Molecular Mechanism Of MdRAV1 Mediated Gibberellin Inhibiting Apple Fruit Ripening

Apple（Malus domestica）is an important horticultural crop,and its post-harvest storage directly affects its economic value.As a climacteric fruit,the ripening of apple fruit is mainly regulated by the plant hormone ethylene.Recent studies have shown that gibberellin can inhibit the synthesis of ethylene in climacteric fruits,but the molecular mechanism is still unclear.In this study,the apple was used as the test material,and exogenous GA₃ was used to treat the ripening fruit.It was found that GA₃ delayed the ripening process of the fruit.A differentially expressed gene MdRAV1 related to the ethylene signal transduction pathway was screened by transcriptome sequencing,and the GA₃ treatment significantly promoted the expression of MdRAV1.Yeast one-hybrid,GUS and other experiments proved that MdRAV1can directly bind to the promoter of the key gene MdACS1 in the ethylene synthesis pathway and inhibit its activity,thereby regulating the ethylene synthesis during fruit ripening.The main findings are as follows:1.Exogenous GA₃ treatment significantly inhibited ethylene production and ripening of apple fruits.The apple fruits at physiological maturity stage were treated with GA₃,and the fruits without any treatment were used as the control,the fruits were stored at room temperature for 20 d.By measuring the amount of ethylene production,firmness and soluble solids content during fruit ripening process,the amount of ethylene production of GA₃treatment fruit was significantly lower than that of the control,and the hardness was higher than control,but there was no significant difference in the soluble solid content between the two groups.2.Exogenous GA₃ treatment significantly inhibited the expression of key genes in ethylene synthesis during apple fruit ripening.Fluorescence quantitative PCR was used to detect the expression of key genes in ethylene synthesis,it was found that the expression pattern of MdACS1 and MdACO1 in the control fruits was consistent with the changing trend of ethylene production,and the expression of MdACS1 and MdACO1 was significantly inhibited by GA₃ treatment.3.Exogenous GA₃ treatment significantly promoted the expression of MdRAV1 during apple fruit ripening.Transcriptome sequencing was performed on the control fruits and GA₃-treated fruits at the 10th day of room temperature storage,and a differentially expressed gene MdRAV1 related to ethylene signal transduction pathway was found.Fluorescence quantitative PCR detection showed that the expression level of MdRAV1 in GA₃-treated fruits was significantly higher than that of the control.4.The treatment of exogenous gibberellin synthesis inhibitor paclobutrazol（PAC）significantly promoted the ethylene production and ripening of apple fruits.In order to further clarify the effect of endogenous gibberellin on the ripening of apple fruits,apple fruits treated with PAC,and the fruits without any treatment were used as the control,the fruits were stored at room temperature for 35 d.By measuring the amount of ethylene production and firmness,it was found that ethylene production of fruits treated with PAC was significantly higher than that of the control.Compared with the control group,PAC treatment significantly promoted the expression of MdACS1 and MdACO1 and inhibited the expression of MdRAV1 in apple fruits.5. MdRAV1 directly binds the promoter of MdACS1 and negatively regulates its transcription.By yeast single hybridization,EMSA and ChIP-PCR,it was found that MdRAV1 could bind to the promoter of MdACS1.GUS reporter assay showed that MdRAV1could inhibit the activity of the MdACS1 promoter,and exogenous GA₃ treatment could further enhance the inhibition of MdRAV1 on the activity of the MdACS1 promoter.6.Interaction between MdRAV1 and MdGAI-like may involve in inhibiting ethylene production during apple fruit ripening.In order to clarify how gibberellin inhibited ethylene production in apple fruits by MdRAV1,apple fruit yeast cDNA library was screened using MdRAV1 as bait,and found a DELLA protein MdGAI-like in gibberellin signal transduction pathway.The results of yeast two-hybrid test showed that MdRAV1 interacted with MdGAI-like.The above results show that exogenous GA₃ treatment of apple fruits significantly promotes the expression of MdRAV1, MdRAV1 binds to the promoter of MdACS1 and inhibits its activity,thereby inhibiting the transcription of MdACS1.The interaction between MdRAV1and MdGAI-like may be involved in GA inhibition of ethylene synthesis in apple fruits.This study initially clarified the molecular mechanism of gibberellin inhibiting the synthesis of ethylene in apple fruits was preliminarily clarified,which provided an important theoretical basis for the research on how to improve the storage of apple fruits.