4 Discussion
TH has abundant resources in China, and the growth and development of TH
is closely related to its hosts. It not only absorbs water, nutrients,
etc. from the hosts, but also contains some characteristic components of
the hosts [29–31]. Proteomic analysis of medicinal plants can
contribute to our understanding of the changes that occur at the protein
level in TH from different hosts and the molecular mechanisms by which
these changes further lead to the accumulation of secondary metabolites.
In this study, proteomics was used to analyze TH samples from common
hosts of SS and FXS. The expression of proteins related to several
pathways, such as amino acid metabolism, glycolysis/gluconeogenesis,
phenylpropanoid biosynthesis, flavonoid biosynthesis, and terpenoid
biosynthesis, have been altered.
Enzymes associated with abiotic stress, such as bglB and bglX, play an
important role in maintaining the normal physiological functions of the
organism [32]. 14 peroxidases were identified in this study, and
their expression was not consistent in TH from SS, so we hypothesized
that the hosts had influence on the activity and quantity of the
enzymes. Most proteins took part in the process from chemical backbone
to modification of flavonoids. PAL, C3’H and 4CL are key enzymes in the
phenylpropanoid biosynthesis. With the exception of one 4CL
(TRINITY_DN6526_c0_g1_i1_orf1), the expression of these proteins
was not noticeably altered. However, minor changes might result in
variations in the accumulation of coumaroyl-CoA in the upstream
flavonoid synthesis in TH from two hosts. Coumaroyl-CoA is a
prerequisite for the formation of flavonoid mother nucleus structure.
The 4CL coding gene has been successfully cloned from SS [33].
According to the research, different 4CL plays the role of rate-limiting
enzyme in lignin monomer biosynthesis pathway, but its specific function
varies with plant species [34]. Most of the differences in protein
expression identified upstream of the pathway in this study were not
significant, while significant differences were mainly found in the
downstream proteins, including CAD (TRINITY_DN6468_c0_g1_i11_orf1),
2 CCRs (TRINITY_DN20850_c0_g2_i1_orf1,
TRINITY_DN8569_c1_g1_i5_orf1), and bglX
(TRINITY_DN1860_c0_g1_i15_orf1). Based on the expression of CAD in
roots, collaterals, leaves and shoots of Pinus massoniana [35], we
speculated that there was a similar phenomenon in TH.
The content of ferulic acid in TH from SS was lower than that of TH from
FXS, presumably as a result of the synergistic effect of forming
multiple enzymes on the ferulic acid pathway that were mostly
under-expressed. In contrast, the chlorogenic acid content showed a
completely opposite trend. Three pathways of chlorogenic acid synthesis
have been reported [24]. Only one of these pathways was activated in
this study, and the content level might be reflected in multiple
pathways jointly. The expression of F3H and FLS were up-regulated in TH
from SS. Meanwhile, based on the content of quercetin, the change trend
of protein level and product content is not consistent. The content of
(+)-Catechin from SS was significantly higher than that in FXS, while
the expression levels of related proteins in the upstream of catechin
synthesis pathway were not different. Therefore, the reasons for this
phenomenon are worth exploring. In terms of the content of flavonoid
aglycones ( (+)-Catechin and isosakuranetin) were higher in TH from SS
than those from FXS, which indicated that the formation of flavonoid
aglycones was positively correlated with the expression of upstream
catalytic enzymes. The synthesis and accumulation of flavonols in plant
tissues are synergistically regulated by various factors such as plant
species, developmental stage, tissue site, and growth environment. The
metabolism of compounds in plants is influenced by plant genetic and
environmental factors, thus the amount and content of bioactive
components can reflect intra-species variation to some extent [36],
which also leads to large differences in the same herb species in terms
of yield and quality.
Proteomics approaches have been demonstrated to be effective in
elucidating the biological changes that accompany plant growth or in
response to environmental stress in a variety of main crop species. In
general, the overall content of flavonoid aglycones in TH from SS was
higher than these from FXS, and the content of flavonoid glycosides was
nearly 7 times that of the latter. The abundance of enzymes encoded by
particular genes in biosynthesis pathways was correlated with the
production and accumulation of these metabolites [37]. Since only
one glycosidase was discovered in our work, we hypothesized that the
ultimate cause of the difference in the content of bioactive
constituents is not only the expression of the relevant synthase, but
also the involvement of glycosyltransferases [38].