Dynamic Characteristics And Regulation Mechanism Of Flower Bud Formation In Spur-type Fuji ’Tianhong 2’

Apple(Malus domestica Borkh.)is the main economic trees in China.There are several problems in production,such as the infrequent formation of flower buds during its growth,biennial bearing due to mismanagement and so on.Therefore,under routine management conditions,mastering the nutritional status,and clarifying the variations of the internal substances and gene expression levels in spur terminal buds at different stages,which could provide a theoretical basis for flower bud differentiation mechanism,and has important significance for regulation on flower bud differentiation and improving the phenomenon of biennial bearing.In this paper,the ’Tianhong 2’ apple was used as the test materials,based on distinguish the physiologically differentiated stages of spur terminal buds,spur flower buds,and spur leaf buds,to analyzed the changes of carbohydrates and mineral elements contents in the short shoot leaves,and the variation of endogenous hormones contents in the spur terminal buds.Meanwhile,high-throughput sequencing technology(RNA-seq)was used to analyze the transcriptome of the physiological differentiation stage spur terminal buds,spur flower buds and spur leaf buds,the expression levels of genes which related with the hormone signaling pathways,starch and sucrose metabolism and flowering were analyzed.The main results were shown as following:1.In the Shennan area of Shunping County,Baoding City,Hebei Province,the flowering period was generally in mid-April of ’Tianhong 2’.The initial stage of flower bud differentiation usually occurs about 40 days after spur terminal buds stop growing,the morphological differentiation of flower buds is divided into seven periods,there are transformation stage,initial differentiation stage,inflorescence primordium stage,sepal primordium stage,petal primordium stage,stamen primordium stage and pistil primordium stage.Each stage occurred successively and overlapped among different buds,and lasted for more than 30 days.2.Analysis of carbon and nitrogen nutrition content in apple leaves showed that:during the physiological differentiation,the sucrose content decreased in the short shoot leaves,and the starch content decreased first and then increased,indicating that the decrease of sucrose content and starch accumulation in leaves were beneficial to induction.The ratio of C to N showed an upward trend during the physiological differentiation,and the C/N ratio in spur flower bud leaves at the transformation stage was higher than that in spur leaf bud leaves at the same period,indicating that within a certain range of ratio,a relatively high C/N ratio was conducive to flower bud formation.3.Analysis of mineral elements content in apple leaves showed that:during the physiological differentiation,the content of P decreased rapidly in the short shoot leaves,the K content decreased first and then increased,the Ca and Mg contents increased,and the Mg reached the peak at transformation stage.The content of Zn in spur flower bud leaves was higher than that in spur leaf bud leaves at the early stage of morphological differentiation,indicating that high content of Zn was beneficial to early morphological differentiation.4.The contents of endogenous hormones in physiological differentiation stage spur terminal buds,spur leaf buds and spur flower buds were determined.The results showed that the content of ZR and the radio of ZR to IAA in spur terminal buds decreased significantly during the physiological differentiation.The contents of ZR,IAA,and GA3 in the leaf buds come to culmination at initial differentiation stage.The content of ZR in flower buds was significantly higher than that in leaf buds after formation of the inflorescence primordium and sepal primordium.Before the appearance of stamen primordium,the content of GA3 in flower buds was parallelly and significantly lower than that in leaf buds.The ratios of ABA/IAA and ZR/IAA in flower buds were significantly higher than those in leaf buds before the appearance of flower organ primordium.Moreover,ABA content.ABA/ZR and ABA/GA3 in flower buds were higher than those in leaf buds through the whole flower bud morphological differentiation process.Therefore,the reduction of ZR content was beneficial to floral induction.The low content of GA3,and high ratios of ABA/IAA and ZR/IAA were conducive to early morphological differentiation.In addition,higher ratios of ABA/GA3 and ABA/ZR were beneficial to morphological differentiation of flower buds.Moreover,the high ABA content was beneficial to floral induction and morphological differentiation of flower buds.5.A transcriptome sequencing was performed on the floral induction phase and flower bud differentiation stage of the spur terminal buds in different years(2014 and 2015).A total of 70,981 genes were obtained,and 53,706 were annotated.The annotation results showed that 5,625 genes were annotated into the 22 functional categories of the COG database;44,564 genes were annotated into the 58 functional groups of the GO classification,total 4,767 GOterms;6,324 genes were annotated into 115 metabolic pathways.A total of 6330 differentially expressed genes were obtained by comparison between samples.Transcription in these differential genes,the number of transcription factor and transcriptional regulator were 622 and 87.6.Analysis showed that 103 differentially expressed genes were enriched into the hormone signal transduction pathway,mainly involved in auxin,cytokinin,gibberellin,abscisic acid,ethylene and jasmonic acid signal transduction,and the number of differentially expressed genes was 31,12,12,18,11 and 13.Cluster analysis showed that there were eight types of expression patterns in 2014 and 2015.Further analysis showed that there were differences in expression patterns and expression levels of genes related hormone signal transduction in different years.This indicates that there are many ways for hormone signaling to regulate floral induction.7.The analysis showed that 80 differentially expressed genes were enriched in the starch and sucrose metabolic pathways,involving 22 related enzymes.13 enzyme genes involved in starch synthesis and degradation pathways and 7 genes involved in sucrose synthesis and transformation.There are 13 enzyme genes related to trehalose synthesis and degradation,14 cellulose degradation-related enzyme genes,14 enzyme genes related to pectin synthesis and degradation,and 7 glucose-1-phosphate adenosyltransferase genes.In 2014 and 2015,the related enzyme genes in the starch and sucrose metabolic pathways were clustered into eight types of expression patterns.Further analysis showed that the expression levels and expression patterns of genes were differentially in different years,which related with β-amylase,trehalose synthase,starch/glycophosphatase,sucrose synthase and β-fructofuranosidase,indicating that starch and sucrose metabolism-related genes provide different signals for floral induction in different years.8.Among the differentially expressed genes,87 were associated with the flowering-related,including integration factor,photoperiod pathway,gibberellin pathway,sugar signaling pathway,age pathway,vernalization pathway,autonomic pathway and temperature-sensitive pathway.Cluster analysis showed that 87 differentially expressed genes were divided into eight types of expression patterns in 2014 and 2015.Further analysis showed that the expression levels and expression patterns of genes related photoperiod pathway,gibberellin pathway,age pathway,vernalization pathway and sugar signaling pathway were different in different years.The expression patterns of integration factors were similar,but the expression levels were different,indicating that different flowering pathways induce flowering by regulating the expression level of integration factors.9.The expression analysis of flower-related genes in spur terminal buds showed that the expression of MdTFL1 continued to decrease during the flower bud differentiation,and the expression of MdCOL1 and MdSOC1 decreased during floral induction phase.During the flower bud differentiation,the expression levels of MdAFL1,MdAFL2,MdMADS2 and MdMADS5 showed an overall upward trend,and the expression in spur flower buds was higher than that in spur leaf buds at the same period during morphological differentiation.The expression analysis in flower organ showed that MdCOL1 was expressed in ovary,style and pedicel,MdCOL2 was expressed in calyx,ovary,style and pedicel,MdFT was expressed in filament and anther,MdTFL1 was expressed in calyx and filament,MdSOC1 was expressed in filament,MdFLC was expressed in anther,MdAFL1 was expressed in filament and ovary,MdAFL2 was expressed in ovary and style.MdMADS2 and MdMADS5 were expressed in calyx,ovary and pedicel.