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.