Several E3 ubiquitin ligases have been associated with the response to abiotic and biotic stresses in higher plants. Here, we report that the hot pepper (Capsicumannuum) abscisic acid (ABA)-InsensitiveRINGprotein1gene(CaAIR1) is essential for a hypersensitive response to drought stress. CaAIR1 contains a C3HC4-type RING finger motif, which plays a role for attachment of ubiquitins to the target protein, and a putative transmembrane domain. The expression levels of CaAIR1 are upregulated in pepper leaves by ABA treatments, drought, and NaCl, suggesting its role in the response to abiotic stress. Our analysis showed that CaAIR1 displays self-ubiquitination and localized in the nucleus. We generated CaAIR1-silenced peppers via virus-induced gene silencing (VIGS) and CaAIR1-overexpressing (OX) transgenic Arabidopsis plants to evaluate their responses to ABA and drought. VIGS of CaAIR1 in pepper plants conferred an enhanced tolerance to drought stress, which was accompanied by low levels of transpirational water loss in the drought-treated leaves. CaAIR1-OX plants displayed an impaired sensitivity to ABA during seed germination, seedling, and adult stages. Moreover, these plants showed enhanced sensitivity to drought stress because of reduced stomatal closure and decreased expression of stress-responsive genes. Thus, our data indicate that CaAIR1 is a negative regulator of the ABA-mediated drought-stress tolerance mechanism.

Drought and high salinity are the most important abiotic factors limiting plant development, growth, and crop productivity in agriculture (Munns and Tester 2008, Sengupta and Majumder 2009, Zhu 2002). As sessile organisms, plants are frequently exposed to drought and high salinity conditions, which alter water potential and cause osmotic stress, leading to serious damage to plant tissues (Bartels and Sunkar 2005, Boudsocq and Lauriere 2005). During exposure to water stress, plants display many physiological changes, such as reduction of water content, closure of stomata, and decreased cell enlargement and growth. In addition, severe and continuous water stress in plants causes the cessation of photosynthesis and disturbance of metabolism, and finally results in death (Nath et al. 2005, Shao et al. 2008). To adapt to these abiotic stress conditions, plants show a variety of responses, including the accumulation of abscisic acid (ABA) and expression of a large number of stress-related proteins (Krasensky and Jonak 2012, Lee and Luan 2012, Skriver and Mundy 1990, Stewart and Lee 1974). Although the cellular and molecular responses to environmental stress are well studied (Hasegawa et al. 2000, Thomashow 1999), the mechanisms underlying the functional modifications caused by osmotic stress are yet to be clarified, because of the complexity at the cellular level as well as at the whole plant level (Ashraf and Harris 2004, Flowers 2004, Foolad et al. 2003a, 2003b, Xiong et al. 2002).

Plants are constantly exposed to a variety of biotic and abiotic stresses, which include pathogens and conditions of high salinity, low temperature, and drought. Abscisic acid (ABA) is a major plant hormone involved in signal transduction pathways that mediate the defense response of plants to abiotic stress. Previously, we isolated Ring finger protein gene (CaRING1) frompepper(Capsicum annuum), which is associated with resistance to bacterial pathogens, accompanied by hypersensitive cell death. Here, we report a new function of the CaRING1 gene product in the ABA-mediated defense responses of plants to drought stress. The expression of the CaRING1 gene was induced in pepper leaves treated with ABA or exposed to drought or NaCl. CaRING1-overexpressing (OX) transgenic plants showed enhanced sensitivity to ABA during the seedling growth and establishment. Furthermore, these plants were more tolerant to drought stress than the wild-type plants because of enhanced stomatal closure and increased expression of stress-responsive genes. Together, these results suggest that the CaRING1 acts as positive factor for drought tolerance in Arabidopsis by modulating ABA-mediated stomatal closing and gene expression.

A pepper bZIP transcription factor gene, CabZIP2, was isolated from pepper leaves infected with an a virulent strain of Xanthomonas campestris pv. vesicatoria (Xcv). Transient expression analysis of the CabZIP2-GFP fusion protein in Nicotiana benthamiana revealed that the CabZIP2 protein is localized in the cytoplasm as well as the nucleus. The acidic domain in the N-terminal region of CabZIP2 that is fused to the GAL4 DNA-binding domain is required to activate the transcription of reporter genes in yeast. Transcription of CabZIP2 is induced in pepper plants inoculated with virulent or avirulent strains of Xcv. The CabZIP2 gene is also induced by defense-related hormones such as salicylic acid, methyl jasmonate, and ethylene. To elucidate the in vivo function of the CabZIP2 gene in plant defense, virus-induced gene silencing (VIGS) in pepper and overexpression in Arabidopsis were used. CabZIP2-silenced pepper plants were susceptible to infection by the virulent strain of Xcv, which was accompanied by reduced expression of defense-related genes such as CaBPR1 and CaAMP1. CabZIP2 overexpression (OX) in transgenic Arabidopsis plants conferred enhanced resistance to Pseudomonas syringae pv. tomato DC3000. Together, these results suggest that CabZIP2 is involved in bacterial disease resistance.

In plants, lipoxygenases (LOXs) are involved in various physiological processes, including defense responses to biotic and abiotic stresses. Our previous study has shown that pepper 9-LOX gene, CaLOX1, plays a crucial role in cell death due to pathogen infection. Here, the function of CaLOX1 in response to osmotic, drought, and high salinity was examined using CaLOX1-overexpressing (CaLOX1-OX) Arabidopsis plants. Changes in the temporal expression pattern of the CaLOX1 gene were observed when pepper leaves were treated with drought and high salinity, but not with abscisic acid (ABA), the primary hormone in response to drought stress. During seed germination and seedling development, CaLOX1-OX plants were more tolerant to ABA, mannitol, and high salinity than wild-type plants. In contrast, expression of the ABA-responsive marker genes RAB18 and RD29B was higher in CaLOX1-OX Arabidopsis plants than in wild-type plants. In response to high salinity, CaLOX1-OX plants exhibited enhanced tolerance, compared with wild-type, which is accompanied by decreased accumulation of H2O2 and high levels of RD20, RD29A, RD29B, and P5CS gene expressions. Similarly, CaLOX1-OX plants were also more tolerant than wild-type plants to severe drought stress. H2O2 production and relative increase of lipid peroxidation were lower, and the expression of COR15A, DREB2A, RD20, RD29A, and RD29B was higher in CaLOX1-OX plants, relative to those of wild-type plants. Taken together, our results indicate that CaLOX1 plays a crucial role in plant stress responses by modulating the expression of ABA- and stress-responsive marker genes, lipid peroxidation, and H2O2 production.

Stomata are natural pores of plants and constitute the entry points for water during transpiration. However, they also facilitate the ingress of potentially harmful bacterial pathogens. The phytohormone abscisic acid (ABA) plays a pivotal role in protecting plants against biotic stress, by regulating stomatal closure. In the present study, we investigated the mechanism whereby ABA influences plant defense responses to Pseudomonas syringae pv. tomato (Pst) DC3000, which is a virulent bacterial pathogen of Arabidopsis, at the pre-invasive stage. We found that overexpression of two ABA receptors, namely, RCAR4/PYL10-OX and RCAR5/PYL11-OX (hereafter referred to as RCARs), resulted in ABA-hypersensitive phenotypes being exhibited during the seed germination and seedling growth stages. Sensitivity to ABA enhanced the resistance of RCAR4-OX and RCAR5-OX plants to Pst DC3000, through promoting stomatal closure leading to the development of resistance to this bacterial pathogen. Protein phosphatase HAB1 is an important component that is responsible for ABA signaling and which interacts with ABA receptors. We found that hab1 mutants exhibited enhanced resistance to Pst DC3000; moreover, similar to RCAR4-OX and RCAR5-OX plants, this enhanced resistance was correlated with stomatal closure. Taken together, our findings demonstrate that alteration of RCAR4- or RCAR5-HAB1 mediated ABA signaling influences resistance to bacterial pathogens via stomatal regulation.