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

Abstract

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.

Unveiling synergistic QTLs associated with slow wilting in soybean (Glycine max [L.] Merr.)

“…Five genes (Glyma.10G017700, Glyma.10G017900, Glyma.10G019000, Glyma.10G019300, and Glyma.10G021800), functionally expressed, showed nonsynonymous missense mutations in exons between the parental lines (Table S6 ): 3 SNPs in Glyma.10G017700 (poly(ADP-ribose) polymerase 2, PARP2), 2 in Glyma.10G021800.1 (degradation of periplasmic proteins 2, DEG2), 1 in each of Glyma.10G017900 (photolyase/blue-light receptor 2, PHR2), Glyma.10G019000 (multidrug resistance-associated protein 4, MRP4) and Glyma.10G019300 (AAA-type ATPase family protein). For only Glyma.10G019000, its Arabidopsis ortholog AT2G27800 has been reported for its function in drought stress (Klein et al 2004 ; Gong et al 2022 ). Compared to SS2-2, we detected multiple variants in the genic region of Glyma.10G019000 in Taekwangkong, including a missense variant in which the amino acid at position 87 changed from Met to Ile and a premature start codon gain variant in 5’ untranslated region (UTR) region (Fig.…”

“…Therefore, Glyma.10G019000 is suggested to be an MRP4 which predominantly functions under drought stress in soybean. AT2G47800, an Arabidopsis ortholog of Glyma.10G019000, was highly expressed in stomata and involved in the regulation of stomatal aperture (Klein et al 2004 ; Gong et al 2022 ). Knockout mutants of AtMRP4 showed larger stomatal aperture regardless of light and wilt faster than wild-type under drought conditions (Klein et al 2004 ).…”

“…Knockout mutants of AtMRP4 showed larger stomatal aperture regardless of light and wilt faster than wild-type under drought conditions (Klein et al 2004 ). Recently, it was discovered that MRP4-dependent eATP release triggered by ethyl vinyl ketone (evk) mediates K + Efflux, leading to stomata closure (Gong et al 2022 ). Both PK1B and MRP4 are known to be involved in the regulation of H + -ATPase activity, a key regulator of stomatal opening (Zhao et al 2019 ).…”

CAM5, WRKY53, and TGA5 regulate defense gene expression mediated by the volatile organic compound ethyl vinyl ketone

“…Cytoplasmic free Ca 2+ concentrations and H 2 O 2 are thought to play crucial roles in the volatile-sensing mechanisms of plants, although these roles have not been directly demonstrated. We previously showed that evk is recognized by plants and induces H 2 O 2 production, which ultimately mediates stomatal closure to reduce pathogen infection 69 . In the current study, we demonstrated that evk, a volatile compound released by plants, increased the levels of H 2 O 2 and intracellular calcium in Arabidopsis mesophyll cells.…”

“…The occurrence of floral end rot was reduced by increasing the evk content of tomatoes (Solanum lycopersicum) by treating the plants with selenium during the green blossom stage 22 . We previously demonstrated that evk-induced stomatal closure requires MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN 4 (MRP4)-dependent extracellular ATP accumulation and subsequent H 2 O 2 accumulation to regulate K + efflux 23 . Evk is involved in many biological processes, but whether it activates defense genes (e.g., PLANT DEFENSIN 1.2 [PDF1.2] and PATHOGENESIS-RELATED 1 [PR1]) remains unknown.…”