Plasma membrane H+-ATPases in mineral nutrition and crop improvement.

Plasma membrane H+-ATPases (PMAs) pump H+ out of the cytoplasm by consuming ATP to generate a membrane potential and proton motive force for the transmembrane transport of nutrients into and out of plant cells. PMAs are involved in nutrient acquisition by regulating root growth, nutrient uptake, and translocation, as well as the establishment of symbiosis with arbuscular mycorrhizas. Under nutrient stresses, PMAs are activated to pump more H+ and promote organic anion excretion, thus improving nutrient availability in the rhizosphere. Herein we review recent progress in the physiological functions and the underlying molecular mechanisms of PMAs in the efficient acquisition and utilization of various nutrients in plants. We also discuss perspectives for the application of PMAs in improving crop production and quality.

Discovery of a Plant 14-3-3 Inhibitor Possessing Isoform Selectivity and In Planta Activity.

Synthetic modulators for plant 14-3-3s are promising chemical tools both for understanding the 14-3-3-related signaling pathways and controlling plant physiology. Here, we describe a novel small-molecule inhibitor for 14-3-3 proteins ofArabidopsis thaliana. The inhibitor was identified from unexpected products in DMSO stock solution of an in-house chemical library. Mass spectroscopy, mutant-based analyses, fluorescence polarization assays, and thermal shift assaysrevealed that the inhibitor covalently binds to an allosteric site of 14-3-3 with isoform selectivity. Moreover, infiltration of the inhibitor to Arabidopsis leaves suppressed the stomatal aperture. The inhibitor should provide a new insight into the design of potent and isoform-selective 14-3-3 modulators.

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.

Phosphorylation of plasma membrane H+-ATPase Thr881 participates in light-induced stomatal opening.

Plasma membrane (PM) H+-ATPase is crucial for light-induced stomatal opening and phosphorylation of a penultimate residue, Thr948 (pen-Thr, numbering according to Arabidopsis AHA1) is required for enzyme activation. In this study, a comprehensive phosphoproteomic analysis using guard cell protoplasts from Vicia faba shows that both red and blue light increase the phosphorylation of Thr881, of PM H+-ATPase. Light-induced stomatal opening and the blue light-induced increase in stomatal conductance are reduced in transgenic Arabidopsis plants expressing mutant AHA1-T881A in aha1-9, whereas the blue light-induced phosphorylation of pen-Thr is unaffected. Auxin and photosynthetically active radiation induce the phosphorylation of both Thr881 and pen-Thr in etiolated seedlings and leaves, respectively. The dephosphorylation of phosphorylated Thr881 and pen-Thr are mediated by type 2 C protein phosphatase clade D isoforms. Taken together, Thr881 phosphorylation, in addition of the pen-Thr phosphorylation, are important for PM H+-ATPase function during physiological responses, such as light-induced stomatal opening in Arabidopsis thaliana.

Accumulation of Phosphorylated SnRK2 Substrate 1 Promotes Drought Escape in Arabidopsis.

Plants adopt optimal tolerance strategies depending on the intensity and duration of stress. Retaining water is a priority under short-term drought conditions, whereas maintaining growth and reproduction processes takes precedence over survival under conditions of prolonged drought. However, the mechanism underlying changes in the stress response depending on the degree of drought is unclear. Here, we report that SNF1-related protein kinase 2 (SnRK2) substrate 1 (SNS1) is involved in this growth regulation under conditions of drought stress. SNS1 is phosphorylated and stabilized by SnRK2 protein kinases reflecting drought conditions. It contributes to the maintenance of growth and promotion of flowering as drought escape by repressing stress-responsive genes and inducing FLOWERING LOCUS T (FT) expression, respectively. SNS1 interacts with the histone methylation reader proteins MORF-related gene 1 (MRG1) and MRG2, and the SNS1-MRG1/2 module cooperatively regulates abscisic acid response. Taken together, these observations suggest that the phosphorylation and accumulation of SNS1 in plants reflect the intensity and duration of stress and can serve as a molecular scale for maintaining growth and adopting optimal drought tolerance strategies under stress conditions.

A Small-Molecule Modulator Affecting the Clock-Associated PSEUDO-RESPONSE REGULATOR 7 Amount.

Circadian clocks are biological timekeeping systems that coordinate genetic, metabolic and physiological behaviors with the external day-night cycle. The clock in plants relies on the transcriptional-translational feedback loops transcription-translation feedback loop (TTFL), consisting of transcription factors including PSUEDO-RESPONSE REGULATOR (PRR) proteins, plant lineage-specific transcriptional repressors. Here, we report that a novel synthetic small-molecule modulator, 5-(3,4-dichlorophenyl)-1-phenyl-1,7-dihydro-4H-pyrazolo[3,4-d] pyrimidine-4,6(5H)-dione (TU-892), affects the PRR7 protein amount. A clock reporter line of Arabidopsis was screened against the 10,000 small molecules in the Maybridge Hitfinder 10K chemical library. This screening identified TU-892 as a period-lengthening molecule. Gene expression analyses showed that TU-892 treatment upregulates CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) mRNA expression. TU-892 treatment reduced the amount of PRR7 protein, a transcriptional repressor of CCA1. Other PRR proteins including TIMING OF CAB EXPRESSION 1 were altered less by TU-892 treatment. TU-892-dependent CCA1 upregulation was attenuated in mutants impaired in PRR7. Collectively, TU-892 is a novel type of clock modulator that reduces the levels of PRR7 protein.

Identification and improvement of isothiocyanate-based inhibitors on stomatal opening to act as drought tolerance-conferring agrochemicals.

Stomatal pores in the plant epidermis open and close to regulate gas exchange between leaves and the atmosphere. Upon light stimulation, the plasma membrane (PM) H+-ATPase is phosphorylated and activated via an intracellular signal transduction pathway in stomatal guard cells, providing a primary driving force for the opening movement. To uncover and manipulate this stomatal opening pathway, we screened a chemical library and identified benzyl isothiocyanate (BITC), a Brassicales-specific metabolite, as a potent stomatal-opening inhibitor that suppresses PM H+-ATPase phosphorylation. We further developed BITC derivatives with multiple isothiocyanate groups (multi-ITCs), which demonstrate inhibitory activity on stomatal opening up to 66 times stronger, as well as a longer duration of the effect and negligible toxicity. The multi-ITC treatment inhibits plant leaf wilting in both short (1.5 h) and long-term (24 h) periods. Our research elucidates the biological function of BITC and its use as an agrochemical that confers drought tolerance on plants by suppressing stomatal opening.

PECT1, a rate-limiting enzyme in phosphatidylethanolamine biosynthesis, is involved in the regulation of stomatal movement in Arabidopsis.

An Arabidopsis mutant displaying impaired stomatal responses to CO2 , cdi4, was isolated by a leaf thermal imaging screening. The mutated gene PECT1 encodes CTP:phosphorylethanolamine cytidylyltransferase. The cdi4 exhibited a decrease in phosphatidylethanolamine levels and a defect in light-induced stomatal opening as well as low-CO2 -induced stomatal opening. We created RNAi lines in which PECT1 was specifically repressed in guard cells. These lines are impaired in their stomatal responses to low-CO2 concentrations or light. Fungal toxin fusicoccin (FC) promotes stomatal opening by activating plasma membrane H+ -ATPases in guard cells via phosphorylation. Arabidopsis H+ -ATPase1 (AHA1) has been reported to be highly expressed in guard cells, and its activation by FC induces stomatal opening. The cdi4 and PECT1 RNAi lines displayed a reduced stomatal opening response to FC. However, similar to in the wild-type, cdi4 maintained normal levels of phosphorylation and activation of the stomatal H+ -ATPases after FC treatment. Furthermore, the cdi4 displayed normal localization of GFP-AHA1 fusion protein and normal levels of AHA1 transcripts. Based on these results, we discuss how PECT1 could regulate CO2 - and light-induced stomatal movements in guard cells in a manner that is independent and downstream of the activation of H+ -ATPases. [Correction added on 15 May 2023, after first online publication: The third sentence is revised in this version.].

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.

PP2C.D phosphatase SAL1 positively regulates aluminum resistance via restriction of aluminum uptake in rice.

Aluminum (Al) toxicity represents a primary constraint for crop production in acidic soils. Rice (Oryza sativa) is a highly Al-resistant species; however, the molecular mechanisms underlying its high Al resistance are still not fully understood. Here, we identified SAL1 (SENSITIVE TO ALUMINUM 1), which encodes a plasma membrane (PM)-localized PP2C.D phosphatase, as a crucial regulator of Al resistance using a forward genetic screen. SAL1 was found to interact with and inhibit the activity of PM H+-ATPases, and mutation of SAL1 increased PM H+-ATPase activity and Al uptake, causing hypersensitivity to internal Al toxicity. Furthermore, knockout of NRAT1 (NRAMP ALUMINUM TRANSPORTER 1) encoding an Al uptake transporter in a sal1 background rescued the Al-sensitive phenotype of sal1, revealing that coordination of Al accumulation in the cell, wall and symplasm is critical for Al resistance in rice. By contrast, we found that mutations of PP2C.D phosphatase-encoding genes in Arabidopsis (Arabidopsis thaliana) enhanced Al resistance, which was attributed to increased malate secretion. Our results reveal the importance of PP2C.D phosphatases in Al resistance and the different strategies used by rice and Arabidopsis to defend against Al toxicity.

Photosynthetic-Product-Dependent Activation of Plasma Membrane H+-ATPase and Nitrate Uptake in Arabidopsis Leaves.

Plasma membrane (PM) proton-translocating adenosine triphosphatase (H+-ATPase) is a pivotal enzyme for plant growth and development that acts as a primary transporter and is activated by phosphorylation of the penultimate residue, threonine, at the C-terminus. Small Auxin-Up RNA family proteins maintain the phosphorylation level via inhibiting dephosphorylation of the residue by protein phosphatase 2C-D clade. Photosynthetically active radiation activates PM H+-ATPase via phosphorylation in mesophyll cells of Arabidopsis thaliana, and phosphorylation of PM H+-ATPase depends on photosynthesis and photosynthesis-related sugar supplementation, such as sucrose, fructose and glucose. However, the molecular mechanism and physiological role of photosynthesis-dependent PM H+-ATPase activation are still unknown. Analysis using sugar analogs, such as palatinose, turanose and 2-deoxy glucose, revealed that sucrose metabolites and products of glycolysis such as pyruvate induce phosphorylation of PM H+-ATPase. Transcriptome analysis showed that the novel isoform of the Small Auxin-Up RNA genes, SAUR30, is upregulated in a light- and sucrose-dependent manner. Time-course analyses of sucrose supplementation showed that the phosphorylation level of PM H+-ATPase increased within 10 min, but the expression level of SAUR30 increased later than 10 min. The results suggest that two temporal regulations may participate in the regulation of PM H+-ATPase. Interestingly, a 15NO3- uptake assay in leaves showed that light increases 15NO3- uptake and that increment of 15NO3- uptake depends on PM H+-ATPase activity. The results opened the possibility of the physiological role of photosynthesis-dependent PM H+-ATPase activation in the uptake of NO3-. We speculate that PM H+-ATPase may connect photosynthesis and nitrogen metabolism in leaves.

Discovery of 2,6-Dihalopurines as Stomata Opening Inhibitors: Implication of an LRX-Mediated H+-ATPase Phosphorylation Pathway.

Stomata are pores in the leaf epidermis of plants and their opening and closing regulate gas exchange and water transpiration. Stomatal movements play key roles in both plant growth and stress responses. In recent years, small molecules regulating stomatal movements have been used as a powerful tool in mechanistic studies, as well as key players for agricultural applications. Therefore, the development of new molecules regulating stomatal movement and the elucidation of their mechanisms have attracted much attention. We herein describe the discovery of 2,6-dihalopurines, AUs, as a new stomatal opening inhibitor, and their mechanistic study. Based on biological assays, AUs may involve in the pathway related with plasma membrane H+-ATPase phosphorylation. In addition, we identified leucine-rich repeat extensin proteins (LRXs), LRX3, LRX4 and LRX5 as well as RALF, as target protein candidates of AUs by affinity based pull down assay and molecular dynamics simulation. The mechanism of stomatal movement related with the LRXs-RALF is an unexplored pathway, and therefore further studies may lead to the discovery of new signaling pathways and regulatory factors in the stomatal movement.

Stomatal properties of Arabidopsis cauline and rice flag leaves and their contributions to seed production and grain yield.

Cauline leaves on the inflorescence stem of Arabidopsis thaliana may play important roles in supplying photosynthetic products to sinks, such as floral organs. Flag leaves in rice (Oryza sativa) have a higher photosynthetic capacity than other leaves, and are crucial for increasing grain yield. However, the detailed properties of stomata in cauline and flag leaves have not been investigated. In Arabidopsis, stomatal conductance and CO2 assimilation rate were higher in cauline leaves under white light than in rosette leaves, consistent with higher levels of plasma membrane (PM) H+-ATPase, a key enzyme for stomatal opening, in guard cells. Moreover, removal of cauline leaves significantly reduced the shoot biomass by approximately 20% and seed production by approximately 46%. In rice, higher stomatal density, stomatal conductance, and CO2 assimilation rate were observed in flag leaves than in fully expanded second leaves. Removal of the flag leaves significantly reduced grain yield by approximately 49%. Taken together, these results show that cauline and flag leaves have important roles in seed production and grain yield through enhanced stomatal conductance and CO2 assimilation rate.

Tryptophan synthase ß subunit 1 affects stomatal phenotypes in Arabidopsis thaliana.

Stomata open in response to several environmental stimuli, such as light and low CO2. Plasma membrane (PM) H+-ATPase in guard cells plays a pivotal role for light-induced stomatal opening. In contrast, stomata close in response to the dark or plant hormone abscisic acid (ABA). However, molecular mechanisms of stomatal movements remain unclear. To elucidate the molecular mechanism of stomatal movements, we performed a genetic screen based on stomatal aperture-dependent weight decrease of detached leaves from EMS-treated Arabidopsis thaliana and isolated a rapid transpiration in detached leaves 2 (rtl2). The rtl2 mutant showed constitutive open-stomata phenotype with lower leaf temperature. ABA had no effect on stomatal aperture in rtl2. The rtl2 mutant also showed increased stomatal density, severe dwarf phenotype with pale green leaves and dark veins. Map-based analysis of the RTL2 locus revealed that the rtl2 mutant possesses a single nucleotide substitution, which induces amino acid substitution Gly162 to Glu in the tryptophan synthase ß subunit 1 (TSB1). The TSB1 encodes an enzyme in tryptophan (Trp) biosynthetic pathway. Amount of TSB1 protein was drastically reduced in rtl2 mutant. A different allele of tsb1 mutant (tsb1-1) also showed constitutive open-stomata phenotype with reduced TSB1 protein as in rtl2. Analyses of test-crossed plants of rtl2 and tsb1-1 showed open-stomata and dwarf phenotypes. These results indicate that a responsible gene for rtl2 is TSB1. We further investigated stomatal phenotype in mutants from Trp biosynthetic pathway, such as wei2-1 wei7-1, trp3-1, and tsb2-1. The trp3-1 mutant showed significant wider stomatal aperture as well as tsb1-1. Trp biosynthetic pathway closely relates to auxin biosynthesis. Then, we investigated auxin responsible genes and found that an expression of AUR3 was up in rtl2. In contrast, auxin had no effect on stomatal aperture in Arabidopsis and the phosphorylation status of PM H+-ATPase in guard cell protoplasts from Vicia faba. In addition, auxin antagonist had no effect on stomatal aperture. Interestingly, tsb1-1 grown under hydroponic culture system showed normal stomatal aperture by exogenously application of Trp. These results suggest that open stomata phenotype in tsb1-1 is due to Trp deficiency but not auxin.

Overexpression of a Plasma Membrane H+-ATPase Gene OSA1 Stimulates the Uptake of Primary Macronutrients in Rice Roots.

Plasma membrane (PM) H+-ATPase is a master enzyme involved in various plant physiological processes, such as stomatal movements in leaves and nutrient uptake and transport in roots. Overexpression of Oryza sativa PM H+-ATPase 1 (OSA1) has been known to increase NH4+ uptake in rice roots. Although electrophysiological and pharmacological experiments have shown that the transport of many substances is dependent on the proton motive force provided by PM H+-ATPase, the exact role of PM H+-ATPase on the uptake of nutrients in plant roots, especially for the primary macronutrients N, P, and K, is still largely unknown. Here, we used OSA1 overexpression lines (OSA1-oxs) and gene-knockout osa1 mutants to investigate the effect of modulation of PM H+-ATPase on the absorption of N, P, and K nutrients through the use of a nutrient-exhaustive method and noninvasive microtest technology (NMT) in rice roots. Our results showed that under different concentrations of P and K, the uptake rates of P and K were enhanced in OSA1-oxs; by contrast, the uptake rates of P and K were significantly reduced in roots of osa1 mutants when compared with wild-type. In addition, the net influx rates of NH4+ and K+, as well as the efflux rate of H+, were enhanced in OSA1-oxs and suppressed in osa1 mutants under low concentration conditions. In summary, this study indicated that overexpression of OSA1 stimulated the uptake rate of N, P, and K and promoted flux rates of cations (i.e., H+, NH4+, and K+) in rice roots. These results may provide a novel insight into improving the coordinated utilization of macronutrients in crop plants.

Photoinduced Synthesis of Thiocyanates through Hydrogen Atom Transfer and One-Pot Derivatization to Isothiocyanates.

Photoinduced benzylic C-H thiocyanation is described. A series of alkyl thiocyanates were efficiently obtained by using Selectfluor as the oxidant. Moreover, we accomplished the one-pot isothiocyanation following the C-H thiocyanation. The thiocyanates and isothiocyanates were applied to the divergent transformation of pharmaceuticals.

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.

ABP1-TMK auxin perception for global phosphorylation and auxin canalization.

The phytohormone auxin triggers transcriptional reprogramming through a well-characterized perception machinery in the nucleus. By contrast, mechanisms that underlie fast effects of auxin, such as the regulation of ion fluxes, rapid phosphorylation of proteins or auxin feedback on its transport, remain unclear1-3. Whether auxin-binding protein 1 (ABP1) is an auxin receptor has been a source of debate for decades1,4. Here we show that a fraction of Arabidopsis thaliana ABP1 is secreted and binds auxin specifically at an acidic pH that is typical of the apoplast. ABP1 and its plasma-membrane-localized partner, transmembrane kinase 1 (TMK1), are required for the auxin-induced ultrafast global phospho-response and for downstream processes that include the activation of H+-ATPase and accelerated cytoplasmic streaming. abp1 and tmk mutants cannot establish auxin-transporting channels and show defective auxin-induced vasculature formation and regeneration. An ABP1(M2X) variant that lacks the capacity to bind auxin is unable to complement these defects in abp1 mutants. These data indicate that ABP1 is the auxin receptor for TMK1-based cell-surface signalling, which mediates the global phospho-response and auxin canalization.

A tonoplast-localized magnesium transporter is crucial for stomatal opening in Arabidopsis under high Mg2+ conditions.

Plant stomata play an important role in CO2 uptake for photosynthesis and transpiration, but the mechanisms underlying stomatal opening and closing under changing environmental conditions are still not completely understood. Through large-scale genetic screening, we isolated an Arabidopsis mutant (closed stomata2 (cst2)) that is defective in stomatal opening. We cloned the causal gene (MGR1/CST2) and functionally characterized this gene. The mutant phenotype was caused by a mutation in a gene encoding an unknown protein with similarities to the human magnesium (Mg2+ ) efflux transporter ACDP/CNNM. MGR1/CST2 was localized to the tonoplast and showed transport activity for Mg2+ . This protein was constitutively and highly expressed in guard cells. Knockout of this gene resulted in stomatal closing, decreased photosynthesis and growth retardation, especially under high Mg2+ conditions, while overexpression of this gene increased stomatal opening and tolerance to high Mg2+ concentrations. Furthermore, guard cell-specific expression of MGR1/CST2 in the mutant partially restored its stomatal opening. Our results indicate that MGR1/CST2 expression in the leaf guard cells plays an important role in maintaining cytosolic Mg2+ concentrations through sequestering Mg2+ into vacuoles, which is required for stomatal opening, especially under high Mg2+ conditions.