The Regulatory Mechanism Of PHL1 Expression In Arabidopsis Under Low Phosphate Stress

PHOSPHATE STARVATION RESPONSE1(PHR1)is key transcription factor of MYB-CC family in the regulation network of phosphorus starvation,which is involved in the regulation of root development,phosphorus absorption and transport.The specific regulatory mechanisms are still being improved.Previous studies have shown that PHL1 and PHR1 of MYB-CC family members have functional redundancy in phosphorus starvation response.But so far,the functional characteristics of PHL1 in the process to low phosphorus responses are not clear.Here,we found that the expression level of PHL1 is regulated by phosphate starvation,auxin signal and salicylic acid signal in Arabidopsis roots.Overexpression of PHL1 inhibits root development of Arabidopsis.The main results obtained as follows:1.PHL1 is localized in the nucleusThe constructed 35S::PHL1-e GFP fusion expression vector was transformed into Arabidopsis by floral dip method.Fluorescence observation of 35S::PHL1-e GFP transgenic Arabidopsis showed that GFP fluorescence signal overlaps with the nuclear localization DAPI signal,indicating that PHL1 was localized in the nucleus.2.PHL1 is highly expressed in the rootThe PHL1p::GUS vector was transformed into Arabidopsis by Agrobacterium-mediated floral immersion.GUS activity analysis showed that PHL1 was actively expressed from the germination of Arabidopsis seeds.At the early stage of seed germination,PHL1 was only expressed in the hypocotyl and root tips of seedlings.The expression level of PHL1 was continuously accumulated in roots and leaves with the growth and development of seedlings.On the 10 th day after germination,the expression level of PHL1 in the root reached a peak,and then began to decline.PHL1 was expressed in almost all parts of 40-day-old Arabidopsis,including rosette leaves,roots,internodes,inflorescences,pedicels and siliques,but the expression level was relatively high in roots.3.Pi starvation inhibited the expression of PHL1 in rootsGUS activity analysis of PHL1p::GUS transgenic Arabidopsis and q RT-PCR analysis showed that Pi starvation inhibits PHL1 expression in Arabidopsis roots under different phosphorus concentrations.4.Exogenous auxin inhibits PHL1 expression in rootsGUS activity analysis of PHL1p::GUS transgenic Arabidopsis and q RT-PCR analysis showed that exogenous auxin NAA inhibited PHL1 expression,and auxin polar transport inhibitor NPA up-regulated PHL1 expression in Arabidopsis roots under high and low phosphorus nutrient conditions.5.ARF7 and ARF19 negatively regulate PHL1The results of q RT-PCR showed that the expression of PHL1 in Arabidopsis roots of arf7,arf19 and arf7 arf19 mutants was significantly up-regulated under different phosphorus nutrient conditions.GUS activity analysis of arf7 PHL1p::GUS,arf19PHL1p::GUS and arf7 arf19 PHL1p::GUS also indicated that the deletion of ARF7 and ARF19 resulted in significantly enhanced PHL1 promoter activity.Therefore,ARF7 and ARF19 negatively regulated PHL1 expression in Arabidopsis roots under low phosphorus stress.6.ARF7 and ARF19 bind to auxin response elements of PHL1 promoterPromoter cis-element analysis revealed two auxin response elements at sites-70(Aux RE)and-814(TGA-element)in the PHL1 promoter.We demonstrated that both ARF7 and ARF19 bind to the two auxin response elements of PHLI promoter by yeast one-hybrid system,electrophoretic mobility-shift assay(EMSA),and the chromatin immunoprecipitation assay(Ch IP)in vitro and in vivo respectively.7.Overexpression of PHL1 inhibits the elongation of primary rootThe statistical analysis of primary root elongation in PHL1 mutants and overexpressed Arabidopsis showed that under normal phosphorus nutrient conditions(HP),The overexpression of PHL1 significantly inhibited the elongation of primary root.Compared with phr1 and phl1,the root elongation of the double mutant phr1 phl1 was significantly inhibited,showing a stronger superposition effect under Pi starvation,suggesting that PHR1 and PHL1 redundantly regulate Pi responses.In addition,low phosphorus stress resulted in changes in physiological indexes related to phosphorus metabolism of OEPHL1,among which MDA content and POD activity levels were significantly increased,indicating that Pi starvation caused greater damage to PHL1 overexpression lines.8.PHL1 is induced by salicylic acidAnalysis of the cis-acting elements of the PHL1 promoter showed that there were many plant hormone response elements in the sequence.Through various hormone treatments,we found that 50 μM salicylic acid(SA)significantly induced the expression of PHL1 and inhibited the elongation of the primary root.On the contrary,0.01 μM auxin(NAA)remarkably inhibited the expression of PHL1 and promoted the elongation of the primary root,suggesting that auxin signal and salicylic acid signal play an antagonistic role in the root growth of Arabidopsis.In conclusion,auxin signals inhibit the expression of PHL1 by activating auxin response factors ARF7 and ARF19,which directly bind to the downstream PHL1 promoter sequence in Arabidopsis roots under low phosphorus stress.In addition,auxin signal and salicylic acid signal play an antagonistic role in the root growth of Arabidopsis.In this paper,we studied the regulatory mechanism of PHL1 in Arabidopsis roots under phosphate starvation.Our results enrich and improve the low phosphorus regulatory network involving auxin signal and salicylic acid signal pathway.