Coordinated Regulation of Anthocyanin Biosynthesis Genes Confers Varied Phenotypic and Spatial-Temporal Anthocyanin Accumulation in Radish (Raphanus sativus L.)

Abstract

Anthocyanins are natural pigments that have important functions in plant growth and development. Radish taproots are rich in anthocyanins which confer different taproot colors and are potentially beneficial to human health. The crop differentially accumulates anthocyanin during various stages of growth, yet molecular mechanisms underlying this differential anthocyanin accumulation remains unknown. In the present study, transcriptome analysis was used to concisely identify putative genes involved in anthocyanin biosynthesis in radish. Spatial-temporal transcript expressions were then profiled in four color variant radish cultivars. From the total transcript sequences obtained through illumina sequencing, 102 assembled unigenes, and 20 candidate genes were identified to be involved in anthocyanin biosynthesis. Fifteen genomic sequences were isolated and sequenced from radish taproot. The length of these sequences was between 900 and 1,579 bp, and the unigene coverage to all of the corresponding cloned sequences was more than 93%. Gene structure analysis revealed that RsF3'H is intronless and anthocyanin biosynthesis genes (ABGs) bear asymmetrical exons, except RsSAM. Anthocyanin accumulation showed a gradual increase in the leaf of the red radish and the taproot of colored cultivars during development, with a rapid increase at 30 days after sowing (DAS), and the highest content at maturity. Spatial-temporal transcriptional analysis of 14 genes revealed detectable expressions of 12 ABGs in various tissues at different growth levels. The investigation of anthocyanin accumulation and gene expression in four color variant radish cultivars, at different stages of development, indicated that total anthocyanin correlated with transcript levels of ABGs, particularly RsUFGT, RsF3H, RsANS, RsCHS3 and RsF3'H1. Our results suggest that these candidate genes play key roles in phenotypic and spatial-temporal anthocyanin accumulation in radish through coordinated regulation and the major control point in anthocyanin biosynthesis in radish is RsUFGT. The present findings lend invaluable insights into anthocyanin biosynthesis and may facilitate genetic manipulation for enhanced anthocyanin content in radish.

Keywords: Raphanus sativus; anthocyanin; color variation; coordinated regulation; gene expression; spatial-temporal; transcriptome.

Figure 1

Four different colored radish genotypes at different developmental stages. (A) “NAU-XBC”, (B) “NAU-XLM”, (C) “NAU-YZH”, (D) “NAU-YH”.

Figure 2

Changes in the concentration of anthocyanin (mg/g Fw) in the leaf and root of four radish cultivars during different stages of growth. XB, YH, YZ and XL represents “NAU-XBC”, “NAU-YH”, “NAU-YZH”, and “NAU-XLM”, respectively. Values are means of three independent replicates. Values not connected by the same letter within the same data point are significantly different according to Tukeys HSD test (P ≥ 0.5).

Figure 3

Spatial temporal transcriptional analysis of genes associated with anthocyanin biosynthesis in four cultivars of R. sativus using qRT-PCR. Four tissues were used: the leaf (L), young root (R), flesh (F) and skin (S). Relative gene expression levels were normalized against actin transcript levels. Values connected by the same letter within the same gene and data collection point are not significantly different at P ≤ 0.05. The transcriptional levels of the same gene in the leaf and root tissues at 10, 30, and 50 DAS are expressed on the y axes while radish genotypes are expressed on the x axis.

Figure 4

Spatial temporal transcriptional analysis of genes associated with anthocyanin biosynthesis in four cultivars of R. sativus using qRT-PCR. Four tissues were used: the leaf (L), young root (R), flesh (F) and skin (S). Relative gene expression levels were normalized against actin transcript levels. Values connected by the same letter within the same gene and data collection point are not significantly different at P ≤ 0.05. The transcriptional levels of the same gene in the leaf and root tissues at 10, 30, and 50 DAS are expressed on the y axes while radish genotypes are expressed on the x axis.

Figure 5

Heat map of the clustered correlations between anthocyanin accumulation patterns and gene expressions in red radish. Three sampling stages, three biological replicates, skin and flesh tissues were treated as independent factors in anthocyanin-transcript pairwise comparisons, which were carried out for 21 transcriptional data points for each gene.