Anthocyanins accumulation analysis of correlated genes by metabolome and transcriptome in green and purple peppers (Capsicum annuum)

In order to clarify the the molecular mechanism of anthocyanin accumulation in green and purple fruits of pepper using metabolomics and transcriptomics,to identify different anthocyanin metabolites,and to analyze the differentially expressed genes involved in anthocyanin biosynthesis.. We analyzed the anthocyanin metabolome and transcriptome data of the fruits of 2 purple pepper and 1 green pepper. A total of 5 anthocyanin metabolites and 2224 differentially expressed genes were identified between the green and purple fruits of pepper. Among the 5 anthocyanin metabolites,delphin chloride was unique to purple pepper fruits,which is the mainly responsible for the purple fruit color of pepper. A total of 59 unigenes encoding 7 enzymes were identified as candidate genes involved in anthocyanin biosynthesis in pepper fruit. The six enzymes (PAL,C4H,CHI,DFR,ANS,UFGT) had higher expression levels except the F3H gene in purple compared with green fruits. In addition,seven transcription factors were also found in this study. These transcription factors may contribute to anthocyanin metabolite biosynthesis in the fruits of pepper. One of differentially expressed gene novel.2098 was founded. It was not annotated in NCBI. Though blast analysis we preliminarily considered that this gene related to MYB transcription factor and was involved in anthocyanin biosynthesis in pepper fruit. Overall, the results of this study provide useful information for understanding anthocyanin accumulation and the molecular mechanism of anthocyanin biosynthesis in peppers.

in seedlings,reduce plant tissue damage caused by high levels of Ultraviolet (UV) light,and improve stress resistance in plants [6].
Pepper (Capsicum annuum L.) is a plant that belongs to the genus Capsicum of the Solanaceae. There are many types of pepper germplasm resources,and purple pepper is one of the rarer types. Purple pepper is rich in anthocyanins and has good physiological tolerance to high temperature and drought stresses. Generally,plant fruit skin has the highest anthocyanin content. However,the fruit skin of most plants such as eggplant and purple sweet potato is inedible,whereas the edible part of purple pepper is the brightly colored pericarp,which is valuable for human health and directly determines its economic value and popularity in the market.
In higher plants,the biosynthesis of most anthocyanins is often regulated in different spatial temporal patterns by combinations of multiple regulatory factors such as R2R3-MYB,MADS-box,bHLH and WD40 [11,12]. To date,several R2R3-MYB genes associated with either positive or negative regulation of the key genes (DFR,ANS,and UFGT) in anthocyanin biosynthesis have been identified. However,their contributions to the color of fruits and other organs vary [13][14][15]. bHLH transcription factor is also a key regulator of anthocyanin biosynthesis,usually independently regulating CHS,DFR,and UFGT [16]. WD40 proteins do not bind to the promoters of the anthocyanin biosynthetic genes. Instead,they interact with bHLH and MYB to regulate anthocyanin biosynthesis [17]. The R2R3MYB,bHLH,and WD40 proteins usually form a transcriptional activation complex (MYBbHLH-WD40,MBW) to regulate the transcription of late anthocyanin biosynthetic genes in most plants [18][19][20].
In recent years, Metabolomics and transcriptomics have been widely used to study the biosynthesis of metabolites and their molecular mechanisms [21,22]. The research on the regulation mechanism of anthocyanin synthesis has been reported in capsicum annuum [23][24][25][26], but different materials of purple pepper contained different anthocyanin metabolites, so the molecular mechanism of anthocyanin biosynthesis was slightly different. Therefore, In this study,2 purple and 1 green pepper (check, CK) were analyzed by means of targeted metabolome and transcriptome technologies to discover the differential genes of enzyme and transcription factors associated with anthocyanin synthesis in peppers, To explore the molecular mechanism of purple pepper fruit formation, provide theoretical and technical support for expanding purple pepper gene resources and promoting purple pepper molecular breeding.

Analysis of pepper anthocyanin metabolome
Anthocyanidins are the most important flavonoid pigments in plants. In order to compare the composition of anthocyanin metabolites between three pepper, L66,L29,and L9(CK),the anthocyanin-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 anthocyanin 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. S1). A total of 5 anthocyanin like compounds were detected in the fruits of three pepper. The contents of delphinidin 3-O-glucoside,delphinidin 3-O-rutinoside,and delphin chloride in L66 and L29 were significantly higher than L9(CK). Delphin chloride was found in the fruits of purple pepper L66 and L29,but not in L9(CK). This suggesting that it is a unique metabolite in purple pepper fruits. Delphin chloride could be mainly responsible for the purple fruit color of pepper. The cyanidin 3-O-galactoside content in the fruits of the green pepper line was higher than that in the purple pepper L29 fruits,indicating that the green pepper fruit also accumulated anthocyanins to a certain extent (Fig. 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 three of the 4 annotated metabolites were significantly upregulated in purple pepper(L29,L66) and they were delphinidin-derived products.

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 of 63.26Gb 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 TrANScriptome sequencing between the fruits of 2 purple pepper (L66 and L29) and 1 green pepper (L9). 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 (Table S3). 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,of which 3 pathways (ko00360,ko00400,and ko00941) were anthocyanin-related (Fig. 2).

Regulation of anthocyanin biosynthetic pathway genes in purple and green pepper
Anthocyanins are one of the natural products synthesized through the metabolic pathways of phenylpropanes and flavonoids. We analyzed the anthocyanin biosynthesis pathway in order to performed the key genes in the metabolism of purple and green fruits pepper. In total,59 unigenes that encoded 9 enzymes in the anthocyanidin biosynthesis pathways were studied. Fifteen unigenes of the seven enzymes had different expression levels,including 13 upregulated and 2 downregulated unigenes (Fig. 3). The six enzymes (PAL、C4H、CHI、DFR、ANS、UFGT) had higher expression levels except the F3H gene in purple compared with green fruits. The 13 unigenes were upregulated by 1.04 to 13.13 fold (Log 2 FC) and 2 unigenes were downregulated by − 1.5 to − 3.5 fold (Table S4).

qRT-PCR validation
We examined differential expression of unigenes between purple and green peppers to identify which unigenes had expression correlating with anthocyanin. The expression levels of anthocyanin biosynthetic genes PAL (LOC1078 71487,LOC107843099,LOC107854291),C4H(LOC10787 5407,LOC107875406),CHI (LOC107852750), F3H(LOC 107843086,LOC107875955),DFR (LOC107850726,LOC were determined by RT-qPCR using leaves of both purple pepper(L66,L29) and green pepper(L9). The results showed that the expression levels of the 6 genes were significantly higher in fruits of purple pepper than that in fruits of the green pepper line,but The expression of two genes in F3H was higher in green pepper. The qRT-PCR results of the expression of 15 genes were highly consistent with the results obtained based on transcriptome sequencing (Fig. 3), indicating that DEG analysis was highly reliable.

Transcription factors regulating anthocyanin biosynthesis in pepper fruits
Transcription factors (TF) are special genes that modulate the expression levels of other genes and represent the main players for the determination of spatiotemporal transcriptional activity [27]. Among the annotated unigenes,transcription factors related to anthocyanin synthesis were MYB,bHLH, WD40,Bzip, WRKY,MA-DS, Zinc-finger and NAC. In the same transcription factor, DEGs in the two kinds of purple pepper are not identical. All DEGs encoding eight TFs were retrieved and the results revealed that bHLH,MYB and NAC were the more DEGs which may differentially regulate structural genes participating in the Anthocyanin formation of pepper (Table S5). Most of these TFs were found up-regulated in purpel peppers(L66 and L29) when compared to the green pepper(L9),suggesting that a high transcriptional activity is required to form the anthocyanin (Table 1). Eight genes were randomly selected from each transcription factor for QRT-PCR verification,and their expression trends were highly consistent with RNA-SEQ results,indicating that DEG analysis was highly reliable (Fig. 4, Table S6).

The gene involved in anthocyanin biosynthesis in fruits of Capsicum annuum
During the biosynthesis of anthocyanin in capsicum annuum,we found a differentially expressed gene novel.2098 that was not annotated in NCBI.  Though blast analysis we preliminarily considered that this gene related to MYB transcription factor and was involved in anthocyanin biosynthesis in pepper fruit (Fig. 5).

Anthocyanin identification in the fruits of Capsicum annuum
Purple pepper had been gaining more popularity because of their antioxidant function,antiaging effects and attractive colors. Anthocyanin as one of the important pigment play a crucial role in the formation of pepper fruit color [28]. The anthocyanin accumulation causes pepper fruit to appear purple and black [29]. According to the studies reported previously,delphinidin is the only anthocyanidin found in peppers [30]. In our study, The accumulation levels of these anthocyanins in particular,delphinidin 3-O-glucoside,delphinidin 3-O-rutinoside and delphin chloride positively correlated with the purple flesh coloration,implying that these compounds partly contribute to the purple color. Among the above three delphinidins,delphin chloride was unique in purple pepper. This result that delphin chloride could be mainly responsible for the purple fruit color of pepper.

Structure genes associated with anthocyanin biosynthesis in fruits of Capsicum annuum
Anthocyanidin biosynthesis belongs to a branch pathway of flavonoids that starts from phenylalanine. After carboxylation,glycosylation,methylation,and acylation,the anthocyanins are transported to and accumulated in vacuoles. A variety of enzymes participate in the biosynthesis of anthocyanins in higher plants [31,32]. It has been reported that there was no significant difference in the expression levels of PAL,C4H,CHS and 4CL between purple and green peppers,while the expression levels of F3H, F3'5'H,DFR,ANS,and UFGT were all significantly higher in purple pepper than in green peppers [33][34][35].However,it was also suggested that the high expression of PAL, C4H and 4CL in purple pepper might be one of the reasons for the high anthocyanin content [29]. In our study,we found that the expressions of CHS and 4CL did not change significantly in purple pepper fruits, while the expressions of DFR,ANS,CHI,PAL,C4H and UFGT were significantly increased during anthocyanin biosynthesis and the expression level of F3H gene was low in purple pepper fruit, which was slightly different with the results of previous studies. Therefore, we think preliminarily DFR,ANS,CHI,PAL,C4H and UFGT play a positive regulatory role in anthocyanin biosynthesis,and their high expression is conducive to the production of anthocyanin derivatives. However,F3H gene was low expressed in purple pepper,and its decrease did not affect the increase of delphinidin products,and played a role in reverse regulation of anthocyanin biosynthesis. The structural genes of anthocyanin biosynthesis have been studied a lot,but the results are slightly different,which may be caused by different experimental materials and environmental conditions.

Transcription factor associated with anthocyanin biosynthesis in fruits of Capsicum annuum
In addition to structural genes,transcription factors are also the key factors in the synthesis of anthocyanins in plants. To date,transcription factors of MYB,bHLH, WD40,NAC,zinc finger, MADs and WRKY proteins have been found to regulate anthocyanin biosynthesis [29,[36][37][38][39]. In peppers,through qRT-PCR and RNA-Seq analysis, Tang et al. [40] found that two MYBs (CaANT1 and CaANT2),one bHLH (CaAN1),and one WD40(CaTTG1) transcription factor participated in the accumulation of anthocyanins in pepper flowers,and might activate anthocyanin accumulation by forming a new MBW complex. In recent studies,it reported that the WRKY transcription factors interacts with the MBW complex regulate the anthocyanin biosynthetic pathway [34] and some MADS-box proteins have involved in the synthesis of flavonoids [41]. It was speculated that NAC and H2C2 transcription factors may regulate the expression levels of these MYB/HD-Zip genes in the ripening periods [42]. In this study,we found that transcription factors related to anthocyanin synthesis of pepper were MYB,bHLH, WD40,Bzip, WRKY,MADS,Zinc-finger and NAC. Among them,transcription factors bHLH NAC and MYB were the more DEGs which may differentially regulate structural genes participating in the anthocyanin formation of pepper. At present,many transcription factors have been found in the process of anthocyanin biosynthesis in pepper,but the relationship between transcription factors is not clear,which needs further research and verification.
Furthermore,In this study,we detected one differentially expressed MYB genes (novel.2098) that were upregulated in green vs purple pepper. Blast analysis indicated that the novel.2098 gene is a new MYB gene in Capsicum annuum. According to the results of the forefathers [43,44], we speculated that the high expression of the MYB gene stimulated the upregulation of structural gene expression, which led to the accumulation of anthocyanin derivatives. Therefore,the high expression level of the MYB gene may have stimulated the formation of anthocyanin, leading to the formation of purple fuirts.
By analyzing the transcriptome and metabolome data of purple and green pepper,we found that the biological metabolism of anthocyanin is complex and affected by multiple transcription factors and enzyme genes. Different gene expression levels lead to changes in the accumulation of anthocyanin metabolites,resulting in different degrees of purple in pepper. This experiment studied the metabolic pathways and key genes for the color of purple pepper fruits,which provided new views into the synthesis and accumulation of anthocyanins in pepper fruits and laid a foundation for more effective cultivation of purple pepper. In addition,the biosynthesis of pepper anthocyanins is not only affected by the key enzyme genes and transcription factors,but also affected by various biochemical reactions and environmental conditions. More in-depth research is needed in the future.

Conclusions
Metabolome and transcriptome data were used to reveal the anthocyanin biosynthesis metabolic pathway in pepper fruit. Changes in anthocyanin metabolites,delphinidin 3-O-glucoside and delphinidin 3-O-rutinoside contribute to the purple color. Delphin chloride could be mainly responsible for the purple fruit color of pepper. In an analysis of the anthocyanin biosynthesis pathway,we identified 59 unigenes encoding 7 enzymes that are involved in anthocyanin biosynthesis in fruits of pepper. In addition,seven transcription factors were also found that could contribute to anthocyanin metabolite biosynthesis in the fruits of pepper. Our results provide valuable information on the anthocyanin metabolites and the candidate genes involved in the anthocyanin biosynthesis pathways in pepper.

Plant materials
Two purple pepper L66 and L29 and a green pepper line L9 were provided by the Economic Crop Research Institute of Hebei Academy of Agricultural and Forestry Sciences. Purple pepper line L66 fruits have deeper purple color than L29 fruits (Fig. 6). The pepper were sown in seedling trays in early January and transplanted. Pericarp tissues of the matured fruits collected from different plants were sampled (each line had three repeats),frozen in liquid nitrogen,and then stored at − 80 °C for subsequent metabolite extraction,transcriptome sequencing (RNA-seq),and real-time quantitative PCR (qRT-PCR) analysis. Fig. 6 The phenotype of purple and green pepper fruits

ESI-q trap-MS/MS
Linear ion trap (LIT) and triple quadrupole (QQQ) scANS were acquired on a triple quadrupole-linear ion trap mass spectrometer (Q TRAP), API 6500 Q TRAP LC/MS/MS System,equipped with an ESI Turbo Ion-Spray interface,operating in a positive ion mode and controlled by Analyst 1.6 software (AB Sciex). The ESI source operation parameters were as follows:ion source,turbo spray,with source temperature at 500 °C;ion spray voltage (IS),5500 V; ion source gas I (GSI),gas II (GSII),and curtain gas (CUR) at 55,60,and 25.0 psi,respectively; and the collision gas (CAD) was 12 psi.Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes,respectively. QQQ scans were acquired as MRM experiments with the collision gas (nitrogen) set to 5 psi. Declustering potential (DP) and collision energy (CE) for individual MRM transitions were done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within that period.

RNA extraction and Illumina sequencing
Total RNAs for constructing nine cDNA libraries of the three pepper,each with three replicates,were isolated from frozen pericarp tissues using the modified CTAB method [47]. mRNAs were used as templates for reverse transcription into cDNAs that were then used to construct cDNA libraries after purification. Agilent 2100 bioanalyzer was used to detect the insert size to control the quality of the libraries. The qualified libraries were sequenced on the Illumina HiSeq 4000 platform. The reads of adapters, N-containing sequences (N indicates ambiguous bases),and low-quality sequences (reads with the bases that have Qphred 20 or less accounting for over 50% of the entire read length) were removed. HISAT2 software was used to quickly and accurately align the clean reads with the reference genome (https:// www. ncbi. nlm. nih. gov/ genome/ term= Capsi cum% 20ann uum) to locate the reads on it. FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) was used to correct the sequencing depth and gene length [48]. DESeq software [49] was employed to analyze and screen the genes differentially expressed between sample groups,adopting |log 2 (Fold Change)| > 1 and padj< 0.05 as the screening standard. The differentially expressed genes (DEGs) were then analyzed through GO enrichment and KEGG [50] functional annotation of metabolic pathways.

Verification of DEGs by qRT-PCR
Real-time fluorescence qRT-PCR was used to verify the results obtained based on transcriptome sequencing. Primer Premier 5.0 software (Primer,Canada) was used to design specific primers for DEGs. The PCR program was as follows: 95 °C for 5 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The PCR was repeated 3 times for each sample. Actin (serial number: GQ339766) was used as the internal reference gene to determine the relative expression level of DEGs.