Functional Analysis Of Purple Acid Phosphatase OsPAP10a And OsPAP10c In Rice

Phosphorus (P) is one of the macronutrients required for plant growth and development. Although P is abundant in the earth’s crust, it is usually present in the form of organophosphate compounds. Inorganic phosphate (Pi) is the only form of phosphorus that can be uptake by plants. The concentration of Pi is well below the level that is required for optimal plant growth. To obtain high crop yield, Pi fertilizer is widely used in farming. Overfertilization not only increases the cost of crop production, but also leads to eutrophication of water bodies. Developing crops with improved P utilization efficiency is important for sustainable agriculture.Under phosphate starvation, plants increase the production and secretion of purple acid phosphatases (PAPs) into rhizosphere to release Pi from these organic sources. Studies in Arabidopsis and other species showed the importance of the la group of PAPs in organic P utilization and tolerance to Pi deficiency. In rice, the la group of PAPs include OsPAP10a, OsPAP10b, OsPAP10c, OsPAP10d,and OsPAP26. Expression profiling analysis showed that OsPAP10a was specifically induced by Pi starvation in both roots and leaves, while OsPAP10c was specifically induced by Pi starvation only in roots. OsPAP26 was expressed constitutively. OsPAP10b and OsPAP10d were expressed at very low level in all tissues and all developmental stages. This study is focused on the functional analysis of OsPAP10a and OsPAP10c.Using Agrobacterium-mediated rice transformation method, transgenic rice plants that expressed the contructs with OsPAP10a or OsPAP10c promoter driven the GUS reporter gene were obtained. GUS staining of the transgenic plants showed similar expression pattern of OsPAP10a and OsPAP10c with those of qRT-PCR. Moreover, OsPAP10c has a basal expression in leaves, young spikes, and pollen. Cross sections of roots showed that OsPAP10a and OsPAP10c were predominantly expressed in epidermis and exodermis cells.The root-associated APase activity was analyzed by 5-bromo-4-chloro-3-indolyl phosphate (BCIP) staining. Compared with OsPAP10a overexpression plants, a more pronounced staining was observed for OsPAP10c overexpression lines. Quantitative analysis of APase activity was conducted on root-associated surface, roots, leaves, and the medium solution grown the rice suspension cells. Results showed that overexpression of OsPAP10c resulted in a 10-folds increase of root-associated APase activity compared to wild type plants, that of OsPAP10a increased root-associated APase activity for 2-folds.Overexpression of OsPAP10c increased APase activity nearly 5-folds in both roots and leaves, and more than 10-folds in the growing medium, whereas overexpression of OsPAP10a only induced the APase activity no more than 2-folds. Thus, OsPAP10c has greater potential for utilization in the improvement of P use efficiency.In-gel APase activity assays found that the A2 band was speficially accumulated to a very high level in leaf and root protein samples extracted from OsPAP10a overexpression plants.The band also existed in samples from WT plants grown under Pi starvation conditions.OsPAP10c overexpression plants induced A1, A3, A4 and A5 bands. In-gel activity assay and Western blot of protein extracts from the growth medium of the resuspension culture showed that OsPAP10c is a secreted APase.To access the function of OsPAP10a and OsPAP10c on the crop improvement of P use efficiency, ATP degradation ability was measured in the WT, OsPAP10a and OsPAP10c overexpression plants grown in solution culture supplied with ATP as the sole P source. Compared with the WT plants, overexpression of OsPAP10c significantly increased the Pi release from ATP. Pi concentrations in leaves of WT were gradually decreased during the treatment with a 56% decline after five days. Pi concentration decreased 25% in OsPAP10a overexpression line, while 8% to 24% decline of the Pi concentrations were observed in different OsPAP10c overexpression lines.Taken together, the study investigated and compared that functions of OsPAP10a and OsPAP10c in hydrolyzation of organic P. Results showed that OsPAP10c is a dominant secreted APase, playing important roles in utilization of external organic phosphorus. OsPAP10c has great potential to ultilize in the crop improvement of P use efficiency.