| Melting flesh(MF)peaches and stony hard(SH)peaches exhibit different softening patterns and texture changes during postharvest ripening,and studies revealed that auxin is necessary to trigger ethylene biosynthesis in peach fruit.micro RNAs(mi RNAs)are short non-coding RNAs that can act as regulators of auxin response pathways and participate in auxin perception and homeostasis maintenance.However,whether mi RNAs are involved in peach auxin signaling and fruit ripening is unclear.In this paper,the MF fruits and SH fruits were used as materials to explore the mi RNAs that regulate peach fruit softening and analyze the regulation mechanism of mi RNA participating in peach fruit softening.The research results enhance the understanding of the regulatory mechanism of auxin-induced peach fruit softening and provide a potential target for preventing peach fruit from softening rapidly and extending its shelf life.The main results are as follows:1.ppe-mi R393 a and b target the auxin receptor Pp TIR1 and are involved in the softening regulation of MF peach and SH peach fruits.By Small RNA sequencing,we identified mi RNA gene family members,ppe-mi R393 a and b,which are differentially expressed in SH and MF fruit.q RT-PCR analysis showed that the transcript levels of ppe-mi R393 was highly expressed in three MF peaches and lowly expressed in four SH peaches.RLM-5’RACE and transient transformation of tobacco revealed Pp TIR1 as a target of ppe-mi R393 a and b.2.PpIAA13,the interacting protein of Pp TIR1,binds and activates the promoter of Pp ACS1.Using tobacco dual luciferase system,the regulatory effects of Pp AUX/IAA and Pp ARF family members,the downstream components of growth hormone signaling Pp TIR1,on the Pp ACS1 promoter were developed and PpIAA13 was found to activate the Pp ACS1 promoter activity.Yeast one-hybrid and EMSA experiments showed that PpIAA13 can bind to Pp ACS1 promoter directly.Transient overexpression and silencing of PpIAA13 in peach fruit induced and inhibited Pp ACS1 expression,respectively.The interaction between the Pp TIR1 and PpIAA13 proteins was confirmed by yeast two-hybrid system and bimolecular fluorescence complementation assay.3.The ppe-mi R393-Pp TIR1-PpIAA13-Pp ACS1 module in regulating auxin-induced ethylene synthesis and peach fruit softening was clarified.Gene expression analysis of ppe-mi R393a/b,Pp TIR1,PpIAA13 and Pp ACS1 in developing MF and SH fruit,combined with postharvest α-Naphthalene acetic acid treatment,supports a role for a ppe-mi R393-Pp TIR1-PpIAA13-Pp ACS1 module in regulating auxin-related differences in ethylene production and softening extent in different types of peach.Following NAA treatment or endogenous IAA increase in peach,ppe-mi R393 a and b levels are increased and promote the cleavage of Pp TIR1,leading to the accumulation of PpIAA13.High levels of PpIAA13 result in high expression of Pp ACS1,which increases ethylene production and peach fruit softening.Transient overexpression and silencing of ppe-mi R393 a in peach fruit induced and inhibited Pp ACS1 expression,respectively,confirming this operation of this module in vivo.In summary,an auxin responsive mi RNA family ppe-mi R393 was identified in peach fruit,which can regulate the interacting protein PpIAA13 of Pp TIR1 via cleaving Pp TIR1 m RNA,thereby activating the promoter activity of Pp ACS1 and promoting ripening and softening in peach fruit. |
