A novel semi-dominant mutation in brassinosteroid signaling kinase1 increases stomatal density.

Stomata play a pivotal role in balancing CO2 uptake for photosynthesis and water loss via transpiration. Thus, appropriate regulation of stomatal movement and its formation are crucial for plant growth and survival. Red and blue light induce phosphorylation of the C-terminal residue of the plasma membrane (PM) H+-ATPase, threonine, in guard cells, generating the driving force for stomatal opening. While significant progress has been made in understanding the regulatory mechanism of PM H+-ATPase in guard cells, the regulatory components for the phosphorylation of PM H+-ATPase have not been fully elucidated. Recently, we established a new immunohistochemical technique for detecting guard-cell PM H+-ATPase phosphorylation using leaves, which was expected to facilitate investigations with a single leaf. In this study, we applied the technique to genetic screening experiment to explore novel regulators for the phosphorylation of PM H+-ATPase in guard cells, as well as stomatal development. We successfully performed phenotyping using a single leaf. During the experiment, we identified a mutant exhibiting high stomatal density, jozetsu (jzt), named after a Japanese word meaning 'talkative'. We found that a novel semi-dominant mutation in BRASSINOSTEROID SIGNALING KINASE1 (BSK1) is responsible for the phenotype in jzt mutant. The present results demonstrate that the new immunohistochemical technique has a wide range of applications, and the novel mutation would provide genetic tool to expand our understanding of plant development mediated by brassinosteroid signaling.

Image-Based Quantification of Arabidopsis thaliana Stomatal Aperture from Leaf Images.

The quantification of stomatal pore size has long been a fundamental approach to understand the physiological response of plants in the context of environmental adaptation. Automation of such methodologies not only alleviates human labor and bias but also realizes new experimental research methods through massive analysis. Here, we present an image analysis pipeline that automatically quantifies stomatal aperture of Arabidopsis thaliana leaves from bright-field microscopy images containing mesophyll tissue as noisy backgrounds. By combining a You Only Look Once X-based stomatal detection submodule and a U-Net-based pore segmentation submodule, we achieved a mean average precision with an intersection of union (IoU) threshold of 50% value of 0.875 (stomata detection performance) and an IoU of 0.745 (pore segmentation performance) against images of leaf discs taken with a bright-field microscope. Moreover, we designed a portable imaging device that allows easy acquisition of stomatal images from detached/undetached intact leaves on-site. We demonstrated that this device in combination with fine-tuned models of the pipeline we generated here provides robust measurements that can substitute for manual measurement of stomatal responses against pathogen inoculation. Utilization of our hardware and pipeline for automated stomatal aperture measurements is expected to accelerate research on stomatal biology of model dicots.

Elevated CO2 induces rapid dephosphorylation of plasma membrane H+ -ATPase in guard cells.

Light induces stomatal opening, which is driven by plasma membrane (PM) H+ -ATPase in guard cells. The activation of guard-cell PM H+ -ATPase is mediated by phosphorylation of the penultimate C-terminal residue, threonine. The phosphorylation is induced by photosynthesis as well as blue light photoreceptor phototropin. Here, we investigated the effects of cessation of photosynthesis on the phosphorylation level of guard-cell PM H+ -ATPase in Arabidopsis thaliana. Immunodetection of guard-cell PM H+ -ATPase, time-resolved leaf gas-exchange analyses and stomatal aperture measurements were carried out. We found that light-dark transition of leaves induced dephosphorylation of the penultimate residue at 1 min post-transition. Gas-exchange analyses confirmed that the dephosphorylation is accompanied by an increase in the intercellular CO2 concentration, caused by the cessation of photosynthetic CO2 fixation. We discovered that CO2 induces guard-cell PM H+ -ATPase dephosphorylation as well as stomatal closure. Interestingly, reverse-genetic analyses using guard-cell CO2 signal transduction mutants suggested that the dephosphorylation is mediated by a mechanism distinct from the established CO2 signalling pathway. Moreover, type 2C protein phosphatases D6 and D9 were required for the dephosphorylation and promoted stomatal closure upon the light-dark transition. Our results indicate that CO2 -mediated dephosphorylation of guard-cell PM H+ -ATPase underlies stomatal closure.

Ethanol treatment enhances drought stress avoidance in cassava (Manihot esculenta Crantz).

External application of ethanol enhances tolerance to high salinity, drought, and heat stress in various plant species. However, the effects of ethanol application on increased drought tolerance in woody plants, such as the tropical crop "cassava," remain unknown. In the present study, we analyzed the morphological, physiological, and molecular responses of cassava plants subjected to ethanol pretreatment and subsequent drought stress treatment. Ethanol pretreatment induced a slight accumulation of abscisic acid (ABA) and stomatal closure, resulting in a reduced transpiration rate, higher water content in the leaves during drought stress treatment and the starch accumulation in leaves. Transcriptomic analysis revealed that ethanol pretreatment upregulated the expression of ABA signaling-related genes, such as PP2Cs and AITRs, and stress response and protein-folding-related genes, such as heat shock proteins (HSPs). In addition, the upregulation of drought-inducible genes during drought treatment was delayed in ethanol-pretreated plants compared with that in water-pretreated control plants. These results suggest that ethanol pretreatment induces stomatal closure through activation of the ABA signaling pathway, protein folding-related response by activating the HSP/chaperone network and the changes in sugar and starch metabolism, resulting in increased drought avoidance in plants.

Ethanol-Mediated Novel Survival Strategy against Drought Stress in Plants.

Water scarcity is a serious agricultural problem causing significant losses to crop yield and product quality. The development of technologies to mitigate the damage caused by drought stress is essential for ensuring a sustainable food supply for the increasing global population. We herein report that the exogenous application of ethanol, an inexpensive and environmentally friendly chemical, significantly enhances drought tolerance in Arabidopsis thaliana, rice and wheat. The transcriptomic analyses of ethanol-treated plants revealed the upregulation of genes related to sucrose and starch metabolism, phenylpropanoids and glucosinolate biosynthesis, while metabolomic analysis showed an increased accumulation of sugars, glucosinolates and drought-tolerance-related amino acids. The phenotyping analysis indicated that drought-induced water loss was delayed in the ethanol-treated plants. Furthermore, ethanol treatment induced stomatal closure, resulting in decreased transpiration rate and increased leaf water contents under drought stress conditions. The ethanol treatment did not enhance drought tolerance in the mutant of ABI1, a negative regulator of abscisic acid (ABA) signaling in Arabidopsis, indicating that ABA signaling contributes to ethanol-mediated drought tolerance. The nuclear magnetic resonance analysis using 13C-labeled ethanol indicated that gluconeogenesis is involved in the accumulation of sugars. The ethanol treatment did not enhance the drought tolerance in the aldehyde dehydrogenase (aldh) triple mutant (aldh2b4/aldh2b7/aldh2c4). These results show that ABA signaling and acetic acid biosynthesis are involved in ethanol-mediated drought tolerance and that chemical priming through ethanol application regulates sugar accumulation and gluconeogenesis, leading to enhanced drought tolerance and sustained plant growth. These findings highlight a new survival strategy for increasing crop production under water-limited conditions.

Type 2C protein phosphatase clade D family members dephosphorylate guard cell plasma membrane H+-ATPase.

Plasma membrane (PM) H+-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg2+- and Mn2+-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates the dephosphorylation of PM H+-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H+-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H+-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H+-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8, and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H+-ATPase in guard cells as Col-0. In contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H+-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H+-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H+-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H+-ATPase in guard cells.

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.

Inhibition of light-induced stomatal opening by allyl isothiocyanate does not require guard cell cytosolic Ca2+ signaling.

The glucosinolate-myrosinase system is a well-known defense system that has been shown to induce stomatal closure in Brassicales. Isothiocyanates are highly reactive hydrolysates of glucosinolates, and an isothiocyanate, allyl isothiocyanate (AITC), induces stomatal closure accompanied by elevation of free cytosolic Ca2+ concentration ([Ca2+]cyt) in Arabidopsis. It remains unknown whether AITC inhibits light-induced stomatal opening. This study investigated the role of Ca2+ in AITC-induced stomatal closure and inhibition of light-induced stomatal opening. AITC induced stomatal closure and inhibited light-induced stomatal opening in a dose-dependent manner. A Ca2+ channel inhibitor, La3+, a Ca2+chelator, EGTA, and an inhibitor of Ca2+ release from internal stores, nicotinamide, inhibited AITC-induced [Ca2+]cyt elevation and stomatal closure, but did not affect inhibition of light-induced stomatal opening. AITC activated non-selective Ca2+-permeable cation channels and inhibited inward-rectifying K+ (K+in) channels in a Ca2+-independent manner. AITC also inhibited stomatal opening induced by fusicoccin, a plasma membrane H+-ATPase activator, but had no significant effect on fusicoccin-induced phosphorylation of the penultimate threonine of H+-ATPase. Taken together, these results suggest that AITC induces Ca2+ influx and Ca2+ release to elevate [Ca2+]cyt, which is essential for AITC-induced stomatal closure but not for inhibition of K+in channels and light-induced stomatal opening.

Fluence rate dependence of red light-induced phosphorylation of plasma membrane H+-ATPase in stomatal guard cells.

Stomatal opening is induced by red light as well as blue light. Recently, we established an immunohistochemical technique using whole leaves to study plasma membrane (PM) H+-ATPase in guard cells, which is an important enzyme driving stomatal opening. Our technique revealed that red light illuminated to whole leaves induces photosynthesis-dependent phosphorylation of C-terminal penultimate residue of PM H+-ATPase, threonine, in guard cells, which has been considered to be important for activation of PM H+-ATPase, and we proposed that red light promotes stomatal opening via activation of PM H+-ATPase in guard cells in whole leaves. Here, using our new immunohistochemical technique, we investigated fluence rate dependence of red light-induced phosphorylation of PM H+-ATPase. We found that illumination of red light at 50 µmol m-2 s-1, which was suggested to initiate photosynthesis, saturates phosphorylation of PM H+-ATPase. Furthermore, we immunohistochemically confirmed decrease in the amount of PM H+-ATPase protein in a knock-out mutant of AHA1, an isogene encoding the major isoform of PM H+-ATPase in guard cells, implying the importance of AHA1 as the major PM H+-ATPase protein in guard cells for light-induced stomatal opening.

Red Light-Induced Phosphorylation of Plasma Membrane H+-ATPase in Stomatal Guard Cells.

Stomatal opening is stimulated by red and blue light. Blue light activates plasma membrane (PM) H+-ATPase by phosphorylating its penultimate residue, threonine, via a blue light photoreceptor phototropin-mediated signaling pathway in guard cells. Blue light-activated PM H+-ATPase promotes the accumulation of osmolytes and, thus, the osmotic influx of water into guard cells, driving stomatal opening. Red light-induced stomatal opening is thought to be dependent on photosynthesis in both guard cell chloroplasts and mesophyll cells; however, how red light induces stomatal opening and whether PM H+-ATPase is involved in this process have remained unclear. In this study, we established an immunohistochemical technique to detect the phosphorylation level of PM H+-ATPase in guard cells using whole leaves of Arabidopsis (Arabidopsis thaliana) and unexpectedly found that red light induces PM H+-ATPase phosphorylation in whole leaves. Red light-induced PM H+-ATPase phosphorylation in whole leaves was correlated with stomatal opening under red light and was inhibited by the plant hormone abscisic acid. In aha1-9, a knockout mutant of one of the major isoforms of PM H+-ATPase in guard cells, red light-dependent stomatal opening was delayed in whole leaves. Furthermore, the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibited red light-induced PM H+-ATPase phosphorylation as well as red light-induced stomatal opening in whole leaves. Our results indicate that red light-induced PM H+-ATPase phosphorylation in guard cells promotes stomatal opening in whole leaves, providing insight into the photosynthetic regulation of stomatal opening.

A flowering integrator, SOC1, affects stomatal opening in Arabidopsis thaliana.

Stomatal movements are regulated by multiple environmental signals. Recent investigations indicate that photoperiodic flowering components, such as CRY, GI, CO, FT and TSF, are expressed in guard cells and positively affect stomatal opening in Arabidopsis thaliana. Here we show that SOC1, which encodes a MADS box transcription factor and integrates multiple flowering signals, also exerts a positive effect on stomatal opening. FLC encodes a potent repressor of FT and SOC1, and FRI acts as an activator of FLC. Thus, we examined stomatal phenotypes in FRI-Col, which contains an active FRI allele of accession Sf-2 by introgression. We found higher expression of FLC and lower expression of FT, SOC1 and TSF in guard cells from FRI-Col than in those from Col. Light-induced stomatal opening was significantly suppressed in FRI-Col. Interestingly, vernalization of FRI-Col partially restored light-induced stomatal opening, concomitant with a decrease of FLC and increase of FT, SOC1 and TSF. Furthermore, we observed the constitutive open-stomata phenotype in transgenic plants overexpressing SOC1-GFP (green fluorescent protein) in guard cells (SOC1-GFP overexpressor), and found that light-induced stomatal opening was significantly suppressed in a soc1 knockout mutant. RNA sequencing using epidermis from the SOC1-GFP overexpressor revealed that the expression levels of several genes involved in stomatal opening, such as BLUS1 and the plasma membrane H(+)-ATPases, were higher than those in background plants. From these results, we conclude that SOC1 is involved in the regulation of stomatal opening via transcriptional regulation in guard cells.

TWIN SISTER OF FT, GIGANTEA, and CONSTANS have a positive but indirect effect on blue light-induced stomatal opening in Arabidopsis.

FLOWERING LOCUS T (FT) is the major regulatory component controlling photoperiodic floral transition. It is expressed in guard cells and affects blue light-induced stomatal opening induced by the blue-light receptor phototropins phot1 and phot2. Roles for other flowering regulators in stomatal opening have yet to be determined. We show in Arabidopsis (Arabidopsis thaliana) that TWIN SISTER OF FT (TSF), CONSTANS (CO), and GIGANTEA (GI) provide a positive effect on stomatal opening. TSF, which is the closest homolog of FT, was transcribed in guard cells, and light-induced stomatal opening was repressed in tsf-1, a T-DNA insertion mutant of TSF. Overexpression of TSF in a phot1 phot2 mutant background gave a constitutive open-stomata phenotype. Then, we examined whether CO and GI, which are upstream regulators of FT and TSF in photoperiodic flowering, are involved in stomatal opening. Similar to TSF, light-induced stomatal opening was suppressed in the GI and CO mutants gi-1 and co-1. A constitutive open-stomata phenotype was observed in GI and CO overexpressors with accompanying changes in the transcription of both FT and TSF. In photoperiodic flowering, photoperiod is sensed by photoreceptors such as the cryptochromes cry1 and cry2. We examined stomatal phenotypes in a cry1 cry2 mutant and in CRY2 overexpressors. Light-induced stomatal opening was suppressed in cry1 cry2, and the transcription of FT and TSF was down-regulated. In contrast, the stomata in CRY2 overexpressors opened even in the dark, and FT and TSF transcription was up-regulated. We conclude that the photoperiodic flowering components TSF, GI, and CO positively affect stomatal opening.