Yuri N. Shkryl

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.

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.

Recent Advances in the Understanding of Agrobacterium rhizogenes-Derived Genes and Their Effects on Stress Resistance and Plant Metabolism

It is commonly accepted that the plant pathogens Agrobacterium rhizogenes and Agrobacterium tumefaciens, acting via their T-DNA oncogenes, disturb hormone metabolism or hormone perception pathways in plants, thereby attaining their aim of successful pathogenesis. In this work, we summarize recent data on the A. rhizogenes rolC and rolB oncogenes in comparison to the A. tumefaciens 6b oncogene with respect to their effects on the physiology of transformed cells. The newly discovered functions of the rol genes include the modulation of secondary metabolism, the modulation of levels of intracellular ROS and stress resistance of transformed cells, changed sucrose metabolism, and the inhibition of programmed cell death. We show that the rol genes do not have suppressive effects on plant innate immunity; rather, these genes activate plant defense reactions. The existence of not only the hormone-related mechanism of pathogenicity but also the defense-related mechanism of pathogenicity during plant-Agrobacterium interactions is suggested.

Engineering high yields of secondary metabolites in Rubia cell cultures through transformation with rol genes.

Among the different methods currently used to improve yields of secondary metabolites in cultured plant cells, the method involving transformation by rol genes represents an example of relatively new technology. These genes, isolated from plasmids of the plant pathogen Agrobacterium rhizogenes, 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 was sufficient to overcome the inability of cultured plant cells to produce large amounts of secondary metabolites. Stimulation of production characteristics of cultured plant cells mediated by the rol genes was shown to be remarkably stable over long-term cultivation. In this chapter, we describe transformation of Rubia cordifolia L. cells with the rol genes as an example of metabolic engineering of secondary metabolites.

Silicatein genes in spicule-forming and nonspicule-forming Pacific demosponges.

Silicatein genes are known to be involved in siliceous spicule formation in marine sponges. Proteins encoded by these genes, silicateins, were recently proposed for nanobiotechnological applications. We studied silicatein genes of marine sponges Latrunculia oparinae collected in the west Pacific region, shelf of Kuril Islands. Five silicatein genes, LoSilA1, LoSilA1a, LoSilA2, and LoSilA3 (silicatein-α group), LoSilB (silicatein-β group), and one cathepsin gene, LoCath, were isolated from the sponge L. oparinae for the first time. The deduced amino acid sequence of L. oparinae silicateins showed high-sequence identity with silicateins described previously. LoCath contains the catalytic triad of amino acid residues Cys-His-Asn characteristic for cathepsins as well as motifs typical for silicateins. A phylogenetic analysis places LoCath between sponge silicateins-β and L-cathepsins suggesting that the LoCath gene represents an intermediate form between silicatein and cathepsin genes. Additionally, we identified, for the first time, silicatein genes (AcSilA and AcSilB) in nonspicule-forming marine sponge, Acаnthodendrilla sp. The results suggest that silicateins could participate also in the function(s) unrelated to spiculogenesis.

CDPK-driven changes in the intracellular ROS level and plant secondary metabolism

Heterologous expression of a constitutively active calcium-dependent protein kinase (CDPK) gene was previously shown to increase secondary metabolite production in cultured cells of Rubia cordifolia, but the critical question of how CDPK activates secondary metabolism remains to be answered. In this article, we report that the expression of the Arabidopsis CDPK gene, AtCPK1, in R. cordifolia cells caused moderate and stable elevation of intracellular reactive oxygen species (ROS) levels. In contrast, the non-active, mutated AtCPK1 gene did not cause such an effect. The active AtCPK1 also increased cell size, likely by restricting cell division. These results are consistent with the model in which constitutive expression of AtCPK1 mimics the effects of elicitors, acting on secondary metabolism via the activation of ROS production.

Occurrence of a Silicatein Gene in Glass Sponges (Hexactinellida: Porifera)

Silicatein genes are involved in spicule formation in demosponges (Demospongiae: Porifera). However, numerous attempts to isolate silicatein genes from glass sponges (Hexactinellida: Porifera) resulted in a limited success. In the present investigation, we performed analysis of potential silicatein/cathepsin transcripts in three different species of glass sponges (Pheronema raphanus, Aulosaccus schulzei, and Bathydorus levis). In total, 472 clones of such transcripts have been analyzed. Most of them represent cathepsin transcripts and only three clones have been found to represent transcripts, which can be related to silicateins. Silicatein transcripts were identified in A. schulzei (Hexactinellida; Lyssacinosida; Rosselidae), and the corresponding gene was called AuSil-Hexa. Expression of AuSil-Hexa in A. schulzei was confirmed by real-time PCR. Comparative sequence analysis indicates high sequence identity of the A. schulzei silicatein with demosponge silicateins described previously. A phylogenetic analysis indicates that the AuSil-Hexa protein belongs to silicateins. However, the AuSil-Hexa protein contains a catalytic cysteine instead of the conventional serine.

The rolB gene activates the expression of genes encoding microRNA processing machinery

The rolB gene of Agrobacterium rhizogenes renders cells more tolerant of environmental stresses and increases their defense potential. However, these effects, coupled with the developmental abnormalities caused by rolB, have not yet been explained. In rolB-transformed Arabidopsis thaliana cells, we detected a 2.2 to 7-fold increase in the expression of genes encoding core and accessory proteins (DCL1, SE, HYL1, AGO1, TGH, DDL, HEN1, AGO4 and RDR2) of the microRNA processing machinery. However, the rolB gene did not affect the expression of DCL2, DCL3 and HST. The diverse and complex effects of rolB on transformed plant cells may be attributable to changes caused by this gene in particular RNA silencing pathways.

Application of Agrobacterium Rol Genes in Plant Biotechnology: A Natural Phenomenon of Secondary Metabolism Regulation

The rolA, rolB and rolC genes are plant oncogenes that are carried in plasmids of the plant pathogen Agrobacterium rhizogenes. Following agrobacterial infection, these genes are transferred into the plant genome and cause tumor formation and hairy root disease. The rolB and rolC genes of Agrobacterium rhizogenes were studied extensively for the past two decades as regulators of cell growth and differentiation. A new function for the rol genes in plant-Agrobacterium interactions became apparent with the discovery that these genes are also potential activators of secondary metabolism in transformed cells in different plant families (reviewed by Bulgakov, 2008). Classically, rolB and rolC have been considered closely related genes, possessing similar biological functions. However, they demonstrated different, or even opposite, effects on cell death processes (Schmulling et al., 1988), calcium balance in transformed cells (Bulgakov et al., 2003), sensitivity to auxin (Maurel et al., 1991), growth of transformed tissues (Capone et al., 1989) and secondary metabolism (Shkryl et al., 2008). Plant-microbe interactions often lead to the development of defense mechanisms in plant cells. Since reactive oxygen species (ROS) play a pivotal role in the regulation of plant defense mechanisms, extensive experiments were performed to study the relationship between secondary metabolism (phytoalexin production) and the production of ROS in cells transformed with rol genes. Here, we summarize these results. Surprisingly, the rolB and rolC genes not only activated phytoalexin production but also suppressed intracellular ROS levels. This combination of defense responses, coupled with the effect of ROS suppression, represents a unique case in plant-microbe interactions. These findings suggest that bypassing upstream cell control mechanisms may be useful in the construction of plant cells possessing stable production of secondary metabolites. This chapter describes the new findings relating to secondary metabolism and ROS production under the individual and combined expression of the rol genes in plant cells.

The rolB gene activates secondary metabolism in Arabidopsis calli via selective activation of genes encoding MYB and bHLH transcription factors

It is known that the rolB gene of Agrobacterium rhizogenes increases the production of secondary metabolites in transformed plant cells, but its mechanism of action remains unclear. In this report, we demonstrate that rolB expression in Arabidopsis thaliana calli led to the activation of most genes encoding secondary metabolism-specific MYB and bHLH transcription factors (TFs), such as MYB11, MYB12, MYB28, MYB76, MYB34, MYB51, MYB122, TT2 and TT8. Accordingly, a higher transcript abundance of main biosynthetic genes related to these factors was detected. The rolB-transformed calli produced 3-fold higher levels of indolic glucosinolates (GSs) compared with normal calli but did not produce secondary metabolites from other groups. Enhanced accumulation of indolic GSs was caused by activation of MYB34, MYB51 and MYB122, and the absence of aliphatic GSs in transformed calli was caused by the inability of rolB to induce MYB29. The inability of rolB-calli to produce flavonoids was caused by the lack of MYB111 expression, induced by the rolB-mediated conversion of MYB expression from cotyledon-specific to root-specific patterns. The high specificity of rolB on secondary metabolism-specific TFs was demonstrated for the first time.