Yury N. Shkryl

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.

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.

Expression profiles of calcium-dependent protein kinase genes (CDPK1-14) in Agrobacterium rhizogenes pRiA4-transformed calli of Rubia cordifolia under temperature- and salt-induced stresses.

Agrobacterium rhizogenes genetically transform plant cells naturally via horizontal gene transfer by the introduction of T-DNA from the Ri plasmid into genomic DNA to create favorable conditions for successful colonization. An intriguing feature of pRiA4-transformed cells is their recently discovered enhanced tolerance to abiotic stress stimuli and activation of antioxidant enzyme expression. The mechanism by which A. rhizogenes modulates the defense responses of transformed cells remains unclear. It has been established that calcium-dependent protein kinase (CDPK) genes mediate crosstalk of signaling pathways in plants, and these genes have been implicated in biotic and abiotic stress signaling. In this study, we identified fourteen CDPK genes from Rubia cordifolia and examined their expression in aerial plant organs as well as in non-transformed and A. rhizogenes A4-transformed calli. Expression of RcCDPK4, RcCDPK5, RcCDPK7, and RcCDPK10 was 1.2- to 3.9-fold higher in pRiA4-transformed cells than in non-transformed cells, whereas expression of RcCDPK1, RcCDPK9, RcCDPK11, and RcCDPK14 was 1.2- to 1.9-fold lower. Agrobacterium transformation substantially modified the transcriptional responses of specific RcCDPK isoforms in pRiA4-transformed cells under conditions of temperature- and salinity-induced stress. On the basis of the results, we suggest that A. rhizogenes T-DNA genes exert their diverse biological functions by altering the expression of various CDPK genes.

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.

Cell-wall polysaccharide composition and glycanase activity of Silene vulgaris callus transformed with rolB and rolC genes

The aim of this research is to investigate the effects of the Agrobacterium rhizogenes rol genes on the composition of cell-wall polysaccharides and glycanase activity in the campion callus. The expression of the rolC gene reduces the yield of campion pectin, while the expression of the rolB or rolC gene inhibits the volumetric production of both pectin and intracellular arabinogalactan. The rol genes are involved in regulating the activity of glycanases and esterases, thereby contributing to the modification of polysaccharide structures, their molecular weight (Mw) and the degree of pectin methyl esterification (DE). The increase in pectin arabinose residue appears to be connected to a decrease in intracellular and extracellular α-l-arabinofuranosidase activity in transgenic campion calluses. In transgenic calluses expressing the rolB and rolC genes, the increase in pectin galactose residue is likely due to a decrease in β-galactosidase activity. The decrease in the Mw of pectin and its d-galacturonic acid content appears to be connected to an increase in extracellular polygalacturonase activity. Finally, the increase in pectinesterase activity causes a decrease in the DE of pectin. Thus, the expression of rolB and rolC genes in campion callus has a considerable effect on pectins sugar composition, DE and Mw, while it appears to have an insignificant influence on intracellular and extracellular arabinogalactans.

Biomimetic Synthesis of Nanosized Silica Structures on a Substrate with Silicatein

Using atomic force microscopy (AFM), the formation of nanosized silica structures on a substrate, catalyzed by the recombinant silicatein LoSilA1 from the marine sponge Latrunculia oparinae, was studied. It has been shown that at room temperature under neutral conditions, recombinant silicatein immobilized on a mica substrate causes the rapid polycondensation of tetrakis(2-hydroxyethyl) orthosilicate to form spherical particles. Thus, immobilized silicatein may acts as a catalyst in the preparation of ordered silica structures on various surfaces.

State of antioxidant systems and ginsenoside contents in the leaves of Panax ginseng in a natural habitat and an artificial plantation

Native Panax ginseng Meyer plants are now quite rare in their natural environment, and thus, artificial plantations are used for commercial purposes. However, ginseng plantations are frequently exposed to abiotic and biotic stress stimuli, which can decrease biomass accumulation and cause plants to wither and die. The antioxidant systems of the plants play a vital role in their defense mechanism against adverse stresses through maintaining the balance between reactive oxygen species generation and detoxification. Here, the adaptability of wild and cultivated P. ginseng was compared with respect to the antioxidant enzyme activities and gene expression, lipid peroxidation, ratio of reduced-to-oxidized glutathione, and ginsenoside content. Several new genes for antioxidant enzymes, including PgApx1, PgApx2, PgCSD2, PgCSD3, PgMSD1, PgGR1, PgPrx2, and PgPrx3, were identified, and their relative expression levels were determined together with previously characterized genes (PgCSD1, PgCat1, and PgPrx1). The relative transcription of PgMSD1 was higher in wild ginseng, whereas PgApx2 was overexpressed in cultivated plants. Expression of other antioxidant genes remained constant. The activity of superoxide dismutase, class III peroxidase, and glutathione reductase was significantly decreased in cultivated P. ginseng, whereas the activity of ascorbate peroxidase and catalase was not changed. Moreover, oxidative stress markers such as malondialdehyde concentration, the ratio of reduced-to-oxidized glutathione, and Rg-type ginsenosides content were elevated in cultivated ginseng plants. Our results indicate that P. ginseng plants grown in their natural habitat or artificial plantations have different antioxidative statuses. The process of domestication appears to have reduced the antioxidant defense system of ginseng.