Protein phosphorylation and dephosphorylation are important mechanisms that regulate many cellular processes. Protein kinases usually function in the regulation of the stress responses by adjusting activity via phosphorylation of target proteins. Here, we isolated CaAIMK1 (Capsicum annuum ABA Induced MAP Kinase 1) from the pepper leaves that had been subjected to drought stress. CaAIMK1 transcripts were induced by drought, abscisic acid (ABA), high salinity, and H 2 O 2 ; further, the CaAIMK1-Green fluorescent protein localized in the nucleus and cytoplasm. We performed genetic studies using CaAIMK1-silenced pepper plants and CaAIMK1-overexpressing (OX) Arabidopsis plants. CaAIMK1-silenced pepper plants showed a drought-sensitive phenotype characterized by altered ABA signaling, including low leaf temperatures, and large stomatal apertures. CaAIMK1-OX plants exhibited a contrasting drought-tolerant phenotype characterized by decreased levels of transpirational water loss and increased expression levels of Arabidopsis stress-related genes. In CaAIMK1 K32N-OX transgenic Arabidopsis plants, sensitivity to ABA and drought was restored. Collectively, these results demonstrate that CaAIMK1 positively regulates the drought stress responses via an ABA-dependent pathway.

Summary Induction of the abscisic acid (ABA) signalling network is associated with various stress conditions, including cold, high salinity and drought. As core ABA signalling components, group A type 2C protein phosphatases (PP2Cs) interact with and inhibit snf1‐related protein kinase2s. Here, we isolated and characterised the pepper mitogen‐activated protein kinase kinase kinase CaADIK1, which interacts with the group A PP2C CaADIP1. CaADIK1 transcripts were induced by abiotic stresses, and CaADIK1 localised in the nucleus and cytoplasm. We verified that CaADIP1 inhibits the autokinase activity of CaADIK1; moreover, the kinase activity of CaADIK1 is enhanced by drought stress. We performed genetic analysis using CaADIK1‐silenced pepper and CaADIK1‐overexpressing (OX) Arabidopsis plants. CaADIK1‐silenced pepper plants showed drought‐sensitive phenotypes, whereas CaADIK1‐OX Arabidopsis plants showed ABA‐sensitive and drought‐tolerant phenotypes. In CaADIK1K32N‐OX Arabidopsis plants mutated at the ATP‐binding site, the ABA‐insensitive and drought‐sensitive phenotypes were restored. Taken together, our findings show that CaADIK1 positively regulates the ABA‐dependent drought stress response and is inhibited by CaADIP1.

Summary The phytohormone abscisic acid (ABA) is important for the plant growth and development, in which it plays a key role in the responses to drought stress. Among the core components of ABA signaling, SnRK2s interact with a range of proteins, including Raf‐like MAP3Ks. In this study, we isolated the pepper MEKK subfamily member CaMEKK23 that interacts with CaSnRK2.6. CaMEKK23 has kinase activity and is specifically trans‐phosphorylated by CaSnRK2.6. Compared with control plants, CaMEKK23‐silenced pepper were found to be sensitive to drought stress and insensitive to ABA, whereas overexpression of CaMEKK23 in both pepper and Arabidopsis plants induced the opposite phenotypes. These altered phenotypes were established to be dependent on the kinase activity of CaMEKK23, which was also shown to interact with CaPP2Cs, functioning upstream of CaSnRK2.6. In addition to inhibiting the kinase activity of CaMEKK23, these CaPP2Cs were found to have inhibitory effects on CaSnRK2.6. Using CaMEKK23‐, CaAITP1/CaMEKK23‐, CaSnRK2.6‐, and CaAITP1/CaSnRK2.6‐silenced pepper, we revealed that CaMEKK23 and CaSnRK2.6 function downstream of CaAITP1. Collectively, our findings indicate that CaMEKK23 plays a positive regulatory role in the ABA‐mediated drought stress responses in pepper plants, and that its phosphorylation status is modulated by CaSnRK2.6 and CaPP2Cs, functioning as core components of ABA signaling.

Mitogen-activated protein kinase kinase kinases (MAPKKKs or MEKKs) are crucial components of the MAPK signaling cascades, which play central roles in the signaling transduction pathways for plant growth, development, and response to abiotic stresses such as drought. The MAPKKK gene families in pepper have not been functionally characterized yet. Here, from the pepper genome, we predicted 27 putative MAPKKK genes belonging to the MEKK subfamily (named CaMEKK1-27), based on in silico analysis. Phylogenetic analysis revealed that 14 CaMEKK genes were clustered into A5 of the five groups (A1-A5), of which 9 genes are primarily on chromosomes 2 and 7, and are located close to each other. These nine genes showed transcriptional regulation by treatment with abscisic acid (ABA) and drought stress in quantitative reverse-transcription PCR analysis. Among the ABA-and/or drought-induced CaMEKK genes, in a previous study, we isolated CaAIMK1 (Capsicum annuum ABA Induced MAP Kinase 1), which plays a positive role in drought resistance via an ABA-dependent pathway. Our expression analysis and functional characterization of the MEKK subfamily genes will provide a better understanding of the functional roles of pepper MAPK cascades in ABA-mediated drought responses.

Protein phosphorylation by kinase is an important mechanism for adapting to drought stress conditions. Here, we isolated the CaDIMK1 (Capsicum annuum drought-induced MAP kinase 1) from dehydrated pepper leaf tissue and functionally characterized it. Subcellular localization analysis revealed that the CaDIMK1 protein was localized in the cytoplasm and nucleus. CaDIMK1-silenced pepper plants exhibited drought-susceptible phenotypes that were characterized by increased transpiration rates, low leaf temperatures, and decreased stomatal closure. In contrast, CaDIMK1-overexpressing (OX) transgenic Arabidopsis plants were hypersensitive to abscisic acid (ABA) from germination to adult growth stages. Furthermore, the CaDIMK1-OX plants were tolerant to drought stress. The transcript levels of several stress-related genes were high in CaDIMK1-OX plants than in wild-type plants. Taken together, our data demonstrate that CaDIMK1 acts as a positive modulator of drought tolerance and ABA signal transduction in pepper plants.