The Mechanism Study Of BpIAA10 Gene Regulates Vegetative Organogenesis By Auxin Signal Transduction Pathway In Betula Platyphylla

Auxin plays a pivotal role in plant development process,which is involved in the initiation and differentiation of root and apex,light tropism,apical dominance and leaf morphogenesis.The Auxin/Indole acetic acid(Aux/IAA)family is an important class in auxin signal transduction pathway.However,the biological function of these genes remains unclear in perennial wood plants.Thus,it is very significative to explore the functions and auxin signal transduction mechanisms of Aux/IAA TFs.According to the previous study of BpIAAs expression pattern and analysis of bioinformatics,and the result showed that BpIAA10 was expressed highly all the time in diploid and tetraploid and BpIAA10 protein includes the four canonical domains.In order to further explore the functions of BpIAA10 gene/BpIAA10 TF(Betulaplatyphylla),we performed a series of molecular biological and physiological experiments which included promoter function analysis,genetic transformation,phenotype observation and transcriptome sequencing,yeast one hybrid,yeast two hybrid in the present study.First,we cloned the promoter of BpIAA10 and constructed a BpProIAA::Luc vector for transformation into Arabidopsis thaliana.The results indicated that BpIAA10 is expressed in the apical meristem,embryonic leaves,and young stem.The tissue-specific expression pattern was same in A.thaliana and B,platyphylla.Additionally,IAA,IB A,GA,SA,and ABA could induce the expression of BpIAA10 while MeJA inhibited the expression,suggesting that plant hormones influence the expression of BpIAA10.Strong light conditions also markedly induced the expression of BpIAA10,indicating that BpIAA10 is sensitive to light intensity and that light may be sufficient but not necessary for the normal expression of BpIAA10.Conversely,IBA-induced expression of BpIAA10 was suppressed with strong light,suggesting that IBA-induced BpIAA10 expression is partly light dependent.Therefore,our study characterized the expression of BpIAA10 and provides a basis for determining the function of BpIAA10.Then,we identified an Aux/IAA10 gene(BpIAA10)from birch(Betula platyphylla)that is associated with organogenesis.The result was confirmed by gain-and loss-of-function analysis of BpIAA10.Transgenic birch overexpressing(OE)and repressing(RE)BpIAA10 exhibited significantly increased numbers of embryonic leaves and stipules than non-transgenic lines(NT).The IAA concentration was increased by 3.50 and 3.61-fold in OE and RE lines than NT,respectively.OE and RE had the defective in dormancy bud formation under the short day condition.BpIAA10 gene involved in the apical dominance and height growth,and the branches were increased in OE and RE,the height of them were lower than NT.In addition,transgenic lines showed smaller leaf and more stoma than NT.In OE and RE,the number of primary root was significantly increased than NT.Then transcriptome sequencing was conducted on shoot apical meristem to explore the related genes involved in embryonic leaf.The results showed that both OE and RE affected the expression of auxin biosynthesis and metabolism genes,auxin response genes,and leaf development genes,which is the reason for increased numbers of embryonic leaves in transgenic lines.We also performed the transcriptome sequencing to reveal the bud dormancy mechanism.The result indicated that many hormones participate in bud dormancy formation,and the auxin throughout the whole dormancy processes.The transgenic lines were insensitive to hormones and the genes related to hormones with few changes.Furthermore,yeast one-hybrid and transient transformation verification in tobacco demonstrated that the bait2(TGA-element)can specifically bind to transcription factors 14-3-3 and Ribosomal_L2,and the bait3(ntBBF1 ARROLB)can specifically bind to FAR1,14-3-3 Like and kinesin-like protein.Yeast two hybrid screening and BiFC verification indicate that BpIAA10 specifically interacts with BpIAA7 and BpARF19 during organogenesis.According to above result,we speculated that six auxin response genes are the targets of BpARF19,which may involve in the regulation of organogenesis.All these results indicated that BpIAA10 is involved in the organogenesis by specific interactions and complex regulation with components of the birch auxin response pathway.Our findings provide insights into organofaction in perennial trees.Taken together,we selected BpIAA10 gene and its corresponding protein BpIAA10 TF as the objects,and orderly conducted bioinformatics analysis and temporal expression pattern analysis in diploid and tetraploid,promoter functional research,gene functional research and auxin signal transduction pathway exploration in the present study.The results revealed the functions of BpIAA10 gene in B.platyphylla,and summarized the auxin signal transduction pathway that BpIAA10 TF participated in.Moreover,the finally achieved results and conclusions in this study not only provide valuable clues for the completion of the huge and intricated auxin signal transduction netwoks but also provide alternative gennplasm resources and theoretical support for growth breeding work in the near future in B.platyphylla.