Deastringent Effects Of High CO₂ Indifferent Persimmon Cultivars And Screening Of Related Differentially Expressed Genes

Persimmon（Diospyros kaki,L.）fruits are generally divided into two categories:sweet persimmon and astringent persimmon according to whether they can deastringent naturally on the tree.The astringent persimmon fruit still contains a large amount of soluble tannins when it matures,which results in the fruits’astringent taste.However,different varieties of persimmon fruits show different deastringency rates during the deastringent treatment,but different temperature,different treatments,different seasons and other reasons make it more difficult to compare the results,and the corresponding mechanism is also less clear.This study focused on the most commonly used high-concentration CO₂ treatment in the industry,using 42 different varieties of mature astringent persimmon fruits as experimental materials,through high-concentration CO₂ treatment（95%CO₂+1%O₂）for post-harvest deastringent treatment,through the single tannin printing,tannin content determination,etc.were used to determine the difficulty of desquamation,and RNA-seq combined with WGCNA correlation analysis was used to screen out the differential genes and to conduct preliminary verification.The main results are as follows:1.The physiological indexes of 42 different varieties of ripe astringent persimmon fruits showed obvious differences among varieties.The ripe astringent persimmon fruits used in this experiment were collected from the Shanxi Germplasm Nursery in October 2017.The hardness and color difference of untreated fresh persimmon fruits were tested.The lowest hardness was‘ZaoGaiShi（ZGS）’,the hardness was less than 20N;the highest hardness was‘YaoXianWuhuaShi（YXWHS）’,the hardness value was higher than 70N.The large differences in fruit hardness of the varieties of persimmon may be related to the maturity and pulp texture.The L value of different varieties of fruit is between 50-70,the difference is not obvious,indicating that there is not much difference in the surface gloss of different varieties of persimmon fruit;a value is between 10-35,b value is between 40-65,indicating different varieties.There are differences among varieties of persimmon fruit peel color,but there is not much difference between fruits of the same variety.2.After treatment with high concentration of CO₂,42 different varieties of ripe astringent persimmon fruits showed different deastringent effects.Further analysis of soluble tannin and insoluble tannin content found that the soluble tannin content of 42different varieties of fruit at the initial point was distributed between 0.44%-1.39%,while the insoluble tannin content ranged from 0.01%-0.18%.The content of soluble tannin and insoluble tannin in fruits after 24 hours of high concentration CO₂treatment were distributed between 0.003%-1.1%and 0.08%-1.01%.Based on the comparison of tannin imprinting,content and deastringency rate,different varieties are divided into three categories.Among them,the more difficult to deastringent variety is defined as the hard-to-deastringent varieties（the content of soluble tannin is higher than 0.6%after 24 hours of high concentration CO₂ treatment,and the deastringency rate less than 40%）,and the easier to deastringent variety is defined as easily deastringent varieties（soluble tannin content<0.2%after high concentration CO₂ treatment for 24h,deastringency rate higher than 70%）.Since the initial soluble tannin content of different varieties of fruits is slightly different,the deastringency rate of the single varieties of fruits is compared.The highest varieties are’YangShuoNiuXinShi（YSNXS）’,’SanYuanShaoShi（SYSS）’,and’MoXinShi（MXS）’,’ChangAnHuoGuanShi（CAHGS）’,’YiDuTuoShi（YDTS）’,etc.The varieties with the lowest decolorization rate are’LuoYangChangTianShengShi（LYCTSS）’,’LiChengMianShi（LCMS）’,’XiaoDiShi（XDS）’and’LaoPiGe（LPG）’.3.Based on the analysis of transcriptome sequencing,14 differentially expressed genes that may be related to the differences in the ease of astringency between varieties were screened.’SiGouShi（SGS）’and’LuoYangFangTianSheng Shi（LYFTSS）’were chosen as representatives of easily deastringent varieties,and’LaoPiGe（LPG）’and’XiaoDiShi（XDS）’were chosen as representatives of difficult-to-astringent varieties.Transcriptome sequencing was performed to obtain information such as gene sequence,gene expression in sequenced varieties,GO、COG、KEGG function prediction,etc.,and to compare and confirm the gene sequence in the transcriptome with the persimmon genome sequence.Furthermore,the correlation analysis of WGCNA was combined with physiological data,and 28differentially expressed genes were preliminarily screened,and 20 differentially expressed genes were confirmed by qPCR verification.4.Previous studies showed that high-concentration CO₂ treatment hasaccelerated deastringency and induced softening in many persimmon varieties.To further eliminate softening-related genes,samples of’mirror persimmon’treated with CO₂+1-MCP（deastringent and keep crisp）to verify,and finally get 14 differentgenes that may be related to the difference between varieties that are difficult to getastringent,such as EVM0027048（polyubiquitin）,EVM0027265（GMP synthase）,EVM0028686（neomenthol dehydrogenase-like）,EVM0031224（lysine-specific demethylase JMJ18-like isoform X1）,EVM0002193（pheophytinase）,EVM0009991（expansin-A1）,EVM0015029（hypothetical protein）,EVM0026845（putative ABC transporter C family member）,EVM0031877（probable fructokinase-6）,EVM0002369（glutathione S-transferase）,EVM0017291（putative 12-oxophytodienoate reductase 11）,EVM0021701（hypothetical protein CICLE）,EVM0023725（UDP-glycosyltransferase）,EVM0029962（carboxylesterase）.Among these 14 differential genes,there are genes related to ubiquitination and methylation,which may be effect aldol conversion or tannin polymerization;genes related to fructose kinase may play a role in upstream of the regulatory network.We concern at the genes such as glutathione S-transferase and UDP-glycosyltransferase,which speculate that they may effect the glycosylationprocess of tannin monomers and the transport and conversion process of tannin,UDP-sugar Glycotransferase may combine with gallic acid to glycosylate gallic acid,participate in the conversion process of tannin,convert the original soluble tannin into insoluble tannin,and then go through the action of glutathione S-transferase,transport insoluble tannin into the vacuole and store it in the tannin cells,and then let the fruits been astringency.In summary,this study compared and analyzed the effect of high-concentration CO₂ treatment on the fruits of 42 astringent persimmon varieties,screened and preliminarily verified the difference in expressed genes related to the different varieties of astringent persimmon.To explore the reasons for the difficulty of removing astringency between different varieties and provide a preliminary data basis.