rolC Gene Of Agrobacterium Rhizogenes Research Articles

Inorganic markers profiling in wild type and genetically modified plants subjected to abiotic stresses

Nicotiana is a well characterized genus of Solanaceae. It is widely studied in literature in different research fields, such as biology, environmental chemistry, etc. In this study Nicotiana langsdorffii wilde type plants and transgenic for the rat glucocorticoid receptor gene (GR) or Agrobacterium rhizogenes rolC gene were used to value the effect of exposure to chemical compounds (Cr) and to harsh physical conditions (dryness, heat stress) on the inorganic compositions in plants. Four markers were selected, namely Na, K, Cl− and NO3−. Different procedures of extraction were tested: water permitted to obtain the best conditions for the determination of four selected markers. The analysis was carried out by atomic emission spectroscopy for the determination of the cations; the anions were determined both by ion chromatography and potentiometry: the results obtained by these two techniques and their potentiality were compared. The total cation content (Al, Ca, Cr, Fe, K, Mg, Mn, Na, P, Si and Zn) was also determined. The effect of the genetic modifications on the capacity of the plant to contrast the stress stimuli was valued: the introduction of GR gene seems to greatly reduce the effect caused by the stresses and the adsorption of the inorganic pollutants. The modification with rolC increases the absorption capacity of the plant toward Cr and at the same time reduces its release: this behavior can be exploited in phytoremediation applications.

Genome of Linaria dalmatica Contains Agrobacterium rhizogenes RolC Gene Homolog

The horizontal transfer of genes into the nuclear genome of plants is a rare phenomenon. Currently, the only known examples of this have been described for several Nicotiana and Linaria species. In the genome of Linaria dalmatica (L.) Mill a nucleotide sequence sharing 94% similarity with the sequence of the Agrobacterium rhizogenes rolC gene have been described for the first time. The significance of the rolC gene and T-DNA for the evolution of Linaria is discussed, as well as their role in the origin of plant species.

Genome of Linaria dalmatica contains the homolog of Agrobacterium rhizogenes. rolC gene

Examples of horizontal gene transfer involving nuclear genomes of plants are rare. Currently, only two examples were detected in certain species of tobaccos and toadflax. The paper firstly describes the genomic sequence of Linaria dalmatica (L.) P. Mill. 94% similar to that Agrobacterium rhizogenes rolC gene. A possible role of rolC gene and T-DNA in the evolution of Linaria as well as their potential role in the origin of plant species are argued.

The insertion of the Agrobacterium rhizogenes rolC gene in tomato ( Solanum lycopersicum L.) affects plant architecture and endogenous auxin and abscisic acid levels

In the present paper we report on the effects of the insertion of the Agrobacterium rhizogenes rolC gene in the tomato ( Solanum lycopersicum L., formerly Lycopersicon esculentum Mill.) cultivar Tondino. Several transgenic lines were successfully obtained, between which two clones, rolC1 and rolC3, were chosen for the analysis of morpho-productive traits as well as of the endogenous levels of auxin and abscisic acid. Consistent with the known phenotypic effect of this gene, the transformed tomato plants were significantly shorter than the corresponding controls. On the other hand, even if yield was not affected by the transformation in terms of average number of fruits produced, fruit weight was significantly lower in the transgenics with respect to the controls. Therefore, insertion of the rolC gene does not lead to an improvement in plant productivity. Furthermore, we have observed alterations in the hormonal levels in the shoot apices of the transgenic plants. In fact, quantifications of free and conjugated forms of indole-3-acetic acid (IAA) indicated a significant reduction of IAA levels in the shoot apical region of the transgenic clone rolC3, in comparison with both the control and the clone rolC1. Abscisic acid (ABA) concentration on the other hand was unchanged in the transgenics compared to the controls, but significantly lower in rolC3 with respect to rolC1 plants. The resulting ABA/IAA ratio was higher in both transgenic clones compared to the untransformed plants, indicating that the rolC gene affects the balance between these hormones in transformed tomato 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.

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.

RolC-transgenic carnation plants: adventitious organogenesis and levels of endogenous auxin and cytokinins

Overexpression of the rolC gene of Agrobacterium rhizogenes in plants alters growth and development. We studied whether the cytokinin (CK)-like and auxin-like effect of rolC on enhanced shoot and root regeneration in explants of three lines of rolC-transgenic carnation plants ( Dianthus caryophyllus L. cv. White Sim) was related to altered levels of the endogenous auxin indole-3-acetic acid (IAA) or isoprenoid-CKs. rolC led to improved shoot and root organogenesis in transgenic petal and leaf explants and differences were observed between the lines. Total CKs and the CK/auxin ratio were not correlated with the organogenesis of these lines. However, zeatin riboside levels were up to 3-fold higher in rolC leaves and N 6-isopentenyladenine levels up to 7-fold higher in transgenic petals. Immunolocalization of the latter showed cytoplasmic but mainly nuclear labelling in rolC and non-transgenic petals. The enhanced adventitious shoot organogenesis could be correlated with higher specific CK levels, although they might be an indirect effect of rolC on hormone metabolism. Since levels of IAA remained unaffected in transgenic explants, we propose that an increase in the auxin sensitivity in rolC carnation tissues is responsible for improved rhizogenesis. However, more studies are needed for clarification of the precise function of the RolC protein in transgenic plants.

Promoter of the rolC gene of Agrobacterium rhizogenes can be strongly regulated in glandular cell of transgenic tobacco.

A 577-bp promoter segment of Agrobacterium rhizogenes rolC, previously known as the phloem-specific gene expression promoter, was fused to the 5' end of a reporter gene, beta-glucuronidase (GUS), uidA. This rolC-promoter-driven expression of the GUS gene was found to be significantly strong in glandular cells in transgenic tobacco plants. Analysis of this segment of the promoter sequence revealed a myb response element.

Gene Stability in Transgenic Aspen‐Populus ‐ III. T‐DNA Repeats Influence Transgene Expression Differentially among Different Transgenic Lines

Abstract: Transgene silencing mechanisms have been confirmed in many crop plants, however, few reports are currently available for long‐lived tree species. Three transgenic aspen lines carrying the rolC gene of Agrobacterium rhizogenes, under control of the 35S promoter of the cauliflower mosaic virus, but showing very different types of reversion from rolC‐phenotype to wild type are investigated in detail. A complete suppression of rolC phenotype was observed after one year of in vitro culturing in one transgenic line, whereas the second transgenic line showed gradual decrease in rolC expression, and the plants were comparable to control plants after three to four years in the greenhouse. In the third transgenic line, however, transgene inactivation was observed only in some shoots of the greenhouse‐grown plants which reverted to the rolC phenotype in subsequent years. Southern blotting experiments using a rolC probe revealed the integration of one or two tandem copies of the foreign genes into the host genome. All three transgenic lines contain in/complete T‐DNA repeats, as indicated by reverse primer (rp) PCR. In the reverted tissues of two transgenic lines loss of rolC protein was observed despite the presence of the rolC transcript. The third transgenic line, showing a completely suppressed rolC phenotype, was found positive for the rolC transcript and protein. On the basis of the molecular data presented for the three unstable lines, including Southern, Northern and Western blotting experiments, as well as a survey of transgenic methylation patterns, new insights were obtained with respect to structure and inactivation of transgenes following their transfer in a host tree species.

Genetic transformation by particle bombardment of cultivated jute, Corchorus capsularis L.

Using a biolistic particle delivery system, we have developed an efficient protocol for the generation of stable genetic transformants in jute (Corchorus capsularis var. JRC 321). The apical, meristematic region of a germinating seedling was used as the explant. Multiple shoots were generated from each explant using a medium supplemented with benzylaminopurine (0.2 mg/l) and indole-3-butyric acid (0.5 mg/l). Cotransformation was carried out with the bialaphos resistance gene bar and the rolC gene of Agrobacterium rhizogenes. The positive transformants containing the bar gene grew in growth medium containing 2 mg/l bialaphos. Southern, Northern, polymerase chain reaction (PCR) and reverse transcriptase-PCR analyses provided evidence of gene integration into the genomic DNA of jute. Transgenic plants were resistant to bialaphos. One plant had an altered growth pattern, most likely due to the expression of the transferred rolC gene. The TO transformants showed a stable inheritance of the trans gene to their progenies.

Gene Expression in Bacteria Directed by Plant-specific Regulatory Sequences

The regulation of gene expression represents a specific process which has different structural and functional requirements in different groups of organisms. It is thus assumed that regulatory sequences of eucaryotes cannot be recognized in procaryotes. This assumption is of interest for risk assessments of the environmental impact of deliberate release experiments with genetically modified organisms. In order to analyse the extent of heterologous gene expression caused by the transfer of plant-specific regulatory sequences into bacteria, we constructed fusions between plant-specific regulatory sequences and the coding regions of the luxAB genes for the luciferase of the bioluminescent bacterium Vibrio harveyi, transferred the fusions into different bacterial species and measured the luminescence to quantify the expression of the luciferase genes. The regulatory sequences investigated included (a) the 35S promoter of the Cauliflower mosaic virus, (b) the B33 promoter of a class I patatin gene of potatoes, (c) the promoter of the ST-LS1 gene of potatoes and (d) the promoter of the rolC gene of Agrobacterium rhizogenes. We could show that in addition to the 35S promoter, which has already been described as being recognized in Escherichia coli, the sequences containing the B33 and the ST-LS1 promoters are recognized in bacteria. Luciferase gene expression promoted by the sequence with the ST-LS1 promoter could be observed in E. coli, Yersinia enterocolitica and Agrobacterium tumefaciens. Comparison of the luminescence caused by fusions between luxAB and different promoters on the chromosome and on an endogenous plasmid of Y. enterocolitica demonstrated that the level of the heterologous gene expression caused by the fragment with the ST-LS1 promoter was within the range of gene expression levels caused by endogenous promoters of Y. enterocolitica.

Expression of the Agrobacterium rhizogenes rolC Gene in a Deciduous Forest Tree Alters Growth and Development and Leads to Stem Fasciation.

We have altered the growth and development of a deciduous forest tree by transforming hybrid aspen (Populus tremula x Populus tremuloides) with the Agrobacterium rhizogenes rolC gene expressed under the strong cauliflower mosaic virus 35S promoter. We demonstrate that the genetically manipulated perennial plants, after a period of dormancy, maintain the induced phenotypical changes during the second growing period. Furthermore, mass-spectrometrical quantifications of the free and conjugated forms of indole-3-acetic acid and cytokinins and several gibberellins on one transgenic line correlate the induced developmental alterations such as stem fasciation to changes in plant hormone metabolism. We also show that the presence of the RolC protein increases the levels of the free cytokinins, but not by a process involving hydrolysis of the inactive cytokinin conjugates.

Chemically induced expression of the rolC‐encoded β‐glucosidase in transgenic tobacco plants and analysis of cytokinin metabolism: rolC does not hydrolyze endogenous cytokinin glucosides in planta

SummaryThe rolC gene of Agrobacterium rhizogenes T‐DNA plays an essential role in the establishment of hairy root disease and its overexpression in transgenic plants causes pleiotropic developmental alterations. This study investigated whether the biological activity of the rolC β‐glucosidase is due to an alteration of the cytokinin balance in planta. HPLC radiocounting assays of [3H]‐labeled cytokinin glucosides fed exogenously to tobacco leaf disks, to rolC expressing Escherichia coli cells or cell‐free extracts showed that cytokinin N3‐ and O‐glucosides are the preferred substrate of the rolC protein. Hydrolysis of N7‐ and N9‐glucosides was not detected at substrate concentrations close to physiological levels. Furthermore, these conjugates were also not active as cytokinins in biotests when fed to rolC‐expressing tissues. For analysis of the rolC activity on endogenous cytokinin conjugates the gene was expressed under the transcriptional control of a modified tetracycline‐inducible 35S promoter. This was done to avoid possible interference with secondary effects or plant homeostatic mechanisms which could mask primary in planta events when transgenes are expressed constitutively. No changes in the endogenous pool of different cytokinin glucosides, as determined by a newly developed electrospray tandem mass spectroscopy directly coupled to high performance liquid chromatography, were found following chemical induction of the rolC gene. Also the levels of free cytokinins remained unchanged after gene induction. Hybrid tobacco plants expressing the cytokinin‐synthezising ipt gene and the rolC gene showed added phenotypes indicating that the rolC phenotype is mediated on a signalling pathway different from those of cytokinins. RolC/ipt hybrids also accumulated high levels of cytokinin O‐glucosides. It is concluded that the phenotypic alterations caused by the rolC gene product are not due to a release of free cytokinins from inactive conjugates, most likely because of subcellular compartmentation of the putative substrate.

Horizontal gene transfer: regulated expression of a tobacco homologue of the Agrobacterium rhizogenes rolC gene.

A tobacco homologue (trolC) of the rolC gene of the Agrobacterium rhizogenes Ri-plasmid was cloned and sequenced from Nicotiana tabacum L. cv. Havana 425. The coding region of trolC is similar in sequence (69-87% for DNA and 54-89% for the deduced amino acid sequence) to rolC genes of the agropine, mannopine, and mikimopine strains of Ri-plasmids and the N. glauca rolC homologue. Southern analyses showed that trolC is encoded by a small gene family derived from the tomentosiformis ancestor of tobacco. This suggests that trolC resulted from an ancient transfer of DNA between A. rhizogenes and a progenitor of modern tobacco. Transcripts of trolC were detected in three morphologically distinct cultivars of tobacco. trolC mRNA accumulated in young leaves and shoot tips, but not in lower leaves and roots of mature plants. Accumulation of trolC mRNA in cultured leaf tissues was strongly down-regulated by auxin and induced by cytokinin. These results are of particular interest because they suggest that a gene of bacterial origin introduced during evolution can have a function in a modern plant.

A 43-kDa nuclear tobacco protein interacts with a specific single-stranded DNA sequence from the 5'-upstream region of the Agrobacterium rhizogenes rolC gene.

We identified in the nuclear extract of tobacco seedlings a 43-kDa DNA-binding protein RCS2 that interacted with the 5'-upstream region of the rolC gene of the Agrobacterium rhizogenes Ri plasmid. DNA-protein gel shift and competition assays demonstrated that RCS2 bound to single-stranded DNA in a sequence-specific manner. A five-base direct repeat is important for the DNA binding of RCS2. South-Western blot analysis was employed to determine the size of RCS2 which appears to be approx. 43 kDa.

Constitutive or light-regulated expression of the rolC gene in transgenic potato plants has different effects on yield attributes and tuber carbohydrate composition

Tetraploid potato clones, transgenic for the rolC gene of Agrobacterium rhizogenes under control of the light-inducible ribulose bisphosphate carboxylase small subunit promoter (rbcS-rolC), were compared, with respect to yield attributes and tuber carbohydrates, with transformed and untransformed controls and with 35S-rolC transgenic potato plants. In rbcS-rolC plants, the expression of the rolC gene was located mainly in leaves, while in 35S-rolC plant transcripts were detected as well in shoots and roots. Phenotypically, rbcS-rolC transgenic plants were found to be slightly reduced in plant size with a few more tillers than control plants. Photosynthetic rate and chlorophyll content were significantly lower in all rolC transgenic plants irrespective of the type of construct used. Tuber yield was not significantly different between controls and rbcS-rolC transgenic plants, but was reduced in the 35S-rolC transformants. Sucrose level was unchanged in all rolC clones investigated, whereas fructose content was significantly enhanced in 35S-rolC transformants, but not in the plants expressing the rolC gene in aerial plant parts only. In both types of rolC transgenic plants, glucose content was lower than in controls, resulting in a significant reduction of reducing sugar in tubers. The results suggest a hormonal influence on the carbohydrate composition of potato tubers.

Hormonal Characterization of Transgenic Tobacco Plants Expressing the rolC Gene of Agrobacterium rhizogenes TL-DNA.

Transgenic tobacco (Nicotiana tabacum L. cv Wisconsin 38) plants expressing the Agrobacterium rhizogenes rolC gene under the control of the cauliflower mosaic virus 35S RNA promoter were constructed. These plants displayed several morphological alterations reminiscent of changes in indole-3-acetic acid (IAA), cytokinin, and gibberellin (GA) content. However, investigations showed that neither the IAA pool size nor its rate of turnover were altered significantly in the rolC plants. The biggest difference between rolC and wild-type plants was in the concentrations of the cytokinin, isopentenyladenosine (iPA) and the gibberellin GA19. Radio-immunoassay and liquid chromatography-mass spectrometry measurements revealed a drastic reduction in rolC plants of iPA as well as in several other cytokinins tested, suggesting a possible reduction in the synthesis rate of cytokinins. Furthermore, gas chromatography-mass spectrometry quantifications of GA19 showed a 5- to 6-fold increase in rolC plants compared with wild-type plants, indicating a reduced activity of the GA19 oxidase, a proposed regulatory step in the gibberellin biosynthesis. Thus, we conclude that RolC activity in transgenic plants leads to major alterations in the metabolism of cytokinins and gibberellins.

Restoration of fertility by antisense RNA in genetically engineered male sterile tobacco plants.

Transgenic tobacco plants (Nicotiana tabacum L.) expressing the rolC gene of Agrobacterium rhizogenes under the transcriptional control of the 35S RNA promoter are male sterile. When these plants are genetically crossed with others containing the rolC gene linked in antisense orientation to the 35S RNA promoter, hybrid progeny display restoration of male fertility. Moreover, hybrid progeny are revertant for other features of the rolC phenotype, such as restoration of plant height, leaf pigment content and female fertility. The level of restoration of the characteristics of untransformed tobacco appeared to be independent of the steady-state level of antisense RNA. Addition of six transcriptional enhancer sequences upstream of the 35S transcriptional start region in the antisense construct led to a higher steady-state level of antisense RNA than that produced using a promoter linked to a single enhancer sequence. However no significant difference was observed in the level of attenuation of the rolC phenotype in the progeny of crosses with either one or six transcriptional enhancers linked to the antisense rolC gene. Antisense constructs comprising only 189 bp of the rolC 5' coding region appeared less efficient in attenuating the rolC phenotype than those including the whole rolC coding region as well as its 3' untranslated region. Furthermore, results from experiments on light-controlled rolC gene expression indicate that microsporogenesis is sensitive to rolC gene action during the early stages of flower development.

Transgenic tobacco plants regenerated from leaf disks can be periclinal chimeras

Amongst rolC transgenic tobacco plants regenerated from leaf disks 6.5% are periclinal chimeras, i.e. plants with genetically different cell populations in different cell layers. The expression of the rolC gene of Agrobacterium rhizogenes causes a reduction in pigment content in leaves. The chimeric composition of the regenerated plants becomes thus apparent as light green leaf tissue in the transgenic region, tissue flanked by dark green wild-type sectors. Southern and northern blot analysis confirmed the chimeric nature of such plants. Investigation of selfed progeny of chimeric plants on selective media indicates that layer invasion in reproductive tissues can occur in tobacco early during the formation of the flower buds. The results show (1) that tobacco plants regenerated from leaf disks and grown on selective media have not necessarily the same clonal origin and (2) that they can give rise to non-transgenic offspring. The chimeric plants provide insight on the effect of rolC gene expression on microsporogenesis.

The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates.

The rolC gene of Agrobacterium rhizogenes, which drastically affects growth and development of transgenic plants, codes for a cytokinin-beta-glucosidase. Indeed, rolC protein expressed in Escherichia coli as a fusion protein hydrolyses cytokinin glucosides, thus liberating free cytokinins. Furthermore, beta-glucosidase activity present in E. coli extracts expressing the rolC protein was inhibited by affinity-purified antibodies specific for the rolC protein. Finally, rolC proteins expressed in transgenic plants were shown to be responsible for cytokinin-beta-glucosidase activity. Morphological and phytohormonal analysis, performed on transgenic plants that are somatic mosaics for the expression of the rolC gene, extend and confirm our interpretation that the developmental, physiological and morphological alterations caused by rolC expression in transgenic plants are primarily due to a modification of the cytokinin balance. These observations shed new light on the control of growth and differentiation in plants by growth factors.