Alexander M. Nosov

Expression of Acyl-lipid Δ12-desaturase Gene in Prokaryotic and Eukaryotic Cells and Its Effect on Cold Stress Tolerance of Potato

We report the expression profile of acyl-lipid Delta12-desaturase (desA) gene from Synechocystis sp. PCC6803 and its effect on cell membrane lipid composition and cold tolerance in prokaryotic (Escherichia coli) and eukaryotic (Solanum tuberosum) cells. For this purpose, a hybrid of desA and reporter gene encoding thermostable lichenase (licBM3) was constructed and used to transform these cells. The expression of this hybrid gene was measured using qualitative (Petri dish test, electrophoregram and zymogram) and quantitative methods (spectrometry and gas liquid chromatography assays). The maximum level of linoleic acid in the bacterial cells containing hybrid gene was 1.9% of total fatty acids. Cold stress tolerance assays using plant damage index and growth parameters showed that cold tolerance was enhanced in primary transgenic lines because of increased unsaturated fatty acid concentration in their lipids. The greatest content of 18:2 and 18:3 fatty acids in primary transgenic plants was observed for lines 2 (73%) and 3 (41%). Finally, our results showed that desaturase could enhance tolerance to cold stress in potato, and desaturase and lichenase retain their functionality in the structure of the hybrid protein where the enzymatic activity of target gene product was higher than in the case of reporter lichenase gene absence in the construction.

Malonyl-ginsenoside content of a cell-suspension culture of Panax japonicus var. repens

The presence of large amounts of ginsenosides malonyl-Rb1, -Rc, -Rb2, and -Rd in a suspension culture of Panax japonicus var. repens cells was demonstrated for the first time. Identification of ginsenoside malonyl-Rb1 was based on chromatographic, chemical, and spectroscopic evidence. Ginsenosides malonyl-Rc, -Rb2, and -Rd were identified on the basis of chromatographic and chemical data. Content and composition of the individual ginsenosides (Rg1, R0, malonyl-Rb1, Rb1, Rc, Rb2, and Rd) were monitored in the suspension culture over 4 years. The RP-HPLC-UV analysis showed that Rg1, R0, and malonyl-Rb1 accounted for more than 75% of the total pool of ginsenosides. In accordance with this result, and data analysis reported in the literature, we propose that ginsenoside formation in the cells of P. japonicus var. repens in vitro is closely related to the cellular compartmentation of these substances. In particular, the accumulation of the 20(S)-protopanaxadiol ginsenosides (especially Rb1) is strongly dependent on their pattern of malonylation, which likely targets them for transport into the vacuole.

Isoprenoid Production via Plant Cell Cultures: Biosynthesis, Accumulation and Scaling-Up to Bioreactors

Plant cell culture is traditionally viewed as a unique artificially created biological system representing a heterogenous population of dedifferentiated cells. This system undergoes a continuous process of autoselection based on the intensity and stability of cell proliferation. We discuss here the details of formation and regulation of isoprenoid biosynthesis in plant cell in vitro based on literature survey and our research. Obviously, secondary metabolism differs in cell culture compared to the plant per se, because in cell culture metabolites are synthesized and compartmentalized within a single heterotrophic cell with sparse or underdeveloped vacuoles and plastids. For example, in plant cell cultures isoprenoid biosynthesis via MVA pathway was found to be more active than via plastid-localized MEP pathway. Also, it was hypothesized that cell cultures preferably produce metabolites, which promote cell proliferation and growth. Indeed, cell cultures of Dioscorea deltoidea produced mainly furostanol glycosides, which promoted cell division. Triterpene glycosides (ginsenosides) in the cell cultures of various Panax species are represented mainly by Rg- and Rb-groups. Rb ginsenosides are predominantly found as malonyl-esters that may influence their intracellular localization.

Rare triterpene glycoside of ginseng (ginsenoside malonyl-Rg 1 ) detected in plant cell suspension culture of Panax japonicus var. repens

This paper reports for the first time about the detection and identification of ginsenoside malonyl-Rg1 (the rare 20(S)-protopanaxatriol-type ginsenoside) in the biomass of plant cell suspension culture of Japanese ginseng (Panax japonicus C.A. Mey. var. repens). Ginsenosides were analyzed by means of high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC-ESI-MS) in positive-ion mode. Malonyl-Rg1 was identified as a result of interpretation of MS spectra obtained upon fragmentation of protonated molecular ion ([M + H]+) of this compound in an ionization source. Chromatographic analysis and MS spectra showed that the cells of P. japonicus var. repens cultivated in vitro contain several isomers of malonyl-Rg1. Thus, we ascertained for the first time that, in addition to malonyl ginsenosides of 20(S)-protopanaxadiol group, the plant cell culture of ginseng P. japonicus var. repens can accumulate glycosides of 20(S)-protopanaxatriol group acylated with a malonic acid residue. The obtained results showed that, in the cells of ginseng cultivated in vitro for a long time (for 10 years and more), the assortment of secondary metabolites (ginsenosides) may be as wide as in intact plants.

ANALYSIS OF GYNZENOSIDES IN THE ROOTS OF PANAX GINSENG INTRODUCED IN THE CENTRAL BOTANICAL GARDEN OF NAS OF BELARUS

For the first time, a detailed study of the qualitative and quantitative composition of ginsenosides in the Panax ginseng roots was carried out with the help of high-performance liquid chromatography combined with mass spectrometry (HPLC-MS). The plants were introduced into the conditions of the Republic of Belarus at the experimental plot of the Central Botanical Garden of the National Academy of Sciences of Belarus. It was found that in the examined roots, all basic neutral glycosides of ginseng (ginsenosides Rb1, Rc, Rb2/Rb3, Rd, Rf, Rg1 and Re), as well as their malonylated derivatives (malonylginsenosides Rb1, Rc, Rb2/Rb3, Rd, Rg1 and Re) and some “minor” ginsenosides (20-gluco-ginsenoside Rf, notoginsenosides R1 and R2, isomers of malonyl-ginsenosides Rb1 and Rd) are present. The research also showed that different parts of the P. ginseng roots differ significantly in a total content of ginsenosides: for the main root, this parameter was 3.3 % of dry mass, and for the lateral roots – 7.8 % of dry mass.

Ion-exchange properties of the cell walls isolated from suspension-cultured plant cells

Ion-exchange capacity of the cell walls isolated from suspension-cultured Panax japonicus, Polyscias filicifolia and Dioscorea deltoidea cells was analyzed at pH 2.8–12 and constant ionic strength (100xa0mM). The cell walls of all cultures contain three types of ion-exchange groups: primary amino groups (pKaxa0<xa03), carboxyl groups of polygalacturonic acid (pKa 3.71), and carboxyl groups of hydroxycinnamic acids (pKa 7.62). Amount of primary amino groups ranges from 500 (D. deltoidea) to 710 (P. japonicus) µmol/g cell wall dry weight, carboxyl groups with pKa 3.71—from 570 (D. deltoidea) to 670 (P. filicifolia), carboxyl groups with pKa 7.62—from 270 (P. filicifolia) to 370 (P. japonicus) µmol/g cell wall dry weight. The comparison of the data obtained by elemental and functional analyses demonstrated that the cell walls of all cultures are characterized by high content of pectins (~40% by weight) and structural proteins (~17–30% by weight), but do not contain phenolic OH–groups, which presumably signifies the absence of lignin in them.

Occurrence of 14-hydroxylated taxoids in the plant in vitro cell cultures of different yew species (Taxus spp.)

This is the first study to show that the formation of 14β-hydroxylated derivatives of taxa-4(20),11-diene is a specific feature of in vitro cultured dedifferentiated yew cells that distinguishes them from intact plant cells. This may be due to a lower toxicity of the 14-OH taxoids for proliferating plant cells compared to the 13-OH derivatives.

Protodioscin in Dioscorea deltoidea Suspension Cell Culture

One of the main furostanol glycosides in Dioscorea deltoidea suspension cell culture, i.e., protodioscin, was isolated and characterized structurally using NMR spectroscopy, high-resolution mass spectrometry, and an analysis of its accumulation.

Study of electron-dense thylakoids in chloroplasts of Stevia rebaudiana Bertoni in relation to the biosynthesis of diterpenoids

Using transmission electronic microscopy and mass spectrometry electron-dense thylakoids of chloroplasts of Stevia rebaudiana leaves during active vegetable growth of this plant were studied in relation to the biosynthesis of diterpenoid glycosides (DGs). It was found that these compounds are absent in these thylakoids, but they contain a water-insoluble weakly polar ent-kauren, a known biosynthetic precursor of DGs as well as gibberellins. This finding provides a base for the suggestion that similar, electron-dense, thylakoids were observed earlier by other authors in other plant species. These data allowed us to conclude that an intensive biosynthesis of ent-kauren is likely related to adaptation of the short-day plants including Stevia rebaudiana to vegetable growth under the long day conditions.

The occurrence of gypenoside XVII in suspension cell culture of ginseng Panax japonicus var. repens

42 One of the outstanding achievements of bioorganic chemistry of low molecular weight compounds in the XX century was the discovery of the major biologically active compounds of ginseng, triterpene glycosides ginsenosides, in 1962. Over the past half of the century, the interest of researchers in this unique group of com pounds not only does not go down but continues to steadily increase, due to the discovery of new pharma cological effects of ginsenosides [1]. Currently, over 300 individual ginsenosides are known, and this number steadily increases [1, 2]. Studies of cell and tissue cultures of ginseng in vitro were started almost simultaneously with the discovery of ginsenosides [3]; however, advances in studying the diversity of these compounds in a sterile culture seem modest. For example, the possibility of formation of various minor ginsenosides in a ginseng culture in vitro remains unexplored to date. At the same time, phar macological studies of the last decade have convinc ingly shown that it is this group of ginsenosides that determines many of the unique biological effects of ginseng [4]. This is the first study to report the isolation of gype noside XVII, a minor ginseng glycoside, from the cell culture of Panax japonicus var. repens. The study was performed with a suspension cell culture derived from the root of intact two year plants (Primorsky Krai, Russia) in 1997 [5]. The strain was registered in the All Russia Collection of Cell Cul tures of Higher Plants under number 62. The growth conditions of the culture were described earlier [6]. The total triterpene glycoside fraction (TGF) was iso lated by the standard methods [2] from 600 g of an air dry biomass of the cell culture grown in 500 L biore actor (1T, Russia). The TGF was purified and individ ual components were isolated by sequential use of low pressure column chromatography and HPLC [7]. The 1H and 13C NMR spectra of the obtained compounds in pyridine d5 were recorded with a Bruker Avance AV600 instrument (Germany) using tetramethylsilane as an internal standard. The high resolution mass spectra with electrospray ionization obtained with Bruker micrOTOF II and maXis instruments (Bruker Daltonics, Germany) [8].