Proteomic Analyses Provide Novel Insights into Plant Growth and Ginsenoside Biosynthesis in Forest Cultivated Panax ginseng (F. Ginseng)

Abstract

F. Ginseng (Panax ginseng) is planted in the forest to enhance the natural ginseng resources, which have an immense medicinal and economic value. The morphology of the cultivated plants becomes similar to that of wild growing ginseng (W. Ginseng) over the years. So far, there have been no studies highlighting the physiological or functional changes in F. Ginseng and its wild counterparts. In the present study, we used proteomic technologies (2DE and iTRAQ) coupled to mass spectrometry to compare W. Ginseng and F. Ginseng at various growth stages. Hierarchical cluster analysis based on protein abundance revealed that the protein expression profile of 25-year-old F. Ginseng was more like W. Ginseng than less 20-year-old F. Ginseng. We identified 192 differentially expressed protein spots in F. Ginseng. These protein spots increased with increase in growth years of F. Ginseng and were associated with proteins involved in energy metabolism, ginsenosides biosynthesis, and stress response. The mRNA, physiological, and metabolic analysis showed that the external morphology, protein expression profile, and ginsenoside synthesis ability of the F. Ginseng increased just like that of W. Ginseng with the increase in age. Our study represents the first characterization of the proteome of F. Ginseng during development and provides new insights into the metabolism and accumulation of ginsenosides.

Keywords: Panax ginseng; energy metabolism; ginsenosides biosynthesis; growth; proteomic analysis.

Figure 1

Different parts of W. Ginseng and F. Ginseng.

Figure 2

Morphological features of F. Ginseng in different growth years: 10 years old (A), 15 years old (B), 20 years old (C) and 25 years old (D), and W. Ginseng (E).

Figure 3

Cluster analysis of spots showing differences in relative volumes among F. Ginseng in different growth years and W. Ginseng. Two-way hierarchical clustering analysis of the 57 spots showed at least a 1.5-fold change in the relative spot volumes (ANOVA, P < 0.01) among the five samples. The clustering analysis was performed with PermutMatrix graphical interface after Z-score normalization of the averages of relative spot values (n = 6). Pearson's distance and Ward's algorithm were used for the analysis.

Figure 4

The clusters of the abundance of different expressed proteins from F. Ginseng in different growth years by MALDI-TOF/TOF-MS/MS. The up-regulated proteins in cluster (A–C); The down-regulated proteins in cluster (D–F).

Figure 5

The clusters of abundance of different expressed proteins from F. Ginseng in different growth years by iTRAQ. The up-regulated proteins in cluster (A–D); The down-regulated proteins in cluster (E–H).

Figure 6

Functional analyses of differentially expressed proteins in the biological process. Percentage distributions of the GO terms were calculated by iProClass GO tool in PIR database.

Figure 7

The key enzyme activities and metabolite contents of F. Ginseng in different growth years. Bars indicate ±SD. (A), Amylase activity; (B), MDH activity; (C), FDPS activity; (D), CAS activity; (E), SE activity; (F), SS activity; (G), SOD activity; (H), CAT activity; (I), POD activity; (J), Starch contents; (K), Pyruvic acid contents; (L), Saponin content; (M), AsA contents; (N), GSH contents.

Figure 8

Expressions (A) and Relative levels of Grx gene (B) and PR5 gene (C) of F. Ginsengs in different growth years and W. Ginseng. Actin gene from ginseng was used as a control.

Figure 9

Schematic overview of the enzymes and metabolites involved in the growth and development of F. Ginseng, reveals its growth and ginsenoside biosynthesis.