Study On The Regulation Mechanism And Related Genes In Flower Bud Differentiation Of Ficus Carica L.

Fig(Ficus carica L.)is a deciduous fruit tree belonging to Ficus of Moraceae,which grows mainly in subtropical and temperate regions.Fig is an anther inflorescence,and its small flowers are born on the inner wall of the receptacle,which is not visible in appearance,so it is called fig.The fig fruit is not only nutritious but also has significant anti-cancer effects.Therefore,it has caused people's universal attention.Compared with other fruit trees,fig has unique flowering and fruit-bearing characteristics.It not only has early flowering,but also has continuous fruiting ability.On a new shoot,floral buds can be formed at almost every leaf axil.Therefore,as the new shoots elongation,the flower bud differentiation gradually proceeds from the bottom to top,and from flower bud differentiation to fruit ripening can be completed in one growing season.However,the regulation mechanism of flower bud differentiation in fig has rarely been reported so far.In this study,two different types of Ficus carica L.Brunswick apical buds(which can form floral primordium,FB and cannot form floral primordium,LB)were used for test materials.Through transcriptome sequencing,proteomic analysis,endogenous hormone content determination,exogenous hormone treatment,gene cloning and genetic transformation,the key metabolic pathways and related differentially expressed genes that may be involved in the process of flower bud differentiation in figs were discovered.The changes of endogenous cytokinin and gibberellin during floral bud differentiation and the effects of exogenous gibberellin and its inhibitor paclobutrazol on flower bud differentiation of fig were understood.Finally,the key gene of floral bud differentiation FcLFY was cloned and expressed analysis,and verified its biological function.These results provide a theoretical basis for elucidating the mechanism of flower bud differentiation in fig.Below are key research findings.1.Using high-throughput sequencing technology,two different types of fig top buds were used as experimental materials to obtain transcriptome data of physiological differentiation process of fig flower buds.After functional analysis and annotation,FB compared with LB,more than 2 fold of 2943 differentially expressed genes were obtained,among them 1239 differential genes were up-regulated and 1704 differential genes were down-regulated,involving in 115 metabolic pathways.Several key metabolic pathways involved in regulating the flower bud differentiation of figs were selected,including transcription factors,plant hormone signal transduction,sugar synthesis,signal transduction and flowering pathways.Among them,the expression levels of related genes in GAs and CTKs pathways are up-regulated,and both of them play an antagonistic role in flower bud differentiation of most fruit trees,which is interesting.Therefore,we will further study their role in fig flower bud differentiation by using them as entry points.2.Using 2-DE gel and mass spectrometry techniques,FB isolated and identified 30 differentially expressed proteins compared to LB.Among them,4 were up-regulated and 26 were down-regulated.These differential proteins are mainly involved in the life activity processes of plant stress response,primary metabolic processes(carbohydrate and energy metabolism,amino acid metabolism,nucleotide metabolism),cell cycle,photosynthesis,oxidation and reduction and so on.Among them,the expression of PR protein,ascorbate peroxidase,Rubisco and cyclin CDC48 in FB was higher than that in LB,indicating that the antioxidant capacity enhanced,photosynthetic product content increased and cell division speed is accelerated in fig flower bud differentiation process,flower bud differentiation was promoted.3.In the transcriptome data,FB compared with LB,all the GA pathway-related and more than half of the CTKs pathway-related genes were up-regulated,and they are antagonistic in fruit tree regulation.This is contradictory and strange,so we focus on the role of CTKs and GAs metabolism and signal transduction pathways in the process of fig flower bud differentiation.According to the different developmental processes of the apical buds,FB and LB were divided into three layers.The content of cytokinin and gibberellin in different stratification of two buds was determined,and the ratio of cytokinin to gibberellin and the expression of genes related to metabolism and signal transduction pathway were analyzed.The results showed that the physiological differentiation of fig flower buds required more cytokinins,especially TZR,and higher TZR/GA₃,ZT/GA₃,(TZR+ZT)/GA₃,(TZR+ZT+GA₄)/GA₃ ratio is favorable for flower bud differentiation of figs.The CTKs synthetic genes IPT4,AHP and ARR-A1 were down-regulated from FB1 to FB3,and the inhibition synthesis gene CKX showed opposite expression,indicating that cytokinin promoted flower bud differentiation of fig.Different types of gibberellins may have different effects.The content of GA₃ was the highest in LB1,but the content in FB was not significant.It indicated that high level of GA₃ in the growth point of apical bud could promote the vegetative growth of growth cone and inhibit flower bud differentiation.The expression of GA₄ was down-regulated in FB,and highest in FB1,indicating that GA₃ may require higher GA₄ during physiological differentiation of flower buds.At the very least,GA₄ does not inhibit the differentiation of fig flower buds like GA₃.And the higher ratio of GA₄/GA₃ is beneficial to the physiological differentiation of the flower buds of the fig.In addition,the expression of genes involved in GA signal transduction pathway increased in the early stage of differentiation of FB and LB,indicating that as the gradually completed of differentiation,the GA content gradually increased and inhibited flower bud differentiation,which was consistent with the change of GA₃ content.4.The exogenous gibberellins(GA₃ and GA₄)and paclobutrazol(PBZ)were used to treat fig plants.The changes of physiological parameters under different treatments were investigated and the different expression levels of gibberellin synthesis and signal transduction pathway genes were analyzed.The results showed that 100 mg/L GA₃ treatment significantly increased the shoot length and leaf length,decreased stem diameter and chlorophyll content,and lightened the leaf color,inhibited the flower bud formation ratio and delayed the flower bud differentiation process.While the treatment with 500 mg/L PBZ was just the opposite result.The 5 and 10 mg/L GA₄ treatments showed no significant changes in shoot length and stem diameter compared with the control,but the leaf become smaller and the color were darker.The 20 and 30 mg/L GA₄ treatments significantly reduced the shoot length and stem diameter.The leaf become smaller and the color become lighter.In flower bud differentiation,GA₃ treatment significantly inhibited the flower bud formation rate and delayed the flower bud differentiation process;while PBZ treatment increased the ratio and promoted flower bud differentiation.At lower concentrations(5 and 10 mg/L)GA₄,the lowest node of fruiting reduced,and the fruit of base first node became larger,especially the 10 mg/L GA₄ treatment was most effective.The higher concentrations(20 and 30 mg/L)of GA₄ treatment were reversed.In addition,compared with control,the expression levels of FcCPS,FcGIDl and GA2ox3 were up-regulated and GA20ox genes expression was down-regulated after GA₃ treatment,while PBZ treatment was reversed.The expression levels of FcCPS,FcGIDl and GA2ox were up-regulated after GA₄ treatment,while the expression levels of GA20ox and GA₃ox genes were increased in 5 and 10 mg/L GA₄ treatments,while these genes expression levels were decreased in 20 and 30 mg/L GA₄ treatments.In addition,the expression level of FcLFY was down-regulated by GA₃ treatment,and was significantly increased in PBZ treatment.The expression levels of FcLFY,FcFT and FcAGL were increased in the 5 and 10 mg/L GA₄ treatments,but decreased in the 20 and 30 mg/L GA₄ treatments.In summary,100 mg/L GA₃ promoted the vegetative growth of fig plants and inhibited flower bud differentiation;500 mg/L PBZ promoted the reproductive growth of figs and promoted the bud differentiation of basal nodes.Different concentrations of GA₄ treatment had different effects on fig plants.GA₄ treatment at lower concentrations(5 and 10 mg/L)reduced the lower node of fruiting,and the fruit became larger,which promoted flower bud differentiation,while higher concentration of GA₄(20 and 30 mg/L),flower bud differentiation of fig plants was inhibited.It is indicated that fig flower bud differentiation is inhibited by GA₃ like many other deciduous fruit trees;however,lower concentrations of GA₄ promote flower bud differentiation.5.The FcLFY gene related to fig flower bud differentiation was cloned and analyzed for biological function.The gene has a length of 1526 bp and contains a 1377 bp open reading frame(ORF),and predicted encoding of 458 amino acids.This amino acid sequence has a typical LFY/FLO family domain,contains leucine repeat and basic regions,and a unique glycine rich region.The homologous multiple sequence alignment analysis showed that the FcLFY amino acid sequence was up to 95%identical to the LFY amino acid sequence of Morus notabilis,and the consistency with other species was over 78%.Phylogenetic analysis showed that the fig was closely related to Morus notabilis,followed by the Rosaceae fruit trees.FcLFY was expressed differentlly in many tissues of fig,and the highest expression level in apical buds,suggested that FcLFY may be involved in the formation of apical bud meristem in fig.The FcLFY gene overexpressed in Arabidopsis thaliana plants prematurely flowered,shorted the vegetative growth time of the plants,induced flower clusters to solitary flowers,partially complemented the late flowering and petal deletion phenotypes of the mutant lfy-15,and inducing secondary branch or inflorescence of stem transformed into a solitary flower,and the leaves become smaller and have fluff.qRT-PCR analysis the expression levels of Arabidopsis flower meristem and floral organ determinant genes in each line,found that the expression levels of flowering promoting genes AP1,LFY,CAL and SEP3 were increased and flowering inhibitory genes TFL1 and FLC were decreased in the FcLFY overexpressing transgenic Arabidopsis plants.The above results indicated that the FcLFY gene is a homologous gene of Arabidopsis thaliana LFY,which can promote the early flowering of Arabidopsis plants and partially complement the phenotypic defects of mutant lfy-15.This gene may be involved in the flower bud differentiation of fig and play an important role in the formation of apical meristem.