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

University Of Massachusetts Amherst, Amherst MA

Investigators

Abstract

Kieliszewski/Cannon Proposal Abstract COLLABORATIVE RESEARCH: Dissecting the role of RSH extensin in assembly of the plant cell wall The 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 us to dissect 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 us to define 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. Finally, as the most abundant biomass, plant cell walls at all levels from forest to food to fibre to fuel, contribute crucially to self-sustaining civilizations. 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. To a greater or lesser extent, both University of Massachusetts and Ohio University have diverse student bodies ensuring that minority students and women form a significant part of the team. Both Dr. Cannon and Dr. Kieliszewski already take part in outreach activities relevant to increasing diversity in the sciences, including outreach to Appalachia.