Glycogen synthase kinase 3/SHAGGY-like kinases in plants: an emerging family with novel functions

Animal glycogen synthase kinase 3 (GSK-3)/SHAGGY kinases have been studied for more than 20 years, whereas plant glycogen synthase kinase 3/SHAGGY-like kinases (GSKs) have only recently entered the scene. Present evidence indicates that plant GSKs are involved in different processes, such as flower development, brassinosteroid signaling, NaCl stress and wound responses. In contrast to mammals, which contain two genes, plants have a multigene family of GSKs. Analysis of the Arabidopsis genome revealed the existence of ten GSK genes that fall into four distinct subfamilies. We discuss the functions and mechanisms of GSK action in plants and other organisms.     

Phylogenetic diversification of glycogen synthase kinase 3/SHAGGY-like kinase genes in plants

Background  

The glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinases (GSKs) are non-receptor serine/threonine protein kinases that are involved in a variety of biological processes. In contrast to the two members of the GSK3 family in mammals, plants appear to have a much larger set of divergent GSK genes. Plant GSKs are encoded by a multigene family; analysis of the Arabidopsis genome revealed the existence of 10 GSK genes that fall into four major groups. Here we characterized the structure of Arabidopsis and rice GSK genes and conducted the first broad phylogenetic analysis of the plant GSK gene family, covering a taxonomically diverse array of algal and land plant sequences.     

Structural and functional characterization of arabidopsis GSK3-like kinase AtSK12

Plant GSK3-like kinases are key regulators that modulate a broad range of physiological processes such as cell growth, stomatal and flower development, responses for abiotic and biotic stress, and carbohydrate metabolism. Arabidopsis Shaggy/GSK3-like kinases (AtSK) consist of ten members that are classified into four subfamilies (I～IV). Only one of these Arabidopsis GSK3s, BIN2 (also named AtSK21), has been characterized by biochemical and genetic studies. BIN2 acts as a negative regulator in brassinosteroid (BR) signaling that controls cell growth and differentiation. Recent studies suggest that at least seven AtSKs are involved in BR signaling. However, specificities for the substrates and the functional differences of each member of the family remain to be determined. Here we report structural characteristics and distinct function of AtSK12 compared with BIN2. AtSK12 has a longer N-terminal extension, which is absent in BIN2. Transgenic plants overexpressing the AtSK12 mutant carrying deletion of Nterminal region display more severe dwarf phenotypes than those of the wild-type AtSK12. Microscopic analysis reveals that N-terminal-deleted AtSK12 accumulates in the nucleus. This implies that structural difference in the Nterminal region of AtSK members contributes to their subcellular localization. In contrast to BIN2, overexpression of AtSK12 does not cause a stomatal cluster. Furthermore, we show that YODA MAPKKK, which controls stomatal development, interacts with BIN2 but not with AtSK12. Our results suggest that AtSK12 mediates BR-regulated cell growth but not stomatal development while BIN2 regulates both processes. Our study provides evidence that different GSK3 members can have overlapping but non-identical functions.     

The UCU1 Arabidopsis gene encodes a SHAGGY/GSK3-like kinase required for cell expansion along the proximodistal axis

Most signal transduction pathways central to development are not shared by plants and animals. Such is the case of the Wingless/Wnt signaling pathway, whose components play key roles in metazoan pattern formation and tumorigenesis, but are absent in plants, with the exception of SHAGGY/GSK3, a cytoplasmic protein kinase represented in the genome of Arabidopsis thaliana by a family of 10 AtSK genes for which mutational evidence is scarce. Here, we describe the characterization of mutant alleles of the Arabidopsis ULTRACURVATA1 (UCU1) gene, the two strongest of which dramatically reduce cell expansion along the proximodistal axis, dwarfing the mutant plants, whose cells expand properly across but not along most organs. Proximodistal expansion of adaxial (dorsal) and abaxial (ventral) leaf cells exhibits a differential dependence on UCU1 function, as suggested by the leaves of ucu1 mutants, which are rolled spirally downward in a circinate manner. We have positionally cloned the UCU1 gene, which encodes an AtSK protein involved in the cross-talk between auxin and brassinosteroid signaling pathways, as indicated by the responses of ucu1 mutants to plant hormones and the phenotypes of double mutants involving ucu1 alleles.     

The Arabidopsis thaliana MEK AtMKK6 activates the MAP kinase AtMPK13

Mitogen-activated protein (MAP) kinases mediate cellular responses to a wide variety of stimuli. Activation of a MAP kinase occurs after phosphorylation by an upstream dual-specificity protein kinase, known as a MAP kinase kinase or MEK. The Arabidopsis thaliana genome encodes 10 MEKs but few of these have been shown directly to activate any of the 20 Arabidopsis MAP kinases. We show here that functional complementation of the cell lysis phenotype of a mutant yeast strain depends on the co-expression of the Arabidopsis MEK AtMKK6 and the MAP kinase AtMPK13. The kinase activity of AtMPK13 is stimulated in the presence of AtMKK6 in yeast cells. RT-PCR analysis showed the co-expression of these two genes in diverse plant tissues. These data show that AtMKK6 can functionally activate the MAP kinase AtMPK13.     

Structural and biochemical investigation of two Arabidopsis shikimate kinases: the heat-inducible isoform is thermostable

The expression of plant shikimate kinase (SK; EC 2.7.1.71), an intermediate step in the shikimate pathway to aromatic amino acid biosynthesis, is induced under specific conditions of environmental stress and developmental requirements in an isoform-specific manner. Despite their important physiological role, experimental structures of plant SKs have not been determined and the biochemical nature of plant SK regulation is unknown. The Arabidopsis thaliana genome encodes two SKs, AtSK1 and AtSK2. We demonstrate that AtSK2 is highly unstable and becomes inactivated at 37 °C whereas the heat-induced isoform, AtSK1, is thermostable and fully active under identical conditions at this temperature. We determined the crystal structure of AtSK2, the first SK structure from the plant kingdom, and conducted biophysical characterizations of both AtSK1 and AtSK2 towards understanding this mechanism of thermal regulation. The crystal structure of AtSK2 is generally conserved with bacterial SKs with the addition of a putative regulatory phosphorylation motif forming part of the adenosine triphosphate binding site. The heat-induced isoform, AtSK1, forms a homodimer in solution, the formation of which facilitates its relative thermostability compared to AtSK2. In silico analyses identified AtSK1 site variants that may contribute to AtSK1 stability. Our findings suggest that AtSK1 performs a unique function under heat stress conditions where AtSK2 could become inactivated. We discuss these findings in the context of regulating metabolic flux to competing downstream pathways through SK-mediated control of steady state concentrations of shikimate.     

Functional characterization of ARAKIN (ATMEKK1): a possible mediator in an osmotic stress response pathway in higher plants.

The Arabidopsis thaliana ARAKIN (ATMEKK1) gene shows strong homology to members of the (MAP) mitogen-activated protein kinase family, and was previously shown to functionally complement a mating defect in Saccharomyces cerevisiae at the level of the MEKK kinase ste11. The yeast STE11 is an integral component of two MAP kinase cascades: the mating pheromone pathway and the HOG (high osmolarity glycerol response) pathway. The HOG signal transduction pathway is activated by osmotic stress and causes increased glycerol synthesis. Here, we first demonstrate that ATMEKK1 encodes a protein with kinase activity, examine its properties in yeast MAP kinase cascades, then examine its expression under stress in A. thaliana. Yeast cells expressing the A. thaliana ATMEKK1 survive and grow under high salt (NaCl) stress, conditions that kill wild-type cells. Enhanced glycerol production, observed in non-stressed cells expressing ATMEKK1 is the probable cause of yeast survival. Downstream components of the HOG response pathway, HOG1 and PBS2, are required for ATMEKK1-mediated yeast survival. Because ATMEKK1 functionally complements the sho1/ssk2/ssk22 triple mutant, it appears to function at the level of the MEKK kinase step of the HOG response pathway. In A. thaliana, ATMEKK1 expression is rapidly (within 5 min) induced by osmotic (NaCl) stress. This is the same time frame for osmoticum-induced effects on the electrical properties of A. thaliana cells, both an immediate response and adaptation. Therefore, we propose that the A. thaliana ATMEKK1 may be a part of the signal transduction pathway involved in osmotic stress.     

Progress in Studies of Plant Homologs of Mitogen-Activated Protein (MAP) Kinase and Potential Upstream Components in Kinase Cascades

Mitogen-activated protein (MAP) kinase cascades were originally identified as protein phosphorylation systems that control the division and the growth of yeast and animal cells. Such cascades consist of MAP kinases, MAP-kinase kinases, and MAP-kinase-kinase kinases. In addition, these organisms have been also shown to have structurally related but functionally different MAP kinase cascades, which are involved in various cellular processes such as a response to osmotic stress and apoptosis. Plants also have been shown to have a number of members of each kinase family. Although physiological and genetic functions of most plant members have yet to be established, some of members have been shown to be responsible for the cellular transmission of signals generated by wounding or a mechanical stress, which predicts that MAP kinase cascades may function in a variety of physiological processes in the plant cells. In the present review, we summarize recent progresses of researches on plant members of each kinase family as well as those of analyses of the cascades in other organisms.     

The Roles of Phosphorylation and SHAGGY-Like Protein Kinases in Geminivirus C4 Protein Induced Hyperplasia

Even though plant cells are highly plastic, plants only develop hyperplasia under very specific abiotic and biotic stresses, such as when exposed to pathogens like Beet curly top virus (BCTV). The C4 protein of BCTV is sufficient to induce hyperplasia and alter Arabidopsis development. It was previously shown that C4 interacts with two Arabidopsis Shaggy-like protein kinases, AtSK21 and 23, which are negative regulators of brassinosteroid (BR) hormone signaling. Here we show that the C4 protein interacts with five additional AtSK family members. Bikinin, a competitive inhibitor of the seven AtSK family members that interact with C4, induced hyperplasia similar to that induced by the C4 protein. The Ser49 residue of C4 was found to be critical for C4 function, since: 1) mutagenesis of Ser49 to Ala abolished the C4-induced phenotype, abolished C4/AtSK interactions, and resulted in a mutant protein that failed to induce changes in the BR signaling pathway; 2) Ser49 is phosphorylated in planta; and 3) plant-encoded AtSKs must be catalytically active to interact with C4. A C4 N-myristoylation site mutant that does not localize to the plasma membrane and does not induce a phenotype, retained the ability to bind AtSKs. Taken together, these results suggest that plasma membrane associated C4 interacts with and co-opts multiple AtSKs to promote its own phosphorylation and activation to subsequently compromise cell cycle control.     

小拟南芥MKK基因家族全基因组鉴定及进化和表达分析

丝裂原活化蛋白激酶激酶(mitogen-activated protein kinase kinase,MAPKK或MKK)是丝裂原活化蛋白激酶(mitogen-activatedproteinkinase,MAPK)级联的重要组成部分,在植物的生长发育和胁迫应答过程中发挥重要作用。目前,已在多种植物中鉴定了MKK基因家族,但在十字花科植物小拟南芥(Arabidopsis pumila)中MKK基因家族的系统鉴定与分析尚未见报道。为了探索小拟南芥MKK基因家族的进化和功能,本研究通过全基因组分析鉴定了小拟南芥中16个MKK基因,散布于小拟南芥的10条染色体上。基于系统发育分析和多重序列比对,将这些基因分为5个亚族:A亚族(5个)、B亚族(2个)、C亚族(4个)、D亚族(3个)和E亚族(2个)。分子进化和共线性分析表明小拟南芥中存在7对复制基因,分别是ApMKK1-1/1-2、ApMKK2-1/2-2、ApMKK3-1/3-2、ApMKK4-1/4-2、ApMKK5-1/5-2、ApMKK9-1/9-2和ApMKK10-1/10-2,其中ApMKK1-1/1-2在复制事件之后发生了加速进化。结合ApMKKs启动子区的顺式元件分布和ApMKKs在成熟叶片、茎、花和果实以及盐胁迫下的表达模式,结果发现复制基因的表达具有组织特异性和功能多样性。部分复制基因在组织中的表达模式存在差异,但在盐胁迫下的表达模式却基本相同。本研究结果为解析MKK介导的小拟南芥发育过程和非生物胁迫信号转导通路的复杂机制奠定了基础。     

拟南芥转录因子GRAS 家族基因群响应渗透和干旱胁迫的初步探索

GRAS家族是一类植物特有的转录调控因子, 已有报道表明该家族基因在植物生长发育和光信号转导过程中具有重要作用。目前在拟南芥(Arabidopsis thaliana)基因组中已鉴定了33个GRAS家族基因。利用功能基因组学和生物信息学手段,通过基因芯片数据挖掘和基因功能预测, 对拟南芥GRAS家族基因在渗透和干旱胁迫过程中的应答模式进行了初步探索, 提出了一类响应渗透胁迫和干旱胁迫的拟南芥GRAS家族基因。以SCL13为例, 利用基因芯片相关性和GO分析, 对其在渗透胁迫信号转导过程中可能的调控机制进行了预测和分析。这一研究将为阐明GRAS家族基因参与水分胁迫的分子机制提供新的思路, 同时也为植物抗逆分子育种提供候选基因。     

Toward understanding the molecular mechanism of a geminivirus C4 protein

Geminiviruses are ssDNA plant viruses that cause significant agricultural losses worldwide. The viruses do not encode a polymerase protein and must reprogram differentiated host cells to re-enter the S-phase of the cell cycle for the virus to gain access to the host-replication machinery for propagation. To date, 3 Beet curly top virus (BCTV) encoded proteins have been shown to restore DNA replication competency: the replication-initiator protein (Rep), the C2 protein, and the C4 protein. Ectopic expression of the BCTV C4 protein leads to a severe developmental phenotype characterized by extensive hyperplasia. We recently demonstrated that C4 interacts with 7 of the 10 members of the Arabidopsis thaliana SHAGGY-like protein kinase gene family and characterized the interactions of C4 and C4 mutants with AtSKs. Herein, we propose a model of how C4 functions.     

Functions of chloroplastic adenylate kinases in Arabidopsis

Adenosine monophosphate kinase (AMK; adenylate kinase) catalyses the reversible formation of ADP by the transfer of one phosphate group from ATP to AMP, thus equilibrating adenylates. The Arabidopsis (Arabidopsis thaliana) genome contains 10 genes with an adenylate/cytidylate kinase signature; seven of these are identified as putative adenylate kinases. Encoded proteins of at least two members of this Arabidopsis adenylate kinase gene family are targeted to plastids. However, when the individual genes are disrupted, the phenotypes of both mutants are strikingly different. Although absence of AMK2 causes only 30% reduction of total adenylate kinase activity in leaves, there is loss of chloroplast integrity leading to small, pale-looking plantlets from embryo to seedling development. In contrast, no phenotype for disruption of the second plastid adenylate kinase was found. From this analysis, we conclude that AMK2 is the major activity for equilibration of adenylates and de novo synthesis of ADP in the plastid stroma.     

大叶落地生根类糖原合成激酶KdSK基因的克隆与表达分析

类糖原合成酶激酶(SKs)属于丝氨酸/苏氨酸类蛋白激酶,在植物器官发育、激素信号传导过程中十分重要,并参与生物胁迫、非生物胁迫的应答过程。大叶落地生根中的胎生苗发育过程,同时具备胚胎发生和器官发生的特征,是研究无性生殖的理想模型。为了更好地理解大叶落地生根中胎生苗发育的分子机制,该研究利用RACE-PCR技术,从大叶落地生根中克隆了1个新的基因KdSK。该基因具有423个氨基酸残基,分子量为47.79 kD,等电点为8.37,其开放阅读框长为1 272 bp。其蛋白与黄瓜的同源性最高,属于植物类GSK3/shaggy蛋白激酶家族的第Ⅳ类,与苜蓿(MSK4)蛋白在进化关系上最近,且与拟南芥(AtSK4-1、AtKSK4-2)聚为一枝。保守域结构分析表明,KdSK蛋白具有明显的蛋白激酶的结构域,包括蛋白激酶的ATP结构域和丝氨酸/苏氨酸蛋白激酶活化结构域。实时荧光定量PCR分析表明,该蛋白基因在大叶落地生根的根中表达量最高,且受渗透胁迫(甘露醇)的诱导上调表达。该研究首次从大叶落地生根中克隆出KdSK基因,该研究结果为进一步研究该基因的功能打下了基础。     

Comprehensive Analysis of Wall-Associated Kinase Genes and Their Expression Under Abiotic and Biotic Stress in Chinese Cabbage (Brassica rapa ssp. pekinensis)

The wall-associated kinase (WAK) gene family, a subfamily of the receptor-like kinase (RLK) gene family, is associated with the cell wall in plants, and has vital functions in cell expansion, pathogen resistance, and heavy metal stress tolerance because of their roles of the extracellular environment sensors to trigger intracellular signals in Arabidopsis. In the present study, 96 Chinese cabbage (Brassica rapa ssp. pekinensis) BrWAK gene family members were identified from the B. rapa genome using a reiterative database search and manual confirmation. The protein domain characterization, gene structure analysis, and phylogenetic analysis of the BrWAKs classified them into three gene groups. Comparative genomic analysis between WAK genes from Chinese cabbage and Arabidopsis revealed that the BrWAK genes have undergone the gene expansion and deletion events during evolution. Furthermore, the conserved motifs in the kinase domains of the WAK proteins and eukaryotic protein kinase family proteins were compared and some non-RD kinase proteins among the BrWAKs were identified. Ultimately, expression analysis of BrWAK genes in six tissues and under various stress conditions revealed that some tissue-specific WAK genes might function in callus cell growth and reproduction process; Bra012273, Bra016426, Bra016427, and Bra025882 might be involved in downy mildew resistance and high humidity stress; Bra012273, Bra025882, and Bra025883 might be responded to drought and heat stress. Taken together, this research was identified and classified the WAK gene family in Chinese cabbage and provided valuable resources to explore the potential roles of BrWAK genes in plant development and stress responses.