Functional Differences Of PHR1 And WRKY75 In Phosphate Starvation Responses Of Arabidopsis

Phosphorus(P)is one of the essential elements for plant growth.P has various biological functions during plant growth and development,including energy production,nucleic acid synthesis(DNA,RNA),glycolysis,respiration,photosynthesis,cell membrane formation,signal transduction,phosphorylation and dephosphorylation,redox Response,carbohydrate metabolism and nitrogen fixation,etc.The P used by plants comes mainly from soils,and its main absorption form is inorganic phosphate(Pi).Most of Pi in soils could not be absorbed and utilized by plants.So,P nutrient deficiency is a limiting factor for crop growth and development,and is also one of the main problems for future agriculture.Therefore,the uncovering of Pi starvation responses and regulatory mechanism is significant theoretically and valuable potentially for agricultural application.A member of the MYB-CC family transcription factors,PHR1(PHOSPHATE STARVATION RESPONSE 1)is a core regulator of Pi starvation responses in Arabidopsis thaliana.In low Pi responses,PHR1 activates downstream target genes by binding to the cis-element P1BS(GNATATNC)in the target gene promoters.In addition,the WRKY family transcription factor,WRKY75,is also involved in the regulation of low Pi responses in Arabidopsis.Previously data showed that WRKY75 is overlapped functionally with PHR1 on regulating low Pi response genes.Whereas,the functional differences between these two transcription factors in low Pi responses are unclear to date.Here,the differences between PHR1 and WRKY75 in tissue expression,phenotypes of loss-of-function and gain-of function mutants are analyzed.The data will be useful for uncovering the mechanism of Pi up-taking and molecular regulatory network of plant Pi deficient responses.The main research results as follows:1.PHR1 and WRKY75 genes have different expression patterns during Arabidopsis growth and developmentThe promoter activity of PHR1 and WRKY75 was analyzed with PHR1 p:GUS and WRKY75p:GUS transgenic Arabidopsis.GUS staining results showed that the expression of PHR1 is higher in seedlings roots and tissues with strong life activity,and lower in senescent tissues.The WRKY75 gene promoter activity is just the opposite.The expression of WRKY75 is very weak at the seedling stage,especially at the root tip,and the activity is stronger in the seedling stage and senescent leaves.The results of qRT-PCR detection of PHR1 and WRKY75 genes during Arabidopsis growth and development were consistent with promoter activity analysis.The experimental results show that PHR1 and WRKY75 may play different functions during the growth and development of Arabidopsis.2.Under low Pi conditions,PHR1 and WRKY75 genes have different expression patternsUnder low Pi conditions,the GUS activity in PHRlp:GUS and WRKY75p:GUS transgenic Arabidopsis was increased obviously indicating the activity of PHR1 and WRKY75 promoters was induced by Pi deficiency in Arabidopsis.The expression of PHR1 was increased in whole roots and the expression of WRKY75 was only enhanced at upper part of roots,and WRKY75 promoter activity in root tip was not detected.The qRT-PCR analysis also showed that PHR1 and WRKY75 gene expression were up-regulated under low Pi stress.These results indicate that although low Pi stress induces both PHR1 and WRKY75 genes,the patterns of them are different.3.PHR1 and WRKY75 have different effects on the growth and development of ArabidopsisThe PHR1,WRKY75 overexpressing transgenic lines and phrl,wrky75 mutants were obtained respectively.Under high and low Pi conditions,overexpression of both genes can promote up-taking of Pi from the outside world,resulting in increased Pi content in Arabidopsis.Under low Pi conditions,PHR1 overexpression promotes anthocyanin accumulation,while WRKY75 overexpression reduces anthocyanin accumulation.The up-taking of Pi and anthocyanin accumulation in phrl mutant were reduced under low Pi conditions.While in wrky75 mutant,Pi absorption had no significant effect and anthocyanin accumulation increased more or less.In the leaves going senescence,the Pi content of wrky75 mutant was higher than that of wild-type plants.Under low Pi conditions,OERHR1 OEWRKY75 double overexpressing transgenic lines showed increased anthocyanin accumulation,whereas phrl wrky75 double mutants showed decreased anthocyanin accumulation.These results show that the effects of WRKY75 on anthocyanin accumulation depending on the function of PHR1.4.PHR1 and WRKY75 regulate different Pi transporters respectively to modulate Pi absorption and homeostasisThe qRT-PCR analysis showed that overexpression of RHR1 and WRKY75 in Arabidopsis can increase the expression of Pht1;1/PT1,Pht1;4/PT2,Pht1;5/PT5.Under low Pi conditions,Pht1;1/PT1,Pht1;4/PT2 expression decreased in phr1 mutant,and Pht1;5/PT5 expression had no significant effect.The expression of Pht1;5/PT5 in wrky75 mutant decreased,especially in senescent leaves,whereas Pht1;1/PT1,Pht1;4/PT2 expression did not change significantly.In phr1 wrky75 double mutant,Pht1:1/PT1,Pht1;4/PT2,Pht1;5/PT5 gene expression were significantly down-regulated.These results indicate that under low Pi conditions PHR1 regulates Pht1;1/PT1,Pht1;4/PT2 genes to promote up-taking of Pi from outside,and WRKY75 may play a role in homeostasis of Pi in Arabidopsis.For example,it promotes the transport of P from senescent leaves to young tissues.5.WRKY75 inhibits the expression of PHR1 gene by promoting SA synthesisThe qRT-PCR analysis showed that the expression of PHR1 gene in OEWRKY75 transgenic lines was down-regulated under low Pi conditions,while the expression of WRKY75 was not affected by PHR1 gene overexpression.Under low Pi conditions,the phenotypes of OEWRKY75 and wrky75 mutants is dependent on the function of PHR1.The SA treatment also showed the activity of PHR1 promoter was inhibited by SA regardless of under high or low Pi conditions.The results of qRT-PCR analysis were consistent with promoter activity analysis.The overexpression of WRKY75 in transgenic Arabidopsis significantly increased the content of SA,while the content of SA in wrky75 mutants decreased significantly,indicating that WRKY75 overexpression in Arabidopsis promotes the synthesis of SA,which inhibit PHR1 gene expression.6.WRKY75 inhibits auxin signal by promoting SA synthesisThe GUS activity in DR5:GUS and IAA:GUS transgenic lines treated with SA showed that auxin signal could be suppressed by SA.The content of auxin in OEWRKY75 transgenic plants is decreased obviously,while the content of auxin in wrky75 mutant is similar to that of wild-type plants.The results indicate that WRKY75 overexpression inhibits PHR1 expression by reducing the auxin signal.7.Auxin inhibits the expression of WRKY75 geneThe GUS activity of WRKY75p:GUS transgenic Arabidopsis treated with auxin and NPA showed that the expression of WRKY75 gene could be inhibited by auxin and promoted by auxin transport inhibitor respectively.The qRT-PCR analysis also showed that auxin is the upstream negative regulator of WRKY75 gene in Arabidopsis.The results are consistent with the expressing patterns of WRKY75 genes witch is inactive in young tissues and active in senescence tissues.In summary,under low Pi conditions,in early stage and in active tissues of Arabidopsis,the high level of auxin promotes PHR1 gene expression and inhibits WRKY75 gene expression.Then,PHR1 activates Phtl;l/PT1,Pht1;4/PT2 and the high-affinity Pi transporters can improve the ability of plants to obtain Pi from the soils.In senescent tissues,WRKY75 expression is up-regulated,which promotes SA production and inhibits auxin signaling,and PHR1 expression is also suppressed.At this time,WRKY75 promotes P nutrients transferred out of aging tissue by positively regulating Pht1;5/PT5.