| Apple(Malus domestica)is a typical climacteric fruit,whose ripening process is mainly regulated by plant hormone ethylene.Controlling ethylene production during fruit ripening is an important means to delay fruit ripening and prolong storage period.Nitric oxide(NO)is a signal free radical in planta,which is involved in many physiological processes of plants directly or indirectly,including fruit growth,development and ripening.As a signal substance,NO can inhibit ethylene biosynthesis in fruits.Therefore,exogenous NO can be used as fruit preservative in production.However,the molecular mechanism in which NO affects ethylene biosynthesis in fruit remains unclear.It is of great significance for rational use of NO in accurately regulating ethylene biosynthesis and prolonging fruit shelf life to clarify this mechanism.In the study,‘Golden Delicious’ fruit was used as the material and the endogenous NO content was detected during fruit development to determine the effect of endogenous NO on fruit ripening.Apple fruit were treated with S-nitrosoglutathione(GSNO),which is a NO donor,and it was found that GSNO significantly inhibited the ethylene production of fruit and effectively prolonged the storage period of fruit.A novel ERF gene significantly induced by GSNO was identified by RNA-Seq and named MdERF5.The molecular mechanism in NO inhibiting ethylene biosynthesis in apple fruit was elucidated by studying the effect of MdERF5 on ethylene biosynthesis gene.The representative results are as follows:1.It was observed that endogenous NO content was higher in the early stage of fruit development,and gradually decreased with fruit development,which was contrary to the ethylene production,indicating a negative correlation between nitric oxide and ethylene in fruit.2.Apple fruit were treated with NO donor GSNO and endogenous NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide(c PTIO),and it was found that GSNO treatment inhibited the ethylene production and ripening of fruit,c PTIO had the opposite effect.3.To identify the molecular mechanism in NO inhibiting ethylene production of apple fruit,we compared the transcriptome of GSNO-treated and control fruits and identified a transcription factor ERF,named MdERF5,that was significantly induced by GSNO.In MdERF5-silenced apple calli,NO treatment could not significantly inhibit ethylene biosynthesis,suggesting that MdERF5 is a NO-activated inhibitor of ethylene biosynthesis.4.To further analyze the function of MdERF5,the subcellular localization of MdERF5 was detected by transient transformation assay in tobacco leaves,and it was found that MdERF5 was in the nucleus.After treatment with GSNO,it was found that GSNO induced MdERF5 to move from the nucleus to the cytoplasm,indicating that MdERF5 is a nucleocytoplasmic shuttling protein.Meanwhile,a Phos-Tag? SDS-Page assay was used to determine the MdERF5 phosphorylation level.GSNO treatment promoted MdERF5 dephosphorylation and MdERF5 accumulation in both nucleus and cytoplasm,suggesting that the nucleocytoplasmic shuttling of MdERF5 may be related to its phosphorylation status.5.In clarify the mechanism in MdERF5 nucleocytoplasmic shuttling,tobacco leaves were treated respectively with protein phosphatase PP1 and PP2 A inhibitor okadaic acid(OA)and PP2 C inhibitor hemirhizine chloride(SC),and the subcellular localization of MdERF5 was detected.It was found that GSNO did not induce MdERF5 to transfer from nucleus to cytoplasm after SC treatment.A protein phosphatase 2C gene,MdPP2C57,was identified.NO induced MdPP2C57 expression,and MdPP2C57 dephosphorylated MdERF5,leading to MdERF5 transfer from nucleus to cytoplasm.6.By analyzing the nuclear localization signal of MdERF5,a suspected phosphorylation site Ser260 was found.By detecting the subcellular localization of stable phosphomimetic form MdERF5S260 D and phosphodeficient form MdERF5S260 A,it was found that MdERF5S260 D was in nucleus,MdERF5S260 A was in nucleus and cytoplasm,and MdPP2C57 did not affect the localization of MdERF5S260 D.These results suggest that MdPP2C57 acts on the Ser260 site of MdERF5 to affect its localization.7.To further study the MdERF5 function,an apple fruit c DNA library in a Y2 H screening system with MdERF5 as the bait was screened and a key enzyme for ethylene biosynthesis,MdACO1 was identified.Pull down,Co IP,Bi FC and Luc complementation imaging assaies were performed and it was found that NO promoted MdERF5-MdACO1 interaction.More importantly,the interaction between MdERF5 and MdACO1 inhibited MdACO1 activity.8.As a transcription factor,MdERF5 regulates the downstream genes transcription.To verify the transcriptional regulation of MdERF5 on key ethylene biosynthesis genes,the results of Y1 H,EMSA and Ch IP-PCR showed that MdERF5 bound to ethylene biosynthetic gene MdACS1 promoter.And MdERF5 down-regulated MdACS1 expression by GUS activity analysis.9.To confirm the role of MdERF5 in NO-suppressed ethylene biosynthesis,a transient expression assay involving A.tumefaciens infiltration was used to transiently silence MdERF5 in apple fruit.MdERF5-silenced apple fruit showed higher ethylene production,up-regulated MdACS1 expression and increased MdACO1 enzyme activity,and NO donor GSNO treatment did not significantly inhibit ethylene production of fruit.These results suggested that NO suppresses ethylene biosynthesis and ripening in apple fruit through promoting negative transcription factor MdERF5.Taken together,in the early stage of apple fruit development,the NO content is high,and MdERF5 is expressed in large quantity.Part of MdERF5 protein is dephosphorylated by protein phosphatase MdPP2C57 and then moves into the cytoplasm,where it suppresses MdACO1 enzyme activity in the cytoplasm via a direct protein interaction.The nucleusretained MdERF5 suppresses the transcription of the ethylene biosynthetic gene MdACS1 in the nucleus by direct promoter binding and so ethylene biosynthesis is limited.With fruit development,endogenous NO content decreases gradually,MdERF5 expression declines and MdERF5 significantly reduces in the nucleus and cytoplasm,which weakens the MdERF5-mediated suppression of MdACO1 enzyme activity and MdACS1 transcription,leading to a burst in ethylene production and fruit ripening. |
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