The influence of nutritional phosphate deprivation on the secreted proteome of Arabidopsis thaliana
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This thesis examines the influence of nutritional phosphate (Pi) deprivation on extracellular proteins secreted by the model plant Arabidopsis thaliana. Initial studies compared the secretome of Pi-sufficient (+Pi) versus Pi-deficient (-Pi) Arabidopsis cell cultures by 2-dimensional gel electrophoresis. Mass spectrometry identified 18 different secreted proteins that were upregulated by at least 2-fold by –Pi Arabidopsis. They were predicted to function in Pi scavenging, cell wall and ROS metabolism, proteolysis, and pathogen responses. The relationship between mRNA levels and relative amounts of selected secretome proteins was assessed. The results indicate that transcriptional control is but one of many factors contributing to Arabidopsis Pi starvation responses and highlight the importance of parallel biochemical and proteomic studies of –Pi plants. Three purple acid phosphatase (APase) isoforms were fully purified from the culture media of –Pi Arabidopsis cells and identified as AtPAP12 (At2g27190) and two AtPAP26 (At5g34850) glycoforms. As each purple APase exhibited broad substrate specificities and pH-activity profiles, it is hypothesized that their combined activities facilitate Pi scavenging from soil-localized organophosphates during nutritional Pi deprivation. AtPAP26 is dual-targeted during Pi stress since an earlier report demonstrated that it is also the principal intracellular (vacuolar) APase upregulated by -Pi Arabidopsis. The results indicate that differential glycosylation influences AtPAP26’s substrate specificity and subcellular targeting. An atpap26 T-DNA insertional mutant lacking AtPAP26 transcripts and immunoreactive AtPAP26 polypeptides exhibited: (i) 9- and 5-fold lower shoot and root APase activity, respectively, which did not change in response to Pi starvation, (ii) a 40% reduction in secreted APase activity during Pi deprivation, (iii) 35 and 50% reductions in free and total Pi concentration, respectively, in shoots of –Pi plants, and (iv) impaired shoot and root development when subjected to Pi deficiency. By contrast, no deleterious influence of AtPAP26 loss of function was apparent in +Pi plants. The results establish a firm role for AtPAP26 in the acclimation of Arabidopsis to Pi deficiency. The identification and functional characterization of secreted proteins upregulated by –Pi Arabidopsis is relevant to applied efforts to engineer Pi-efficient transgenic plants, needed to minimize the input of expensive, unsustainable, and polluting Pi fertilizers in crop production.