Integrated Transcriptomic and Metabolomic analysis reveals a transcriptional regulation network for the biosynthesis of carotenoids and flavonoids in 'Cara cara' navel Orange

Abstract

Background: Carotenoids and flavonoids are important secondary metabolites in plants, which exert multiple bioactivities and benefits to human health. Although the genes that encode carotenogenesis and flavonoid biosynthetic enzymes are well characterized, the transcriptional regulatory mechanisms that are related to the pathway genes remain to be investigated. In this study, 'Cara cara' navel orange (CNO) fruit at four development stages were used to identify the key genes and TFs for carotenoids and flavonoids accumulation.

Results: In this study, CNO was used to investigate the profiles of carotenoids and flavonoids by a combination of metabolomic and transcriptomic analyses. The important stage for the accumulation of the major carotenoid, lycopene was found to be at 120 days after florescence (DAF). The transcripts of five carotenogenesis genes were highly correlated with lycopene contents, and 16, 40, 48, 24 and 18 transcription factors (TFs) were predicted to potentially bind 1-deoxy-D-xylulose-5-phosphate synthase (DXS1), deoxyxylulose 5-phosphate reductoisomerase (DXR), geranylgeranyl diphosphate synthase (GGPPS2), phytoene synthase (PSY1) and lycopene β-cyclase (LCYB) promoters, respectively. Narirutin was the most abundant flavonoid in the flesh at the early stages, 60 DAF was the most important stage for the accumulation of flavonoids, and 17, 22, 14, 25, 24 and 16 TFs could potentially bind phenylalanine ammonia-lyase (PAL-1 and PAL-4), 4-Coumarate-CoA ligase (4CL-2 and 4CL-5), chalcone synthase (CHS-1) and chalcone isomerase (CHI) promoters, respectively. Furthermore, both sets of 15 candidate TFs might regulate at least three key genes and contribute to carotenoids/flavonoids accumulation in CNO fruit. Finally, a hierarchical model for the regulatory network among the pathway genes and TFs was proposed.

Conclusions: Collectively, our results suggest that DXS1, DXR, GGPPS2, PSY1 and LCYB genes were the most important genes for carotenoids accumulation, while PAL-1, PAL-4, 4CL-2, 4CL-5, CHS-1 and CHI for flavonoids biosynthesis. A total of 24 TFs were postulated as co-regulators in both pathways directly, which might play important roles in carotenoids and flavonoids accumulation in CNO fruit.

Keywords: Carotenoid; Citrus; Flavonoid; Metabolomics; Transcription factor; Transcriptome.

Fig. 1

The main carotenoids and flavonoids identified in CNO juice sacs during fruit development. a, The main carotenoids in CNO fruit (μg/g, FW); b, The main flavonoids in CNO fruit (μg/g, FW)

Fig. 2

Basic information obtained from transcriptomic data. a, Expression levels of genes based on RNA-Seq data from juice sacs of CNO at 4 different development stages. b, The specially expressed genes in juice sacs of CNO at 4 different development stages. c: Correlation analysis matrix among CNO at 4 different development stages based on RNA-Seq data. d: PCA analysis on CNO at 4 different development stages based on RNA-Seq data

Fig. 3

Expressed genes were clustered into nine expression patterns using Mfuzz in CNO at four developmental stages

Fig. 4

Heatmap representation of the expression levels of carotenoid biosynthesis-related genes in CNO fruit based on transcriptome data. a, Carotenoid metabolism pathway in citrus. b, Expression level of carotenoid metabolism pathway genes in CNO fruit at four developmental stages

Fig. 5

The relationships between DXS1 (Cs1g20530), DXR (Cs5g05440), GGPPS2 (Cs8g02140), PSY1 (Cs6g15910) and LCYB (orange1.1 t00772) and its potential TFs based on transcriptome data

Fig. 6

The relationships between PAL-1 (Cs6g11940), PAL-4 (Cs8g16290), 4CL-2 (Cs5g24900), 4CL-5 (orange1.1 t04489), CHS-1 (Cs2g14720) and CHI (Cs7g28130) genes and its potential TFs based on transcriptome data