Transcriptome Changes During Fruit Development And Ripening Of Sweet Orange (Citrus Sinensis) And Functional Analysis Of CsASR Gene

Fruit ripening is a highly coordinated and complicated biological process. Great importance has been gradually attached to the study on the fruit development and ripening of the non-climacteric fruit-citrus. However, an interpretation of the molecular mechanism of fruit development is far from enough. Sugars, organic acids, and carotenoids attained during fruit ripening are major components of citrus fruit quality. It was very important to study the molecular mechanism regulating fruit ripening and carotenoid accumulation for the improvement of citrus fruit quality. The red-fleshed mutant’Hong Anliu’, characterized as high sucrose and low citric acid, was ideal for the study on the molecular mechanism of the formation of fruit quality straits. This study provided a description of the transcriptomic changes occurring during fruit development and ripening in sweet orange, along with a dynamic view of the gene expression differences between the wild type ’Anliu’(WT) and the mutant ’Hong Anliu’(MT). An important candidate gene CsASR was functionally characterized. The main results were as follows:1The WT and MT fruit pulp harvested at120,150,190, and220days after flowering (DAF) was subjected to RNA-seq using an Illumina sequencing platform. The average number of tags produced for each library was4.01million. Mapping tags to a reference citrus unigene dataset identified between68.1%and76.2%of the tags (46,328-76,424) were homologous to sequences with known or unknown function.We solely used WT as a model to demonstrate the transcriptome changes during fruit development and ripening. A total of18,829genes were detected in at least one of the four stages in WT, of which8,825genes were expressed in all the four stages. The cluster analysis of gene expression patterns during fruit development and ripening arranged18,829genes into22groups. It also revealed that the abundance of89.7%of the transcripts detected in the WT pulp varied over the course of fruit development and ripening. A comparison of expression patterns between WT and MT revealed that20of the groups were common to both.A comparison of the transcriptomes of different developmental stages in WT identified9,377,7,886, and7,757were differentially expressed between120DAF and150DAF,150DAF and190DAF, and190DAF and220DAF, respectively. Of these,36.7%were assigned to one of18GO categories. The categories "metabolic process","cellular process","establishment of localization","localization","biological regulation","pigmentation", and "response to stimulus" based on biological process captured most of these genes. Many genes were associated with cell wall metabolism and softening, sucrose metabolism, the TCA cycle, carotenoid biosynthesis, and stress response.The comparison between the transcriptomes of WT and MT identified634,568,540, and616genes were significantly differentially expressed at p<0.05and|log2Ratio|>1in the four developmental stages, respectively. Many encode stress-related products. At all the four developmental stages the number of up-regulated genes was less than that of down-regulated genes. Only883genes were differentially expressed between WT and MT after the removal of the redundant. The cluster analysis of these genes showed over one half (492) turned out to be up-regulated in MT at all the developmental stages except150DAF. Only five genes were detected as differentially expressed at all four stages. The GO categories of differentially expressed genes based on the molecular function revealed that most encoded products associated with "protein binding","hydrolase activity","transferase activity" and "transporter activity". GO enrichment analysis revealed carotenoid metabolic process and capsanthin/capsorubin synthase activity were enriched at150DAF in MT.Q-Real-Time PCR validation of the transcription profiles for22of the differentially expressed genes indicated a good correlation between transcript abundance assayed by real-time PCR and RNA-seq data, with an overall correlation coefficient of0.8379.The dynamics of pulp soluble sugar, organic acid, carotenoid and H2O2content were monitored during fruit development and ripening in WT and MT. The content of soluble sugars increased markedly during the late stages of fruit development and ripening in both WT and MT. The concentration of sucrose was higher but the citris acid content was much lower in MT than in WT throughout fruit development and ripening. Carotenoids and lycopene both accumulated over time in MT, but remained at a low level in WT. H2O2content fell as the fruit developed and ripened, but was higher in MT than in WT at120DAF.2Our transcriptomic analysis and previous reports indicated ASR gene was significantly differentially expressed. The full-length cDNA of sweet orange ASR, designated as CsASR, was cloned based on the EST sequence (FE659120) and deposited in Genbank (accession number HQ398364). CsASR cDNA was929bp long and contained an open reading frame of540bp. The deduced protein contained179amino acids. Sequence homology analysis of amino acid showed CsASR shared considerable identity with other ASR proteins from various plant species. CsASR contained two highly conserved regions as other ASRs. The function prediction suggested that the CsASR protein might be involved in transcription regulation (0.241), growth factor (0.110), transcription (0.072), and signal transducer (0.063).Southern blot analysis suggested CsASR belonged to a small multi-gene family in most citrus species. Subcellular localization analysis revealed CsASR was localized in the cell nucleus.CsASR mRNA accumulation was detected in various tissues and fruits during fruit development and ripening. The result showed CsASR was mainly expressed in mature tissues, especially in mature fruits. The expression pattern of CsASR was the same in the pulp and peel of WT, but different in the pulp and peel of MT. CsASR transcription level was higher in’Hong Anliu’ than in’Anliu’ throughout fruit development except at120DAF. CsASR was significantly induced under cold, heat and salt stress, and ABA treatment.Exogenous ABA was applied to the pulp of WT and MT before colour break. All detected soluble sugars and organic acid content was reduced in WT fruit pulp but increased in MT fruit pulp after ABA treatment. We just detected two carotenoid compositions:Antheraxanthin and a-carotene in treated pulp. Although the content of carotenoid was lower in ABA treated WT pulp than that in the control, there is no difference in MT after treatment.We overexpressed CsASR in tomato via Agrobacterium tumefaciens-mediated transformation, and eleven independent transgenic lines were obtained. The mature fruit of the T1segregation developed to a different red colour, however, there was not other visual significant difference in phenotype and fruit maturity compared to the wild type. Differential interference microscope results showed that pericarp cells of CsASR overexpression tomato fruits appeared red, while the control was yellow. Frozen section watching under a bright field revealed the cell structure of transgenic tomato fruits was different from that in the wild type, and the cell wall of the former was thickening; further the cell wall of the transgenic tomato fruits presents strong blue fluorescence under fluorescence, suggesting the lignin content is high. While, ittle blue fluorescence was observed in the control.HPLC analysis revealed the content of violaxanthin, β-carotene, lycopene and phytoene all increased significantly in CsASR overexpressed fruits. No difference in the lutein content was observed. The expression level of all detected carotenoid metabolism-related genes was up-regulated in transgenic fruits compared to the wild type.GC-MS profiles revealed that the CsASR overexpression fruits displayed substantial changes in the level of primary metabolites. The content of almost all detected sugars and organic acids including the tricarboxylic acid cycle intermediates was lower in the CsASR overexpression fruits than in the wild type. Many amino acids content was also altered. Analysis of the expression profiles of related genes suggested the changes of expression levels of these genes were in agreement with the observed levels of metabolites. The result of LC-MS showed CsASR overexpression fruits had lower ABA level than that in the wild type.The transcriptome sequencing data showed401genes were significantly differentially expressed (FDR≤0.001and|log2Ratio|≥1) in CsASR overexpression fruits compared to the wild type. Of these180genes were up-regulated and221genes down-regulated in transgenic fruits, including NCED gene, APETALA2(AP2) domain-containing proteins, and ring-finger domain-containing protein. Analysis of the RNA-seq data using KEGG database revealed primary metabolism, biosynthesis of secondary metabolites, plant hormone signal transduction, and carotenoid biosynthesis were significantly changed in transgenic fruits.