Transcriptome Profiling And Physio-chemical Analysis Reveals The Biosynthesis Of Anthocyanin In Sweet Cherry Fruit

In order to preliminarily reveal the mechanism of the red pigment development in sweet cherry(Prunus avium L.) fruits, a pure yellow cultivar ‘13-33’ and a red cultivar ‘Tieton’ were used as the experimental materials in this study. The composition and the content of anthocyanin, soluble sugars and organic acids as well as the changes of the activity of the enzymes involved in anthocyanin biosynthesis were analyzed. Transcriptome analyses and DGE profiling based on deep sequencing were used to estimate of overall gene expressions at different developmental stages between these two cultivars and to identify the structural genes and transcription factors genes involved to anthocyanin biosynthesis. Furthermore, several transcription factor genes were cloned and the expression patterns analysis was also conducted. The main results were as follows:1. The changes of the contents of anthocyanin and the activities of the enzymes involved in anthocyanin biosynthesis including plenylalanine ammonia lyase(PAL), chalcone ismoerase(CHI), dihydroflavonol 4-reductase(DFR) and flavonol 3-O-glucosyltransferase(UFGT) were detected in the red cultivar of ‘Tieton’ and the yellow cultivar ‘13-33’ during the fruit development. The result showed that, the content of anthocyanin in ‘Tieton’ increased gradually, and then increased sharply after the period of pigmention. The anthocyanin accumulation in ‘Tieton’ were not correlative with the activity of PAL and DFR, but significantly correlative with the activities of CHI and UFGT. The content of anthocyanin in the yellow sweet cherry ‘13-33’ fruit was almost unchanged and remained at a very low level during fruit ripening.2. A method of ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) for the detection of anthocyanins in sweet cherry was developed by optimizing the extraction solvent, purification conditions, chromatography and mass spectrometry parameters. The composition and the content of anthocyanin in ‘Tieton’ and ‘13-33’ were analyzed by this method. The result showed that seven anthocyanins were detected from the red cultivar ‘Tieton’. The content of each component increased gradualy during the fruit development. However, only four anthocyanins were detected in the yellow cultivar ‘13-33’. The content of each component decreased gradualy during the fruit development, and the level was very low at the fruit maturity period. Among these compounds, cyanidin-3-xyloside and delphinidin-3-glucoside were detected for the first time in sweet cherry.3. The composition and the content of the soluble sugar and organic acid in red and yellow cultivar were measured by HPLC. It was found that fructose, glucose and sorbitol were detected from ‘Tieton’ and ‘13-33’. The content of each component and the total soluble sugar were increased during fruit development in these two cultivars. The content of fructose, glucose and sorbitol were significantly positive correlation with the synthesis of anthocyanin in red cultivar ‘Tieton’. The orgnic acid composition were similar in the two cultivars, but the changes of each component and the total orgnic acid content during the fruit development were different. The content of Malic acid reached to the highest level during the whole period of fruit development, which was the primary factor responsible for the acidity of sweet cherry.4. Normalized cDNA libraries from red cultivar ‘Tieton’ and yellow cultivar ’13-33’ fruits were sequenced using the next-generation Illumina/Solexa sequencing platform and de novo assembly. 66,392,570 high-quality reads were assembled into 43,128 unigenes using a combined assembly strategy. The results of gene functional annotation showed that 22,452 unigenes were annotated from at least one database. Approximately 20,095(46.59%) unigenes showed significant BLAST hits against known sequences in the Nr databases. Sequence homology revealed that 16,853 of the assembled unigenes were assigned at least one GO term and 8,645 unigenes were assigned to 26 KOG categories. To identify the biological pathways activated in sweet cherry fruit, we mapped the annotated sequences to the canonical reference pathways in the KEGG database. Significant matches were found for 4,035 unigenes, which were assigned to 242 KEGG pathways. A total of 72 unigenes encoding 11 enzymes were assigned to the anthocyanin biosynthetic pathway based on a KEGG pathway assignment.5. To investigate the gene expression patterns in red and yellow sweet cherry fruits during the ripening process, eight DGE libraries(20 DAF, 35 DAF, 45 DAF and 55 DAF fruits from ‘Tieton’ and ‘13-33’) were constructed and sequenced using Illumina deep sequencing technology. Over 7.0 million clean reads were obtained from each library. Analysis on genes expression levels involved in anthocyanin biosynthesis through DGE profiles, it finds that the expression of unigenes encoding PAL, 4CL, CHS, CHI, F3 H, F3’H, DFR, ANS and UFGT in ‘Tieton’ showed significantly upregulated during the ripening process, particularly when the fruit turned red. In contrast, all of the DEGs encoding anthocyanin biosynthesis in fruit of the yellow cultivar ‘13-33’ showed significantly downregulated expression. These results are consistent with the accumulation of anthocyanin in these two cultivars. Furthermore, we identified that 4 candidate MYB genes, 2 candidate bHLH genes and 1 candidate WD40 gene among the DEGs that may regulate anthocyanin biosynthesis. We confirmed the altered expression levels of eighteen unigenes that encode anthocyanin biosynthetic enzymes and transcription factors using qRT-PCR.6. The cDNA sequences of 6 transcription factor genes which were involved in anthocyanin biosynthesis were cloned from the red cultivar ‘Tieton’ by RT-PCR. They are 3 MYB genes PaMYB10, PaMYB11 and PaMYB111 classified as R2R3 MYB, 2 b HLH genes PabHLH3 and PabHLH13 and one WD40 gene PaWD40. Sequence analysis indicated that 6 transcription factor genes was highly homologous with the orthologous genes in other specie. PaMYB10, PaMYB11, PaMYB111, PabHLH3, PabHLH13 and Pa WD40 genes expressed in stem, young leaf, flowver and fruit. The higher expression level of these genes were found in young leaf and mature fruit, which contained anthocyanins.