Molecular Mechanism Of Violet-red Changes In Minimally Processed Lily Bulbs Based On Transcriptomic And Metabolomic

Lilium davidii var.unicolor is one of the characteristic crops in Lanzhou City,Gansu Province,and is the representative of edible sweet lily in China,with high nutritional and economic value.However,during the post-harvest processing and storage of Lilium davidii var.unicolor,violet-red changes often occur on the surface of the bulbs,which affects its shelf life and product quality,limiting its economic value.Many studies on the surface discoloration of lily bulbs have focused on their phenotype and color protection methods,with little research on the molecular mechanism of violet-red changes.Firstly,the environmental factors responsible for the violet-red changes on the surface of minimally processed lily bulbs were explored.The main focus was on the effects of light and temperature conditions on the surface discoloration of minimally processed lily bulbs.By measuring indicators（such as L^*,a^*,b^*,BD,TPC,TAC,and PPO,POD,PAL,CHS,CHI,F3H,DFR,and ANS enzyme activities）,the key influencing factors of violet-red changes on the minimally processed lily bulbs were explored.Secondly,transcriptomics was used to analyze and screen differentially expressed genes of lily bulbs with violet-red changes,and metabolomics was used to analyze the differential metabolites of lily bulbs violet-red changes for combined analysis.Finally,bioinformatics methods were used to screen and predict the key transcription factors responsible for the violet-red changes phenomenon in lily bulbs.The main research results are as follows:（1）Effect of light on discoloration of minimally processed lily bulbs:Under the conditions of 20°C/light,the surface of lily bulbs showed obvious violet-red color from the 6d and deepened during storage late period.The enzyme activities of CHS,CHI,DFR and ANS,which are related to anthocyanin synthesis was significantly increased,and reached the highest level at 6 d.q RT-PCR detection showed that the expression trends of CHS,CHI,DFR and ANS genes was consistent with enzyme activities.TAC of lily bulbs was significantly increased,reaching the maximum of 9.55 g L^-1at 6 d,which was 5.08 times higher than that of the 20°C/shade group.The a^*value of the surface color index increased significantly.Under the conditions of 20°C/shade,no obvious violet-red changes was observed on the surface of minimally processed lily bulbs.（2）Effect of temperature on discoloration of minimally processed lily bulbs:In the35℃/shading group,the browning degree of lily bulbs was the highest.The activities of PPO and POD were increased,and the TPC was increased,but there was no obvious violet-red.In the 20℃/shading group,the TAC in lily bulbs was the highest,reaching 5.99 g L^-1at 12 d.But there was no significant correlation between the TAC trend and the enzyme activities trend,and no obvious violet-red changes was found on the lily bulbs.In the 4℃/shading group,there was no obvious color changes on the surface of lily bulbs.（3）Transcriptomic study of violet-red lily bulbs:A total of 8244 differentially expressed genes were screened by analyzing the transcriptome data of violet-red lily bulbs（LZ）and initial lily bulbs at 0 d（CK）.Compared with the CK group,4753 genes were up-regulated and3491 genes were down-regulated genes in violet-red lily bulbs.Enrichment analysis found that the flavonoid synthesis pathway was the most significant,and the number of differentially expressed genes/total genes detected was 29/36 in this pathway.Further analysis showed that anthocyanin synthesis related genes were highly expressed,including 10 CHS,1CHI,1 F3’H,1 F3H,1 DFR,and 1 ANS gene.And 1 CHS gene was found to be lowly expressed.Finally,one BZ1 gene was found to be highly expressed and played an important role in the conversion of anthocyanins to stable anthocyanidins.（4）Metabolomics study and correlation analysis of violet-red lily bulbs:A total of 106different metabolites were screened by analyzing the metabolome data of violet-red lily bulbs（LZ）and initial lily bulbs（CK）.Compared with the CK group,71 metabolites were up-regulated and 35 metabolites were down-regulated of the violet-red changes in lily bulbs.Enrichment analysis showed that there were significant differences in the flavonoid synthesis pathway,and a variety of flavonoid metabolites up-regulated,including chalconaringenin,naringenin,cyanidin 3-rutinoside,and rutin,etc.The combined analysis of transcriptomics and metabolomics revealed that 1 CHS gene（Cluster-4138.13786）in the flavonoid synthesis pathway was negatively correlated with different metabolites,while other differentially expressed genes related to anthocyanin synthesis were significantly positively correlated with differentially metabolites related to anthocyanin synthesis.These results indicated that the expression levels of multiple structural genes（CHS,CHI,DFR,and ANS,etc.）related to anthocyanin synthesis were increased in the violet-red lily bulbs,leading to the accumulation of intermediate metabolites related to anthocyanin synthesis（such as chalconaringenin,naringenin,eriodictyol and cyanidin）,and promoting the increase in anthocyanin content.（5）Prediction of transcription factors for violet-red changes of minimally processed lily bulbs:In the transcriptome data of Lilium davidii var.unicolor bulbs,5 MYB and 2 b HLH transcription factor related to anthocyanin synthesis were screened and predicted.Among them,there was no significant difference in the expression of 1 b HLH gene（Cluster-18294.0）,while the other 6 transcription factors showed differential expression.After correlation analysis with differentially expressed structural genes related to anthocyanin synthesis,3MYB genes（Cluster-4138.3405,Cluster-4138.19860,and Cluster-4138.19859）and 1 b HLH gene（Cluster-4138.9158）showed significant positive correlation with the expression of most structural genes,which played a promoting role in the accumulation of anthocyanins in lily bulbs.One MYB gene（Cluster-4138.14450）showed significant negative correlation with the expression of most structural genes,which played a negative regulatory role in anthocyanin accumulation of lily bulbs.One MYB gene（Cluster-668.0）showed a weak correlation with structural genes,exerting a weaker influence on anthocyanin accumulation.In conclusion,this study found that under appropriate light conditions,MYB and b HLH transcription factors play a regulatory role in lily bulbs,the expression levels of structural genes（CHS,CHI,F3H,DFR and ANS）were significantly increased,which promoted the corresponding enzyme activities of CHS,CHI,DFR,and ANS.It enhanced the growth of upstream metabolites（chalconaringenin,naringenin,eriodictyol and taxifolin）related to anthocyanin synthesis.thereby increasing the accumulation of anthocyanins（cyanidin）,and finally forming stable anthocyanins（cyanidin 3-rutinoside）,resulting in the violet-red changes of minimally processed lily bulb surfaces.The results of this study are of great significance for exploring the molecular mechanism of the violet-red changes of minimally processed lily bulb surfaces.