Mohammad Anowar Hossain

The Arabidopsis Calcium Dependent Protein Kinase, CPK6, functions as a Positive Regulator of Methyl Jasmonate Signaling in Guard Cells.

Previous studies have demonstrated that methyl jasmonate (MeJA) induces stomatal closure dependent on change of cytosolic free calcium concentration in guard cells. However, these molecular mechanisms of intracellular Ca2+ signal perception remain unknown. Calcium-dependent protein kinases (CDPKs) function as Ca2+ signal transducers in various plant physiological processes. It has been reported that four Arabidopsis (Arabidopsis thaliana) CDPKs, CPK3, CPK6, CPK4, and CPK11, are involved in abscisic acid signaling in guard cells. It is also known that there is an interaction between MeJA and abscisic acid signaling in guard cells. In this study, we examined the roles of these CDPKs in MeJA signaling in guard cells using Arabidopsis mutants disrupted in the CDPK genes. Disruption of the CPK6 gene impaired MeJA-induced stomatal closure, but disruption of the other CDPK genes did not. Despite the broad expression pattern of CPK6, we did not find other remarkable MeJA-insensitive phenotypes in the cpk6-1 mutant. The whole-cell patch-clamp analysis revealed that MeJA activation of nonselective Ca2+-permeable cation channels is impaired in the cpk6-1 mutant. Consistent with this result, MeJA-induced transient cytosolic free calcium concentration increments were reduced in the cpk6-1 mutant. MeJA failed to activate slow-type anion channels in the cpk6-1 guard cells. Production of early signal components, reactive oxygen species and nitric oxide, in guard cells was elicited by MeJA in the cpk6-1 mutant as in the wild type. These results provide genetic evidence that CPK6 has a different role from CPK3 and functions as a positive regulator of MeJA signaling in Arabidopsis guard cells.

Involvement of Endogenous Abscisic Acid in Methyl Jasmonate-Induced Stomatal Closure in Arabidopsis

In this study, we examined the involvement of endogenous abscisic acid (ABA) in methyl jasmonate (MeJA)-induced stomatal closure using an inhibitor of ABA biosynthesis, fluridon (FLU), and an ABA-deficient Arabidopsis (Arabidopsis thaliana) mutant, aba2-2. We found that pretreatment with FLU inhibited MeJA-induced stomatal closure but not ABA-induced stomatal closure in wild-type plants. The aba2-2 mutation impaired MeJA-induced stomatal closure but not ABA-induced stomatal closure. We also investigated the effects of FLU and the aba2-2 mutation on cytosolic free calcium concentration ([Ca2+]cyt) in guard cells using a Ca2+-reporter fluorescent protein, Yellow Cameleon 3.6. In wild-type guard cells, FLU inhibited MeJA-induced [Ca2+]cyt elevation but not ABA-induced [Ca2+]cyt elevation. The aba2-2 mutation did not affect ABA-elicited [Ca2+]cyt elevation but suppressed MeJA-induced [Ca2+]cyt elevation. We also tested the effects of the aba2-2 mutation and FLU on the expression of MeJA-inducible VEGETATIVE STORAGE PROTEIN1 (VSP1). In the aba2-2 mutant, MeJA did not induce VSP1 expression. In wild-type leaves, FLU inhibited MeJA-induced VSP1 expression. Pretreatment with ABA at 0.1 μm, which is not enough concentration to evoke ABA responses in the wild type, rescued the observed phenotypes of the aba2-2 mutant. Finally, we found that in wild-type leaves, MeJA stimulates the expression of 9-CIS-EPOXYCAROTENOID DIOXYGENASE3, which encodes a crucial enzyme in ABA biosynthesis. These results suggest that endogenous ABA could be involved in MeJA signal transduction and lead to stomatal closure in Arabidopsis guard cells.

Cooperative Function of PLDδ and PLDα1 in Abscisic Acid-Induced Stomatal Closure in Arabidopsis

Phospholipase D (PLD) is involved in responses to abiotic stress and abscisic acid (ABA) signaling. To investigate the roles of two Arabidopsis (Arabidopsis thaliana) PLDs, PLDα1 and PLDδ, in ABA signaling in guard cells, we analyzed ABA responses in guard cells using Arabidopsis wild type, pldα1 and pldδ single mutants, and a pldα1 pldδ double mutant. ABA-induced stomatal closure was suppressed in the pldα1 pldδ double mutant but not in the pld single mutants. The pldα1 and pldδ mutations reduced ABA-induced phosphatidic acid production in epidermal tissues. Expression of either PLDα1 or PLDδ complemented the double mutant stomatal phenotype. ABA-induced stomatal closure in both pldα1 and pldδ single mutants was inhibited by a PLD inhibitor (1-butanol ), suggesting that both PLDα1 and PLDδ function in ABA-induced stomatal closure. During ABA-induced stomatal closure, wild-type guard cells accumulate reactive oxygen species and nitric oxide and undergo cytosolic alkalization, but these changes are reduced in guard cells of the pldα1 pldδ double mutant. Inward-rectifying K+ channel currents of guard cells were inhibited by ABA in the wild type but not in the pldα1 pldδ double mutant. ABA inhibited stomatal opening in the wild type and the pldδ mutant but not in the pldα1 mutant. In wild-type rosette leaves, ABA significantly increased PLDδ transcript levels but did not change PLDα1 transcript levels. Furthermore, the pldα1 and pldδ mutations mitigated ABA inhibition of seed germination. These results suggest that PLDα1 and PLDδ cooperate in ABA signaling in guard cells but that their functions do not completely overlap.

Roles of AtTPC1, Vacuolar Two Pore Channel 1, in Arabidopsis Stomatal Closure

Abscisic acid (ABA) induces production of reactive oxygen species (ROS) and nitric oxide (NO), elevation of the cytosolic free calcium ion concentration ([Ca(2+)](cyt)) and cytosolic pH (pH(cyt)), and activation of S-type anion channels in guard cells, causing stomatal closure. To investigate whether Arabidopsis Two pore channel 1 (AtTPC1) that encodes the slow vacuolar (SV) channel is involved in stomatal closure, we examined stomatal movements and mobilization of second messengers in the attpc1-2 loss-of-function mutant in response to ABA, methyl jasmonate (MeJA) and Ca(2+). Both ABA and MeJA elicited production of ROS and NO, [Ca(2+)](cyt) oscillations, cytosolic alkalization and activation of S-type anion channel currents to lead to stomatal closure in the attpc1-2 mutant as well as the wild type. Unlike the wild type, in the attpc1-2 mutant exogenous Ca(2+) neither induced stomatal closure nor activated plasma membrane S-type anion channel currents despite [Ca(2+)](cyt) elevation. These results indicate that AtTPC1 functions in response to external Ca(2+) but not to ABA and MeJA in Arabidopsis guard cells and suggest that AtTPC1 could be involved in priming of plasma membrane S-type anion channels by external Ca(2+) in Arabidopsis guard cells.

Cytosolic Alkalization and Cytosolic Calcium Oscillation in Arabidopsis Guard Cells Response to ABA and MeJA

Abscisic acid (ABA)- and methyl jasmonate (MeJA)-induced stomatal closure are accompanied by cytosolic alkalization in guard cells. However, it remains to be clarified how the alkalization functions in not only ABA signaling but also MeJA. We investigated cytosolic alkalization in guard cells during ABA-, MeJA- and Ca(2+)-induced stomatal closure of wild type, abi1-1, abi2-1, ost1-2 and coi1 using a pH-sensitive fluorescent dye, BCECF-AM. ABA induced cytosolic alkalization in guard cells of wild-type and coi1 but not in ost1-2 guard cells whereas MeJA elicited cytosolic alkalization in wild-type and ost1-2 guard cells but not in coi1. Neither ABA nor MeJA induced cytosolic alkalization in abi1-1 and abi2-1 guard cells. Exogenous Ca(2+) induced stomatal closure accompanied by cytosolic alkalization in guard cells of wild-type, abi1-1, abi2-1, ost1-2 and coi1 plants. An agent to acidify cytosol, butyrate, suppressed Ca(2+)-induced cytosolic alkalization and ABA-, MeJA- and Ca(2+)-induced cytosolic Ca(2+) oscillation in wild-type guard cells to prevent stomatal closure. These results indicate that cytosolic alkalization and cytosolic Ca(2+) oscillation coordinately function in ABA and MeJA signaling in Arabidopsis guard cells.

Yeast Elicitor-Induced Stomatal Closure and Peroxidase-Mediated ROS Production in Arabidopsis

Yeast elicitor (YEL) induces stomatal closure. We investigated reactive oxygen species (ROS) production, nitric oxide (NO) production and [Ca(2+)](cyt) oscillations to clarify YEL signaling in Arabidopsis guard cells. YEL induced ROS accumulation in guard cells. A peroxidase inhibitor [salicylhydroxamic acid (SHAM)] inhibited the stomatal closure and the ROS accumulation, but neither the atrbohD atrbohF mutation nor an NADPH oxidase inhibitor [diphenylene iodonium chloride (DPI)] had any effect. An NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)] inhibited the YEL-induced stomatal closure and SHAM abolished NO production. YEL-elicited [Ca(2+)](cyt) oscillations were inhibited by SHAM but not by the atrbohD atrbohF mutation. These results indicate that YEL induces stomatal closure accompanied by ROS production mediated by peroxidases and NO production.

Mg-chelatase H subunit affects ABA signaling in stomatal guard cells, but is not an ABA receptor in Arabidopsis thaliana.

Mg-chelatase H subunit (CHLH) is a multifunctional protein involved in chlorophyll synthesis, plastid-to-nucleus retrograde signaling, and ABA perception. However, whether CHLH acts as an actual ABA receptor remains controversial. Here we present evidence that CHLH affects ABA signaling in stomatal guard cells but is not itself an ABA receptor. We screened ethyl methanesulfonate-treated Arabidopsis thaliana plants with a focus on stomatal aperture-dependent water loss in detached leaves and isolated a rapid transpiration in detached leaves 1 (rtl1) mutant that we identified as a novel missense mutant of CHLH. The rtl1 and CHLH RNAi plants showed phenotypes in which stomatal movements were insensitive to ABA, while the rtl1 phenotype showed normal sensitivity to ABA with respect to seed germination and root growth. ABA-binding analyses using 3H-labeled ABA revealed that recombinant CHLH did not bind ABA, but recombinant pyrabactin resistance 1, a reliable ABA receptor used as a control, showed specific binding. Moreover, we found that the rtl1 mutant showed ABA-induced stomatal closure when a high concentration of extracellular Ca2+ was present and that a knockout mutant of Mg-chelatase I subunit (chli1) showed the same ABA-insensitive phenotype as rtl1. These results suggest that the Mg-chelatase complex as a whole affects the ABA-signaling pathway for stomatal movements.

Allyl isothiocyanate (AITC) induces stomatal closure in Arabidopsis

Isothiocyanates (ITCs) are degradation products of glucosinolates in crucifer plants and have repellent effect on insects, pathogens and herbivores. In this study, we report that exogenously applied allyl isothiocyanate (AITC) induced stomatal closure in Arabidopsis via production of reactive oxygen species (ROS) and nitric oxide (NO), and elevation of cytosolic Ca(2+) . AITC-induced stomatal closures were partially inhibited by an inhibitor of NADPH oxidase and completely inhibited by glutathione monoethyl ester (GSHmee). AITC-induced stomatal closure and ROS production were examined in abscisic acid (ABA) deficient mutant aba2-2 and methyl jasmonate (MeJA)-deficient mutant aos to elucidate involvement of endogenous ABA and MeJA. Genetic evidences have demonstrated that AITC-induced stomatal closure required MeJA priming but not ABA priming. These results raise the possibility that crucifer plants produce ITCs to induce stomatal closure, leading to suppression of water loss and invasion of fungi through stomata.

Negative regulation of abscisic acid-induced stomatal closure by glutathione in Arabidopsis.

We found that glutathione (GSH) is involved in abscisic acid (ABA)-induced stomatal closure. Regulation of ABA signaling by GSH in guard cells was investigated using an Arabidopsis mutant, cad2-1, that is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). Glutathione contents in guard cells decreased along with ABA-induced stomatal closure. Decreasing GSH by both the cad2-1 mutation and CDNB treatment enhanced ABA-induced stomatal closure. Glutathione monoethyl ester (GSHmee) restored the GSH level in cad2-1 guard cells and complemented the stomatal phenotype of the mutant. Depletion of GSH did not significantly increase ABA-induced production of reactive oxygen species in guard cells and GSH did not affect either activation of plasma membrane Ca(2+)-permeable channel currents by ABA or oscillation of the cytosolic free Ca(2+) concentration induced by ABA. These results indicate that GSH negatively modulates a signal component other than ROS production and Ca(2+) oscillation in ABA signal pathway of Arabidopsis guard cells.

Methylglyoxal-induced stomatal closure accompanied by peroxidase-mediated ROS production in Arabidopsis.

Methylglyoxal (MG) is an oxygenated short aldehyde and a glycolytic intermediate that accumulates in plants under environmental stresses. Being a reactive α-oxoaldehyde, MG may act as a signaling molecule in plants during stresses. We investigated whether MG induces stomatal closure, reactive oxygen species (ROS) production, and cytosolic free calcium concentration ([Ca²⁺](cyt)) to clarify roles of MG in Arabidopsis guard cells. MG induced production of ROS and [Ca²⁺](cyt) oscillations, leading to stomatal closure. The MG-induced stomatal closure and ROS production were completely inhibited by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), but were not affected by an NAD(P)H oxidase mutation, atrbohD atrbohF. Furthermore, the MG-elicited [Ca²⁺](cyt) oscillations were significantly suppressed by SHAM but not by the atrbohD atrbohF mutation. Neither endogenous abscisic acid nor endogenous methyl jasmonate was involved in MG-induced stomatal closure. These results suggest that intrinsic metabolite MG can induce stomatal closure in Arabidopsis accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca²⁺](cyt) oscillations.