Studies On Molecular Mechanism Of Loquat Flowering Time Regulation

Loquat(Eriobotrya Lindl.) is a subtropical evergreen fruit tree in the apple subfamily(Maloideae, Rosaceae). The cultivated loquat(Eriobotrya japonica Lindl.), which is the only edible specie of genus Eriobotrya, blooms in fall or early winter. Besides the cultivated loquat, there are a lot of wild species in Eriobotrya that demonstrate different flowering time, such as E. deflexa Nakai flowers in spring. In addition, our group found that blooming period of Eriobotrya plants may be plastic. For example, E. deflexa Nakai f. koshunensis flowers in spring in the original place(Taiwan), whereas it flowers in autumn-winter after transplanted to Guangzhou. However, so far molecular studies on loquat flowering are still limited, and few focused on wild species and different flowering time. Thus, we aimed to study the flowering mechanisms of loquat to understand the molecular basis of various flowering patterns. On the one hand, several flowering genes were cloned, and their function and expression patterns were analyzed. On the other hand, transcriptome of loquat leaves before and during flower bud differentiation period were sequenced, which provided abundant information for molecular study of flowering time control. By above-mentioned studies, we hope to estblish molecular foundation for revealing the molecular mechanism of loquat flowering time regulation, provide theoretical basis for loquat year-round-production, and support reference for flowering time mechanism research of the other woody fruit trees. The main results were as follows:(1) E. deflexa Nakai and E. deflexa f. koshunensis Nakai bloom in different season in the loquat germplasm resource preservation garden, South China Agricultural University, Guangzhou. Anatomical observation of shoot apex slices showed that E. deflexa f. koshunensis started flower bud differentiation from late September or early October, while E. deflexa began at November, suggest that they flower at different seasons is due to the different time to start flower bud differentiation. Shoot apex of E. japonica Lindl. cv ‘Jiefangzhong’ and ‘Zaozhong No.6’ which have divergent flowering period were also observed at different stages, and results implied that the ratio of inflorescence development is probably the main reason for flowering time diversity.(2) One FT, CO, GI, SOC1, PIF4 and two FD, SVP genes were cloned from the loquat(E. deflexa f. koshunensis) and referred as Ed FT, Ed CO, Ed GI, Ed SOC1, Ed PIF4, Ed FD1, Ed FD2, Ed SVP1 and Ed SVP2, respectively. Phylogenetic analysis of the deduced amino IVacids indicated they are conserved during the evolution process.(3) Ed FT, Ed SVP1 and Ed SVP2 were localized in both the cytoplasm and nucleus, while Ed FD1, Ed FD2, Ed CO, Ed GI, Ed SOC1 and Ed PIF4 were only localized in the nucleus.(4) Ectopic overexpression of Ed FT, Ed FD1, Ed FD2, Ed CO, Ed GI or Ed SOC1 promoted Arabidopsis flowering. Gene expression analysis of Ed GI, Ed CO and Ed FT showed their expression levels were influenced by daily oscillations in photoperiod. Moreover, Ed FT interacted with two Ed FDs in the nucleus in Nicotiana benthamiana. These data suggested that they are the loquat orthologs of Arabidopsis FT, FD, CO, GI and SOC1.(5) Spatial and temporal expression patterns of these flowering genes in E. deflexa f. koshunensis indicated that Ed FD1, Ed FD2, Ed SOC1 may promote flowering, whereas Ed SVP1 may prevent flowering. While, in E. japonica Lindl. cv ‘Jiefangzhong’ and ‘Zaozhong No.6’, besides Ed FD1 and Ed SOC1, Ed CO and Ed FT may also promote flowering, with Ed FD2 possibly have no effect.(6) E. deflexa and E. deflexa f. koshunensis were used as materials for de novo transcriptome sequencing. 74314777320 nt bases were generated totally. In the results of assembly, 116674 unigenes were detected. For function annotation analysis, we got 87777, 86984, 58438, 56099, 39221, 66514 unigenes which annotated to the NR, NT, Swiss-Prot, KEGG, COG, GO database, respectively, the total annotation unigenes were 94459. For protein coding region prediction analysis, 88481 CDS were predicted.(7) Unigene expression difference analysis identified quite a lot of differentially expressed genes between samples. WGCNA was adopted for coexpression network analysis, and resulted in 28 distinct modules. Pathway enrichment analysis of lightcyan module found 8 pathways significantly enriched, that were: circadian rhythm-plant, pentose and glucuronate interconversions, phenylalanine metabolism, biosynthesis of secondary metabolites, sesquiterpenoid and triterpenoid biosynthesis, phenylpropanoid biosynthesis, carotenoid biosynthesis, cysteine and methionine metabolism. Circadian rhythm-plant and carotenoid biosynthesis may play important role on loquat flowering regulation, and their functions and requlatory mechanisms would be investigated in future studies.(8) 56 homologous genes about 193 sequences involved in flowering regulation were identified by alignment of their counterparts in Arabidopsis, including a large number of photoperiodic genes, and several thermosensory and autonomous pathway genes. In addition, expression levels of a few photoperiodic genes incresesed during floral transition in both E. deflexa Nakai and E. deflexa Nakai f. koshunensis, implied that these genes may have positive effect in flowering time regulation of E. deflexa and E. deflexa f. koshunensis.