Functional Analysis Of Purple Acid Phosphatase Genes OsPAP26、ZmPAP10a And ZmPAP26

Phosphorus (P) deficiency is an important limiting factor affecting crop growth and yield. Development of new germplasm with enhanced P use efficiency based on the understanding of rice phosphorus deficiency adapt mechanisms is important for reducing the amount of phosphorus fertilizer in crop production and alleviating environment pollution. Over the past decades, many scientific studies aimed at elucidating the complex molecular mechanisms and crucial regulators underlying phosphate (Pi) signaling and Pi homeostasis in plants have been performed and a preliminary Pi signal regulation network has been constructed. However, genetic improvement using the known genes in the network failed to achieve the expected effect in terms of Pi use efficiency, suggesting the current Pi signal network may have missed important members.Our previous study analyzed the purple acid phosphates gene family in rice. Through the quantitative RT-PCR and promoter analysis of these PAP genes, it was found that the rice orthologue of AtPAP26, OsPAP26, is not directly regulated by the central transcription factor OsPHR2. In this study, the role of OsPAP26 in maintaining Pi homeostasis was studied through characterization of the expression of the gene, and the physiological and biochemical properties of the transgenic rice plants that overexpressed or suppressed the expression of the gene. Quantitative RT-PCR (qRT-PCR) showed that the transcript abundance of OsPAP26 is not affected by Pi deficiency or leaf senescence.The Western blot and enzymatic analysis showed that protein accumulation and enzyme activity of OsPAP26 increased under Pi deficiency condition, suggesting that OsPAP26 is up-regulated by Pi deficiency signal at post transcription level. Using the construct containing the expression cassette of the OsPAP26 promoter fusing the GUS reporter gene, it was found that OsPAP26 was constitutively expressed in roots, shoots, and leaves. There is no significant difference between the GUS densities of the roots from Pi-deficiency and Pi-sufficiency plants. Western blotting analysis showed that Pi starvation and leaf senescence increased the accumulation of OsPAP26 protein. Transgenic rice lines that overexpressed the OsPAP26 increased intracellular and secreted APase activity in the transgenic lines. Organic P utilization efficiency of the transgenic lines was assessed by using ATP as the only P source. Results showed that overexpression of OsPAP26 increased organic P utilization efficiency. Furthermore, the in-gel activity assay determined the specific band of OsPAP26.The study analyzed the functions of two maize PAP genes encoding la family PAPs, including ZmPAP10a and ZmPAP26. Through agrobacterium-mediated immature embryo transformation, the transgenic lines that overexpressed the ZmPAP10a or ZmPAP26 were obtained. The overexpression effect was verified by qRT-PCR. In-gel activity assay showed that overexpression of the ZmPAP10a and ZmPAP26 led to significant increase of a specific band, respectively. Intracellular and root-associated acid phosphatase activity of the ZmPAP10a overexpression lines was significantly increased compared to that in the wild type plants (WT). As a result, the ZmPAP10a overexpression plants had a significant higher Pi concentration than that of WT. Among the three ZmPAP26 overexpression lines, lines OE-15 and OE-17 showed increased root and shoot intracellular and root-associated acid phosphatase activity, and Pi concentrations than these of WT. Line OE-10 showed increased root-associated acid phosphatase activity, but not the intracellular acid phosphatase activity and Pi concentration. Field experiments showed that both ZmPAP10a and ZmPAP26 overexpression lines showed enhanced 1000 seed weight and seed weight per plant, suggesting that overexpression of ZmPAP10a and ZmPAP26 might be a useful strategy for the improvement of P use efficiency in maize.