Transcriptomic analyses of cacao cell suspensions in light and dark provide target genes for controlled flavonoid production

Abstract

Catechins, including catechin (C) and epicatechin (E), are the main type of flavonoids in cacao seeds. They play important roles in plant defense and have been associated with human health benefits. Although flavonoid biosynthesis has been extensively studied using in vitro and in vivo models, the regulatory mechanisms controlling their accumulation under light/dark conditions remain poorly understood. To identify differences in flavonoid biosynthesis (particularly catechins) under different light treatments, we used cacao cell suspensions exposed to white-blue light and darkness during 14 days. RNA-Seq was applied to evaluate differential gene expression. Our results indicate that light can effectively regulate flavonoid profiles, inducing a faster accumulation of phenolic compounds and shifting E/C ratios, in particular as a response to switching from white to blue light. The results demonstrated that HY5, MYB12, ANR and LAR were differentially regulated under light/dark conditions and could be targeted by overexpression aiming to improve catechin synthesis in cell cultures. In conclusion, our RNA-Seq analysis of cacao cells cultured under different light conditions provides a platform to dissect key aspects into the genetic regulatory network of flavonoids. These light-responsive candidate genes can be used further to modulate the flavonoid production in in vitro systems with value-added characteristics.

Figure 1

Experimental setup and chemical quantifications for cacao cell suspensions under light treatments and polyphenol content. (a) Diagram showing treatments with white - blue LED lights (top) versus dark (bottom) along the 14-day time course. (b) Measurements of total polyphenol content. (c) Measurements of catechin and epicatechin contents during the time course experiment. d: day. The graph shows the average values from three independently sampled cell suspensions. Error bars indicate the standard deviation. Different letters between each treatment indicate p < 0.05.

Figure 2

Total number of DEGs in pairwise comparisons over the time course for all cell cultures sequenced under both W-B and D conditions. Values above columns represent total DEGs, up and down regulated genes. d: day.

Figure 3

Diagrams of changes in expression levels of differentially expressed flavonoid genes. (a) Number of up and down regulated structural genes in the flavonoid pathway. (b–d) Fold changes for flavonoid genes at d0-VS-d1, d7-VS-d8 and d8-VS-d14. Blue dots for treatment W-B. Gray triangles for treatment D. Abbreviations: day (d), phenylalanine ammonia lyase (PAL); cinnamate 4-hydroxylase (C4H); 4-coumarate coenzyme A ligase (4CL); chalcone synthase (CHS); chalcone isomerase (CHI); flavanone 3-hydroxylase (F3H); flavanone 3′-hydroxylase (F3′H); Flavonoid-3′,5′-hydroxylase (F3′5′H); Flavonol synthase (FLS); dihydroflavonol-4-reductase (DFR); anthocyanidin synthase (ANS); anthocyanidin reductase (ANR); leucoanthocyanidin reductase (LAR); Glycosiltransferases (GLY); Cytochrome P450 enzymes (P450).

Figure 4

Co-expression network using late genes of flavonoid pathway ANS, ANR and LAR as seed nodes. (a) Subnetwork for W-B in the “Indianred4” module. (b) Subnetwork for D in the “Darkgreen” module. In both cases using top 30 of genes co-expressing with late biosynthetic genes.

Figure 5

Expression patterns of MYB, bHLH and WD40 transcription factors and phylogeny of selected MYBs in cacao. (a) Heat map illustrating expression patterns of selected transcription factors of MBW complex at W-B and D treatment. (b) Phylogenetic analysis of selected MYBs genes associated with flavonoid pathway for cacao, Arabidopsis and grape. Abbreviations: day (d). Dotted line: shortened evolutionary distance.

Figure 6

Heat map of selected light signaling genes over time in white-blue light versus dark. Treatments are summarized at the bottom. To the right are the codes assigned for each gene. Abbreviations: day (d).