Ethyl vinyl ketone activates oxidative and calcium burst and CML8-ACA8 participates in calcium recovery in Arabidopsis leaves.

Plants produce ethyl vinyl ketone (evk) in response to biotic stress, but the evk's identification and downstream defense response remain unclear. In this paper, it is predicted by docking for the first time that evk can be recognized by RBOH protein and assist the electron transfer of RBOHD/RBOHF by binding to its FAD or NADPH binding site. Surface plasmon resonance (SPR) binding assay shows that evk indeed bind to RBOHD. Here, we show that evk treatment increased H2O2 and intracellular calcium concentrations in Arabidopsis thaliana mesophyll cells, as observed by confocal laser scanning microscopy and non-invasive micro-test technology, and that H2O2 signaling functioned upstream of Ca2+ signaling. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that the ACA8 (AUTOINHIBITED Ca2+-ATPASE, ISOFORM 8)-CML8 (CALMODULIN-LIKE 8) interaction promoted Ca2+ efflux to return Ca2+ levels to the resting state.

Ethyl Vinyl Ketone Activates K+ Efflux to Regulate Stomatal Closure by MRP4-Dependent eATP Accumulation Working Upstream of H2O2 Burst in Arabidopsis.

Plants regulate stomatal mobility to limit water loss and improve pathogen resistance. Ethyl vinyl ketone (evk) is referred to as a reactive electrophilic substance (RES). In this paper, we found that evk can mediate stomatal closure and that evk-induced stomatal closure by increasing guard cell K+ efflux. To investigate the role of eATP, and H2O2 in evk-regulated K+ efflux, we used Arabidopsis wild-type (WT), mutant lines of mrp4, mrp5, dorn1.3 and rbohd/f. Non-invasive micro-test technology (NMT) data showed that evk-induced K+ efflux was diminished in mrp4, rbohd/f, and dorn1.3 mutant, which means eATP and H2O2 work upstream of evk-induced K+ efflux. According to the eATP content assay, evk stimulated eATP production mainly by MRP4. In mrp4 and mrp5 mutant groups and the ABC transporter inhibitor glibenclamide (Gli)-pretreated group, evk-regulated stomatal closure and eATP buildup were diminished, especially in the mrp4 group. According to qRT-PCR and eATP concentration results, evk regulates both relative gene expressions of MRP4/5 and eATP concentration in rbohd/f and WT group. According to the confocal data, evk-induced H2O2 production was lower in mrp4, mrp5 mutants, which implied that eATP works upstream of H2O2. Moreover, NADPH-dependent H2O2 burst is regulated by DORN1. A yeast two-hybrid assay, firefly luciferase complementation imaging assay, bimolecular fluorescence complementation assay, and pulldown assay showed that the interaction between DORN1 and RBOHF can be realized, which means DORN1 may control H2O2 burst by regulating RBOHF through interaction. This study reveals that evk-induced stomatal closure requires MRP4-dependent eATP accumulation and subsequent H2O2 accumulation to regulate K+ efflux.

CML8 and GAD4 function in (Z)-3-hexenol-mediated defense by regulating γ-aminobutyric acid accumulation in Arabidopsis.

(Z)-3-hexenol, a small gaseous molecule, is produced in plants under biotic stress and induces defense responses in neighboring plants. However, little is known about how (Z)-3-hexenol induces plant defense-related signaling. In this study, we uncovered how (Z)-3-hexenol treatment enhances plant resistance to insect attacks by increasing γ-aminobutyric acid (GABA) contents in Arabidopsis leaves. First, (Z)-3-hexenol increases the intracellular content of calcium as secondary messenger in Arabidopsis leaf mesophyll cells. Both intracellular and extracellular calcium stores regulate changes in calcium content. Then, CML8 and GAD4 transmit calcium signaling to affect (Z)-3-hexenol induced GABA content and plant resistance. Herein, CML8 interaction with GAD4 was examined via yeast two-hybrid assays, firefly luciferase complementation imaging, and GST pull-down assays. These results indicate that (Z)-3-hexenol treatment increased the GABA contents in Arabidopsis leaves based on CML8 and GAD4, thus increasing plant resistance to the insect Plutella xylostella. This study revealed the mechanism of activating plant insect defense induced by (Z)-3-hexenol, which guides the study of volatiles as biological pest control.

CALMODULIN1 and WRKY53 Function in Plant Defense by Negatively Regulating the Jasmonic Acid Biosynthesis Pathway in Arabidopsis.

Jasmonic acid (JA) is an important hormone that functions in plant defense. cam1 and wrky53 mutants were more resistant to Spodoptera littoralis than in the wild-type (WT) Arabidopsis group. In addition, JA concentration in cam1 and wrky53 mutants was higher compared with the WT group. To explore how these two proteins affect the resistance of Arabidopsis plants, we used a yeast two-hybrid assay, firefly luciferase complementation imaging assay and in vitro pull-down assay confirming that calmodulin 1 (CAM1) interacted with WRKY53. However, these two proteins separate when calcium concentration increases in Arabidopsis leaf cells. Then, electrophoretic mobility shift assay and luciferase activation assay were used to verify that WRKY53 could bind to lipoxygenases 3 (LOX3) and lipoxygenases 4 (LOX4) gene promoters and negatively regulate gene expression. This study reveals that CAM1 and WRKY53 negatively regulate plant resistance to herbivory by regulating the JA biosynthesis pathway via the dissociation of CAM1-WRKY53, then the released WRKY53 binds to the LOXs promoters to negatively regulate LOXs gene expression. This study reveals WRKY53's mechanism in insect resistance, a new light on the function of WRKY53.

Linalool Activates Oxidative and Calcium Burst and CAM3-ACA8 Participates in Calcium Recovery in Arabidopsis Leaves.

Plants produce linalool to respond to biotic stress, but the linalool-induced early signal remains unclear. In wild-type Arabidopsis, plant resistance to diamondback moth (Plutella xylostella) increased more strongly in a linalool-treated group than in an untreated control group. H2O2 and Ca2+, two important early signals that participated in biotic stress, burst after being treated with linalool in Arabidopsis mesophyll cells. Linalool treatment increased H2O2 and intracellular calcium concentrations in mesophyll cells, observed using a confocal microscope with laser scanning, and H2O2 signaling functions upstream of Ca2+ signaling by using inhibitors and mutants. Ca2+ efflux was detected using non-invasive micro-test technology (NMT), and Ca2+ efflux was also inhibited by NADPH oxidase inhibitor DPI (diphenyleneiodonium chloride) and in cells of the NADPH oxidase mutant rbohd. To restore intracellular calcium levels, Ca2+-ATPase was activated, and calmodulin 3 (CAM3) participated in Ca2+-ATPase activation. This result is consistent with the interaction between CAM7 and Ca2+-ATPase isoform 8 (ACA8). In addition, a yeast two-hybrid assay, firefly luciferase complementation imaging assay, and an in vitro pulldown assay showed that CAM3 interacts with the N-terminus of ACA8, and qRT-PCR showed that some JA-related genes and defense genes expressions were enhanced when treated with linalool in Arabidopsis leaves. This study reveals that linalool enhances H2O2 and intracellular calcium concentrations in Arabidopsis mesophyll cells; CAM3-ACA8 reduces intracellular calcium concentrations, allowing cells to resume their resting state. Additionally, JA-related genes and defense genes' expression may enhance plants' defense when treated with linalool.

Plant ion channels and transporters in herbivory-induced signalling.

In contrast to many biotic stresses that plants face, feeding by herbivores produces unique mechanical and chemical signatures. Plants have evolved effective systems to recognise these mechanical stimuli and chemical elicitors at the plasma membrane (PM), where this recognition generates ion fluxes, including an influx of Ca2+ that elicits cellular Ca2+ signalling, production of reactive oxygen species (ROS), and variation in transmembrane potential. These signalling events also function in propagation of long-distance signals (Ca2+ waves, ROS waves, and electrical signals), which contribute to rapid, systemic induction of defence responses. Recent studies have identified several candidate channels or transporters that likely produce these ion fluxes at the PM. Here, we describe the important roles of these channels/transporters in transduction or transmission of herbivory-induced early signalling events, long-distance signals, and jasmonic acid and green leaf volatile signalling in plants.