DNA mutagenesis in 2- and 20-yr-old Panax ginseng cell cultures

Previously, Panax ginseng var. Mimaki C.A. Meyer had been shown to accumulate genetic mutations during long-term propagation of a callus culture over a period of 20 yr. In this study, we analyzed the mutation types and frequency in a 2-yr-old P. ginseng callus culture and compared it with the 20-yr-old callus culture, and leaves of cultivated plants. We analyzed the sequence variability between the Actin genes, which are a family of housekeeping genes; phenylalanine ammonia-lyase (PAL) and dammarenediol synthase (DDS), which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinases (SERK), which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-yr-old P. ginseng callus culture was markedly higher than in cultivated plants, but lower than in the 20-yr-old callus culture. Most of the mutations in the 2-yr-old P. ginseng calli were A?G and T?C transitions, as in the 20-yr-old calli and intact P. ginseng plants. The number of nonsynonymous mutations was higher in the 2- and 20-yr-old callus cultures than the number of nonsynonymous mutations in cultivated P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using a methylation-sensitive DNA fragmentation assay, we showed the level of methylcytosine to be higher in the DNA of the 20-yr-old P. ginseng calli that than in the DNA of the 2-yr-old cultures.     

Regulation of somatic embryogenesis in Panax ginseng C. A. Meyer cell cultures by PgCDPK2DS1

We isolated the full-length cDNA of PgCDPK2DS1 gene whose expression was significantly increased at early stages of embryo development in cell cultures of ginseng P. ginseng 2c3. Interest in this gene also was supported by its nonstandard structure: the amino acid sequence of the PgCDPK2DS1 gene contained only the N-terminal domain and 80% of the kinase domain. Overexpression of the PgCDPK2DS1 gene in nonembryonic calli 1c resulted in the appearance of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture 1c-2d. Also, expression of the plant embryogenesis marker genes WUS and SERK significantly increased in cell culture 1c-2d. The observed embryo-like structures were at early stages of embryo development; attempts to obtain adult plants from these embryo-like structures were unsuccessful. Overexpression of PgCDPK2DS1 gene in the embryonic cell culture PG resulted in a decrease of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture PG-2d. Moreover, expression of plant embryogenesis marker genes WUS and SERK and expression of the endogenous PgCDPK2DS1 significantly decreased in the cell culture PG-2d. Thus, for the first time it was shown that the PgCDPK2DS1 gene is involved in the regulation of somatic embryogenesis in P. ginseng cell cultures.     

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 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.     

Structure and expression profiling of a novel calcium-dependent protein kinase gene <Emphasis Type="Italic">PgCDPK1a</Emphasis> in roots,leaves, and cell cultures of <Emphasis Type="Italic">Panax ginseng</Emphasis>

Calcium-dependent protein kinases (CDPKs) are proposed to play an essential role in plant defense responses. In this study, we aimed to define the full sequence of a CDPK gene of Panax ginseng and analyze its expression in roots, leaves, and cell cultures of P. ginseng, one of the most valuable Chinese traditional medicinal herbs. We isolated the full-length cDNA of a P. ginseng CDPK gene, which was designated PgCDPK1a. PgCDPK1a shares high sequence identity at the amino acidic level with previously reported CDPK sequences for other plant species. We analyzed PgCDPK1a expression in the leaves of wild-growing P. ginseng plants, and in the roots and leaves of cultivated P. ginseng plants growing in an open experimental nursery at a natural ginseng habitat. PgCDPK1a was more actively expressed in the young leaves of cultivated P. ginseng plants than in that of wild-growing ones. Finally, we analyzed the expression of the gene in control GV and five rolC and rolB transgenic callus cultures of P. ginseng with different levels of fresh biomass accumulation, pathogen-related gene expression, and ginsenoside production. We observed a strong positive correlation between fresh biomass accumulation of P. ginseng cell cultures and expression of the PgCDPK1a gene. There was a less clear negative correlation between the expression of pathogen-related genes and the content of ginsenosides with the PgCDPK1a expression in cell cultures of P. ginseng. Perhaps, PgCDPK1a is involved in ginseng growth, as a positive regulator.     

RAPD analysis of genome variability of planted ginseng,Panax ginseng

Genome variability of 23 ginseng plants (Panax ginseng) grown in culture in Primorskii Krai was studied by RAPD method. Eleven arbitrary chosen primers were used to analyze 138 loci of DNA samples, 17 of which appeared to be polymorphic. The OPD-11-1000 fragment was found to be a RAPD marker allowing plants to be differentiated according to their morphotype. Using five primers, it was demonstrated that the genetic polymorphism of the cultivated plants is lower than that in nature (7.6% and 10.6%, respectively). Dendrograms of genetic relatedness are in accord with genetic differences between individuals of plantedP. ginseng belonging to different morphotypes, and demonstrate close relatedness of one of the morphotypes to wild plants. This morphotype could be recommended for reintroduction into natural habitats.     

RAPD and Allozyme Analysis of Genetic Diversity in Panax ginseng C.A. Meyer and P. quinquefolius L.

Inter- and intraspecific variation of two ginseng species Panax ginseng and P. quinquefolius was estimated by studying 159 RAPD and 39 allozyme loci. Parameters of polymorphism and genetic diversity were determined and a tree was constructed to characterize the differences between individual plants, samples, and species. Genetic variation in P. ginseng proved to be lower than in P. quinquefolius. Gene diversity in the total P. ginseng sample was comparable with the mean expected heterozygosity of herbaceous plants. This suggests that wild P. ginseng plants in various areas of the currently fragmented natural habitat and cultivated plants of different origin have retained a significant proportion of their gene pool. The mean heterozygosity calculated per polymorphic locus for the RAPD phenotypes is similar to that of the allozyme loci and may be helpful in estimating gene diversity in populations of rare and endangered plant species.     

Cell culture system versus adventitious root culture system in Asian and American ginseng: a collation

Panax ginseng and Panax quinquefolius of Panax genus are valuable as health foods as well as pharmaceuticals for the treatment of cancer, diabetes and ageing as these plants possess saponins. In the current study, Cell and adventitious root cultures of P. ginseng and P. quinquefolius were investigated for the biomass, cell division, saponin content and ginsenosides profile from four lines namely P. quinquefolius (AM), P. ginseng mountain (Mt.) Baekdu line, P. ginseng Cheong-sol line (CS) and P. ginseng CBN line (CBN) with the objective of comparing cell and adventitious root systems to check their efficacy for the production of ginseng saponins. Additionally, genes related to ginsenoside biosynthesis were also analyzed concerning to cell and adventitious root lines. The results indicated that various cell lines were better in multiplication and growth compared to adventitious root lines. However, adventitious root lines showed higher accumulation of dry biomass (1.5–2 fold) than that of cell lines. CS adventitious root line showed higher saponin content and ginsenoside productivity (10.48 mg·g^?1 DW, 12.88 mg·L^?1, respectively) than that of CS cell line (9.50 mg·g^?1 DW, 2.39 mg·L^?1, respectively). Especially, Rd ginsenoside productivity of CS adventitious root line recorded fourfold higher than CS cell line. Genes which are related to ginsenoside biosynthesis such as P. ginseng squalene synthase (PgSS2), P. ginseng squalene epoxidase (PgSE2), P. ginseng protopanaxadial synthase (PgPPDS) and P. ginseng protopanaxatriol synthase (PgPPTS) were analyzed by real time quantitative polymerase chain reaction to support ginsenoside production. The adventitious root culture system described in this study is useful system for biomass and ginsenoside production.     

Transcriptome profiling characterizes phosphate deficiency effects on carbohydrate metabolism in rice leaves

Phosphorus (P) is a structural component of nucleic acids and phospholipids and plays important roles in plant growth and development. P accumulation was significantly reduced (about 35%) in rice leaves from plants grown under low (32 μM) P compared to 320 μM P grown plants. Genome response to low P was examined using the rice 60K oligonucleotide DNA microarrays. At the threshold significance of |log₂| fold > 2.0, 21,033 genes (about 33.7% of all genes on the microarray) were affected by P deficiency. Among all genes on the microarray, 4271 genes were sorted into 51 metabolic pathways. Low P affected 1494 (35.0%) genes and the largest category of genes was related to sucrose degradation to ethanol and lactate pathway. To survey the role of P in rice, 25 pathways were selected based on number of affected genes. Among these pathways, cytosolic glycolysis contained the least number of upregulated but most down-regulated genes. Low P decreased glucose, pyruvate and chlorophyll, and genes related to carbon metabolism and chlorophyllide a biosynthesis. However, sucrose and starch levels increased. These results indicate that P nutrition affects diverse metabolic pathways mostly related to glucose, pyruvate, sucrose, starch, and chlorophyll a.     

Nucleotide substitutions in <Emphasis Type="Italic">rolC</Emphasis> and <Emphasis Type="Italic">nptII</Emphasis> gene sequences during long-term cultivation of <Emphasis Type="Italic">Panax ginseng</Emphasis> cell cultures

It has been shown previously that the rolC gene from Agrobacterium tumefaciens gene was stably and highly expressed in 15-year-old Panax ginseng transgenic cell cultures. In the present report, we analyze in detail the nucleotide composition of the rolC and nptII (neomycin phosphotransferase) genes, which is the selective marker used for transgenic cell cultures of P. ginseng. It has been established that the nucleotide sequences of the rolC and nptII genes underwent mutagenesis during cultivation. Particularly, 1–4 nucleotide substitutions were found per sequence in the 540 and 798 bp segments of the complete rolC and nptII genes, respectively. Approximately half of these nucleotide substitutions caused changes in the structure of the predicted gene product. In addition, we attempted to determine the rate of accumulation of these changes by comparison of DNA extracted from P. ginseng cell cultures from 1995 to 2007. It was observed that the frequency of nucleotide substitutions for the rolC and nptII genes in 1995 was 1.21 ± 0.02 per 1,000 nucleotides analyzed, while in 2007, the nucleotide substitutions significantly increased (1.37 ± 0.07 per 1,000 nucleotides analyzed). Analyzing the nucleotide substitutions, we found that substitution to G or to C nucleotides significantly increased (in 1.9 times) in the rolC and nptII genes compared with P. ginseng actin gene. Finally, the level of nucleotide substitutions in the rolC gene was 1.1-fold higher when compared with the nptII gene. Thus, for the first time, we have experimentally demonstrated the level of nucleotide substitutions in transferred genes in transgenic plant cell cultures.     

cDNA-AFLP analysis of salt-inducible genes expression in Chrysanthemum lavandulifolium under salt treatment

Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is a halophyte species that belongs to the Asteraceae family, and the genus Chrysanthemum. It is one of the ancestors of C. × morifolium Ramatella. Understanding the tolerance mechanism associated with salt stress in C. lavandulifolium could provide important information for explaining the salt tolerance of higher plants and could also help enhancing breeding programs of cultivated Chrysanthemum. In this study, cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to detect differential gene expression in leaves of C. lavandulifolium in response to NaCl treatment. The determination of membrane permeablility, peroxidase activity (POD), malon-dialdehyde (MDA), as well as proline and leaf chlorophyll contents under different NaCl concentrations showed that a 200 mM NaCl treatment was an optimal condition for the cDNA-AFLP experiment. Using this concentration during different times (0, 3 h, 12 h, 24 h and 48 h), we obtained 1930 cDNA fragments using 64 primers. After sequencing 234 randomly chosen cDNA clones and BLASTx analyzing, we got 129 expressed sequence tags (ESTs) which had no significant homology with other sequences, 85 ESTs were homologous to genes with known functions, whereas the rest of ESTs showed homology to unclassified or putative proteins. 25 ESTs that were similar to known functional genes involved in several abiotic and biotic stresses were confirmed by semi-quantitative RT-PCR and qRT-PCR. The expression patterns of these salt-responsive genes not only responded to salt stress but also to plant hormones, such as abscisic acid (ABA), and to other abiotic stresses such as drought and cold. These results indicate an extensive cross-talk among several stresses. Our results provide interesting information for further understanding the molecular mechanisms of salt tolerance in C. lavandulifolium.     

Molecular evolution and nucleotide diversity of nuclear plastid phosphoglycerate kinase (PGK) gene in Triticeae (Poaceae)

Levels of nucleotide divergence provide key evidence in the evolution of polyploids. The nucleotide diversity of 226 sequences of pgk1 gene in Triticeae species was characterized. Phylogenetic analyses based on the pgk1 gene were carried out to determine the diploid origin of polyploids within the tribe in relation to their A^u, B, D, St, Ns, P, and H haplomes. Sequences from the Ns genome represented the highest nucleotide diversity values for both polyploid and diploid species with π = 0.03343 and θ = 0.03536 for polyploid Ns genome sequences and π = 0.03886 and θ = 0.03886 for diploid Psathyrostachys sequences, while Triticum urartu represented the lowest diversity among diploid species at π = 0.0011 and θ = 0.0011. Nucleotide variation of diploid Aegilops speltoides (π = 0.2441, presumed the B genome donor of Triticum species) is five times higher than that (π = 0.00483) of B genome in polyploid species. Significant negative Tajima's D values for the St, A^u, and D genomes along with high rates of polymorphisms and low sequence diversity were observed. Origins of the A^u, B, and D genomes were linked to T. urartu, A. speltoides, and A. tauschii, respectively. Putative St genome donor was Pseudoroegneria, while Ns and P donors were Psathyrostachys and Agropyron. H genome diploid donor is Hordeum.     

Effects of exogenous hormones on leaf photosynthesis of Panax ginseng

Ginseng (Panax ginseng) is a typical perennial shade plant. Aim of this study was to investigate the effects of exogenous hormones on photosynthesis of P. ginseng. At different growth stages, the aerial parts of P. ginseng plants were cut at the stem base and they were inserted into the nutrient solutions containing different exogenous hormones. Then the leaf photosynthesis and water absorbing capacity (absorbing water mass) of the excised plants were measured. The results showed that exogenous abscisic acid (ABA) decreased significantly net photosynthetic rate (P _N), stomatal conductance, transpiration rate, and absorbed water mass of excised P. ginseng at all growth stages, while both cytokinin (CTK) and indole-3-acetic acid (IAA) enhanced those parameters. Comparing different growth stages, ABA caused more severe inhibition of leaf photosynthesis at the early growth stage, while CTK and IAA showed significant enhancement of leaf photosynthesis at later growth stage. ABA reduced highly intercellular CO₂ concentration of P. ginseng at the flowering and green fruit stages, but it had only a small effect at red fruit early and red fruit stages. During the early growth stage, the inhibitory effect of ABA on leaf P _N might be caused mainly due to the stomatal limitation. However, the reason for this reduction was complex at the later growth stage and it included stomatal and other factors.     

Cytological analysis of ginseng carpel development

Panax ginseng Meyer, commonly known as ginseng, is considered one of the most important herbs with pharmaceutical values due to the presence of ginsenosides and is cultivated for its highly valued root for medicinal purposes. Recently, it has been recognized that ginseng fruit contains high contents of triterpene such as ginsenoside Re as pharmaceutical compounds. However, it is unclear how carpel, the female reproductive tissue of flowers, is formed during the three-year-old growth before fruit is formed in ginseng plants. Here, we report P. ginseng carpel development at the cytological level, starting from the initial stage of ovule development to seed development. The carpel of P. ginseng is composed of two free stigmas, two free styles, and one epigynous bilocular ovary containing one ovule in each locule. Based on our cytological study, we propose that the female reproductive development in P. ginseng can be classified into seven stages: early phase of ovule development, megasporogenesis, megagametogenesis, pre-fertilization, fertilization, post-fertilization, and seed development. We also describe the correlation of the female and male gametophyte development and compare morphological differences in carpel development between ginseng and other higher plants. One unique feature for ginseng seed development is that it takes 40 days for the embryo to develop to the early torpedo stage and that the embryo is small relative to the seed size, which could be a feature of taxonomic importance. This study will provide an integral tool for the study of the reproductive development and breeding of P. ginseng.     

De novo assembly and characterization of the complete chloroplast genome of radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) is an edible root vegetable crop that is cultivated worldwide and whose genome has been sequenced. Here we report the complete nucleotide sequence of the radish cultivar WK10039 chloroplast (cp) genome, along with a de novo assembly strategy using whole genome shotgun sequence reads obtained by next generation sequencing. The radish cp genome is 153,368 bp in length and has a typical quadripartite structure, composed of a pair of inverted repeat regions (26,217 bp each), a large single copy region (83,170 bp), and a small single copy region (17,764 bp). The radish cp genome contains 87 predicted protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Sequence analysis revealed the presence of 91 simple sequence repeats (SSRs) in the radish cp genome.     

Protein-Precursor tRNA Contact Leads to Sequence-Specific Recognition of 5′ Leaders by Bacterial Ribonuclease P

Bacterial ribonuclease P (RNase P) catalyzes the cleavage of 5′ leader sequences from precursor tRNAs (pre-tRNAs). Previously, all known substrate nucleotide specificities in this system are derived from RNA-RNA interactions with the RNase P RNA subunit. Here, we demonstrate that pre-tRNA binding affinities for Bacillus subtilis and Escherichia coli RNase P are enhanced by sequence-specific contacts between the fourth pre-tRNA nucleotide on the 5′ side of the cleavage site (N(− 4)) and the RNase P protein (P protein) subunit. B. subtilis RNase P has a higher affinity for pre-tRNA with adenosine at N(− 4), and this binding preference is amplified at physiological divalent ion concentrations. Measurements of pre-tRNA-containing adenosine analogs at N(− 4) indicate that specificity arises from a combination of hydrogen bonding to the N6 exocyclic amine of adenosine and steric exclusion of the N2 amine of guanosine. Mutagenesis of B. subtilis P protein indicates that F20 and Y34 contribute to selectivity at N(− 4). The hydroxyl group of Y34 enhances selectivity, likely by forming a hydrogen bond with the N(− 4) nucleotide. The sequence preference of E. coli RNase P is diminished, showing a weak preference for adenosine and cytosine at N(− 4), consistent with the substitution of Leu for Y34 in the E. coli P protein. This is the first identification of a sequence-specific contact between P protein and pre-tRNA that contributes to molecular recognition of RNase P. Additionally, sequence analyses reveal that a greater-than-expected fraction of pre-tRNAs from both E. coli and B. subtilis contains a nucleotide at N(− 4) that enhances RNase P affinity. This observation suggests that specificity at N(− 4) contributes to substrate recognition in vivo. Furthermore, bioinformatic analyses suggest that sequence-specific contacts between the protein subunit and the leader sequences of pre-tRNAs may be common in bacterial RNase P and may lead to species-specific substrate recognition.