Apple MdACS6 Regulates Ethylene Biosynthesis During Fruit Development Involving Ethylene-Responsive Factor.

Ethylene biosynthesis in plants involves different 1-aminocyclopropane-1-carboxylic acid synthase (ACS)genes. The regulation of each ACS gene during fruit devel-opment is unclear. Here, we characterized another apple(Malus domestica) ACS gene, MdACS6. The transcript ofMdACS6 was observed not only in fruits but also in othertissues. During fruit development, MdACS6was initiated at amuch earlier stage, whereas MdACS3a and MdACS1 began tobe expressed at 35 d before harvest and immediateley afterharvest, respectively. Moreover, the enzyme activity ofMdACS6 was significantly lower than that of MdACS3aand MdACS1, accounting for the low ethylene biosynthesisin young fruits. Overexpression of MdACS6 (MdACS6-OE) bytransient assay in apple showed enhanced ethylene produc-tion, and MdACS3a was induced in MdACS6-OE fruits butnot in control fruits. In MdACS6 apple fruits silenced by thevirus-induced gene silencing (VIGS) system (MdACS6-AN),neither ethylene production nor MdACS3a transcript wasdetectable. In order to explore the mechanism throughwhich MdACS3a was induced in MdACS6-OE fruits, weinvestigated the expression of apple ethylene-responsivefactor (ERF) genes. The results showed that the expressionof MdERF2 was induced in MdACS6-OE fruits and inhibitedin MdACS6-AN fruits. Yeast one-hybrid assay showed thatMdERF2 protein could bind to the promoter of MdACS3a.Moreover, down-regulation of MdERF2 in apple flesh callusled to a decrease of MdACS3a expression, demonstrating theregulation of MdERF2on MdACS3a. The mechanism throughwhich MdACS6 regulates the action of MdACS3a wasdiscussed.Keywords: ACS Apple Ethylene biosynthesis Ethyleneresponsive factor Fruit.Abbreviations: ACC, 1-aminocyclopropane-1-carboxylate;ACO, ACC oxidase; ACS, ACC synthase; CDS, coding se-quence; CTR1, constitutive triple response 1; DAFB, daysafter full bloom; ERF, ethylene responsive factor; GD,Golden Delicious; 1-MCP, 1-methylcyclopropene; ORF, openreading frame; qRT–PCR, quantitative reverse transcription–PCR; RACE, rapid amplification of cDNA ends; RT–PCR,reverse transcription–PCR; UTR, untranslated region; VIGS,virus-induced gene silencing.