Physiologicaland Molecular Responses Of “Fuji” Apple （Malus Domestica Borkh.） To GA₃ Application And Alternate Bearing Signal During Floral Bud Differentiation

Fuji apple is the major apple cultivar in China, however, there are several severe problems in its cultivation, such as long juvenile phase, low floral bud rate, alternate bearing(AB) and so on. Gibberellin(GA) promotes floral induction in Arabidopsis while it inhibits floral bud formation in apple; besides, it is reported that AB in apple has a lot to do with GA derived from seeds in immature fruits. To explore the physiological and molecular mechanisms of flowering, we studied the hormonal and genome-wide responses in terminal buds to the applications of exogenous GA3 and its inhibitor paclobutrazol(PAC), and to the AB signal in ‘Changfu 2’ apple.To gain a better insight into gibberellin-regulated flowering, the responses of hormones,and gibberellin- and flowering-associated genes, to applications of gibberellin acid(GA3) and its inhibitor, paclobutrazol(PAC) were explored. We studied the patterns of phytohormones[GA1+3, GA4+7, indole-3-acetic acid, zeatin-riboside(ZR), and abscisic acid] and expression profiles of Md KO, Md GA20 ox, Md RGL1a/b(repressor of ga1-3-like gene), Md SPLs(SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE), and the floral integrators [Md FT1/2(FLOWERING LOCUS T), Md SOC1(SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1), Md LEAFY, and Md AP1(APETALA1)]. Results showed that:1. GA3 stimulated vegetative growth, to some extent, and delayed floral induction,which consequentially led to fewer flowers the next year.2. Moreover, GA3 spraying instantly increased the content of endogenous GAs and reduced that of ZR, as well as the ZR/GAs ratio, which constituted an unfavorable factor for flower formation.3.The GA-induced genes Md KO and Md GA20 ox were significantly repressed by a high level of GAs through the negative feedback regulation of GA. Additionally, the Md SPLs were all significantly repressed by GA3 but promoted by PAC.4.The expression of Md FT1/2, Md SOC1 and Md AP1 in GA3-treated buds changed inthe same way, and they were retarded in floral induction.5. It suggested that Md SPLs might be involved in regulating Md FT, Md SOC1 and Md AP1, which, along with the low ZR/GAs rate, may be responsible for the negative effects of GAs on apple flowering. Thus, we propose that GA3 spraying indirectly disrupts the balance between ZR and GAs, and inhibits floral induction by suppressing Md SPLs and the floral integrators in flower induction, which ultimately contributed to inhibiting flower formation.Additionally, RNA-Seq was used to explore the responsive pathways in floral induction to AB signal. To narrow down the possible involved pathways and key genes in AB, ten uniform alternate-bearing Fuji apple trees was used to analyze the genome-wide gene expression in buds during floral induction by RNA-Seq and the contents of carbohydrates and hormones, as well as the expression profile of the selected DEGs(differentially expressed genes) and flowering genes during bud differentiation. The results showed that:1. 1027 DEGs between ON- and OFF-year terminal buds were screened out and grouped into several pathways or processes according to GO and Pathway annotation. Results showed that biosynthesis of secondary metabolites, metabolic pathways and plant hormone(especially auxin, CTK, GA and ABA) biosynthesis and signal transduction were the major enriched pathways or processes of the screened-out DEGs.2.The content of ZR(CTK) in OFF-year buds was significantly higher than that in ON-year buds and continuously increased during bud physiological differentiation(47d and75 d after fullbloom). ON-Year buds had more GAs than OFF-Year ones, but no significant difference was detected. Levels of IAA and GA were dramatically reduced during bud physiological differentiation, especially in floral induction(47d). Surprisingly, more ABA was detected in OFF-year buds than ON-year ones during bud differentiation.3. It’s worth noting that the expression of the repressor in GA signaling—DELLA(Md RGL2) was strongly depressed in OFF-year buds at 47 d and 75 d, while the expression of another repressor Md SPY was also depressed at 25 d but then promoted in OFF-year buds,which might resulted from/in the high level of CTK.4.More sucrose was detected in OFF-year buds during bud physiological differentiation and the expression of Md TPS1 was promoted, which suggests the sucrose signal in OFF-year buds was stronger than that in ON-year ones.5.We found the expression of Md LFY strongly responded to the AB signal at 75 d but other floral integrators(Md FT and Md SOC1) didn’t.