Pepper G-type Lectin Receptor-like Kinase, CaRLK1, Modulates ABA-Mediated Stomatal Closure and Drought Tolerance.

Abscisic acid (ABA) is a plant hormone that plays a key role in the environmental stress response, especially the induction of ABA-responsive and stress-responsive genes and modulation of the stomatal aperture in response to drought stress. Here, we identified CaDILZ1 (Capsicum annuum Drought-Induced Leucine Zipper 1) belonging to subgroup D of the bZIP protein family; gene functions of this family in response to ABA and drought signaling still remain unknown. CaDILZ1 expression was significantly induced in pepper leaves after exposure to ABA, drought, and NaCl. The CaDILZ1 protein localized in the nucleus of plant cells. In response to drought stress, CaDILZ1-silenced pepper and CaDILZ1-overexpressing Arabidopsis plants exhibited drought-sensitive and drought-tolerant phenotypes, respectively, via altered ABA content, stomatal closure, and expression of ABA-responsive and drought-responsive marker genes. We isolated the RING finger protein CaDSR1 (Capsicum annuum Drought Sensitive RING finger protein 1), which interacted with CaDILZ1 in the nucleus. The CaDSR1 protein exhibited E3 ligase activity and promoted CaDILZ1 degradation via the 26S proteasome pathway. Under drought stress conditions, CaDSR1-silenced pepper and CaDSR1-overexpressing Arabidopsis plants exhibited contrasting phenotypes to those of CaDILZ1-silenced pepper and CaDILZ1-overexpressing Arabidopsis plants. Taken together, our data suggest that CaDSR1 and CaDILZ1 function in ABA-mediated drought stress signaling in pepper plants.

In response to drought stress, land plants close their stomata to minimize transpiration. This action precedes a gradual accumulation of the stress hormone abscisic acid (ABA) that enhances plant drought tolerance. However, the molecular mechanisms that cause the time lag between the onset of stomatal closure and ABA accumulation and coordinate these two phases remain unexplained. Here, we found that Arabidopsis thaliana loss-of-function CLAVATA3/ENDOSPERM SURROUNDING REGION 5 (CLE5) mutants are less tolerant to drought. The CLE5 dodecapeptide (CLE5p) acts as a local signal to induce stomatal closure by binding to the LEUCINE-RICH REPEAT RECEPTOR-LIKE KINASE (LRR-RLK) receptor complex, BARELY ANY MERISTEM 1 (BAM1)–GUARD CELL HYDROGEN PEROXIDE-RESISTANT 1 (GHR1), in guard cells. The BAM1–GHR1–CLE5p module directly phosphorylates two SNF1-related protein kinases, OPEN STOMATA1 (SRK2E) and SRK2D, the central regulators of drought responses in plants, to regulate stomatal movement and drought-responsive gene expression without stimulating ABA biosynthesis or ROS accumulation. Our findings mark a critical step in understanding how plants promptly counteract environmental stresses. The CLEp–LRR-RLK signalling components are highly conserved across plant phyla, suggesting that peptide-mediated rapid stomatal closure is a widespread survival strategy and can be exploited to generate drought-resistant crops.

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.

Drought is one of the most important abiotic stress factors impeding crop productivity. With the uncovering of their role as potential regulators of gene expression, microRNAs (miRNAs) have been recognized as new targets for developing stress resistance. MicroRNAs are small noncoding RNAs whose abundance is significantly altered under stress conditions. Interestingly, plant miRNAs predominantly targets transcription factors (TFs), and some of which are also the most critical drought-responsive genes that in turn could regulate the expression of numerous loci with drought-adaptive potential. The phytohormone ABA plays important roles in regulating stomatal conductance and in initiating an adaptive response to drought stress. miRNAs are implicated in regulating ABA-(abscisic acid) and non-ABA-mediated drought resistance pathways. For instance, miR159-MYB module and miR169-NFYA module participates in an ABA-dependent pathway, whereas several other ABA-independent miRNA-target modules (miR156-SPL; miR393-TIR1; miR160-ARF10, ARF16, ARF17; miR167-ARF6 and ARF8; miR390/TAS3siRNA-ARF2, ARF3, ARF4) collectively regulate drought responses in plants. Overall, miRNA-mediated drought response manifests diverse molecular, biochemical and physiological processes. Because of their immense role in controlling gene expression, miRNA manipulation has significant potential to augment plant tolerance to drought stress. This review compiles the current understanding of drought-responsive miRNAs in major cereals. Also, potential miRNA manipulation strategies currently in use along with the challenges and future perspectives are discussed.

Drought is one of the major constraints to rain-fed agricultural production, especially under climate change conditions. Plants evolved an array of adaptive strategies that perceive stress stimuli and respond to these stress signals through specific mechanisms. Abscisic acid (ABA) is a premier signal for plants to respond to drought and plays a critical role in plant growth and development. ABA triggers a variety of physiological processes such as stomatal closure, root system modulation, organizing soil microbial communities, activation of transcriptional and post-transcriptional gene expression, and metabolic alterations. Thus, understanding the mechanisms of ABA-mediated drought responses in plants is critical for ensuring crop yield and global food security. In this review, we highlighted how plants adjust ABA perception, transcriptional levels of ABA- and drought-related genes, and regulation of metabolic pathways to alter drought stress responses at both cellular and the whole plant level. Understanding the synergetic role of drought and ABA will strengthen our knowledge to develop stress-resilient crops through integrated advanced biotechnology approaches. This review will elaborate on ABA-mediated drought responses at genetic, biochemical, and molecular levels in plants, which is critical for advancement in stress biology research.

Water limitation of plants causes stomatal closure to prevent water loss by transpiration. For this purpose, progressing soil water deficit is communicated from roots to shoots. Abscisic acid (ABA) is the key signal in stress-induced stomatal closure, but ABA as an early xylem-delivered signal is still a matter of debate. In this study, poplar plants (Populus × canescens) were exposed to water stress to investigate xylem sap sulfate and ABA, stomatal conductance, and sulfate transporter (SULTR) expression. In addition, stomatal behavior and expression of ABA receptors, drought-responsive genes, transcription factors, and NCED3 were studied after feeding sulfate and ABA to detached poplar leaves and epidermal peels of Arabidopsis (Arabidopsis thaliana). The results show that increased xylem sap sulfate is achieved upon drought by reduced xylem unloading by PtaSULTR3;3a and PtaSULTR1;1, and by enhanced loading from parenchyma cells into the xylem via PtaALMT3b. Sulfate application caused stomatal closure in excised leaves and peeled epidermis. In the loss of sulfate-channel function mutant, Atalmt12, sulfate-triggered stomatal closure was impaired. The QUAC1/ALMT12 anion channel heterologous expressed in oocytes was gated open by extracellular sulfate. Sulfate up-regulated the expression of NCED3, a key step of ABA synthesis, in guard cells. In conclusion, xylem-derived sulfate seems to be a chemical signal of drought that induces stomatal closure via QUAC1/ALMT12 and/or guard cell ABA synthesis.

Drought is a major environmental stress that threatens crop production. Therefore, identification of genes involved in drought stress response is of vital importance to decipher the molecular mechanism of stress signal transduction and breed drought tolerance crops, especially for maize. Clade A PP2C phosphatases are core abscisic acid (ABA) signaling components, regulating ABA signal transduction and drought response. However, the roles of other clade PP2Cs in drought resistance remain largely unknown. Here, we discovered a clade F PP2C, ZmPP84, that negatively regulates drought tolerance by screening a transgenic overexpression maize library. Quantitative RT-PCR indicates that the transcription of ZmPP84 is suppressed by drought stress. We identified that ZmMEK1, a member of the MAPKK family, interacts with ZmPP84 by immunoprecipitation and mass spectrometry analysis. Additionally, we found that ZmPP84 can dephosphorylate ZmMEK1 and repress its kinase activity on the downstream substrate kinase ZmSIMK1, while ZmSIMK1 is able to phosphorylate S-type anion channel ZmSLAC1 at S146 and T520 invitro. Mutations of S146 and T520 to phosphomimetic aspartate could activate ZmSLAC1 currents in Xenopus oocytes. Taken together, our study suggests that ZmPP84 is a negative regulator of drought stress response that inhibits stomatal closure through dephosphorylating ZmMEK1, thereby repressing ZmMEK1-ZmSIMK1 signaling pathway.

Membranes are primary sites of perception of environmental stimuli. Polyunsaturated fatty acids are major structural constituents of membranes that also function as modulators of a multitude of signal transduction pathways evoked by environmental stimuli. Different stresses induce production of a distinct blend of oxygenated polyunsaturated fatty acids, "oxylipins." We employed three Arabidopsis (Arabidopsis thaliana) ecotypes to examine the oxylipin signature in response to specific stresses and determined that wounding and drought differentially alter oxylipin profiles, particularly the allene oxide synthase branch of the oxylipin pathway, responsible for production of jasmonic acid (JA) and its precursor 12-oxo-phytodienoic acid (12-OPDA). Specifically, wounding induced both 12-OPDA and JA levels, whereas drought induced only the precursor 12-OPDA. Levels of the classical stress phytohormone abscisic acid (ABA) were also mainly enhanced by drought and little by wounding. To explore the role of 12-OPDA in plant drought responses, we generated a range of transgenic lines and exploited the existing mutant plants that differ in their levels of stress-inducible 12-OPDA but display similar ABA levels. The plants producing higher 12-OPDA levels exhibited enhanced drought tolerance and reduced stomatal aperture. Furthermore, exogenously applied ABA and 12-OPDA, individually or combined, promote stomatal closure of ABA and allene oxide synthase biosynthetic mutants, albeit most effectively when combined. Using tomato (Solanum lycopersicum) and Brassica napus verified the potency of this combination in inducing stomatal closure in plants other than Arabidopsis. These data have identified drought as a stress signal that uncouples the conversion of 12-OPDA to JA and have revealed 12-OPDA as a drought-responsive regulator of stomatal closure functioning most effectively together with ABA.

Raising crops for dry and saline lands: Challenges and the way forward.

Ethylene, a plant hormone that significantly influences both plant growth and response to stress, plays a well-established role in stress signaling. However, its impact on stomatal opening and closure during dehydration and rehydration remains relatively unexplored and is still debated. Exogenous ethylene has been proven to induce stomatal closure through a series of signaling pathways, including the accumulation of reactive oxygen species, subsequent synthesis of nitric oxide and hydrogen sulfide, and SLOW ANION CHANNEL-ASSOCIATED 1 activation. Thus, it has been suggested that ethylene might function to induce stomatal closure synergistically with abscisic acid (ABA). Furthermore, it has also been shown that increased ethylene can inhibit ABA- and jasmonic acid-induced stomatal closure, thus hindering drought-induced closure during dehydration. Simultaneously, other stresses, such as chilling, ozone pollution, and K+ deficiency, inhibit drought- and ABA-induced stomatal closure in an ethylene synthesis-dependent manner. However, ethylene has been shown to take on an opposing role during rehydration, preventing stomatal opening in the absence of ABA through its own signaling pathway. These findings offer novel insights into the function of ethylene in stomatal regulation during dehydration and rehydration, giving a better understanding of the mechanisms underlying ethylene-induced stomatal movement in seed plants.

Nature provides all necessary components for healthy growth and development of plants in the form of air, water, light, nutrients, and soil. Any imbalance in the environmental harmony may cause stress to them. Stresses encountered by plants can broadly be categorized into biotic and abiotic stresses. Biotic stresses are mainly caused by pathogens and herbivory, whereas abiotic stresses include the threat imposed by drought, salinity, and extremes of temperature, heavy metals, and pollution. Drought stress is a major cause of yield instability in crops across diverse eco-geographic regions worldwide. A variety of biochemical, molecular, and physiological changes are manifested by plants in response to drought stress. The cellular abscisic acid (ABA) concentration increases on water deficit leading to the activation of a number of stress-responsive genes and the patterns of expression of these genes are very complex, with some genes being induced early while others respond slowly. In general, drought-responsive genes respond to salt and cold stresses as well as to exogenous ABA treatment. However, there are several genes, which express themselves in an ABA-independent manner suggesting that both ABA-dependent and -independent signal transduction cascades exist for drought stress perception, response, and adaptation. Drought stress response and adaptation in plants involves an array of pathways for signal perception, transduction, gene expression and synthesis of proteins, and other stress metabolites. Drought-responsive genes can mainly be classified into two groups. First group constitutes genes whose products provide osmotolerance and protection to plants thus directly functioning in tolerance to stress, while the second group includes genes playing a role in signal transduction as well as regulation of gene expression. This chapter summarizes the complex molecular mechanisms of drought stress response and adaptation in plants, highlighting the transcriptional regulation of stress-responsive gene expression. It also focuses on the recent advances in analyzing various stress-responsive pathways with prime emphasis on ABA-dependent and -independent pathways.

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.

Multiprotein bridging factor 1 (MBF1) is an evolutionarily conserved transcriptional co-activator in archaea and eukaryotes that has been demonstrated previously to play an important role in various types of stress response. In this study, a full-length MBF1 cDNA sequence (VvMBF1) was isolated from grape (Vitis labrusca × V. vinifera) and was found to be up-regulated in the leaves of grape plants following both drought and abscisic acid (ABA) treatments. Furthermore, constitutive expression of VvMBF1 in Arabidopsis thaliana enhanced drought stress tolerance in transgenic plants. To gain further insight into the role of VvMBF1 in drought resistance, we analyzed various physiological parameters related to stress response in transgenic Arabidopsis lines and found that transgenic plants were better able to prevent water loss under stress conditions than wild-type (WT) plants. This was likely due to an increase in the sensitivity of stomata to ABA, which is a well-known signaling molecule in plant drought response. In addition, dehydration stress yielded less cell damage to transgenic plants than WT plants. We also found that VvMBF1-expressing transgenic lines exhibited up-regulation of two drought-responsive genes that are known to function in the ABA-dependent drought-response pathway. Taken together, these results reveal that VvMBF1 is likely involved in drought-responsiveness in grape, and confers increased drought tolerance in transgenic plants, possibly through an ABA-dependent signal transduction pathway.

Chapter 13 - Drought Stress: Molecular Genetics and Genomics Approaches

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 tohave 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.