RolB gene-induced production of isoflavonoids in transformed Maackia amurensis cells.

Maackia amurensis Rupr. et Maxim is a valuable leguminous tree grown in the Russian Far East, in China, and in Korea. Polyphenols from the heartwood of this species (primarily stilbenes and isoflavonoids) possess strong hepatoprotective activity. Callus culture of M. amurensis produced isoflavonoids and their derivatives. In pharmacological experiments, the callus complex was at least as effective, as the plant complex. To increase the yield of isoflavonoids, calli were transformed with the rolB gene of Agrobacterium rhizogenes. Neomycin phosphotransferase (nptII) gene was used for transgenic cell selection. Three rolB transgenic callus lines with different levels of the rolB gene expression were established. Insertion of the rolB gene caused alterations in callus structure, growth, and isoflavonoid production, and stronger alterations were observed with higher expression levels. MB1, MB2, and MB4 cultures accumulated 1.4, 1.5, and 2.1 % of dry weight (DW) isoflavonoids, respectively. In contrast, the empty vector-transformed MV culture accumulated 1.22 % DW. Isoflavonoid productivity of the obtained MB1, MB2, and MB4 cultures was equal to 117, 112, and 199 mg/L of medium, respectively, comparing to 106 mg/L for the MV culture. High level of expression of the rolB gene in MB4 culture led to a 2-fold increase in the isoflavonoid content and productivity and reliably increased dry biomass accumulation. Lower expression levels of the rolB gene in MB1 and MB2 calli did not significantly enhance biomass accumulation and isoflavonoid content, although the rolB gene activated isoflavonoid biosynthesis during the early growth stages and caused the increased content of several distinct compounds.

The rolC gene increases caffeoylquinic acid production in transformed artichoke cells.

Caffeoylquinic acids are found in artichokes, and they are currently considered important therapeutic or preventive agents for treating Alzheimer's disease and diabetes. We transformed artichoke [the cultivated cardoon or Cynara cardunculus var. altilis DC (Asteraceae)] with the rolC gene, which is a known inducer of secondary metabolism. High-performance liquid chromatography with UV and high-resolution mass spectrometry (HPLC-UV-HRMS) revealed that the predominant metabolites synthesized in the transgenic calli were 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, and chlorogenic acid. The rolC-transformed calli contained 1.5% caffeoylquinic acids by dry weight. The overall production of these metabolites was three times higher than that of the corresponding control calli. The enhancing effect of rolC remained stable over long-term cultivation.

The production of class III plant peroxidases in transgenic callus cultures transformed with the rolB gene of Agrobacterium rhizogenes.

The production of plant peroxidases by plant cell cultures is of great interest because of the potential for industrial applications. We used plant cell cultures overexpressing the rolB gene to produce increased amounts of plant class III peroxidases. The rolB gene ensured the stable and permanent activation of peroxidase activity in the transformed callus cultures of different plants. In particular, the total peroxidase activity in transformed Rubia cordifolia cells was increased 23-86-fold, and the abundance of the major peroxidase gene transcripts was increased 17-125-fold (depending on the level of rolB expression) compared with non-transformed control calli. The peroxidase-activating effect of rolB was greater than that of other peroxidase inducers, such as external stresses and methyl jasmonate.

Structural and electronic properties of perylene from first principles calculations.

The electronic structure of crystalline perylene has been investigated within the framework of density functional theory including van der Waals interactions. The computations of the lattice parameters and cohesive energy have good agreement with experimental values. We have also calculated the binding distance and energy of perylene dimers, using different schemes, which include van der Waals interactions.

Molecular cloning and characterization of seven class III peroxidases induced by overexpression of the agrobacterial rolB gene in Rubia cordifolia transgenic callus cultures.

Here, seven new class III peroxidase genes of Rubia cordifolia L., RcPrx01-RcPrx07, were isolated and characterized. Expression of the Prx genes was studied in R. cordifolia aerial organs as well as in cells transformed with the rolB and rolC genes of Agrobacterium rhizogenes and cells transformed with the wild-type A. rhizogenes A4 strain. In rolC- and rolB-transformed cells, the rol genes were expressed under the control of the 35S promoter, whereas in A. rhizogenes A4-transformed cells the rol genes were expressed under the control of their native promoters. All studied peroxidase genes were greatly upregulated in rolB-overexpressing cells. In contrast, overexpression of the rolC gene and expression of the rol genes under the control of their native promoters had little effect on the abundance of peroxidase transcripts. In accordance with this observation, peroxidase activity was substantially increased in rolB cells and was slightly affected in other transformed cells. Our results indicate that rolB strictly affects the regulation of a set of seven R. cordifolia class III peroxidases.

Catechin production in cultured cells of Taxus cuspidata and Taxus baccata.

The main polyphenols in callus and cell suspension cultures of Taxus cuspidata and T. baccata were (+)-catechin and (-)-epicatechin, while lignans, such as (+)-taxiresinol, (+)-isotaxiresinol, (+)-isolariciresinol and (-)-secoisolariciresinol, were present in trace amounts. T. cuspidata cells contained 1.7% (+)-catechin and 2.4% (-)-epicatechin on dry wt basis but when stimulated with methyl jasmonate produced 3.4% catechin and 5.2% epicatechin. These are the highest levels of these metabolites obtained in plant cell cultures.

Induction of anthraquinone biosynthesis in Rubia cordifolia cells by heterologous expression of a calcium-dependent protein kinase gene.

Calcium-dependent protein kinases (CDPKs) play an important role in plant cell responses to stress and pathogenic attack. In this study, we investigated the effect of heterologous expression of the Arabidopsis CDPK gene, AtCPK1, on anthraquinone production in transgenic Rubia cordifolia cells. AtCPK1 variants (a constitutively active, Ca(2+) -independent form and a non-active form used as a negative control) were transferred to callus cells by agrobacterial transformation. Overexpression of the constitutively active, Ca(2+) -independent form in R. cordifolia cells caused a 10-fold increase in anthraquinone content compared with non-transformed control cells, while the non-active form of AtCPK1 had no effect on anthraquinone production. Real-time PCR measurements showed that the activation of anthraquinone biosynthesis in transgenic calli correlated with the activation of isochorismate synthase gene expression. The activator effect of AtCPK1 was stable during prolonged periods of transgenic cell cultivation (more than 3 years) and the transgenic cultures exhibited high growth. Our results provide the first evidence that a CDPK gene can be used for the engineering of secondary metabolism in plant cells.

High chloroplast haplotype diversity in the endemic legume Oxytropis chankaensis may result from independent polyploidization events.

Oxytropis chankaensis Jurtz. (Fabaceae) is an endangered perennial tetraploid species endemic to the Khanka Lake coast. In Russia, O. chankaensis is distributed across a very restricted zone along the western shore of this lake. To characterise all known populations of this species, we assessed the genetic diversity of four noncoding regions of chloroplast DNA (cpDNA). Variable sites detected within the trnL-trnF, the petG-trnP, and the trnS-trnG regions allowed the identification of seven haplotypes. On the other hand, no variation was found in the trnH-psbA region. O. chankaensis exhibited an overall low level of nucleotide diversity (π = 0.00052) but a marked haplotype diversity (h = 0.718). A combination of three or four haplotypes was found in each population, and most of the cpDNA variation (above 90%) was distributed within populations. The level of genetic structure that we detected in O. chankaensis using maternal plastid DNA markers was much lower (G (ST) = 0.037) than the average that is estimated for angiosperms. We found no evidence for isolation by distance or for phylogeographic structuring in O. chankaensis. Our data suggest that autopolyploidy has arisen more than once in the evolutionary history of this species. Repetitive expansion and contraction during past and ongoing demographic events both seem to be involved in shaping the current genetic structure of O. chankaensis. This study provides valuable information for developing the most appropriate strategy for conserving this endemic species with a narrow habitat range.

Decreased ROS level and activation of antioxidant gene expression in Agrobacterium rhizogenes pRiA4-transformed calli of Rubia cordifolia.

Microbe-plant interactions often lead to a decrease in the reactive oxygen species (ROS) level of plant cells, which allows pathogen survival through the suppression of plant immune responses. In the present investigation, we tested whether transformation of Rubia cordifolia cells by Agrobacterium rhizogenes had a similar effect. We isolated partial cDNA sequences of ascorbate peroxidase, catalase and Cu/Zn superoxide dismutase genes (RcApx1, RcApx2, RcApx3, RcCAT1, RcCAT2, RcCSD1, RcCSD2 and RcCSD3) from plant tissues, as well as pRiA4-transformed and normal calli of Rubia cordifolia, and studied their expression by real-time PCR. Transcription profiling revealed that ascorbate peroxidase (RcApx1) and Cu/Zn superoxide dismutase (RcCSD1) were the most abundant transcripts present in both plant tissues and non-transformed calli. Catalase genes were weakly expressed in these samples. The pRiA4-transformed calli showed enhanced expression of several genes encoding ROS-detoxifying enzymes. Confocal microscopy imaging revealed decreased ROS level in pRiA4-transformed calli compared to the control. These results demonstrate that A. rhizogenes, like other plant pathogens, uses a strategy aimed at decreasing ROS levels in host cells through the general upregulation of its antioxidant genes.

The rolB gene-induced overproduction of resveratrol in Vitis amurensis transformed cells.

Resveratrol is a stilbene, which prevents carcinogenesis at stages of tumor initiation, promotion and progression. In the present investigation, we developed cell cultures of wild-growing grape (Vitis amurensis Rupr.). The cultures produced low levels of resveratrol, up to 0.026% dry wt., i.e., comparable to levels reported for other plant cell cultures previously established. Different methods commonly used to increase secondary metabolite production (cell selection, elicitor treatments and addition of a biosynthetic precursor) only slightly enhanced cell productivity. Transformation of V. amurensis V2 callus culture by the rolB gene of Agrobacterium rhizogenes resulted in more than a 100-fold increase in resveratrol production in transformed calli. The rolB-transformed calli are capable of producing up to 3.15% dry wt. of resveratrol. We show that the capability to resveratrol biosynthesis is tightly correlated with the abundance of rolB mRNA transcripts. Tyrosine phosphatase inhibitors abolished the rolB-gene-mediated stimulatory effect, thus documenting for the first time the involvement of tyrosine phosphorylation in plant secondary metabolism.

Effects of Ca(2+) channel blockers and protein kinase/phosphatase inhibitors on growth and anthraquinone production in Rubia cordifolia callus cultures transformed by the rolB and rolC genes.

The transformation of Rubia cordifolia L. cells by the 35S- rolB and 35S- rolC genes of Agrobacterium rhizogenes caused a growth inhibition of the resulting cultures and an induction of the biosynthesis of anthraquinone-type phytoalexins. Inhibitor studies revealed a striking difference between the rolC- and rolB-gene-transformed cultures in their sensitivity to verapamil, an L-type Ca(2+) channel blocker. The rolC culture possessed a 2-fold lowered resistance to the inhibitor than the normal culture, while the rolB culture was 4-fold more resistant to the treatment. Additionally, growth of the rolC culture was totally inhibited when the culture was grown in Ca(2+)-free medium, whereas growth of the rolB culture was reduced by less than half. We interpreted these results as evidence for a lack of calcium homeostasis in both transgenic cultures. Anthraquinone (AQ) production was not inhibited in the normal or transformed cultures by the Ca(2+) channel blockers verapamil and LaCl(3), or by diphenylene iodonium, an inhibitor of NADPH oxidase, or by the protein kinase inhibitor staurosporine. These results indicate that the induction of AQ production in non-transgenic and transgenic cultures does not proceed through the activation of the common Ca(2+)-dependent NADPH oxidase pathway that mediates signal transduction between an elicitor-receptor complex via transcriptional activation of defense genes. Okadaic acid and cantharidin, inhibitors of protein phosphatases 1 and 2A, caused an increase in AQ production in transgenic cultures. Okadaic acid stimulated AQ accumulation in the non-transformed culture, whereas cantharidin had no effect. These results show that different phosphatases are involved in AQ synthesis in normal and transgenic cultures of R. cordifolia.

Gal4-gene-dependent alterations of embryo development and cell growth in primary culture of sea urchins.

Primary cell cultures from sea urchins have a low proliferative level that prevents the establishment of long-term cultures. To increase expression levels of the genes regulating cell growth in sea urchins, and thus enhance cell growth, we used the transcriptional activator gene Gal4 found earlier in yeast. Sea urchin embryos were treated with plasmid DNA containing the Gal4 gene. Expression of the transgene was confirmed by reverse transcriptase polymerase chain reaction. When the fully functional gene was used, embryos effectively formed teratoma-like structures after 50 to 55 hours of cultivation. In contrast, the Gal4 gene, devoid of acidic activating regions, possessed little activity as a teratogen. The Gal4-treated cells in blastula-derived culture showed higher DNA synthesis and higher proliferative activity than control cells. We suggest that formation of the teratoma-like structures in embryos, activation of DNA synthesis, and significant increase of cell number in embryo-derived cell cultures could be attributed to Gal4 gene action.

Shikonin production by p-fluorophenylalanine resistant cells of Lithospermum erythrorhizon.

Studies were conducted with a BK-39 callus culture of Lithospermum erythrorhizon, which produced seven shikonin derivatives (acetylshikonin, propionylshikonin, isobutyrylshikonin, beta,beta-dimethylacrylshikonin, isovalerylshikonin, beta-hydroxyisovalerylshikonin and alpha-methyl-n-butyrylshikonin). A selection of cell aggregates of BK-39 culture on a medium containing p-fluorophenylalanine (PFP) yields a cell line possessing a higher resistance to the inhibitor than the initial culture. Selected BK-39F cultures produced almost the same profile of shikonin naphthoquinones as the initial culture. The shikonin derivative content of PFP-resistant culture was approximately two times higher than that of the control, reaching 12.6% of DW cell biomass.

Isoflavonoid production by callus cultures of Maackia amurensis.

Callus cultures were established from the different parts of Maackia amurensis plants and analyzed for isoflavonoids. The isoflavones daidzein, retuzin, genistein and formononetin and the pterocarpans maakiain and medicarpin were found to be produced by these cultures. The content of isoflavones and pterocarpans was essentially the same in cultures derived from leaf petioles, inflorescences and apical meristems of the plant. The maximal yield of isoflavones and pterocarpans in calluses was 20.8 mg/g cell dry wt., approximately four times higher than the content of the heartwood of M. amurensis plants. Unlike wild-growing plants, none of the cell cultures had the ability to accumulate stilbenes.

Primary structures of two ribonucleases from ginseng calluses. New members of the PR-10 family of intracellular pathogenesis-related plant proteins.

The amino acid sequences of two ribonucleases from a callus cell culture of Panax ginseng were determined. The two sequences differ at 26% of the amino acid positions. Homology was found with a large family of intracellular pathogenesis-related proteins, food allergens and tree pollen allergens from both dicotyledonous and monocotyledonous plant species. There is about 30% sequence difference with proteins from species belonging to the same plant order (Apiales: parsley and celery), 60% with those from four other dicotyledonous plant orders and about 70% from that of the monocotyledonous asparagus. More thorough evolutionary analyses of sequences lead to the conclusion that the general biological function of members of this protein family may be closely related to the ability to cleave intracellular RNA and that they have an important role in cell metabolism. As the three-dimensional structure of one of the members of this protein family has been determined recently [Gajhede et al., Nature Struct Biol 3 (1996) 1040-1045], it may be possible to assign active-site residues in the enzyme molecule and make hypotheses about its mode of action. Structural features in addition to the cellular site of biosynthesis indicate that this family of ribonucleases is very different from previously investigated ones.

Uptake of labelled tobacco mosaic virus by tobacco protoplasts in the presence of metabolic inhibitors and at low temperature.

In the presence of inhibitors of protein synthesis and energy metabolism, tobacco protoplasts were shown to retain 14C-labelled tobacco mosaic virus (14C-TMV) with the same intensity as in control. At 2 degrees C, the protoplasts retained 14C-TMV at approximately the same rate as at 25 degrees C. In protoplasts inocculated at different temperatures, approximately equal amounts of infectious virus were produced, this being possibly indicative of a non-physiological nature of the first stages of virus penetration into the protoplast.