Somatic embryogenesis in the Panax ginseng cell culture induced by the rolC oncogene is associated with increased expression of WUS and SERK genes

The embryogenic cell culture 2c3 was previously obtained by the transfer of the rolC gene from Agrobacterium rhizogenes into ginseng callus cells. It was found by us that the expression of SERK and WUS genes in the embryogenic culture 2c3 is increased. These genes are known to be responsible for the initial stimulus to the development of embryogenesis in plants. Taking into consideration earlier data, we suppose that the development of somatic embryos in the ginseng cell culture 2c3 is associated with changes in the work of the calcium signaling system and with the activation of expression of SERK and WUS genes. Original Russian Text ? K.V. Kiselev, G.K. Tchernoded, 2009, published in Genetika, 2009, Vol. 45, No. 4, pp. 511–518.     

Calcium-dependent mechanism of somatic embryogenesis in oncogene rolC expressing cell cultures of Panax ginseng

It was shown earlier, that ginseng embryogenic cell culture 2c3 was obtained as a result of callus cells transformation with the Agrobacterium rhizogenes rolC oncogene. In the present report we determine that inhibitors of Ca2+-channels (LaCl3, verapamil, niflumic acid) certainly lowered the quantity of somatic embryos in the 2c3 cell culture. This is the evidence of the influence of calcium-dependent signal system on plant embryogenesis. Protein kinases inhibitors W7 and H7 also caused the lowering of somatic embryos quantity in the 2c3 cell culture. We analysed changes of CDPK genes expression in embryogenic 2c3 cell culture. Total expression decreased 1.2-1.5 times comparing with the control callus culture. CDPK expression in the 2c3 embryogenic culture lowered by the inhibition of expression of the gene subfamilies PgCDPK1 (PgCDPK1a and PgCDPK1b) and PgCDPK3 (PgCDPK3a). At the same time, expression of PgCDPK2 gene subfamily (PgCDPK2b and PgCDPK2d) was increased. We suppose that genes of PgCDPK2 subfamily might be responsible for the embryogenesis initiation in the 2c3 ginseng cell culture. It was shown for the first time that the rolC gene and the process of embryogenesis could change expression of particular forms of CDPK genes.     

Calcium-dependent mechanism of somatic embryogenesis in Panax ginseng cell cultures expressing the rolC oncogene

The Panax ginseng 2c3 embryogenic cell culture was earlier obtained by callus cell transformation with Agrobacterium rhizogenes rolC. Calcium channel blockers (LaCl₃, verapamil, and niflumic acid) reduced the production of somatic embryos in the 2c3 culture, implicating the Ca²⁺ signaling system in plant somatic embryogenesis. The protein kinase inhibitors W7 and H7 also decreased the yield of somatic embryos in the 2c3 culture. The total CDPK expression in the 2c3 culture was 1.2-to 1.5-fold lower than in a control callus culture as a result of a silencing of the genes belonging to the PgCDPK1 (PgCDPK1a and PgCDPK1b) and PgCDPK3 (PgCDPK3a) subfamilies. Expression of the PgCDPK2 subfamily genes (PgCDPK2b and PgCDPK2d) was increased. It was assumed that some genes of the PgCDPK1, PgCDPK2, and PgCDPK3 subfamilies were responsible for generation of embryogenic cells in the 2c3 culture. For the first time, rolC expression and embryogenesis were associated with changes in the expression of certain CDPK genes.     

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.     

Mutation of Panax ginseng genes during long-term cultivation of ginseng cell cultures

It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during the long-term cultivation of transgenic cell cultures of Panax ginseng. In the present report, we analyzed the nucleotide sequences of selected plant gene families in the 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We sequenced the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase (PAL) and dammarenediol synthase genes (DDS), which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase (SERK) genes, which control plant development. We demonstrate that the plant genes also developed mutations during long-term cultivation. The highest level of nucleotide substitution was detected in the sequences of the SERK genes (2.00 ± 0.11 nt per 1000 nt), and the level was significantly higher when compared with the cultivated P. ginseng plant. Interestingly, while the diversity of Actin genes was similar in the P. ginseng cell culture and the cultivated plants, the diversity of the DDS and SERK genes was less in the 20-year-old cell culture than in the cultivated plants. In this work, we detail the level of nucleotide substitutions in different plant genes during the long-term culture of plant cells.     

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.     

The Agrobacterium rhizogenes rolC-gene-induced somatic embryogenesis and shoot organogenesis in Panax ginseng transformed calluses

Expression of the Agrobacterium rhizogenes rolC gene in Panax ginseng callus cells results in formation of tumors that are capable to form roots. The selection of non-root forming tumor clusters yielded the embryogenic 2c3 callus line, which formed somatic embryos and shoots independently of external growth factors. Although the 2c3 somatic embryos developed through a typical embryogenesis process, they terminated prematurely and repeatedly formed adventitious shoot meristems and embryo-like structures. A part of the shoots and somatic embryos formed enlarged and fasciated meristems. This is the first indication of the rolC gene embryogenic effect and, to our knowledge, the first indication that a single gene of non-plant origin can induce somatic embryogenesis in plants.     

Microspore embryogenesis in wheat: new marker genes for early, middle and late stages of embryo development

Microspore embryogenesis involves reprogramming of the pollen immature cell towards embryogenesis. We have identified and characterized a collection of 14 genes induced along different morphological phases of microspore-derived embryo development in wheat (Triticum aestivum L.) anther culture. SERKs and FLAs genes previously associated with somatic embryogenesis and reproductive tissues, respectively, were also included in this analysis. Genes involved in signalling mechanisms such as TaTPD1-like and TAA1b, and two glutathione S-transferase (GSTF2 and GSTA2) were induced when microspores had acquired a ‘star-like’ morphology or had undergone the first divisions. Genes associated with control of plant development and stress response (TaNF-YA, TaAGL14, TaFLA26, CHI3, XIP-R; Tad1 and WALI6) were activated before exine rupture. When the multicellular structures have been released from the exine, TaEXPB4, TaAGP31-like and an unknown embryo-specific gene TaME1 were induced. Comparison of gene expression, between two wheat cultivars with different response to anther culture, showed that the profile of genes activated before exine rupture was shifted to earlier stages in the low responding cultivar. This collection of genes constitutes a value resource for study mechanism of intra-embryo communication, early pattern formation, cell wall modification and embryo differentiation.     

Characterization and expression analysis of SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) genes in sexual and apomictic Paspalum notatum

The SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene plays a fundamental role in somatic embryogenesis of angiosperms, and is associated with apomixis in Poa pratensis. The objective of this work was to isolate, characterize and analyze the expression patterns of SERK genes in apomictic and sexual genotypes of Paspalum notatum. A conserved 200-bp gene fragment was amplified from genomic DNA with heterologous primers, and used to initiate a chromosomal walking strategy for cloning the complete sequence. This procedure allowed the isolation of two members of the P. notatum SERK family; PnSERK1, which is similar to PpSERK1, and PnSERK2, which is similar to ZmSERK2 and AtSERK1. Phylogenetic analyses indicated that PnSERK1 and PnSERK2 represent paralogous sequences. Southern-blot hybridization indicated the presence of at least three copies of SERK genes in the species. qRT-PCR analyses revealed that PnSERK2 was expressed at significantly higher levels than PnSERK1 in roots, leaves, reproductive tissues and embryogenic calli. Moreover, in situ hybridization experiments revealed that PnSERK2 displayed a spatially and chronologically altered expression pattern in reproductive organs of the apomictic genotype with respect to the sexual one. PnSERK2 is expressed in nucellar cells of the apomictic genotype at meiosis, but only in the megaspore mother cell in the sexual genotype. Therefore, apomixis onset in P. notatum seems to be correlated with the expression of PnSERK2 in nucellar tissue.     

Regeneration of Plants from Protoplasts of Arabidopsis thaliana L. cv. Columbia (C24), Via Direct Embryogenesis

This report describes a protocol for the regeneration of fertileplants from mesophyll protoplasts of Arabidopsis thaliana raceColumbia (C24). Regeneration was rapid and reproducible. Theprotocol is especially novel in that a large proportion of regeneratingprotoplasts regenerated via direct somatic embryogenesis. Protoplastsisolated from in vitro-grown plants entered sustained divisionafter 3–5 d in culture medium and over a period of severaldays 6–22% of protoplasts underwent at least one celldivision. Approximately 2–16% of these protoplasts continuedto divide and after 3 weeks in culture had formed macroscopiccolonies, of which 70–80% were regular embryo-like structures.Four weeksafter release from the alginate culture matrix andtransfer to solid medium in the light, 68–88% of thesestructures had produced well-developed shoots. Shoots couldbe maintained in culture or established in peat blocks. Theregenerated plants were fertile. Key words: Arabidopsis thaliana, protoplast, regeneration, embryogenesis, dicamba     

Draft Genome Sequencing of Bacillus sp. Strain M2-6, Isolated from the Roots of Korean Ginseng,Panax ginseng C. A. Meyer,after High-Hydrostatic-Pressure Processing

A bacterium, designated M2-6, was isolated from Korean ginseng, Panax ginseng C. A. Meyer, roots after high-hydrostatic-pressure processing. On the basis of 16 rRNA gene phylogeny, the isolate was presumptively identified as a Bacillus sp. Here we report the draft genome sequence of Bacillus sp. strain M2-6 (= KACC 16563).     

FISH and GISH analysis of the genomic relationships amongPanax species

Ginseng is a well-known medicinal plant that has been used as an anti-aging agent for many years in East Asia. In the genusPanax, only three species,P. ginseng (Oriental ginseng),P. quinquefolius (American ginseng) andP. notoginseng (Chinese ginseng), are currently considered to be important medicinal herbs. Despite the increase in their breeding value, molecular cytogenetic information on the species is not available. To analyze the genomic relationships among thePanax species, FISH (fluorescencein situ hybridization) and GISH (genomicin situ hybridization) techniques were applied. FISH analysis revealed that the 45S and 5S rRNA genes ofP. notoginseng (2n=2x=24) andP. ginseng (2n=4x=48) cluster on a single locus on different chromosomes, whileP. quinquefolius (2n=4x=48),P. japonicus (2n=4x=48), and Korean wild ginseng (2n =4x= 48) had one locus of the 45S rRNA gene and two loci of the 5S rRNA gene, respectively. GISH analysis using genomic DNA as a probe detected strong cross-hybridization of genomes betweenP. ginseng andP. quinquefolius. GISH analysis of other species showed weak or no distinct signals on the chromosomes. Our data revealed thatP. ginseng andP. quinquefolius showed the highest degree of homology, indicating that these species diverged in most recent years.     

Two new dammarane-type triterpene saponins from Korean red ginseng and their anti-inflammatory effects

Panax ginseng has been the subject of extensive research on potential medicinal materials. The goal of this study was search the chemical constituents and biological activities of processed Panax ginseng, Korean red ginseng. Our efforts led to the isolation eleven compounds (1–11) including two new compounds 1 and 2 from Korean red ginseng using various chromatographic techniques. Chemical structures of isolated compounds were demonstrated by spectroscopic methods (1D-, 2D-NMR, and HR-ESI-MS). The anti-inflammatory effects of the compounds were investigated by inhibiting IL-6 and TNF-α secretion in LPS-activated RAW264.7 cells. Additionally, the effects of the compounds on the expression of COX-2 and iNOS were examined by Western blotting. Compound 1 significantly reduced the level of proinflammatory cytokines IL-6 and TNF-α secretion in LPS-activated RAW264.7 cells and the expression of COX-2 and iNOS inflammatory enzymes in the cells. These results suggested that compound 1, a new ginsenoside might useful in treatment of inflammation.