RUI: Cell Wall Associated Protein Kinases and Cell Elongation

RUI：细胞壁相关蛋白激酶和细胞伸长

• 批准号: 0235023
• 负责人: Bruce Kohorn
• 金额: $ 33.78万
• 依托单位: Bowdoin College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2006-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0235023&HistoricalAwards=false
• 关键词: RUI; Cell; Wall; Associated; Protein

The plant Wall Associated Kinases,WAKs, which were discovered by Dr. Kohorn, are phosphorylating enzyme proteins that physically link the plasma membrane to the carbohydrate and protein matrix that comprises the plant cell wall. WAKs are of importance as they have the potential to directly signal cellular events through their cytoplasmic kinase domain. There are five Arabidopsis WAKs that vary primarily in their extracellular domain and that collectively are expressed in most tissues. WAK mRNA and protein are present in vegetative meristems, junctions of organ types and areas of cell expansion. They are also induced by pathogen infection and wounding. Recent experiments indicate that WAKs are also involved in pollen tube growth in the gynoecium, and may possibly be involved in pollen tube orientation. WAKs can be found bound to pectin in the cell wall, and at least one isoform is associated with a secreted glycine rich protein (GRP). Disruption by antisense and RNAi of WAK expression in leaves leads to the loss of cell expansion, indicating that WAKs play an important role in plant growth and development. It remains to be discovered how WAKs, pectins and GRPs combine to regulate the patterns of cell expansion in coordination with the complex architecture and mechanical constraints of the plant cell wall.This project will explore the role WAKs play in cell expansion and in pollen tube growth. A set of WAK specific antibodies will be generated to allow a thorough characterization of the protein expression of each family member and to serve as tools in the analysis of plants with mutations in individual WAKs. Arabidopsis lines carrying an insertion mutation in a WAK gene are unable to produce progeny homozygous for the mutation because fertilization is affected (mutant pollen grains are able to germinate but do not reach the egg). The exact nature of the defect will be determined using microscopy, antibodies and markers specific for the mutant pollen.The pollen mutant phentoype will be complemented such that the progeny can be made homozygous for the WAK mutation. The resulting vegetative mutant tissue will be analyzed to understand how WAKs mediate cell expansion. Tests for altered turgor, cell wall extension and altered expansin and cell wall enzyme expression will be performed. Application of Gibberellin (GA) rescues plants that are rendered dwarf by WAK mutations. Therefore, the relationship between the GA and WAK signaling pathways will be explored through a series of crosses with mutants in the GA pathway and through the analysis of GA biosynthetic enzyme expression.Finally, the interaction of WAKs with both extracellular glycine rich proteins and with cytoplasmic transcriptional adaptor proteins will be explored using genetics and biochemical analysis.The broader impacts of this project will be manifested primarily through the teaching and training of undergraduates. Bowdoin College is a predominantly undergraduate institution, and undergraduate student researchers will be extensively involved in performing these experiments.

Kohorn博士发现的植物壁相关激酶（WAK）是磷酸化酶蛋白，它将质膜与构成植物细胞壁的碳水化合物和蛋白质基质物理连接起来。WAK是重要的，因为它们具有通过其胞质激酶结构域直接信号传导细胞事件的潜力。有五种拟南芥WAK主要在其细胞外结构域中变化，并且在大多数组织中共同表达。WAK mRNA和蛋白质存在于营养分生组织、器官类型的连接处和细胞扩展区域。它们也由病原体感染和创伤引起。 最近的实验表明，WAKs也参与了雌蕊中花粉管的生长，并可能参与了花粉管的定向。可以发现WAK与细胞壁中的果胶结合，并且至少一种同种型与分泌的富含甘氨酸的蛋白（GRP）相关。 WAK在植物叶片中的表达被反义和RNAi干扰导致细胞扩增的丧失，表明WAK在植物生长发育中起重要作用。WAKs、果胶和GRP如何结合联合收割机来调节细胞的扩张模式，以协调植物细胞壁的复杂结构和机械约束，这一点还有待发现。 将产生一组WAK特异性抗体，以允许对每个家族成员的蛋白质表达进行彻底表征，并用作分析在单个WAK中具有突变的植物的工具。 在WAK基因中携带插入突变的拟南芥品系不能产生突变纯合的后代，因为受精受到影响（突变花粉粒能够萌发，但不能到达卵）。 将使用显微镜、抗体和突变花粉特异性标记来确定缺陷的确切性质。将补充花粉突变表型，使得可以使后代对于WAK突变是纯合的。将分析所得的营养突变组织以了解WAK如何介导细胞扩增。将对改变的膨压、细胞壁延伸和改变的扩张蛋白和细胞壁酶表达进行检测。 赤霉素（GA）的应用挽救了由于WAK突变而变得矮小的植物。 因此，GA和WAK信号通路之间的关系将通过与GA通路中的突变体的一系列杂交以及通过分析GA生物合成酶表达来探索。WAKs与细胞外富含甘氨酸的蛋白质以及与细胞质转录衔接蛋白的相互作用将通过遗传学和生物化学分析进行探索。通过本科生的教学和培训。 鲍登学院是一个主要的本科院校，本科生研究人员将广泛参与执行这些实验。