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

Kieliszewski/Cannon Proposal AbstractCOLLABORATIVE RESEARCH：剖析RSH extensiin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，其性质来自于正确的组装。 细胞壁组装的故障会导致植物的早期死亡，就像拟南芥rsh胚胎致死突变体一样，这是本提案的重点。rsh突变体不能在分裂细胞中组装功能性细胞壁，因为它缺乏特异性富含羟脯氨酸的糖蛋白，特别是称为RSH（根-芽-下胚轴-缺陷）的交联延伸蛋白。这种突变体将能够在分子水平上解剖壁自组装的精确规则。通过转移一系列新的RSH基因，突变植物将被治愈，这些基因可能取代缺失基因的功能。用一系列RSH类似物进行的这些互补实验将能够定义RSH对细胞壁组装的贡献。该策略涉及操纵野生型基因和将通过合成基因产生的类似物。RSH非常适合合成基因的方法，因为它是一种简单，高度重复的蛋白质。 某些氨基酸，包括赖氨酸、酪氨酸、丝氨酸和组氨酸，对于组装RSH并帮助其交联以在植物细胞壁中形成支架可能是至关重要的，因此将制备引起RSH蛋白质在这些氨基酸的含量方面改变的基因，并将评估这些基因治愈突变体的能力。 其他设计的RSH分子将缺乏蛋白质的一个末端（N-末端）或另一个末端（C-末端）的一部分，因为初步结果表明，RSH的一个末端，特别是C-末端，是壁组装所需的。更广泛的影响：长期目标是确定RSH在植物细胞壁中的分子相互作用，以及RSH中不同部分和氨基酸所起的确切作用，这与理解细胞壁如何自组装有关，包括理解植物防御反应和植物形态是如何形成的。更广泛的生物技术价值包括对超分子化学规则的新见解;在分子水平上指导物质自组装的多功能分子的设计是纳米技术的一个关键目标。这项工作还整合了细胞壁生物学和生物化学的方式，教导初级研究人员识别一个重要的生物学问题，并通过合作解决它，整合了从蛋白质设计和表征到分子遗传学和细胞生物学的方法。 两位研究人员都参加了与增加科学多样性有关的外展活动，包括对阿巴拉契亚的外展。

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.