| Flowering is a crucial biological process for plants,which marks the transition from vegetative to reproductive growth.This transition only happens when environmental and endogenous signals are most favorable for reproductive success.Floral transition is a complex process that under strict genetic control,in which MADS-box transcription factors plays important roles.APETALA1(API)is a typical MADS-box transcription factor that has been videly studied in the transition from shoot apical meristem(SAM)to floral organs.Although flowering pattern has been extensively studied in perfect flower plant Arabidopsis thaliana,studies on the mechanism of this process in imperfect flower plants are still poorly understood.Birch(Betula)is an imperfect flower plant which develops unisexual flowers on the same tree but with separate male and female inflorescences.The flower structure and floral organ composition of birch are quite different from that of the typical perfect flower plant Arabidopsis.Therefore,studying the molecular mechanism of BpAP1 on regulating birch flower development has important reference significance for revealing flower development in imperfect flower plants.Our previous studies suggested that overexpression of BpAP1 caused early flowering in birch.In order to investigate the molecular mechanism of BpAP1 on regulating flower development in birch,we conducted this study.The research results are as follows:To investigate the molecular events that lead to the conversion of the SAM into male inflorescence in 35S::BpAP1 transgenic birch,we used RNA-seq to explore the global changed genes during this transition from vegetative to reproductive growth.The results indicated that overexpression of BpAP1 affected many flowering-related genes differentially expressed,and found that BpAP1 had significant effect on B class MADS-box genes.We reconstructed a BpAPl-mediated gene regulatory network to predict BpAP1 target genes,and BpDEF was finally predicted as a putative key target gene of BpAP1 in the process of the transition from SAM into male inflorescence in birch.Our previous studies found that another B class MADS-box gene BpPI might be a putative key target gene of BpAP1.Therefore,we finally predicted BpDEF and BpPI as putative key target genes of BpAP 1.In order to validate if BpDEF and BpPI were authentic target genes of BpAP1,we performed chromatin immunoprecipitation(ChIP)assay by using 35S::BpAPl-GFP transgenic birch.ChIP-PCR and ChIP-qPCR results showed that BpAP1 could directly bind to the promoter sequence of BpDEF and BpPI,indicating that BpDEF and BpPI were authentic target genes of BpAPl in birch.Yeast one-hybrid(Y1H)and transient transformation of constructs into tobacco results indicated that BpAP1 regulated BpDEF and BpPI expression via directly binding to the CArG box motifs in BpDEF and BpPI promoters.BpDEF function was further analyzed by generating BpDEF transgenic birch.We totally obtained 17 35S::BpDEF transgenic lines(D1-D17)and six BpDEF-RNAi transgenic lines(iD-iD6).We found that D1 flowered earlier than non-transgenic birch and other BpDEF transgenic lines.Although D1 produced male inflorescence in birch,the male inflorescences in D1 degenerated during the further development and finally fell off in autumn.We further observed flowering pattern of BpDEF transgenic birch for three consecutive years,and found that D1 only produced male inflorescence while without any female inflorescence formation.In contrast,the three-year old BpDEF-RNAi transgenic birch(iD6)only produced female inflorescence for the first time in April,while without any male inflorescence formation in June.Our existing results indicated that BpDEF may function in sex-determination,and specially specify the identity of male inflorescence in birch.Therefore,we conjectured that BpAPl may directly regulate BpDEF to promote male inflorescence formation in birch.BpPI function was further analyzed by generating BpPI transgenic birch,and we totally obtained five 35S::BpPI transgenic lines(P1-P5)and seven BpPI-RNAi transgenic lines(iPl-iP7).We found that the vegetative growth of 35S::BpPI transgenic birch was significantly lower than that of non-transgneic birch and BpPI-RArAi transgenic birch.We observed the flowering pattern of BpPI transgenic birch for four consecutive years,and we found that overexpression of BpPI may delay flowering in birch.Transcriptome analysis of BpPI transgenic birch showed that BpPI restricted some flowering activators expression,such asBpAPl,LFY,BpMADS4 and BpMADS5.Meanwhile,BpPI also activated the expression of owering repressor FLC.Our results indicate that BpPI may delay flowering by affecting these flowering-related genes in birch.Studies on regulatory relationship and protein interaction between BpAPl and its key target genes were further performed.We found that BpPI and BpDEF could form heterodimer to perform the function of B class genes in birch.BpPI/BpDEF heterodimer could directly restrict BpAPl expression via binding to the promoter sequence of BpAPl.BpDEF and BpPI could also regulate each other directly or indirectly.In addition,BpAP1,BpPI and BpDEF also have autoregulatory effects to activate or restrict themselves.Yeast two-hybrid results showed that BpAPl could interact with BpPI to form heterodimer.In addition,BpAP1 and BpPI themselves could also form homodimers.However,BpDEF could not interact with BpAP1 to form heterodimer,and BpDEF also could not form homodimer in yeast.Our results indicate that there is a complex regulatory relationship and protein interaction between BpAPl and its key target genes,and they may interact with other MADS-box transcription factors to form higher-structure regulatory network to control flower development in birch.Taken together,in order to investigate events that lead to the conversion of the SAM into male inflorescence in 35S::BpAPl transgenic birch,we used RNA-seq to predict putative target genes of Bp API during the process of male inflorescence formation in birch.A series of studies were carried out around Bp AP1 and its target gene BpDEF and BpPI,including validation of Bp AP1 key target genes,BpAPl key target genes function analysis,and regulatory relationship and protein interaction between BpAP1 and its key target genes.Our results provided valuable clues for our understanding of the molecular mechanism of BpAP1 during the process of male inflorescence formation in birch,and bring new evidences for further analysis of the molecular mechanism of flower development in imperfect flower plants. |
