Influence of calcium influx induced by the calcium ionophore, A23187, on resveratrol content and the expression of CDPK and STS genes in the cell cultures of Vitis amurensis

The present study examines the effect of calcium influx induced by the calcium ionophore (CI) on the biosynthesis of resveratrol and the expression of stilbene synthase (STS) and calcium-dependent protein kinase (CDPK) genes in cell cultures of Vitis amurensis, which have different levels of resveratrol production. The present study utilized the control cell culture V2 of V. amurensis, which contains no more than 0.02?% dry weight (DW) of resveratrol, in addition to rolB transgenic cell cultures VB1 and VB2, which have increased resveratrol contents (0.1–0.8?% DW). Treatment with the CI at a 1?μM concentration significantly increased STS gene expression (6 of 10 analyzed STS genes) and resveratrol production in the control V2 cell culture by fourfold; however, use of the CI at 10?μM significantly decreased resveratrol production by 2–4 fold in all cell cultures tested. In the control V2 grape cell culture, treatment with the CI increased expression of all of the CDPK genes except VaCDPK1a and VaCDPK3a. In the rolB transgenic VB2 grape cell culture treated with the CI, we detected alterations in expression of several CDPK genes, but these changes in gene expression were not significant. Our results indicated that treatment with 1?μM of the CI increased resveratrol content and production in control grape cells by selectively increasing the expression of STS genes. Conversely, the CI treatment did not significantly increase resveratrol content and production, or the expression of CDPK or STS genes in the rolB transgenic cells. Likely, untreated VB2 cells have increased concentrations of cytoplasmic calcium, and therefore, treatment with the CI did not significantly change CDPK expression. These results suggest that the rolB gene has an important role in the regulation of calcium-dependent transduction pathways in transformed cells.     

Effect of the rol genes from Agrobacterium Rhizogenes on the content and structure of pectic substances and glycanase activity in Rubia Cordifolia transgenic cell cultures

The expression of the rolB gene was found to increase the pectic yield in Rubia cordifolia cells, while the rolC gene inhibited the pectin production, which correlated with its expression level. The expression of the rolA, rolB, and rolC genes led to an increase in the content of arabinogalactan (AG) in cells. The increase in the expression of the rolB and rolC genes resulted in a more significant reduction in the content of arabinose residues in pectin, which was accompanied by an increased activity of α-L-arabinofuranosidase in cells. Moreover, the amount of galactose residues in pectin increased with the enhancement of the rolB expression due to a decrease in the activity of β-galactosidase in cells. The content of galacturonic acid residues in pectin from transgenic cultures decreased in the following order: rolC > rolB > rolA. The amount of arabinose residues in AG decreased independently of the gene type. The amount of arabinose residues in AG was found to be considerably reduced when the rolB expression level was increased.     

Can plant oncogenes inhibit programmed cell death? The rolB oncogene reduces apoptosis-like symptoms in transformed plant cells

The rolB oncogene was previously identified as an important player in ROS metabolism in transformed plant cells. Numerous reports indicate a crucial role for animal oncogenes in apoptotic cell death. Whether plant oncogenes such as rolB can induce programmed cell death (PCD) in transformed plant cells is of particular importance. In this investigation, we used a single-cell assay based on confocal microscopy and fluorescent dyes capable of discriminating between apoptotic and necrotic cells. Our results indicate that the expression of rolB in plant cells was sufficient to decrease the proportion of apoptotic cells in steady-state conditions and diminish the rate of apoptotic cells during induced PCD. These data suggest that plant oncogenes, like animal oncogenes, may be involved in the processes mediating PCD.     

Effects of 5-azacytidine induced DNA demethylation on methyltransferase gene expression and resveratrol production in cell cultures of Vitis amurensis

DNA becomes methylated in vivo through the action of a specific group of enzymes known as methyltransferases or methylases. Plants are known to possess the methyltransferases (Met), chromo methyltransferases (CMT), and domainrearranged methyltransferases (DRM) methylase families, which affect cytosine methylation within different contexts. DNA methylation has been proposed to play a role in secondary plant metabolism, but there is a lack of valid data connecting these two processes. In this study, we treated control and transformed with rolB gene from Agrobacterium rhizogenes cell cultures of Vitis amurensis with the demethylation agent 5-azacytidine (azaC). The purpose of the current investigation was to study effects of induced DNA demethylation on methyltransferase gene expression in connection to resveratrol production, a naturally occurring polyphenol that has a wide range of intriguing biological properties. Using semi-quantitative and real-time PCR, we showed that rolB gene transformation of V. amurensis cells decreased Met and CMT expression, but significantly increased DRM expression. AzaC treatment of the control and the rolB-transgenic calli significantly increased expression of all methylases (excluding Met). Following 3 months of azaC treatment, we detected significantly elevated levels of rolB gene expression in the transgenic calli. In current paper, we discuss how methylase expression may influence resveratrol biosynthesis and rolB transgene expression. Effects of azaC application are discussed.     

Agrobacterium-Mediated Transformation of Tomato with rolB Gene Results in Enhancement of Fruit Quality and Foliar Resistance against Fungal Pathogens

Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens.     

Efficiency of the tetracycline-dependent gene expression system: complete suppression and efficient induction of the rolB phenotype in transgenic plants

We have investigated the use of the tetracycline-dependent gene expression system to regenerate and propagate tobacco plants transformed with a gene whose product — when highly expressed — interferes with regeneration and/or further reproduction. Plants transformed with the Agrobacterium rhizogenes rolB gene under the control of the tetracycline-dependent expression system were phenotypically indistinguishable from wild type owing to efficient repression of the promoter. Induction of the rolB gene with tetracycline led to high-level expression of the rolB mRNA, which resulted in extremely stunted plants with necrotic and wrinkled leaves that did not develop a floral meristem. Upon cessation of tetracycline treatment healthy shoots developed even from severely affected meristems. Data on the dose response of the rolB phenotype as a function of tetracycline concentration demonstrate that the tetracycline-dependent gene expression system can be used to modulate the manifestation of a particular phenotype.     

Involvement of DNA methylation in the regulation of STS10 gene expression in Vitis amurensis

DNA methylation is known to play an important role in various developmental processes and defense mechanisms in plants and other organisms. However, it is not known whether DNA methylation is implicated in the genetic regulation of plant secondary metabolism, including resveratrol biosynthesis. Resveratrol is a naturally occurring polyphenol that is present in grapes, peanuts, and other plant sources, and it exhibits a wide range of valuable biologically active properties. The transformation of the wild-growing grape Vitis amurensis with the oncogene rolB from Agrobacterium rhizogenes has been demonstrated to considerably increase resveratrol production. To investigate whether DNA methylation regulates resveratrol biosynthesis, we treated both rolB transgenic and empty vector control V. amurensis cell cultures with the DNA demethylation agent 5-azacytosine (azaC). The azaC treatment significantly increased stilbene synthase 10 gene (VaSTS10) expression and resveratrol content in the V. amurensis cell cultures. Using bisulfite sequencing, we examined the methylation status of VaSTS10 in cell cultures under normal conditions and after azaC treatment. Both the promoter and 3′-end of the protein coding region of the VaSTS10 gene were hypermethylated (54–67 %) in the control cell culture. The rolB transgenic cell culture had high levels of resveratrol and lower hypermethylation levels of the VaSTS10 gene (20–47 %). The azaC treatment resulted in reduction in the DNA methylation levels in the promoter and coding regions of the VaSTS10 gene in both cell cultures. These data suggest that the DNA methylation may be involved in the control of resveratrol biosynthesis via the regulation of STS genes expression.     

Sequence of the cellular T-DNA in the untransformed genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and analysis of its expression in genetic tumors of N. glauca x N. langsdorffii

A region homologous to the TL-DNA of Agrobacterium rhizogenes was previously detected in the genome of untransformed Nicotiana glauca and designated cellular T-DNA (cT-DNA). Subsequently, part of this region was sequenced and two genes, which corresponded to rolB and rolC and were named NgrolB and NgrolC, were found. We have now sequenced a region of the cT-DNA other than the region that includes NgrolB and C and we have found two other open reading frames (ORFs), NgORF13 and NgORF14. These ORFs correspond to ORFs 13 and 14 of the TL-DNA of A. rhizogenes and exhibit a high degree of homology to these ORFs, without having a nonsense codon. We have not found any sequence homologous to rolD (ORF15). The two genes, NgORF13 and 14, as well as the NgrolB and C genes, are expressed in genetic tumors of hybrids between N. glauca and N. langsdorffii but not in leaf tissues of the hybrid.     

Effect of <Emphasis Type="Italic">Rol</Emphasis> Transgenes,IAA, and Kinetin on Starch Content and the Size of Starch Granules in Tubers of <Emphasis Type="Italic">In Vitro</Emphasis> Potato Plants

Stem cuttings were produced from Solanum tuberosum L., cv. Desiree, plants and their transgenic forms harboring rolB and rolC genes from Agrobacterium rhizogenes. Plants were cultured on hormone-free Murashige and Skoog nutrient medium (MS) and on MS supplemented with IAA or kinetin. In microtubers developed on these cuttings, we estimated the content of starch and the number and size of starch granules. Expression of rol genes changed these indices: in tubers of rolC transformants, a greater number of small granules were produced, whereas in tubers of rolB transformants, a fewer number of large granules were developed as compared with wild-type plants. Expression of rol genes did not affect starch content during the first three weeks of cutting culturing but increased it by 15–30% in five-week-old tubers. IAA addition to MS medium increased starch content and the size of starch granules in control plants and rolB tubers by 10–30%, whereas kinetin did not exert any significant influence. The effects of rol transgenes on the initiation and termination of starch granule development are discussed.     

Expression of <Emphasis Type="Italic">rol</Emphasis> genes in transgenic soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) leads to changes in plant phenotype,leaf morphology,and flowering time

Soybean (Glycine max L.) cultivar NARC-4 was transformed with constructs carrying rolA, rolB, or rolC genes, each under the control of the Cauliflower Mosaic Virus 70S promoter. Cotyledonary nodes of soybean seeds were infected with Agrobacterium tumefaciens strain LBA4404 carrying one of the three rol genes, along with nptII in the plasmid pLBR. The efficiency of the transformation varied slightly among the three constructs, with frequencies of 6, 7, and 5% for the rolA, rolB, and rolC genes, respectively, being observed. Southern blot analysis confirmed the integration of rol genes in the soybean genome with varying numbers of copies of the transgene. All transformed plants showed enhanced rooting, but the number of adventitious roots was higher for transformants carrying the rolB transgene. rolA and rolC transformants showed dwarf phenotypes, clustered branching, and variations in leaf morphology. Furthermore, these plants flowered within a short period of time and produced lower numbers of flowers. rolB transformants showed variations in phenotype, including dwarf to semi-dwarf and shrubby growth, abnormal stem growth, short internodes, variations in leaf morphology, and greenish to yellowish-green colored leaves. These plants also flowered early, but dwarf plants produced low numbers of flowers, while shrubby plants produced high numbers of flowers, but these were mostly infertile.     

Nucleolus-like bodies in micronuclei of cultured Xenopus cells

Two of the 36 chromosomes in Xenopus laevis are known to carry nucleolar organizer loci. Partitioning of the chromosomes of cultured, early-passage Xenopus cells among variable numbers of micronuclei could be induced by extended colcemid treatment. A large, obvious nucleolus occurred in a maximum of 4 micronuclei per colcemid-induced tetraploid cell. The large, deeply-stained nucleoli incorporated [³H]uridine and appeared by electron microscopy to have typical nucleolar morphology with fibrillar and granular areas disposed in nucleolonema. In situ hybridization to radioactive ribosomal RNA (rRNA) resulted in heavy labelling of nucleoli in no more than 4 micronuclei per cell. The other micronuclei generally contained small bodies (blobs) which stained for RNA and protein as well as with ammoniacal silver. In the electron microscope, these appeared as round, dense bodies resembling nucleoli segregated by actinomycin D treatment. Nucleoplasmic RNA synthesis occurred in all micronuclei regardless of whether they contained definitive nucleoli. These observations suggest that micronuclei which formed large, typical, RNA-synthesizing nucleoli contained nucleolar organizer chromosomes, while the other micronuclei, which contained nucleolus-like “blobs” probably lacked nucleolar organizer loci. It is possible that the nucleolus-like bodies may have been aggregates of previously synthesized nucleolar RNA and protein trapped in micronuclei after mitosis.     

The Plant Oncogene rolB Alters Binding of Auxin to Plant Cell Membranes

We measured auxin-binding capacity of the membrane preparationsfrom tobacco cells transformed by rolB as compared to untransformedcontrols. In the transformed cells, the overall auxin-bindingactivity is severalfold enhanced through an increase in a bindingactivity removable from the membranes at 0.5 M salt, while thebinding activity still attached to the membranes after saltwashes remains unchanged. Antibodies against the 22 kDa maizeauxin binding protein (ABP) depress most of the membrane-attachedbinding activity in both normal and rolB-transformed cells,while they do not affect the salt-washable binding activity.In contrast, antibodies against the RolB protein prevent completelybinding of auxin to the latter activity in both normal and transformedcells, while substantially unaffecting the membrane-associatedbinding. These results point to the presence, in untransformedmembranes, of an auxin-binding activity associated with a proteinimmunologically related to RolB. This activity is much increasedin rolB cells. In contrast, the auxin-binding protein analogousto maize ABP present in tobacco membranes does not increasein the rolB-transformed cells. (Received October 1, 1993; Accepted April 22, 1993)     

Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd2+ tolerance and accumulation but not translocation to the shoot

Phytochelatins (PCs) are metal binding peptides involved in heavy metal detoxification. To assess whether enhanced phytochelatin synthesis would increase heavy metal tolerance and accumulation in plants, we overexpressed the Arabidopsis phytochelatin synthase gene (AtPCS1) in the non-accumulator plant Nicotiana tabacum. Wild-type plants and plants harbouring the Agrobacterium rhizogenes rolB oncogene were transformed with a 35S AtPCS1 construct. Root cultures from rolB plants could be easily established and we demonstrated here that they represent a reliable system to study heavy metal tolerance. Cd²⁺ tolerance in cultured rolB roots was increased as a result of overexpression of AtPCS1, and further enhanced when reduced glutathione (GSH, the substrate of PCS1) was added to the culture medium. Accordingly, HPLC analysis showed that total PC production in PCS1-overexpressing rolB roots was higher than in rolB roots in the presence of GSH. Overexpression of AtPCS1 in whole seedlings led to a twofold increase in Cd²⁺ accumulation in the roots and shoots of both rolB and wild-type seedlings. Similarly, a significant increase in Cd²⁺ accumulation linked to a higher production of PCs in both roots and shoots was observed in adult plants. However, the percentage of Cd²⁺ translocated to the shoots of seedlings and adult overexpressing plants was unaffected. We conclude that the increase in Cd²⁺ tolerance and accumulation of PCS1 overexpressing plants is directly related to the availability of GSH, while overexpression of phytochelatin synthase does not enhance long distance root-to-shoot Cd²⁺ transport.     

A Stereoselective Synthesis of dl-C17-Cecropia Juvenile Hormone

The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.     

Accessory nucleoli in microsporocytes of hybrid Phlox

Studies were conducted on the number of nucleoli present during diplotene and diakinesis in plants adjacent to, at the edge, and at the center of a zone of secondary intergradation between Phlox pilosa subsp. pilosa and P. pilosa subsp. fulgida. Assessory nucleoli were present in some cells of about 75% of the plants examined. Nucleolar numbers varied from 1 to 5. Where 2 or more nucleoli were present they were usually attached to different chromosomes. As the number of nucleoli increased, the volume of single nucleoli decreased so that volume of all nucleoli was roughly that of a normal single nucleolus. — Only 1% of the microsporocytes from populations adjacent to the zone had accessory nucleoli as compared to 4.6% of the cells in populations at the edge of the zone, and 7.7% in populations in the center. The correlation between population hybridity and incidence of accessory nucleoli is r = 0.80.     

Effect of red- and blue-light-emitting diodes on growth and morphogenesis of grapes

The effect of red- and blue-light-emitting diodes on shoot and root growth of Hybrid Franc grape, a rootstock cultivar, along with two other grape genotypes, Kadainou R-1 and Vitis ficifolia var. ganebu, were investigated in vitro. Plants cultured under red-light-emitting diodes produced the longest shoots with longer internodes for all genotypes. The chlorophyll content measured as SPAD value and leaf number per explant were highest on plants cultured under blue-light-emitting diodes in all the genotypes. Blue light was also responsible for a higher number of stomata in all the genotypes; however, there was no significant difference in size of stomata in all genotypes under the different light conditions tested. Different light-emitting diodes did not affect the rooting percentage of Hybrid Franc but red-light-emitting diodes gave a higher rooting percentage along with higher root numbers for the two other grape genotypes.     

Agrobacterial <Emphasis Type="Italic">rol</Emphasis> genes modify thermodynamic and structural properties of starch in microtubers of transgenic potato

Wild-type (WT) plants of potato (Solanum tuberosum L.) and their transgenic forms carrying agrobacterial genes rolB or rolC under the control of B33 class I patatin promoter were cultured in vitro on MS medium with 2% sucrose in a controlled-climate chamber at 16-h illumination and 22°C. These plants were used as a source of single-node stem cuttings, which were cultured in darkness on the same medium supplemented with 8% sucrose. The tubers formed on them were used for determination of the structure of native starch using the methods of differential scanning microcalorimetry (DSC), X-ray scattering, and scanning electron microscopy. It was found that, in starch from the tubers of rolB-plants, the temperature of crystalline lamella melting was lower and their thickness was less than in WT potato. In tubers of rolC plants, starch differed from starch in WT plants by a higher melting temperature, considerably reduced melting enthalpy, and a greater thickness of crystalline lamellae. Deconvolution of DSC thermogram makes it possible to interpret the melting of starch from the tubers of rolC plants as the melting of two independent crystalline structures with melting temperatures of 65.0 and 69.8°C. Electron microscopic examination confirmed the earlier obtained data indicating that, in the tubers of rolC plants, starch granules are smaller and in the tubers of rolB plants larger than in WT plants. Possible ways of influence of rol transgenes on structural properties of starch in amyloplasts of potato tubers are discussed.     

Stimulation of RNA synthesis in nucleoli by inorganic phosphate in vitro

The biological function of a phosphoprotein with a molecular weight of 120 000 daltons localized in the nucleoli of mouse ascites sarcoma cells was studied by examining the effect of the phosphoprotein on RNA synthesis in the nucleoli in vitro. The phosphoprotein did not stimulate ribosomal RNA synthesis in vitro. During this study, it was observed that inorganic phosphate enhanced RNA synthesis in the nucleoli in vitro in the presence of either Mn²⁺ or Mn²⁺ plus Mg²⁺ as divalent cations. Inorganic phosphate stimulated the rate of the chain elongation reaction in RNA synthesis.