Transcriptome and metabolome analysis reveals the effect of flavonoids on flower color variation in Dendrobium nobile Lindl

Abstract

Introduction: Dendrobium nobile L. is a rare orchid plant with high medicinal and ornamentalvalue, and extremely few genetic species resources are remaining in nature. In the normal purple flower population, a type of population material with a white flower variation phenotype has been discovered, and through pigment component determination, flavonoids were preliminarily found to be the main reason for the variation.

Methods: This study mainly explored the different genes and metabolites at different flowering stages and analysed the flower color variation mechanism through transcriptome- and flavonoid-targeted metabolomics. The experimental materials consisted of two different flower color phenotypes, purple flower (PF) and white flower (WF), observed during three different periods.

Results and discussion: The results identified 1382, 2421 and 989 differentially expressed genes (DEGs) in the white flower variety compared with the purple flower variety at S1 (bud stage), S2 (chromogenic stage) and S3 (flowering stage), respectively. Among these, 27 genes enriched in the ko00941, ko00942, ko00943 and ko00944 pathways were screened as potential functional genes affecting flavonoid synthesis and flower color. Further analysis revealed that 15 genes are potential functional genes that lead to flavonoid changes and flower color variations. The metabolomics results at S3 found 129 differentially accumulated metabolites (DAMs), which included 8 anthocyanin metabolites, all of which (with the exception of delphinidin-3-o-(2'''-o-malonyl) sophoroside-5-o-glucoside) were found at lower amounts in the WF variety compared with the PF variety, indicating that a decrease in the anthocyanin content was the main reason for the inability to form purple flowers. Therefore, the changes in 19 flavone and 62 flavonol metabolites were considered the main reasons for the formation of white flowers. In this study, valuable materials responsible for flower color variation in D. nobile were identified and further analyzed the main pathways and potential genes affecting changes in flavonoids and the flower color. This study provides a material basis and theoretical support for the hybridization and molecular-assisted breeding of D. nobile.

Keywords: Dendrobium nobile; anthocyanin; flavonoids; flower variation; transcriptomics.

Figure 1

Floral morphology, dendrobine content and genomic identification of the PF and WF varieties. (A) Color differences at different flower development stages. (B) Determination of the dendrobine content by gas chromatography. (C) ITS2 sequence alignment. HM590382, HQ114218, JN388579 and KC205193 were the local IDs of D. nobile collected in the NCBI database. Three biological replicates were used for dendrobine determination. ^** p<0.01.

Figure 2

Color indices and contents of anthocyanins and flavonoids in the PF and WF varieties. (A) Color indices of different floral tissues. (B) Determination of the anthocyanin and flavonoid contents. The error bars represent the SEs of three biological replicates.

Figure 3

Heatmaps of flavonoid metabolites in two different colored flowers of D. nobile. PF, purple flower; WF, white flower. Each individual graph represents different subclasses of flavonoids.

Figure 4

Analysis of differentially expressed genes (DEGs) derived from comparative transcriptomics among the different colors and periods of experimental D. nobile materials. (A) Numbers of total, downregulated, and upregulated DEG. (B) Venn diagram displaying the up/downregulated DEGs. Three comparison groups were established to reveal the flavonoid accumulation differences: PF-S1 vs. WF-S1, PF-S2 vs. WF-S2, and PF-S3 vs. WF-S3. (C) Volcano map diagram of DEGs in the different colored flowers of D. nobile. Red dots indicate upregulated DEGs, green dots indicate downregulated DEGs, and blue dots indicate genes that did not show differential expression. (D) Number of DEGs involved in the flavonoid biosynthesis (ko00941), anthocyanin biosynthesis (ko00942), isoflavonoid biosynthesis (ko00943) and flavone and flavonol biosynthesis (ko00944) pathways.

Figure 5

Metabolic regulatory network and gene expression analysis of the flavonoid synthesis pathway in D. nobile. (A) Central metabolic network dominating the gene expression and metabolite flux differences in the overall flavonoid biosynthesis pathway in the two D. nobile materials. Key regulatory genes catalyzing each enzymatic step are marked in solid font, and their expression differences in the three pairwise comparisons are reflected by the comparison of the calibrated FPKM values of each unigene. For a unigene, a positive value indicates upregulated expression, whereas a negative value indicates downregulated expression. The color change in the gene expression heatmap from green to red indicates low to high expression. The changes in crucial metabolic nodes are represented by different colors: red indicates upregulation, and green indicates downregulation. Important derivatives of the isoflavonoid biosynthesis, flavone and flavonol biosynthesis, and anthocyanin biosynthetic pathways are collectively integrated into the three dashed boxes marked. (B) Relative expression levels of 15 genes involved in the flavonoid biosynthesis pathway at three stages analyzed by qRT-PCR.