The AtMYB60 transcription factor regulates stomatal opening by modulating oxylipin synthesis in guard cells

Stomata are epidermal pores formed by pairs of specialized guard cells, which regulate gas exchanges between the plant and the atmosphere. Modulation of transcription has emerged as an important level of regulation of stomatal activity. The AtMYB60 transcription factor was previously identified as a positive regulator of stomatal opening, although the details of its function remain unknown. Here, we propose a role for AtMYB60 as a negative modulator of oxylipins synthesis in stomata. The atmyb60-1 mutant shows reduced stomatal opening and accumulates increased levels of 12-oxo-phytodienoic acid (12-OPDA), jasmonic acid (JA) and jasmonoyl-l-isoleucine (JA-Ile) in guard cells. We provide evidence that 12-OPDA triggers stomatal closure independently of JA and cooperatively with abscisic acid (ABA) in atmyb60-1. Our study highlights the relevance of oxylipins metabolism in stomatal regulation and indicates AtMYB60 as transcriptional integrator of ABA and oxylipins responses in guard cells.

Interactive Effects of Intraspecific Competition and Drought on Stomatal Conductance and Hormone Concentrations in Different Tomato Genotypes

Plant physiological responses to various stresses are characterized by interaction and coupling, while the intrinsic mechanism remains unclear. The effects of intraspecific competition on plant growth, stomatal opening, and hormone concentrations were investigated with three tomato genotypes (WT-wild type, Ailsa Craig; FL-a abscisic acid (ABA) deficient mutant, flacca; NR-a partially ethylene-insensitive genotype) under two water regimes (full irrigation, irrigation amount = daily transpiration; deficit irrigation, 60% of irrigation amount in full irrigation) in this study. Three kinds of competitions were designed, i.e., root and canopy competition, non-root competition, and non-canopy competition, respectively. Intraspecific competition reduced plant leaf area and stomatal conductance (gs) of wild-type tomato, accompanied by ABA accumulation and ethylene evolution. Intraspecific competition-induced decrease in gs was absent in FL and NR, indicating ABA and ethylene involved in plant response to intraspecific competition. As soil water becomes dry, the competition decreased gs by elevating ABA and ethylene accumulations. Under severe drought, the competition-induced decline in gs was covered by the severe drought-induced decrease in gs, as hydraulic signals most probably dominate. The absence of canopy competition insignificantly influenced plant stomatal opening of well-watered tomato, as canopy separation minimized the plant neighbor sensing by ethylene and other signals. Whereas under water deficit condition, the absence of canopy competition significantly reduced ABA accumulation in roots and then stomatal conductance, indicating the belowground neighbor detection signals maybe enhanced by soil drought. The absence of root competition increased ethylene evolution, confirming the importance of ethylene in neighbor detection and plant response to environmental stress.

Guard-Cell-Specific Expression of Phototropin2 C-Terminal Fragment Enhances Leaf Transpiration

Phototropins (phot1 and phot2) are plant-specific blue light receptors that mediate chloroplast movement, stomatal opening, and phototropism. Phototropin is composed of the N-terminus LOV1 and LOV2 domains and the C-terminus Ser/Thr kinase domain. In previous studies, 35-P2CG transgenic plants expressing the phot2 C-terminal fragment–GFP fusion protein (P2CG) under the control of 35S promoter showed constitutive phot2 responses, including chloroplast avoidance response, stomatal opening, and reduced hypocotyl phototropism regardless of blue light, and some detrimental growth phenotypes. In this study, to exclude the detrimental growth phenotypes caused by the ectopic expression of P2C and to improve leaf transpiration, we used the PHOT2 promoter for the endogenous expression of GFP-fused P2C (GP2C) (P2-GP2C) and the BLUS1 promoter for the guard-cell-specific expression of GP2C (B1-GP2C), respectively. In P2-GP2C plants, GP2C expression induced constitutive phototropin responses and a relatively dwarf phenotype as in 35-P2CG plants. In contrast, B1-GP2C plants showed the guard-cell-specific P2C expression that induced constitutive stomatal opening with normal phototropism, chloroplast movement, and growth phenotype. Interestingly, leaf transpiration was significantly improved in B1-GP2C plants compared to that in P2-GP2C plants and WT. Taken together, this transgenic approach could be applied to improve leaf transpiration in indoor plants.

Promotion and Upregulation of a Plasma Membrane Proton-ATPase Strategy: Principles and Applications

Plasma membrane proton-ATPase (PM H+-ATPase) is a primary H+ transporter that consumes ATP in vivo and is a limiting factor in the blue light-induced stomatal opening signaling pathway. It was recently reported that manipulation of PM H+-ATPase in stomatal guard cells and other tissues greatly improved leaf photosynthesis and plant growth. In this report, we review and discuss the function of PM H+-ATPase in the context of the promotion and upregulation H+-ATPase strategy, including associated principles pertaining to enhanced stomatal opening, environmental plasticity, and potential applications in crops and nanotechnology. We highlight the great potential of the promotion and upregulation H+-ATPase strategy, and explain why it may be applied in many crops in the future.

A tonoplast-localized magnesium transporter is involved in stomatal opening in Arabidopsis

Plant stomata play an important role in CO2 uptake for photosynthesis and transpiration, but the mechanisms underlying stomatal opening and closing are still not completely understood. Here, through large-scale screening, we identified an Arabidopsis mutant (cst2 for closed stomata2) defective in stomatal opening under light condition. A map-based cloning combined with complementation test revealed that the mutant phenotype was caused by a nucleotide substitution of a gene, which domains show similarity to human Mg efflux transporter ACDP/CNNM. Functional analysis showed that CST2 encodes a tonoplast-localized transporter for Mg. This protein is constitutively and highly expressed in the guard cells. Furthermore, CST2 is phosphorylated by calcineurin B-like protein (CBL)-interacting protein kinases 26 (CIPK26) in vitro, which is probably required for its activation. Knockout of this gene resulted in stomatal closing and growth retardation under high Mg concentration conditions, while over-expression of this gene increased tolerance to high Mg. Our results indicate that CST2 plays an important role in maintaining Mg homeostasis in plant cells through sequestering Mg into vacuoles especially in guard cells and that this homeostasis is required for stomatal opening, which provide a novel insight into mechanism of stomatal opening in plants.

Overexpression of Plasma Membrane H+-ATPase in Guard Cells Enhances Light-Induced Stomatal Opening, Photosynthesis, and Plant Growth in Hybrid Aspen

Stomata in the plant epidermis open in response to light and regulate CO2 uptake for photosynthesis and transpiration for uptake of water and nutrients from roots. Light-induced stomatal opening is mediated by activation of the plasma membrane (PM) H+-ATPase in guard cells. Overexpression of PM H+-ATPase in guard cells promotes light-induced stomatal opening, enhancing photosynthesis and growth in Arabidopsis thaliana. In this study, transgenic hybrid aspens overexpressing Arabidopsis PM H+-ATPase (AHA2) in guard cells under the strong guard cell promoter Arabidopsis GC1 (AtGC1) showed enhanced light-induced stomatal opening, photosynthesis, and growth. First, we confirmed that AtGC1 induces GUS expression specifically in guard cells in hybrid aspens. Thus, we produced AtGC1::AHA2 transgenic hybrid aspens and confirmed expression of AHA2 in AtGC1::AHA2 transgenic plants. In addition, AtGC1::AHA2 transgenic plants showed a higher PM H+-ATPase protein level in guard cells. Analysis using a gas exchange system revealed that transpiration and the photosynthetic rate were significantly increased in AtGC1::AHA2 transgenic aspen plants. AtGC1::AHA2 transgenic plants showed a>20% higher stem elongation rate than the wild type (WT). Therefore, overexpression of PM H+-ATPase in guard cells promotes the growth of perennial woody plants.

Identification of Genes Preferentially Expressed in Stomatal Guard Cells of Arabidopsis thaliana and Involvement of the Aluminum-Activated Malate Transporter 6 Vacuolar Malate Channel in Stomatal Opening

Stomatal guard cells (GCs) are highly specialized cells that respond to various stimuli, such as blue light (BL) and abscisic acid, for the regulation of stomatal aperture. Many signaling components that are involved in the stomatal movement are preferentially expressed in GCs. In this study, we identified four new such genes in addition to an aluminum-activated malate transporter, ALMT6, and GDSL lipase, Occlusion of Stomatal Pore 1 (OSP1), based on the expression analysis using public resources, reverse transcription PCR, and promoter-driven β-glucuronidase assays. Some null mutants of GC-specific genes evidenced altered stomatal movement. We further investigated the role played by ALMT6, a vacuolar malate channel, in stomatal opening. Epidermal strips from an ALMT6-null mutant exhibited defective stomatal opening induced by BL and fusicoccin, a strong plasma membrane H+-ATPase activator. The deficiency was enhanced when the assay buffer [Cl–] was low, suggesting that malate and/or Cl– facilitate efficient opening. The results indicate that the GC-specific genes are frequently involved in stomatal movement. Further detailed analyses of the hitherto uncharacterized GC-specific genes will provide new insights into stomatal regulation.

Interactive Effects of Intraspecific Competition and Drought on Stomatal Conductance and Hormone Concentrations in Different Tomato Genotypes

We elucidated the effects of intraspecific competition on plant growth, stomatal opening and hormone concentrations in different tomato genotypes under different water regimes. Intraspecific competition reduced plant leaf area and stomatal conductance (gs) of wild-type tomato (Ailsa Craig), which was accompanied by abscisic acid (ABA) accumulation and ethylene evolution. Intraspecific competition-induced decrease in gs was absent in flacca, an ABA-deficient mutant, and in never-ripe, a partially ethylene-insensitive genotype, indicating ABA and ethylene involved in plant response to intraspecific competition. As soil water becomes dry, the competition decreased gs by elevating ABA and ethylene accumulations. Under severe drought, the competition-induced decline in gs was covered by the severe drought-induced decrease in gs, as hydraulic signals most probably dominate. Absence of canopy competition had no significant influence on plant stomatal opening of well-watered tomato, due to canopy separation minimized the plant neighbor sensing by ethylene and other signals. Whereas under water deficit condition, absence of canopy competition significantly reduced ABA accumulation in roots and then stomatal conductance, indicating the belowground neighbour detection signals maybe enhanced by soil drought. Absence of root competition increased ethylene evolution, confirming the importance of ethylene in neighbor detection and plant response to environmental stress.

Protease Inhibitor-Dependent Inhibition of Light-Induced Stomatal Opening

Stomata in the epidermis of plants play essential roles in the regulation of photosynthesis and transpiration. Stomata open in response to blue light (BL) by phosphorylation-dependent activation of the plasma membrane (PM) H+-ATPase in guard cells. Under water stress, the plant hormone abscisic acid (ABA) promotes stomatal closure via the ABA-signaling pathway to reduce water loss. We established a chemical screening method to identify compounds that affect stomatal movements in Commelina benghalensis. We performed chemical screening using a protease inhibitor (PI) library of 130 inhibitors to identify inhibitors of stomatal movement. We discovered 17 PIs that inhibited light-induced stomatal opening by more than 50%. Further analysis of the top three inhibitors (PI1, PI2, and PI3; inhibitors of ubiquitin-specific protease 1, membrane type-1 matrix metalloproteinase, and matrix metalloproteinase-2, respectively) revealed that these inhibitors suppressed BL-induced phosphorylation of the PM H+-ATPase but had no effect on the activity of phototropins or ABA-dependent responses. The results suggest that these PIs suppress BL-induced stomatal opening at least in part by inhibiting PM H+-ATPase activity but not the ABA-signaling pathway. The targets of PI1, PI2, and PI3 were predicted by bioinformatics analyses, which provided insight into factors involved in BL-induced stomatal opening.