| The plant root system is not only responsible for absorbing water and nutrients from the soil to ensure the normal growth of plants,but also responds to soil environment changes.Lateral roots are an important part of the plant root system and play a key role in root function.Lateral root development is regulated by many factors,and auxin plays an important role in each stage of lateral root development.To date,SLR(Solitary Root)-ARF7/19(Auxin Response Factors)-LBDs(Lateral Organ Boundaries)pathway has been widely studied.LBD transcription factor family member LBD16/18/29 is the direct target genes of ARF7 and play an important role in lateral root development,but the molecular mechanism remains to be elucidated.In order to explore the molecular mechanism of LBDs-mediated auxin signaling in regulating lateral root development,we screened the Arabidopsis root cDNA library by yeast two-hybrid method to identify its interacting proteins.TCP(Teosinte Branched 1,Cycloidea 1,Pcf 1)transcription factor TCP 14/15/20 interact with LBD 16/18/29,respectively.TCP s are a class of transcription factors in plants,which play key regulatory roles in plant immunity,flower development,leaf development,and seed germination.In this study,we used genetics,cell biology,and molecular biology methods to investigate the mechanism of the interaction between TCPs and LBDs in mediating auxin signaling to regulate lateral root formation.Firstly,the lateral root phenotypes of tcp15-1,tcp20-1,tcp20-2,tcp15-1/20-2,and tcp14/15/20 mutants were analyzed,and the results showed that the number of lateral roots primordia of tcp15-1,tcp20-1,tcp20-2,and tcp15-1/20-2 is reduced.The number of emerged lateral roots is not changed,the total number of lateral roots is decreased.The tcp 14/15/20 mutant shows an enhanced reduction of lateral roots,suggesting that TCPs are functional redundancy in regulating lateral roots development.The number of lateral root primordium,emerged lateral roots,and the total lateral roots of TCP overexpression lines is increased.The GUS staining of pTCP15:GUS transgenic lines showed that TCP15 is mainly expressed in the pericycle,and the expression is significantly enhanced in lateral root primordium,which is consistent with the function of TCP 15 in regulating lateral root development.qRT-PCR analysis showed that the expression of TCP20 is significantly induced in the wild-type under exogenous auxin treatment,but not in the arf7 mutant,indicating that the transcription of TCP20 is induced by auxin and in an ARF7 dependent manner.Overexpression of TCP15 or TCP20 partially restored the deficiency of lateral roots development in arf7 mutant,suggesting that TCPs act downstream of ARF7 to regulate lateral root development.The lateral root phenotypes of tcp20/lbd16、tcp20/lbd18、tcp20/lbd29 double mutants are similar to those of lbd16,lbd18 and lbd29 mutants.Moreover,when TCP15 and TCP20 are overexpressed in the lbd16/18/33 mutant,the phenotype of increased lateral roots is significantly weaker than that in the wild-type background,suggesting that the TCP15/20-induced lateral roots require LBDs.In order to further explore the molecular mechanism of the lateral root development by TCPs and LBDs,we analyze the known downstream genes of LBDs and TCPs.Exp14/17(Expansina 14/17)is a direct target gene of LBD18,and its promoter region contains several TCP-binding elements,suggesting that it may be a common target gene of TCPs and LBDs.qRTPCR analysis showed that the expression levels of EXP14 and EXP 17 are downregulated in tcp14/15/20 mutants.Transient protoplast transformation experiments showed that the co-expression of TCPs and LBDs significantly enhanced the transcription level ofEXP14 compared with TCPs or LBDS only.The transcription level of EXP14 is significantly lower when LBDs or TCPs are expressed in the protoplasts of tcp14/15/20 and lbd16/18/33 mutants than that in the wild-type background,suggesting that TCPs and LBDs interaction enhances the transcription of the downstream target genes related to lateral root development and positively regulate lateral root development during lateral root growth and development.Taken together,these results suggest that TCP 14/15/20 is regulated by the auxin that mediated by ARF7,TCP14/15/20 can also increase the transcriptional regulation of LBDs on its downstream genes through interaction with LBDs,positively regulating the development of lateral roots. |
