以‘宝石无核’（Ruby Seedless）葡萄试管苗为材料，采用RT-PCR技术，克隆得到8个葡萄SnRK2家族基因。序列分析发现，该家族基因蛋白结构在N端相对保守，而在C端极其特异；聚类结果表明葡萄SnRK2基因家族可以分为3个亚家族；对这8个基因所编码的蛋白质进行分析发现，其富含酸性氨基酸，且均为亲水蛋白；基因组结构分析发现VvSnRK2.2和VvSnRK2.8含有10个外显子，其他6个均含9个外显子；对蛋白二级结构分析发现8个基因编码的蛋白主要以α–螺旋、β–转角和不规则卷曲为主；亚细胞定位预测，8个基因主要定位于细胞质中。顺式作用元件分析表明，除VvSnRK2.1、VvSnRK2.2、VvSnRK2.6外，其他基因顺式作用元件包含ABRE、DRE/CRT、LRTE中的一个或多个。定量PCR分析表明，VvSnRK2的表达存在组织差异性，VvSnRK2.7在根中表达水平最高，是叶片的3.8倍，VvSnRK2.8在茎中表达水平最高，是叶片的5.0倍。0 ~–4 ℃处理后，表达水平下调幅度最小的为VvSnRK2.2，VvSnRK2.7下调幅度较大，VvSnRK2.8的表达水平为0；30 ℃处理后VvSnRK2.2和VvSnRK2.5上调表达，分别为对照的3.8倍和3.6倍；VvSnRK2.1和VvSnRK2.2与盐胁迫调节紧密相关，VvSnRK2.5与干旱胁迫调节密切相关。

Increases in the plant hormone abscisic acid (ABA) initiate water-stress responses in plants. We present evidence that a transcript with homology to protein kinases is induced by ABA and dehydration in wheat. A 1.2-kilobase cDNA clone (PKABA1) was isolated from an ABA-treated wheat embryo cDNA library by screening the library with a probe developed by polymerase chain reaction amplification of serine/threonine protein kinase subdomains VIb to VIII. The deduced amino acid sequence of the PKABA1 clone contains the features of serine/threonine protein kinases, including homology with all 12 conserved regions of the catalytic domain. PKABA1 transcript levels are barely detectable in growing seedlings but are induced dramatically when plants are subjected to dehydration stress. The PKABA1 transcript can also be induced by supplying low concentrations of ABA, and coordinate increases in ABA levels and PKABA1 mRNA occur when seedlings are water-stressed. Identification of this ABA-inducible transcript with homology to protein kinases provides a basis for examining the role of protein phosphorylation in plant responses to dehydration.

The mechanisms of salt stress response and tolerance have eluded definition despite reasonable success in defining their physiological manifestations. In this review, we consider the integrated salt metabolism of plants, essentially as a problem in meganutrient physiology. Two critical aspects of cellular and organismal metabolism are given particular attention-those involved in the control and integration of Na(+) acquisition and allocation in plants and those involved in readjustment of other aspects of metabolism, especially those involving carbon as a resource.

\n SnRK2.8 is a member of the sucrose nonfermenting-related kinase family that is down-regulated when plants are deprived of nutrients and growth is reduced. When this kinase is over expressed in\n Arabidopsis\n, the plants grow larger. To understand how this kinase modulates growth, we identified some of the proteins that are phosphorylated by this kinase. A new phosphoproteomic method was used in which total protein from plants overexpressing the kinase was compared with total protein from plants in which the kinase was inactivated. Protein profiles were compared on two-dimensional gels following staining by a dye that recognizes phosphorylated amino acids. Candidate target proteins were confirmed with\n in vitro\n phosphorylation assays, using the kinase and target proteins that were purified from\n Escherichia coli\n. Seven target proteins were confirmed as being phosphorylated by SnRK2.8. Certain targets, such as 14-3-3 proteins, regulate as yet unidentified proteins, whereas other targets, such as glyoxalase I and ribose 5-phosphate isomerase, detoxify byproducts from glycolysis and catalyze one of the final steps in carbon fixation, respectively. Also, adenosine kinase and 60S ribosomal protein were confirmed as targets of SnRK2.8. Using mass spectrometry, we identified phosphorylated residues in the SnRK2.8, the 14-3-3κ, and the 14-3-3χ. These data show that the expression of\n SnRK2.8\n is correlated with plant growth, which may in part be due to the phosphorylation of enzymes involved in metabolic processes.\n

Plants encode a large number of leucine-rich repeat receptor-like kinases. Legumes encode several LRR-RLK linked to the process of root nodule formation, the ligands of which are unknown. To identify ligands for these receptors, we used a combination of profile hidden Markov models and position-specific iterative BLAST, allowing us to detect new members of the CLV3/ESR (CLE) protein family from publicly available sequence databases.

\n Protein phosphorylation/dephosphorylation are major signaling events induced by osmotic stress in higher plants. Here, we showed that a SNF1-related protein kinase 2 (SnRK2), SRK2C, is an osmotic-stress-activated protein kinase in\n Arabidopsis thaliana\n that can significantly impact drought tolerance of\n Arabidopsis\n plants. Knockout mutants of SRK2C exhibited drought hypersensitivity in their roots, suggesting that SRK2C is a positive regulator of drought tolerance in\n Arabidopsis\n roots. Additionally, transgenic plants with\n CaMV35S\n promoter::\n SRK2C\n -\n GFP\n displayed higher overall drought tolerance than control plants. Whereas stomatal regulation in\n 35S\n ::\n SRK2C\n -\n GFP\n plants was not altered, microarray analysis revealed that their drought tolerance coincided with up-regulation of many stress-responsive genes, for example,\n RD29A\n,\n COR15A\n, and\n DREB1A\n /\n CBF3\n. From these results, we concluded that SRK2C is capable of mediating signals initiated during drought stress, resulting in appropriate gene expression. Our present study reveals new insights around signal output from osmotic-stress-activated SnRK2 protein kinase as well as supporting feasibility of manipulating SnRK2 toward improving plant osmotic-stress tolerance.\n

To date, a large number of sequences of protein kinases that belong to the sucrose nonfermenting1–related protein kinase2 (SnRK2) family are found in databases. However, only limited numbers of the family members have been characterized and implicated in abscisic acid (ABA) and hyperosmotic stress signaling. We identified 10 SnRK2 protein kinases encoded by the rice (Oryza sativa) genome. Each of the 10 members was expressed in cultured cell protoplasts, and its regulation was analyzed. Here, we demonstrate that all family members are activated by hyperosmotic stress and that three of them are also activated by ABA. Surprisingly, there were no members that were activated only by ABA. The activation was found to be regulated via phosphorylation. In addition to the functional distinction with respect to ABA regulation, dependence of activation on the hyperosmotic strength was different among the members. We show that the relatively diverged C-terminal domain is mainly responsible for this functional distinction, although the kinase domain also contributes to these differences. The results indicated that the SnRK2 protein kinase family has evolved specifically for hyperosmotic stress signaling and that individual members have acquired distinct regulatory properties, including ABA responsiveness by modifying the C-terminal domain.

As sessile organisms, plants must adapt to variations in the environment. Environmental stress triggers various responses, including growth inhibition, mediated by the plant hormone abscisic acid (ABA). The mechanisms that integrate stress responses with growth are poorly understood. Here, we discovered that the Target of Rapamycin (TOR) kinase phosphorylates PYL ABA receptors at a conserved serine residue to prevent activation of the stress response in unstressed plants. This phosphorylation disrupts PYL association with ABA and with PP2C phosphatase effectors, leading to inactivation of SnRK2 kinases. Under stress, ABA-activated SnRK2s phosphorylate Raptor, a component of the TOR complex, triggering TOR complex dissociation and inhibition. Thus, TOR signaling represses ABA signaling and stress responses in unstressed conditions, whereas ABA signaling represses TOR signaling and growth during times of stress. Plants utilize this conserved phospho-regulatory feedback mechanism to optimize the balance of growth and stress responses.Copyright © 2017 Elsevier Inc. All rights reserved.

\n The plant hormone abscisic acid (ABA) helps plants to respond to changes in the environment, such as drought. Physiological responses are initiated when ABA binds to its receptor. In the absence of ABA, downstream kinases are held inactive by phosphatases.\n \n Soon\n et al.\n \n (p.\n 85\n, published online 24 November; see the Perspective by\n \n Leung\n \n ) now show that both the hormone-receptor complex and the downstream kinase bind to the same site on the phosphatase. Thus, in the presence of hormone, the phosphatase is occupied and unable to interfere with downstream kinase activity.\n

Abiotic stresses, including drought and salinity, trigger a complex osmotic-stress and abscisic acid (ABA) signal transduction network. The core ABA signalling components are snf1-related protein kinase2s (SnRK2s), which are activated by ABA-triggered inhibition of type-2C protein-phosphatases (PP2Cs). SnRK2 kinases are also activated by a rapid, largely unknown, ABA-independent osmotic-stress signalling pathway. Here, through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant MAPKK-kinases (M3Ks) that are necessary for activation of SnRK2 kinases. These M3Ks phosphorylate a specific SnRK2/OST1 site, which is indispensable for ABA-induced reactivation of PP2C-dephosphorylated SnRK2 kinases. ABA-triggered SnRK2 activation, transcription factor phosphorylation and SLAC1 activation require these M3Ks in vitro and in plants. M3K triple knock-out plants show reduced ABA sensitivity and strongly impaired rapid osmotic-stress-induced SnRK2 activation. These findings demonstrate that this M3K clade is required for ABA- and osmotic-stress-activation of SnRK2 kinases, enabling robust ABA and osmotic stress signal transduction.

Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments.

Rapid changes in messenger RNA population are vital for plants to properly exert multiple adaptive responses under continuously changing stress conditions. Transcriptional activation mediated by the 'abscisic acid (ABA)-activated SnRK2 protein kinases-ABA-responsive element (ABRE)-binding proteins/ABRE-binding factors (AREB/ABFs)' signalling module is a crucial step in the expression of stress-inducible genes under osmotic stress conditions in Arabidopsis. In addition to transcriptional control, proper transcript levels of individual genes can be achieved by post-transcriptional regulation, but how this regulation functions under stress conditions and the underlying molecular mechanisms remain elusive. Here, we show that ABA-unresponsive osmotic stress-activated subclass I SnRK2s and their downstream substrate, VARICOSE (VCS), an mRNA decapping activator, regulate mRNA decay under osmotic stress conditions. The expression of many stress-responsive genes was similarly misregulated in a mutant lacking all functional subclass I SnRK2s and in VCS-knockdown plants. Additionally, the mRNA decay of the transcripts of these genes was impaired in these plants under osmotic stress conditions. Furthermore, these plants showed growth retardation under osmotic stresses. Notably, subclass I-type SnRK2s have been identified in seed plants but not in lycophytes or mosses. Therefore, the post-transcriptional regulation mediated by the 'subclass I SnRK2s-VARICOSE' signalling module represents an additional mechanism of gene expression control that facilitates drastic changes in mRNA populations under osmotic stresses and might enhance the adaptability of seed plants to stress conditions.

Environmental stresses such as drought, salinity, and cold are major adverse factors that significantly affect agricultural productivity. Protein phosphorylation/dephosphorylation is a major signalling event induced by osmotic stress in higher plants. Sucrose non-fermenting 1-related protein kinase 2 (SnRK2) family members play essential roles in the response to hyperosmotic stresses in plants. In this study, the TaSnRK2.3 gene, a novel SnRK2 member was cloned, and three copies located on chromosomes 1A, 1B, and 1D were identified in common wheat. TaSnRK2.3 was strongly expressed in leaves, and responded to polyethylene glycol, NaCl, abscisic acid, and cold stresses. To characterize its function, transgenic Arabidopsis overexpressing TaSnRK2.3-GFP controlled by the cauliflower mosaic virus 35S promoter was generated and subjected to severe abiotic stresses. Overexpression of TaSnRK2.3 resulted in an improved root system and significantly enhanced tolerance to drought, salt, and freezing stresses, simultaneously demonstrated by enhanced expression of abiotic stress-responsive genes and ameliorative physiological indices, including a decreased rate of water loss, enhanced cell membrane stability, improved photosynthetic potential, and significantly increased osmotic potential and free proline content under normal and/or stressed conditions. These results demonstrate that TaSnRK2.3 is a multifunctional regulator, with potential for utilization in transgenic breeding for improved abiotic stress tolerance in crop plants.

Water availability is one of the main limiting factors for plant growth and development. The phytohormone abscisic acid (ABA) fulfills a critical role in coordinating the responses to reduced water availability as well as in multiple developmental processes. Endogenous ABA levels increase in response to osmotic stresses such as drought and high salinity, and ABA activates the expression of many genes via ABA-responsive elements (ABREs) in their promoter regions. ABRE-binding protein/ABRE-binding factor (AREB/ABF) transcription factors (TFs) regulate the ABRE-mediated transcription of downstream target genes. Three subclass III sucrose non-fermenting-1 related protein kinase 2 (SnRK2) protein kinases (SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3) phosphorylate and positively control the AREB/ABF TFs. Substantial progress has been made in our understanding of the ABA-sensing system mediated by Pyrabactin resistance1/PYR1-like/regulatory components of ABA receptor (PYR/PYL/RCAR)-protein phosphatase 2C complexes. In addition to PP2C-PYR/PYL/RCAR ABAreceptor complex, the AREB/ABF-SnRK2 pathway, which is well conserved in land plants, was recently shown to play a major role as a positive regulator of ABA/stress signaling through ABRE-mediated transcription of target genes implicated in the osmotic stress response. This review focuses on current progress in the study of the AREB/ABF-SnRK2 positive regulatory pathway in plants and describes additional signaling factors implicated in the AREB/ABF-SnRK2 pathway. Moreover, to help promote the link between basic and applied studies, the nomenclature and phylogenetic relationships between the AREB/ABFs and SnRK2s are summarized and discussed.Copyright © Physiologia Plantarum 2012.

Members of the sucrose non-fermenting related kinase Group2 (SnRK2) subclasses are implicated in both direct and indirect abscisic acid (ABA) response pathways. We have used phylogenetic, biochemical, and transient in vivo approaches to examine interactions between Triticum tauschii protein kinase 1 (TtPK1) and an interacting protein, Oryza sativa SnRK2-calcium sensor (OsSCS1). Given that TtPK1 has 100% identity with its rice ortholog, osmotic stress/ABA-activated protein kinase (OsSAPK2), we hypothesized that the SCS and TtPK1 interactions are present in both wheat and rice. Here, we show that SnRK2s are clearly divided into four pan-angiosperm clades with those in the traditionally defined Subclass II encompassing two distinct clades (OsSAPK1/2 and OsSAPK3), although OsSAPK3 lacks an Arabidopsis ortholog. We also show that SCSs are distinct from a second lineage, that we term SCSsister, and while both clades pre-date land plants, the SCSsister clade lacks Poales representatives. Our Y2H assays revealed that the removal of the OsSCS1 C-terminal region along with its N-terminal EF-hand abolished its interaction with the kinase. Using transient in planta bimolecular fluorescence complementation experiments, we demonstrate that TtPK1/OsSCS1 dimerization co-localizes with DAPI-stained nuclei and with FM4-64-stained membranes. Finally, OsSCS1- and OsSAPK2-hybridizing transcripts co-accumulate in shoots/coleoptile of drying seedlings, consistent with up-regulated kinase transcripts of PKABA1 and TtPK1. Our studies suggest that interactions between homologs of the SnRK2 and SCS lineages are broadly conserved across angiosperms and offer new directions for investigations of related proteins.

SNF1-RELATED PROTEIN KINASES 2 (SnRK2) are important components of early osmotic and salt stress signaling pathways in plants. The Arabidopsis () SnRK2 family comprises the abscisic acid (ABA)-activated protein kinases SnRK2.2, SnRK2.3, SnRK2.6, SnRK2.7, and SnRK2.8, and the ABA-independent subclass 1 protein kinases SnRK2.1, SnRK2.4, SnRK2.5, SnRK2.9, and SnRK2.10. ABA-independent SnRK2s act at the posttranscriptional level via phosphorylation of VARICOSE (VCS), a member of the mRNA decapping complex, that catalyzes the first step of 5'mRNA decay. Here, we identified VCS and VARICOSE RELATED (VCR) as interactors and phosphorylation targets of SnRK2.5, SnRK2.6, and SnRK2.10. All three protein kinases phosphorylated Ser-645 and Ser-1156 of VCS, whereas SnRK2.6 and SnRK2.10 also phosphorylated VCS Ser-692 and Ser-680 of VCR. We showed that subclass 1 SnRK2s, VCS, and 5' EXORIBONUCLEASE 4 (XRN4) are involved in regulating root growth under control conditions as well as modulating root system architecture in response to salt stress. Our results suggest interesting patterns of redundancy within subclass 1 SnRK2 protein kinases, with SnRK2.1, SnRK2.5, and SnRK2.9 controlling root growth under nonstress conditions and SnRK2.4 and SnRK2.10 acting mostly in response to salinity. We propose that subclass 1 SnRK2s function in root development under salt stress by affecting the transcript levels of aquaporins, as well as CYP79B2, an enzyme involved in auxin biosynthesis.© 2020 American Society of Plant Biologists. All Rights Reserved.

Knowledge on the responses of woody plants to abiotic stress can inform strategies to breed improved tree varieties and to manage tree species for environmental conservation and the production of lignocellulosic biomass. In this study, we examined the expression patterns of poplar (Populus trichocarpa) genes encoding members of the sucrose nonfermenting1-related protein kinase 2 (SnRK2) family, which are core components of the abiotic stress response. The P. trichocarpa genome contains twelve SnRK2 genes (PtSnRK2.1- PtSnRK2.12) that can be divided into three subclasses (I-III) based on the structures of their encoded kinase domains. We found that PtSnRK2s are differentially expressed in various organs. In MS medium-grown plants, all of the PtSnRK2 genes were significantly upregulated in response to abscisic acid (ABA) treatment, whereas osmotic and salt stress treatments induced only some (four and seven, respectively) of the PtSnRK2 genes. By contrast, soil-grown plants showed increased expression of most PtSnRK2 genes under drought and salt treatments, but not under ABA treatment. In soil-grown plants, drought stress induced SnRK2 subclass II genes in all tested organs (leaves, stems, and roots), whereas subclass III genes tended to be upregulated in leaves only. These results suggest that the PtSnRK2 genes are involved in abiotic stress responses, are at least partially activated by ABA, and show organ-specific responses.

Sucrose non-fermenting-1 related protein kinase 2 (SnRK2) is a unique family of protein kinases associated with abiotic stress signal transduction in plants. In this study, a maize SnRK2 gene ZmSnRK2.11 was cloned and characterized. The results showed that ZmSnRK2.11 is up-regulated by high-salinity and dehydration treatment, and it is expressed mainly in maize mature leaf. A transient expression assay using onion epidermal cells revealed that ZmSnRK2.11-GFP fusion proteins are localized to both the nucleus and cytoplasm. Overexpressing-ZmSnRK2.11 in Arabidopsis resulted in salt and drought sensitivity phenotypes that exhibited an increased rate of water loss, reduced relative water content, delayed stoma closure, accumulated less free proline content and increased malondialdehyde (MDA) content relative to the phenotypes observed in wild-type (WT) control. Furthermore, overexpression of ZmSnRK2.11 up-regulated the expression of the genes ABI1 and ABI2 and decreased the expression of DREB2A and P5CS1. Taken together, our results suggest that ZmSnRK2.11 is a possible negative regulator involved in the salt and drought stress signal transduction pathways in plants.