Root-Expressed Rice PAP3b Enhances Secreted APase Activity and Helps Utilize Organic Phosphate.

Phosphate (Pi) deficiency leads to the induction of purple acid phosphatases (PAPs) in plants, which dephosphorylate organic phosphorus (P) complexes in the rhizosphere and intracellular compartments to release Pi. In this study, we demonstrate that OsPAP3b belongs to group III low-molecular weight PAP and is low Pi-responsive, preferentially in roots. The expression of OsPAP3b is negatively regulated with Pi resupply. Interestingly, OsPAP3b was found to be dual localized to the nucleus and secretome. Furthermore, OsPAP3b is transcriptionally regulated by OsPHR2 as substantiated by DNA-protein binding assay. Through in vitro biochemical assays, we further demonstrate that OsPAP3b is a functional acid phosphatase (APase) with broad substrate specificity. The overexpression (OE) of OsPAP3b in rice led to increased secreted APase activity and improved mineralization of organic P sources, which resulted in better growth of transgenics compared to the wild type when grown on organic P as an exogenous P substrate. Under Pi deprivation, OsPAP3b knock-down and knock-out lines showed no significant changes in total P content and dry biomass. However, the expression of other phosphate starvation-induced genes and the levels of metabolites were found to be altered in the OE and knock-down lines. In addition, in vitro pull-down assay revealed multiple putative interacting proteins of OsPAP3b. Our data collectively suggest that OsPAP3b can aid in organic P utilization in rice. The APase isoform behavior and nuclear localization indicate its additional role, possibly in stress signaling. Considering its important roles, OsPAP3b could be a potential target for improving low Pi adaptation in rice.

Identification of Purple Acid Phosphatases in Chickpea and Potential Roles of CaPAP7 in Seed Phytate Accumulation.

Purple acid phosphatases (PAPs) play important roles in phosphate (Pi) acquisition and utilization. These PAPs hydrolyze organic Phosphorus (P) containing compounds in rhizosphere as well as inside the plant cell. However, roles of PAPs in one of the most widely cultivated legumes, chickpea (Cicer arietnum L.), have not been unraveled so far. In the present study, we identified 25 putative PAPs in chickpea (CaPAPs) which possess functional PAP motifs and domains. Differential regulation of CaPAPs under different nutrient deficiencies revealed their roles under multiple nutrient stresses including Pi deficiency. Interestingly, most of the CaPAPs were prominently expressed in flowers and young pods indicating their roles in flower and seed development. Association mapping of SNPs underlying CaPAPs with seed traits revealed significant association of low Pi inducible CaPAP7 with seed weight and phytate content. Biochemical characterization of recombinant CaPAP7 established it to be a functional acid phosphatase with highest activity on most abundant organic-P substrate, phytate. Exogenous application of recombinant CaPAP7 enhanced biomass and Pi content of Arabidopsis seedlings supplemented with phytate as sole P source. Taken together, our results uncover the PAPs in chickpea and potential roles of CaPAP7 in seed phytate accumulation.

OsHAD1, a Haloacid Dehalogenase-Like APase, Enhances Phosphate Accumulation.

Phosphorus (P) deficiency limits plant growth and yield. Since plants can absorb only the inorganic form of P (Pi), a large portion of soil P (organic and inorganic P complexes) remains unused. Here, we identified and characterized a PHR2-regulated, novel low-Pi-responsive haloacid dehalogenase (HAD)-like hydrolase, OsHAD1 While OsHAD1 is a functional HAD protein having both acid phosphatase and phytase activities, it showed little homology with other known low-Pi-responsive HAD superfamily members. Recombinant OsHAD1 is active at acidic pH and dephosphorylates a broad range of organic and inorganic P-containing substrates, including phosphorylated serine and sodium phytate. Exogenous application of recombinant OsHAD1 protein in growth medium supplemented with phytate led to marked increases in growth and total P content of Pi-deficient wild-type rice (Oryza sativa) seedlings. Furthermore, overexpression of OsHAD1 in rice resulted in enhanced phosphatase activity, biomass, and total and soluble P contents in Pi-deficient transgenic seedlings treated with phytate as a restricted Pi source. Gene expression and metabolite profiling revealed enhanced Pi starvation responses, such as up-regulation of multiple genes involved in Pi uptake and solubilization, accumulation of organic acids, enhanced secretory phosphatase activity, and depletion of ATP in overexpression lines as compared with the wild type. To elucidate the underlying regulatory mechanisms of OsHAD1, we performed in vitro pull-down assays, which revealed the association of OsHAD1 with protein kinases such as OsNDPKs. We conclude that, besides dephosphorylation of cellular organic P, OsHAD1 in coordination with kinases may regulate the phosphorylation status of downstream targets to accomplish Pi homeostasis under limited Pi supply.

Improvement in phosphate acquisition and utilization by a secretory purple acid phosphatase (OsPAP21b) in rice.

Phosphate (Pi) deficiency in soil system is a limiting factor for rice growth and yield. Majority of the soil phosphorus (P) is organic in nature, not readily available for root uptake. Low Pi-inducible purple acid phosphatases (PAPs) are hypothesized to enhance the availability of Pi in soil and cellular system. However, information on molecular and physiological roles of rice PAPs is very limited. Here, we demonstrate the role of a novel rice PAP, OsPAP21b in improving plant utilization of organic-P. OsPAP21b was found to be under the transcriptional control of OsPHR2 and strictly regulated by plant Pi status at both transcript and protein levels. Biochemically, OsPAP21b showed hydrolysis of several organophosphates at acidic pH and possessed sufficient thermostability befitting for high-temperature rice ecosystems with acidic soils. Interestingly, OsPAP21b was revealed to be a secretory PAP and encodes a distinguishable major APase (acid phosphatase) isoform under low Pi in roots. Further, OsPAP21b-overexpressing transgenics showed increased biomass, APase activity and P content in both hydroponics supplemented with organic-P sources and soil containing organic manure as sole P source. Additionally, overexpression lines depicted increased root length, biomass and lateral roots under low Pi while RNAi lines showed reduced root length and biomass as compared to WT. In the light of these evidences, present study strongly proposes OsPAP21b as a useful candidate for improving Pi acquisition and utilization in rice.