Y. N. Zhuravlev

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.

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.

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 Agrobacteriumrhizogenes 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.

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.

The effect of salicylic acid on phenylalanine ammonia-lyase and stilbene synthase gene expression in Vitis amurensis Cell Culture

Resveratrol is a phytoalexin with antibacterial, antiviral and cancer-preventing effects. The objective of the study was to identify PAL and STS genes of Vitis amurensis Rupr. encoding the phenylalanine ammonia-lyases (PAL) and stilbene synthases (STS), the key enzymes involved in the resveratrol biosynthesis. A V. amurensis Rupr. cell culture characterized by low resveratrol level was chosen as a model object. Salicylic acid (SA), a known secondary metabolism inducing agent, was used for enhancement of resveratrol production in this culture. PAL and STS gene expression was analyzed using the reverse transcription PCR and real-time PCR techniques. SA was originally found to specifically enhance the expression of VaPAL3, VaSTS2, VaSTS3, VaSTS4, VaSTS5, VaSTS6, and VaSTS8 of multigene families VaPAL and VaSTS. The results obtained were compared with the earlier published data on PAL and STS gene expression in the rolB transformed V. amurensis cell cultures characterized by high levels of resveratrol. The effects of SA treatment and the rolB transformation on VaPAL and VaSTS gene expression were found to be considerably different.

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.

CDPK gene expression in salt tolerant rolB and rolC transformed cell cultures of Panax ginseng

CDPKs (calcium-depended protein kinases) are of great importance for the activation of defense reactions in plants. In this study, we aimed to find a connection between CDPK expression and increased salt tolerance in Panax ginseng. Treatment of P. ginseng cell cultures with W7 (CDPK protein inhibitor) showed that CDPK proteins were necessary for salt tolerance. Expression of PgCDPK1c, PgCDPK2c and PgCDPK4a was significantly increased in the cells treated with 60 mM NaCl compared to control cells, whereas expression of PgCDPK1b and PgCDPK3a was decreased. In the NaCl-treated cells, new CDPK transcripts also appeared (PgCDPK3c, PgCDPK4as). We also used rolC and rolB transformed cultures and the effects of the rol genes on CDPK expression were similar to the effects of salt stress: they caused a significant increase in the expression of PgCDPK1c, PgCDPK2c, and PgCDPK4a and decreased expression of PgCDPK3a, in addition to the appearance of the “short” CDPK transcripts.

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.

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.

Green synthesis of silver nanoparticles using transgenic Nicotiana tabacum callus culture expressing silicatein gene from marine sponge Latrunculia oparinae

Abstract In the present investigation, transgenic tobacco callus cultures and plants overexpressing the silicatein gene LoSilA1 from marine sponge Latrunculia oparinae were obtained and their bioreduction behaviour for the synthesis of silver nanoparticles (AgNPs) was studied. Synthesized nanoparticles were characterized using UV–visible spectroscopy, Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic flame electron microscopy (AFM) and nanoparticle tracking analysis (NTA). Our measurements showed that the reduction of silver nitrate produced spherical AgNPs with diameters in the range of 12–80 nm. The results of XRD analysis proved the crystal nature of the obtained AgNPs. FTIR analysis indicated that particles are reduced and stabilized in solution by the capping agent, which is likely to be proteins present in the callus extract. Interestingly, the reduction potential of LoSiLA1-transgenic callus line was increased three-fold compared with the empty vector-transformed calli. The synthesized AgNPs were found to exhibit strong antibacterial activity against Escherichia coli and Agrobacterium rhizogenes. The present study reports the first evidence for using genetic engineering for activation of the reduction potential of plant cells for synthesis of biocidal AgNPs.