions in transgenic Arabidopsis. We further elucidated the function of this gene in transgenic cotton using virus-induced gene silencing (VIGS) techniques. We hypothesized that GhSnRK2 participates in the stress signaling pathway and elucidated its role in enhancing stress tolerance in plants via various stress-related pathways and stress-responsive genes. We determined that the subcellular localization of the GhSnRK2-green fluorescent protein (GFP) was localized in the nuclei and cytoplasm. In contrast to wild-type plants, transgenic plants overexpressing GhSnRK2 exhibited increased tolerance to drought, cold, abscisic acid and salt stresses, suggesting that GhSnRK2 acts as a positive regulator in response to cold and drought stresses. Plants overexpressing GhSnRK2 displayed evidence of reduced water loss, turgor regulation, elevated relative water content, biomass, and proline accumulation. qRT-PCR analysis of GhSnRK2 expression suggested that this gene may function in diverse tissues. Under normal and stress conditions, the expression levels of stress-inducible genes, such as AtRD29A, AtRD29B, AtP5CS1, AtABI3, AtCBF1, and AtABI5, were increased in the GhSnRK2-overexpressing plants compared to the wild-type plants. GhSnRK2 gene silencing alleviated drought tolerance in cotton plants, indicating that VIGS technique can certainly be used as an effective means to examine gene function by knocking down the expression of distinctly expressed genes. The results of this study suggested that the GhSnRK2 gene, when incorporated into Arabidopsis, functions in positive responses to drought stress and in low temperature tolerance.     

Differences in growth and water relations among Phaseolus vulgaris cultivars in response to induced drought stress

Relatively little ecophysiological research has been conducted to determine the responses to drought of Phaseolus vulgaris. Four bean cultivars (cvs.) from Brazil, A320, Carioca, Ouro Negro and Xodó were submitted to an imposed water deficit in order to evaluate the importance of some adaptive mechanisms of drought resistance through the analysis of growth parameters, water status, gas exchange and indicators of tolerance mechanisms at the cellular level. During the drought treatment, relative growth rates were more reduced for A320 and Xodó than Carioca and Ouro Negro. A320 closed its stomata very rapidly and complete stomatal closure was obtained at Psi(w)=-0.6 MPa, in contrast to the other cvs. where stomata were fully closed only at Psi(w)=-0.9 MPa. Net assimilation rates were closely related to stomatal conductances. Mechanisms at the cellular level appeared to be mostly important for higher tolerance. Carioca and Ouro Negro, when compared to A320 and Xodó, were characterized by having better drought tolerance mechanisms and higher tissue water retention capacity leading to a better growth under water deficits. The leaf dehydration rates of those cvs. were slow whereas those of the drought sensitive cvs. were rapid. The results were confirmed by the electrolyte leakage test and leaf osmotic potential measurements, which indicated higher membrane resistance and osmotic adjustment in the two tolerant cvs. Carioca and Ouro Negro. It appears from this study that despite being cultivated in the same geographical region, the four cvs. of P. vulgaris displayed somewhat different drought adaptive capacities for prolonged drought during the vegetative phase.     

Seedling response to water stress in valley oak (Quercus lobata) is shaped by different gene networks across populations

Drought is a major stress for plants, creating a strong selection pressure for traits that enable plant growth and survival in dry environments. Many drought responses are conserved species‐wide responses, while others vary among populations distributed across heterogeneous environments. We tested how six populations of the widely distributed California valley oak (Quercus lobata) sampled from contrasting climates would differ in their response to soil drying relative to well‐watered controls in a common environment by measuring ecophysiological traits in 93 individuals and gene expression (RNA‐seq) in 42 individuals. Populations did not differ in their adjustment of turgor loss point during soil drying, suggesting a generalized species‐wide response. Differential expression analysis identified 689 genes with a common response to treatment across populations and 470 genes with population‐specific responses. Weighted gene co‐expression network analysis (WGCNA) identified groups of genes with similar expression patterns that may be regulated together (gene modules). Several gene modules responded differently to water stress among populations, suggesting regional differences in gene network regulation. Populations from sites with a high mean annual temperature responded to the imposed water stress with significantly greater changes in gene module expression, indicating that these populations may be locally adapted to respond to drought. We propose that this variation among valley oak populations provides a mechanism for differential tolerance to the increasingly frequent and severe droughts in California.