The Investigation Of Auxin Response Factor ARFX Regulated Lateral Root Development In Arabidopsis Thaliana

Auxin plays an important role in plant growth and development,especially in lateral root development.The perception of the auxin promoted the combination of Aux/IAAs and SCF-TIR1/AFB,thus induced the degradation of Aux/IAAs which repressed ARFs activity through the direct interaction.In Arabidopsis,there were 23 ARFs members,of which the structure of MR domain influenced their transcription activation or repression activity.By cluster analysis,ARFX and other ARFs including ARF1,ARF2,ARF9 and ARF11 with transcriptional repression,clustered together.However,the function on ARFX in Arabidopsis has not been understood well.In the present study,using the CRISPR/Cas9 technology,we have generated two null alleles of arfx mutant,named arfx-1 and arfx-2,which had no obvious visible phenotype.Detailed analysis of the lateral root development showed that the arfx mutant didn't affect the total number of lateral roots.However,the arfx mutants displayed decreased LR initiation and increased LR emergence.Meanwhile,the constitutive expression of ARFX with 35S promoter and pericycle specific up-regulation of ARFX with UAS-GAL4 system both caused a decreased lateral root density,as well as lateral root emergence.The protoplast transient assays indicated that ARFX was a repressor in auxin signal,which was consistent with the decreased DR5rev:GFP signals in the Pro35S:ARFX line.However,compared with the DR5rev:GFP control,the arfx-1 mutant didn't show the changed DR5rev:GFP signals,suggesting a redundant role between ARFX and other ARFs.Yeast two hybrid assay and BiFC experiments both showed that the interaction between ARFX and IAA28.The QRT-PCR showed that ARFX repressed the GATA23 expression under auxin treatment.QRT-PCR and GUS staining indicated that ARFX and ARF7 repressed each other at the transcription level.arfx-2/arf7 could partially rescuse decreased LR emergence of arf7.Therefore,the ARFX was a repressor in auxin signal to antagonize the effect of ARF7 in lateral root development.Based on the microarray analysis,we found that Qua-Quine Starch(QQS)was a differentially regulated gene between arfx and arf7,with an increased expression in arfx,and a decreased expression in arf7.The protoplast transient assays,yeast one hybrid and ChIP-PCR demonstrated that ARFX could inhibit the expression of QQS directly.I2-KI staining and quantitative measurement showed that the starch and soluble sugar content were reduced in the leaves and roots of the arfx mutant.Our experiments showed that the high C/N ratio supplemented MS medium inhibited lateral root emergence,and the increased endogenous C/N ratio of QQS RNAi also repressed LR emergence.arfx-1/QQS RNAi can rescuse the increased LR emergence of the arfx mutant,indicating that the increased LR emergence of the arfx mutant was caused by low C/N ratio.Zhang et al.predicted that ARFX was the target gene of PIF1,PIF3 and PIF5.We also detected the reduced expression level of ARFX in the pifq mutant(pif1/3/4/5),while increased expression level in Pro35S:PIF1,Pro35S:PIF4.The protoplast transient assay,yeast one hybrid and ChIP-PCR demonstrated that PIFs could directly bind the promoter of ARFX.We further detected that ARFX was inhibited by light,which was mediated through PIFs.Using D-Root device,we didn't detect the significant difference of ARFX expression with root exposed or not to light.Under shade condition,we also detected that the up-regulation of ARFX expression,and down-regulation of QQS expression,in accordance with the PIFs accumulation.In pifq,arfx and QQS RNAi mutants,the inhibition of LR emergence induced by the shade condition was reduced.Further,we also demonstrated that PIFs could inhibit the expression of the QQS.Yeast one hybrid also showed that PIF1 can bind directly on the promoter of QQS.Consistently,the pifq mutant had reduced starch content and increased LR emergence.In nature,the shade condition sensed by the light receptor was transduction by the PIFs.The accumulated PIFs under the shade condition induced the expression of auxin repressor ARFX,which could inhibit LR emergence.On the other hand,PIFs and ARFX inhibited the expression of QQS together,caused a high C/N ratio,which resulted in reduced LR emergence to reduce the consumption of photosynthetic carbohydrate for better adapt to the shade environment.In summary,our experiment showed that the Arabidopsis ARFX was an auxin signal repressor,and regulated by PIFs-mediated light signaling.Moreover,the ARFX and PIFs all could repress the expression of the QQS,thus regulated endogenous C/N ratio to adjust LR emergence.Meanwhile,ARFX also inhibited auxin signal to antagonise auxin positive regulation factor ARF7 to participate in the regulation of lateral root development.Therefore,the Arabidopsis ARFX integrated the auxin,light signal and C/N ratio to regulalte lateal root emergence.