| Flowering is influenced by nutrition,hormone level,gene expression and environmental condition.Compared with annual ones,flowering is more individual diverse and the mechanism is more complicated in perennial woody plants.Salix viminalis,a shrub in the genus Salix,is distributed worldwide in cold regions of northern hemisphere high latitudes.It can produce a high biomass within a short period,with fast initial growth,and are well suited to production of energy forest and phytoremediation of heavy metals.However,fluffy catkins and pollen dispersal from blooming willows are an annual annoyance for many people who suffer from respiratory diseases and allergies in spring.Consequently,the use of this species in new landscaping has been greatly limited.it is an ideal material of dioecious woody plants,because of the short juvenile stage,floral buds of cutting formation in that year,gender stability with only 450 M genome.So far,as dioecious perennial woody plant,the flowering mechanism is indistinct in S.viminalis.In this study,2-3 cm length shoot tips were used as the materials at three stages of vegetative growth,floral bud physiological differentiation and floral bud morphological differentiation in male and female S.viminalis.First of all,the process of floral bud differentiation and shoot growth dynamics were observed.Then,the contents of endogenous substances such as carbohydrate,hormone and polyamine were determined.Finally,we constructed the sequencing libraries and obtained genes expression data at different developmental stages of floral bud differentiation using RNA-seq high-throughput sequencing.It is necessary to further validate the differential gene expression pattern by qRT-PCR.Based on the studies,the mechanisms of floral bud differentiation were analyzed and sex determining genes were explored at the morphological,physiological and molecular levels of male and female in S.viminalis.The results could not only enrich the basic theory of trees floral bud differentiation and illuminate the mechanism of sex determination in dioecious plants,but also provide a practical basis for shortening the breeding period of willows and solve the problems of fluffy catkins and pollen dispersal from blooming willows.The main results were summarized as follows:1.According to the phenological period,new shoots and buds growth dynamic observation,the process of floral bud differentiation was divided into 3 stages include vegetative stage(floral bud undifferentiated),floral bud physiological differentiation and floral bud differentiation in male and female of S.viminalis.Most floral buds appeared in the middle and upper part of annual branches,and the important symbol of initiation of the floral bud differentiation was the growth rate of annual shoot slowed down.The beginning time of floral bud physiological differentiation was about 40 days after full bloom in female of S.viminalis,while the male was about 30 days.At this time,there were at least 8 unfolding leaves in the annual branches and 13 visible buds in female,while 10 unfolding leaves and 15 visible buds in male respectively.2.The changes of endogenous substances contents were similar at three stages of floral bud differentiation in males and females.The contents of soluble sugar,ABA,ZT and IAA increased first and then decreased.Total C decreased slightly,total N and GA3 continued to decline significantly.The contents of starch,Put,Spd,Spm and C/N all increased continuously.The rapid accumulation of soluble sugar,ABA,ZT,IAA and starch in the shoot tip was beneficial to the floral bud differentiation,while it was necessary floral bud differentiation when total N and GA3 decreased in S.viminalis.The effects of endogenous substances,such as sugars,hormones and polyamines,on floral bud differentiation of male and female S.viminalis were similar.3.The transcriptome of 18 samples in males and females were investigated by RNA-seq technologies.More than 50 million raw reads,7.5 G nt sequencing bases and 46 million clean reads were obtained in each sample.De novo assembly yielded 124,353 All-Unigenes with average length of 676-904 nt.A total of 107,161 ungenes were annotated to NR,Nt,Swiss-Prot,KEGG and COG database with more than 86% of annotation rate.4.A total of 9,064 DEGs were obtained during the floral bud differentiation process in females,and 6,914 of them were up regulated and down regulated by 2,150.The number of DEGs up-regulated were 60,3,277 and 3,577,and down regulation DEGs were 121,664 and 1,365 in FS1-VS-FS2?FS2-VS-FS3 and FS1-VS-FS3 respectively.A total of 9,156 DEGs were obtained during the floral bud differentiation process in males,and 6,428 of them were up regulated and down regulated by 2,728.The number of DEGs up-regulated were 889,2,907 and 2,632,and down regulation DEGs were 887,861 and 980 in MS1-VS-MS2?MS2-VS-MS3 and MS1-VS-MS3 respectively.The differentially expressed genes related to floral bud differentiation of male and female S.viminalis were analyzed,such as sucrose and starch metabolism pathway,hormone metabolism,hormone signal transduction pathway,polyamine metabolism pathway and plant biological rhythm.5.Genes of SS and TPS which related sucrose synthesis were up-regulated during floral bud differentiation in male and female of S.viminalis.T-6-P,one of sugars,as a signal molecule,mediated FT to regulated flowering induction.Cytokinin signaling can be negative feedback regulation of ARR in SvSOC1 through WUS/CLV3 control system.Key genes of SvRCOM and SvKO were down-regulated in GA synthesis pathway,and DELLA protein,a negative regulator of GA signal pathway,increased significantly,which regulation of SvLFY to integrate into the flowering networks.The process of ABA induction of plant flowering was positively correlated with the transcription level of SvCO,a key gene of photoperiod.Genes related to ABA synthesis pathway,such as SvNCED5,SvZEP,SvCCD4 were up regulation,and SvACS3,SvHMT1,SvGELP were down regulation during floral induction of S.viminalis.Expression of SvPAO was down-regulated,and SvSPD1,SvSPD2 and SvCAT1 were up-regulated during floral induction of S.viminalis respectively.Polyamines can positively regulate SvFT and integrate into the flowering network.6.A total of 1,325 DEGs were identified between the two sexualities,among which,743 DEGs were up-regulated and 582 DEGs were down-regulated as male-biased expression relative to females.A total of 488 DEGs were obtained,and the expression of DEGs was up-regulated by 293,and the number of 195 was down regulated at the vegetative growth stage.There were 263 up-regulated and 182 down-regulated in the 445 DEGs at the floral bud physiological differentiation stage.There were 185 up-regulated and 205 down-regulated in the 390 DEGs at the floral bud morphological differentiation.7.There were no gender differences in the contents of endogenous substances and genes expression at the vegetative growth stage.Genes with male-specific expression in S.viminalis included homologs of SvPGLR,SvPME,SvLAC male gametophyte development genes.Several other genes such as SvNPF related oligopeptide transport,SvRKS related to pollen identification,and SvECI2,SvCIPK11,SvFLA2,SvCYP707A1,SvCYP87A3 related to JA and BR were significantly expressed more abundant in males relative to females.Genes specifically expressed involved in pollen microspore and tapetum development were identified in males which may promote male flower formation.8.A number of genes influencing ovule development exhibited female-biased expression relative to males,which included SvTOGT1 and SvHST related to phenylpropanoid and flavonoid metabolic pathways,SvANT1,SvAAP3,SvBCAT2 related to amino acid transport and the formation,AP2 homologous gene SvAIL6 related to ethylene signal in response to the female gametophyte development and flowering and the and ethylene signaling response transcription factor ERF expression inhibition,stamen growth such as SvRPN10,SvFDX3,SvSAUR,SvGID1 B,SvAK7,SvJMJD6 expression;SvGAMMACA1,SvSPL6,SvSPL13 B,SvSPL2.These results suggested that mitochondrial coding genes,histone methylation,and post transcriptional regulation of miRNA may play important roles in sexual determination. |
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