Transcriptome Analysis Of Stylo In Response To Phosphorus Deficiency And Functional Study Of Candidate Gene SgPAP15a

Phosphorus(P)is one essential nutrient for plants growth and development.P deficiency is a common issue remains in acid soil,which limits crop and forage production.Although the content of soluble inorganic phosphate(Pi)which can be directly absorbed by plant roots in soil is low,there are a large number of organic P that can not be directly absorbed by roots,mainly exists in the form of phytate-P(Phy)in soil.Stylo(Stylosanthes)is an important kind of tropical forage and green manure crop.It has strong adaptability to acid soil and can efficiently utilize Phy.In our previous study,a purple acid phosphatase(PAP),SgPAP23 was identified from stylo.Functional analysis demonstrated that SgPAP23 participate in the utilization of Phy.However,the efficient utilization of Phy may depend on other PAP members in stylo.This study was based on the materials of Reyan No.5 Stylo,which has completed whole genome sequencing.We carried out transcriptome analysis,whole genome identification of PAP family members,functional analysis of candidate genes,and other researches,which aimed at exploring the potential PAP members involved in Phy utilization in stylo.Mechanism of stylo adaptation to P deficiency were further revealing.The main findings are as follows:(1)Hydroponic experiments showed that P deficiency significantly decreased plant dry weight and total phosphorus content,but significantly increased root surface area,root volume,total root length,and root acid phosphatase(APase)activity by 42.6%,40.8%,44.6%,and 314.3%,respectively.(2)Transcriptome analysis showed that low P treatment resulted in 1145,874 and2844 genes significant up-regulation in the leaves,stems and roots of stylo,and 277,172 and 1624 genes significant down-regulation in the leaves,stems and roots of stylo.GO enrichment revealed that the genes up-regulated by P deficiency in leaves,stems and roots were significantly enriched to 15 terms,15 terms and 20 terms of molecular functions,the differentially expressed genes in leaves,roots and stems of stylo were significantly enriched in the term "APase activity".(3)Through genome-wide analysis,28 PAP genes were identified in the genome of stylo.Among them,14,12 and 11 PAPs were up-regulated by P deficiency in leaves,stems and roots,respectively.Through protein evolution tree analysis,3 stylo PAP members(SgPAP15 a,SgPAP15 b and SgPAP23)were classified into PAP Ib-1 subfamily(with potential function in Phy degradation).SgPAP15 a and SgPAP15 b are one pairs of tandem repeat genes,and SgPAP15 a in P-deficient stylo roots was significantly up-regulated.Furthermore,genotypic variation for exogenous ADP utilization had a significant positive correlation with the difference of SgPAP15 a expression among 20 genotypes roots.Thus,the SgPAP15 a was selected as a candidate gene for further functional analysis.(4)The functional analysis showed that over-expression of SgPAP15 a in transgenic Arabidopsis thaliana significantly increased the root APase activity.When phytate was added to 1/2 MS solid medium or soil medium,the dry weight and total P content of SgPAP15 a overexpression lines were significantly higher than that of wild type(WT),indicating that SgPAP15 a had the biological function of utilizing Phy.In conclusion,a new stylo PAP member,SgPAP15 a,was identified in this study.Under low P stress,stylo increased cc APase activity by up-regulating the expression of SgPAP15 a in roots,thus promoting the utilization of exogenous Phy.The results further explain the molecular mechanism of stylo adaptation to P deficiency,and SgPAP15 a could be a useful candidate for genetic engineering of new stylo with high P efficiency.