Plants have evolved complex defense mechanisms to adapt and survive under adverse growth conditions. Abscisic acid (ABA) is a phytohormone that plays a pivotal role in the stress response, especially regulation of the stomatal aperture in response to drought. Here, we identified the pepper CaASRF1 (Capsicum annuum ABA Sensitive RING Finger E3 ligase 1) gene, which modulates drought stress tolerance via ABAmediated signaling. CaASRF1 contains a C3H2C3-type RING finger domain, which functions as an E3 ligase by attaching ubiquitins to the target proteins. CaASRF1 expression was enhanced after exposure to ABA, drought and NaCl. Loss-of-function in pepper plants and gain-of-function in Arabidopsis plants revealed that CaASRF1 positively modulates ABA signaling and the drought stress response. Moreover, CaASRF1 interacted with and was associated with degradation of the bZIP transcription factor CaAIBZ1 (Capsicum annuum ASRF1-Interacting bZIP transcription factor 1). Contrary to CaASRF1 phenotypes, CaAIBZ1-silenced pepper and CaAIBZ1-overexpressing Arabidopsis exhibited drought-tolerant and drought-sensitive phenotypes, respectively. Taken together, our data indicate that CaASRF1 positively modulates ABA signaling and the drought stress response via modulation of CaAIBZ1 stability.

Ubiquitination is a eukaryotic protein modulation system for identifying and affecting proteins that are no longer needed in the cell. In a previous study, we elucidated the biological function of CaASRF1, which contains a RING finger domain and functions as an E3 ligase. We showed that CaASRF1 positively modulates abscisic acid (ABA) signalling and drought stress tolerance by modulating the stability of subgroup D bZIP transcription factor CaAIBZ1. We performed yeast two-hybrid (Y2H) screening to identify an additional target protein of CaASRF1. In this study, we identified pepper CaATBZ1 (Capsicum annuum ASRF1 target bZIP transcription factor 1), which belongs to the subgroup A bZIP transcription factors. We investigated the biological function of this protein using virus-induced gene silencing (VIGS) in pepper plants and by generating overexpressing transgenic Arabidopsis plants. Our loss-of-function and gain-of-function studies revealed that CaATBZ1 negatively modulates ABA signalling and drought stress response. Consistent with CaATBZ1silenced pepper plants, CaASRF1/CaATBZ1-silenced pepper plants displayed drought-tolerant phenotypes via ABA-mediated signalling. Our results demonstrated that CaASRF1-mediated ubiquitination plays a crucial role in regulating the stability of CaATBZ1. These findings provide valuable insight into the post-translational regulation of transcriptional factors.

Protein degradation by the ubiquitin/26S proteasome system is a critical process that modulates many eukaryotic cellular processes. E3 ligase usually modulates stress response by adjusting the stability of transcription factors. Previous studies have shown that a RING‐type E3 ligase, CaASRF1, positively modulates abscisic acid (ABA) signalling and ABA‐mediated drought response by modulating the stability of CaAIBZ1 and CaATBZ1. In this study, we conducted yeast two‐hybrid (Y2H) screening with CaATBZ1 to isolate an additional modulator, identified as CaATIR1 (Capsicum annuum ATBZ1 Interacting RING finger protein 1). CaATIR1 has E3 ligase activity and promoted CaATBZ1 degradation using the 26S proteasome system. We investigated the loss‐of and gain‐of functions of this E3 ligase by using silencing pepper and overexpressing (OX) Arabidopsis plants, respectively. In response to ABA and drought treatments, CaATIR1‐silenced pepper plants showed ABA insensitive and drought‐sensitive phenotypes, while CaATIR1‐OX plants showed the opposite phenotypes. Additionally, CaATBZ1‐silencing rescued the ABA insensitive and drought‐sensitive phenotypes of CaATIR1‐silencing pepper plants. Taken together, these data demonstrate that the stability of CaATBZ1 mediated by CaATIR1 has a crucial role in drought stress signalling in pepper plants.

: Under drought stress, plants have developed various mechanisms to survive in the reduced water supply, of which the regulation of stress-related gene expression is responsible for several transcription factors. The basic leucine zippers (bZIPs) are one of the largest and most diverse transcription factor families in plants. Among the 10 Arabidopsis bZIP groups, group A bZIP transcription factors function as a positive or negative regulator in ABA signal transduction and drought stress response. These bZIP transcription factors, which are involved in the drought response, have also been isolated in various plant species such as rice, pepper, potato, and maize. Recent studies have provided substantial evidences that many bZIP transcription factors undergo the post-translational modifications, through which the regulation of their activity or stability affects plant responses to various intracellular or extracellular stimuli. This review aims to address the modulation of the bZIP proteins in ABA signaling and drought responses through phosphorylation, ubiquitination, and sumoylation.

Drought negatively affects plant growth and development, thereby leading to loss of crop productivity. Several plant E3 ubiquitin ligases act as positive or negative regulators of abscisic acid (ABA) and thus play important roles in the drought stress response. Here, we show that the C3HC4-type RING finger E3 ligase, CaDTR1, regulates the drought stress response via ABA-mediated signalling. CaDTR1 contains an amino-terminal RING finger motif and two carboxyl-terminal hydrophobic regions; the RING finger motif functions during attachment of ubiquitins to the target proteins, and the carboxyl-terminal hydrophobic regions function during subcellular localisation. The expression of CaDTR1 was induced by ABA, drought, and NaCl treatments. CaDTR1 localised in the nucleus and displayed in vitro E3 ubiquitin ligase activity. CaDTR1-silenced pepper plants exhibited a drought-sensitive phenotype characterised by high levels of transpirational water loss. On the other hand, CaDTR1-overexpressing (OX) Arabidopsis plants exhibited an ABA-hypersensitive phenotype during the germinative and post-germinative growth stages. Moreover, in contrast to CaDTR1-silenced pepper plants, CaDTR1-OX plants exhibited a drought-tolerant phenotype characterised by low levels of transpirational water loss via increased stomatal closure and high leaf temperatures. Our data indicate that CaDTR1 functions as a positive regulator of the drought stress response via ABA-mediated signalling.