Study Of Protein Profile, Organ Specific, And UB-B Induction Mechanism Of Four Medicinal Plants By Proteomic Approach

Medicinal plant is an important tool for preventing and treatment of human diseases. It is also the source of new drug discovery. The resources of wild medicinal plant are depleted, because of people’s increasing demand. The quality of cultivated medicinal plant is different, which influence the research of Traditional Chinese Medicine. Recently, a method established by our group can increase the content of active ingredients through improving the metabolic pathway in medicinal plant. However, the mechanism of this method is unknown until now, because of the specificity of resource distribution and imperfection of genetic information. Proteomics is a hotspot of post genome era and is an important part of life characteristics of the particular implementation. In this paper, proteomic analysis of protein profile, organ specific, and UV-B induction mechanism of four medicinal plants (Lonicera, Mahonia, Catharanthus, and Morus) were performed. The research revealed the molecular mechanism of the production or variation of active consituents in four medicinal plants. At the sam time, it also laid the foundation for application of proteomics in medicinal plants. The main contents and results of this paper are as follows:(1) Proteomic analysis of Lonicera japonica Thunb. Immature flower buds using combinatorial peptide ligand libraries and polyethylene glycol fractiation.Lonicera japonica is a well-known medicinal plant, which is widely used a traditional medicinal plants due to their various biological activities such as anti-virus, anti-inflammatory, and antioxidant. The main active ingredients in Lonicera are chlorogenic acid, flavonoid, and iridoid. To explore the molecular mechanism of the production of active ingredients in L. japonica, a label-free proteomic technique was used in combination with combinatorial peptide ligand libraries (CPLL) and polyethylene glycol (PEG) fractionation for the enrichment of low-abundance proteins and removal of high-abundance proteins, respectively. A total of 177,614, and 529 proteins were identified in crude protein extraction, CPLL fractions, and PEG fractions, respectively. Among the identified proteins,283 and 239 proteins were specifically identified by CPLL and PEG methods, respectively. In particular, proteins related to the oxidative pentose phosphate pathway, signaling, hormone metabolism, and transport were highly enriched by CPLL and PEG fractionation compared to crude protein extraction. A total of 28 secondary metabolism-related proteins were identified in L. japonica and to determine the specificity of the identified proteins for L. japonica, Cerasus was used for a comparison, resulting that the abundance of hydroxymethylbutenyl 4-diphosphate synthase in L. japonica was ten-fold higher than that in Cerasus. These results suggest that proteins related to secondary metabolism might be responsible for the biological activities of L. japonica. The findings also provided a new method for identification of low-abundance proteins in medicinal plants using CPLL and PEG methods.(2) Organ-specific analysis of Mahonia using gel-free/label-free proteomic techniqueMahoina is an important medicnal plant used for the treatment of human diseases. Studies showed that it has anti-bacterial, anti-inflammatory, anti-virus, and anti-hypotensive activites. The levels of alkaloids in Mahonia, which were columbamine, jatrorrhizine, palmatine, tetrandrine, and berberine, were markedly higher in roots compared to those detected in stems and leaves. In the present study, organ-specific proteomics was performed in Mahonia. A total of 304,314, and 182 proteins were identified in leaf, stem, and root, respectively. Many root-specific proteins, including S-adenosylmethionine synthetase and (S)-tetrahydroprotoberberine oxidase, involved in the biosynthesis of alkaloids, were identified. The abundance of calreticulin was markedly higher in roots than that detected in stems and leaves. Taken together, these results suggest that alkaloid biosynthesis is an important function in Mahonia roots. The multifunctional protein calreticulin might affect the transport benzylisoquinoline alkaloid from roots to other organs in Mahonia. The finding revealed the molecular mechanism of different pharmacological functions in the roots, stems and leaves of Mahonia for the first time.(3) Binary stress induces an increase in indole alkaloid biosynthesis in Catharanthus roseus.Catharanthus roseus is an important medicinal plant, which produces a variety of indole alkaloids of significant pharmaceutical relevance. Previously, our research showed that the contents of the detectable alkaloids ajmalicine, vindoline, catharanthine, and strictosidine in C. roseus were significantly increased under binary stress. In the present study, we aimed to investigate the potential stress-induced increase of indole alkaloid biosynthesis in C. roseus using proteomic technique. A total of 87 porteins were identified as being differentially changed, including 21 proteins down-regulated and 66 proteins up-regulated in C. roseus leaves upon binary stress. The abundance of proteins related to tricarboxylic acid cycle and cell wall was largely increased; while, that of proteins related to tetrapyrrole synthesis and photosynthesis was decreased. Of note,10-hydroxygeraniol oxidoreductase, which is involved in the biosynthesis of indole alkaloid was two-fold more abundant in treated group compared to the control. In addition, mRNA expression levels of genes involved in the indole alkaloid biosynthetic pathway indicated an up-regulation in their transcription in C. roseus under UV-B irradiation. These results suggest that binary stress might negatively affect the process of photosynthesis in C. roseus. In addition, the induction of alkaloid biosynthesis appears to be responsive to binary stress. This finding illustrated the molecular mechanism of increased alkaloid in C. roseus under UV-B stress, and also provided a theoretical basis for the light-enzyme induction method.(4) Study on the biosynthesis pathway of Diels-Alder adducts in Morus leaf by proteomic approachMorus is an imporotant medicinal plant, its dried roots contained trace of Diels-Alder adducts. These compounds have anti-inflammotary, anticancer, and anti-virus activities. In the present study, Diels-Alder adducts were induced in mulberry leaf by UV-B irradiation and dark incubation. Nine compounds including chlorogenic acid, luteolin-7-glycoside, isoquercitrin, moracin C, moracin N, morachalcone, guangsangon E, chalcomoracin, and moracin M were separated and identified in induced mulberry leaf. To investigate the mechanism of Diels-Alder adduct biosynthesis in mulberry leaf under UV-B irradiation and dark incubation, proteomic approach was performed. A total of 123 proteins were identified as being differentially changed, including 16 proteins down-regulated and 107 proteins up-regulated in mulberry leaves after UV-B irradiation. While, a total of 169 differential expression proteins were identified, including 40 proteins up-regulated and 129 proteins down-regulated under UV-B irradiation and dark incubation. In addition, mRNA expression levels of genes involved in the Diels-Alder adduct biosynthetic pathway indicated an up-regulation in their transcription in mulberry under UV-B irradiation. These results uncovered the mechanism of Diels-Alder adducts biosynthesis under UV-B irradiation and dark incubation at the first time.