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.

Role of Gamma-Aminobutyric Acid in Plant Defense Response.

Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid that acts as a defense substance and a signaling molecule in various physiological processes, and which helps plants respond to biotic and abiotic stresses. This review focuses on the role of GABA's synthetic and metabolic pathways in regulating primary plant metabolism, redistributing carbon and nitrogen resources, reducing the accumulation of reactive oxygen species, and improving plants' tolerance of oxidative stress. This review also highlights the way in which GABA maintains intracellular pH homeostasis by acting as a buffer and activating H+-ATPase. In addition, calcium signals participate in the accumulation process of GABA under stress. Moreover, GABA also transmits calcium signals through receptors to trigger downstream signaling cascades. In conclusion, understanding the role of GABA in this defense response provides a theoretical basis for applying GABA in agriculture and forestry and feasible coping strategies for plants in complex and changeable environments.

DORN1 and GORK regulate stomatal closure in Arabidopsis mediated by volatile organic compound ethyl vinyl ketone.

Evk (ethyl vinyl ketone) is a signal substance for plant defense, but little is known about how evk mediates stomatal closure. Through stomatal biology experiments, we found that evk can mediate stomatal closure, and stomatal closure is weakened when DORN1 (DOES NOT RESPOND TO NUCLEOTIDES 1) and GORK (GATED OUTWARDLY-RECTIFYING K+ CHANNEL) are mutated. In addition, it was found by non-invasive micro-test technology (NMT) that the K+ efflux mediated by evk was significantly weakened when DORN and GORK were mutated. Yeast two-hybrid (Y2H), firefly luciferase complementation imaging (LCI), and in vitro pull-down assays demonstrated that DORN1 and GORK could interact in vitro and in vivo. It was found by molecular docking that evk could combine with MRP (Multidrug Resistance-associated Protein), thus affecting ATP transport, promoting eATP (extracellular ATP) concentration increase and realizing downstream signal transduction. Through inoculation of botrytis cinerea, it was found that evk improved the antibacterial activity of Arabidopsis thaliana. As revealed by reverse transcription quantitative PCR (RT-qPCR), the expression of defense related genes was enhanced by evk treatment. Evk is a potential green antibacterial drug.

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.

Herbivore exposure alters ion fluxes and improves salt tolerance in a desert shrub.

Plants have evolved complex mechanisms that allow them to withstand multiple environmental stresses, including biotic and abiotic stresses. Here, we investigated the interaction between herbivore exposure and salt stress of Ammopiptanthus nanus, a desert shrub. We found that jasmonic acid (JA) was involved in plant responses to both herbivore attack and salt stress, leading to an increased NaCl stress tolerance for herbivore-pretreated plants and increase in K+ /Na+ ratio in roots. Further evidence revealed the mechanism by which herbivore improved plant NaCl tolerance. Herbivore pretreatment reduced K+ efflux and increased Na+ efflux in plants subjected to long-term, short-term, or transient NaCl stress. Moreover, herbivore pretreatment promoted H+ efflux by increasing plasma membrane H+ -adenosine triphosphate (ATP)ase activity. This H+ efflux creates a transmembrane proton motive force that drives the Na+ /H+ antiporter to expel excess Na+ into the external medium. In addition, high cytosolic Ca2+ was observed in the roots of herbivore-treated plants exposed to NaCl, and this effect may be regulated by H+ -ATPase. Taken together, herbivore exposure enhances A. nanus tolerance to salt stress by activating the JA-signalling pathway, increasing plasma membrane H+ -ATPase activity, promoting cytosolic Ca2+ accumulation, and then restricting K+ leakage and reducing Na+ accumulation in the cytosol.

Duplication and Divergence of Leucine-Rich Repeat Receptor-Like Protein Kinase (LRR-RLK) Genes in Basal Angiosperm Amborella trichopoda.

Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases, which are one of the largest protein superfamilies in plants, and play crucial roles in development and stress responses. Although the evolution of LRR-RLK families has been investigated in some eudicot and monocot plants, no comprehensive evolutionary studies have been performed for these genes in basal angiosperms like Amborella trichopoda. In this study, we identified 94 LRR-RLK genes in the genome of A. trichopoda. The number of LRR-RLK genes in the genome of A. trichopoda is only 17-50% of that of several eudicot and monocot species. Tandem duplication and whole-genome duplication have made limited contributions to the expansion of LRR-RLK genes in A. trichopoda. According to the phylogenetic analysis, all A. trichopoda LRR-RLK genes can be organized into 18 subfamilies, which roughly correspond to the LRR-RLK subfamilies defined in Arabidopsis thaliana. Most LRR-RLK subfamilies are characterized by highly conserved protein structures, motif compositions, and gene structures. The unique gene structure, protein structures, and protein motif compositions of each subfamily provide evidence for functional divergence among LRR-RLK subfamilies. Moreover, the expression data of LRR-RLK genes provided further evidence for the functional diversification of them. In addition, selection analyses showed that most LRR-RLK protein sites are subject to purifying selection. Our results contribute to a better understanding of the evolution of LRR-RLK gene family in angiosperm and provide a framework for further functional investigation on A. trichopoda LRR-RLKs.