Vacuolar CAX1 and CAX3 influence auxin transport in guard cells via regulation of apoplastic pH.

CATION EXCHANGERs CAX1 and CAX3 are vacuolar ion transporters involved in ion homeostasis in plants. Widely expressed in the plant, they mediate calcium transport from the cytosol to the vacuole lumen using the proton gradient across the tonoplast. Here, we report an unexpected role of CAX1 and CAX3 in regulating apoplastic pH and describe how they contribute to auxin transport using the guard cell's response as readout of hormone signaling and cross talk. We show that indole-3-acetic acid (IAA) inhibition of abscisic acid (ABA)-induced stomatal closure is impaired in cax1, cax3, and cax1/cax3. These mutants exhibited constitutive hypopolarization of the plasma membrane, and time-course analyses of membrane potential revealed that IAA-induced hyperpolarization of the plasma membrane is also altered in these mutants. Both ethylene and 1-naphthalene acetic acid inhibited ABA-triggered stomatal closure in cax1, cax3, and cax1/cax3, suggesting that auxin signaling cascades were functional and that a defect in IAA transport caused the phenotype of the cax mutants. Consistent with this finding, chemical inhibition of AUX1 in wild-type plants phenocopied the cax mutants. We also found that cax1/cax3 mutants have a higher apoplastic pH than the wild type, further supporting the hypothesis that there is a defect in IAA import in the cax mutants. Accordingly, we were able to fully restore IAA inhibition of ABA-induced stomatal closure in cax1, cax3, and cax1/cax3 when stomatal movement assays were carried out at a lower extracellular pH. Our results suggest a network linking the vacuolar cation exchangers to apoplastic pH maintenance that plays a crucial role in cellular processes.

MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling.

Reactive oxygen species (ROS) mediate abscisic acid (ABA) signaling in guard cells. To dissect guard cell ABA-ROS signaling genetically, a cell type-specific functional genomics approach was used to identify 2 MAPK genes, MPK9 and MPK12, which are preferentially and highly expressed in guard cells. To provide genetic evidence for their function, Arabidopsis single and double TILLING mutants that carry deleterious point mutations in these genes were isolated. RNAi-based gene-silencing plant lines, in which both genes are silenced simultaneously, were generated also. Mutants carrying a mutation in only 1 of these genes did not show any altered phenotype, indicating functional redundancy in these genes. ABA-induced stomatal closure was strongly impaired in 2 independent RNAi lines in which both MPK9 and MPK12 transcripts were significantly silenced. Consistent with this result, mpk9-1/12-1 double mutants showed an enhanced transpirational water loss and ABA- and H(2)O(2)-insensitive stomatal response. Furthermore, ABA and calcium failed to activate anion channels in guard cells of mpk9-1/12-1, indicating that these 2 MPKs act upstream of anion channels in guard cell ABA signaling. An MPK12-YFP fusion construct rescued the ABA-insensitive stomatal response phenotype of mpk9-1/12-1, demonstrating that the phenotype was caused by the mutations. The MPK12 protein is localized in the cytosol and the nucleus, and ABA and H(2)O(2) treatments enhance the protein kinase activity of MPK12. Together, these results provide genetic evidence that MPK9 and MPK12 function downstream of ROS to regulate guard cell ABA signaling positively.

De-regulated expression of the plant glutamate receptor homolog AtGLR3.1 impairs long-term Ca2+-programmed stomatal closure.

Cytosolic Ca(2+) ([Ca(2+)](cyt)) mediates diverse cellular responses in both animal and plant cells in response to various stimuli. Calcium oscillation amplitude and frequency control gene expression. In stomatal guard cells, [Ca(2+)](cyt) has been shown to regulate stomatal movements, and a defined window of Ca(2+) oscillation kinetic parameters encodes necessary information for long-term stomatal movements. However, it remains unknown how the encrypted information in the cytosolic Ca(2+) signature is decoded to maintain stomatal closure. Here we report that the Arabidopsis glutamate receptor homolog AtGLR3.1 is preferentially expressed in guard cells compared to mesophyll cells. Furthermore, over-expression of AtGLR3.1 using a viral promoter resulted in impaired external Ca(2+)-induced stomatal closure. Cytosolic Ca(2+) activation of S-type anion channels, which play a central role in Ca(2+)-reactive stomatal closure, was normal in the AtGLR3.1 over-expressing plants. Interestingly, AtGLR3.1 over-expression did not affect Ca(2+)-induced Ca(2+) oscillation kinetics, but resulted in a failure to maintain long-term 'Ca(2+)-programmed' stomatal closure when Ca(2+) oscillations containing information for maintaining stomatal closure were imposed. By contrast, prompt short-term Ca(2+)-reactive closure was not affected in AtGLR3.1 over-expressing plants. In wild-type plants, the translational inhibitor cyclohexamide partially inhibited Ca(2+)-programmed stomatal closure induced by experimentally imposed Ca(2+) oscillations without affecting short-term Ca(2+)-reactive closure, mimicking the guard cell behavior of the AtGLR3.1 over-expressing plants. Our results suggest that over-expression of AtGLR3.1 impairs Ca(2+) oscillation-regulated stomatal movements, and that de novo protein synthesis contributes to the maintenance of long-term Ca(2+)-programmed stomatal closure.

In vitro evaluation of new functional properties of poly-γ-glutamic acid produced by Bacillus subtilis D7.

We investigated the functionality of poly-γ-glutamic acid (γ-PGA), which is produced by Bacillus subtilis D7, for its potential applications in medicine and cosmetics. The γ-PGA had angiotensin-converting enzyme (ACE) inhibition activity. ACE inhibition activity was dependent on the γ-PGA concentration; the highest ACE inhibition activity was observed at 1.25 mg/l of γ-PGA. IC50 (0.108 mg/ml) of the γ-PGA was lower than that of standard ACE inhibitory drug, N-[(S)-mercapto-2-methylpropionyl]-L-proline (0.247 mg/ml). The γ-PGA also had water-holding capacity and hygroscopicity. Furthermore, the γ-PGA inhibited growth of some pathogenic bacteria, including Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Klebsiella pneumonia and Esherichia coli. The γ-PGA exhibited a good metal adsorption capacity; Cr (VI) adsorption capacity of γ-PGA increased with decreasing pH, and the maximal adsorption was observed at pH 2. Our results suggest that γ-PGA may be expected to be widely applied in cosmetics, biomedical and environmental industries with the feature of being less harmful to humans and the environment.

Improved production of poly-γ-glutamic acid by Bacillus subtilis D7 isolated from Doenjang, a Korean traditional fermented food, and its antioxidant activity.

The objectives of this study was to improve poly-γ-glutamic acid (γ-PGA) production by Bacillus subtilis D7 isolated from a Korean traditional fermented food and to assess its antioxidant activity for applications in the cosmetics and pharmaceutical industries. Strain D7 produced γ-PGA in the absence of L-glutamic acid, indicating L-glutamic acid-independent production. However, the addition of L-glutamic acid increased γ-PGA production. Several tricarboxylic acid cycle intermediates and amino acids could serve as the metabolic precursors for γ-PGA production, and the addition of pyruvic acid and D-glutamic acid to culture medium improved the yield of γ-PGA markedly. The maximum yield of γ-PGA obtained was 24.93 ± 0.64 g/l in improved medium, which was about 5.4-fold higher than the yield obtained in basal medium. γ-PGA was found to have 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (46.8 ± 1.5 %), hydroxyl radical scavenging activity (52.0 ± 1.8 %), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) radical scavenging activity (42.1 ± 1.8 %), nitric oxide scavenging activity (35.1 ± 1.3 %), reducing power (0.304 ± 0.008), and metal chelating activity (91.3 ± 3.5 %). These results indicate that γ-PGA has a potential use in the food, cosmetics, and biomedical industries for the development of novel products with radical scavenging activity. As far as we are aware, this is the first report to describe the antioxidant activityof γ-PGA produced by bacteria.

Phosphorylation of the Arabidopsis AtrbohF NADPH oxidase by OST1 protein kinase.

The plant hormone abscisic acid (ABA) triggers production of reactive oxygen species (ROS) in guard cells via the AtrbohD and AtrbohF NADPH oxidases, leading to stomatal closure. The ABA-activated SnRK2 protein kinase open stomata 1 (OST1) (SRK2E/SnRK2.6) acts upstream of ROS in guard cell ABA signaling. Here, we report that OST1 phosphorylates Ser13 and Ser174 on AtrbohF. In addition, substitution of Ser174 to Ala results in a approximately 40% reduction in the phosphorylation of AtrbohF by OST1. We also show that OST1 physically interacts with AtrbohF. These results provide biochemical evidence suggesting that OST1 regulates AtrbohF activity.