Photosynthetic Pigment and anthocyanin Content of H11 and H12 Leaves
The detection revealed that chlorophyll a, b, a + b and carotenoid contents of the H12 leaves were lower than those of H11 leaves. And the chlorophyll a, b, a + b and carotenoid contents of the H12 were 0.54-, 0.71-, 0.58-, 0.54- fold compared with H11. However, the total anthocyanin content in H12 leaves was 4.06 times higher than that of H11. The results suggested that anthocyanin was responsible for the redness of H12 leaves (Fig. 2).
Secondary Metabolite Assay and Differential Metabolite Assay Filter
In this experiment, 556 metabolites were detected, including 14 anthocyanins, 40 flavonoids, 38 flavonols, 19 flavonoid glycosides, 17 flavanones, 19 saccharides, 28 hydroxycinnamoyl derivatives, 17 quinic acid derivatives, 10 catechin derivatives, 5 proanthocyanidins, 55 amino acids, 54 organic acid derivatives, 46 amino acid derivatives, 46 nucleotide derivatives, 15 benzoic acid derivatives, 13 kinds of cinnamon derivatives and other substances.
Based on the criteria of VIP ≥ 1 and fold change ≤ 0.5 or fold change ≥ 2, 165 differential metabolites were screened. Among them, 96 and 69metabolites were up-regulated and down-regulated, respectively, in H12 compared with H11. The 96 differentially up-regulated metabolites mainly contained 4 anthocyanins, 2 proanthocyadins, 20 flavones, 19 flavonols, isoflavones. The contents of peonidin O-hexoside, cyanidin 3-O- malonylhexoside, cyanidin O-syringic acid, cyanidin 3,5-O-diglucoside and cyanidin in H12 were 137.38-, 2.30-, 57.39-, 45.70- and 10.50-fold higher than in H11, respectively. Type A and B proanthocyanidins are dimeric flavonoids, and the contents of proanthocyanidin A1 and A2 in H12 were 4.15 and 89.46 times higher than in H11. The 69 differentially down-regulated metabolites mainly included 6 amino acids, 9 amino acid derivatives, 15 organic acid derivatives lipid-fatty acids and so on. 18 selected differential metabolites are listed in Table 1. In addition, several important mass spectrograms are shown in Figure S1.
KEGG Enrichment Analyses of Differential Metabolites in H11 and H12
The KEGG pathway enrichment analysis revealed that the biosynthesis of flavone and flavonol, flavonoid, anthocyanin, as well as that of amino acids, changed significantly in H12 compared with H11. First, in the flavone and flavonol biosynthesis pathway (ko00941), 17 flavone substances were enriched in this pathway and 10 metabolites were highly expressed in H12. Notably, dihydroquercetin was up-regulated, with its content being7.99 times higher in H12 than in H11. Most importantly, in the anthocyanin biosynthesis pathways (ko00942), the contents of cyanidins and cyanidin 3,5-glucoside in H12 were 10.51 and 45.71 times higher than in H11, respectively. Thus, cyanidins and cyanidin 3,5-glucoside appear to be substances involved in the purple-red appearance of H12 leaves. Moreover, in biosynthesis of amino acids pathway (ko01230), the contents of 7 metabolites including tryptophan and glutamine decreased significantly. But in the glutathione metabolism pathway (ko00480), the contents of glutathione disulfide and glutathione were up-regulated in H12. The pathway maps mentioned above were shown in Figure S2.
For the transcriptome sequencing, the average H12 sample produced 594,236,10 clean reads, the base error distribution rate was 0.02%, and the GC content was 46.57%. The average H11 sample produced 60,047,905 clean reads, the base error distribution rate was 0.02%, and the GC content was 46.76%. And the clean data have been submitted to SRA database of NCBI (Accession ID: SRR12569328 and SRR12569327). The sequenced reads were compared with the reference genome of E. ulmoides. The average rates of total mapping for H12 and H11 were 95.53% and 95.36%, respectively. There were 17,503 genes expressed in H12, 17,765 genes in H11, and 16,687 genes were expressed in both leaf types. DEGs met the criteria of log2 (fold change) ≥ 1 and corrected P ≤ 0.005. In total, 8,368 DEGs in H12 vs H11 were identified.
The GO analysis annotated 12,893 genes that mapped to the biological process, cell component and molecular functional classes. The COG annotation placed 24,851 genes into 25 COG categories. The KEGG database annotated 11,104 genes, the Swiss-Prot database annotated 20,382 genes, the Pfam database annotated 20,000 genes, and the Nr database annotated 25,233 genes. The number of genes annotated in the Nr database was the highest, followed by the COG database. The DEGs were subjected to a GO enrichment analysis (Figure S3). The KEGG enrichment of DEGs was shown in Figure S4, which reflected the differential metabolic processes of H11 and H12 during the same period in leaf tissues.
Photosynthesis Biosynthetic Pathways
To figure out the relationship between anthocyanin and photoprotection, the expression of photosynthetic genes in E. ulmoides red leaves were analyzed. Photosynthesis is related to photosynthesis pathways (ko00195) and photosynthesis antenna proteins pathway (ko00196), which had 5 parts, including photosystem II, photosystem I, cytochrome b6/f complex, photosynthethic electron transport and F-type ATPase. They were shown in Figure S5. Thus, the 13 DEGs related to photosynthesis in H11 was significantly up-regulated than that in H12. And the 5 genes in H11 is down-regulated than that in H12 in Table 2. This result showed that most of photosynthesis DEGs in red leaves of H12 were down-regulated at that time.
Combined analysis of key metabolites and corresponding genes related to Flavonoid and Anthocyanidin Biosynthetic Pathways
In order to speculate the regulation of metabolites and genes in the red leaves of E. ulmoides, metabolome and transcriptome were used to analyze the anthocyanins and flavonoids biosynthetic pathways of H12. Firstly, CHI gene (EUC08195) was up-regulated 1.06-fold in H12 compared with H11. Next, the F3'5'H (EUC01284) was down-regulated − 3.34-fold while F3'H (EUC03526) was up-regulated 1.22-fold, which lift the content of dihydroquercetin 10.51-fold up. Then, DFR (EUC00272, MSTRG.13706, MSTRG.13719, MSTRG.15870, MSTRG.15871) were up-regulated 2.57-, 1.72-, 1.69-, 1.86-, 1.34-fold, respectively. The corresponding result was dihydroquercetin was more transformed to promote the synthesis of cyanidin, and cyanidin was up-regulated 7.99-fold. Finally, the structural gene 3MaT1 (EUC11625) was up-regulated 1.22-fold. Cyanidin 3-malonyl-glucoside and cyanidin 3, 5-glucoside were up-regulated 1.58- and 5.51-fold, respectively. The results of the combined analysis are shown in Fig. 3. Therefore, cyanidin, cyanidin 3-malonyl-glucoside and cyanidin 3, 5-glucoside were the key substances for the red color of H12 leaves. And EuCHI, EuF3'H, EuF3'5'H, EuDFR and Eu3MaT1 were the key regulatory genes. Notably, the content of procyandinA1 and procyandinA2 of H12 were up-regulated 4.15-fold and 89.46-fold, respectively. This is the first time to detect proanthocyanidins in H12 leaves, and the antioxidant activity of them provided a basis for the development and utilization of E. ulmoides.
Candidate Transcription Factors
Among all of the transcripts, a total of 1931 transcripts were annotated as transcription factors (TFs) which were divided into 44 TF families. Among these TF families, 123 MYB transcripts was the largest, followed by 118 MYB-related and 100 bHLHs. And differential expressed transcripts were screen from MYBs, MYB-related family, bHLHs and WD40 TFs. Then, the phylogenetic analysis and alignment were performed to find the candidate MYBs and bHLHs involved in anthocyanin biosynthesis. Moreover, the matched sequences were picked from published paper and NCBI.
The phylogenetic tree and alignment analysis results in Fig. 4 indicated EUC13670 (up-regulated, 2.05-fold) was probably involved in the synthesis of anthocyanin and proanthocyanin[21, 22]. Like other anthocyanin MYBs, EUC13670 contained the highly-conserved R2R3 domain and bHLH binding motifs at the N-terminus. The motif [D/E]Lx2[R/K] x3Lx6Lx3R is important for interaction with bHLH TFs. In addition, the conservative [K/R]Pxxx[K/T][F/Y] sequence in the C-terminal was part of the signature for MYBs that positively regulated anthocyanin biosynthesis. Intriguingly, EUC13670 was also likely to regulate procyanidins synthesis in E. ulmoides.
The deduced amino acid sequence of EUC06643, EUC01933, EUC18920 and EUC04483 were align with other bHLHs regulating anthocyanin biosynthesis. However, EUC04483 (down-regulated, -1.47-fold) was proved to be bHLH-MYC TF with helix-loop-helix DNA-binding domain and R2R3-MYB transcription factors in N-terminal. As shown in Fig. 5, the box 11, 18 and 13 domain at the N-terminus were key to the interaction of bHLH and MYB.
Previous studies showed that GST transported anthocyanins from endoplasmic reticulum to vacuolar membrane as a carrier. AcGST1 promoter was directly bound with AcMYB110 to regulate anthocyanins in kiwifruit (Actinidia chinensis). The transcriptome analysis indicated GST (EUC05510, EUC04745, EUC12154, EUC0882, EUC12155) were up-regulated 2.47-, 1.29-, 1.11-, 1.17-, 2.02-fold, respectively. The expression of GST genes was significantly increased in the red leaves of E. ulmoides.
To verify the RNA-Seq results, we selected 10 genes (2 flavonoids, 4 anthocyanin and 3 photosynthetic metabolism pathway genes). The RT-qPCR results showed the up-regulated and down-regulated gene expression levels in the leaves of H11 and H12 were consistent with RNA-Seq (Fig. 6).