Here, we show that pepper (Capsicum annuum) CaWRKY27 negatively regulates basal thermotolerance mediated by H2O2 signaling. CaWRKY27 expression increased during heat stress and persisted during recovery. CaWRKY27 overexpression impaired basal thermotolerance in tobacco (Nicotiana tabacum) and Arabidopsis thaliana, CaWRKY27-overexpressing plants had a lower survival rate under heat stress, accompanied by decreased expression of multiple thermotolerance-associated genes. Accordingly, silencing of CaWRKY27 increased basal thermotolerance in pepper plants. Exogenously applied H2O2 induced CaWRKY27 expression, and CaWRKY27overexpression repressed the scavenging of H2O2 in Arabidopsis, indicating a positive feedback loop between H2O2accumulation and CaWRKY27 expression. Consistent with this, CaWRKY27 expression was repressed under heat stress in the presence H2O2 scavengers and CaWRKY27 silencing decreased H2O2 accumulation in pepper leaves. These changes may result from changes in levels of reactive oxygen species (ROS)-scavenging enzymes, since the heat stress-challenged CaWRKY27-silenced pepper plants had significantly higher expression of multiple genes encoding ROS-scavenging enzymes, such as CaCAT1, CaAPX1, CaAPX2, CaCSD2, and CaSOD1. Therefore, CaWRKY27 acts as a downstream negative regulator of H2O2-mediated heat stress responses, preventing inappropriate responses during heat stress and recovery.
FIGURE 1. CaWRKY27 was transcriptionally induced by heat treatment in pepper plants. (A) CaWRKY27 expression in pepper leaves was determined with qRT-PCR at the indicated time points during or after heat treatment. Relative transcript levels of CaWRKY27 in heat-treated pepper plants were compared to the control, which was set to a relative expression level of ‘1.’ Data represent the mean ± SE of three biological replicates, and asterisks indicate significant difference compared to control plants (SNK-test, ∗P < 0.05 or ∗∗P < 0.01). H, heat; R, recovery. (B) GUS expression in transgenic Arabidopsis plants carrying the pCaWRKY27::GUS construct. Figures a, b, c1, and c2, show a seedling of 3 (a), 5 (b) and 7 (c1 and c2) days after germination (DAG). In figures d1 and d2 transgenic pCaWRKY27::GUS seedlings were heat treated. In figures d1-shoot, d1-root, d2-shoot, and d2-root the shoot or root was magnified from the corresponding d1 or d2 figures, respectively. These seedlings or plants were grown on ½ MS media under 16 h light/8 h dark conditions.