Analysis of pepper anthocyanidin metabolome
Anthocyanidins are the most important flavonoid pigments in plants. In order to compare the composition of anthocyanidin metabolites between three pepper lines, L66, L29, and L9 (CK), the anthocyanidin-like substances in pepper fruits were examined. Principal component analysis (PCA) showed that the samples between the groups scattered and the samples within the groups clustered, indicating that the anthocyanidin metabolome data was reliable. On the principal component PC1 and PC2, L9 (CK) was clearly separated from L29 and L66 and the difference was significant (Fig. 1). A total of 5 anthocyanidin-like compounds were detected in the fruits of three pepper lines. The contents of delphinidin 3-O-glucoside, delphinidin 3-O-rutinoside, and delphin chloride in lines L66 and L29 were significantly higher than that in line L9 (CK), showing that they were significantly up-regulated. Delphin chloride was found in the fruits of purple pepper lines L66 and L29, but not in L9 (CK), suggesting that it is a unique metabolite in purple pepper fruits. The cyanidin 3-O-galactoside content in the fruits of the green pepper line was higher than that in the purple pepper line L29 fruits, indicating that the green pepper fruit also accumulated anthocyanidins to a certain extent (Table 1). The KEGG database was used to annotate the differential metabolites. No annotation result was obtained for cyanidin 3-O-galactoside. The rest of the detected metabolites were found to share the metabolic pathway ko00942. It can be seen from the KEGG pathway map that one of the 4 annotated metabolites remained at an unchanged level while the rest were delphinidin-derived products and up-regulated.
Table1
Type and content of anthocyanins in two purple and one green pepper cultivars.
Compound
|
Molecular weight
|
L29
|
L66
|
L9 (CK)
|
Kegg map
|
Cyanidin 3-O-glucoside
|
449.38
|
N/A
|
1.10E+05
|
N/A
|
Ko00942
|
Delphinidin 3-O-glucoside
|
500.84
|
4.55E+06
|
3.33E+06
|
3.45E+04
|
Ko00942
|
Delphinidin 3-O-rutinoside
|
611.53
|
6.24E+06
|
8.26E+06
|
5.00E+04
|
Ko00942
|
Cyanidin 3-O-galactoside
|
484.838
|
1.04E+04
|
2.65E+06
|
7.22E+04
|
—
|
Delphin chloride
|
627.52
|
4.83E+04
|
9.77E+04
|
N/A
|
Ko00942
|
Statistics of purple and green pepper transcriptome sequencing data
We further investigated the differences in gene expression among the three samples. With three biological replicates, the transcriptome sequencing of the 9 samples yielded a total of63.26 Gb clean data with 94.14% of bases scoring Q30 (Table S1). The transcriptome sequencing reads were aligned with the reference genome with efficiencies ranged from 86.14% to 95.04% (Table S2), which showed a normal rate of data utilization, suggesting that the selected reference genome was suitable for subsequent analysis.
DEGs between purple and green peppers and KEGG enrichment analysis
In order to clarify DEGs and their biological pathways between purple and green peppers, we analyzed the DEGs between the fruits of 2 purple pepper lines (L66 and L29) and 1 green pepper line (L9) and performed KEGG enrichment. The number of DEGs were 6567 (L66 vs. L9) and 5091 (L29 vs. L9). A total of 2224 DEGs were common in both of the purple pepper lines(Fig S1). The 2224 DEGs were annotated and 111 KEGG pathways were found. Eight of the top 20 KEGG pathways were shared by the two purple pepper lines, of which 3 pathways (ko00360, ko00400, and ko00941) were anthocyanidin-related (Fig.2).
Regulation of anthocyanidin biosynthetic pathway genes in purple and green pepper
Anthocyanins are one of the natural products synthesized through the metabolic pathways of phenylpropanes and flavonoids. A number of studies have revealed that the biosynthesis of anthocyanins is completed under the co-catalysis of different enzymes. Through metabolome and transcriptome analysis, we obtained 4 pathway diagrams. We mapped these biosynthesis pathway diagrams of pepper and found that three enzymes, namely DFR, ANS, and UFGT, in the anthocyanidin biosynthetic pathway were up-regulated in the fruits of purple pepper lines. DFR can selectively catalyze the formation of colorless leucopelargonidin and leucocyanidin from dihydrokaempferol and dihydroquercetin, respectively. ANS catalyzes the formation of colored pelargonidin, delphinidin, and cyanidin from leucopelargonidin and leucocyanidin. After that, mediated by UFGT, the metabolites delphinidin 3-O-glucoside, delphinidin 3-O-rutinoside, and delphin chloride were all up-regulated (Fig3). In this process, as the three enzymes DFR, ANS, and UFGT in fruits of purple pepper lines were up-regulated, those metabolites were also increased, resulting in an increase in the content of colored anthocyanidins. As a result, the degree of the purple color in the two purple pepper lines varied.
qRT-PCR validation
The transcriptome sequencing results showed that three enzymes, DFR, ANS, and UFGT, were up-regulated during anthocyanin formation in fruits of purple pepper lines. The expression of DFR coding genes LOC107850726 and LOC107860031, ANS coding gene LOC107866341, and UFGT coding genes LOC107843659, LOC107861697, and LOC107860695 were significantly up-regulated. In order to verify the results obtained from transcriptome sequencing, those 6 genes that were significantly up-regulated in the fruits of purple peppers (Line L29 and L66) compared with the fruits of green pepper (Line L9) were further analyzed using qRT-PCR. The results showed that the expression levels of the 6 genes were significantly higher in fruits of purple pepper lines than that in fruits of the green pepper line, and their expression levels in line L66 fruits were higher than in line L29 fruits, which may be due to the difference in anthocyanin content in the fruits of different pepper lines. Line L66 fruits had a higher colored anthocyanin content and deeper purple color than line L29 fruits. The qRT-PCR results of the expression of 6 genes were highly consistent with the results obtained based on transcriptome sequencing (Fig. 4), indicating that DEG analysis was highly reliable.
Transcription factors regulating anthocyanidin biosynthesis in pepper fruits
Previous studies have shown that in most plant species a protein complex formed by three types of transcription factors from the MYB family, BHLH family, and WD40 family can bind to the promoter of a structural gene or genes involved in the anthocyanidin biosynthesis pathway to activate or inhibit expression22. In our study, analysis of the key genes for anthocyanidin biosynthesis in pepper fruits showed that the transcription factors MYB, BHLH, and WD40 were up-regulated in purple fruits (lines L29 and L66) compared with green fruits (line L9) (Fig. 5A), which was consistent with the increase of the expression level of the structural genes involved in anthocyanidin biosynthesis, indicating that MYB, BHLH, and WD40 positively regulated anthocyanidin biosynthetic genes. We proposed a model of anthocyanidin biosynthesis in pepper fruits: MYB, BHLH, and WD40 formed a ternary protein complex and bound to the specific cis-acting elements in the promoter of the structural genes related to anthocyanidin biosynthesis to regulate their expression, which altered the metabolites and resulted in color change in pepper fruits (Fig. 5B).