Identification of candidate genes involved in anthocyanin biosynthesis to improve the pigment content in carrot taproots
Research output: Book/Report › Ph.D. thesis › Research
Carrot (Daucus carota), an important root vegetable, is rich in many nutrients. Black carrots, inparticular, have attracted increasing attention, due to their high anthocyanin contents, which can beused as natural food colorants as well as antioxidant components that provide health benefits tohumans. There are many factors which may influence anthocyanin accumulation in carrots besidesthe structural genes encoding enzymes involved in the anthocyanin synthesis pathway such as thetranscription factors (TFs), anthocyanin transporters and peroxidase (Prx). In order to unravel themechanism of coloration of carrots, three different colored cultivars of taproots were used as materialin this study. Candidate R2R3MYB genes related to anthocyanin biosynthesis were identified bysearching the carrot genome database with Pfam domain, and their gene expression levels in fourgrowth stages of three carrot cultivars were determined. Subsequently, RNA-sequence (RNA-seq)analysis was used to evaluate differentially expressed genes between different tissues of three carrotcultivars, to identify which genes are upregulated in purple tissues, and downregulated or notexpressed in orange tissue and their relative expression level with particular focus on the TFs (MYBand bHLH), glutathione S-transferase (GST) and Prx genes. The anthocyanin profile in differentpurple tissues of two purple cultivars were determined and specific anthocyanin in the profileidentified using high-performance liquid chromatography (HPLC) and (liquid chromatography-massspectrometry) LC-MS analysis. Moreover, to facilitate the use of CRISPR/Cas9 technology inverification of candidate genes, the methods for plant regeneration and Agrobacterium-mediatedtransformation were optimized for carrots. Hypocotyls were used as the explants and Agrobacteriumdelivered CRISPR/Cas9 system was used to knock out candidate MYB genes that might be related to anthocyanin biosynthesis in order to verify their function. Moreover, the large class III peroxidase gene family was analysed in order to determine the phylogeny and duplication of the Prx genes in carrot. Corroborating the total anthocyanin and lignin contents in xylem and phloem of two purple carrot cultivars with peroxidase expression, Prx genes that might be involved in either anthocyanin or lignin biosynthesis were identified.In total, 224 R2R3MYB genes were identified in the carrot genome and by phylogenetic analysis of these genes including well-known R2R3MYB genes from other species, they could all be dividedinto four subgroups based on the R2R3MYB gene classification information. By using RT-qPCR andRNA-seq analysis, two MYB genes (LOC108192278 and LOC108213488) were identified to beinvolved in genotype-dependent regulation of anthocyanin biosynthesis and they were specifically upregulated in solid black colored taproots including both black phloem and xylem. One MYB gene (LOC108208100) belongs to subgroup 4 of R2R3MYB genes and its expression is positively correlated with anthocyanin accumulation. DcbHLH3 (LOC108204485) and another bHLH (LOC108225265) were upregulated in all black tissue samples compared to the orange tissue. Asingle GST (LOC108205254) was highly expressed in all the purple-pigmented tissues comparedwith orange tissue. The up-regulated GST (LOC108205254) might be responsible for increasing the vacuolar anthocyanin levels in purple tissue through anthocyanin transportation. In order to facilitate validation of candidate genes methods for site-directed mutagenesis in carrots were implemented. Here, 1 mg/L NAA supplied on MS medium was found to be appropriate for carrot calli induction, and 2 mg/L BA added on MS medium was suitable for shoot regeneration. The highest GUS transient expression was obtained when hypocotyls were immersed in the Agrobacterium with OD600 = 0.6for 30 min. CRISPR/Cas9 were used with aim to identify the functions of two MYB TFs, in whichgenes no mutants have been identified so far. In total 75 Prx genes (DcPrxs) were identified in thecarrot genome sequence and classified into seven subgroups based on the phylogenetic analysis. Theexpression levels of DcPrx34, DcPrx35, DcPrx51, DcPrx64, and DcPrx67 were strongly and negatively correlated to the lignin content, while DcPrx49 were positively correlated with lignin accumulation. DcPrx5, DcPrx23 and DcPrx32 were hypothesized to be positively involved inanthocyanin metabolism based on these studies. For the analysis of the compounds, the feruloylderivative of cyanidin xylosylglucosylgalactoside was the predominant pigment in all the samples, cyanidin 3-xylosylgalactoside was the second-highest anthocyanin in the phloem of Purple 68 cultivar, while cyanidin 3-xylosyl (sinapoylglucosyl) galactoside was the second one in the xylem of Purple 68 and phloem of Purple Haze cultivar. The Ph.D. thesis aims at providing a better understanding o f the mechanism of anthocyanin biosynthesis in genotypic and tissue-specific manner. A similar approach of identifying the candidate gene related to anthocyanin biosynthesis in other plant species could be used to identify related species-specific genes involved in pigmentation. In particular modifications of individual anthocyanin chemistry could increase the stability of these during extraction, processing and use as food colorant. Eventually, combining all the identified genes may help to improve the colorant food production and thereby, meet the increasing demand for future food cultivars.
|Publisher||Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen|
|Publication status||Published - 2019|