OsASR5 enhances drought tolerance through a stomatal closure pathway associated with ABA and H2O2 signalling in rice

Drought is one of the major abiotic stresses that directly implicate plant growth and crop productivity. Although many genes in response to drought stress have been identified, genetic improvement to drought resistance especially in food crops is showing relatively slow progress worldwide. Here, we reported the isolation of abscisic acid, stress and ripening (ASR) genes from upland rice variety, IRAT109 (Oryza sativa L. ssp. japonica), and demonstrated that overexpression of OsASR5 enhanced osmotic tolerance in Escherichia coli and drought tolerance in Arabidopsis and rice by regulating leaf water status under drought stress conditions. Moreover, overexpression of OsASR5 in rice increased endogenous ABA level and showed hypersensitive to exogenous ABA treatment at both germination and postgermination stages. The production of H₂O₂, a second messenger for the induction of stomatal closure in response to ABA, was activated in overexpression plants under drought stress conditions, consequently, increased stomatal closure and decreased stomatal conductance. In contrast, the loss‐of‐function mutant, osasr5, showed sensitivity to drought stress with lower relative water content under drought stress conditions. Further studies demonstrated that OsASR5 functioned as chaperone‐like protein and interacted with stress‐related HSP40 and 2OG‐Fe (II) oxygenase domain containing proteins in yeast and plants. Taken together, we suggest that OsASR5 plays multiple roles in response to drought stress by regulating ABA biosynthesis, promoting stomatal closure, as well as acting as chaperone‐like protein that possibly prevents drought stress‐related proteins from inactivation.     

PeCHYR1, a ubiquitin E3 ligase from Populus euphratica,enhances drought tolerance via ABA‐induced stomatal closure by ROS production in Populus

Drought, a primary abiotic stress, seriously affects plant growth and productivity. Stomata play a vital role in regulating gas exchange and drought adaptation. However, limited knowledge exists of the molecular mechanisms underlying stomatal movement in trees. Here, PeCHYR1, a ubiquitin E3 ligase, was isolated from Populus euphratica, a model of stress adaptation in forest trees. PeCHYR1 was preferentially expressed in young leaves and was significantly induced by ABA (abscisic acid) and dehydration treatments. To study the potential biological functions of PeCHYR1, transgenic poplar 84K (Populus alba × Populus glandulosa) plants overexpressing PeCHYR1 were generated. PeCHYR1 overexpression significantly enhanced H₂O₂ production and reduced stomatal aperture. Transgenic lines exhibited increased sensitivity to exogenous ABA and greater drought tolerance than that of WT (wild‐type) controls. Moreover, up‐regulation of PeCHYR1 promoted stomatal closure and decreased transpiration, resulting in strongly elevated WUE (water use efficiency). When exposed to drought stress, transgenic poplar maintained higher photosynthetic activity and biomass accumulation. Taken together, these results suggest that PeCHYR1 plays a crucial role in enhancing drought tolerance via ABA‐induced stomatal closure caused by hydrogen peroxide (H₂O₂) production in transgenic poplar plants.     

Evolution of the bHLH Genes Involved in Stomatal Development: Implications for the Expansion of Developmental Complexity of Stomata in Land Plants

Stomata play significant roles in plant evolution. A trio of closely related basic Helix-Loop-Helix (bHLH) subgroup Ia genes, SPCH, MUTE and FAMA, mediate sequential steps of stomatal development, and their functions may be conserved in land plants. However, the evolutionary history of the putative SPCH/MUTE/FAMA genes is still greatly controversial, especially the phylogenetic positions of the bHLH Ia members from basal land plants. To better understand the evolutionary pattern and functional diversity of the bHLH genes involved in stomatal development, we made a comprehensive evolutionary analysis of the homologous genes from 54 species representing the major lineages of green plants. The phylogenetic analysis indicated: (1) All bHLH Ia genes from the two basal land plants Physcomitrella and Selaginella were closely related to the FAMA genes of seed plants; and (2) the gymnosperm ‘SPCH’ genes were sister to a clade comprising the angiosperm SPCH and MUTE genes, while the FAMA genes of gymnosperms and angiosperms had a sister relationship. The revealed phylogenetic relationships are also supported by the distribution of gene structures and previous functional studies. Therefore, we deduce that the function of FAMA might be ancestral in the bHLH Ia subgroup. In addition, the gymnosperm “SPCH” genes may represent an ancestral state and have a dual function of SPCH and MUTE, two genes that could have originated from a duplication event in the common ancestor of angiosperms. Moreover, in angiosperms, SPCHs have experienced more duplications and harbor more copies than MUTEs and FAMAs, which, together with variation of the stomatal development in the entry division, implies that SPCH might have contributed greatly to the diversity of stomatal development. Based on the above, we proposed a model for the correlation between the evolution of stomatal development and the genes involved in this developmental process in land plants.     

Down‐regulation of CBP80 gene expression as a strategy to engineer a drought‐tolerant potato

Developing new strategies for crop plants to respond to drought is crucial for their innovative breeding. The down‐regulation of nuclear cap‐binding proteins in Arabidopsis renders plants drought tolerant. The CBP80 gene in the potato cultivar Desiree was silenced using artificial microRNAs. Transgenic plants displayed a higher tolerance to drought, ABA‐hypersensitive stomatal closing, an increase in leaf stomata and trichome density, and compact cuticle structures with a lower number of microchannels. These findings were correlated with a higher tolerance to water stress. The level of miR159 was decreased, and the levels of its target mRNAs MYB33 and MYB101 increased in the transgenic plants subjected to drought. Similar trends were observed in an Arabidopsis cbp80 mutant. The evolutionary conservation of CBP80, a gene that plays a role in the response to drought, suggests that it is a candidate for genetic manipulations that aim to obtain improved water‐deficit tolerance of crop plants.     

Ecological interactions and the fitness effect of water‐use efficiency: Competition and drought alter the impact of natural MPK12 alleles in Arabidopsis

The presence of substantial genetic variation for water‐use efficiency (WUE) suggests that natural selection plays a role in maintaining alleles that affect WUE. Soil water deficit can reduce plant survival, and is likely to impose selection to increase WUE, whereas competition for resources may select for decreased WUE to ensure water acquisition. We tested the fitness consequences of natural allelic variation in a single gene (MPK12) that influences WUE in Arabidopsis, using transgenic lines contrasting in MPK12 alleles, under four treatments; drought/competition, drought/no competition, well‐watered/competition, well‐watered/no competition. Results revealed an allele × environment interaction: Low WUE plants performed better in competition, resulting from increased resource consumption. Contrastingly, high WUE individuals performed better in no competition, irrespective of water availability, presumably from enhanced water conservation and nitrogen acquisition. Our findings suggest that selection can influence MPK12 evolution, and represents the first assessment of plant fitness resulting from natural allelic variation at a single locus affecting WUE.     

PdEPF1 regulates water‐use efficiency and drought tolerance by modulating stomatal density in poplar

Water deficiency is a critical environmental condition that is seriously reducing global plant production. Improved water‐use efficiency (WUE) and drought tolerance are effective strategies to address this problem. In this study, PdEPF1, a member of the EPIDERMAL PATTERNING FACTOR (EPF) family, was isolated from the fast‐growing poplar clone NE‐19 [Populus nigra × (Populus deltoides × Populus nigra)]. Significantly, higher PdEPF1 levels were detected after induction by dehydration and abscisic acid. To explore the biological functions of PdEPF1, transgenic triploid white poplars (Populus tomentosa ‘YiXianCiZhu B385’) overexpressing PdEPF1 were constructed. PdEPF1 overexpression resulted in increased water deficit tolerance and greater WUE. We confirmed that the transgenic lines with greater instantaneous WUE had approximately 30% lower transpiration but equivalent CO₂ assimilation. Lower transpiration was associated with a 28% reduction in abaxial stomatal density. PdEPF1 overexpression not only strongly enhanced WUE, but also greatly improved drought tolerance, as measured by the leaf relative water content and water potential, under limited water conditions. In addition, the growth of these oxPdEPF1 plants was less adversely affected by reduced water availability than plants with a higher stomatal density, indicating that plants with a low stomatal density may be well suited to grow in water‐scarce environments. Taken together, our data suggest that PdEPF1 improves WUE and confers drought tolerance in poplar; thus, it could be used to breed drought‐tolerant plants with increased production under conditions of water deficiency.     

ABA inhibits entry into stomatal‐lineage development in Arabidopsis leaves

The number and density of stomata are controlled by endogenous and environmental factors. Despite recent advances in our understanding of stomatal development, mechanisms which prevent stomatal‐lineage entry remain unclear. Here, we propose that abscisic acid (ABA), a phytohormone known to induce stomatal closure, limits initiation of stomatal development and induces enlargement of pavement cells in Arabidopsis cotyledons. An ABA‐deficient aba2‐2 mutant had an increased number/proportion of stomata within a smaller cotyledon, as well as reduced expansion of pavement cells. This tendency was reversed after ABA application or in an ABA over‐accumulating cyp707a1cyp707a3 doublemutant. Our time course analysis revealed that aba2‐2 shows prolonged formation of meristemoids and guard mother cells, both precursors of stoma. This finding is in accordance with prolonged gene expression of SPCH and MUTE, master regulators for stomatal formation, indicating that ABA acts upstream of these genes. Only aba2‐2 mute, but not aba2‐2 spch double mutant showed additive phenotypes and displayed inhibition of pavement cell enlargement with increased meristemoid number, indicating that ABA action on pavement cell expansion requires the presence of stomatal‐lineage cells.