Functional Characterization And Regulatory Mechanism Analysis Of Sweet Orange CsMYBF1 Gene

Citrus is one of the most important fruit crops worldwide, which are abundant with the phenylpropanoid compounds. The phenylpropanoid compound, as one important type of secondary metabolites in plants, plays key roles in many aspects like the formation of fruit quality and stress resistance. To exert the effective usage of the phenylpropanoid compounds, it is essential to understand the relevant regulation mechanism. However, few studies about the citrus phenylpropanoid regulation mechanism have been reported, as influence the progress of the related genetic improvement programs. MYB transcription factor represents one of the largest gene families in plant genomes, and is usually involved in the regulation of many biological progresses. There have been many studies about the MYB genes that regulate the phenylpropanoid metabolism in different plant species. Regarding the MYB transcription factors in citrus as the breakthrough point, this study identified the MYB gene family members in sweet orange genome with the methods of bioinformatics. The candidate gene, CsMYBF1, was selected out of the phenylpropanoid-related MYB genes, and its potential roles that regulate phenylpropanoid metabolism was further researched in this study. The main results were as follows:1. Genome-wide analysis of R2R3-MYB transcription factors in sweet orange. A total of 100 R2R3-MYB transcription factors were identified in sweet orange genome.Out of them, 87 genes were unevenly distributed across all 9 chromosomes, and chromosome 2 had the largest number of MYB genes; 21 MYB genes with segmental duplication and 8 with tandem duplication. The phylogenetic tree was constructed using R2R3-MYB protein sequences from sweet orange and Arabidopsis, and the 100 MYB proteins in sweet orange were classified into 29 subfamilies; the gene structure and conserved motif analyses further validated the classification. The expression patterns of each sweet orange MYB gene were provided according to the transcriptome data and the quantitative RT-PCR results. Combined with the phylogenetic analysis, the MYB gene functions were predicted, which could provide valuable information for further functional characterization of the citrus MYB genes.2. Functional characterization and regulatory mechanism analysis of CsMYBF1 gene. The CsMYBF1, which may be correlated with the regulation of flavonol biosynthesis, was selected for further functional analysis among the characterized putative phenylpropanoid-related MYB genes. The full-length sequence of CsMYBF1 was obtained by RACE PCR. Phylogenetic analysis indicated that CsMYBF1 exhibited close relationship with the characterized flavonol-related MYB genes. Gene expression analysis showed that CsMYBF1 was preferentially expressed in the peel, pulp in immature fruits and young leaves. The subcellular distribution analysis indicated that CsMYBF1 was localized in the nucleus. Southern blot analysis suggested that only one copy of CsMYBF1 was detected in many different citrus species. Transactivation assays indicated CsMYBF1 may interact with other potenial proteins to exert its transactivation function.The CsMYBF1-overexpressing tomato lines were successfully obtained. Secondary metabolites analysis indicated that the levels of hydroxycinnamic acids were substantially increased and the flavonol contents were not significantly altered in transgenic tomato fruits. The digital gene expression analysis characterized a series of differentially expressed genes including genes involved in the glycolysis, shikimate pathway and the phenylpropanoid pathway. Suppression of CsMYBF1 in citrus callus significantly reduced the contents of the hydroxycinnamic acids, flavonols and the flavones. The q RT-PCR results revealed that the down-regulation of CsMYBF1 accompanied with proportionally reduced expression of most phenylpropanoid pathway genes in transgenic callus lines. Similar results were also observed in the transgenic Hongkong kumquat leaves.The promoter activation assays indicated that CsMYBF1 could activate several promoters of genes in the phenypropanoid pathway of tomato and citrus. Specially, CsMYBF1 could activate the CHS gene promoter in citrus, but not in tomato. Further work indicated that CsMYBF1 could activate phenypropanoid pathway gene promoters through the functional MYBPLANT cis-elements. The yeast one-hybrid and EMSA experiment further proved that CsMYBF1 could directly bind to the MYBPLANT cis-elements. The results also indicated that the exogenous glycerol treatment could regulate the phenypropanoid metabolism through the induction of CsMYBF1 gene expression.The function of CsMYBF1 that regulate the hydroxycinnamic acids and flavonol metabolism was identified, and the regulatory network was initially established in this study. It was proposed that the unaltered flavonol contents in CsMYBF1-overexpressing tomato fruits may be due to the failure of CsMYBF1 to activate the tomato CHS promoters, and the sequence differences of the MYBPLANT cis-elements was one possible reason.