Analysis Of Anthocyanin And Lignin Metabolism In Toona Sinensis Sprouts During Cold Storag

Toona sinensis is a deciduous tree belongs to the Meliaceae family.The young buds are edible and are considered as a wild woody vegetable in China.However,during storage T.sinensis buds display phenomena such as color change,water loss,wilting,rotting,browning,and lignification etc.,which will directly affect its vegetable quality.The color change and lignification of T.sinensis buds undergoes a complex process that involves anatomical changes,biomass accumulation,enzyme and gene regulation.At present,comprehensive analysis of the anthocyanin and lignin content of T.sinensis buds during storage have not been comprehensively reported.In the present study,we analyzed the content changes of anthocyanin and lignin in red T.sinensis buds,green T.sinensis buds and red T.sinensis buds from different harvest periods during storage based on physiological,biochemical,cellular and molecular methods.The major findings are as following:1)Transcriptome sequencing analysis of T.sinensis budsWe collected red T.sinensis buds and performed transcriptome sequencing to analysis the molecular mechanism of lignin and anthocyanin in T.sinensis buds.A total of 297,838 transcripts were generated from our transcriptome,which were assembled into 32,716 unigenes with a mean length of 887 bp.A total of 307 unigenes involved in phenylpropanoid biosynthesis using KEGG database,10 anthocyanin-related genes(Ts PAL,Ts C4 H,Ts4CL,Ts CHS,Ts CHI,Ts F3 H,Ts F3’5’H,Ts DFR,Ts ANS,Ts3GT)and11 lignin-related genes(Ts PAL,Ts C4 H,Ts4CL,Ts CCR,Ts CAD,Ts HCT,Ts C3’H,Ts COMT,Ts F5 H,Ts CCo AOMT,Ts LAC)were identified.2)Color and tissue structure changes of T.sinensis budsRed T.sinensis buds are bright and purple in spring;red T.sinensis buds are green in summer and autumn.During the refrigeration process of T.sinensis buds,the leaves lose water and wilt,and become dull in color,among which the purple color of the leaves gradually became lighter during the refrigeration process of spring red T.sinensis buds after harvesting,while black spots started to appear on the petiole of green T.sinensis buds after 1 d of refrigeration,and the black color gradually deepened and the number of black spots gradually increased with extension of storage time.In addition,the petiole xylem cells of the T.sinensis buds were larger than the vein xylem cells,and the secondary cell walls of the vein and petiole xylem cells gradually thickened with increasing storage time.The red T.sinensis buds showing the greatest lignification in autumn,followed by the green T.sinensis buds.3)Differential analysis of anthocyanin and lignin content of T.sinensis budsThe anthocyanin content of red T.sinensis buds was higher than that of green T.sinensis buds,which was about 1.70 times higher than that of green T.sinensis buds;while the lignin content was lower than that of green T.sinensis buds,the lignin content of petioles in T.sinensis buds was about 1.1 times higher than the lignin content of leaves.In spring,red T.sinensis buds had the highest anthocyanin content and the lowest lignin content;in summer,the anthocyanin and lignin content of red T.sinensis buds were the second highest;in autumn,red T.sinensis buds had the lowest anthocyanin content and the highest lignin content.With the increase of storage time,the anthocyanin content of T.sinensis buds gradually decreased and the lignin content gradually increased.4)Analysis of enzyme activity of T.sinensis buds.Phenylalanine ammonia lyase(PAL),4-Coumarate-Co A ligase(4CL)and Cinnamyl alcohol dehydrogenase(CAD)are key enzymes of phenylpropane metabolism in plants,which closely related to the synthesis of secondary metabolites,such as lignin.There were significant differences of lignin synthesis enzyme activities in red T.sinensis buds at different harvesting periods.In order to investigate the relationship between lignin accumulation and enzyme activities related to the lignin metabolic pathway.PAL,4CL and CAD enzyme activities were measured during the refrigeration of red T.sinensis buds in spring,summer and autumn.The results showed an overall increasing trend of PAL,4CL and CAD enzyme activities during the refrigeration of red T.sinensis buds.5)Anthocyanin-related and lignin-related genes expression analysis of T.sinensis budsThe qPCR results showed that the expression levels of Ts PAL,Ts CHS,Ts CHI,Ts F3 H,Ts F3’5’H,Ts DFR,Ts ANS,Ts3 GT genes were significantly down-regulated during the cold storage of red and green T.sineneis buds;Ts CAD,Ts HCT,and Ts COMT genes were up-regulated in red T.sinensis buds;Ts4CL,Ts CCR,Ts HCT,and Ts LAC genes were significantly up-regulated in green T.sinensis buds.In addition,the expression levels of anthocyanin-related and lignin-related genes were significantly different during the cold storage of red T.sinensis buds at different harvesting stages.The expression levels of Ts ANS genes were significantly down-regulated in the leaves;Ts F3 H and Ts ANS genes were significantly down-regulated in the petioles;Ts4CL,Ts CCR and Ts CAD genes were significantly up-regulated in the leaves;Ts CCR,Ts C3’H and Ts CCo AOMT genes were significantly up-regulated in petioles.