Molecular characterization of lipoxygenase genes and their expression analysis against biotic and abiotic stresses in Panax ginseng Abstract Lipoxygenase (LOX) belongs to a family of non-heme-iron-containing fatty acid dioxygenases that are widely distributed in plants and animals. LOX involved in the synthesis of jasmonic acid and six-carbon (C6) volatiles which is necessary for plant growth and responses to a wide range of biotic and abiotic stresses. We have isolated and characterized LOX cDNA clones from Panax ginseng Meyer. From their deduced amino acid sequences, two diverse classes of 9-LOX (LOX1, LOX2, and LOX3) and 13-LOX (LOX4, LOX5) are defined in P. ginseng. A GenBank Blast X search revealed that the deduced amino acid of PgLOXs share a high degree of homology with LOX from other plants and mammals especially in three distinct motifs; motif1 harboring iron binding regions, motif2 and motif3.Chloroplast localization was predicted for PgLOX5. PgLOXs displayed organ-specific expression, highly expressed in aerial parts of the plant such as 3-year old flower, stem and leaf tissues. PgLOXs mRNAs were elevated strongly by bacterial infection. Expression of PgLOXs was differentially induced in ginseng not only by mechanical damage and methyl jasmonate but also after exposure to withholding water. Ginseng 13-LOXs positively respond to wounding that may involve in production of C6 volatiles and jasmonic acid at the wounded sites. However, the higher expression of PgLOX3 by water deficit and 82 % of the nucleotide sequence identity with the EST from severe drought-stressed leaves of Populus (CU229089.1) at +6371 bp downstream of PgLOX3 genomic DNA structure can suggest drought tolerance role for PgLOX3. Ginseng LOX genes have different expression pattern which may suggest different specific function against various environmental stresses.

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The overall survival mechanism and secondary metabolite synthesis under cold stress conditions (4°C) at exogenous supply with KNO 3 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 spe cific 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 peroxi dase, 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 KNO 3 . 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.

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.