Regulatory Mechanism Of Apple Ethylene Response Factor ERF In Ethylene Biosythesis During Fruit Ripening

Apple is one of the fruit with the largest cultivation area and production in the world.The economic value of mature apple fruit is determined by its shelf-life that was regulated by ethylene,a major plant growth regulator and plant honnone.Apple is a typical climacteric fruit with flaring up respiration rate and ethylene release rate.Climacteric and ethylene production peaks are an important symbol of fruit ripening and fruit shelf-time is determined by ethylene production during ripening.Although ethylene signaling has been well studied,knowledge of the transcriptional regulation of ethylene biosynthesis is still limited.Our previous study revealed that MdERF2 is involved in apple fruit ripening.We performed yeast two-hybrid(Y2H)screening using an apple fruit cDNA library.With the full-length cDNA of MdERF2 as bait,MdERF3 was a positive clone identified from the screening.Here we show that how an apple(Malus domestica)ethylene response factor,MdERF2,MdERF3 and interation MdERF2-MdERF3 to negatively affect ethylene biosynthesis and fruit ripening by suppressing the transcription of MdACS1,We found MdERF2 negatively regulates ethylene biosynthesis through three mechanisms.The results are following:1.Ethylene treatment can inhibite expression of MdERF2 protein and promote expression of MdERF3 protein during apple fruit ripening.2.Yeast one-hybrid(Y1H)assay shown MdERF2 can bind the promoter of MdACSl in vitro;ChIP-PCR shown MdERF2 can bind the promoter of MdACS1 in planta.GUS activity assay shown MdERF2 can inhibite the transcription of MdACSl.3.Yeast one-hybrid(Y1H)assay shown MdERF2 can bind the promoter of MdERF3 in vitro;ChIP-PCR shown MdERF2 can bind the promoter of MdERF3 in planta.GUS activity assay shown MdERF2 can inhibite the transcription of MdERF3.4.Yeast one-hybrid(Y1H)assay shown MdERF3 can bind the promoter of MdACSl in vitro;ChIP-PCR shown MdERF3 can bind the promoter of MdACS1 in planta.GUS activity assay shown MdERF3 promote the transcription of MdACS1.So MdERF2 can indirectly inhibite the expression of MdACSl by negatively regulate the transcription of MdERF3.5.Yeast two-hybrid(Y2H),Pull down and Co1P shown MdERF2 can directly interact with MdERF3.EMSA and GUS activity assay shown MdERF2 can compete with binding of MdERF3 to the MdACSl promoter by interaction with MdERF3.So MdERF2 can indirectly inhibite the expression of MdACSl by interaction with MdERF3.6.After MdERF2 gene was overexpression or silenced in apple fruit by VIGS,the results shown ethylene production was decreased or increased in apple fruit.So we found apple fruit ripening was inhibited by MdERF2.Take together,these data indicate that MdERF2 negatively regulates ethylene biosynthesis through three mechanisms:(Ⅰ)suppressing the activity of MdACS1 by binding to its promoter;(Ⅱ)suppressing the promoter of MdERF3,which can promote the transcription of MdACS1;(Ⅲ)MdERF2 can compete with binding of MdERF3 to the MdACSl promoter by interacting with MdERF3 Regulations the promoter of MdACSl isoforms at both transcriprion and posttranslational levels by MdERF2 and MdERF3 inhibite ethylene production.The results of this studay will be greatly helpful in clarifying the role of ERF in regulating apple fruit ripening and in integrating the research on fruit ripening,as well as in supporting the technology of fruit storage and this is also new highlight in research of functional genomics.