Study on the Relationship Between the Ploidy Level of Microspore-Derived Plants and the Number of Chloroplast in Stomatal Guard Cells in Brassica oleracea

The relationship between the ploidy level of microspore-derived plants and chloroplast number in stomatal guard cells was studied in cabbage, broccoli, and Chinese kale. In the experiment, distribution statistics analysis and t-test were used to perform statistical analysis on chloroplast number of different ploidy level in those stomatal guard cells mentioned above, and morphology identifying and chromosome counting were used to test accuracy of counting chloroplast number in stomatal guard cells. The chloroplast average number in stomatal guard cells was very similar among the different leaf positions on the same plant and among significantly among the different ploidy the different locations in the same stoma in the same variety. All the leaf, while the chloroplast number varied distributions of the chloroplast number in different ploidy stoma were normal distribution fitted. A correlation has been established between ploidy and chloroplast number in the stomatal guard cells. In every single stoma of microspore-derived plants, the chloroplast number for a haploid should not be more than 10, diploids 11 to 15, and polyploids more than 15. The accuracy of this method for identification of different ploidy plants was 93.93%. Furthermore, the accuracy of this method was reliable and did not vary with the plants growth conditions. Therefore, the chromosome ploidy of plants derived from microspore culture in cabbage, broccoli, and Chinese kale can be identified by simply counting the chloroplast number in stomatal guard cells.

Effects of Exterior Abscisic Acid on Calcium Distribution of Mesophyll Cells and Calcium Concentration of Guard Cells in Maize Seedlings

In this study, the direct effects of exterior abscisic acid （ABA） on both calcium distribution of mesophyll cells and cytosolic calcium concentration of guard cells were examined. The distribution of Ca^2＋ localization were observed with calcium antimonate precipitate-electromicroscopic-cyto-chemical methods after treated with ABA and pretreated with ethylene glycol-bis-（2-aminoethylether）-N,N,N＇,N＇-tetraacetic acid （EGTA）, verapamil （Vp）, and trifluoperazine （TFP）. The laser scanning confocal microscopy was used to measure the cytosolic calcium concentrations of guard cells under different treatments. The results showed that the cytosolic Ca^2＋ concentration of mesophyll ceils was induced to increase by ABA, but to decrease in both outside cell and the vacuoles within 10 rain after treatments. The cytosolic calcium concentration of guard cells was increased gradually with the lag in treatment time. However, both EGTA and TFP could inverse those effects, indicating that the increase of cytosolic calcium induced by exterior ABA was mainly caused by calcium influx. The results also showed that calmodulin could influence both the calcium distribution of mesophyll cells and calcium concentration of guard cells. It shows that calmodulin participates in the process of ABA signal transduction, but the mechanism is not known as yet. The changes both calcium distribution of mesophyll cells and calcium concentration of guard cells further proved that the variations of cytosolic Ca^2＋ concentration induced by ABA were involved in the stomatal movements of maize seedlings.

Hydrogen peroxide-induced changes in intracellular pH of guard cells precede stomatal closure

Epidermal bioassay demonstrated that benzylamine, a membrane-permeable weak base, can mimick hydrogen peroxide (H2O2) to induce stomatal closure, and butyric acid, a membrane-permeable weak acid, can partly abolish the H2O2-induced stomatal closure. Confocal pH mapping with the probe 5-(and-6)- carboxy seminaphthorhodafluor- 1 - acetoxymethylester (SNARF-1-AM) revealed that H2O2 leads to rapid changes in cytoplasmic and vacuolar pH in guard cells of Viola faba L, i. e. alkalinization of cytoplasmic areas occur red in parallel with a decrease of the vacuolar pH, and that butyric acid pretreatment can abolish alkalinization of cytoplasmic areas and acidification of vacuolar areas of guard cells challenged with H2O2. These results imply that the alkalinization of cytoplasm via efflux of cytosol protons into the vacuole in guard cells challenged with H2O2 is important at an early stage in the signal cascade leading to stomatal closure.

K^+channels inhibited by hydrogen peroxide mediate abscisic acid signaling in Vicia guard cells

A number of studies show that environmental stress conditions increase abscisic acid (ABA) and hydrogen peroxide (H2O2) levels in plant cells. Despite this central role of ABA in altering stomatal aperture by regulating guard cell ion transport, little is known concerning the relationship between ABA and H2O2 in signal transduction leading to stomatal movement. Epidermal strip bioassay illustrated that ABA- inhibited stomatal opening and ABA-induced stomatal closure were abolished partly by externally added catalase (CAT) or diphenylene iodonium (DPl), which are a H2O2 scavenger and a NADPH oxidase inhibitor respectively. In contrast, internally added CAT or DPI nearly completely or partly reversed ABA-induced closure in half-stoma. Consistent with these results, whole-cell patch-clamp analysis showed that intracellular application of CAT or DPI partly abolished ABA-inhibited inward K+ current across the plasma membrane of guard cells. H2O2 mimicked ABA to inhibit inward K+ current, an effect which was reversed by the addition of ascorbic acid (Vc) in patch clamping micropipettes. These results suggested that H2O2 mediated ABA-induced stomatal movement by targeting inward K+ channels at plasma membrane.

Cell type-specific proteomics uncovers a RAF15-SnRK2.6/OST1 kinase cascade in guard cells

Multicellular organisms such as plants contain various cell types with specialized functions.Analyzing the characteristics of each cell type reveals specific cell functions and enhances our understanding of organization and function at the organismal level.Guard cells(GCs)are specialized epidermal cells that regulate the movement of the stomata and gaseous exchange,and provide a model genetic system for analyzing cell fate,signaling,and function.Several proteomics analyses of GC are available,but these are limited in depth.Here we used enzymatic isolation and flow cytometry to enrich GC and mesophyll cell protoplasts and perform in-depth proteomics in these two major cell types in Arabidopsis leaves.We identified approximately 3,000 proteins not previously found in the GC proteome and more than600 proteins that may be specific to GC.The depth of our proteomics enabled us to uncover a guard cell-specific kinase cascade whereby Raf15and Snf1-related kinase2.6(SnRK2.6)/OST1(open stomata 1)mediate abscisic acid(ABA)-induced stomatal closure.RAF15 directly phosphorylated SnRK2.6/OST1 at the conserved Ser175 residue in its activation loop and was sufficient to reactivate the inactive form of SnRK2.6/OST1.ABAtriggered SnRK2.6/OST1 activation and stomatal closure was impaired in raf15 mutants.We also showed enrichment of enzymes and flavone metabolism in GC,and consistent,dramatic accumulation of flavone metabolites.Our study answers the long-standing question of how ABA activates SnRK2.6/OST1 in GCs and represents a resource potentially providing further insights into the molecular basis of GC and mesophyll cell development,metabolism,structure,and function.

Carbon Monoxide-induced Stomatal Closure Involves Generation of Hydrogen Peroxide in Vicia faba Guard Cells

Here the regulatory role of CO during stomatal movement in Vicia faba L. was surveyed. Results indicated that, like hydrogen peroxide （H2O2）, CO donor HemaUn induced stomatal closure in dose- and time-dependent manners. These responses were also proven by the addition of gaseous CO aqueous solution with different concentrations, showing the first time that CO and H2O2 exhibit the similar regulation role in the stomatal movement. Moreover, our data showed that ascorbic acid （ASA, an important reducing substrate for H2O2 removal） and diphenylene iodonium （DPI, an inhibitor of the H2O2-generating enzyme NADPH oxidase） not only reversed stomatal closure by CO, but also suppressed the H2O2 fluorescence induced by CO, implying that CO induced-stomatal closure probably involves H2O2 signal. Additionally, the CO/NO scavenger hemoglobin （Hb） and CO specific synthetic inhibitor ZnPPIX, ASA and DPI reversed the darkness-induced stomatal closure and H2O2 fluorescence. These results show that, perhaps like H2O2, the levels of CO in guard cells of V. faba are higher in the dark than in light, HO-1 and NADPH oxidase are the enzyme systems responsible for generating endogenous CO and H2O2 in darkness respectively, and that CO is involved in darkness-induced H2O2 synthesis in V. faba guard cells.

NRGA1, a Putative Mitochondrial Pyruvate Carrier, Mediates ABA Regulation of Guard Cell Ion Channels and Drought Stress Responses in Arabidopsis

Abscisic acid （ABA） regulates ion channel activity and stomatal movements in response to drought and other stresses. Here, we show that the Arabidopsis thaliana gene NRGA1 is a putative mitochondrial pyruvate carrier which negatively regulates ABA-induced guard cell signaling. NRGA1 transcript was abundant in the A. thaliana leaf and par- ticularly in the guard cells, and its product was directed to the mitochondria. The heterologous co-expression of NRGA1 and AtMPC1 in yeast complemented a loss-of-function mitochondrial pyruvate carrier （MPC） mutant. The nrgal loss-of- function mutant was very sensitive to the presence of ABA in the context of stomatal movements, and exhibited a height- ened tolerance to drought stress. Disruption of NRGA1 gene resulted in increased ABA inhibition of inward K＋ currents and ABA activation of slow anion currents in guard cells. The nrgal/NRGA1 functional complementation lines restored the mutant＇s phenotypes. Furthermore, transgenic lines of constitutively overexpressing NRGA1 showed opposite stomatal responses, reduced drought tolerance, and ABA sensitivity of guard cell inward K＋ channel inhibition and anion channel activation. Our findings highlight a putative role for the mitochondrial pyruvate carrier in guard cell ABA signaling in response to drought.

Modulation of Guard Cell Turgor and Drought Folerance by a Peroxisomal Acetate-Malate Shunt

In plants, stomatal movements are tightly controlled by changes in cellular turgor pressure. Carbohydrates produced by glycolysis and the tricarboxylic acid cycle play an important role in regulating turgor pressure. Here, we describe anArabidopsis mutant, bzul, isolated in a screen for elevated leaf temperature in response to drought stress, which displays smaller stomatal pores and higher drought resistance than wild-type plants. BZU1 encodes a known acetyl-coenzyme A synthetase, ACN1, which acts in the first step of a metabolic pathway converting acetate to malate in peroxisomes. We showed that BZUl/ACNl-mediated acetate-to-malate conversion provides a shunt that plays an important role in osmoregulation of stomatal turgor. We found that the smaller stomatal pores in the bzul mutant are a consequence of reduced accumu- lation of malate, which acts as an osmoticum and/or a signaling molecule in the control of turgor pressure within guard cells, and these results provided new genetic evidence for malate-regulated stomatal movement. Collectively, our results indicate that a peroxisomal BZUl/ACNl-mediated acetate--malate shunt regulates drought resistance by controlling the turgor pressure of guard cells in Arabidopsis.

Direct Modulation of the Guard Cell Outward- Rectifying Potassium Channel （CORK） by Abscisic Acid

Dear Editor,Abscisic acid （ABA） induces turgot loss and hence stomatal closure by promoting rapid net K^＋ efflux from guard cells （GCs） through outward-rectifying K^＋ （K^＋out） channels （Schroeder et al., 1987; Blatt, 1990）. The mechanisms of ABA signaling in GCs are detailed elsewhere （see Munemasa et al., 2015; Weiner et al., 2010; Pandey et al., 2007）. Briefly, ABA binds to the PYR/ PYL/RCARs, a family of soluble steroidogenic acute regulatory- related lipid transfer （START） proteins, and, in turn, inactivates the downstream PP2C （type 2C protein phosphatase）, leading to the activation of SnRK2.6 （SNF1 [sucrose non-fermenting-1- related protein kinase]/OST1 [open stomata 1]） protein kinases.

Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

Free cytosolic Ca^2＋ （[Ca^2＋]cyt） is an ubiquitous second messenger in plant cell signaling, and [Ca^2＋]cyt elevation is associated with Ca^2＋-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca^2＋ channels and their regulation remains limited in planta. A type of voltage- dependent Ca^2＋-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba^2＋ and Ca^2＋, and their activities can be inhibited by micromolar Gd^3＋. The unitary conductance and the reversal potential of the channels depend on the Ca^2＋ or Ba^2＋ gradients across the plasma membrane. The inward whole-cell Ca^2＋ （Ba^2＋） current, as well as the unitary current amplitude and NPo of the single Ca^2＋ channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NPo of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.

Interplay between hydrogen sulfide and other signaling molecules in the regulation of guard cell signaling and abiotic/biotic stress response

Stomatal aperture controls the balance between transpirational water loss and photosynthetic carbon dioxide(CO2)uptake.Stomata are surrounded by pairs of guard cells that sense and transduce environmental or stress signals to induce diverse endogenous responses for adaptation to environmental changes.In a recent decade,hydrogen sulfide(H2S)has been recognized as a signaling molecule that regulates stomatal movement.In this review,we summarize recent progress in research on the regulatory role of H2S in stomatal movement,including the dynamic regulation of phytohormones,ion homeostasis,and cell structural components.We focus especially on the cross talk among H2S,nitric oxide(NO),and hydrogen peroxide(H2O2)in guard cells,as well as on H2S-mediated post-translational protein modification(cysteine thiol persulfidation).Finally,we summarize the mechanisms by which H2S interacts with other signaling molecules in plants under abiotic or biotic stress.Based on evidence and clues from existing research,we propose some issues that need to be addressed in the future.

Current Injection Provokes Rapid Expansion of the Guard Cell Cytosolic Volume and Triggers Ca^2＋ Signals

High-resolution microscopy opens the door for detailed single-cell studies with fluorescent reporter dyes and proteins. We used a confocal spinning disc microscope to monitor fluorescent dyes and the fluorescent protein Venus in tobacco and Arabidopsis guard cells. Multi-barreled microelectrodes were used to inject dyes and apply voltage pulses, which provoke transient rises in the cytosolic Ca^2＋ level. Voltage pulses also caused changes in the distribution of Lucifer Yellow and Venus, which pointed to a reversible increase of guard cell cytosolic volume. The dynamic cytosolic volume changes turned out to be provoked by current injection of ions. A reduction of the clamp current, by blocking K^＋ uptake channels with Cs^＋, strongly suppressed the cytosolic volume changes. Cs^＋ not only inhibited the expansion of the cytosol, but also inhibited hyperpolarization-induced elevations of the cytosolic Ca^2＋ concentration. A complete loss of voltage-induced Ca^2＋ signals occurred when Ca^2＋-permeable plasma membrane channels were simultaneously blocked with La^3＋. This shows that two mechanisms cause hyperpolarization-induced elevation of the cytosolic Ca^2＋-concentration： （i） activation of voltage-dependent Ca^2＋-permeable channels, （ii） osmotically induced expansion of the cytosol, which leads to a release of Ca^2＋ from intracellular stores.

MAP kinase specifically mediates the ABA-induced H_2O_2 generation in guard cells of Vicia faba L.

Mitogen-activated protein (MAP) kinase is involved in ABA- or H2O2-signaling, and H2O2 acts as inter-mediate in the downstream of ABA signal transduction pathway, which has recently emerged as a secondary mes-senger of ABA signaling. Using an epidermal strip bioassay and laser scanning confocal microscope, we provided the first evidence that MAP kinase plays an important role in H2O2 signal initial, amplification and specific targeting in response to stimuli in guard cells. ABA- or H2O2-induced Vicia faba stomatal closure was inhibited or reversed by the specific inhibitor PD98059 of MEK1/2; the guard cells were pre-incubated or -microinjected by 10 mmol·L-1 PD98059, ABA could not enhance the fluorescence intensity of H2O2 probe dichlorofluorescein (DCF). Meanwhile, after ABA induced the H2O2 accumulation in guard cells, the exogenous or intracellular PD98059 could reduce the DCF fluorescence intensity. Most interestingly, on the contrary to ABA, the DCF fluorescence intensity of guard cells treated by 100 mmol·L-1 salicylic acid (SA) was not down-regulated by PD98059, yet PD98059 did not regulate the stomatal move-ment being induced by light, dark or salicylic acid. These results suggest that MEK1/2 could mediate stomatal closure by abolishing the ABA-induced H2O2 generation/accumula- tion in the specific manner.

ATHK1 acts downstream of hydrogen peroxide to mediate ABA signaling through regulation of calcium channel activity in Arabidopsis guard cells

Plants gradually develop their ability to tolerate environmental water deficit as part of the evolutionary process.Abscisic acid(ABA) plays a critical role during drought and osmotic stress.Several histidine protein kinases are regarded as osmotic sensors or regulators in the adaptive response of plants to water deficit.In this study,we report that ATHK1,which was previously shown to function as an osmotic regulator,is involved in ABA-induced stomatal signaling in Arabidopsis.Mutants null for ATHK1 expression were unable to transmit normal ABA responses in guard cells,including inducing stomatal closure,producing hydrogen peroxide and activating calcium influx.Moreover,patch clamp and confocal analysis demonstrated that ATHK1 may function downstream of hydrogen peroxide in ABA-induced stomatal closure,by regulating calcium channel activity and calcium oscillation in Arabidopsis guard cells.

Effects of La^(3+) and Eu^(3+) on outward potassium channels in Vicia guard cells

Effects of La3+ and Eu3+ on outward potassium channels(K+out) in Vicia guard cells have been studied by patch clamping technique.Extracellular La3+ inhibited K+out currents with a half-inhibition concentration(IC50) of 81 μmol·L-1.Interestingly,intracellular La3+ activated K+out currents at a free concentration of 1.13 × 10-14 mol·L-1,and inhibited K+out currents at a free concentration of 5.86 × 10-14 mol·L-1.Extracellular Eu3+ also activated K+out currents at concentrations of 10 μmol·L-1 and 50 μmol·L-1,and inhibited K+out currents at concentrations of more than 1 mmol·L-1.The effects of La3+ and Eu3+ on K+out currents may contribute to regulation of the plant water status,which may be one of the mechanisms of the biological effect of rare earth elements.

Effects of La^3+ on inward K^+ channels at plasma membrane in guard cells

The effects of La3+ on inward K+ channels at plasma membrane in vicia guard cells are investigated using the whole-cell patch-clamp recording mode. It is shown that La3+ on both sides of plasma membrane blocks inward K+ currents in a concentration- dependent manner, indicating that La3+ binding sites may exist on both sides of plasma membrane in guard cells in vicia. The dose response is fitted by the Michaelis-Menten relation characterized by an inhibitor constant Ki of 2.56±0.25 μmol·L?1 (outside membrane) and (1.18±0.11)×10?15 mol·L?1 (inside membrane). Intracellular La3+ has much stronger inhibitory effect on inward K+ currents than ex- tracellular La3+ does, suggesting there may exist stronger binding sites inside membrane than outside membrane. Since ion channel activities of guard cells directly affect plant stomatal movement and water status, our results imply that rare earth elements might have potential practical values in regulating plant water status and strengthening plant drought endurance.

Localization of muscarinic acetylcholine receptor in plant guard cells

Acetylcholine (ACh), as an important neuro-transmitter in animals, also plays a significant role in various kinds of physiological functions in plants. But relatively little is known about its receptors in plants. A green fluorescence BODIPY FL-labeled ABT, which is a high affinity ligand of muscarinic acetylcholine receptor (mAChR), was used to localize mAChR in plant guard cells. In Vicia faba L. and Pisum sativum L., mAChR was found both on the plasma membrane of guard cells. mAChR may also be distributed on guard cell chloroplast membrane of Vicia faba L. The evidence that mAChR localizes in the guard cells provides a new possible signal transduction pathway in ACh mediated stomata movement.

DGP1,a drought-induced guard cell-specific promoter and its function analysis in tobacco plants

The genetic regulation of stomatal movement mainly depends on an efficient control system of gene expression, and guard cell-specific promoter is becoming the best choice. Here we combined the dehydration responsive element (DRE) with guard cell specific element (GCSE) to construct a novel promoter, DGP1. Histochemical assays in transgenic tobacco carrying β -glucuronidase (gus) gene fused to DGP1 demonstrated that GUS activity was found to be highly inducible by drought treatment and specifically restricted to guard cells. No GUS activity was detected in roots, stems or flowers after treatment. Further quantitative analysis showed that GUS activity in the epidermal strips was apparently induced by dehydration and dramatically increased with the elongation of treatment. The GUS activity after 8 h treatment was 179 times that of those without treatment. Although GUS activity in roots, stems or mesophyll increased after treatment, no great changes were observed. These results suggested that DGP1 could drive target gene expressed in guard cells when plant is subjected to drought stress. And this gets us prepared to control opening and closing of stomata through plant gene engineering.

Regulating role of acetylcholine and its antagonists in inward rectified K^+ channels from guard cell protoplasts of Vicia faba

The inward rectified potassium current of Vicia faba guard cell protoplasts treated with acetylcholine (ACh) or the antagonists of its receptors were recorded by employing the patch clamp technique. The results show that ACh at lower concentrations increases the inward K+ current, in contrast, ACh at higher concentrations inhibits it. Treated with d-Tubocurarine (d-Tub), an antagonist of the nicotine ACh receptor (nAChR) inhibits the inward K+ current by 30%. Treated with atropine (Atr), an antagonist of the muscarine (Mus) ACh receptor (mAChR) also inhibits it by 36%. However, if guard cell protoplasts are treated with d-Tub and Atr together, the inward K+ current is inhibited by 60%-75%. Tetraethylammonium chloride (TEA), a strong inhibitor of K+ channels has no effect on the inward K+ current regulated by ACh, suggesting that there are inward K+ channels modulated by AChRs on the membrane of the guard cell protoplasts. These data demonstrate an ACh-regulated mechanism for stomatal movement.

pH induced elastic modulus of guard cell wall in stomatal movement

Guard cell wall properties are important in stomatal movement. Previous research focused on the structure and anatomy of guard cell walls, but little is known about the physical changes that take place within the walls during stomatal opening and closure. In this work, we investigate the volumetric elastic modulus (ε) of the guard cell wall at different pH values during stomatal opening in Vicia faba epidermal strips using a cell pressure probe. The volumetric elastic modulus of the guard cell wall decreased from 7.098 to 5.690 MPa when the pH of the cell wall decreased from 8.60 to 6.50. It is easier for guard cells to change their volume at a lower volumetric elastic modulus during stomatal movement. The results are suggestive of a putative role for pH in the regulation of stomatal movement.