Functional Study Of BpCUC2 Regulating Leaf Shape And Internode Development In Betula Platyphylla

The CUP-SHAPED COTYLEDON 2(CUC2)gene is one of the plant NAC(NAM/ATAF/CUC)family transcription factors,which plays an important role in the formation of apical meristem,the establishment of organ borders,leaf margin morphogenesis,and flower morphology growth.At present,most of the functional excavation of this gene is concentrated in model plants,and studies in woody plants are rarely reported.In the early stage of the research team,we screened a number of key genes,including BpCUC2,when screening genes related to the morphological morphogenesis of Betula platyphylla.In order to reveal the molecular function of BpCUC2,the study carried out tissue expression characteristics,bioinformatics analysis and genetic transformation,and performed phenotypic analysis and anatomical observation on the obtained transgenic lines.The upstream and downstream regulatory genes were screened and verified by transcriptome sequencing,yeast one-hybridization and ChIP-PCR.In addition,the upstream regulatory gene BpmiR164(BpmicroRNA164)was analyzed for tissue expression and bioinformatics,and genetic transformation was performed.The research results are as follows:(1)Bioinformatics and tissue expression characterization analysis indicated that BpCUC2 has a conserved subdomain of A,B,C,D and E typical of the NAC family,and is highly expressed in buds.Subcellular localization results showed that the BpCUC2 gene was localized in the nucleus.Transcriptional activation domain analysis indicated that the transcriptional activation domain of the BpCUC2 transcription factor is located between 748 and 906 bp of the C-terminus of the gene.(2)Three BpCUC2 overexpressing lines(OE1-OE3)were obtained by genetic transformation of Betula platyphylla.Compared with wild type birch(WT),overexpressing lines showed obvious defects in seedling height,internode and leaf shape development.For example,the overexpression lines have a lower seedling height,which is only 50%of WT.with a shorter fast-growing period and a slower growth rate.In the internode traits,the number of internodes in the overexpressed lines decreased and the internode length was shortened.In the internode epidermal cells length.the stem epidermal and internal cells length of the overexpressing lines were about 50%of WT.In the morphology of the apical buds,the overexpressing lines were nearly 90° hook-shaped due to the fusion of the petioles and stems at the buds.On the leaf-shaped traits,compared with WT,the leaf margin of the overexpressed lines decreased,the number of primary and secondary teeth of the leaves decreased,and even the third-order teeth disappeared,and the leaf aspect ratio increased,the leaf base angle reduced,however,the leaf net photosynthetic rate did not change compared to WT.Anatomical analysis showed that the size of the apical meristem did not change between the transgenic lines and WT.(3)Endogenous hormone content assay results showed that auxinwas reduced in overexpressing lines compared to WT,while cytokinin was elevated in overexpressing lines.Immunohistochemical localization results showed that the distribution of auxin and cytokinin did not change in the apical buds and stems of the overexpressing lines compared with WT.(4)According to the BpCUC2 overexpressing transcriptome sequencing results,compared with WT,there are 688 differential genes(533 up-regulated expression and 155 down-regulated expression)in overexpressing lines,which are mainly related to plant hormone signal transduction,hormone metabolism and growth regulation and et al.Among them,many genes related to cyclin,auxin response and synthesis showed significant up-regulated expression.Further,from the TF-Centered Yeast One Hybrid and the yeast one-hybrid transient transformation verification experiment,the cis-acting elements that BpCUC2 may bind to are CGT[A/G],LTRECOREATCOR15,CARESOSREP1 and BIHDIOS.In addition.ChIP-PCR(Chromatin Immunoprecipitation-PCR)results show that BpCUC2 can directly regulate a series of gene expression related to cyclin,auxin response and synthesis.(5)The 5’ RACE experiment indicated that BpCUC2 is the target gene of BpmiR164 and the mRNA cleavage site is located between the 11th and 12th positions of its complementary sequence.In the present study.BpmiR164 has four precursor sequences(BpmiR164a,BpmiR164b,BpmiR164c and BpmiR164d)and two mature sequences(BpmiR164-1,BpmiR164-2).Analysis of gene expression characteristics indicated that BpmiR164d was mainly expressed in the tip of young leaves and mature leaves.Three BpmiR164 inhibitory expression lines were obtained by genetic transformation.Real-time quantitative PCR results showed that BpCUC2 expression was increased in BpmiR164-inhibited expression lines compared with WT.The plant heights were 61.3%,87.2%and 68.6%of WT,respectively;the internode numbers were 73.4%,80%and 70%of WT,respectively;the average internode length was 80%、98%and 84.7%,respectively.In summary,BpCUC2 is an important player in regulating leaf shape and internode development of birch and BpmiR164 is a negative regulator of BpCUC2.BpCUC2 acts primarily on a range of genes involved in cyclin,auxin response,and synthesis.Therefore,the regulation relationship exists to regulate the leaf shape and internode development of birch by regulating the homeostasis of endogenous hormone content and the size of internode epidermal cells.The results of this study reveal the important role of BpCUC2 in the leaf shape and internode development of Betula platyphylla,and provide a theoretical reference for the design breeding and variety improvement of Betula platyphylla.