Effects Of High Temperature Stress On Anthocyanin Biosynthesis In Eggplant Peel

Eggplant（Solanum melongena L.）is popular as a functional food because of its rich antioxidant anthocyanin in its peel.The optimum temperature for the flowering and fruiting stage of eggplant is 25～30℃.The temperature above 35℃ will have adverse effects on the growth and development of eggplant.In this experiment,purple eggplant peel from the cultivar ’Tewangda’ was used as the experimental material to analyze the following parameters:anthocyanin and flavonoid contents in eggplant peel under the control and high temperature treatment,the change of enzyme activity and gene expression related to anthocyanin synthesis process,the CHS gene family and the molecular regulatory mechanism of anthocyanin biosynthesis pathway under high temperature stress.The above analysis is useful to further understand the effects of high temperature stress on anthocyanin biosynthesis in eggplant peel.The main results are as follows:（1）Using purple eggplant ’Tewangda’ as plant material,plants were treated under high temperature of 38℃ and 45℃ in phytotron.The peel of eggplant was taken as sample at treatment time of 1 h,3 h,6 h,12 h,24 h,48 h and 60 h.The concentrations of anthocyanin and flavonoids were measured.The activities of phenylalanine ammonialyase（PAL）,chalcone synthase（CHS）,chalcone isomerase（CHI）,dihydroflavonol 4-reductase（DFR）,anthocyanidin synthase（ANS）,and flavonoid 3-O-glucosyltransferase（UFGT）were determined.The expression levels of genes encoding key enzymes were analyzed at 3 h and 6 h.Attempts were made to explore the effects of high temperature stress on the synthesis and accumulation of anthocyanins and flavonoids,activities of key enzymes and the expression levels of related genes during anthocyanin biosynthesis.The results showed that the concentrations of flavonoids and anthocyanin decreased significantly compared with those of the control.With the increase of stress time,the activity of PAL increased at first and then decreased;activities of CHS,CHI,DFR,ANS,UFGT showed a continuous descending trend,and at 38℃their activities were significantly lower than those of the control,while at 45℃ their activities were lower than those of the control and at 38℃.The relative expression levels of SmPAL and Sm4CL were increased gradually with the increase of temperature in 3 h treatment.When the treatment time reached 6 h,the relative expression levels under 38℃ and 45℃ were lower than those of the control.The relative expression level of SmCH1 increased significantly at 38℃,but decreased at 45℃ to the level of the control.The relative expression levels of SmF3H,SmANS and SmUFGT showed the same trend.The higher the temperature,the lower the expression level.These results indicated that high temperature stress reduced the activities of key enzymes of anthocyanin synthesis and relative expression levels of genes encoding key enzymes for anthocyanin synthesis which results in the decrease of the concentrations of flavonoids and anthocyanin in eggplant peel.（2）Eggplant is a vegetable with high anthocyanin content.Anthocyanin synthesis is affected by many factors.Among them,temperature is an important environmental factor.In some areas of China,eggplants are often exposed to high temperature environmental conditions during the cultivation season.Therefore,this experiment aims to explore the expression mechanism of anthocyanin biosynthesis-related genes in eggplant peels under high temperature stress.The eggplant variety ’Tewangda’ was subjected to high temperature treatment at 38℃ and 45℃.The optimum temperature for eggplant growth was 27℃ as a control.The treatment time was 3 h and 6 h.The peel of the eggplant was subjected to transcriptome sequencing and biological analysis.The results showed that there were 770 genes that were differentially expressed between different high temperature treatments.By comparing with the GO database and the KEGG database,14 genes related to anthocyanin biosynthesis were screened out,among which Sme2.5₀0283.1_g00002.1was up-regulated,other 13 genes including Sme2.5₀1638.1_g00005.1,Sme2.5₀0188.1_g00020.1,Sme2.5₀4313.1_g00001.1,Sne2.5₀1193.1_g00009.1,Sme2.5₀0461.1_g00010.1,Sme2.5₀4555.1_g00001.1,Sme2.5 00015.1_g00020.1,Sme2.5₀9880.1_g00001.1Sme2.5₀6210.1_g00004.1,Sme2.5₀5988.1_g00001.1.Sme2.5₀0749.1_g00006.1,Sme2.5₀0081.1_g00022.1,Sme2.5₀0029.1_g00004.1 were down-regulated.The above results indicate that the genes related to anthocyanin biosynthesis in eggplant peels are mostly down-regulated and a few are up-regulated under high temperature stress,which has a reference value for further understanding and improvement of eggplant heat tolerance mechanisms.（3）The chalcone synthase（CHS）family is involved in a series of secondary metabolic processes in plants.The secondary metabolites have many biological functions.Anthocyanin is one of the secondary metabolites in plants.It plays a role in resisting biotic and abiotic stresses.The bioinformatics method was used to identify the CHS gene family of eggplant and its expression analysis under high temperature stress.The results showed that seven CHS（SmCHSl-7）genes were identified in the whole genome of eggplant.The SmCHS gene were distributed on 7 branches and 3 clusters.Phylogenetic analysis showed that 73 CHS genes from 7 species of Solanaceae were divided into 10 groups.At 38℃ and 45℃SmCHS1,SmCHS2,and SmCHS3 were continuously down-regulated,while SmCHS4 was up-regulated at 38℃,the change was not obvious at 45℃.The expression profile of key anthocyanin biosynthesis gene families showed that PAL,4CL and ANT1 were expressed in five tissues:root,stem,leaf,flower and peel.CHI,F3H,F3’5’H,DFR,3GT and bHLH1 were expressed only in flower and peel.The results show that the SmCHS gene family has conserved genetic structure and functional diversity.SmCHS displays two or more expression patterns and performs multiple functions to regulate anthocyanin content.Combined with the regulatory network,we can further understand the regulation mechanism of eggplant peel color.（4）Through the bioinformatics method,combining the STRING database information and the data obtained from the transcriptome under high temperature stress,a gene regulatory network for eggplant under high temperature stress was constructed.KEGG（ko00941）was screened for 73 genes related to the flavonoid biosynthetic pathway,and the interaction map was drawn.GO and KEGG enrichment analyses were performed on 148 genes.The results showed that there were 34250 pairs of interactions in the intersections of the four sets of gene regulatory networks of eggplant,tomato,potato,arabidopsis and rice.Three differentially expressed regulatory genes were selected based on the interaction map,they were Sme2.5₀0670.1_g00012.1,Sme2.5₀0029.1_g00004.1 and Sme2.5 00749.1_g00006.1.A total of 148 genes were mainly enriched in the flavonoid biosynthesis process（GO:0009813）,the lignin biosynthesis process（GO:0009809）,Prp19 complex（GO:0000974）and naringenin-chalcone synthase activity（GO:0016210）.The KEGG pathway is mainly enriched in ko00941（flavonoid biosynthesis）and ko00940（phenylpropanoid biosynthesis）.The above results clarify the molecular regulatory mechanism of key genes in anthocyanin biosynthesis of eggplant peel under high temperature stress.