Molecular Mechanism Of Aconitase ZjACO3 Regulating Citric Acid Accumulation In Jujube Fruit

Jujube(Ziziphus jujuba Mill.)is the most important economic forest tree species of Rhamnaceae.It is domesticated from wild jujube.During the domestication process,a large amount of citric acid is lost,which affects the flavor and quality of fruits.Studying the molecular mechanism of citric acid loss during jujube domestication is not only of great significance to domestication research,but also provides theoretical support for jujube variety improvement and new variety breeding,which is conducive to promoting the jujube breeding process in China.Previous studies have shown that aconitase(ACO)affects citric acid accumulation,but its molecular regulation mechanism is still unclear.In this study,the citric acid content,ZjACO3 expression level and ACO enzyme activity in wild jujube population samples and cultivated jujube population samples were analyzed,and the negative regulation relationship between ZjACO3 and citric acid was proved.Through spatiotemporal expression analysis,transient overexpression,virus-induced gene silencing and subcellular localization,it was confirmed that ZjACO3 degraded citric acid in the cytoplasm and regulated the accumulation of citric acid in jujube fruit.By cloning and sequencing the ZjACO3 promoter of wild jujube and cultivated jujube population samples,the SNP mutation sites related to domestication were screened,and the relevant KASP molecular markers were developed.The regulation of domestication sites on ZjACO3 and citric acid was further analyzed by molecular biotechnology such as yeast single hybrid and dual luciferase.The specific results are as follows:1.Through the determination of citric acid content in 27 cultivated jujube and 47 wild jujube fruits,it was found that the citric acid content of wild jujube fruits was significantly higher than that of cultivated jujube fruits.The expression level of ZjACO3 was detected by q RT-PCR.The results showed that the expression level of ZjACO3 was significantly negatively correlated with citric acid content.The activity of ACO enzyme in wild jujube and cultivated jujube fruit was determined,and it was found that the activity of ACO enzyme in jujube fruit was significantly higher than wild jujube fruit.2.Subcellular localization results showed that ZjACO3 was localized in the cytoplasm.Transient overexpression of ZjACO3 could significantly reduce the citric acid content in jujube fruit and lead to the expression levels of citric acid degradation downstream genes Zj IDH1,Zj IDH2,Zj IDH3 and Zj GAD1 were significantly increased.Transient silencing of ZjACO3 significantly increased the content of citric acid in fruits,and the expression levels of Zj IDH5 and Zj GAD1 were significantly decreased.These results indicated that ZjACO3 had the function of degrading citric acid.3.The results of cloning and sequencing of ZjACO3 promoter sequence showed that there was a SNP mutation site(A/G)at-484 bp upstream of ZjACO3 initiation codon.By analyzing the relationship between mutation types and citric acid content in different samples,it was found that there was a significant difference in citric acid content among AA type,AG type and GG type.The SNP locus was highly linked to citric acid content,and KASP molecular markers related to citric acid were developed for this SNP locus.4.The results of cis-acting element analysis showed that the mutation at-484 bp upstream of the start codon of ZjACO3 was E-box element mutation(CAG/AGTG).The transcription factor Zjb HLH113 bound to this element was screened by yeast one-hybrid,and the binding ability of Zjb HLH113 to ZjACO3 promoter sequence(CAGGTG)of cultivated jujube was stronger than the ZjACO3 promoter sequence(CAAGTG)of wild jujube.Further through the double luciferase assay showed that Zjb HLH113 had a positive regulatory effect on ZjACO3,and the regulatory effect on ZjACO3 promoter of cultivated jujube was stronger than ZjACO3 promoter of wild jujube.Transient overexpression experiments also showed that Zjb HLH113 positively regulated the expression of ZjACO3 to promote citric acid degradation.In summary,this study showed that ZjACO3 regulated the degradation of citric acid in fruits.In wild jujube fruits,the binding ability of Zjb HLH113 to the E-box(CAAGTG)cis-acting element in the ZjACO3 promoter was weak,resulting in low transcription level of ZjACO3 and hindering the degradation of citric acid.The mutation of the E-box(CAGGTG)site in the cultivated jujube fruit enhanced the binding ability of Zjb HLH113 to the ZjACO3 promoter and activated its transcription,thereby promoting the degradation of citric acid.