K. V. Kiselev

Perspectives for production and application of resveratrol

The polyphenol trans-resveratrol (3,5,4′-trihydroxy-trans-stilbene) is one of the best known plant secondary metabolites. The number of articles devoted to trans-resveratrol has been steadily increasing. Trans-resveratrol is a molecule that is beneficial to human health; this explains the high level of interest in trans-resveratrol among different research groups. Therefore, it is important to develop an effective method to produce this compound commercially. The applicability of biotechnology for trans-resveratrol extraction is still uncertain. This review describes and compares the available biotechnological methods of trans-resveratrol production, focusing on their advantages and disadvantages.

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.

Phenylalanine ammonia-lyase and stilbene synthase gene expression in rolB transgenic cell cultures of Vitis amurensis

Transformation of Vitis amurensis callus culture by the plant oncogene rolB of Agrobacterium rhizogenes results in high (up to 3.15% dry wt.) levels of resveratrol in the transformed culture. The present study deals with the effect of rolB on phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) gene expression in two rolB transgenic V. amurensis callus cultures with different levels of rolB expression and resveratrol production. The total expression of PALs and STSs in rolB transgenic cultures increased 1.3–3.8 times compared with the control culture. In the rolB transgenic cultures expression of VaPAL1, VaPAL2, and six STS genes was increased, while expression of VaPAL3 and VaSTS6 was not significantly changed. These results suggest that rolB increases resveratrol production via selective enhancement of expression of individual genes from PAL and STS gene families. We propose that increase of VaPAL3, VaSTS1, and VaSTS6 transcript levels is not strongly required for high resveratrol production by rolB transgenic cell cultures.

Expression of calcium-dependent protein kinase (CDPK) genes under abiotic stress conditions in wild-growing grapevine Vitis amurensis.

Calcium-dependent protein kinases (CDPKs), which are important sensors of Ca(2+) flux in plants, are known to play essential roles in plant development and adaptation to abiotic stresses. In the present work, we studied expression of CDPK genes under osmotic and temperature stress treatments in wild-growing grapevine Vitis amurensis Rupr., which is known to possess high adaptive potential and a high level of resistance against adverse environmental conditions. In this study, using RT-PCR with degenerate primers, DNA sequencing and frequency analysis of RT-PCR products, we identified 13 CDPK genes that are actively expressed in healthy V. amurensis cuttings under high salt, high mannitol, desiccation, and temperature stress conditions. 12 CDPKs, namely VaCPK1, VaCPK2, VaCPK3, VaCPK9, VaCPK13, VaCPK16, VaCPK20, VaCPK21, VaCPK25, VaCPK26, VaCPK29 and VaCPK30, were novel for Vitaceae, and their full cDNAs were obtained and described. Quantitative real-time RT-PCR demonstrated that mRNA levels of 10 VaCPK genes were differentially up-regulated under the osmotic and temperature stress treatments, while the abundance of 3 VaCPK transcript variants, VaCPK3a, VaCPK25, and VaCPK30, was not markedly changed. Expression profiling of the VaCPK genes in leaves, leaf petioles, stems, inflorescences, berries, and seeds of V. amurensis revealed that the genes exhibit different organ-specific expression patterns. The stimulatory effect of abiotic stress on the expression of the VaCPK1, 2, 3, 9, 13, 16, 20, 21, 26, and VaCPK29 genes is suggestive of their implication in the grapevine response to osmotic and temperature stresses, while the variability in their organ-specific expression patterns indicates that the enzymes perform distinct biological functions.

Resveratrol content and expression of phenylalanine ammonia-lyase and stilbene synthase genes in rolC transgenic cell cultures of Vitis amurensis

Resveratrol, a naturally occurring polyphenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In the present investigation, we show that transformation of Vitis amurensis Rupr. with the oncogene rolC of Agrobacterium rhizogenes increased resveratrol production in the two transformed callus cultures 3.7 and 11.9 times. The rolC-transformed calli were capable of producing 0.099% and 0.144% dry weight of resveratrol. We characterized phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) gene expression in the two rolC transgenic callus cultures of V. amurensis. In the rolC transgenic culture with higher resveratrol content, expression of VaPAL3, VaSTS3, VaSTS4, VaSTS5, VaSTS6, VaSTS8, VaSTS9, and VaSTS10 was increased; while in the rolC culture with lower resveratrol content, expression of VaPAL3 and VaSTS9 was increased. We suggest that transformation of V. amurensis calli with the rolС gene induced resveratrol accumulation via selective enhancement of expression of individual PAL and STS genes involved in resveratrol biosynthesis. We compared the data on PAL and STS gene expression in rolC transgenic calli with the previously obtained results for rolB transgenic calli of V. amurensis. We propose that the transformation of V. amurensis with the rolC and rolB genes of A. rhizogenes increased resveratrol accumulation through different regulatory pathways.

The Agrobacterium rhizogenes rolC-gene-induced somatic embryogenesis and shoot organogenesis in Panax ginseng transformed calluses

Expression of the Agrobacterium rhizogenes rolC gene in Panax ginseng callus cells results in formation of tumors that are capable to form roots. The selection of non-root forming tumor clusters yielded the embryogenic 2c3 callus line, which formed somatic embryos and shoots independently of external growth factors. Although the 2c3 somatic embryos developed through a typical embryogenesis process, they terminated prematurely and repeatedly formed adventitious shoot meristems and embryo-like structures. A part of the shoots and somatic embryos formed enlarged and fasciated meristems. This is the first indication of the rolC gene embryogenic effect and, to our knowledge, the first indication that a single gene of non-plant origin can induce somatic embryogenesis in plants.

Calcium-dependent mechanism of somatic embryogenesis in Panax ginseng cell cultures expressing the rolC oncogene

The Panax ginseng 2c3 embryogenic cell culture was earlier obtained by callus cell transformation with Agrobacterium rhizogenes rolC. Calcium channel blockers (LaCl3, verapamil, and niflumic acid) reduced the production of somatic embryos in the 2c3 culture, implicating the Ca2+ signaling system in plant somatic embryogenesis. The protein kinase inhibitors W7 and H7 also decreased the yield of somatic embryos in the 2c3 culture. The total CDPK expression in the 2c3 culture was 1.2-to 1.5-fold lower than in a control callus culture as a result of a silencing of the genes belonging to the PgCDPK1 (PgCDPK1a and PgCDPK1b) and PgCDPK3 (PgCDPK3a) subfamilies. Expression of the PgCDPK2 subfamily genes (PgCDPK2b and PgCDPK2d) was increased. It was assumed that some genes of the PgCDPK1, PgCDPK2, and PgCDPK3 subfamilies were responsible for generation of embryogenic cells in the 2c3 culture. For the first time, rolC expression and embryogenesis were associated with changes in the expression of certain CDPK genes.

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.

Structure and expression profiling of a novel calcium-dependent protein kinase gene, CDPK3a, in leaves, stems, grapes, and cell cultures of wild-growing grapevine Vitis amurensis Rupr.

Key messageVaCDPK3ais actively expressed in leaves, stems, inflorescences, and berries ofVitis amurensisand may act as a positive growth regulator, but is not involved in the regulation of resveratrol biosynthesis.AbstractCalcium-dependent protein kinases (CDPKs) are known to play important roles in plant development and defense against biotic and abiotic stresses. It has previously been shown that CDPK3a is the predominant CDPK transcript in cell cultures of wild-growing grapevine Vitis amurensis Rupr., which is known to possess high resistance against environmental stresses and to produce resveratrol, a polyphenol with valuable pharmacological effects. In this study, we aimed to define the full cDNA sequence of VaCDPK3a and analyze its organ-specific expression, responses to plant hormones, temperature stress and exogenous NaCl, and the effects of VaCDPK3a overexpression on biomass accumulation and resveratrol content in V. amurensis calli. VaCDPK3a was actively expressed in all analyzed V. amurensis organs and tissues and was not transcriptionally regulated by salt and temperature stresses. The highest VaCDPK3a expression was detected in young leaves and the lowest in stems. A reduction in the VaCDPK3a expression correlated with a lower rate of biomass accumulation and higher resveratrol content in calli of V. amurensis under different growth conditions. Overexpression of the VaCDPK3a gene in the V. amurensis calli significantly increased cell growth for a short period of time but did not have an effect on resveratrol production. Further subculturing of the transformed calli resulted in cell death and a decrease in expression of the endogenous VaCDPK3a. The data suggest that while VaCDPK3a acts as a positive regulator of V. amurensis cell growth, it is not involved in the signaling pathway regulating resveratrol biosynthesis and resistance to salt and temperature stresses.

A new method for analyzing gene expression based on frequency analysis of RT-PCR products obtained with degenerate primers

This paper presents a new method of gene expression analysis: frequency analysis of RT-PCR products obtained with degenerate primers (FAPP). The main advantage of the new approach compared to the present methods of gene expression analysis is that it is applicable to non-model plant objects whose gene sequences are unknown. The advantages and disadvantages of FAPP are described in detail using data on calcium-dependent protein kinase, stilbene synthase, and phenylalanine ammonia-lyase gene expression in two cell cultures of Vitis amurensis. We compared the expression profiles obtained by FAPP to those obtained by real-time PCR and expressed sequence tags.