Abstract In silico analysis showed that Arabidopsis thaliana gene AT5G09530 encodes a uniquely repetitive, proline-enriched protein that is conserved across species, and is likely secreted to the cell wall. Based on its most common amino acid repeat motif, I named the gene PELPK1 and its putative paralog PELPK2 (AT5G09520). Reporter (GUS) expression showed that the PELPK1 upstream genomic region is sufficient for expression in the aleurone layer during seed germination, and is induced throughout the plant by biotic factors (especially Pseudomonas syringae infection), defense chemicals (MeJa, salicylic acid), and mechanical wounding, consistent with the presence of conserved regulatory elements. Sub-cellular localization of a translational fusion of PELPK1 with GFP showed that the protein was secreted into seed-coat aleurone cells and to the cell walls of other tissues. Based on these results, it was concluded that the PELPK1 is a cell wall-associated protein and is most actively transcribed during radicle penetration of the seed coat and during pathogen and wounding responses. A proteomic survey of aleurone proteins failed to identify PELPK1, although several proteins not previously associated with this tissue were identified. Mutational analysis demonstrated that RNAi silencing significantly down-regulated the transcript abundance of PELPK1. Phenotypic analysis showed that RNAi plants exhibited significantly slower germination and root growth when the medium was supplemented with sucrose (100mM). Conversely, constitutive overexpression (OX) of PELPK1 enhanced seed germination and root elongation as compared to wild-type (WT). Analysis of soil-grown plants showed slower emergence and slower vegetative growth for RNAi lines, while OX plants exhibited faster emergence and enhanced vegetative growth and flowering as compared to WT. However, PELPK1 RNAi and OX lines did not differ from WT in response to treatment with pathogens. These results show that the abundance of PELPK1 is positively correlated with plant growth rate under some conditions. PELPK1 may influence growth through CW modification or other independent pathways. / Plant Biology

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Histidine-rich glycoprotein (HRG) is a heparin-binding plasma protein modulating immune, hemostatic and vascular functions. I have studied the antiangiogenic functions of HRG in vitro and in vivo in order to understand the molecular mechanisms of action of HRG as an angiogenesis inhibitor. Angiogenesis is the formation of new blood vessels from the pre-existing vasculature. It is a central rate-limiting step of tumor development and thus a possible target for cancer therapeutics. Previous studies have shown that HRG has antiangiogenic functions in vivo and that the antiangiogenic effects are mediated via the proteolytically released His/Pro-rich domain of HRG. In this thesis we demonstrate that HRG can inhibit endothelial cell migration by interfering with focal adhesion and cytoskeletal turnover. Moreover we have identified the minimal active domain of HRG, a 35 amino acid peptide derived from the histidine- and proline-rich domain of HRG. Analyzing human tumor tissue samples, we have found that a His/Pro-rich fragment of HRG is bound to the vasculature from cancer patients but not to the vasculature from healthy individuals. The fragment is found in association with platelets, and we show that activated platelets can induce a functional microenvironment for the His/Pro-rich fragment. Cancer patients often display an increased coagulation and our data describe a new mechanism to confer specificity of an angiogenesis inhibitor for situations with enhanced platelet activation, as in the tumor. We have further studied the role of HRG in tumor growth by crossing HRG-deficient mice with a transgenic mouse model of pancreatic insulinoma. We show that mice lacking HRG display an elevated “angiogenic switch” and that the total tumor volume is larger in these mice than in wild type mice. HRG is also involved in regulation of platelet function and platelets can stimulate angiogenesis in various ways. We have depleted mice of platelets to study the possible connection between the function of HRG in angiogenesis and platelet regulation. Our data suggest an involvement of platelets in the antiangiogenic activities of HRG.

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