3.3 | Metabolomic comparison of fenced and unfencedN. sylvatica plants
Comparison of the global metabolome of fenced and unfenced N.
sylvatica plants resulted in the identification of 1,025 metabolite
features. To assess the overall metabolomic accumulation pattern of the
samples from the fenced and open areas, PCA was conducted using the 1025
metabolite features, which clustered the N. sylvatica samples
into two separate groups that correspond with the treatment (presence or
absence of fence); the first two principal components (PC1 and PC2)
explained 47.5% of the total variability (Figure 4A ). The top
15 metabolite features that contributed to the separation of the samples
into the two distinct clusters were identified using PLS-DA
(Figure 4B ), and their accumulation pattern varied based on
treatment (fence or no fence) (Figure 4C ). Hierarchical cluster
analysis (HCA) was conducted to identify the 25 top significant
metabolite features and to cluster the samples based on the top features
(Figure 4D ). The PLS-DA (Figure 4C ) and HCA
(Figure 4D ) analyses identified similar metabolite features
whose accumulation patterns were consistent with the presence/absence of
fences (Figure 4D ).
We obtained putative prediction on the chemical identity of the 1,025
metabolites and their associated metabolic pathways using the GSEA
functional analysis tool. Among the identified metabolic pathways are
the pentose phosphate pathway (P = 0.01), starch and sucrose metabolism
(P = 0.02), pyrimidine metabolism (P = 0.054), cyanoamino acid
metabolism (P = 0.054), riboflavin metabolism (P = 0.054) and
monoterpenoid biosynthesis (P = 0.058) (Appendix Table 3 ).
Out of the identified metabolic pathways, the monoterpenoid biosynthetic
pathway produces metabolites that mediate indirect defenses in many
plant species , while the cyanoamino acid pathway is implicated in
detoxification .