Pepper SUMO protease CaDeSI2 positively modulates the drought responses via deSUMOylation of clade A PP2C CaAITP1.

Posttranslational modification of multiple ABA signaling components is an essential process for the adaptation and survival of plants under stress conditions. In our previous study, we established that the pepper group A PP2C protein CaAITP1, one of the core components of ABA signaling, undergoes ubiquitination mediated by the RING-type E3 ligase CaAIRE1. In this study, we discovered an additional form of regulation mediated via the SUMOylation of CaAITP1. Pepper plants subjected to drought stress were characterized by reductions in both the stability and SUMOylation of CaAITP1 protein. Moreover, we identified a SUMO protease, Capsicum annuum DeSUMOylating Isopeptidase 2 (CaDeSI2), as a new interacting partner of CaAITP1. In vitro and in vivo analyses revealed that CaAITP1 is deSUMOylated by CaDeSI2. Silencing of CaDeSI2 in pepper plants led to drought-hypersensitive and ABA-hyposensitive phenotypes, whereas overexpression of CaDeSI2 in transgenic Arabidopsis plants resulted in the opposite phenotypes. Importantly, we found that the CaAITP1 protein was stabilized in response to the silencing of CaDeSI2, and CaDeSI2 and CaAITP1 co-silenced pepper plants were characterized by drought-tolerant phenotypes similar to those observed in CaAITP1-silenced pepper. Collectively, our findings indicate that CaDeSI2 reduces the stability of CaAITP1 via deSUMOylation, thereby positively regulating drought tolerance.

Roles of the pepper JAZ protein CaJAZ1-03 and its interacting partner RING-type E3 ligase CaASRF1 in regulating ABA signaling and drought responses.

Plants have developed various defense mechanisms against environmental stresses by regulating hormone signaling. Jasmonic acid (JA) is a major phytohormone associated with plant defense responses. JASMONATE ZIM-DOMAIN (JAZ) proteins play a regulatory role in repressing JA signaling, impacting plant responses to both biotic and abiotic stresses. Here, we isolated 7 JAZ genes in pepper and selected CA03g31030, a Capsicum annuum JAZ1-03 (CaJAZ1-03) gene, for further study based on its expression level in response to abiotic stresses. Through virus-induced gene silencing (VIGS) in pepper and overexpression in transgenic Arabidopsis plants, we established the functional role of CaJAZ1-03. Functional studies revealed that CaJAZ1-03 dampens abscisic acid (ABA) signaling and drought stress responses. The cell-free degradation assay showed faster degradation of CaJAZ1-03 in drought- or ABA-treated pepper leaves compared to healthy leaves. Conversely, CaJAZ1-03 was completely preserved under MG132 treatment, indicating that CaJAZ1-03 stability is modulated via the ubiquitin-26s proteasome pathway. We also found that the pepper RING-type E3 ligase CaASRF1 interacts with and ubiquitinates CaJAZ1-03. Additional cell-free degradation assays revealed a negative correlation between CaJAZ1-03 and CaASRF1 expression levels. Collectively, these findings suggest that CaJAZ1-03 negatively regulates ABA signaling and drought responses and that its protein stability is modulated by CaASRF1.

Pepper SUMO E3 ligase CaDSIZ1 enhances drought tolerance by stabilizing the transcription factor CaDRHB1.

Small ubiquitin-like modifier (SUMO) conjugation (SUMOylation) is a reversible post-translational modification associated with protein stability and activity, and modulates hormone signaling and stress responses in plants. Previously, we reported that the pepper dehydration-responsive homeobox domain transcription factor CaDRHB1 acts as a positive modulator of drought response. Here, we show that CaDRHB1 protein stability is enhanced by SUMO E3 ligase Capsicum annuum DRHB1-interacting SAP and Miz domain (SIZ1) (CaDSIZ1)-mediated SUMOylation in response to drought, thereby positively modulating abscisic acid (ABA) signaling and drought responses. Substituting lysine (K) 138 of CaDRHB1 with arginine reduced CaDSIZ1-mediated SUMOylation, indicating that K138 is the principal site for SUMO conjugation. Virus-induced silencing of CaDSIZ1 promoted CaDRHB1 degradation, suggesting that CaDSIZ1 is involved in drought-induced SUMOylation of CaDRHB1. CaDSIZ1 interacted with and facilitated SUMO conjugation of CaDRHB1. CaDRHB1, mainly localized in the nucleus, but also in the cytoplasm in the SUMOylation mimic state, suggesting that SUMOylation of CaDRHB1 promotes its nuclear export, leading to cytoplasmic accumulation. Moreover, CaDSIZ1-silenced pepper plants were less sensitive to ABA and considerably sensitive to drought stress, whereas CaDSIZ1-overexpressing plants displayed ABA-hypersensitive and drought-tolerant phenotypes. Collectively, our data indicate that CaDSIZ1-mediated SUMOylation of CaDRHB1 functions in ABA-mediated drought tolerance.

Post-translational Modifications of bZIP Transcription Factors in Abscisic Acid Signaling and Drought Responses.

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 evidence 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.

The pepper RING-type E3 ligase, CaATIR1, positively regulates abscisic acid signalling and drought response by modulating the stability of CaATBZ1.

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.

Roles of pepper bZIP transcription factor CaATBZ1 and its interacting partner RING-type E3 ligase CaASRF1 in modulation of ABA signalling and drought tolerance.

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 CaATBZ1-silenced 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.

A pepper RING-type E3 ligase, CaASRF1, plays a positive role in drought tolerance via modulation of CaAIBZ1 stability.

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 ABA-mediated 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.

The Pepper RING Finger E3 Ligase, CaDIR1, Regulates the Drought Stress Response via ABA-Mediated Signaling.

Drought stress from soil or air limits plant growth and development, leading to a reduction in crop productivity. Several E3 ligases positively or negatively regulate the drought stress response. In the present study, we show that the pepper (Capsicum annuum) Drought Induced RING type E3 ligase 1, CaDIR1, regulates the drought stress response via abscisic acid (ABA)-mediated signaling. CaDIR1 contains a C3HC4-type RING finger domain in the N-terminal region; this domain functions during protein degradation via attachment of ubiquitins to the substrate target proteins. The expression levels of the CaDIR1 gene were suppressed and induced by ABA and drought treatments, respectively. We conducted loss-of-function and gain-of function genetic studies to examine the in vivo function of CaDIR1 in response to ABA and drought stress. CaDIR1-silenced pepper plants displayed a drought-tolerant phenotype characterized by a low level of transpirational water loss via increased stomatal closure and elevated leaf temperatures. CaDIR1-overexpressing (OX) Arabidopsis plants exhibited an ABA-hypersensitive phenotype during the germination stage, but an ABA-hyposensitive phenotype-characterized by decreased stomatal closure and reduced leaf temperatures-at the adult stage. Moreover, adult CaDIR1-OX plants exhibited a drought-sensitive phenotype characterized by high levels of transpirational water loss. Our results indicate that CaDIR1 functions as a negative regulator of the drought stress response via ABA-mediated signaling. Our findings provide a valuable insight into the plant defense mechanism that operates during drought stress.

Pepper CaREL1, a ubiquitin E3 ligase, regulates drought tolerance via the ABA-signalling pathway.

Drought stress conditions in soil or air hinder plant growth and development. Here, we report that the hot pepper (C apsicum a nnuum) RING type E3 Ligase 1 gene (CaREL1) is essential to the drought stress response. CaREL1 encodes a cytoplasmic- and nuclear-localized protein with E3 ligase activity. CaREL1 expression was induced by abscisic acid (ABA) and drought. CaREL1 contains a C3H2C3-type RING finger motif, which functions in ubiquitination of the target protein. We used CaREL1-silenced pepper plants and CaREL1-overexpressing (OX) transgenic Arabidopsis plants to evaluate the in vivo function of CaREL1 in response to drought stress and ABA treatment. CaREL1-silenced pepper plants displayed a drought-tolerant phenotype characterized by ABA hypersensitivity. In contrast, CaREL1-OX plants exhibited ABA hyposensitivity during the germination, seedling, and adult stages. In addition, plant growth was severely impaired under drought stress conditions, via a high level of transpirational water loss and decreased stomatal closure. Quantitative RT-PCR analyses revealed that ABA-related drought stress responsive genes were more weakly expressed in CaREL1-OX plants than in wild-type plants, indicating that CaREL1 functions in the drought stress response via the ABA-signalling pathway. Taken together, our results indicate that CaREL1 functions as a negative regulator of ABA-mediated drought stress tolerance.

Identification and functional expression of the pepper RING type E3 ligase, CaDTR1, involved in drought stress tolerance via ABA-mediated signalling.

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.