Study On Molecular Mechanism Of Floral Development In Populus Tomentosa

Populus tomentosa is an important commercial tree species in our country,which is widely applied in greening urban and rural areas,timber forest construction and pulp industry.The research about molecular mechanism of flowering regulation in P.tomentosa is meaningful in below three points:First,it plays an important role in promoting flowering,shortening breeding cycle,and accelerating genetic improvement.Second,it establishes the theoretical basis for seeking a solution to the problem of the trichomes of female floral buds and the allergenic properties of poplar pollen in spring.Third,it has a positive effect on in-depth analysis of the molecular regulatory mechanism of flowering in P.tomentosa,and building a genetic regulation network,and also provides references for studies on floral regulation mechanism of other perennial woody plants.We take P.tomentosa as test materials in our study,and choose eight samples in the representative stages during the process of floral development,on the basis of the rule of flower bud morphological differentiation,to carry out the transcriptomics research using NGS.We annotate the unigenes using data of P.trichocarpa's genome and relevant databases,and on this basis,we analyze the change of the transcriptome programs in the process of floral bud development,screen differential expression genes,and make GO/KEGG enrichment analysis of them.We analyze the crucial gene and gene family which regulate the floral development in P.tomentosa,and combine with RNA-seq and RT-qPCR to analyze the expression pattern.We identify the genes which are related to floral meristen and floral ogran development in P.tomentosa,to establish co-expresssion network,and identify hub genes.We comprehensively analyze the invertase gene family in poplar,including genetic structure,phylogenetic analysis,chromosome location,expression patterns,etc.We determine the activity of invertase and the content of sugar in various tissues and organs in P.tomentosa.On basis of morphology,and the activity of invertase and the content of sugar,we make associatively analysis from three aspects,including morphology,physiology and biochemistry and gene expression,and clarify the process of floral bud development in poplar.We clone key genes and promoter which is related to flowering in poplar,build vector,and transform Arabidopsis and P.tomentosa,to get transgenic plants.We evaluate gene function by making a series of molecular analysis and phenotype observation.The study achieves the following main results:(1)To obtain a comprehensive overview of the dynamic transcriptome during male floral bud development in P.tomentosa,high-throughput RNA-seq was conducted during eight flowering-related stages.Among the 109,212 de novo assembled unigenes,6,959 were differentially expressed during the eight stages.The overrepresented classed of genes identified by Gene Ontology(GO)enrichment included 'response to environmental stimuli' and 'plant-type spore development'.One-third of the differentially expressed genes were transcription factors(TFs).Several genes and gene families were analyzed in depth,including MADS-box TFs,Squamosa promoter binding protein-like family,receptor-like kinases,FLOWERING LOCUS T/TERMINAL-FLOWER-LIKE 1 family,key genes involved in anther and tapetum development,as well as LEAFY,WUSCHEL and CONSTANS.The results provided new insights into the roles of these and other well known gene families during the annual flowering cycle.RNA-seq data was validated for 32 floral genes by reverse-transcription quantitative PCR.To explore the mechanisms regulating poplar flowering,a weighted gene co-expression network was constructed using 98 floral-related genes involved in flower meristem identity and flower development.Many modules of co-expressed genes and hub genes were identified,such as APETALA1 and HUAI.This work provides many new insights on the annual flowering cycle in a perennial plant,and a major new resource for plant biology and biotechnology.(2)Invertase plays a crucial role in carbohydrate partitioning and plant development as it catalyses the irreversible hydrolysis of sucrose into glucose and fructose.The invertase family in plants is composed of two sub-families:acid invertases,which are targeted to the cell wall and vacuole;and neutral/alkaline invertases,which function in the cytosol.In this study,5 cell wall invertase genes(PtCWINV1-5),3 vacuolar invertase genes(PtVINV1-3)and 16 neutral/alkaline invertase genes(PtNINV1-16)were identified in the Populus genome and found to be distributed on 14 chromosomes.A comprehensive analysis of poplar invertase genes was performed,including structures,chromosome location,phylogeny,evolutionary pattern and expression profiles.Phylogenetic analysis indicated that the two sub-families were both divided into two clades.Segmental duplication is contributed to neutral/alkaline sub-family expansion.Furthermore,the Populus invertase genes displayed differential expression in roots,stems,leaves,leaf buds,floral buds and in response to salt/cold stress and pathogen infection.In addition,the analysis of enzyme activity and sugar content revealed that invertase genes play key roles in the sucrose metabolism of various tissues,organs and floral buds in poplar.This work lays the foundation for future functional analysis of the invertase genes in Populus and other woody perennials.(3)In order to study the expression and regulation of API homologous gene in poplar,a 2,103 bp 5' flanking sequence of PtrAP1-2 gene was isolated by PCR from genomic DNA of P.trichocarpa.Promoter sequence analyzed by PLACE and PlantCARE showed that the sequence contains TATA-box,CAAT-box and many light-responsive elements,such as MRE?GT1-motif?AE-box?TCT-motif and Box4.It was supposed that PtrAP1-2 was closely related to flower development.Based on sequence analysis,the PtrAP1-2 promoter was fused to the GUS reporter gene to characterize its expression pattern in tobacco.The result of Agrobacterium-mediated transient expression showed that the promoter could direct transgene expression in sepals and petals of tobacco,no detection of GUS was found in root,stem,leaves,stamens and carpels.It indicated that the PtrAP1-2 promoter was a floral organ specific promoter.API plays a crucial role in floral transition from vegetative to reproductive phase and in flower development.In this study,a comprehensive analysis of API homologues in poplar was performed by describing the gene structure and chromosomal location.The phylogenetic relationship of the deduced amino acid sequences of Arabidopsis API and API homologues from Populus,to other API-like proteins was analyzed.The expression of PtAP1-1 and PtAP1-2 in P.tomentosa was examined by using RT-qPCR.Expression profiles were similar and both genes exhibited a high expression level in the reproductive phase.Seasonal expression profiles in floral buds indicated that the pattern of PtAP1-1 and PtAPl-2 expression in male and female floral buds was different.The trends of the PtAP1-1 and PtAPl-2 transcript levels in both sex floral buds were similar,but the peak of expression of the two genes in male buds was earlier than in female buds.This work would be of value to future functional analysis of AP1 homologues in poplar.(4)AP1 plays a crucial role in the transition from vegetative to reproductive phase and in floral development.In this study,to determine the effect of API expression on flowering time and floral organ by dominant negative mutation)were generated.Transgenic Arabidopsis with e35Spro::AtAPIM3 displayed phenotypes with delayed-flowering compared to wild-type and flowers with abnormal sepals,petals and stamens.In addition,transgenic Arabidopsis plants exhibited reduced growth vigor compared to the wild-type plants.Ectopic expression of AtAP1M3 in poplar resulted in up-or down-regulation of some endogenous key flowering-related genes,including floral meristems identity gene PtLFY,B-class floral organ identity genes PtAP3 and PtPI,flowering pathway integrator PtFT1 and flower repressors PtTFLl and PtSVP.These results suggest that AtAP1M3 regulates flowering time and floral organ development in plants.(5)Two FT-like genes named PtFT1 and PtFT2 were cloned by RT-PCR from P.tomentosa.Sequencing results indicated that the length of both PtFT1 and PtFT2 is 525 bp encoding 174 amino acids.Blast analysis showed that PtFTl and PtFT2 respectively share over or more than 75%homology in amino acid sequence with Arabidopsis(A.thaliana)FT(BAA77838.1),grape(Vitis vinifera)FT(ABI99465.1)and other FT homologues.Thedevelopment,transgenic Arabidopsis and poplar overexpressing of AtAP1M3(Arabidopsis API mutant deduced amino acid sequence contained one conserved motifs and two critical residues.Phylogenetic tree analysis suggested both PtFT1 and PtFT2 were classified into the FT-clade.Furthermore,the expression patterns of PtFTl and PtFT1 in different tissues were examined using real-time quantitative RT-PCR.It showed that the constitutive and stable transcripts of PtFT1 and PtFT2 were detected in each tissue,but the expression levels between PtFTl and PtFT1 were different.In addition,PtFT1 is up-regulated in response to winter temperatures,while PtFT2 is up-regulated in response to the long-days and warm temperatures of the growing season.Overexpression of PtFTl and PtFT2 in Arabidopsis induce early flowering,while the effect of PtFT1 is stronger.This study will contribute to understanding the molecular mechanism of PtFT1 and PtFT2 in light-dependent pathways and also will benefit to regulation of flowering.The study will establish the theoretical basis for clarifying the molecular mechanism of flowering regulation in P.tomentosa,and at the same time provide references for researches into regulation mechanism of other perennial woody plants.