During growth, plants integrate cell division with cell elongation and cell differentiation. We used proteomics to identify proteins from auxin (indole-3-acetic acid; IAA) -induced rapidly growing corn (Zea mays) coleoptiles to find candidates controlling this growth as well as the underlying cell wall and cuticle biosynthesis. Of 86 proteins identified, 15 showed a predicted association with cell wall/cuticle biosynthesis or trafficking machinery; the analysis also revealed four proteins of unknown function. Parallel real-time PCR indicated that the steady-state mRNA levels of genes with a known or predicted role in cell-wall biosynthesis increase upon auxin-treatment. Importantly, genes encoding two of the hypothetical proteins (ARRP1 and Hypothetical protein 3, a putative endoglucanase) also showed higher levels of mRNA; their gene expression was directly correlated with coleoptile and leaf growth. This suggested a role of these two novel proteins in the regulation of processes related to cell and organ expansion and thus plant growth. These data are described in chapter 2.Chapter 3 focuses on ARRP1 (Auxin rapid response protein 1), one of the proteins of unknown function associated with cell and organ growth. Upon auxin treatment ARRP1 mRNA and protein increased within 30 minutes – prior to measurable growth and increased expression of genes encoding cell wall biosynthetic enzymes, suggesting an intracellular signaling function. This was further supported by the localization of ARRP1 at the cell periphery as well as in the nucleus. Arabidopsis mutants lacking the ARRP1 homologue grew normally under standard conditions, but showed delayed growth and delayed flowering when germinated in the dark and transferred to light. This phenotype that could be reversed by expressing ZmARRP1 in the Arabidopsis mutant. ZmARRP1 expression was also induced by red and blue light, indicating a possible role in phytochrome and cryptochrome signaling at the interface with the auxin response. In situ hybridization demonstrated that ARRP1 mRNA is enriched in the vasculature of growing corn tissues. In addition, the corn ARRP1 interacted with the cytoplasmic domain of both Arabidopsis and Zea mays VH1/BRL2 which plays a role in the signaling of vascular development. Based on the fact that ARRP1 protein is involved in light-induced growth responses as well as with components of auxin signaling, we propose that it may integrate several environmental and hormonal signals affecting plant development.Chapter 4 describes the initiation of a series of experiments investigating addtional proteins that interact with ARRP1 to elucidate other components of the signaling pathway that use ARRP1. Using in vitro pull-down assays and mass spectrometry, I identified 32 putative ARRP1-interacting proteins in the developing corn leaf. Some of these proteins appear to be involved in cell wall biosynthesis, including a beta-glucosidase homologue. Others are predicted to be signal molecules, including a tetratricopeptide repeat domain containing protein and a CobW/P47K family protein. Using semiquantitative RT-PCR, I showed that several of these genes have a similar expression pattern compared to ARRP1, suggesting a function in the same signaling/response path.
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