Molecular Mechanisms Underlying Purple Acid Phosphatases Involved In Organic Phosphorus Utilization In Stylosanthes

Phosphorus(P)is an essential macronutrient for plant growth and development.However,phosphate(Pi)is easily fixed by soil components into unavailable forms,included insoluble inorganic P and organic P.Plant acid phosphatase(APase)and phytase are generally considered to involve in P scavenging and recycling from organic P.Stylo(Stylosanthes spp.)is an important tropical forage legume and widely grown in tropical and subtropical areas,where acid soils are widely distributed.Stylo is considered to be a pioneer pasture on acid soils due to its superior capability in adaptation to low P acid soils.However,molecular mechanisms underlying utilization of organic P remain largely unknown in stylo.In this study,53 stylo genotypes were used to examine variations of root intra-and extra-cellular APase or phytase activities.Subsequently,a genotype with superior ability to utilize phytate,TPRC2001-1,was used to investigate APase isozyme profiles.Four purple acid phosphatases(PAPs)were identified through mass spectrometry analysis of one Pi-starvation increased APase isozyme in TPRC2001 roots,and their encoding genes were separately cloned.Functional analysis of the four SgPAPs resulted in clarification of molecular mechanism underlying stylo utilization of external phytate and deoxy-ribonucleotide triphosphate(dNTP).The main results were shown as follows:(1)Genotypic variations of root intra-and extra-cellular APase or phytase activities were observed among the testes 53 stylo genotypes.The coefficient of variation(CV)of root extracellular APase activities(CV = 0.335)and root extracellular phytase activities(CV = 0.228)was higher than that of root intracellular APase activities(CV = 0.165)and root intracellular phytase activities(CV-0.139).(2)Ten genotypes contrasting in root extracellular phytase activities were selected to examine their abilities to utilize exogenous phytate.The results showed that there were genotypic variations for their capabilities to utilize exogenous phytate in stylo,which was mainly dependent on activities of root extracellular phytase.Among them,TPRC2001-1 exhibited superior ability to utilize phytate.(3)One APase isozyme was significantly accumulated by P deficiency in TPRC2001-1 roots.Furthermore,four PAPs were identified from the APase isozyme through mass spectrometry,and their corresponding genes were separately cloned,including SgPAP7,SgPAP10,SgPAP23,and SgPAP26.(4)Expression patterns of SgPAPs were analyzed by qRT-PCR.Results showed that SgPAP7,SgPAP10,SgPAP23,and SgPAP26 transcripts were enhanced by P deficiency in roots of stylo.Transient expression of 35S:SgPAPs-GFP in tobacco leaf epidermal cells verified that these four SgPAPs were co-localized in plasma membrane and cytoplasm,but mainly on the plasma membrane,which was consistent with results from western blot analysis in transgenic bean hairy roots with SgPAPs-GFP overexpression.(5)Overexpressing SgPAP23 could result in significant increases of root extracellular phytase activities in transgenic bean hairy roots and Arabidopsis thaliana.Furthermore,plant biomass and total P content was significantly increased in SgPAP23-OX lines,and higher than those in control lines,when the phytate was supplied as the sole external P source.(6)Overexpression of SgPAP7,SgPAP10,and SgPAP26 led to significant increases of extracellular APase activities in transgenic bean hairy roots compared to those in control lines.Furthermore,with dNTP application,plant biomass and total P content in transgenic bean hairy roots with SgPAP7,SgPAP10 or SgPAP26 overexpression were significantly higher than those in control lines.Taken together,results herein suggest that stylo capacities to utilize exogenous organic P had genotypic variations,and were positively correlated with activities of root extracellular APase and phytase.Furthermore,P deficiency increased extracellular APase and phytase activities in roots of the P-efficient genotype,TPRC2001-1,which might be caused by enhanced expression of SgPAP7,SgPAP10,SgPAP23,and SgPAP26,and thus facilitate utilization of exogenous phytate or dNTP,respectively.Therefore,our results not only dissect molecular mechanisms underlying stylo adaptation to P deficiency on acid soils,but also provide candidate genes for developing cultivars with high P efficiency through traditional or modern breeding approaches.