CaADIP1‐dependent CaADIK1‐kinase activation is required for abscisic acid signalling and drought stress response in Capsicum annuum

Abstract

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.

Pepper SnRK2.6‐activated MEKK protein CaMEKK23 is directly and indirectly modulated by clade A PP2Cs in response to drought stress

“…We have previously demonstrated that the kinase activity of CaADIK1, a homolog of CaMEKK23, contributes to ABA and drought stress responses (Jeong et al ., 2021), and thus, we predicted that drought stress‐promoted CaMEKK23 kinase activity might be closely associated with the enhanced drought tolerance and ABA sensitivity of CaMEKK23 ‐OX plants. To verify this assumption, we generated transgenic Arabidopsis plants overexpressing the kinase activity deficient CaMEKK23 K32N mutant (Figs 6, S3C), which were then exposed to drought stress.…”

“…This identical function tends to limit the scope of VIGS in elucidating differences between the influence of these two genes on pepper plant phenotypes. As an alternative approach to assess the putative association between CaSnRK2.6 and CaMEKK23, we adopted a strategy based on our findings that CaADIK1, a homolog of CaMEKK23, interacts with and is inhibited by CaADIP1, a group A PP2C (Jeong et al ., 2021). Specifically, we pondered as to whether CaMEKK23 might interact with pepper PP2Cs including CaADIP1.…”

“…MAPKKK18 also interacts with SnRK2.6, although the outcome of this interaction has yet to be ascertained (Tajdel et al ., 2016). Moreover, the pepper clade A PP2C CaADIP1 inhibits CaADIK1 kinase activity, which is an essential process for the drought stress responses (Jeong et al ., 2021). To date, however, the combined influence of MEKK and core components on the ABA signaling pathway remains insufficiently determined.…”

“…Previously, we have reported that pepper MEKK subfamily genes are differentially expressed in response to ABA and drought treatments (Lim et al ., 2020), among which, CaAIMK1, CaDIMK1, and CaADIK1 play positive roles in ABA signaling and the drought stress responses (Jeong et al ., 2020, 2021; Kim et al ., 2021). In this study, we identified CaMEKK23, which physically interacts with both pepper SnRK2.6 and group A PP2Cs, with CaMEKK23 phosphorylation being promoted by CaSnRK2.6 and inhibited by CaPP2Cs.…”

Pepper stress‐associated protein 14 is a substrate of CaSnRK2.6 that positively modulates abscisic acid‐dependent osmotic stress responses

“…For in vitro phosphorylation analyses, we used kinase buffer (1 mM CaCl 2 , 1 mM dithiothreitol, 2.5 mM MnCl 2 , 2.5 mM MgCl 2 , and 20 mM Tris-HCl, at pH 7.5) and 15 lCi [c-32 P]-ATP, with assays being performed using a previously described protocol (Jeong et al, 2020(Jeong et al, , 2021Kim et al, 2021).…”