The Molecular Mechanism Of ARF7,ARF19 Regulating PHOSPHATE STARVATION RESPONSE 1(PHR1) In Arabidopsis Roots

In Arabidopsis,PHOSPHATE STARVATION RESPONSE 1(PHR1)is a key regulatory component of responses to phosphate(Pi)starvation.As an important hormone for plant growth and development,auxin also plays an important role in response to Pi starvation in Arabidopsis.To date,the relationship between auxin signaling and PHR1-regulon in low Pi responses is still unclear.Here,this relationship is uncovered by our work,which indicates that PHR1 is the target gene of ARF7 and ARF19,and is positively regulated by auxin signaling in Arabidopsis roots.The main results obtained as follows:1.PHRI is induced by Pi starvation and exogenous auxinHistochemical assay of GUS activity with PHR1p:GUS transgenic Arabidopsis show the expression of PHR1 is high under high Pi conditions and increased in Arabidopsis roots under low Pi conditions.The qRT-PCR analysis show that whether on 1 mM Pi or 1 ?M Pi,the expression of PHR1 is obviously increased in Arabidopsis roots,which are exposed to IAA or NAA.The promoter activity analysis also show that whether on 1 mM Pi or 1 ?M Pi,the activity of PHR1 promoter is obviously increased in Arabidopsis roots,which are exposed to IAA or NAA.On the contrary,expression of PHR1 is significantly decreased in upper region of Arabidopsis roots that are exposed to auxin-transport inhibitors.These results show that PHR1 is regulated by auxin in Arabidopsis roots on transcirption level.2.Analysis of auxin responsive elements of PHR1 promoterBy bioinformatic analysis,we found several cis-elements in PHR1 promoter.Among them,there are three auxin responsive elements,namely one copy of AuxRE(GAGACA)and two copies of TGA elements(AACGAC).Histochemical staining of GUS activity revealed that PHR1 promoter activity is decreased in roots of transgenic Arabidopsis with the deletion mutation of auxin responsive elements.The results suggest that the three elements are essential but that the TGA-1 element is the most important of them for PHRI transcription in Arabidopsis roots.3.ARF7 and ARF19 is induced by phosphate starvationThe qRT-PCR analysis also indicates that ARF7 and ARF19 are induced by low Pi in Arabidopsis roots.And GUS activity is increased in roots of ARF7p:GUS and ARF19p:GUS transgenic plants by low Pi.The results indicate that expression patterns of ARF7 and ARF19 similar to that of PHR1 in Arabidopsis roots.4.ARF7 and ARF19 binding to auxin responsive elements of PHR1 PromoterYeast one-hybrid assay indicates that both ARF7 and ARF19 are able to bind with PHR1 promoter and initiate the expression of reporter gene in yeast cells.The electrophoretic mobility-shift assays(EMSAs)and the chromatin immunoprecipitation assay(ChIP)indicate ARF7 and ARF19 can directly bind to auxin responsive elements of PHR1 promoter in vitro and in vivo respectively.Our data firstly suggests that TGA-element as auxin responsive element can be bound by ARFs in plants.5.PHR1 positively regulated by ARF7 and ARF19In the arf7,arf79 and arfparf19 mutants,PHR1 and its downstream PSI genes were obviously down regulated in roots.Besides,the shoots of arf7arfl9 double mutants showed defective Pi uptake in plants and over accumulation of anthocyanin.PHR1p:GUS was introduced into arf7,arf19 and arf7arfl9 mutants and the GUS activity analysis of transgenic Arabidopsis showed that GUS signal was detectable in roots of all kinds of transgenic Arabidopsis.The results suggest that the transcription of PHR1 is positively regulated by ARF7 and ARF19 in Arabidopsis roots.6.Genes encoding MYB-CC family members regulated by ARF7 and ARF19The MYB-CC family codes 15 genes in Arabidopsis thaliana.Besides the promoter of PHR1,the promoters of other member genes also have auxin response elements.The ChIP assay analysis shows ARF7 and ARF19 are able to interact with the auxin response elements in the promoters(At5929000/PHL1,At5g06800,At3913040,At3912730,At4g13640/PHL3,Atlg69580,At3g04030).These results demonstrate that ARF7 and ARF19,as the transcription factors,are the upstream regulators of the genes encoding MYB-CC family members.7.PHR1 is independent of LBD16 and LBD29 in regulating lateral root formation under low Pi conditionsARF7 and ARF19 regulate lateral roots formation by directly regulating the downstream target genes LBD16 and LBD29.The number of lateral roots is decreased in phrl mutant,while increased in PHR1 overexpressing transgenic plants under low Pi conditions.The qRT-PCR analysis show LBD16 and LBD29 genes are not changed obviously in phrl mutants or OEPHR1 under low Pi conditions.It seems that PHR1 is independent of LBD16 and LBD29 for regulating lateral root formation in responses to low Pi.Based on the obtained results,we reveal the new mechanism for coordinating auxin signaling and PHR1-regulon responses to Pi deficiency in Arabidopsis roots.