The structure of Arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases.     

Cloning and characterization of the SnRK2 gene family from <Emphasis Type="Italic">Zea mays</Emphasis>

The SnRK2 gene family is a group of plant-specific protein kinases that has been implicated in ABA and abiotic stress signaling. We found 11 SnRK2s in maize, assigned names from ZmSnRK2.1 to ZmSnRK2.11 and cloned ten of them. By analyzing the gene structure of all the SnRK2s from Arabidopsis, rice, and maize, we found seven exons that were conserved in length among most of the SnRK2s. Although the C-terminus was divergent, we found seven conserved motifs. Of these, motif 1 was common to all of the SnRK2 genes. Based on phylogenetic analysis using the kinase domain and motif 1, the SnRK2s were divided into three groups. Motifs 4 and 5 were found specifically in group I, and many genes of this group have been confirmed to be induced by ABA. This result suggests that these two motifs mediate the ABA response. The expression patterns of ZmSnRK2 genes were characterized by using quantitative real-time RCR, which revealed that ZmSnRK2 genes were induced by one or more abiotic stress treatments and therefore may play important roles in maize stress responses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Huai and M. Wang have contributed equally to this paper.     

Genome-wide identification,characterization, and expression analysis of <Emphasis Type="Italic">SnRK2</Emphasis> family in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis>

The sucrose non-fermenting 1-related protein kinase 2 (SnRK2) gene family belongs to a group of plant-specific serine/threonine kinase family involved in abscisic acid (ABA) signaling and biotic and abiotic stress response. Although genome-wide analyses of the SnRK2 gene family have been conducted in some species, little is known about the SnRK2 gene family in rubber tree (Hevea brasiliensis). In this study, we identified 10 SnRK2s designated as HbSnRK2.1 to HbSnRK2.10 in the rubber tree genome. The subsequently constructed phylogenetic tree demonstrated that HbSnRK2s have three subfamilies that correlate well with those of Arabidopsis sp. and rice subfamilies. All SnRK2 genes contained nine exons and eight introns. Although the C-terminus was divergent, eight conserved motifs were found. Motifs 1–6 were common to all HbSnRK2s. Expression analysis results showed that 7 of the 10 HbSnRK2s were highly expressed in latex. HbSnRK2.7 was predominantly expressed and simultaneously regulated by abscisic acid, jasmonic acid, and ethylene treatment in laticifers. HbSnRK identification and characterization provided further understanding on the role of ABA signal in the rubber tree.     

Phospho‐site mapping,genetic and in planta activation studies reveal key aspects of the different phosphorylation mechanisms involved in activation of SnRK2s

Snf1‐related protein kinases 2 (SnRK2s) are major positive regulators of drought stress tolerance. The kinases of this family are activated by hyperosmotic stress, but only some of them are also responsive to abscisic acid (ABA). Moreover, genetic evidence has indicated the ABA‐independence of SnRK2 activation in the fast response to osmotic stress. Although phosphorylation was demonstrated to be crucial for the activation or activity of the kinases of both subgroups, different phosphorylation mechanisms were suggested. Here, using one kinase from each subgroup (SnRK2.6 and SnRK2.10), two phosphorylation sites within the activation loop were identified by mass spectrometry after immunoprecipitation from Arabidopsis cells treated by ABA or osmolarity. By site‐directed mutagenesis, the phosphorylation of only one of the two sites was shown to be necessary for the catalytic activity of the kinase, whereas both sites are necessary for the full activation of the two SnRK2s by hyperosmolarity or ABA. Phosphoprotein staining together with two‐dimensional PAGE followed by immunoblotting indicated distinct phosphorylation mechanisms of the two kinases. While SnRK2.6 seems to be activated through the independent phosphorylation of these two sites, a sequential process occurs in SnRK2.10, where phosphorylation of one serine is required for the phosphorylation of the other. In addition, a subgroup of protein phosphatases 2C which interact and participate in the regulation of SnRK2.6 do not interact with SnRK2.10. Taken together, our data bring evidence for the involvement of distinct phosphorylation mechanisms in the activation of SnRK2.6 and SnRK2.10, which may be conserved between the two subgroups of SnRK2s depending on their ABA‐responsiveness.     

The role of ABA and MAPK signaling pathways in plant abiotic stress responses

As sessile organisms, plants have developed specific mechanisms that allow them to rapidly perceive and respond to stresses in the environment. Among the evolutionarily conserved pathways, the ABA (abscisic acid) signaling pathway has been identified as a central regulator of abiotic stress response in plants, triggering major changes in gene expression and adaptive physiological responses. ABA induces protein kinases of the SnRK family to mediate a number of its responses. Recently, MAPK (mitogen activated protein kinase) cascades have also been shown to be implicated in ABA signaling. Therefore, besides discussing the role of ABA in abiotic stress signaling, we will also summarize the evidence for a role of MAPKs in the context of abiotic stress and ABA signaling.     

Activation of SnRK2 by Raf-like kinase ARK represents a primary mechanism of ABA and abiotic stress responses

The Raf-like protein kinase abscisic acid (ABA) and abiotic stress-responsive Raf-like kinase (ARK) previously identified in the moss Physcomitrium (Physcomitrella) patens acts as an upstream regulator of subgroup III SNF1-related protein kinase2 (SnRK2), the key regulator of ABA and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2, including the role of ABA receptor-associated group A PP2C (PP2C-A), are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic P. patens ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance, and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in the wild type (WT). The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking protein phosphatase 2C-A (PP2C-A) was similar to that in the WT, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense mutant lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. Therefore, ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototype mechanism for ABA and abiotic stress responses in plants.

Phosphorylation in the activation loop of the Raf-like kinase ARK is critical for SNF1-related protein kinase2 regulation during abscisic acid responses in the moss Physcomitrium (Physcomitrella) patens.     

苹果SRS基因家族的鉴定与功能分析北大核心CSCD

SHI-related sequence(SRS)基因家族通过介导激素变化以调控植物成花及生长发育,并且在适应环境胁迫中起重要调控作用。该研究基于苹果(Malus domestica Borkh.)基因组数据,通过生物信息学手段鉴定苹果SRS基因家族成员,并分析SRS基因家族特点与功能及表达情况。结果表明:(1)苹果MdSRS基因家族共包含11个成员,分别命名为MdSRS1-MdSRS11,不均匀地分布在苹果的9条染色体上。(2)MdSRS蛋白包含229~414个不等的氨基酸残基,等电点分布在6.38~9.36之间;亚细胞定位结果表明,MdSRS蛋白大多分布于细胞膜,在细胞核、叶绿体中也有分布。(3)通过引入拟南芥、水稻、番茄及杨树的SRS基因进行系统发育分析表明,将11个MdSRSs分成5个亚族(A-A),在A4中分布最多。(4)顺式作用元件分析表明,11个MdSRSs启动子上游2 000 bp序列分布有激素、环境适应性和逆境诱导等响应元件。(5)荧光定量PCR结果显示,苹果MdSRS基因家族在盐胁迫和干旱胁迫下总体呈下调表达,在ABA胁迫后大多呈上调表达,是具有很大潜力的抗性候选基因,说明SRS家族对ABA调节等非生物胁迫具有调控作用。研究认为,SRS家族的11个成员均参与了调控干旱、盐及ABA胁迫多种逆境的响应,推测在实际苹果生产中对抵御不良环境具有重要作用。     

Genome-wide identification and expression analysis of MAPK and MAPKK gene family in Malus domestica

MAPK signal transduction modules play crucial roles in regulating many biological processes in plants, which are composed of three classes of hierarchically organized protein kinases, namely MAPKKKs, MAPKKs, and MAPKs. Although genome-wide analysis of this family has been carried out in some species, little is known about MAPK and MAPKK genes in apple (Malus domestica). In this study, a total of 26 putative apple MAPK genes (MdMPKs) and 9 putative apple MAPKK genes (MdMKKs) have been identified and located within the apple genome. Phylogenetic analysis revealed that MdMAPKs and MdMAPKKs could be divided into 4 subfamilies (groups A, B, C and D), respectively. The predicted MdMAPKs and MdMAPKKs were distributed across 13 out of 17 chromosomes with different densities. In addition, analysis of exon–intron junctions and of intron phase inside the predicted coding region of each candidate gene has revealed high levels of conservation within and between phylogenetic groups. According to the microarray and expressed sequence tag (EST) analysis, the different expression patterns indicate that they may play different roles during fruit development and rootstock–scion interaction process. Moreover, MAPK and MAPKK genes were performed expression profile analyses in different tissues (root, stem, leaf, flower and fruit), and all of the selected genes were expressed in at least one of the tissues tested, indicating that the MAPKs and MAPKKs are involved in various aspects of physiological and developmental processes of apple. To our knowledge, this is the first report of a genome-wide analysis of the apple MAPK and MAPKK gene family. This study provides valuable information for understanding the classification and putative functions of the MAPK signal in apple.     

Differential expression of poplar <Emphasis Type="Italic">sucrose nonfermenting1-related protein kinase 2</Emphasis> genes in response to abiotic stress and abscisic acid

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.     

Molecular dissection of the response of a rice leucine-rich repeat receptor-like kinase (LRR-RLK) gene to abiotic stresses

Leucine-rich repeat (LRR) receptor-like kinase (RLK) proteins play key roles in a variety of biological pathways. In a previous study, we analyzed the members of the rice LRR-RLK gene family using in silico analysis. A total of 23 LRR-RLK genes were selected based on the expression patterns of a genome-wide dataset of microarrays. The Oryza sativa gamma-ray induced LRR-RLK1 (OsGIRL1) gene was highly induced by gamma irradiation. Therefore, we studied its expression pattern in response to various different abiotic and phytohormone treatments. OsGIRL1 was induced on exposure to abiotic stresses such as salt, osmotic, and heat, salicylic acid (SA), and abscisic acid (ABA), but exhibited downregulation in response to jasmonic acid (JA) treatment. The OsGIRL1 protein was clearly localized at the plasma membrane. The truncated proteins harboring juxtamembrane and kinase domains (or only harboring a kinase domain) exhibited strong autophosphorylation. The biological function of OsGIRL1 was investigated via heterologous overexpression of this gene in Arabidopsis plants subjected to gamma-ray irradiation, salt stress, osmotic stress, and heat stress. A hypersensitive response was observed in response to salt stress and heat stress, whereas a hyposensitive response was observed in response to gamma-ray treatment and osmotic stress. These results provide critical insights into the molecular functions of the rice LRR-RLK genes as receptors of external signals.     

Regulation of growth by the trehalose pathway: Relationship to temperature and sucrose

Carbon signaling can override carbon supply in the regulation of growth. At least some of this regulation is imparted by the sugar signal trehalose 6-phosphate (T6P) through the protein kinase, SnRK1. This signaling pathway regulates biosynthetic processes involved in growth under optimal growing conditions. Recently, using a seedling system we showed that under sub-optimal conditions, such as cold, carbon signaling by T6P/ SnRK1 enables recovery of growth following relief of the stress. The T6P/ SnRK1 mechanism thus could be selected as a means of improving low temperature tolerance. High-throughput automated Fv/Fm measurements provide a potential means to screen for T6P/ SnRK1, and here we confirm through measurements of Fv/Fm in rosettes that T6P promotes low temperature tolerance and recovery during cold to warm transfer. Further, to better understand the coordination between sugars, trehalose pathway, and temperature-dependent growth, we examine the interrelationship between sugars, trehalose phosphate synthase (TPS), and trehalose phosphate phosphatase (TPP) gene expression and T6P content in seedlings. Sucrose, particularly when fed exogenously, correlated well with TPS1 and TPPB gene expression, suggesting that these enzymes are involved in maintaining carbon flux through the pathway in relation to sucrose supply. However, when sucrose accumulated to higher levels under low temperature and low N, TPS1 and TPPB expression were less directly related to sucrose; other factors may also contribute to regulation of TPS1 and TPPB expression under these conditions. TPPA expression was not related to sucrose content and all genes were not well correlated with endogenous glucose. Our work has implications for understanding acclimation to sink-limited growth conditions such as low temperature and for screening cold-tolerant genotypes with altered T6P/ SnRK1 signaling.