Sergey A. Fedoreyev

Effect of salicylic acid, methyl jasmonate, ethephon and cantharidin on anthraquinone production by Rubia cordifolia callus cultures transformed with the rolB and rolC genes

It has been suggested that the rol genes of Agrobacterium rhizogenes could play an essential role in the activation of secondary metabolite production in plant transformed cultures. This study investigated whether the content of anthraquinone phytoalexins was changed in callus cultures of Rubia cordifolia transgenic for the 35S-rolB and 35S-rolC genes in comparison with a non-transformed callus culture. The anthraquinone content was shown to be significantly increased in transgenic cultures, thus providing further evidence that the rol-gene transformation can be used for the activation of secondary metabolism in plant cells. Methyl jasmonate and salicylic acid strongly increased anthraquinone accumulation in both transgenic and non-transgenic R. cordifolia calluses, whereas ethephon did not. A treatment of the cultures by cantharidin, the protein phosphatase 2A inhibitor, resulted in massive induction of anthraquinone accumulation in the transgenic cultures only. We suggest the involvement of a cantharidin-sensitive protein phosphorylation mechanism in anthraquinone biosynthesis in transgenic cultures.

Rosmarinic acid and its derivatives: biotechnology and applications

Rosmarinic acid (RA) is one of the first secondary metabolites produced in plant cell cultures in extremely high yields, up to 19% of the cell dry weight. More complex derivatives of RA, such as rabdosiin and lithospermic acid B, later were also obtained in cell cultures at high yields. RA and its derivatives possess promising biological activities, such as improvement of cognitive performance, prevention of the development of Alzheimer’s disease, cardioprotective effects, reduction of the severity of kidney diseases and cancer chemoprevention. The TNF-α-induced NF-κB signaling pathway has emerged as a central target for RA. Despite these impressive activities and high yields, the biotechnological production of these metabolites on an industrial scale has not progressed. We summarized data suggesting that external stimuli, the Ca2+-dependent NADPH oxidase pathway and processes of protein phosphorylation/dephosphorylation are involved in the regulation of biosynthesis of these substances in cultured plant cells. In spite of growing information about pathways regulating biosynthesis of RA and its derivatives in cultured plant cells, the exact mechanism of regulation remains unknown. We suggest that further progress in the biotechnology of RA and its derivatives can be achieved by using new high-throughput techniques.

Inhibitory effect of the Agrobacterium rhizogenes rolC gene on rabdosiin and rosmarinic acid production in Eritrichium sericeum and Lithospermum erythrorhizon transformed cell cultures

Rabdosiin and related caffeic acid metabolites have been proposed as active pharmacological agents demonstrating potent anti-HIV and antiallergic activities. We transformed Eritrichium sericeum and Lithospermum erythrorhizon seedlings by the rolC gene, which has been recently described as an activator of plant secondary metabolism. Surprisingly, the rolC-transformed cell cultures of both plants yielded two- to threefold less levels of rabdosiin and rosmarinic acid (RA) than respective control cultures. This result establishes an interesting precedent when the secondary metabolites are differently regulated by a single gene. We show that the rolC gene affects production of rabdosiin and RA irrespective of the methyl jasmonate (MeJA)-mediated and the Ca2+-dependent NADPH oxidase pathways. Cantharidin, an inhibitor of serine/threonine phosphatases, partly diminishes the rolC-gene inhibitory effect that indicates involvement of the rolC-gene-mediated signal in plant regulatory controls, mediated by protein phosphatases. We also show that the control MeJA-stimulated E. sericeum root culture produces (−)-rabdosiin up to 3.41% dry weight, representing the highest level of this substance for plant cell cultures reported so far.

Enhanced resveratrol accumulation in rolB transgenic cultures of Vitis amurensis correlates with unusual changes in CDPK gene expression.

It has been established that transformation of Vitis amurensis callus culture with the plant oncogene rolB of Agrobacterium rhizogenes results in a high level of resveratrol production in the transformed culture. In the present report, we investigated two rolB transgenic V. amurensis cell cultures with different levels of rolB expression and resveratrol production. We examined whether the calcium ion flux and later steps of the calcium-mediated signal transduction pathway play a role in resveratrol biosynthesis in the rolB transgenic cultures. It has been shown that the calcium channel blockers, LaCl(3), verapamil, and niflumic acid, significantly reduced the accumulation of resveratrol in the rolB transgenic cultures. The number of the calcium-dependent protein kinase (CDPK) transcript variants and abundance of some of the transcripts were considerably altered in the rolB transgenic cell cultures, as revealed by frequency analysis of RT-PCR products and real-time PCR. Some unusual CDPK transcripts with deletions and insertions in the kinase domain were isolated from cDNA probes of rolB-transformed cells. These results suggest that active resveratrol biosynthesis in rolB transgenic cultures of V. amurensis is Ca2+ dependent. We propose that the rolB gene has an important role in regulation of calcium-dependent transduction pathways in transformed cells.

Anthraquinone production by callus cultures of Rubia cordifolia

Abstract Munjistin and purpurin were identified as the major components of anthraquinone pigments produced by callus cultures of R. cordifolia. Anthraquinone content in calluses was 0.62–1.22% (by dry wt.) depending on the source of explants. Selection of coloured aggregates yielded a cell line with twofold increase in anthraquinone production.

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.

High Rabdosiin and Rosmarinic Acid Production in Eritrichium sericeum Callus Cultures and the Effect of the Calli on Masugi-Nephritis in Rats

During an investigation of plant cell cultures that might be useful in the treatment of renal disorders, we established a vigorously-growing E-4 callus culture of Eritrichium sericeum that produced large amounts of caffeic acid metabolites, (−)-rabdosiin (1.8% dry wt) and rosmarinic acid (4.6% dry wt). Elicitation of the calli by methyl jasmonate induced a 38% increase in total polyphenol production. The most efficient method of eliciting (−)-rabdosiin biosynthesis was through the treatment of E-4 calli with cuprum glycerate, which induced an increase in (−)-rabdosiin production of as much as 4.1% dry wt. Oral administration of E-4 callus biomass (100 mg/kg/d for 30 d) to rats with induced Masugi-nephritis caused an increase in diuresis and lowered creatinine excretion and proteinuria levels as compared with Masugi-nephritis untreated rats. While all of the Masugi-nephritis untreated rats began to suffer, near a quarter of the E-4 treated rats remained in good health. This result indicates that the E-4 culture has the potential to alleviate the symptoms associated with nephritis.

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.

Echinochrome A Increases Mitochondrial Mass and Function by Modulating Mitochondrial Biogenesis Regulatory Genes

Echinochrome A (Ech A) is a natural pigment from sea urchins that has been reported to have antioxidant properties and a cardio protective effect against ischemia reperfusion injury. In this study, we ascertained whether Ech A enhances the mitochondrial biogenesis and oxidative phosphorylation in rat cardio myoblast H9c2 cells. To study the effects of Ech A on mitochondrial biogenesis, we measured mitochondrial mass, level of oxidative phosphorylation, and mitochondrial biogenesis regulatory gene expression. Ech A treatment did not induce cytotoxicity. However, Ech A treatment enhanced oxygen consumption rate and mitochondrial ATP level. Likewise, Ech A treatment increased mitochondrial contents in H9c2 cells. Furthermore, Ech A treatment up-regulated biogenesis of regulatory transcription genes, including proliferator-activated receptor gamma co-activator (PGC)-1α, estrogen-related receptor (ERR)-α, peroxisome proliferator-activator receptor (PPAR)-γ, and nuclear respiratory factor (NRF)-1 and such mitochondrial transcription regulatory genes as mitochondrial transcriptional factor A (TFAM), mitochondrial transcription factor B2 (TFB2M), mitochondrial DNA direct polymerase (POLMRT), single strand binding protein (SSBP) and Tu translation elongation factor (TUFM). In conclusion, these data suggest that Ech A is a potentiated marine drug which enhances mitochondrial biogenesis.

CDPK-driven changes in the intracellular ROS level and plant secondary metabolism

Heterologous expression of a constitutively active calcium-dependent protein kinase (CDPK) gene was previously shown to increase secondary metabolite production in cultured cells of Rubia cordifolia, but the critical question of how CDPK activates secondary metabolism remains to be answered. In this article, we report that the expression of the Arabidopsis CDPK gene, AtCPK1, in R. cordifolia cells caused moderate and stable elevation of intracellular reactive oxygen species (ROS) levels. In contrast, the non-active, mutated AtCPK1 gene did not cause such an effect. The active AtCPK1 also increased cell size, likely by restricting cell division. These results are consistent with the model in which constitutive expression of AtCPK1 mimics the effects of elicitors, acting on secondary metabolism via the activation of ROS production.