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 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.
Nature of the Project: RSH (Root-Shoot-Hypocotyl-defective) is a protein that is required for proper embryo development in Arabidopsis plants. RSH is a glycoprotein that is analogous to animal collagen in structure and function. It is a rodlike protein that self-assembles to form networks in the extracellular matrix. RSH has unique amino acid sequences at either end (designated the N-terminus and the C-terminus sequences) and sandwiched between these unique ends is a repeating amino acid sequence that occurs eleven times. Without RSH present cells cannot divide properly, the embryo is defective and seedlings die within three weeks of germination. The work proposed in our grant aimed to elucidate the features of RSH that are crucial for its function in cell division processes. Such knowledge bears directly on our understanding of how plants grow and develop. It also has ramifications for aspects of plant disease resistance. We approached this problem by designing novel genes that encoded RSH analogs with altered structure. Then we inserted the genes into mutant Arabidopsis plants (lacking a functioning RSH gene) and determining how the RSH glycoprotein analogs behaved biochemically and, in collaboration with colleague Maura Cannon from The University of Massachusetts at Amherst, if the RSH analogs would give rise to ‘normal’ plants. Outcomes of the Project: We constructed a wide range of genes (~2 sets of 12) encoding RSH glycoprotein analogs with alterations in amino acids that we suspected played crucial roles in RSH contributions to cell wall formation and therefore in growth and development. One set of genes were sent to collaborator Maura Cannon to determine if they could impart normal growth to Arabidopsis plants that had lost the function of the normal RSH gene. The other set we kept to transform plant cells to allow us to isolate the RSH analog proteins for biochemical analysis. As the unique N-terminus and C-terminus amino acid sequences that sandwich the eleven internal repeats were required for RSH function, we focused on isolating the RSH analog proteins that lacked the N-terminus sequence, the C-terminus sequence, or both. We also established a new assay for RSH network self-assembly. The assay uses atomic force microscopy to visualize the networks formed and we anticipate the assay can be used to determine how changes in the amino acid sequence of RSH affects the fidelity and efficiency of network formation. Educational aspects of this work included undergraduate researchers: Ben Wegenhart, won a Goldwater Fellowship for his work imaging RSH self-assembling scaffolds by atomic force microscopy and is in graduate school at Purdue. Undergraduates who participated in the project include: Jessica Cross, who aided in the construction of genes encoding RSH analogs and is currently pursuing a medical degree; Steven Cummings, who worked in the lab for more than 2 years and also aided in the construction of genes encoding RSH analogs, maintaining transformed plant cell lines, propagation of Arabidopsis seedlings for wall analysis and cell wall isolations; Christina Ufholz who maintained cell lines transformed with RSH. Christina is pursuing a career in research.
