br Materials and methods br Results br

Materials and methods
Results
Discussion Intracellular metabolic pathways of sugar, such as glycolysis and TCA cycle, provide material and energy for the synthesis of other substances, including amino acids. Thus, the content of sugars and their metabolic pathways are important for amino NLG-8189 synthesis. We demonstrated that the contents of starch and polysaccharide in wild ginseng were lower compared to cultivated ginseng (0.41- and 0.80-fold, respectively, Fig. 2A, 2B). Conversely, some proteins involved in sugar catabolism had high levels of relative abundance in wild ginseng, including sucrose synthase (Q3 in iTRAQ results), amylase (Spot 10 in 2DE results), ADP-glucose pyrophosphorylase large subunit 1 (Spot 13 in 2DE results) and some glycolysis related proteins, such as phosphofructokinase (Spot 4 and 5 in 2DE results), aldolase (Spot 11 in 2DE results), glyceraldehyde-3-phosphate dehydrogenase (Spot 3 in 2DE results), phosphoglyceromutase (Spot 9 in 2DE results), and enolase (Spot 1 and 2 in 2DE results). Thus, it can be deduced that in wild ginseng, most of the starch and other polysaccharides were degraded into other intermediate metabolites that provide material and energy for subsequent glycolysis and other metabolic signaling processes. Glycolysis is a catabolic anaerobic pathway that oxidizes hexoses to generate adenosine triphosphate (ATP), a reductant, and pyruvate and produces building blocks for anabolism [28]. It has been suggested that enhancing the expression of glycolysis-related enzymes could increase the synthesis of some amino acids, such as glutamate, threonine, glycine, and cysteine [29]. Thus, the upregulation of glycolysis-related proteins could promote the generation of amino acids in wild ginseng. These findings were consistent with those obtained from our amino acid analysis results (Table 1). In plants, pyruvate is one of the most critical metabolites of glycolysis. It is also the starting material for alcohol fermentation (glycolysis branch). In this study, we found a higher accumulation of pyruvate in cultivated ginseng compared to wild ginseng (up to 2.78-fold, Fig. 2C). However, short-chain alcohol dehydrogenase (Q9 in iTRAQ results), which catalyzes the synthesis of alcohol, was upregulated (4.12-fold compared to wild ginseng as assessed using iTRAQ) in cultivated ginseng using 2DE and iTRAQ analysis. These findings could indicate that the metabolic signaling pathway of alcohol fermentation is more active in cultivated ginseng compared to wild ginseng. Most of the pyruvate could be used to produce ATP and other substances via the TCA cycle in wild ginseng. With regard to the canonical metabolic pathways, the TCA cycle is extremely important in oxidizing acetyl-CoA into CO2 to produce hydroxylamine reductase, flavin adenine dinucleotide, and ATP and carbon skeletons for use in several other metabolic processes, such as amino acid metabolism [30,31]. Fumarase is a TCA enzyme, and the activity of this enzyme in wild ginseng is higher compared to cultivated ginseng (3.51 times, Fig. 2D). Malate dehydrogenase, which converts malate into oxaloacetate (via nicotinamide adenine dinucleotide) and provides precursors for the synthesis of aspartate and alanine, was upregulated in wild ginseng (Fig. 2E). Thus, these findings could indicate that a higher level of glycolysis and the TCA cycle in wild ginseng could provide material and energy for the synthesis of amino acids. These findings could underlie the difference in the medical effects between wild and cultivated ginseng. Unlike cultivated ginseng, wild ginseng generally suffers from various biotic and abiotic stresses during growth and development. Evidence has emerged that several nonprotein and protein thiols, together with a network of sulfur-containing molecules and related compounds, fundamentally contribute to plant stress tolerance [32,33]. A growing number of studies have demonstrated various protective mechanisms of sulfur-containing amino acids, such as cysteine (Cys) and methionine (Met) [34–37]. Cys, the by-product of a cysteine synthase-catalyzed reaction, is a precursor for glutathione synthesis, which in turn is a key water-soluble antioxidant and plays a central part in reactive oxygen species scavenging via the GSH-ascorbate cycle and as an electron donor to glutathione peroxidase [38]. We demonstrated that the content of Cys (Table 1) and the accumulation of cysteine synthase (Q8 in iTRAQ results) in wild ginseng are higher compared to cultivated ginseng. In most studies, these increases were reported together with increased GSH. We also demonstrated that the GSH content was consistent with previous reports. The content of GSH in wild ginseng was 1.6-fold greater compared to cultivated ginseng (Fig. 2F). Furthermore, free Cys is often irreversibly oxidized to different by-products [39], such as cysteine (CySS). The redox potential of the CySS/2Cys complex is regarded as an important biochemical marker for early stages of various human diseases [40] and as an important antioxidant system and regulator of the redox state in parasites [41]. Moreover, the CySS/2Cys redox state could also have an important role in the plant stress response [42]. Thus, the difference in Cys metabolism between wild and cultivated ginseng could be one reason for the difference in medicinal functions between wild and cultivated ginseng.