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.

Pepper U-Box E3 ubiquitin ligase 24, CaPUB24, negatively regulates drought stress response.

Under stress conditions, plants modulate their internal states and initiate various defence mechanisms to survive. The ubiquitin-proteasome system is one of the critical modules in these mechanisms, and Plant U-Box proteins play an important role in this process as E3 ubiquitin ligases. Here, we isolated the Plant U-box 24 gene CaPUB24 (Capsicum annuum Plant U-Box 24) from pepper and characterized its functions in response to drought stress. We found that, compared to the other CaPUBs in the same group, the expression of CaPUB24 was significantly induced by drought stress. We also found that CaPUB24 was localized to the nucleus and cytoplasm and had E3 ubiquitin ligase activity. To investigate the biological role of CaPUB24 in response to drought stress further, we generated CaPUB24-silenced pepper plants and CaPUB24-overexpressing Arabidopsis transgenic plants. CaPUB24-silenced pepper plants exhibited enhanced drought tolerance compared to the control plants due to reduced transpirational water loss and increased abscisic acid (ABA) sensitivity. In contrast, CaPUB24-overexpressing Arabidopsis transgenic plants exhibited reduced drought tolerance and ABA-insensitive phenotypes. Our findings suggest that CaPUB24 negatively modulates drought stress response in an ABA-dependent manner.

A pepper RING-finger E3 ligase, CaFIRF1, negatively regulates the high-salt stress response by modulating the stability of CaFAF1.

Controlling protein stability or degradation via the ubiquitin-26S proteasome system is a crucial mechanism in plant cellular responses to stress conditions. Previous studies have revealed that the pepper FANTASTIC FOUR-like gene, CaFAF1, plays a positive role in salt tolerance and that, in this process, CaFAF1 protein degradation is delayed. Here, we sought to isolate the E3 ligases potentially responsible for modulating CaFAF1 protein stability in response to salt stress. The pepper RING-type E3 ligase CaFIRF1 (Capsicum  annuum  FAF1  Interacting  RING  Finger protein  1) was found to interact with and ubiquitinate CaFAF1, leading to the degradation of CaFAF1 proteins. In response to high-salt treatments, CaFIRF1-silenced pepper plants exhibited tolerant phenotypes. In contrast, co-silencing of CaFAF1 and CaFIRF1 led to increased sensitivity to high-salt treatments, revealing that CaFIRF1 functions upstream of CaFAF1. A cell-free degradation analysis showed that high-salt treatment suppressed CaFAF1 protein degradation via the 26S proteasome pathway, in which CaFIRF1 is functionally involved. In addition, an in vivo ubiquitination assay revealed that CaFIRF1-mediated ubiquitination of CaFAF1 proteins was reduced by high-salt treatment. Taken together, these findings suggest that the degradation of CaFAF1 mediated by CaFIRF1 has a critical role in pepper plant responses to high salinity.

Tomato salt-responsive pseudo-response regulator 1, SlSRP1, negatively regulates the high-salt and dehydration stress responses.

Under severe environmental stress conditions, plants inhibit their growth and development and initiate various defense mechanisms to survive. The pseudo-response regulator (PRRs) genes have been known to be involved in fruit ripening and plant immunity in various plant species, but their role in responses to environmental stresses, especially high salinity and dehydration, remains unclear. Here, we focused on PRRs in tomato plants and identified two PRR2-like genes, SlSRP1 and SlSRP1H, from the leaves of salt-treated tomato plants. After exposure to dehydration and high-salt stresses, expression of SISRP1, but not SlSRP1H, was significantly induced in tomato leaves. Subcellular localization analysis showed that SlSRP1 was predominantly located in the nucleus, while SlSRP1H was equally distributed in the nucleus and cytoplasm. To further investigate the potential role of SlSRP1 in the osmotic stress response, we generated SISRP1-silenced tomato plants. Compared to control plants, SISRP1-silenced tomato plants exhibited enhanced tolerance to high salinity, as evidenced by a high accumulation of proline and reduced chlorosis, ion leakage, and lipid peroxidation. Moreover, SISRP1-silenced tomato plants showed dehydration-tolerant phenotypes with enhanced abscisic acid sensitivity and increased expression of stress-related genes, including SlRD29, SlAREB, and SlDREB2. Overall, our findings suggest that SlSRP1 negatively regulates the osmotic stress response.

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 homeobox abscisic acid signalling-related transcription factor 1, CaHAT1, plays a positive role in drought response.

Abscisic acid (ABA) signalling triggers drought resistance mediated by SNF1-related kinase 2s (SnRK2s), which transmits stress signals through the phosphorylation of several downstream factors. However, these kinases and their downstream targets remain elusive in pepper plants. This study aimed to isolate interacting partners of CaSnRK2.6, a homologue of Arabidopsis SnRK2.6/OST1. Among the candidate proteins, we identified a homeodomain-leucine zipper (HD-Zip) class II protein and named it CaHAT1 (Capsicum annuum homeobox ABA signalling related- transcription factor 1). CaHAT1-silenced pepper and -overexpression (OE) transgenic Arabidopsis plants were generated to investigate the in vivo function of CaHAT1 in drought response. Following the application of drought stress, CaHAT1-silenced pepper plants exhibited drought-sensitive phenotypes with reduced ABA-mediated stomatal closure and lower expression of stress-responsive genes compared with control plants. In contrast, CaHAT1-OE transgenic Arabidopsis plants showed the opposite phenotypes, including increased drought resistance and ABA sensitivity. CaHAT1, particularly its N-terminal consensus sequences, was directly phosphorylated by CaSnRK2.6. Furthermore, CaSnRK2.6 kinase activity and CaSnRK2.6-mediated CaHAT1 phosphorylation levels were enhanced by treatment with ABA and drought stress. Taken together, our results indicated that CaHAT1, which is the target protein of CaSnRK2.6, is a positive regulator of drought stress response. This study advances our understanding of CaHAT1-CaSnRK2.6 mediated defence mechanisms in pepper plants against drought stress.

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

The phytohormone abscisic acid (ABA) plays a prominent role in various abiotic stress responses of plants. In the ABA-dependent osmotic stress response, SnRK2.6, one of the subclass III SnRK2 kinases, has been identified as playing a key role by phosphorylating and activating downstream genes. Although several modulatory proteins have been reported to be phosphorylated by SnRK2.6, the identities of the full spectrum of downstream targets have yet to be sufficiently established. In this study, we identified CaSAP14, a stress-associated protein in pepper (Capsicum annuum), as a downstream target of CaSnRK2.6. We elucidated the physical interaction between SnRK2.6 and CaSAP14, both in vitro and in vivo, and accordingly identified a C-terminal C2H2-type zinc finger domain of CaSAP14 as being important for their interaction. CaSAP14-silenced pepper plants showed dehydration- and high salt-sensitive phenotypes, whereas overexpression of CaSAP14 in Arabidopsis conferred tolerance to dehydration, high salinity, and mannitol treatment, with plants showing ABA-hypersensitive phenotypes. Furthermore, an in-gel kinase assay revealed that CaSnRK2.6 phosphorylates CaSAP14 in response to exogenous ABA, dehydration, and high-salinity stress. Collectively, these findings suggest that CaSAP14 is a direct substrate of CaSnRK2.6 and positively modulates dehydration- and high salinity-induced osmotic stress responses.

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

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.

Role of pepper MYB transcription factor CaDIM1 in regulation of the drought response.

Abscisic acid (ABA) is a major phytohormone that plays important roles in stress responses, including regulation of gene expression and stomatal closure. Regulation of gene expression by transcription factors is a key cellular process for initiating defense responses to biotic and abiotic stresses. Here, using pepper (Capsicum annuum) leaves, we identified the MYB transcription factor CaDIM1 (Capsicum annuum Drought Induced MYB 1), which was highly induced by ABA and drought stress. CaDIM1 has an MYB domain in the N-terminal region and an acidic domain in the C-terminal region, which are responsible for recognition and transactivation of the target gene, respectively. Compared to control plants, CaDIM1-silenced pepper plants displayed ABA-insensitive and drought-sensitive phenotypes with reduced expression of stress-responsive genes. On the other hand, overexpression of CaDIM1 in Arabidopsis exhibited the opposite phenotypes of CaDIM1-silenced pepper plants, accompanied by enhanced ABA sensitivity and drought tolerance. Taken together, we demonstrate that CaDIM1 functions as a positive regulator of the drought-stress response via modulating ABA-mediated gene expression.

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.

Core Components of Abscisic Acid Signaling and Their Post-translational Modification.

Abscisic acid (ABA) is a major phytohormone that regulates plant growth, development, and abiotic/biotic stress responses. Under stress, ABA is synthesized in various plant organs, and it plays roles in diverse adaptive processes, including seed dormancy, growth inhibition, and leaf senescence, by modulating stomatal closure and gene expression. ABA receptor, clade A protein phosphatase 2C (PP2C), and SNF1-related protein kinase 2 (SnRK2) proteins have been identified as core components of ABA signaling, which is initiated via perception of ABA with receptor and subsequent activation or inactivation by phosphorylation/dephosphorylation. The findings of several recent studies have established that the post-translational modification of these components, including phosphorylation and ubiquitination/deubiquitination, play important roles in regulating their activity and stability. In this review, we discuss the functions of the core components of ABA signaling and the regulation of their activities via post-translational modification under normal and stress conditions.

Genome-wide identification and expression analysis of Raf-like kinase gene family in pepper (Capsicum annuum L.).

As highly conserved signaling pathway modules, mitogen-activated protein kinase (MAPK) cascades play vital roles in a diverse range of stress and hormonal responses in plants. Among the established components of MAPK cascades, Raf-like MAPK kinase kinases (MAPKKKs) are associated with abscisic acid (ABA) signaling and osmotic stress responses. However, despite the availability of a pepper reference genome, few of the Raf-like kinases in pepper plants have been functionally characterized. In this study, we isolated 47 putative Raf-like kinase genes from the pepper genome based on in silico analysis, which were classified into two major categories, namely, groups B and C (further sub-grouped into B1-B4 and C1-C7, respectively) and named sequentially as CaRaf1 to CaRaf47. Subcellular localization prediction analysis revealed that most of the group B CaRaf-like kinases are probably nuclear-localized, whereas a majority of group C members targeted into the cytoplasm. Transcriptional regulation of the 47 CaRaf genes in response to treatment with ABA, drought, NaCl, and mannitol was quantitatively analyzed by reverse-transcription PCR analysis. This revealed a significant induction of subgroup B3, C2, C3, and C5 members, indicating that these genes may be functionally associated with the response to osmotic stress, mediated via both ABA-dependent and -independent pathways. The findings of this study can accordingly serve as a basis for the identification of CaRaf genes associated with the regulation of ABA signaling and osmotic stress response and thus contribute to enhancing our understanding of the biological functions of CaRaf kinases in the responses of plants to different abiotic stresses.

Differential Functions of Pepper Stress-Associated Proteins in Response to Abiotic Stresses.

Stress-associated proteins (SAPs), a group of zinc-finger-type proteins, have been identified as novel regulators of plant abiotic and biotic stresses. However, although they have been discovered in different plant species, their precise functional roles remain unclear. Here, we identified 14 SAP subfamily genes in the pepper genome. An investigation of the promoter regions of these genes for cis-regulatory elements associated with abiotic stress responses revealed the presence of multiple stress-related elements. Domain and phylogenetic analyses using the corresponding protein sequences revealed that the CaSAP genes can be classified into six groups (I-VI) and sorted into two broad types. Expression levels of the CaSAP genes were found to be differentially induced by low temperature, the dehydration stress, or exogenous abscisic acid. Group II and IV genes were highly induced by the low temperature and dehydration treatments, respectively. Moreover, subcellular localization analysis indicated that the proteins in these two groups are distributed in the nucleus, cytoplasm, and plasma membrane. Among the pepper plants silenced with the three identified group II CaSAP genes, the CA02g10410-silenced plants showed tolerance to low temperature, whereas the CA03g17080-silenced plants were found to have temperature-sensitive phenotypes. Interestingly, group IV CaSAP-silenced pepper plants showed drought-tolerant phenotypes. These findings contribute to a preliminary characterization of CaSAP genes and provide directions for future research on the biological role of CaSAPs in response to different abiotic stresses.

Pepper Novel Pseudo Response Regulator Protein CaPRR2 Modulates Drought and High Salt Tolerance.

Plants modify their internal states to adapt to environmental stresses. Under environmental stress conditions, plants restrict their growth and development and activate defense responses. Abscisic acid (ABA) is a major phytohormone that plays a crucial role in the osmotic stress response. In osmotic stress adaptation, plants regulate stomatal closure, osmoprotectant production, and gene expression. Here, we isolated CaPRR2 - encoding a pseudo response regulator protein - from the leaves of pepper plants (Capsicum annuum). After exposure to ABA and environmental stresses, such as drought and salt stresses, CaPRR2 expression in pepper leaves was significantly altered. Under drought and salt stress conditions, CaPRR2-silenced pepper plants exhibited enhanced osmotic stress tolerance, characterized by an enhanced ABA-induced stomatal closing and high MDA and proline contents, compared to the control pepper plants. Taken together, our data indicate that CaPRR2 negatively regulates osmotic stress tolerance.

Tobacco ubiquitin-specific protease 12 (NbUBP12) positively modulates drought resistance.

Deubiquitination, a type of post-translational modification, cleaves ubiquitin from target proteins, thereby regulating their stability or activity. Deubiquitination enzymes, ubiquitin-specific proteases (UBP/USP), have been reported to be involved in numerous cellular processes in plants, including meristem development, circadian clock regulation, and immunity. In contrast to model plants, however, the functions of UBP in other higher plants remain poorly understood. Here, we isolated a deubiquitination enzyme, ubiquitin-specific protease 12 (NbUBP12), from Nicotiana benthamiana, which shows high sequence homology with the core enzyme regions of UBP12 from other plants. Quantitative reverse-transcription PCR analysis revealed that NbUBP12 gene expression was significantly induced after drought treatment, and its level was higher in seed than in other tissues. Using a virus-induced gene silencing technique, we generated NbUBP12-silenced tobacco plants to analyze NbUBP12 gene function in response to drought stress and found that compared with control plants, NbUBP12-silenced plants exhibited a lower survival rate after exposure to drought stress. In addition, they were characterized by lower leaf surface temperatures and larger stomatal pore size following abscisic acid (ABA) treatment. On the basis of these observations, we suggest that NbUBP12 is involved in modulating drought resistance in N. benthamiana, which is associated with ABA-mediated stomatal closure.

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

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 ubiquitin-specific protease, CaUBP12, positively modulates dehydration resistance by enhancing CaSnRK2.6 stability.

Abscisic acid (ABA) is a plant hormone that activates adaptive mechanisms to environmental stress conditions. Plant adaptive mechanisms are complex and highly modulated processes induced by stress-responsive proteins; however, the precise mechanisms by which these processes function under adverse conditions remain unclear. Here, we isolated CaUBP12 (Capsicum annuum ubiquitin-specific protease 12) from pepper (C. annuum) leaves. We show that CaUBP12 expression is significantly induced after exposure to abiotic stress treatments. We conducted loss-of-function and gain-of-function genetic studies to elucidate the biological functions of CaUBP12 in response to ABA and dehydration stress. CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants displayed dehydration-sensitive and dehydration-tolerant phenotypes, respectively; these phenotypes were characterized by regulation of transpirational water loss and stomatal aperture. Under dehydration stress conditions, CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants exhibited lower and higher expression levels of stress-related genes, respectively, than the control plants. We isolated a CaUBP12 interaction protein, CaSnRK2.6, which is a homolog of Arabidopsis OST1; degradation of this protein was partially inhibited by CaUBP12. Similar to CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants, CaSnRK2.6-silenced pepper plants and CaSnRK2.6-overexpressing Arabidopsis displayed dehydration-sensitive and dehydration-tolerant phenotypes, respectively. Our findings suggest that CaUBP12 positively modulates the dehydration stress response by suppressing CaSnRK2.6 protein degradation.

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.

Mitogen-Activated Protein Kinase CaDIMK1 Functions as a Positive Regulator of Drought Stress Response and Abscisic Acid Signaling in Capsicum annuum.

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.

Pepper E3 ligase CaAIRE1 promotes ABA sensitivity and drought tolerance by degradation of protein phosphatase CaAITP1.

Plants have developed defense mechanisms to survive in extreme environmental conditions. Abscisic acid (ABA) is a key phytohormone associated with plant adaptation to environmental stress. In this study, we isolated and functionally characterized the pepper RING-type E3 ligase CaAIRE1 (Capsicum annuum ABA Induced RING-type E3 ligase 1) containing the C3HC4-type RING domain. CaAIRE1 was induced by ABA and drought, and CaAIRE1 had E3 ligase activity. CaAIRE1-silenced pepper and CaAIRE1-overexpressing Arabidopsis presented drought-sensitive and drought-tolerant phenotypes, respectively, which were accompanied by altered transpiration water loss and ABA sensitivity. Moreover, we found that CaAIRE1 interacts with and ubiquitinates the pepper type 2C protein phosphatase, CaAITP1 (Capsicum annuum CaAIRE1 Interacting Target Phosphatase 1). A cell-free degradation assay with CaAIRE1-silenced peppers and CaAIRE1-overexpressing Arabidopsis plants revealed that the CaAITP1 protein level was negatively modulated by the expression level of CaAIRE1. In contrast to CaAIRE1, CaAITP1-silenced pepper showed ABA-sensitivity phenotypes. CaAITP1-overexpressing Arabidopsis plants were the most insensitive phenotypes to ABA compared with the wild type and other pepper PP2C-overexpressing plants. Taken together, our data indicate that CaAITP1 plays a major role as a negative modulator in ABA signaling, and CaAIRE1 regulates the ABA signaling and drought response through modulation of CaAITP1 stability.