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Aims: Nitrogen, especially NH, can stimulate the uptake of phosphorus in plants, but the underlying mechanisms have not been clearly elucidated. Because phosphate is taken up via an anion/H co-transport process, we propose that the stimulated uptake of phosphorus by NH versus NO nutrition may be related to the activity of plasma membrane H ATPase. In the present study, we investigated the effect of NH and NO on phosphorus uptake and plasma membrane H ATPase activity in rice. Methods: Rice plants were cultivated in a hydroponic solution with NH or NO. After 15 days of cultivation, phosphorus content was determined. Root plasma membrane was isolated using a two-phase partitioning system and hydrolytic H-ATPase activity was determined by measuring the P concentration after a 30-min hydrolysis reaction. Relative expression of plasma membrane H ATPase genes was analyzed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). H ATPase enzyme concentration in the plasma membrane was detected by western blot. For P uptake experiments, rice roots were incubated in the nutrient solution with addition of HPO. Results: P content in both the roots and shoots of rice plants supplemented with NH was significantly higher than P content in plants grown with NO. Plasma membrane H ATPase activity in NH-fed rice roots was significantly higher than that in NO-fed rice roots. Real-time qRT-PCR and western blot results indicated that the higher activity of plasma membrane H ATPase in NH-fed rice roots could be attributed to increased expression of the OSA1, OSA3, OSA7, OSA8 and OSA9 genes and an increase in H ATPase enzyme concentration in the plasma membrane. Results from P uptake experiments showed that rice roots incubated with NH absorbed more P during the four-hour incubation than did rice roots incubated with NO. Vanadate inhibited P uptake in rice roots supplied with NH, while fusicoccin stimulated P uptake under NO nutrition. Conclusions: Taken together, these results suggest an involvement of plasma membrane H ATPase in the stimulated uptake of phosphorus by rice roots supplemented with NH. [ABSTRACT FROM AUTHOR]

The plasma membrane H+-ATPase generates an electrochemical gradient of H+ across the plasma membrane that provides the driving force for solute transport and regulates pH homeostasis and membrane potential in plant cells. Recent studies have demonstrated that phosphorylation of the penultimate threonine in H+ ATPase and subsequent binding of a 14-3-3 protein is the major common activation mechanism for H+-ATPase in vascular plants. However, there is very little information on the plasma membrane H+-ATPase in nonvascular plant bryophytes. Here, we show that the liverwort Marchantia polymorpha, which is the most basal lineage of extant land plants, expresses both the penultimate threonine-containing H+-ATPase (pT H+-ATPase) and non-penultimate threonine-containing H+-ATPase (non-pT H+-ATPase) as in the green algae and that pT H+-ATPase is regulated by phosphorylation of its penultimate threonine. A search in the expressed sequence tag database of M. polymorpha revealed eight H+-ATPase genes, designated MpHA (for M. polymorpha H+-ATPase). Four isoforrns are the pT H+-ATPase; the remaining isoforms are non-pT H+-ATPase. An apparent 95-kD protein was recognized by anti-H+-ATPase antibodies against an Arabidopsis (Arabidopsis thaliana) isoform and was phosphorylated on the penultimate threonine in response to the fungal toxin fusicoccin in thalli, indicating that the 95-kD protein contains pT H+-ATPase. Furthermore, we found that the pT H+-ATPase in thalli is phosphorylated in response to light, sucrose, and osmotic shock and that light-induced phosphorylation depends on photosynthesis. Our results define physiological signals for the regulation of pT H+-ATPase in the liverwort M. polymorpha, which is one of the earliest plants to acquire pT H+-ATPase. [ABSTRACT FROM AUTHOR]

The phytohormone auxin is a major regulator of diverse aspects of plant growth and development. The ubiquitin-ligase complex SCFTIR1/AFB (for Skp1-Cul1-F-box protein), which includes the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) auxin receptor family, has recently been demonstrated to be critical for auxin-mediated transcriptional regulation. Early-phase auxin-induced hypocotyl elongation, on the other hand, has long been explained by the acid-growth theory, for which proton extrusion by the plasma membrane H+-ATPase is a functional prerequisite. However, the mechanism by which auxin mediates H+-ATPase activation has yet to be elucidated. Here, we present direct evidence for H+-ATPase activation in etiolated hypocotyls of Arabidopsis (Arabidopsis thaliana) by auxin through phosphorylation of the penultimate threonine during early-phase hypocotyl elongation. Application of the natural auxin indole-3-acetic acid (IAA) to endogenous auxin-depleted hypocotyl sections induced phosphorylation of the penultimate threonine of the H+-ATPase and increased H+-ATPase activity without altering the amount of the elzzyme. Changes in both the phosphorylation level of H+- ATPase and IAA-induced elongation were similarly concentration dependent. Furthermore, IAA-induced H+-ATPase phosphorylation occurred in a tir1-1 afb2-3 double mutant, which is severely defective in auxin-mediated transcriptional regulation. In addition, α-(phenylethyl-2-one)-IAA, the auxin antagonist specific for the nuclear auxin receptor TIR1/AFBs, had no effect on IAA-induced H+-ATPase phosphorylation. These results suggest that the TIR1/AFB auxin receptor family is not involved in auxin-induced H+-ATPase phosphorylation. Our results define the activation mechanism of H+-ATPase by auxin during early-phase hypocotyl elongation; this is the long-sought-after mechanism that is central to the acid-growth theory. [ABSTRACT FROM AUTHOR]

Phototropins (phot1 and phot2) are autophosphorylating blue-light receptor kinases that mediate blue-light responses such as phototropism, chloroplast accumulation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Only phot2 induces the chloroplast avoidance response under strong blue light. The serine (Ser) residues of the kinase activation loop in phot1 are autophosphorylated by blue light, and autophosphorylation is essential for the phot1-mediated responses. However, the role of autophosphorylation in phot2 remains to be determined. In this study, we substituted the conserved residues of Ser-761 and Ser-763 with alanine (S761A S763A) in the phot2 activation loop and analyzed their function by investigating the phot2-mediated responses after the transformation of phot1 phot2 double mutant with this mutant phot2 gene. Transgenic plants expressing the mutant phot2 protein exhibited impaired responses in chloroplast movement, stomatal opening, phototropic bending, leaf flattening, and plant growth; and those expressing phot2 with S761D S763D mutations showed the normal responses. Substitution of both Ser-761 and Ser-763 with alanine in phot2 did not significantly affect the kinase activity in planta. From these results, we conclude that phosphorylation of Ser-761 and Ser-763 in the activation loop may be a common primary step for phot2-mediated responses. [ABSTRACT FROM AUTHOR]

Phototropins are autophosphorylating proteinkinases of plant-specific blue light receptors. They regulate various blue light responses, including phototropism, chloroplast movements, hypocotyl growth inhibition, leaf flattening, and stomatal opening. However, the physiological role of autophosphorylation remains unknown. Here, we identified phosphorylation sites of Ser or Thr in the N terminus, Hingel region, kinase domain, and C terminus in Arabidopsis phototropini (phot1) by liquid chromatography-tandem mass spectrometry in vivo. We substituted these Ser or Thr residues with Ala in phot1 and analyzed their functions by inspecting the phot1-mediated responses of stomatal opening, phototropism, chloroplast accumulation, and leaf flattening after the transformation of the phot1 phot2 double mutant. Among these sites, we found that autophosphorylation of Ser-851 in the activation loop of the kinase domain was required for the responses mentioned above, whereas the phosphorylation of the other Ser and Thr, except those in the activation loop, was not. Ser-849 in the loop may have an additional role in the responses. Immunological analysis revealed that Ser-851 was phosphorylated rapidly by blue light in a fluence-dependent manner and dephosphorylated gradually upon darkness. We conclude that autophosphorylation of Ser-851 is a primary step that mediates signaling between photo-chemical reaction and physiological events. [ABSTRACT FROM AUTHOR]

Phototropins mediate various blue-light responses such as phototropism, chloroplast relocation, stomatal opening and leaf flattening in plants. Phototropins are hydrophilic chromoproteins that are mainly bound to the plasma membrane. One of two phototropins in Arabidopsis thaliana, phot2, associates with the Golgi apparatus in a light-dependent manner. In this study, we analyzed the biological activities of the N-terminal photosensory and C-terminal kinase domains of phot2. For this purpose, these domains were fused to green fluorescent protein (GFP) and ectopically expressed in the wild-type and a phot1 phot2 double mutant of Arabidopsis. The kinase domain fused to GFP (P2CG) was localized to the plasma membrane and the Golgi apparatus, whereas the photosensory domain fused to GFP (P2NG) was uniformly localized in the cytosol. Hence, the kinase domain rather than the photosensory domain is responsible for the membrane association. Interestingly, the P2CG plants exhibited constitutive blue-light responses even in dark conditions, i.e. stomata were open and chloroplasts were in the avoidance position. By contrast, P2CG with a mutation that abolishes the kinase activity (P2C[D720/N]G) failed to exhibit these responses. phot2 kinase is therefore suggested to be correctly localized to functional sites in the cell and to trigger light signal transduction through its kinase activity. In contrast to P2CG, P2NG did not affect the phot2 responses, except for partial inhibition of the phototropic response caused by the endogenous phototropins. [ABSTRACT FROM AUTHOR]

Stomatal pores, each surrounded by a pair of guard cells, regulate CO2 uptake and water loss from leaves. Stomatal opening is driven by the accumulation of K+ salts and sugars in guard cells, which is mediated by electrogenic proton pumps in the plasma membrane and/or metabolic activity. Opening responses are achieved by coordination of light signaling, light-energy conversion, membrane ion transport, and metabolic activity in guard cells. In this review, we focus on recent progress in blue- and red-light-dependent stomatal opening. Because the blue-light response of stomata appears to be strongly affected by red light, we discuss underlying mechanisms in the interaction between blue-light signaling and guard cell chloroplasts. [ABSTRACT FROM AUTHOR]

Phototropins, plant blue light receptors, mediate stomatal opening through the activation of the plasma membrane H+-ATPase by unknown mechanisms. Here we report that type 1 protein phosphatase (PP1) positively regulates the blue light signaling between phototropins and the H+-ATPase in guard cells of Viola faba. We cloned the four catalytic subunits of PP1 (PP1c) from guard cells and determined the expression of the isoforms in various tissues. Transformation of Viola guard cells with PP1c isoforms that had lost enzymatic activity by one amino acid mutation, or with human inhibitor-2, a specific inhibitor protein of PP1c, suppressed blue light-induced stomatal opening. Addition of fusicoccin, an activator of the plasma membrane H+-ATPase, to these transformed guard cells induced normal stomatal opening, suggesting that the transformations did not affect the basic mechanisms for stomatal opening. Tautomycin, an inhibitor of PP1, inhibited blue light-induced H+ pumping, phosphorylation of the plasma membrane H+-ATPase in guard cell protoplasts, and stomatal opening. However, tautomycin did not inhibit the blue light-dependent phosphorylation of phototropins. We conclude that PP1 functions downstream of phototropins and upstream of the H+-ATPase in the blue light signaling pathway of guard cells. [ABSTRACT FROM AUTHOR]

Recent genetic analysis showed that phototropins (phot1 and phot2) function as blue light receptors in stomatal opening of Arabidopsis thaliana, but no biochemical evidence was provided for this. We prepared a large quantity of guard cell protoplasts from Arabidopsis. The immunological method indicated that phot1 was present in guard cell protoplasts from the wild-type plant and the phot2 mutant, that phot2 was present in those from the wild-type plant and the phot1 mutant, and that neither phot1 nor phot2 was present in those from the phot1 phot2 double mutant. However, the same amounts of plasma membrane H+-ATPase were found in all of these plants. H+ pumping was induced by blue light in isolated guard cell protoplasts from the wild type, from the single mutants of phototropins (phot1-5 and phot2-1), and from the zeaxanthin-less mutant (npq1-2), but not from the phot1 phot2 double mutant. Moreover, increased ATP hydrolysis and the binding of 14–3-3 protein to the H+-ATPase were found in response to blue light in guard cell protoplasts from the wild type, but not from the phot1 phot2 double mutant. These results indicate that phot1 and phot2 mediate blue light-dependent activation of the plasma membrane H+-ATPase and illustrate that Arabidopsis guard cell protoplasts can be useful for biochemical analysis of stomatal functions. We determined isogenes of the plasma membrane H+-ATPase and found the expression of all isogenes of functional plasma membrane H+-ATPases (AHA1-11) in guard cell protoplasts. [ABSTRACT FROM AUTHOR]

Blue light (BL)-dependent H+ pumping by guard cells, which drives stomatal opening, is inhibited by abscisic acid (ABA). We investigated this response with respect to the activity of plasma membrane H+-ATPase using Vicia guard cell protoplasts. ATP hydrolysis by the plasma membrane H+-ATPase, phosphorylation of the H+-ATPase, and the binding of 14-3-3 protein to the H+-ATPase stimulated by BL were inhibited by ABA at 10 µM. All of these responses were similarly inhibited by hydrogen peroxide (H202) at 1 mM. The ABA-induced inhibitions of BL-dependent H+ pumping and phosphorylation of the H+-ATPase were partially restored by ascorbate, an intracellular H2O2 scavenger. A single-cell analysis of the cytosolic H2O2 using 2',7'-dichlorofluorescin revealed that H2O2 was generated by ABA in guard cell protoplasts. We also indicated that H+ pumping induced by fusicoccin and the binding of 14-3-3 protein to the H+-ATPase were inhibited slightly (approximately 20%) by both ABA and H2O2. By contrast, H2O2 at 1 mM did not affect H+ pumping by the H+-ATPase in microsomal membranes. From these results, we concluded that inhibition of BL-dependent H+ pumping by ABA was due to a decrease in the phosphorylation levels of H+-ATPase and that H2O2 might be involved in this response. Moreover, there are at least two inhibition sites by ABA in the BL signaling pathway of guard cells. [ABSTRACT FROM AUTHOR]

Phototropins (phot1 and phot2) are suggested to be multifunctional blue‐light (BL) receptors mediating phototropism, chloroplast movement, stomatal opening, and leaf expansion. The Arabidpsis phot1 phot2 double mutant lacks all of these responses. To confirm the requirement of phototropins in BL responses, the Arabidopsis phot1 phot2 double mutant was transformed with PHOT1 cDNA and the phenotypic restoration was analysed in the transformants. It was found that all BL responses were restored, although differentially, by the transformation of the Arabidopsis phot1 phot2 double mutant with PHOT1 cDNA. The results showed that phot1 was an essential component for all these BL responses in planta, and that the cellular level of phot1 might determine the individual BL responses. [ABSTRACT FROM PUBLISHER]

Phototropins are blue-light (BL) receptor serine (Ser)/threonine kinases, and contain two light, oxygen, and voltage (LOV) domains, and are members of the PAS domain superfamily. They mediate phototropism, chloroplast movement, leaf expansion, and stomatal opening of higher plants in response to BL. In stomatal guard cells, genetic analysis has revealed that phototropins mediate activation of the plasma membrane H[sup +]-ATPase by phosphorylation and drive stomatal opening. However, biochemical evidence for the involvement of phototropins in the BL response of stomata is lacking. Using guard cell protoplasts, we showed that broad bean (Vicia faba) phototropins (Vfphots) were phosphorylated by BL, and that this phosphorylation of Vfphots reached to the maximum level earlier than that of the H[sup +]-ATPase. Phosphorylation of both Vfphots and H[sup +]-ATPase showed similar sensitivity to BL and were similarly suppressed by protein kinase and flavoprotein inhibitors. We found that a 14-3-3 protein was bound to Vfphots upon phosphorylation, and this binding occurred earlier than the H[sup +]-ATPase phosphorylation. Vfphots (Vfphot1a and Vfphot1b) were expressed in Escherichia coli, and phosphorylation sites were determined to be Ser-358 for Vfphot1a and Ser-344 for Vfphot1b, which are localized between LOV1 and LOV2. We conclude that Vfphots act as BL receptors in guard cells and that phosphorylation of a Ser residue between LOV1 and LOV2 and subsequent 14-3-3 protein binding are likely to be key steps of BL response in stomata. The binding of a 14-3-3 protein to Vfphot was found in etiolated seedlings and leaves in response to BL, suggesting that this event was common to phototropin-mediated responses. [ABSTRACT FROM AUTHOR]

Subfractionation studies showed that cytochrome b5 (cyt b5), which has been considered to be a typical ER protein, was localized in both the endoplasmic reticulum membrane (ER) and the outer membrane of mitochondria in cauliflower (Brassica olracea) cells and was a component of antimycin A–insensitive NADH–cytochrome c reductase system in both membranes. When cDNA for cauliflower cyt b5 was introduced into mammalian (COS-7) and yeast cells as well as into onion cells, the expressed cytochrome was localized both in the ER and mitochondria in those cells. On the other hand, rat and yeast cyt b5s were specifically localized in the ER membranes even in the onion cells. Mutation experiments showed that cauliflower cyt b5 carries information that targets it to the ER and mitochondria within the carboxy-terminal 10 amino acids, as in the case of rat and yeast cyt b5s, and that replacement of basic amino acids in this region of cauliflower cyt b5 with neutral or acidic ones resulted in its distribution only in the ER. Together with the established findings of the importance of basic amino acids in mitochondrial targeting signals, these results suggest that charged amino acids in the carboxy-terminal portion of cyt b5 determine its location in the cell, and that the same mechanism of signal recognition and of protein transport to organelles works in mammalian, plant, and yeast cells. [ABSTRACT FROM AUTHOR]

Shows that abscisic-acid-activated protein kinase (AAPK)-interacting protein 1 (AKIP1), with sequence homology to heterogeneous nuclear RNA-binding protein A/B, is a substrate of AAPK. AAPK-dependent phosphorylation for the interaction of AKIP1 with messenger RNA that encodes dehydrin; Partitioning of AKIP1 into subnuclear foci; Interaction cloning and yeast two-hybrid analysis.

The author comments on the stomatal functions of plants. The authors mention that despite the inability of plants to move from the stress of their environments, they can they respond to the negative influences with their stomata. Moreover, they said that plants can boost their tolerance stress level when they encountered biotic stress and this process is known as hysteresis.

Plasma membrane H+-ATPases (PMAs) provide an H+ electrochemical gradient to establish a proton motive force and membrane potential for the transport of various nutrients across the plasma membrane. PMAs are regulated at multiple levels, and genetic modulation of PMA expression could enhance nutrient acquisition and use efficiency in plants. PMAs are involved in the coordination of the uptake and assimilation of both nitrate and ammonium. PMAs are involved in nutrient utilization by regulating root growth, facilitating organic anion exudation, and promoting the acquisition of nutrients through H+ extrusion-mediated apoplast and rhizosphere acidification. PMAs play essential roles in mycorrhizal symbioses in plant roots for nutrient acquisition. 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. [ABSTRACT FROM AUTHOR]

Synthetic modulators of plant 14‐3‐3s are promising chemical tools both for understanding the 14‐3‐3‐related signaling pathways and controlling plant physiology. Herein, we describe a novel small‐molecule inhibitor for 14‐3‐3 proteins of Arabidopsis thaliana. The inhibitor was identified from unexpected products in a stock solution in dimethyl sulfoxide (DMSO) of an in‐house chemical library. Mass spectroscopy, mutant‐based analyses, fluorescence polarization assays, and thermal shift assays revealed 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 new insight into the design of potent and isoform‐selective 14‐3‐3 modulators. [ABSTRACT FROM AUTHOR]

[Display omitted] • Potent ABA agonists developed via structure-guided designs aided by virtual screens. • Unbiased chemical screen identified ABA-independent anti-transpirant compounds. • Chemical compounds disrupting cell polarity during stomatal development identified. • Adverse side effects of a hit compound may be removed via derivatization. Stomata control trade-offs for plants: carbon dioxide uptake for photosynthetic growth and water loss via transpiration. While agrochemical control of transpiration is an old concept, recent discoveries of the core signaling components controlling stomatal function and numbers opened the door to develop chemical compounds with high potency and specificity. ABA agonists with potent anti-transpiration activity have been developed via in silico virtual screens and structure guided design and synthesis. Library-based chemical screens identified new compounds that influence stomatal movement in ABA-independent manners as well as those affecting stomatal numbers and division polarity. Subsequent hit compound derivatization can be employed to separate adverse side effects. Ultimately, such chemicals might help in optimizing plant productivity and water use in agriculture and florist industries. [ABSTRACT FROM AUTHOR]

The effect of immunosuppressant cyclosporin A (CsA) on inward-rectifying K+-channels and biochemical analysis have indicated the presence of cyclophilin in guard cells of Vicia faba. In this study, we identified a full-length cDNA sequence, vcCyP, encoding cyclophilin (CyP), a peptidyl-prolyl cis-trans isomerase of guard cell protoplasts (GCPs) from Vicia faba L. The deduced amino acid sequence revealed that vcCyP contained 171 amino acid residues and exhibited a strong similarity to previously described cytosolic CyP isoforms from other plants. vcCyP had seven extra amino acid residues, which is a characteristic of the cytosolic form of plant CyPs. A complex of recombinant vcCyP and CsA inhibited the phosphatase activity of bovine calcineurin, a type 2B protein phosphatase, with a half-inhibitory concentration of 0.2 μM. Protein phosphatase activity was measured in the cytosolic fraction of GCPs using a 32P-labeled myelin basic protein (32P-MBP) and the activity was increased by a physiological concentration of Ca2+ (1 μM). This Ca2+-stimulated phosphatase activity was inhibited by CsA, suggesting the presence of both cytosolic CyP and calcineurin-like protein phosphatase in guard cells. Northern blot analysis showed that the transcription level of vcCyP was much higher in GCPs than in root and leaf tissues of Vicia. [ABSTRACT FROM AUTHOR]

The opening of stomata, which is driven by the accumulation of K+ salt in guard cells, is induced by blue light (BL). The BL activates the H+ pump; however; the mechanism by which the perception of BL is transduced into the pump activation remains unknown. We present evidence that the pump is the plasma membrane H+-ATPase and that BL activates the H+-ATPase via phosphorylation. A pulse of BL (30 s, 100 µmol/m2/s) increased ATP hydrolysis by the plasma membrane H+-ATPase and H+ pumping in Vicia guard cell protoplasts with a similar time course. The H+-ATPase was phosphorylated reversibly by BL, and the phosphorylation levels paralleled the ATP hydrolytic activity. The phosphorylation occurred exclusively in the C-termini of H+-ATPases on both serine and threonine residues in two isoproteins of H +-ATPase in guard cells. An endogenous 14-3-3 protein was co-precipitated with H+-ATPase, and the recombinant 14-3-3 protein bound to the phosphorylated C-termini of H+-ATPases. These findings demonstrate that BL activates the plasma membrane H+-ATPase via phosphorylation of the C-terminus by a serine/threonine protein kinase, and that the 14-3-3 protein has a key role in the activation. [ABSTRACT FROM AUTHOR]

Calyculin A (CA) and okadaic acid (OA), inhibitors of protein phosphatases, inhibited blue light (BL)-dependent H+pumping in Vicia guard cell protoplasts at half-inhibitory concentrations of 4.5 nM and 400 nM, respectively. Light-induced stomatal opening in Viciaepidermis was completely suppressed by CA at 100 nM and by OA at 1 μM. These results suggest that CA- and OA-sensitive protein phosphatase is involved in the BL response of stomatal guard cells. [ABSTRACT FROM AUTHOR]

Stomatal movements are regulated by many environmental signals, such as light, CO2, temperature, humidity, and drought. Recently, we showed that photoperiodic flowering components have positive effects on light-induced stomatal opening in Arabidopsis thaliana. In this study, we determined that light-induced stomatal opening and increased stomatal conductance were larger in plants grown under long-day (LD) conditions than in those grown under short-day (SD) conditions. Gene expression analyses using purified guard cell protoplasts revealed that FT and SOC1 expression levels were significantly increased under LD conditions. Interestingly, the enhancement of light-induced stomatal opening and increased SOC1 expression in guard cells due to LD conditions persisted for at least 1 week after plants were transferred to SD conditions. We then investigated histone modification using chromatin immunoprecipitation–PCR, and observed increased trimethylation of lysine 4 on histone 3 (H3K4) around SOC1. We also found that LD-dependent enhancement of light-induced stomatal opening and H3K4 trimethylation in SOC1 were suppressed in the ft-2 mutant. These results indicate that photoperiod is an important environmental cue regulating stomatal opening, and that LD conditions enhance light-induced stomatal opening and epigenetic modification (H3K4 trimethylation) around SOC1, a positive regulator of stomatal opening, in an FT-dependent manner. Thus, this study provides novel insights into stomatal responses to photoperiod. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The author discusses the phytohormone abscisic acid (ABA) that regulates various developmental processes in a variety of plays including crops and mentions that the ABA has a major role in adaptive responses to abiotic stress such as high salt conditions, cold temperatures and drought. The author further reflects on a study that designed an antagonist of ABA signaling, which is expected to provide a useful tool for manipulating ABA responses in plants.

SUMMARY: 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.] Significance Statement: Our study reveals that phosphatidylethanolamine (PE) levels are elevated in guard cells, and that reduction in PE levels affect stomatal movement and response to changes in CO2 concentration. Defects in PE synthesis reflect altered stomatal responses, indicating that PE is essential for the regulation of stomatal movement. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

The circadian clock, an internal time-keeping system with a period of about 24 h, coordinates many physiological processes with the day–night cycle. We previously demonstrated that BML-259 [ N -(5-isopropyl-2-thiazolyl) phenylacetamide], a small molecule with mammal CYCLIN DEPENDENT KINASE 5 (CDK5)/CDK2 inhibition activity, lengthens Arabidopsis thaliana (Arabidopsis) circadian clock periods. BML-259 inhibits Arabidopsis CDKC kinase, which phosphorylates RNA polymerase II in the general transcriptional machinery. To accelerate our understanding of the inhibitory mechanism of BML-259 on CDKC, we performed structure–function studies of BML-259 using circadian period-lengthening activity as an estimation of CDKC inhibitor activity in vivo. The presence of a thiazole ring is essential for period-lengthening activity, whereas acetamide, isopropyl and phenyl groups can be modified without effect. BML-259 analog TT-539, a known mammal CDK5 inhibitor, did not lengthen the period nor did it inhibit Pol II phosphorylation. TT-361, an analog having a thiophenyl ring instead of a phenyl ring, possesses stronger period-lengthening activity and CDKC;2 inhibitory activity than BML-259. In silico ensemble docking calculations using Arabidopsis CDKC;2 obtained by a homology modeling indicated that the different binding conformations between these molecules and CDKC;2 explain the divergent activities of TT539 and TT361. [ABSTRACT FROM AUTHOR]

The circadian clock is an internal timekeeping system that governs about 24 h biological rhythms of a broad range of developmental and metabolic activities. The clocks in eukaryotes are thought to rely on lineage-specific transcriptional–translational feedback loops. However, the mechanisms underlying the basic transcriptional regulation events for clock function have not yet been fully explored. Here, through a combination of chemical biology and genetic approaches, we demonstrate that phosphorylation of RNA polymerase II by CYCLIN DEPENDENT KINASE C; 2 (CDKC;2) is required for maintaining the circadian period in Arabidopsis. Chemical screening identified BML-259, the inhibitor of mammalian CDK2/CDK5, as a compound lengthening the circadian period of Arabidopsis. Short-term BML-259 treatment resulted in decreased expression of most clock-associated genes. Development of a chemical probe followed by affinity proteomics revealed that BML-259 binds to CDKC;2. Loss-of-function mutations of cdkc;2 caused a long period phenotype. In vitro experiments demonstrated that the CDKC;2 immunocomplex phosphorylates the C-terminal domain of RNA polymerase II, and BML-259 inhibits this phosphorylation. Collectively, this study suggests that transcriptional activity maintained by CDKC;2 is required for proper period length, which is an essential feature of the circadian clock in Arabidopsis. [ABSTRACT FROM AUTHOR]