Victor P. Bulgakov

Functions of rol genes in plant secondary metabolism

For a long time, the Agrobacterium rhizogenes rolA, rolB and rolC oncogenes have been considered to be modulators of plant growth and cell differentiation. A new function of the rol genes in plant-Agrobacterium interaction became apparent with the discovery that these genes are potential activators of secondary metabolism in transformed cells from the Solanaceae, Araliaceae, Rubiaceae, Vitaceae and Rosaceae families. In some cases, the activator effect of individual rol genes is sufficient to overcome the inability of cultured plant cells to produce large amounts of secondary metabolites. Here, I summarize the available evidence that shows that genetic transformation by single Agrobacterium rol genes may be used as a powerful tool to manipulate secondary metabolites in cultured plant cells. Although it is known that the rol genes act via transcriptional activation of defense genes, the mechanism of activation is unclear. In this review, evidence is presented to support the hypothesis that the rol genes mediate uncommon signal transduction pathways in plants.

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.

The impact of plant rolC oncogene on ginsenoside production by ginseng hairy root cultures

Plasmid constructions containing rolA, rolB and rolC genes, isolated earlier from the TL-DNA of Agrobacterium rhizogenes were used to transform a cell culture (strain 1c) of Panax ginseng. The levels of ginsenosides were measured in the resulting transgenic tissues to evaluate the possible role of rol genes in ginsenoside formation. The ginsenoside content of the hairy root culture of P. ginseng, transformed by wild type A4 plasmid DNA and containing all rol loci, was higher than that of the control 1c culture (5.12-8.92 mg g ˇ1 dry wt), being in the range of 13.23-21.27 mg g ˇ1 dry wt. Ginseng tissue, transgenic for the rolA gene appeared to lose the ability to synthesize ginsenosides since only a trace amount of Re ginse- noside was found in 1c-rolA tissue. 1c-rolB cultures contained at least five times lower ginsenoside levels compared to the initial 1c culture. The ginsenoside content of rolC transgenic roots was about three times higher than that of the respective control. Taking into account the diAerences in cell diAerentiation levels in tissues transformed by rol genes, we compared the ginsenoside levels in rolC roots and tumours. It was found that ginsenoside production in tissues with diAerent levels of diAerentiation is nearly the same. We have concluded that the plant oncogene rolC is responsible for increased ginsenoside formation in ginseng hairy root cultures. # 1998 Elsevier Science Ltd. All rights reserved

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.

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.

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.

Suppression of Reactive Oxygen Species and Enhanced Stress Tolerance in Rubia cordifolia Cells Expressing the rolC Oncogene

It is known that expression of the Agrobacterium rhizogenes rolC gene in transformed plant cells causes defense-like reactions, such as increased phytoalexin production and expression of pathogenesis-related proteins. In the present study, we examined whether this phenomenon is associated with increased production of reactive oxygen species (ROS). Single-cell assays based on confocal microscopy and fluorogenic dyes (2,7-dichlorofluorescein diacetate and dihydrorhodamine 123) showed reduced steady-state levels of ROS in rolC-expressing Rubia cordifolia cells as compared with normal cells. Paraquat, a ROS inducer, caused significant ROS elevation in normal cells but had little effect on rolC-transformed cells. Likewise, ROS elevation triggered by a light stress was suppressed in transformed cells. Our results indicate that the rolC gene acts as a ROS suppressor in unstressed cells and its expression prevents stress-induced ROS elevations. We detected a two- to threefold increase in tolerance of rolC-transformed cells to salt, heat, and cold treatments. Simultaneously, rolC-transformed cells maintained permanently active defensive status, as found by measuring isochorismate synthase gene expression and anthraquinone production. Thus, the oncogene provoked multiple effects in which ROS production and phytoalexin production were clearly dissociated.

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.

The rolB Gene Suppresses Reactive Oxygen Species in Transformed Plant Cells through the Sustained Activation of Antioxidant Defense

The rolB (for rooting locus of Agrobacterium rhizogenes) oncogene has previously been identified as a key player in the formation of hairy roots during the plant-A. rhizogenes interaction. In this study, using single-cell assays based on confocal microscopy, we demonstrated reduced levels of reactive oxygen species (ROS) in rolB-expressing Rubia cordifolia, Panax ginseng, and Arabidopsis (Arabidopsis thaliana) cells. The expression of rolB was sufficient to inhibit excessive elevations of ROS induced by paraquat, menadione, and light stress and prevent cell death induced by chronic oxidative stress. In rolB-expressing cells, we detected the enhanced expression of antioxidant genes encoding cytosolic ascorbate peroxidase, catalase, and superoxide dismutase. We conclude that, similar to pathogenic determinants in other pathogenic bacteria, rolB suppresses ROS and plays a role not only in cell differentiation but also in ROS metabolism.