Molecular Mechanisms Associated With Red-flesh Mutation In Sweet Orange Based On Omics

Enhancing fruit quality is an urgent breeding target for citrus production and marketing in China. Fruit color is an important factor influencing fruit quality. Most citrus fruits have yellow pulps, but occasionally, red flesh fruits with high lycopene content are produced due to bud mutations. Many red-flesh orange fruits possess higher nutritional and market value. Such red-flesh mutants also are ideal materials for the study of carotenoid biosynthesis and its regulation. Therefore understanding the molecular mechanisms underlying the red-flesh mutation in citrus fruits is of importance relevant to both basic research and translation application in citrus industry. In this study, the modern ’omics’technologies were used to analyze the proteomic, transcriptomic, genomic and metabolomic differences in two red-flesh citrus mutants (Cara Cara and’Hong Anliu’) and their respective wild types. The main results were as follows:1. Protocols for protein extraction from different citrus tissues were developed. It was found that an improved TCA method, the TCA-phenol method, and the phenol extraction method were suitable for protein extraction from citrus callus, leaves and fruits, respectively.2. The proteomic alterations in the pulp of’Hong Anliu’versus its wild type’Anliu’ at four maturing stages were determined by Two Dimentional Electrophoresis (2DE) combined with MALDI-TOF-TOF Mass Spectrum. Among the74differentially expressed proteins identified, the majority are predicted to be involved in stress response, carbohydrate/energy metabolism and regulation, or protein modification and degradation. Particularly, expression levels of six anti-oxidative enzymes were altered by the mutation; and assays of their respective enzymatic activities indicated an enhanced level of oxidative stress in’Hong Anliu’, implying a regulatory role of oxidative stress on carotenogenesis. This result was further confirmed by our observation that ROS treatment of fruit pulps induced lycopene accumulation in’Hong Anliu’only. Gene expression showed that genes predicted to function upstream of lycopene biosynthesis were generally upregulated in juice sacs, but downregulated in segment membranes in both ’Hong Anliu’ and its WT. This result suggests that oxidative stress plays an important role in carotenogenesis and that carotenogenesis in juice sacs and segment membranes of citrus fruits may be regulated by different mechanisms.3. Comparative proteomic analysis of leaves from ’Hong Anliu’ and Cara Cara and their wild types revealed that proteins involved in photosynthesis were generally up-regulated in the red-flesh mutants. Forty-eight differentially expressed proteins were identified between the pulps from Cara Cara and Newhall sweet oranges. The majority of these proteins are heat shock proteins, proteins involved in oxidative stress and other abiotic stress responses, and carbohydrate/energy metabolism. The plastid proteome of citrus fruits was also investigated. Although unsuccessful in achieving the original goal, we identified80novel proteins in citrus fruits, which may be of help towards understanding citrus fruit metabolism in general.4. The transcriptomic difference of Cara Cara and Newhall was investigated by Massively Parallel Sinature Sequencing (MPSS) technology. In total,6099788and5645571sequences were obtained which were further assigned to12482and11784unigenes in Cara Cara and Newhall, respectively. A total of622differentially expressed genes were identified (using the threshold of fold change≥2and FDR≤0.001), of which307were up-regulated and315down-regulated in Cara Cara versus Newhall. Some differentially expressed genes may be associated with the red-flesh mutation. These include genes encoding transcription factors, transporters and enzymes likely involved in carotenoid biosynthesis and oxidative stress response. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis assigned these genes to39metabolic pathways (each one of which contains3or more differentially expressed genes). Notably, ten of the differentially expressed genes were predicted to be involved in the starch and sucrose metabolic pathway.5. To assess the genetic difference between’Hong Anliu’and its wild type’Anliu’, we first surveyed the two genomes by Simple Sequence Repeat (SSR), Inter-Retrotransposon Amplied Polymorphism (IRAP) and Amplified Fragment Length Polymorphism (AFLP) and identified two polymorphic AFLP fragments. We then employed Comparative Genome Hybridization (CGH) to investigate the genome-wide variations between the two genotypes. Surprisingly,25genes were shown to exhibit gene dosage variation, with18being hemizygously deleted and7 duplicated in the bud mutant. This gene-dosage variation was validated by quantitative PCR, however, the transcription of some of the corresponding genes was not significantly affected. The detection of gene dosage variation in at least25genes between’Hong Anliu’ and ’Anliu’ suggests that the genomes of these two sweet orange genotypes are genetically unstable, which may provide the genetic basis for the phenotypic deviation of bud mutants from their parental cultivars. Our findings also suggest that CGH is a powerful technology that can be used for detection of genome-wide variations among near-identical genotypes such as bud mutants and their parental cultivars.6. The metabolomic difference of’Hong Anliu’ and ’Anliu’ at three stages of fruit development was analyzed by DART-AccuTOF MS. Forty-three metabolites were identified under the DART negative mode, among which17metabolites showed quantitative difference between’Hong Anliu’ and ’Anliu’. Differences in eleven organic acid were detected, of which malic acid, citric acid, quinic acid,2-furoic acid and cis-aconitic acid were decreased, while lactic acid, acetoacetic acid, glyceric acid, succinic acid, naphthalenedicarboxylic acid and diphenylhydantoic acid were increased in’Hong Anliu’ versus ’Anliu’. Glucose and sucrose were significantly increased in’Hong Anliu’versus’Anliu’. However, no significant difference was detected under the DART positive mode. PCA analysis showed that the6citrus samples tested could be well classified under the negative mode but not under the positive mode.