Spatiotemporal Expression Analysis Of Pht1 Family Genes And Functionalanalysis Of OsPT4 In Rice

Rice(Oryza sativa L.)is the staple food for nearly half-world people.Therefore,it is very important for most agriculture scientific researchers to improve the quality and production of rice.Phosphorus(P),one of the most important major elements,plays an indispensable role in the growth and development of rice.Rice uptake and transport P through phosphate transporters,including Pht1 family genes.In addition,arsenic(As)is a highly toxic metalloid and has a toxic effect on rice.The predominant form of As in soil under aerobic conditions is arsenate,which has a chemical structure similar to that of PO43-.So far,there are many researchers reported that rice roots take up arsenate by phosphate transporters.In our thesis,we studied the spatiotemporal expression patterns of Pht1 family genes and characterized the exact role of OsPT4 in P and As absorption and translocation.There are 13 members in Pht1 family,which are distributed on chromosome 1,3,4,6,8 and 10,respectively.Bioinformatics analysis showed that the protein sequences of Pht1 family were conserved and closely related.GUS staining showed that all genes expressed in rice roots when rice grown in P-deficient culture,although the regions of each gene expression were different.Yeast complementation assay showed that Pht1 genes could complementary to the mutant which defective in high-affinity phosphate transport PHO84,indicating that all genes have P transport activity.OsPT4,one of the Pht1 family genes in rice,was expressed in vegetative organs,such as flag leaves and roots.Moreover,strong GUS activity was specially detected in roots epidermis.The GUS activity in roots were enhanced under P-deprived condition,especially in the root epidermis and cortex of plants.The green fluorescence of OsPT4 was observed in the plasma membrane.Under the normal P(0.3 mmol/L)and high P(1.5 mmol/L)conditions,overexpressing OsPT4 resulted in 1.6 and 2.1 times P accumulation in roots compared with wild-type,and the number of tillers and main roots of OsPT4-RNAi plants significantly decreased.We concluded that OsPT4 is a functional P-influx transporter involved in P absorption in rice.It was reported that many phosphate transporters contribute to As uptake and transport.The transcript levels of OsPT4 both in root and shoot increased gradually when rice grown in hydroponic culture with arsenate.The OsPT4-overexpressing plants,which germinated in hydroponic culture with 25 ?mol/L and 50 ?mol/L arsenate,showed sensitivity to arsenate stress with aboveground parts showing delayed growth and the roots stunted.The As concentration of OsPT4-overexpressing rice in roots was 33% higher than wild type.However,the OsPT4-CRISPR lines showed the opposite phenotype and 30% lower As concentration in roots.Furthermore,the Vmax of arsenate uptake in OsPT4-overexpressing lines increased23-45% compared with Nipponbare.The present study indicates that phosphate transporter OsPT4 is involved in rice arsenate uptake.In flooded soil,OsPT4-overexpressing plants significantly increased total P concentration in both grain and straw.Moreover,no difference was observed in arsenate accumulation between OsPT4-overexpressing plants and wild-type.Therefore,OsPT4 could be a good candidate gene to generate high P-accumulating and low As-accumulating rice.