Physiological And Molecular Mechanism Of Anthocyanin Synthesis And Regulation In Mango(Mangifera Indica Linn)

China is the major mango producer in the world. Mango has diverse cultivars with different fruit colors. Mango is an important tropical fruit with high commercial potential, and many studies have focused on fruit physiological characterization and germplasm resouce research. However, little studies have been done on molecular mechanism of fruit coloration in mango, and genomic sequence information for this species is quite limited in public databases. Thus, application of RNA-Seq transcriptome sequencing and high throughput proteomics technology would be helpful to better understand functional genes and proteins correlate with fruit coloration during fruit development and ripening, Effects of different cultivars, bagging and debagging on pigment, coloration, and gene expression of structure genes and MYB involved in anthocyanin synthesis pathway were studied in this experiment, At the same time, differential gene expression profile was analyzed between two red mango cultivars.The main results of this study are listed as below:1. We performed transcriptome sequencing of mixed fruit sample for ’Zill’ mango (Mangifera indica Linn) including pericarp and pulp during fruit development and ripening stage using Illumina RNA-seq technology. RNA-seq generated 68,419,722 sequence reads with 13× sequence depth and each sequence read averaging 90 bp in length. All the sequence read datasets were deposited at the NCBI Sequence Read Archive (SRA) (GenBank accession SRP035450). All high-quality reads were assembled into 124,002 contigs and 54,207 unigene, including 26,413 culusters and 27,794 singletons. A total of 42,515 unigenes were annotated using public protein databases with a cut-off E-value above 10-5. Out of these,35,198 unigenes and 14619 unigenes were assigned to Gene Ontology terms including biological process, molecular function, cell component and Clusters of Orthologous groups. Functional annotation against Kyoto Encyclopedia of Genes and Genomes pathway database identified 23,741 unigenes which were mapped to 128 pathways, The pathway revealed many transcripts that were previously unknown, which will provide abundant biological information for the development and ripe of mango fruit and provide useful information for further studying gene function and whole genome sequencing. We performed proteomics analysis of mixed fruit sample for’Zill’mango including pericarp and pulp during fruit ripening stage. We applied a mass spectrometry-based transcriptome data to characterize its proteome. LC-MS/MS analysis of the mango fruit proteome was performed using tandem mass spectrometry (MS/MS) in an LTQ Orbitrap Velos (Thermo) coupled online to the HPLC. This approach enabled the identification of 7586 peptides that matched to 2754 proteins. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with identifier PXD000808 and DOI (http://dx.doi.org/10.6019/PXD000808). The theoretical mass distribution for the identified proteins ranged from 3.72 kDa (CL11262.Contig2, unknown [M. truncatula]) to 401 kDa (transcript 679, predicted:E3 ubiquitin-protein ligase UPL1-like [V. vinifera]).Of these proteins,17 were mapped to mango and were mostly involved in carotenoid metabolism, sucrose metabolism, fruit softening, signal transduction, cell cycle control and development. These proteins were found to be submitted to NCBI as conceptual translations, and include well characterized proteins such as phytoene synthase, sucrose phosphate synthase, ripening-related pectate lyase,14-3-3, succinic dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase, and a small heat shock protein.2. Based on results of transcription sequencing in zill mango, structure genes and MYB genes involved in anthocyanins synthesis were selected and used in this experiment. Fourteen mango cultivars with different pericarp color were used in this experiment to evaluate the component of pigments, coloration and mRNA level of anthocyanin synthesis gene including structure gene encoding MiPAL, MiCHI, MiUFGT and MiMYB gene. The concentration of chlorophyll and flavonoids in non-red cultivars were significantly higher than that of red cultivars. Anthocyanin concentration varied from none to 15.96/100g FW among the 14 cultivars, which were divided into three coloration types, i.e. non-red (Deshehari, white ivovy, Guire No.82, Guixiang, Lingshui, Qingpi and Yuexi No.1), medium type (Tainong No.1 and Chinhuang) and red (Renong No.2, Renong No.1, hongwacheng, Zillate, R2E2). From the results of expression of structure and MiMYB gene correlated with anthocyanin synthesis, the expression of the four genes, especially MiC4H, Mi4CL2, MiANS, MiDFR and MiUFGT 2 in red cultivars were significantly higher than that of non-red cultivars. In the medium and red cultivars, anthocyanin concentration was significantly positively correlated with the expression of MiPAL. While in non-red cultivars, the expression of MiC4H, Mi4CL2 and MiUFGT 2 were positively correlated with anthocyanin concentration, and the expression of Mi4CL and MiMYB were significantly and positively correlated with anthocyanin concentration.3.’Zill’(Red-peel) and ’Dashehari’(green-peel) were used to study the effect of debagging treatments (removing bag 10 days prior to harvest and fruits completely re-exposed to sunlight until harvest) on pigment, coloration and gene expression in the experiment. The results showed that the contents of chlorophyll and carotenoids in unbagged fruits decreased during the fruit development and riping, while bagged fruits increased after the bag removal. The content of anthocyanin has an obvious ascend trend, while the concent of flavonoids increased to a peak then decreased. The expression of MiC4H, Mi4CL2, MiF3’H, MiUFGT 1 and MiMYB were inhibited by two-layer bags in’Zill’mango. After bagged fruits exposed to the sunlight, the expression of Mi4CL2, MiF3H2, MiDFR, MiANS, MiUFGT 2 and MiMYB gene increased fast and reached the highest at 4 days after bag removal, then declined, MiF3’H, Mi4CL, MiCHSl and MiF3H1 reached the highest at 7 days, while the expression of genes especially MiPAL, MiC4H, MiCHI and UFGT 1 reached a maximum at 10 days after bag removal, which was concurrent with anthocyanin accumulation after bag removal. Further correlation analysis indicated that the concentration of anthocyanins have positive correlation with the expression of MiPAL and MiCHI. For’Dashehari’mango, the pericarp light and chrome of bagged fruits were significant higher than that of unbagged fruits, while hue angle significantly lower than that of unbagged fruits from 0 days to ripe after bag removal. The expression of Mi4CL, MiDFR, MiANS, MiUFGT 2 increased and expression of MiC4H, MiF3’ H, MiCHI, MiMYB decreased in fruits bagged double-layer bags. However, the expression of MiPAL, MiC4H, Mi4CL2, MiF3Hl, MiF3’H, MiUFGT 1, MiUFGT 2 decreased first, then increased to the highest value at 10 days after bag removal after when bagged fruits were exposed to sunlight, Further correlation analysis indicated that the concentration of chlorophylls and carotenoids have significant negatively correlated with expression of MiC4H and MiF3H1, and flavonoids positively correlated with expression of Mi4CL, MiCHS1 and Mi F3H2.4. Based on results of transcription sequencing in ’Zill’ mango, gene expression profile was analyzed in two red mango cultivars, which can (Ono) or cannot (Renong No.1) synthesize anthocyanins after bagging with double-layer bags in mature fruit. Studies have shown that gene transcripts responding to bagging in Ono were more complex than in Renong No.1, 1349 genes were induced and 2148 genes repressed in the former, whereas 355 genes were induced and 481 suppressed in the latter. They were regulated either in the same or opposing manner in two cultivars, or in only one cultivar. Some candidate genes (PAL, CHS, C4H, F3H, CHI, F3’H, LDOX, UFGT) were identified in the differential responses of’Ono’and Renong No.1 to bagging. In addition,26 MYBs, four bHLHs and one WD40 gene responded differently to bagging in two cultivars. The expression of some LHY/CCA1 family genes in Plant physiology rhythm, FBPase/SBPase, PRK, GAPDH in Carbon fixation in photosynthetic organisms, COP1 ubiquitin mediated proteolysis and correlated gene in Photosynthesis were differentially expressed in two cultivars. The present study provides a valuable comprehensive gene expression profiles and an insight into the coloration difference that may be responsible for unbagged and bagged fruits anthocyanin biosynthesis in mango.