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.

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.

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.

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. Here, we show that evk treatment increased H ₂ O ₂ and intracellular calcium concentrations in Arabidopsis thaliana mesophyll cells, as observed by confocal laser scanning microscopy and non-invasive micro-test technology, and that H ₂ O ₂ signaling functioned upstream of Ca ²⁺ signaling. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that the ACA8 (AUTOINHIBITED Ca ²⁺ -ATPASE, ISOFORM 8)–CML8 (CALMODULIN-LIKE 8) interaction promoted Ca ²⁺ efflux to return Ca ²⁺ levels to the resting state. Evk treatment led to the antagonism of salicylic acid (SA) and jasmonic acid (JA). CALMODULIN 5 (CAM5) positively regulates WRKY53 expression, and CAM5 and WRKY53 positively regulate SA-related gene expression. These proteins physically interact and form a complex that is unlocked by Ca ²⁺ to release WRKY53. An electrophoretic mobility shift assay and dual-luciferase reporter assay demonstrated that WRKY53 and TGA5 cooperate to enhance the expression of the defense gene PATHOGENESIS-REALTED 1 ( PR1 ) and that WRKY53 enhances the binding of TGA5 to the PR1 promoter. This paper proposes a framework that evk, as a RES substance, can achieve plant’s ‘REScue’ through complete defense response.