Shohana Parvin

Spermidine alleviates the growth of saline-stressed ginseng seedlings through antioxidative defense system

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 7d exhibited reduced relative water content, plant growth, increased free radicals, and showing elevated lipid peroxidation. Application of Spd (0.01, 0.1, and 1mM) 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 1mM 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.

Isolation of Sesquiterpene Synthase Homolog from Panax ginseng C.A. Meyer

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.

Influence of potassium nitrate on antioxidant level and secondary metabolite genes under cold stress in Panax ginseng

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.

Classification and characterization of putative cytochrome P450 genes from Panax ginseng C. A. Meyer

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.

Isolation of S-adenosyl-L-methionine synthetase gene from Panax ginseng C.A. meyer and analysis of its response to abiotic stresses.

A cDNA clone containing a S-adenosyl-L-methionine synthetase (SAMS) gene, named as PgSAM, was isolated from a commercial medicinal plant Panax ginseng. PgSAM is predicted to encode a precursor protein of 307 amino acid residues, and its sequence shares high homology with a number of other plant SAMS. PgSAM is expressed at different levels in various organs of ginseng. The expression of PgSAM in adventitious roots and hairy roots of P. ginseng were analyzed using reverse transcriptase (RT)-PCR and real-time PCR under various abiotic stresses. Salt, salicylic acid, abscisic acid and chilling stresses induced PgSAM significantly at different time points within 2–72 h post-treatment. This study revealed that PgSAM may help to protect the plants against various abiotic stresses.

Isolation and Characterization of Cinnamoyl-CoA Reductase Gene from Panax ginseng C. A. Meyer

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44) catalyses the reduction of cinnamic acid CoA esters into their corresponding aldehydes, the first step of the phenylpropanoid pathway specially dedicated to monolignol biosynthesis. A cDNA clones encoding CCR have been isolated from Panax ginseng C.A. Meyer and its expression was investigated in response to abiotic stresses. The cDNA, designated PgCCR which is 865 nucleotides long and has an open reading frame of 590 bp with a deduced amino acid sequence of 176 residues. The PgCCR encoded protein possesses substantial homology with CCRs isolated and cloned from other sources; the highest identity (51.8%) was observed with CCR from Tomato (Lycopersicon esculentum). Under various stress conditions, expression patterns of the PgCCR were highly induced in adventitious and hairy roots by several abiotic stresses. These results indicated that PgCCR plays protective role against diverse environmental stresses.

Isolation and Characterization of Calmodulin Gene from Panax ginseng C. A. Meyer

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.

Isolation and Characterization of Glycolate Oxidase Gene from Panax ginseng C. A. Meyer

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.

Modulation of polyamine levels in ginseng hairy root cultures subjected to salt stress

Modulation of differential gene expression and change of polyamine content by salt stress are analyzed for the first time in a well-known medicinal plant, Panax ginseng C.A. Meyer. Three ginseng genes (PgSPD, PgSAMDC, and PgADC) involved in polyamine biosynthesis showed differential up-regulation patterns after 1 and 7 days of salt treatments. The modulation of gene expression resulted in the elevation of total polyamine content with relatively high levels of spermidine and spermine, while putrescine level diminished depending on the salt concentration. Conversely, salt stress led to a significant increase in diamine oxidase and subsequent decline in polyamine oxidase. The proline content caused by salinity follows a similar pattern as the total polyamine content and exogenous spermidine also resulted in the alleviation of proline content under salinity. Further, polyamine biosynthesis inhibitors, such as cyclohexylamine and methylglyoxal bis-(guanylhydrazone) mediated down-regulation of PgSPD and PgSAMDC, and affected cellular polyamine levels. Thus, polyamines may enhance the ginseng plant tolerance in response to the salt stress by increasing the levels of endogenous polyamines.