The Role Of OsWRKY76 And OsPHO1 In Regulating Phosphorus Homeo Stasis In Rice

Phosphorus is an essential element for plant growth and participates in many important biological processes.In higher plants,phosphorus is taken up by roots in the form of inorganic phosphate(Pi:PO₄^3-,HPO₄^2-,H₂PO₄^-)from soil solutions.Phosphate in the soil can be precipitated by some metal ions,resulting in the reduction of phosphate content that can be easily used by plants.To adapt to the low available phosphate in soil,plants have formed a set of molecular regulation mechanisms to enhance phosphate absorption and utilization efficiency during evolution.The rice root mainly absorbs phosphate from soil through plasmembrane localized Phosphate Transporter1（PHT1）.After the phosphate is absorbed into the root cells,it will be transported to the root vascular bundles through the apoplast and symplast pathways.Then,the phosphate can be loaded to shoot through PHO1.In addition to the MYB transcription factors（PHRs）,WRKY transcription factors are also involved in the low phosphate responses in plants,such as WRKY6,WRKY45,WRKY42,WRKY75 in Arabidopsis thaliana and WRKY74 in rice.For example,WRKY6and WRKY42 regulate the phosphate homostasis in plants by positively regulating the expression PT and negatively regulating the expression of PHO1.However,it is still unclear whether other WRKY transcription factors are involved in regulating phosphate homeostasis in plants.Previously,we have found that OsWRKY76（LOC＿Os09g25060）,a WRKY transcription factor different from the reported WRKY genes,might also involve in phosphate homeostasis.Here,the current work showed that the expression of OsWRKY76 was induced by phosphate deficiency.The shoot inorganic phosphate content of the OsWRKY76 knockout mutant was significantly higher than that of the wild type,while the root inorganic phosphate content of the mutant was comparable to that in wild type.The RT-PCR results showed that the expression of OsWRKY76 in the old leaves is higher than that in the new leaves.Similarly,we found that the inorganic phosphate contents of old leave were higher than that in young leave in the OsWRKY76mutant.Furthermore,we found that the expression levels of OsPHO1;1,and OsPHO1;2 were up-regulated in Oswrky76 mutant,while the expression level of OsPHO1;3 was not significantly changed in the Oswrky76 mutant.It suggested that OsWRKY76 could affects the phosphate accumulation and homeostasis through negatively regulating the expression of OsPHO1;1 and OsPHO1;2.There are three PHO1 homologs in rice.Similar to AtPHO1,OsPHO1;2 is responsible for loading phosphate from roots to shoots,However,the function of OsPHO1;1 and OsPHO1;3 is not yet clear.In this paper,the expression of PHO1 family genes in rice was systematically studied.We found that the expression of OsPHO1;2and OsPHO1;3 was induced by phosphate deficiency,while the expression of OsPHO1;1 was not affected.All the OsPHO1 genes were expressed in phloem and the transverse vascular bundles in leaves.However,the expression of OsPHO1;1 and OsPHO1;2 were detected in both the xylem and phloem of the root vascular bundles.The expression of OsPHO1;3 in the root is very low.Using yeast heterologous expression system,we found that OsPHO1;1,OsPHO1;2 and OsPHO1;3 could catalyze phosphate transport in yeast.Different from the OsPHO1;2 loss-of-function mutant,the shoot inorganic phosphate content of OsPHO1;1 and OsPHO1;3 knockout mutants were significantly higher than that in the wild type.However,the root inorganic phosphate content of Opho1;1 and OsPHO1;3 knockout mutants were similar to that of the wild type.We also noticed that the inorganic phosphate contents in old leaves were much higher in the mutants than that in wild type.This indicated that all the rice PHO1 family genes were involved in the phosphate accumulation and homeostasis with different ways.In summary,we demonstrated that the OsWRKY76 and OsPHO1s participate in the Pi accumulation and homeostasis in rice,and the regulatory mechanism between OsWRKY76 and OsPHO1s need to be further studied in future.