Accumulation and regulation of anthocyanins in white and purple Tibetan Hulless Barley (Hordeum vulgare L. var. nudum Hook. f.) revealed by combined de novo transcriptomics and metabolomics

Abstract

Background: Colored barley, which may have associated human health benefits, is more desirable than the standard white variety, but the metabolites and molecular mechanisms underlying seedcoat coloration remain unclear.

Results: Here, the development of Tibetan hulless barley was monitored, and 18 biological samples at 3 seedcoat color developmental stages were analyzed by transcriptomic and metabolic assays in Nierumuzha (purple) and Kunlun10 (white). A total of 41 anthocyanin compounds and 4186 DEGs were identified. Then we constructed the proanthocyanin-anthocyanin biosynthesis pathway of Tibetan hulless barley, including 19 genes encoding structural enzymes in 12 classes (PAL, C4H, 4CL, CHS, CHI, F3H, F3'H, DFR, ANS, ANR, GT, and ACT). 11 DEGs other than ANR were significantly upregulated in Nierumuzha as compared to Kunlun10, leading to high levels of 15 anthocyanin compounds in this variety (more than 25 times greater than the contents in Kunlun10). ANR was significantly upregulated in Kunlun10 as compared to Nierumuzha, resulting in higher contents of three anthocyanins compounds (more than 5 times greater than the contents in Nierumuzha). In addition, 22 TFs, including MYBs, bHLHs, NACs, bZips, and WD40s, were significantly positively or negatively correlated with the expression patterns of the structural genes. Moreover, comparisons of homologous gene sequences between the two varieties identified 61 putative SNPs in 13 of 19 structural genes. A nonsense mutation was identified in the coding sequence of the ANS gene in Kunlun10. This mutation might encode a nonfunctional protein, further reducing anthocyanin accumulation in Kunlun10. Then we identified 3 modules were highly specific to the Nierumuzha (purple) using WGCNA. Moreover, 12 DEGs appeared both in the putative proanthocyanin-anthocyanin biosynthesis pathway and the protein co-expression network were obtained and verified.

Conclusion: Our study constructed the proanthocyanin-anthocyanin biosynthesis pathway of Tibetan hulless barley. A series of compounds, structural genes and TFs responsible for the differences between purple and white hulless barley were obtained in this pathway. Our study improves the understanding of the molecular mechanisms of anthocyanin accumulation and biosynthesis in barley seeds. It provides new targets for the genetic improvement of anthocyanin content and a framework for improving the nutritional quality of barley.

Keywords: ANS; Proanthocyanin-anthocyanin biosynthesis; Seedcoat color; Tibetan Hulless Barley; Transcriptomic and metabolomic.

Fig. 1

Changes in anthocyanin content of white (Kunlun 10) and purple (Nierumuzha) Tibetan hulless barley grains. Grains were analyzed at the early milk (PC1/WC1), late milk (PC2/WC2), and soft dough stages (PC3/WC3). A Grain phenotype. B Mean total anthocyanin content. Data are shown as the mean ± standard error (SE) of three biological replicates

Fig. 2

Differentially abundant anthocyanin compounds between Kunlun10 and purple Nierumuzha at three stages. A Numbers of differentially abundant anthocyanin compounds between varieties at each stage. B Heatmap showing the patterns of differential abundance across the 41 anthocyanin compounds at each stage. Cell colors correspond to the log10 magnitude of the difference in abundance (fold change values + 1): redder cells indicate higher abundance, and bluer cells indicate lower abundance. C Numbers of differentially accumulated anthocyanins shared and unique between the two varieties at the three developmental stages. D Content of four anthocyanin compounds absent from all stages of Kunlun10 and the early milk stage of Nierumuzha

Fig. 3

Identification of DEGs between Kunlun10 and Nierumuzha at three stages. A–C Venn diagrams showing DEGs shared and unique between the two barley varieties at each developmental stage: downregulated, upregulated, and all. D Veen analysis of DEGs among PC1vsWC1, PC2vsWC2 and PC2vsWC2

Fig. 4

Genes differentially expressed between the two barley varieties encoding transcription factors (TFs). A Differentially expressed TFs. B Distribution of the identified differentially expressed TFs (Top 10). C Heatmap showing the 40 differentially expressed TFs between the two varieties at each stage. Cell colors correspond to the log10 magnitude of the difference in expression level [log10 (fold change values + 1)]: redder cells indicate upregulation, while bluer cells indicate downregulation. D Numbers of differentially expressed TFs between varieties at each stage

Fig. 5

Heatmaps showing correlations between the DEGs and the anthocyanin compounds or TFs. A The DEGs associated with anthocyanin synthesis and the differentially abundant anthocyanin compounds. B The DEGs associated with anthocyanin synthesis and TFs. Redder cells correspond to stronger positive correlations, while bluer cells correspond to stronger negative correlations. Significant correlations in each cell are indicated using asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 6

Proposed model of the molecular mechanisms and the proanthocyanin-anthocyanin biosynthesis pathways in Tibetan hulless barley. The structural enzymes are identified using large red capital letters; the encoding gene IDs are shown in small capital letters and are also in red if these genes are significantly differentially expressed between the two varieties. Red boxes and arrows correspond to TFs that are significantly positively correlated with, and thus may positively regulate, genes encoding structural enzymes. Green boxes and arrows correspond to TFs that are significantly negatively correlated with, and thus may negatively regulate, genes encoding structural enzymes

Fig. 7

Point-nonsynonymous mutation at 1,195 of the coding sequences of ANS (HORVU5Hr1G094280) between Kunlun10 and Nierumuzha

Fig. 8

Gene co-expression networks and hub genes in the different modules. A Brown. B Yellow. C Turquoise. D Black. E Red. F Green. Red circles correspond to hub genes. Purple diamonds correspond to both genes associated with anthocyanin synthesis and genes from PPI network. Orange circles correspond to genes from PPI network

Fig. 9

Expression patterns of the 11 structural genes and one transcription factor in Kunlun10 and Nierumuzha. The TC139057 were used as internal gene. A The relative expression of PAL (HORVU1Hr1G022060). B The relative expression of PAL (HORVU2Hr1G089540). C The relative expression of C4H (HORVU3Hr1G080830). D The relative expression of 4CL (HORVU4Hr1G072130). E The relative expression of CHI (HORVU5Hr1G046480). F The relative expression of CHS (HORVU2Hr1G116390). G The relative expression of and CHS (HORVU2Hr1G004170). H The relative expression of F3H (HORVU2Hr1G110130). I The relative expression of F3’H (HORVU1Hr1G094880). J The relative expression of DFR (HORVU3Hr1G056560). K The relative expression of ANR (HORVU2Hr1G108250). L The relative expression of WD40-like TF LEC14B (HORVU0Hr1G000250). The data displayed in the histograms are expressed as the means ± SD