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.

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.

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.