| Color change is an important event during fruit ripening in blueberry.During the ripening process of blueberry,the chlorophyll content gradually decreases and anthocyanin accumulate in large quantities.Due to the high health and economic value of anthocyanin,many studies have confirmed that multiple regulatory factors can affect the accumulation of anthocyanin in fruits.However,there are few reports on the decrease process of chlorophyll in fruits,and this field is still worth further research.At the same time,we still know little about the relationship between anthocyanin and chlorophyll accumulation during fruit ripening.It is well known that miR156/SPLs act as regulatory modules mediating anthocyanin biosynthesis.It has been confirmed in many species that SPLs inhibit the accumulation of anthocyanin like Arabidophsis,blueberry,apple and pear.Previously,we demonstrated that blueberry VcMIR156a/VcSPL12 affects the accumulation of anthocyanin and chlorophyll in tomato and Arabidopsis.However,the molecular mechanism by which it functions is still unclear.Ethylene,as one of the five major plant hormones,plays a crucial role in the process of color change,and it can promotes anthocyanin accumulation and chlorophyll degradation during fruit ripening.People have noticed an inherent connection between miR156/SPL and ethylene signaling,but the intrinsic connections between the two pathways remains poorly understood.Previous studies have shown that multiple SPLs have been found in crops such as tomatoes,wheat,and cotton to increase ethylene levels in plants or upregulate the expression of ethylene responsive genes,affecting fruit ripening,wheat spike count,callus development,and other processes.However,this promoting effect conflicts the functions of SPL and ethylene signaling in regulating plant color development,requiring us to explore more regulatory pathways and delve deeper into their molecular mechanisms.In this study,we obtained stable VcMIR156a and VcSPL12 transgenic blueberry lines,as well as fruits with transient overexpression and silencing of VcSPL12.We investigated the impact of VcMIR156a/VcSPL12 on color change and discovered the mechanism that VcmiR156/VcSPL12 control ethylene signaling.The following conclusions were obtained:1.Firstly,this research confirmed that VcMIR156a promoted anthocyanin accumulation and reduced chlorophyll content in stem and leaf tissues of blueberry by overexpression of VcMIR156a in blueberry.Through transcriptomic and metabolomic analysis,we found that overexpression of VcMIR156a led to changes in the expression levels of genes related to chlorophyll and anthocyanin accumulation and the downstream target VcSPL12.Through genetic transformation in blueberry,transient overexpression and RNA silencing of VcSPL12 in blueberry fruit,we confirmed that the VcmiR156/VcSPL12 module controls pigment accumulation in fruit.2.In this study,ethylene substitutes（ACC and ethephon）and ethylene inhibitors（1-MCP and CoCl2）were used to treat tissue cultured seedlings and fruits of blueberry.The results confirmed that ACC and ethephon can indeed lead to a decrease in chlorophyll content and an increase in anthocyanin content in stems,leaves,and fruits;on the contrary,treatments of 1-MCP and CoCl2increased chlorophyll accumulation and decreased anthocyanin content.3.Compare to WT,this study found that the ethylene synthesis pathway genes VcSAMS,VcACSs and VcACOs were significantly up-regulated in VcMIR156a overexpressed blueberry,and the accumulation of ethylene precursors SAM and ACC was also significantly increased,reaching about 1.5 times of that in the wild type.VcSAMS and VcACS1 were significantly down-regulated in blueberries with VcSPL12overexpression.Through transient overexpression and RNA silencing experiments in fruits,we found that overexpression of VcSPL12 could inhibit the expression of VcACS1 and VcACO6 in fruits,and the ACC content in peel decreased by more than 2times.At the same time,treatments of ethylene substitutes simulated the inhibition of chlorophyll and induction of anthocyanin by VcMIR156a.Ethylene inhibitor treatment produced a phenotype similar to VcSPL12.However,there was no obvious additive effect between ethylene and VcMIR156a,so the chlorophyll and anthocyanin levels in different blueberry strains were regulated to similar levels by different treatments.4.The yeast single hybrid（Y1H）and dual luciferase reporter gene system（LUC）confirmed that VcSPL12 can recognize and bind to the promoters of VcACS1 and VcACO6,inhibiting gene expression and directly reducing ethylene biosynthesis.When we knocked out the SPL binding sites on the promoters of VcACS1 and VcACO6,the binding ability and transcriptional inhibition effect of VcSPL12disappeared,indicating that VcSPL12 specifically inhibited the expression of VcACS1and VcACO6.The expression of VcSPL12 and VcACS1/VcACO6 has always been negatively correlated in injected fruits and transgenic blueberries,which also proves the inhibitory ability of VcSPL12.5.There is a significant ethylene triple reaction in VcACS1 and VcACO6transgenic Arabidopsis that root shortened,hypocotyl shortened and apex hook intensified,which promote the release of more ethylene gas,indicating that VcACS1and VcACO6 are key enzyme genes for ethylene synthesis in blueberry fruits.These findings established an intrinsic relationship between the miR156/SPL regulatory module and the ethylene pathway,and for the first time confirmed that VcmiR156/VcSPL12 can directly inhibit ethylene synthesis by the key enzyme genes VcACS1 and VcACO6,which inhibit ethylene synthesis in blueberries,and proved that this pathway was conserved in two blueberry varieties（Northland and Bluetta）and Arabidopsis.This study fills the gap between the miR156/SPL regulatory module and ethylene synthesis,further revealing the signal transmission network in plants.In addition,this study confirmed that VcmiR156/SPL12 module promotes chlorophyll synthesis in fruits by inhibiting ethylene synthesis,inhibits anthocyanin accumulation,and associates changes in chlorophyll and anthocyanin content during fruit ripening.This study expanded the understanding of fruit ripening mechanism,opened up new ideas for the study of fruit ripening and plant signal transmission network,and laid a molecular basis for breeding better fruit varieties. |
