Carotenoids and Flavonoids Profiles in Juice Sacs of CNO during Fruit Development
Juice sacs at six different developmental stages of CNO were determined for variation in carotenoids and flavonoids profiles using HPLC. The contents of both carotenoids and flavonoids varied greatly at different developmental stages.
Seven major carotenoids were identified, including lycopene, phytoene, phytofluene, lutein, violaxanthin, β-cryptoxanthin and β-carotene, with the level of lycopene being the highest (Additional file 1: Table S1; Fig. 1A). The content of lycopene was low at 60 DAF, and significantly increased from 90 to 120 DAF, reached to a maximum at 150 DAF, then significantly decreased at the mature stage (Fig. 1A). The increases of lycopene levels were 2.65μg/g, 23.23μg/g and 14.19μg/g from 60 to 90, 90 to 120 and 120 to 150 DAF, respectively, indicating that the stage from 90 to 120 DAF might be the most important for the lycopene biosynthesis (Additional file 1: Table S1; Fig. 1A). For the other carotenoids identified, phytoene and phytofluene were also significantly increased from 90 to 120 DAF, then maintained or slightly decreased at the mature stage (Fig. 1A). Lutein was found at 180 DAF and its level increased at 210 DAF; β-cryptoxanthin and β-carotene were detected at 150 DAF and tended to increase from 150 to 210 DAF; violaxanthin was detected at 120 DAF, then significantly increased from 150 to 180 DAF (Additional file 1: Table S1).
A total of 11 flavonoids were identified in CNO fruit at four development stages, with narirutin as the dominant one at 60 and 90 DAF (Additional file 2: Table S2). The contents of total flavonoids were the highest and lowest at 60 DAF and 210 DAF, respectively. The results suggested that the 60 DAF stage might be the important stage for the accumulation of flavonoids (Fig. 1B).
Pearson correlation coefficient was -0.797 between total carotenoids and total flavonoids levels in CNO fruit at six development stages, while -0.719 was at 60 to 150 DAF, suggesting a negative correlation between total contents of carotenoids and flavonoids. Furthermore, before 150 DAF, the level of carotenoids constantly increased, whereas the content of flavonoids sharply decreased at 90 DAF with a slight increase at 120 DAF due to the increase of hesperidin content. After 150 DAF, the level of lycopene significantly decreased, whilst the levels of the other main carotenoids and all flavonoids decreased or kept at stable levels (Fig. 1). These results showed that there were collaborative changes in carotenoids and flavonoids biosynthesis, with 150 DAF as the turning stage.
Differentially Expressed Genes in CNO during Fruit Development
To further determine the expression levels of the genes related to lycopene over-accumulation in CNO, the juice sacs at four developmental stages (60, 90, 120 and 150 DAF) were subjected to transcriptomic analysis. Total of 282.14 M high-quality base pairs were obtained, with an average of 23.51 M data per sample. Approximately 94.68 – 97.57% reads were mapped to the Valencia sweet orange genome, among them, 87.22 – 93.13% was uniquely aligned, and 4.44 – 7.46% was aligned to multiple loci (Additional file 3: Table S3). Based on their expression levels, the average fragments per kilobase of transcript per million fragments mapped (FPKM) higher than 0.5 were selected for further analysis (Additional file 4: Table S4). Totally there were 17,190 genes expressed in juice sacs at four development stages of CNO, with 16,454, 16,170, 15,937 and 15,911 expressed at 60, 90, 120 and 150 DAF, respectively (Fig. 2A), while 178, 32, 20 and 187 genes were specially expressed at 60, 90, 120 and 150 DAF, respectively (Fig. 2B). Furthermore, 88.61%, 84.62%, 85.18% and 87.93% of expressed genes ranged from 1 to 100 FPKM in 60, 90, 120 and 150 DAF, respectively (Fig. 2A). Principal component analysis (PCA) and correlation analysis showed good repeatability among biological replicates (Fig. 2 CD).
A total of 2482, 4059, 5565, 3738, 5671 and 3555 differentially expressed genes (DEGs) at 60 DAF vs 90 DAF, 60 DAF vs 120 DAF, 60 DAF vs 150 DAF, 90 DAF vs 120 DAF, 90 DAF vs 150 DAF and 120 DAF vs 150 DAF with the fold change (|log2FC| ≥ 1) and FDR ≤ 0.05 were obtained, respectively. For instance, compared with the fruit at 90 DAF, 2063/1675 genes were significantly up-regulated/down-regulated in the fruit at 120 DAF (Additional file 5: Figure S1 A).
Important Pathway Genes for Lycopene Accumulation
The stage from 90 to 120 DAF was the most important stage for lycopene accumulation based on metabolic data. Thus, the DEGs were subjected to KEGG enrichment analysis, and the DEGs were found to be mainly enriched in metabolic pathways, biosynthesis of secondary metabolites and plant hormone signal transduction (Additional file 5: Figure S1 B). Among the DEGs, some genes were clustered into carotenoids biosynthetic and terpenoid backbone biosynthesis, including DXS1 (Cs1g20530), DXS2 (Cs9g05150), HDS (Cs8g16700), HDR (Cs8g07020) and ZDS4 (Cs3g11060), and their expression levels in the fruit at 120 DAF were significantly higher than thoseat90 DAF. Furthermore, KEGG enrichment analysis on the DEGs at 90-120 DAF found that some genes were clustered into plant hormone signal transduction, such as Cs9g08850 (ethylene signal transduction), Cs1g17210 (jasmonate signal transduction) and Cs9g18020 (abscisic acid signal transduction), etc: which might also play important roles in lycopene over-accumulation (Additional file 5: Figure S1 B).
To mine the most important genes for the over-accumulation of lycopene during fruit development, all the expressed genes were grouped using Mfuzz analysis based on their expression pattern changes, which yielded nine clusters (Fig. 3; Additional file 6: Table S5). Notably, Cluster 2, 4 and 7 showed a good positive correlation with the content of lycopene, and 1,804, 1,604 and 1,546 genes were grouped in Cluster 2, 4 and 7, respectively (Fig. 3). Further gene function annotation found that DXS1, PSY1 and ZDS2 (Cs3g11060) were in Cluster4, DXR and GGPPS2 were in Cluster2, and MDS (Cs5g03050) was in Cluster7, implying their importance as candidate genes accounting for lycopene over-accumulation. In addition, LCYB (orange1.1t00772) with consistently low expression levels was in Cluster 6 (Fig. 3).
Next, correlation analysis between the genes within carotenoid biosynthetic pathway and the content of lycopene showed that DXS1, DXR, GGPPS2 and PSY1 were highly and positively related to lycopene levels, with correlation coefficients of 0.997, 0.893, 0.834 and 0.964, respectively, while the values of the other lycopene biosynthesis related genes were lower than 0.800 (Table 1; Fig. 4). Herein, DXS1, DXR, GGPPS2 and PSY1 might be the key biosynthetic pathway genes accounting for the high level of lycopene in juice sacs of CNO fruits.
TFs Related to Key Carotenoid Biosynthetic Genes
To better understand the potential role of TFs in the regulation of DXS1, DXR, GGPPS2, LCYB and PSY1, and thus contributing to lycopene accumulation, their promoters were used to predict the possible binding sites using PlantRegMap (Plant TF Database). Potentially 83, 147, 183, 104 and 228 TF binding site in DXS1, DXR, GGPPS2, LCYB and PSY1 promoters were predicted, respectively (Additional file 7: Table S6). However, 16, 40, 48, 18 and 24 TFs had high Pearson correlation coefficients (|r|>0.85), with 63 and 83 as potential positive and negative regulators respectively (Fig. 5; Additional file 8: Table S7). Among the 146 potential regulators, there were 93 TFs (17 TFs from AP2 family, 12 from MADS, 11 from GRAS, eight from MYB, five from TCP, WRKY and bZIP, four from SBP, three from ERF, HD-ZIP and NAC) (Additional file 8: Table S7).
Some TFs were highly positively or negatively correlated with DXS1, DXR, GGPPS2, LCYB and PSY1 genes that contribute to carotenoids accumulation. These TFs might regulate the expression levels of five important genes that contribute to lycopene accumulation in CNO fruit. For example, DXS1 was positively correlated with 10 TFs, and negatively correlated with six TFs, and the coefficients of seven TFs were higher than 0.900, while four were lower than -0.900 (Fig. 5; Additional file 8: Table S7).
Some potential TFs bind to multiple genes, with high Pearson correlation coefficients. For example, Cs3g19420, Cs3g23270, Cs5g26720, Cs7g11810 and Cs7g26660 might positively regulate the four important pathway genes, while Cs9g16575 might be a negative regulator. Some TFs could regulate three of five pathway genes, such as Cs5g23210, Cs5g33540, Cs9g03820 and orange1.1t02314 which might be potential positive regulators, while Cs4g09270, Cs6g17530, orange1.1t00456, orange1.1t02436 and Cs9g07440 might be negative ones (Fig. 5; Additional file 8: Table S7).
Important Pathway Genes and TFs for Flavonoids Biosynthesis
The contents of flavonoids tended to decrease during fruit development (Fig. 1 B). The expression levels of the most flavonoids biosynthetic genes were found to be high at 60 DAF in CNO, while dropped at 120 and 150 DAF, including PAL-1, PAL-3, 4CL-2, 4CL-5, CHS-1 and CHI (Additional file 9: Figure S2). Results of Mfuzz analysis of Cluster1 showed that their expression tended to decrease (Fig. 3), and KEGG annotation found that the genes in the cluster were mainly involved in metabolic pathways, biosynthesis of secondary metabolites and phenylpropanoid biosynthesis (Additional file 10: Figure S3). Coincidently, PAL-1, PAL-3, 4CL-2, 4CL-5, CHS-1 and CHI grouped into Cluster1 were identified via gene annotation.
To further identify the potential TFs that may regulate the above six pathway genes and the accumulation of flavonoids, TFs were predicted using PlantRegMap. The results showed that there were potentially 213, 229, 95, 262, 227 and 102 TF binding sites in PAL-1, PAL-3, 4CL-2, 4CL-5, CHS-1 and CHI promoters, respectively (Additional file 11: Table S8). The pathway genes with its potential 17, 22, 14, 25, 24 and 16 TFs were found to have high Pearson correlation coefficients (|r|>0.85), with 111 and seven as potential positive and negative regulators, respectively (Fig. 6; Additional file 12: Table S9). Among the 118 potential regulators, there were 64 TFs, mainly including nine TFs from WRKY family, eight from MYB, six from bHLH and four from ERF (Additional file 12: Table S9).
Some TFs were highly positively or negatively correlated with PAL-1, PAL-3, 4CL-2, 4CL-5, CHS-1 and CHI genes that contribute to flavonoids accumulation. These TFs might regulate the expression levels of the six important pathway genes that contribute to flavonoids accumulation in CNO fruit. For instance, PAL-1 was highly and positively correlated with 16 TFs, while negatively correlated with one TF, among them, the Pearson correlation coefficients of 12 TFs were higher than 0.900 (Fig. 6; Additional file 12: Table S9).
Among the potential TFs that had high Pearson correlation coefficients with the key pathway genes, Cs1g23760 and Cs3g16330 might positively regulate the five of six key pathway genes, while Cs2g16940, Cs7g04700, Cs6g01750 and orange1.1t01021 might positively regulate the four out of six pathway genes, and nine TFs (Cs1g08440, Cs3g23190, Cs4g06100, Cs5g12070, Cs5g33880, Cs6g16070, Cs7g03980, Cs9g02700 and orange1.1t00472) could bind three out of six pathway genes as positive regulators (Fig. 6; Additional file 12: Table S9). These TFs might play important roles in flavonoids biosynthesis.
Postulated Hierarchical Network in Regulating Carotenoids and Flavonoids Profiles in CNO
Both sets of fifteen candidate TFs could bind at least three carotenoids and flavonoids biosynthesis genes, respectively. Further analysis identified 24 TFs that could regulate important genes involved in both carotenoids and flavonoids biosynthesis pathways. Twenty of these TFs might positively and negatively regulate flavonoid biosynthesis and carotenogenesis genes, respectively, whereas two of them had the opposite effects and the other two were positively correlated with the important genes in both biosynthetic pathways (Additional file 13: Figure S4). Thus, these TFs might directly regulate the genes related to the biosynthesis pathways for both carotenoids and flavonoids.