Collaborative Research: Dissecting the Role of RSH Extensin in Assembly of the Plant Cell Wall

Kieliszewski/Cannon建议摘要COLLABORATIVE研究：剖析RSH伸展蛋白在植物细胞壁组装中的作用植物细胞壁是一种自组装结构，其性质源于正确的组装。壁组装的故障可能导致植物的早期死亡，就像拟南芥RSH胚胎致命突变体所发生的那样，这是本提案的重点。RSH突变体不能在细胞分裂中组装功能细胞壁，因为它缺乏一种特定的富含羟脯氨酸的糖蛋白，特别是一种名为RSH(根-枝-下胚轴缺陷)的交联性延伸蛋白。这种突变体将能够在分子水平上剖析壁自组装的精确规则。突变植物将通过转移一系列新的RSH基因来治愈它们的缺陷，这些基因可能会取代缺失的基因的功能。这些与一系列RSH类似物的互补实验将使我们能够定义RSH对细胞壁组装的贡献。该策略包括操纵野生型基因和将通过合成基因产生的类似物。RSH是一种简单的、高度重复的蛋白质，非常适合于合成基因方法。某些氨基酸，包括赖氨酸、酪氨酸、丝氨酸和组氨酸，可能是组装RSH并帮助它在植物细胞壁中形成支架的关键，因此将产生引起RSH蛋白质含量改变的基因，并评估这些基因治愈突变的能力。其他设计的RSH分子将缺少蛋白质一端(N-端)或另一端(C-端)的一部分，因为初步结果表明，RSH的一端，特别是RSH的C-端，是壁组装所必需的。最后，对自然产生的RSH、RSH类似物和拟南芥细胞壁进行生化表征。广泛影响：定义RSH在植物细胞壁中的分子相互作用的长期目标，以及不同部分和氨基酸在RSH中所起的确切作用，与理解壁如何自组装有关，包括理解植物的防御反应和植物形式是如何创建的。更广泛的生物技术价值包括对超分子化学规则的新见解；设计在分子水平上指导物质自组装的多功能分子是纳米技术的关键目标。这项工作还将细胞壁生物学和生物化学结合在一起，教会初级研究人员识别重大的生物学问题，并通过合作解决它，合作的方法包括从蛋白质设计和表征到分子遗传学和细胞生物学。这两名研究人员都参加了与增加科学多样性有关的外联活动，包括到阿巴拉契亚的外联活动。

Kieliszewski/Cannon Proposal AbstractCOLLABORATIVE RESEARCH: Dissecting the role of RSH extensinin assembly of the plant cell wallThe plant cell wall is a self-assembling structure whose properties arise from correct assembly. Malfunctions in wall assembly can result in the early death of the plant, as occurs with the Arabidopsis rsh embryo lethal mutant, the focus of this proposal. The rsh mutant cannot assemble a functional cell wall in dividing cells because it lacks a specific hydroxyproline-rich glycoprotein, notably a crosslinking extensin designated RSH (ROOT-SHOOT-HYPOCOTYL-DEFECTIVE). This mutant will enable dissection of the precise rules for wall self-assembly at a molecular level. Mutant plants will be cured of their defect by transferring a range of new RSH genes that might replace the function of the gene that is missing. These complementation experiments with a series of RSH analogs will enable definition of the contributions RSH makes to cell wall assembly. The strategy involves manipulations of the wild-type gene and analogs that will be produced through synthetic genes. RSH is ideally suited for a synthetic gene approach as it is a simple, highly repetitive protein. Certain amino acids, including lysine, tyrosine, serine and histidine may be crucial to assemble RSH and help it crosslink it to form a scaffold in the plant cell wall, therefore genes will be made that give rise to RSH proteins altered in their content of these amino acids and the ability of these genes to cure the mutant will be evaluated. Other designer RSH molecules will lack part of one extreme end of the protein (N-terminus) or the other (C-terminus) as preliminary results indicate one end in particular, the C-terminus of RSH, is required for wall assembly. Finally naturally-occurring RSH, the RSH analogs and Arabidopsis cell walls will be characterized biochemically.Broader Impact: The long term goal of defining the molecular interactions of RSH in the plant cell wall, and the precise roles played by the different sections and amino acids in RSH are relevant to understanding how the wall self-assembles, including understanding plant defense responses and how plant form is created. A broader biotechnological value includes new insights into the rules of supramolecular chemistry; the design of versatile molecules that direct the self-assembly of matter at a molecular level is a key goal of nanotechnology. This work also integrates cell wall biology and biochemistry in a way that teaches junior researchers to identify a significant biological problem and set about solving it through collaboration that integrates methods ranging from protein design and characterization to molecular genetics and cell biology. Both investigators take part in outreach activities relevant to increasing diversity in the sciences, including outreach to Appalachia.