Genome-wide analysis of the PYL-PP2C-SnRK2s family in the ABA signaling pathway of pitaya reveals its expression profiles under canker disease stress.

BACKGROUND: Abscisic acid (ABA) plays a crucial role in seed dormancy, germination, and growth, as well as in regulating plant responses to environmental stresses during plant growth and development. However, detailed information about the PYL-PP2C-SnRK2s family, a central component of the ABA signaling pathway, is not known in pitaya. RESULTS: In this study, we identified 19 pyrabactin resistance-likes (PYLs), 70 type 2 C protein phosphatases (PP2Cs), and 14 SNF1-related protein kinase 2s (SnRK2s) from pitaya. In pitaya, tandem duplication was the primary mechanism for amplifying the PYL-PP2C-SnRK2s family. Co-linearity analysis revealed more homologous PYL-PP2C-SnRK2s gene pairs located in collinear blocks between pitaya and Beta vulgaris L. than that between pitaya and Arabidopsis. Transcriptome analysis showed that the PYL-PP2C-SnRK2s gene family plays a role in pitaya's response to infection by N. dimidiatum. By spraying ABA on pitaya and subsequently inoculating it with N. dimidiatum, we conducted qRT-PCR experiments to observe the response of the PYL-PP2C-SnRK2s gene family and disease resistance-related genes to ABA. These treatments significantly enhanced pitaya's resistance to pitaya canker. Further protein interaction network analysis helped us identify five key PYLs genes that were upregulated during the interaction between pitaya and N. dimidiatum, and their expression patterns were verified by qRT-PCR. Subcellular localization analysis revealed that the PYL (Hp1879) gene is primarily distributed in the nucleus. CONCLUSION: This study enhances our understanding of the response of PYL-PP2C-SnRK2s to ABA and also offers a new perspective on pitaya disease resistance.

APETALA2 is involved in ABA signaling during seed germination.

APETALA2 (AP2) is well known for regulating the development of floral organs, ovules, seed coats, and the mass of seeds, but the role of AP2 in seed germination remains unclear. Here, we report that AP2 interacts with ABI5 in nuclear speckles and functions in controlling seed germination. Genetic study showed that the abi5 mutation could restore the ABA-sensitive phenotype of ap2 mutants, supporting that AP2 antagonizes ABI5 in ABA signaling and ABA-mediated inhibition of seed germination. In addition, we observed the interactions of AP2 with SnRK2.2, SnRK2.3, and SnRK2.6 in nuclear speckles, suggesting that AP2 plays a multifaceted role in the ABA signaling pathway. Our findings revealed that the interactions of AP2 with SnRK2s and ABI5 are critical for ABA signaling in control of seed germination.

Protein Interactomic Analysis of SAPKs and ABA-Inducible bZIPs Revealed Key Roles of SAPK10 in Rice Flowering.

As core components of ABA signaling pathway, SnRK2s (Sucrose nonfermenting1⁻Related protein Kinase 2) bind to and phosphorylate AREB/ABF (ABA responsive element binding protein/ABRE-binding factor) transcriptional factors, particularly bZIPs (basic region-leucine zipper), to participate in various biological processes, including flowering. Rice contains 10 SnRK2 members denoted as SAPK1-10 (Stress-Activated Protein Kinase) and dozens of bZIPs. However, which of the SAPKs and bZIPs pair and involve in ABA signaling remains largely unknown. In this study, we carried out a systematical protein-protein interactomic analysis of 10 SAPKs and 9 ABA-inducible bZIPs using yeast-two-hybrid technique, and identified 14 positive interactions. The reliability of Y2H work was verified by in vitro pull-down assay of the key flowering regulator bZIP77 with SAPK9 and SAPK10, respectively. Moreover, SAPK10 could phosphorylate bZIP77 in vitro. Over-expression of SAPK10 resulted in earlier flowering time, at least partially through regulating the FAC-MADS15 pathway. Conclusively, our results provided an overall view of the SAPK-bZIP interactions, and shed novel lights on the mechanisms of ABA-regulated rice flowering.

CcRR5 interacts with CcRR14 and CcSnRK2s to regulate the root development in citrus.

Response regulator (RR) is an important component of the cytokinin (CK) signal transduction system associated with root development and stress resistance in model plants. However, the function of RR gene and the molecular mechanism on regulating the root development in woody plants such as citrus remain unclear. Here, we demonstrate that CcRR5, a member of the type A RR, regulates the morphogenesis of root through interacting with CcRR14 and CcSnRK2s in citrus. CcRR5 is mainly expressed in root tips and young leaves. The activity of CcRR5 promoter triggered by CcRR14 was proved with transient expression assay. Seven SnRK2 family members with highly conserved domains were identified in citrus. Among them, CcSnRK2.3, CcSnRK2.6, CcSnRK2.7, and CcSnRK2.8 can interact with CcRR5 and CcRR14. Phenotypic analysis of CcRR5 overexpressed transgenic citrus plants indicated that the transcription level of CcRR5 was associated with root length and lateral root numbers. This was also correlated to the expression of root-related genes and thus confirmed that CcRR5 is involved in the root development. Taken together, the results of this study indicate that CcRR5 is a positive regulator of root growth and CcRR14 directly regulates the expression of CcRR5. Both CcRR5 and CcRR14 can interact with CcSnRK2s.

Genome-wide investigation of SnRK2 gene family in two jute species: Corchorus olitorius and Corchorus capsularis.

BACKGROUND: Sucrose non-fermenting-1 (SNF1)-related protein kinase 2 (SnRK2), a plant-specific serine/threonine kinase family, is associated with metabolic responses, including abscisic acid signaling under biotic and abiotic stresses. So far, no information on a genome-wide investigation and stress-mediated expression profiling of jute SnRK2 is available. Recent whole-genome sequencing of two Corchorus species prompted to identify and characterize this SnRK2 gene family. RESULT: We identified seven SnRK2 genes of each of Corchorus olitorius (Co) and C. capsularis (Cc) genomes, with similar physico-molecular properties and sub-group patterns of other models and related crops. In both species, the SnRK2 gene family showed an evolutionarily distinct trend. Highly variable C-terminal and conserved N-terminal regions were observed. Co- and CcSnRK2.3, Co- and CcSnRk2.5, Co- and CcSnRk2.7, and Co- and CcSnRK2.8 were upregulated in response to drought and salinity stresses. In waterlogging conditions, Co- and CcSnRk2.6 and Co- and CcSnRK2.8 showed higher activity when exposed to hypoxic conditions. Expression analysis in different plant parts showed that SnRK2.5 in both Corchorus species is highly expressed in fiber cells providing evidence of the role of fiber formation. CONCLUSION: This is the first comprehensive study of SnRK2 genes in both Corchorus species. All seven genes identified in this study showed an almost similar pattern of gene structures and molecular properties. Gene expression patterns of these genes varied depending on the plant parts and in response to abiotic stresses.

Comprehensive functional analysis of the PYL-PP2C-SnRK2s family in Bletilla striata reveals that BsPP2C22 and BsPP2C38 interact with BsPYLs and BsSnRK2s in response to multiple abiotic stresses.

As the core regulation network for the abscisic acid (ABA) signaling pathway, the PYL-PP2C-SnRK2s family commonly exists in many species. For this study, a total of 9 BsPYLs, 66 BsPP2Cs, and 7 BsSnRK2s genes were identified based on the genomic databases of Bletilla striata, which were classified into 3, 10, and 3 subgroups, respectively. Basic bioinformatics analysis completed, including the physicochemical properties of proteins, gene structures, protein motifs and conserved domains. Multiple cis-acting elements related to stress responses and plant growth were found in promoter regions. Further, 73 genes were localized on 16 pseudochromosomes and 29 pairs of paralogous genes were found via intraspecific collinearity analysis. Furthermore, tissue-specific expression was found in different tissues and germination stages. There were two BsPYLs, 10 BsPP2Cs, and four BsSnRK2 genes that exhibited a difference in response to multiple abiotic stresses. Moreover, subcellular localization analysis revealed six important proteins BsPP2C22, BsPP2C38, BsPP2C64, BsPYL2, BsPYL8, and BsSnRK2.4 which were localized in the nucleus and plasma membrane. Finally, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays suggested that BsPP2C22 and BsPP2C38 could interact with multiple BsPYLs and BsSnRK2s proteins. This study systematically reported on the identification and characterization of the PYL-PP2C-SnRK2s family in B. striata, which provided a conceptual basis for deep insights into the functionality of ABA core signal pathways in Orchidaceae.

Plant Osmotic Stress Signaling: MAPKKKs Meet SnRK2s.

Osmotic stress signaling in higher plants is crucial to cope with abiotic stress. RAF-like MAPKKKs are activated by hyperosmotic stress and activate downstream ABA-unresponsive and ABA-activated SnRK2s, integrating early osmotic stress and ABA signaling cascades. The connection of B2/B3/B4 RAF-like MAPKKKs with SnRK2s is a new paradigm in signal transduction.

HOS15: A missing link that fine-tunes ABA signaling and drought tolerance in Arabidopsis.

Among the phytohormones, abscisic acid (ABA) specifically regulates plant adaptation to osmotic stresses, such as drought and high salinity, by controlling the internal water status in plants. A significant accumulation of ABA occurs in response to conditions of water deficit; this is followed by a sophisticated signaling relay, known as the ABA signaling pathway, which decreases the rate of transpiration through stomatal closure, thereby suppressing photosynthetic activity. Snf1-related kinases (SnRK2s) are the major components regulating the ABA signaling pathway. Of these, SnRK2.6 (OST1) and SnRK2.3 are negatively regulated by HOS15 (HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE15), in an ABA-dependent manner, to cease the signaling relay. HOS15 is a WD40-repeat protein that regulates several physiological processes, including plant growth and development, freezing stress responses, and ABA signaling. Here, we provide a brief overview of the functional importance of HOS15 in the regulation of ABA signaling and drought stress.

Arabidopsis Raf-Like Kinase Raf10 Is a Regulatory Component of Core ABA Signaling.

Abscisic acid (ABA) is a phytohormone essential for seed development and seedling growth under unfavorable environmental conditions. The signaling pathway leading to ABA response has been established, but relatively little is known about the functional regulation of the constituent signaling components. Here, we present several lines of evidence that Arabidopsis Raf-like kinase Raf10 modulates the core ABA signaling downstream of signal perception step. In particular, Raf10 phosphorylates subclass III SnRK2s (SnRK2.2, SnRK2.3, and SnRK2.6), which are key positive regulators, and our study focused on SnRK2.3 indicates that Raf10 enhances its kinase activity and may facilitate its release from negative regulators. Raf10 also phosphorylates transcription factors (ABI5, ABF2, and ABI3) critical for ABAregulted gene expression. Furthermore, Raf10 was found to be essential for the in vivo functions of SnRK2s and ABI5. Collectively, our data demonstrate that Raf10 is a novel regulatory component of core ABA signaling.

Salinity and ABA Seed Responses in Pepper: Expression and Interaction of ABA Core Signaling Components.

Abscisic acid (ABA) plays an important role in various aspects of plant growth and development, including adaptation to stresses, fruit development and ripening. In seeds, ABA participates through its core signaling components in dormancy instauration, longevity determination, and inhibition of germination in unfavorable environmental conditions such as high soil salinity. Here, we show that seed germination in pepper was delayed but only marginally reduced by ABA or NaCl with respect to control treatments. Through a similarity search, pepper orthologs of ABA core signaling components PYL (PYRABACTIN RESISTANCE1-LIKE), PP2C (PROTEIN PHOSPHATASE2C), and SnRK2 (SUCROSE NONFERMENTING1 (SNF1)-RELATED PROTEIN KINASE2) genes were identified. Gene expression analyses of selected members showed a low abundance of PYL and SnRK2 transcripts in dry seeds compared to other tissues, and an up-regulation at high concentrations of ABA and/or NaCl for both positive and negative regulators of ABA signaling. As expected, in hydroponically-grown seedlings exposed to NaCl, only PP2C encoding genes were up-regulated. Yeast two hybrid assays performed among putative pepper core components and with Arabidopsis thaliana orthologs confirmed the ability of the identified proteins to function in ABA signaling cascade, with the exception of a CaABI isoform cloned from seeds. BiFC assay in planta confirmed some of the interactions obtained in yeast. Altogether, our results indicate that a low expression of perception and signaling components in pepper seeds might contribute to explain the observed high percentages of seed germination in the presence of ABA. These results might have direct implications on the improvement of seed longevity and vigor, a bottleneck in pepper breeding.

The Antagonistic Action of Abscisic Acid and Cytokinin Signaling Mediates Drought Stress Response in Arabidopsis.

As sessile organisms, plants encounter a variety of environmental stresses and must optimize their growth for survival. Abscisic acid (ABA) and cytokinin antagonistically regulate many developmental processes and environmental stress responses in plants. However, the molecular mechanism underlying this antagonism remains poorly defined. In this study, we demonstrated that Sucrose nonfermenting1-related kinases SnRK2.2, SnRK2.3, and SnRK2.6, the key kinases of the ABA signaling pathway, directly interact with and phosphorylate type-A response regulator 5 (ARR5), a negative regulator of cytokinin signaling. The phosphorylation of ARR5 Ser residues by SnRK2s enhanced ARR5 protein stability. Accordingly, plants overexpressing ARR5 showed ABA hypersensitivity and drought tolerance, and these phenotypes could not be recapitulated by overexpressing a non-phosphorylated ARR5 mimic. Moreover, the type-B ARRs, ARR1, ARR11 and ARR12, physically interacted with SnRK2s and repressed the kinase activity of SnRK2.6. The arr1,11,12 triple mutant exhibited hypersensitivity to ABA. Genetic analysis demonstrated that SnRK2s act upstream of ARR5 but downstream of ARR1, ARR11 and ARR12 in mediating ABA response and drought tolerance. Taken together, this study unravels the antagonistic actions of several molecular components of the ABA and cytokinin signaling pathways in mediates drought stress response, providing significant insights into how plants coordinate growth and drought stress response by integrating multiple hormone pathways.

Structural basis and functions of abscisic acid receptors PYLs.

Abscisic acid (ABA) plays a key role in many developmental processes and responses to adaptive stresses in plants. Recently, a new family of nucleocytoplasmic PYR/PYL/RCAR (PYLs) has been identified as bona fide ABA receptors. PYLs together with protein phosphatases type-2C (PP2Cs), Snf1 (Sucrose-non-fermentation 1)-related kinases subfamily 2 (SnRK2s) and downstream substrates constitute the core ABA signaling network. Generally, PP2Cs inactivate SnRK2s kinases by physical interaction and direct dephosphorylation. Upon ABA binding, PYLs change their conformations and then contact and inhibit PP2Cs, thus activating SnRK2s. Here, we reviewed the recent progress in research regarding the structures of the core signaling pathways of ABA, including the (+)-ABA, (-)-ABA and ABA analogs pyrabactin as well as 6AS perception by PYLs, SnRK2s mimicking PYLs in binding PP2Cs. PYLs inhibited PP2Cs in both the presence and absence of ABA and activated SnRK2s. The present review elucidates multiple ABA signal perception and transduction by PYLs, which might shed light on how to design small chemical compounds for improving plant performance in the future.