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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.

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.