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. 2021 Oct 2;22(19):10697.
doi: 10.3390/ijms221910697.

Molecular and Metabolic Insights into Anthocyanin Biosynthesis for Leaf Color Change in Chokecherry (Padus virginiana)

Xiang Li  1 , Yan Li  1 , Minghui Zhao  1 , Yanbo Hu  1 , Fanjuan Meng  1 , Xingshun Song  1 , Mulualem Tigabu  2 , Vincent L Chiang  1   3 , Ronald Sederoff  3 , Wenjun Ma  4 , Xiyang Zhao  1   5
Affiliations

Molecular and Metabolic Insights into Anthocyanin Biosynthesis for Leaf Color Change in Chokecherry (Padus virginiana)

Xiang Li et al. Int J Mol Sci. .

Abstract

Chokecherry (Padus virginiana L.) is an important landscaping tree with high ornamental value because of its colorful purplish-red leaves (PRL). The quantifications of anthocyanins and the mechanisms of leaf color change in this species remain unknown. The potential biosynthetic and regulatory mechanisms and the accumulation patterns of anthocyanins in P. virginiana that determine three leaf colors were investigated by combined analysis of the transcriptome and the metabolome. The difference of chlorophyll, carotenoid and anthocyanin content correlated with the formation of P. virginiana leaf color. Using enrichment and correlation network analysis, we found that anthocyanin accumulation differed in different colored leaves and that the accumulation of malvidin 3-O-glucoside (violet) and pelargonidin 3-O-glucoside (orange-red) significantly correlated with the leaf color change from green to purple-red. The flavonoid biosynthesis genes (PAL, CHS and CHI) and their transcriptional regulators (MYB, HD-Zip and bHLH) exhibited specific increased expression during the purple-red periods. Two genes encoding enzymes in the anthocyanin biosynthetic pathway, UDP glucose-flavonoid 3-O-glucosyl-transferase (UFGT) and anthocyanidin 3-O-glucosyltransferase (BZ1), seem to be critical for suppressing the formation of the aforesaid anthocyanins. In PRL, the expression of the genes encoding for UGFT and BZ1 enzymes was substantially higher than in leaves of other colors and may be related with the purple-red color change. These results may facilitate genetic modification or selection for further improvement in ornamental qualities of P. virginiana.

Keywords: Padus virginiana; anthocyanin biosynthesis; leaf color; metabolomic; transcriptomics.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could appear to influence the work reported in this paper.

Figures

Figure 1
Figure 1
Phenotypes of P. virginiana green (GL), purple (RL) and purple-red (PRL) leaf sides on the adaxial (A) and abaxial (B).
Figure 2
Figure 2
The content of metabolites detected in this study among different samples. The x-axis represents the anthocyanin levels (μg/g). The y-axis represents the anthocyanin composition obtained by high-performance liquid chromatography (HPLC). Error bars show standard error (SE) of the mean. GL, green leaves; PL, purple leaves; PRL, purple-red leaves.
Figure 3
Figure 3
Heatmap of metabolites related to cyanidin, peonidin, delphinidin, pelargonidin, procyanidin, flavonoid, malvidin and petunidin in GL, PL and PRL. The marker on the right side of heatmap represents the names of each anthocyanin composition obtained by high–performance liquid chromatography (HPLC). Color scale from green to red in the heatmap represents the normalized metabolite contents (from low to high) using Row Z-score. GL, green leaves; PL, purple leaves; PRL, purple—red leaves.
Figure 4
Figure 4
Heatmaps of differentially accumulated metabolites (DAMs) in GL vs. PL (A), GL vs. PRL (B), PL vs. PRL (C) and GL vs. PL–PRL. (D,E) Venn diagram of DAMs in P. virginiana. GL, green leaves; PL, purple leaves; PRL, purples-red leaves. The color scale from Min (green) to Max (red) refer to the metabolite contents from low to high. Identification of differentially accumulated metabolites (DAMs) between four comparison groups was performed by variable importance in projection (VIP) values (VIP ≥ 1) and fold change ≥ 2 or ≤ 0.5. Venn diagram representing overlap between DAMs identified in P. virginiana at GL vs. PL, GL vs. PRL and PL vs. PRL.
Figure 5
Figure 5
Gene regulation during leaf color change. (A) K-means cluster analysis of co-expression genes and their expression patterns. (B) The DEGs involved in transcriptome factor enriched in cluster 4. (C) KEGG enrichment bar plot of DEGs in cluster 4. Cluster 4 represents the expression pattern of 1232 co-expression genes identified in the K-means cluster analysis. FPKM represents the fragments per kilobase per million.
Figure 6
Figure 6
The expression of genes in the phenylpropanoid and flavonoid biosynthetic pathways in P. virginiana leaf. (A) Reconstruction of the anthocyanins biosynthetic pathway with the differentially expressed structural genes and their regulators. The differentially expressed genes (DEGs) were identified by an adjusted p-value < 0.05 and |log2 fold change (FC)| ≥ 1. (B) The differentially accumulated metabolites (DAMs) in the anthocyanin biosynthetic pathway. The DAMs were identified by projection (VIP) values (VIP ≥ 1) and fold change ≥ 2 or ≤ 0.5. The color scale from Min (blue) to Max (red) refer to the metabolite contents from low to high. The cluster marker on the right side of heatmap represents the names of each gene. GL, green leaf; PL, purple leaf; PRL, purple-red leaf; PAL, phenylalanine ammonia-lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol 4-reductase; F3H, lavanone 3-hydroxylase; F3′H, lavonoid 3′-hydroxylase; ANS, anthocyanidin synthase; UFGT, UDP glucose-flavonoid 3-O-glcosyl-transferase; BZ1, anthocyanidin 3-O-glucosyltransferase; 3GGT, 3-O-glucoside 2″-O-glucosyltransferase; 3AT, 3-O-glucoside-6″-O-malonyltransferase; MYB, v-myb avian myeloblastosis viral oncogene homolog; bHLH, basic helix-loop-helix.
Figure 7
Figure 7
Biosynthetic pathway of chlorophyll in P. virginiana leaf color change. (A) Reconstruction of the chlorophyll biosynthetic pathway with the differentially expressed structural genes (B). Heatmap of the differentially expressed genes (DEGs) involved in chlorophyll biosynthetic pathway. Gene expression was scaled using Z-scores of fragments per kilobase of exon per million fragments mapped (FPKM) for mean valued of three biological replicates in heatmaps. The cluster marker on the right side of heatmap represents the names of each gene. GL, green leaf; PL, purple leaf; PRL, purple-red leaf; HemA, glutamyl-tRNA reductase; HemB, prophobilinogen synthase; HemE, uroporphyrinogen decarboxylase; HemY, copropophyrinogen III oxidase; HemH, ferrochelatase; chlH, magnesium chelatase subunit H; chlE, anaerobic magnesium-protoporphyrin IX monomethyl ester cyclase; chlG, chlorophyll a synthase; chlP, geranylgeranyl reductase; CLH, chporophyllase.
Figure 8
Figure 8
Correlation network of metabolites and genes (15 key genes) involved in phenylpropanoid and flavonoid biosynthesis in GL vs. PRL. Abs_rho represents the Pearson correlation coefficient (r). Degree represents the gene number. Relation represents the correlations with a coefficient (r) value > 0.8 (positive) or < −0.8 (negative). PAL, phenylalanine ammonia-lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol 4-reductase; F3H, lavanone 3-hydroxylase; F3′H, lavonoid 3′-hydroxylase; ANS, anthocyanidin synthase; UFGT, UDP glucose-flavonoid 3-O-glcosyl-transferase; MYB, v-myb avian myeloblastosis viral oncogene homolog; bHLH, basic helix-loop-helix; WRKY, “WRKY” domain genes; AP2/ERF, the APETALA2/Ethylene-responsive factor; Mal-3,5-O-diglu, malvidin 3,5-diglucoside; Mal-3-O-glu, malvidin 3-O-glucoside; Cya-3-O-gal, cyanidin 3-O-galactoside; Pet-3-O-(6-O-malonyl)-glu, petunidin 3-O-(6-O-malonyl-beta-D-glucoside); Pel-3-O-glu, pelargonidin 3-O-glucoside; Pet-3-O-gal, petunidin 3-O-galactoside; Cya-3-O-ara, cyanidin 3-O-arabinoside; del-3-O-(6-O-malonyl)-glu, delphinidin 3-O-(6′′-O-malonyl)-beta-D-glucoside; Peo-3-O-rut, peonidin 3-O-rutinoside; Cya-3,5-O-diglu, cyanidin 3,5-O-diglucoside; Cya-3-O-glu, cyanidin 3-O-glucoside; Cya-3-O-rut, cyanidin 3-O-rutinoside; Del-3-O-glu, delphinidin 3-O-glucoside; Del-3-O-rut, delphinidin 3-O-rutinoside; Peo-3-O-ara, peonidin 3-O-arabinoside; Peo-3-O-glu, peonidin 3-O-glucoside; Cya-3-O-(6-O-malonyl)-glu, cyanidin 3-O-(6-O-malonyl-beta-D-glucoside); Pel-3-O-rut, pelargonidin 3-O-rutinoside.
Figure 9
Figure 9
Quantitative real-time PCR verification of expression levels of 12 DEGs identified by RNA sequencing. The y-axis on the left represents the FPKM value obtained by RNA-seq. The y-axis on the right shows the relative gene expression levels (2−ΔΔCt) analyzed by qRT-PCR. The x-axis represents the different leaves samples. Bars with different lowercase letters are significantly different (p < 0.05). GL, green leaves; PL, purple leaves; PRL, purple-red leaves. FPKM represents the fragments per kilobase per million.
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