Identification Of The BpSPL Gene Family In Betula Platyphylla Suk. And BpSPL2 Function Analysis In Adventitious Root Formation

Betula platyphylla Suk.,a fast-growing native broadleaf tree species,is widely planted in the three northeastern provinces.However,its hardwood cuttings are difficult to rooting and survival.Therefore,it is essentially important for B.platyphylla to improve the capacity of rooting via illustration of the molecular mechanism behind it.This research has very important scientific significance for perfecting theory and technology popularization and application to vegetative propagation.Previous studies have shown that SPL transcription factors play important regulatory roles in plant growth,development and stress response;however the molecular mechanism involved in adventitious root(AR)formation is still not very clear,especially for woody plants.In this study,the BpSPLs gene family in B.platyphylla was identificated.Furthermore,the functions of BpSPL2 and its molecular mechanisms involved in AR formation were explored using molecular biological methods.(1)20 BpSPLs and 4 miR156 precursor sequences were identified by B.platyphylla genome combined with a transcriptome data.The basic characteristics of the BpSPLs were clarified by phylogenetic relationships,conserved motifs and post-transcriptional regulation mediated by miR156.The expression patterns of tissue-specific,female,male and leaves development were analyzed by quantitative real-time PCR(qRT-PCR).Interestingly,the expression level of BpSPL2 increased significantly during wound-induced ARs formation.Strikingly,the BpSPL2 expression level increased by 60-80 times at 5-12 h after cutting,suggesting that it played an important role in the AR formation of birch.(2)BpSPL2 was cloned in B.platyphylla,which comprised an open reading frame of 1566 bp and encoded 521 amino acids.The gene contained three exons and two introns.The encoded protein had an SBP domain,which contained two zinc finger structures and a nuclear localization signal.The results of subcellular localization revealed that BpSPL2 protein was localized in the nucleus and cytoplasm.(3)The promoter sequences analysis of BpSPL2(1956 bp upstream of the start codon)found that this region contained root-specific expression-related elements and a variety of hormones and wounding induced elements.The result suggested that the BpSPL2 gene may be involved in root development and induced by wounding and hormone.The promoter was cloned and further inserted into a plant expression vector.BpSPL2pro::GUS transgenic B.platyphylla and Arabidopsis thaliana were generated to further study.The promoter activity of BpSPL2 was overwhelmingly expressed in the root meristems and the apical buds.GUS staining analysis of BpSPL2pro::GUS transgenic birch during AR formation showed that the BpSPL2 promoter was activated in at the stem base and mainly induced in the early stage of AR formation.(4)The overexpressed(35S::BpSPL2)and suppressed BpSPL2(35S::BpSPL2-SRDX)transgenic birch were generated.Compared with wild-type(WT)plants,BpSPL2-suppresion plants showed 2-3 days earlier root emergence,and the number of ARs and total root length significantly had increased by 41.6%-66.9%.On the contrary,transgenic lines that overexpressed BpSPL2 showed a delay of 3-4 days in AR formation,and the number of ARs had decreased by 46.7%-47.6%.Meanwhile,total length of ARs were obviously decreased by 46.9-50.3%.The results indicated that BpSPL2 was a negative regulator of adventitious rooting in B.platyphylla.Compared with WT,35S::BpSPL2 plants showed dwarfing,reduced stem diameter,and decreased internode numbers.While the plant height,the number of internodes and stem diameter in 35S::BpSPL2-SRDX were significantly higher than those of wild-type and BpSPL2-overexpressed plants.It suggested that BpSPL2 not only regulated the emergence of ARs,but also affected the growth and development of vegetative organs.(5)Transcriptome sequencing was performed using the stem base tissues of WT,35S::BpSPL2 and 35S::BpSPL2-SRDX at different stages of ARs formation.In six comparison groups,2637,2129,1554,1754,2044 and 2047 differentially expressed genes(DEGs)were obtained from WT-0.5h vs OE-0.5h,WT-0.5h vs R-0.5h,WT-24h vs OE-24h,WT-24h vs R-24h,WT-96h vs OE-96h and WT-96h vs R-96h,respectively.GO analysis found that DEGs were enriched in oxidation-reduction process,nucleotide binding and oxidoreductase activity.Among the DEGs of the six comparison groups,many transcription factors involved in auxin synthesis and signal transduction,GA signal pathway,JA signal pathway,and ERF,WOX,LBD were identified.Phenotypic changes of ARs induced by BpSPL2 may be related to the differential expression of the above genes.(6)The target genes directly regulated by BpSPL2 were predicted using bioinformatics methods,among which DOT2 and CTL1 genes are related to root development.qRT-PCR analysis of DOT2 and CTL1 genes showed that the opposite expression patterns during adventitious rooting in 35S::BpSPL2 and 35S::BpSPL2-SRDX transgenic plants.The results indicated that DOT2 and CTL1 genes may be the direct target genes of BpSPL2 involved in ARs formation.However,this needs further experimental verification.Taken together,bioinformatics analysis and expression characteristics of BpSPL gene family were carried out.Further research on promoter activity,gene function and RNA-seq related to BpSPL2 were performed.This paper revealed the role of BpSPL2 in ARs formation and provided genetic resources for ARs formation mechanism in B.platyphylla and some difficult-to-root hardwood tree species.