Mechanisms Of The Signal Transduction For Ethylene Production In Apple(Malus×domestica) Fruit Development And Ripening

Regulation of fruit development and ripening is one of the most important questions in pomology science. It has been well established that ripening of climacteric fruit is controlled by phytohormone ethylene, but mechanisms of the signal production for ethylene production is largely unknown. Apple is well known to be one of the most important, and also, typical climacteric species of fruit tresses. Using’Golden Delicious’apple as material, this study aims to probe into the mechanisms of signal transduction for the ethylene production during apple fruit development and ripening. The major results are summarized as follows:1. Bioinformatics analysis identified nine members of sucrose non-fermenting protein kinase subfamily, SnRK2s, in ’Golden Delicious’ apple. The SnRK2s consists of nine members, all of which contain ATP-binding sites and the Ser/Thr catalytic domain of PKc. Analysis for the temporospatial characteristics of the gene expression of SnRK2s indicated that, MdSnRK2.4is closely correlated to apple fruit development and ripening, implying that MdSnRK2.4may play an important role in the regulation of apple fruit development and ripening.2. Stable transformation of MdSnRK2.4driven by CaM35S in ’Micro Tom’ not only promoted fruit ripening, but also affected plant phenotypes, e.g. plant dwarfing, increase in chlorophyll contents, long epidermal hairs and lower cell density and so on. All these phenotypes are correlated with function of ethylene, demonstrating a role of MdSnRK2.4in the regulation of ethylene production.3. Over-expression of MdSnRK2.4promoted ethylene production and the expression of MdACOl, a gene encoding the key enzyme in ethylene biosynthesis pathway, in apple fruit callus, and by contrast, inhibition of MdSnRK2.4by RNAi inhibited ethylene production and the expression of MdACO1. Additionally, transient expression of MdSnRK2.4in ’micro torn’ fruit also promoted ethylene production and fruit ripening. Maize protoplast transient expression analysis demonstrated that expression of MdSnRK2.4dramatically promoted the activity of the reporter gene GUS driven by MdACO1protmoter. All these results strongly indicated that MdSnRK2.4is a key signal controlling MdACO1expression thereby controlling ethylene production.4. Yeast two-hybrid, BiFC and full-down experiments all demonstrated that MdSnRK2.4could physically interact with MdACO1, and more over, the interaction domain took place between MdSnRK2.4catalytic domain STKC and MdHB functional Homeobox domain. Using prokaryotic expression system and mass spectrum analysis, it was demonstrated that MdSnRK2.4phosphorylated MdHB at three sites of Thr, i.e. T82, T95and T131. Mutation of those three phosphorylation sites in MdHB caused a decrease of interaction strength between MdHB and MdSnRK2.4in the yeast two-hybrid, and failed to activate transcription for MdACO1promoter detected by the yeast one-hybrid. Those showed that the expression regulation of MdSnRK2.4to MdHB is through phosphorylation mechanism.Collectively, this study demonstrated that MdSnRK2.4is a key signal controlling ethylene production. The mechanisms for the MdSnRK2.4-contrlled ethylene production is that MdSnRK2.4could phosphorylate MdHB transcription factor thereby regulating MdACO1expression and finally controlling the ethylene production. The present study is of great significance not only in the understanding of the molecular basis of ethylene signal origination but also in the molecular breeding of apple trees.