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The overall survival mechanism and secondary metabolite synthesis under cold stress conditions (4°C) at exogenous supply with KNO3 were studied in mountain ginseng adventitious root cultures. Expressed sequence tags encoding antioxidant enzymes, such as catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and also ginsenoside-related secondary metabolites were obtained, and expression studies were carried out using quantitative real-time PCR. Chilled adventitious root cultures grown in vitro with or without an exogenous supply with K+ were differentially regulated. Expressions of PgCAT, PgAPX, and PgGPX-1 were increased, whereas PgGPX-2 and all ginsenoside-related secondary metabolite genes showed slight down-regulation upon chilling stress. Interestingly, the most genes were up-regulated at the increased potassium supply except the cytochrome P450 gene, which shows clearly the specific effect of potassium on the antioxidant level and secondary metabolite gene expression involved in the survival mechanism. In addition, we studied the activities of catalase, ascorbate peroxidase, guaiacol peroxidase, and phenylalanine ammonia-lyase, which showed similar pattern of changes. Adventitious root dry weight and relative water content were found to increase at 0.05 mM KNO3. The high level of potassium is needed for Panax ginseng under chilling conditions to increase its cold tolerance through activating the anti-oxidant system as well as to increase ginsenoside-related secondary metabolite transcripts.

Calmodulin (CaM), the predominant Ca(2+) receptors, is one of the best-characterized Ca(2+) sensors in all eukaryotes. In this study the role of CaM and the possible interrelationship between CaM and hydrogen peroxide (H(2)O(2)) in abscisic acid (ABA) induced antioxidant defense were investigated in the seedling of Panax ginseng. Treatment of ABA (100 μM) and H(2)O(2) (10 mM) increased the expression of Panax ginseng calmodulin gene (PgCaM) and significantly enhanced the expression of the antioxidant marker genes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase and the activities of chloroplastic and cytosolic antioxidant enzymes. Pretreatments with two CaM antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W7) and inhibitor or scavenger, diphenyleneiodonium chloride, and dimethylthiourea of reactive oxygen species almost completely suppressed the up-regulation of antioxidant and PgCaM gene. Moreover, H(2)O(2) production and CaM content was almost completely inhibited by pretreatments with two CaM antagonists. In addition, the expressions of PgCaM gene under different biotic stress were analyzed at different time intervals. Thus it may suggests that CaM are involved in ABA-induced increased expression of PgCaM which triggers H(2)O(2) production through activating trans-plasma membrane NADPH oxidase, resulting in up-regulation of defense related antioxidant gene and also plays a pivotal role in defense response against pathogens.

In plants heme containing cytochrome P450 (P450) is a superfamily of monooxygenases that catalyze the addition of one oxygen atom from O2 into a substrate, with a substantial reduction of the other atom to water. The function of P450 families is attributed to chemical defense mechanism under terrestrial environmental conditions; several are involved in secondary and hormone metabolism. However, the evolutionary relationships of P450 genes in Panax ginseng remain largely unknown. In the present study, data mining methods were implemented and 116 novel putative P450 genes were identified from Expressed Sequence Tags (ESTs) of a ginseng database. These genes were classified into four clans and 22 families by sequence similarity conducted at amino acid level. The representative putative P450 sequences of P. ginseng and known P450 family from other plants were used to construct a phylogenetic tree. By comparing with other genomes, we found that most of the P450 genes from P. ginseng can be found in other dicot species. Depending on P450 family functions, seven P450 genes were selected, and for that organ specific expression, abiotic, and biotic studies were performed by quantitative reverse transcriptase-polymerase chain reaction. Different genes were found to be expressed differently in different organs. Biotic stress and abiotic stress transcript level was regulated diversely, and upregulation of P450 genes indicated the involvement of certain genes under stress conditions. The upregulation of the P450 genes under methyl jasmonate and fungal stress justifies the involvement of specific genes in secondary metabolite biosynthesis. Our results provide a foundation for further elucidating the actual function and role of P450 involved in various biochemical pathways in P. ginseng.

Sesquiterpenes are found naturally in plants and insects as defensive agents or pheromones. They are produced in the cytosolic acetate/mevalonate pathway for isoprenoid biosynthesis. The inducible sesquiterpene synthases (STS), which are responsible for the transformation of the precursor farnesyl diphosphate, appear to generate very few olefinic products that are converted to biologically active metabolites. In this study, we isolated the STS gene from Panax ginseng C.A. Meyer, designated PgSTS, and investigated the correlation between its expression and various abiotic stresses using real-time PCR. PgSTS cDNA was observed to be 1,883 nucleotides long with an open reading frame of 1,707 bp, encoding a protein of 568 amino acids. The molecular mass of the mature protein was determined to be 65.5 kDa, with a predicted isoelectric point of 5.98. A GenBank BlastX search revealed the deduced amino acid sequence of PgSTS to be homologous to STS from other plants, with the highest similarity to an STS from Lycopersicon hirsutum (55% identity, 51% similarity). Real-time PCR analysis showed that different abiotic stresses triggered significant induction of PgSTS expression at different time points.

The oxidation of glycolate to glyoxylate, a key step in plant photorespiration, is carried out by the peroxisomal flavoprotein glycolate oxidase (EC 1.1.3.15). To investigate the altered gene expression and the role of GOX in ginseng plant defense system, a cDNA clone containing a GOX gene designated as PgGOX was isolated and sequenced from Panax ginseng. The cDNA was 692 nucleotides long and have an open reading frame of 552 bp with a deduced amino acid sequence of 183 residues. A GenBank BlastX search revealed that the deduced amino acid of PgGOX shares a high degree homology with the Glycine max (95% identity). In the present study we analyzed the expression of PgGOX under various environmental stresses at different times using real time-PCR. The results showed that the expressions of PgGOX increased after various treatments involving salt, light, cold, ABA, SA, and copper treatment.

Ca 2+ and calmodulin (CaM), a key Ca 2+ sensor in all eukaryotes, have been implicated for defense responses of plants. Eukaryotic CaM contains four structurally and functionally similar Ca 2+ domains named Ⅰ, Ⅱ, Ⅲ and IV. Each Ca 2+ binding loop consists of 12 amino acid residues with ligands arranged spatially to satisfy the octahedral symmetry of Ca 2+ binding. To investigate the altered gene expression and the role of CaM in ginseng plant defense system, cDNA clone containing a CaM gene, designated PgCaM was isolated and sequenced from Panax ginseng. PgCaM, which has open reading frame of 450 nucleotides predicted to encode a precursor protein of 150 amino acid residues. Its sequence shows high homologies with a number of other CaMs, with more similarity to CaM of Daucus carota (AAQ63461). The expression of PgCaM in different P. ginseng organs was analyzed using real time PCR. The results showed that PgCaM expressed at different levels in young leaves, shoots, and roots of 3-week-old P. ginseng. In addition, the expressions of PgCaM under different abiotic stresses were analyzed at different time intervals.

Protective effects of exogenous spermidine (Spd), activity of antioxygenic enzymes, and levels of free radicals in a well-known medicinal plant, Panax ginseng was examined. Seedlings grown in salinized nutrient solution (150 mM NaCl) for 7 d exhibited reduced relative water content, plant growth, increased free radicals, and showing elevated lipid peroxidation. Application of Spd (0.01, 0.1, and 1 mM) to the salinized nutrient solution showed increased plant growth by preventing chlorophyll degradation and increasing PA levels, as well as antioxidant enzymes such as CAT, APX, and GPX activity in the seedlings of ginseng. During salinity stress, Spd was effective for lowering the accumulation of putrescine (Put), with a significant increase in the spermidine (Spd) and spermine (Spm) levels in the ginseng seedlings. A decline in the Put level ran parallel to the higher accumulation of proline (Pro), and exogenous Spd also resulted in the alleviation of Pro content under salinity. Hydrogen peroxide (H2O2) and superoxide (O2−) production rates were also reduced in stressed plants after Spd treatment. Furthermore, the combined effect of Spd and salt led to a significant increase in diamine oxidase (DAO), and subsequent decline in polyamine oxidase (PAO). These positive effects were observed in 0.1 and 1 mM Spd concentrations, but a lower concentration (0.01 mM) had a very limited effect. In summary, application of exogenous Spd could enhance salt tolerance of P. ginseng by enhancing the activities of enzyme scavenging system, which influence the intensity of oxidative stress.

A full length spermidine synthase (PgSPD) cDNA was isolated and characterized from the root of Panax ginseng C. A. Meyer. The cDNA was 1,188 nucleotides long and had an open reading frame of 1,002 bp with a deduced amino acid sequence of 333 residues. The calculated molecular mass of the matured protein is approximately 36.38 kDa with a predicated isoelectric point of 5.02. A GenBank BlastX search revealed that the deduced amino acid of PgSPD shares a high degree homology with the Lotus japonicas (78.5% identity, 84% similarity). In the present study we analyzed the expression of PgSPD under various environmental stresses at different time points using real time-PCR. We also determined polyamine content in adventitious roots under salt and chilling stress using HPLC. Our results reveal that PgSPD is slightly induced by mannitol and CuSO4. Otherwise, salt, chilling, abscisic acid and jasmonic acid triggered a significant induction (more than tenfold) of PgSPD within 12-24 h post-treatment, especially; PgSPD was prominently induced by salt (41.5-fold). These results suggest that the transcript of Spd gene involved in PA biosynthesis shows different profiles of expression in response to environmental stress.

The role of plant chitinases in protecting plants against a variety of fungal pathogens is well established. In the present study, a cDNA clone containing a class I chitinase (Chi-1) gene, designated as PgChi-1, has been isolated from the oriental medicinal plant Panax ginseng. PgChi-1 is predicted to encode a protein of 34.9 kDa consisting of 323 amino acid residues. PgChi-1 was found to be expressed constitutively in all of the studied organs of ginseng plant. Under various abiotic stress treatments including Cu, H2O2, mannitol, SA, JA, and NaCl, the expression of PgChi-1 in plantlets and hairy roots increased significantly compared to the control. When different parts of root were analyzed, maximum level was observed in taproot. In addition, levels of PgChi-1 expression were compared between healthy root and fungal, bacterial, and nematode infected root. Significant increase of PgChi-1 was noticed in pathogen infected roots than healthy roots. This study revealed that PgChi-1 may protect the P. ginseng under both biotic and abiotic stress conditions.