Functional Analysis Of Genes Involving In Pi Uptake And Homeostasis In Rice

Although phosphorus is one of the most abundant elements in the earth's crust, it often presents in organic forms that are not readily available for plant. Plants can only take up the inorganic form of phosphorus, phosphate (Pi). Thus, Pi limitation is one of the most important soil stresses for crop production. To obtain high productivity, huge amount of P fertilizer is required. The excessive use of fertilizer not only increases the cost of crop production, but also causes severe environmental pollution in freshwater duo to leakage of Pi from the field. Therefore, isolation of the key regulator for Pi efficient use is of important. In this study, several important genes involving in Pi starvation signaling in rice were cloned and functionally analyzed using reverse genetic methods.In yeast, several genes containing SPX domain are involved in Pi untake and signaling regulation. The SPX domain genes of plant can be divided into four sub-families. The expression of several members of the second families can be induced by phosphate starvations, including OsSPX1, OsSPX2, OsSPX3, OsSPX5 and OsSPX6. Overexpression of OsSPX1 decreased Pi uptake while knocking down of the OsSPX1 increased it. A further analysis of PSI genes in OsSPX1 transgenic lines indicated that OsSPX1 is a negative regulator of phosphate starvation signaling. Using OsSPX1/OsPHR2 double overexpression lines, we found that OsSPX1 could inhibit the induction of gene expression by OsPHR2. As OsSPX1 is an OsPHR2 regulated gene, we proposed that OsSPX1 suppresses Pi starvation signaling through a feedback pathway.The expression of OsPSSl, OsPSS2 and OsPSS3, the member of the third subfamily of SPX domain genes, were inhibited by Pi starvation with different patterns. The transcripts of OsPSS1 decreased in the roots of Pi starvation seedlings while expressions of OsPSS2 and OsPSS3 were suppressed in both roots and leaves. An increased Pi content was observed in both roots and leaves of OsPSSl mutant plants. However, the Pi content shown no significant different in OsPSS2 and OsPSS3 mutants. Therefore, the OsPSS1 suppress Pi uptake in rice.There is a miR827 binding site in the 5'UTR regions of OsPSS1. miR827 can be markably induced by Pi starvation in root and leaves. To elucidate the relationship of miR827 and OsPSS1, we constructed miR827 overexpression plants. The expression of OsPSS1 was suppressed in the miR827 overexpression lines. Moreover, Pi content was increased in miR827 overexpression lines. Thus, we concluded that miR287 could be involved in Pi starvation signaling by regulating the expression of OsPSS1. We further found that the inducation of miR827 and suppression of OsPSS1 expression is under the contrl of OsPHR2. Therefore, OsPHR2, miR827 and OsPSS1 constitute a new pathway in Pi starvation signaling network.Beside SPX domain genes, we also cloned the homologue gene of AtPHO2. The mutants of OsPHO2 showed Pi toxicity as pho2 in Arabidopsis. To elucidate the molecular function of this gene, we screen OsPHO2 interaction proteins by yeast two hybridization systems. Several proteins have been identified, including two homologues of thioredoxins (OsTRX h2a and OsTRX h4). Studying the functions of these proteins will add our knowledge to Pi starvation signaling network in rice.Finally, we analyzed the purple acid phosphates gene family in rice. The expression levels of ten PAP genes were up-regulated by both phosphate deprivation and overexpression of the transcription factor OsPHR2. There PAP genes all contained one or two OsPHR2 binding elements in their promoter regions, implying that they are directly regulated by OsPHR2. Both acid phosphatase (AP) and surface secretory acid phosphatase (SAP) activity assay showed that the up-regulation of PAPs by Pi starvation, OsPHR2 overexpression, PH02 knockout, or OsSPX1 RNA interference led to an increase in AP and SAP activity in rice roots. However, the different patterns of these materials suggested the PAP genes were regulated by different signaling pathways.