3.2 Differential expression analysis between light treatments and enrichment (GO, KEGG) analysis
The gene expression levels quantified from the read counts generated from the transcriptome showed 14,105 differentially expressed genes (DEGs). The FPKM counts (fragments per kilobase of transcript per Million mapped reads) were aggregated from three replicates of each light treatment (Figures S3, S4). Hierarchical clustering analysis showed clear differences in the expression patterns between the light treatments (Figure 3a). In red light treated berries, high number of DEGs corresponding to 7327 up-regulated genes and 1545 down-regulated genes (Figure 3b) were detected when compared with blue light treatment yielding 3686 up-regulated and 1547 down-regulated genes (Figure 3c) as visualized using volcano plots.
GO Enrichment analysis classified the DEGs according to their functions and properties into three major categories Biological process (BP), Molecular function (MF) and Cellular process (CC) (Figure 4a). An average of 60-65% of unigenes were assigned GO terms either through one of the homology searches from Pfam, BLASTx and BLASTp databases. The top significantly enriched GO terms across these three categories from our two comparison subsets showed that in CC ‘intracellular’ and ‘cellular anatomical entity’ were the top sub-categories (Figure 4a). In the MF category ‘catalytic activity’, ‘binding’ and ‘transporter’ activities were found to be abundant (Figure 4a). Both these categories have similar number of GO terms assigned from the sequences but with two contrasting results. In the BP category, the sequences assigned to ‘metabolic’ and ‘cellular’ process were relatively higher in red light than blue light treatment. The GO terms assigned to ‘localization’, ‘signaling’ and ‘response to stimulus’ sub-categories were contrasting between the light treatments (Figure 4a). Some of the unigenes were also classified in ‘rhythmic processes’ and ‘pigmentation’ in the BP category. Hence, we further investigated the biological process (BP) category by direct count of sequence distribution among the top DEGs. GO terms, such as ”oxidation-reduction process”, ”protein phosphorylation”, and ”regulation of transcription”, were the top ones with relatively higher number of assigned sequences found in red light treated samples than blue light treatment. Some annotated sequences related to sugar metabolism, shikimate, chorismate, lignin, cutin and sterol biosynthetic process were also determined with additional GO terms assigned to anthocyanin containing compounds and flavonoid biosynthetic process were obtained from red light treatment (Figure 4b). The KEGG pathways significantly enriched by adjusting FDR corrected/p-value to <0.05 using Benjamini-Hochberg method showed that a higher number of gene ratio fell on metabolic pathways and secondary metabolite biosynthesis predominantly the phenylpropanoid biosynthesis followed by terpenoid biosynthesis with considerable number of gene counts (Figure 4c, d). In addition, the red light treatment has also significantly enriched the fatty acid, galactose, starch and sucrose metabolism related pathways (Tables S4, S5, S6, S7). All the DEGs and corresponding unigene IDs analyzed throughout this study is provided in Table S8.