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].