Effect of the rol genes from Agrobacterium rhizogenes on polyamine metabolism in tobacco roots

Plants of the mangrove species Pelliciera rhizophoreae and Avicennia germinans, exhibit pronounced oscillations in stomatal aperture under certain climatic conditions. During these oscillations, changes in transpirational water loss were closely followed by those in leaf water potential (ψ1) as indicated by continuous monitoring with an in situ dewpoint hygrometer. With this instrument, it was possible to measure dynamic changes in ψ1 for several days under constant conditions. Subsequently, the leaf was detached from the shoot and a pressure-volume (PV) curve was established by repeatedly weighing the leaf, still attached to the hygrometer during short interruptions of the water potential recordings. The pressure-volume relationship was then used to derive other water relations parameters from these water potential data. Thus, the procedure described herein allows a continuous analysis of the relevant components of bulk leaf water relations. Oscillations in water potential were also measured with single leaves using a pressure chamber. Water relations data obtained with these two different methods were in good agreement. In addition, osmotic potentials derived from the PV-analysis were well within the range of those determined cryoscopically using extracted cell sap.     

Hormonal content and sensitivity of transgenic tobacco and potato plants expressing single rol genes of Agrobacterium rhizogenes T-DNA

The expression of single rol genes of the T_L-DNA of Agrobacterium rhizogenes strain A4 in transgenic tobacco (Nicotiana tabacum L.) and potato (Solanum tuberosum L.) plants alters the internal concentrations of, and the sensitivity to, several plant hormones. The levels of immunoreactive cytokinins, abscisic acid, gibberellins and indole-3-acetic acid were analysed in tissues of the apical shoots, stems, leaves, roots and undifferentiated callus tissue. The addition of the dominant and morphogenetically active rolA, rolB, or rolC genes resulted in alterations in the content of several hormones. rolC overexpression in particular led to an up to fourfold increase in the content of isopentenyladenosine, dihydrozeatin riboside and trans-zeatin riboside-type cytokinins in potato plants. This increase correlated well with different levels of expression of the rolC gene in different transgenic plants. Furthermore it was shown that the dwarfism of P_35s-rolC transgenic tobacco and potato plants is correlated with a 28–60% reduction of gibberellic acid A₁ concentration in apical shoots. Exogenous addition of gibberellic acid completely restored stem elongation in P_35s-_rolC transgenic plants. Apical shoots of dwarf rolA transgenic tobacco plants also contained 22% less gibberellic acid A₁ than control plants, but growth cannot be restored completely by exogenously added gibberellic acid. Similarly, the sensitivity of transgenic tobacco seedlings or callus tissues towards different phytohormone concentrations can be altered by the expression of single rol genes. The overexpression of the rolC gene in seedlings led to an altered response to auxins, cytokinins, abscisic acid, gibberellic acid and the ethylene precursor 1-aminocyclopropane-carboxylic acid. The overexpression of the rolB gene in tobacco calli led to necrosis at lower auxin concentrations than in the wild-type, while other parameters of auxin action, like the induction of cell growth, remained unchanged.     

Increase in anthraquinone content in Rubia cordifolia cells transformed by rol genes does not involve activation of the NADPH oxidase signaling pathway

It has been reported that rol plant oncogenes located in Ri-plasmids of Agrobacterium rhizogenes activated synthesis of secondary metabolites in the transformed plant cells. The activator mechanism is still unknown. In this work, we studied whether the NADPH oxidase-signaling pathway, which regulates the synthesis of defense metabolites in plants, is involved in the activator function of the rol genes. It was demonstrated that the transformation of Rubia cordifolia cells by the rolB and rolC genes caused an induction of biosynthesis of anthraquinone-type phytoalexins. Inhibition studies revealed a striking difference between the rolC and rolB transformed cultures in their sensitivity to Ca²⁺ channel blockers and calcium deficiency. The rolC culture displayed lowered resistance to the inhibitors compared to the non-transformed culture, while the rolB culture was more resistant to the treatment. The assumption was made that the oncogenic potential of rol genes is realized through the alteration of calcium balance in the plant cells. Anthraquinone production was not inhibited in the non-transformed and transformed cultures by Ca²⁺ channel blockers, as well as by diphenylene iodonium, an inhibitor of NADPH oxidase, and by the protein kinase inhibitor staurosporine. These results indicate that the induction of anthraquinone production in transgenic cultures does not involve the activation of Ca²⁺-dependent NADPH oxidase pathway.     

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.     

Changes in the structure of apple pectic substances during ripening and storage

During ripening, the degree of polymerization, the degree of esterification, the neutral sugar content and the neutral sugar composition of extractable apple pectic substances did not change. Some xylose and glucose containing polysaccharides can be extracted from the ripe cell walls suggesting that changes in the hemicelluloses take place. In senescent apples, significant changes in the structure of apple pectic substances could be observed. The degree of polymerization of both the galacturonan chains and the arabinogalactan side chains decreased. The amount of water-extractable pectin molecules carrying 1,3/1,6-linked galactans increased. The degree of esterification and the distribution of the methoxyl groups in the apple pectic substances did not change very much.     

Purification, structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds

The structure and bioactivity of a polysaccharide extracted and purified from a 4M KOH + H3BO3 solution from Prunus dulcis seed cell wall material was studied. Anion-exchange chromatography of the crude extract yielded two sugar-rich fractions: one neutral (A), the other acidic (E). These fractions contain a very similar monosaccharide composition: 5:2:1 for arabinose, uronic acids and xylose, respectively, rhamnose and galactose being present in smaller amounts. As estimated by size-exclusion chromatography, the acidic fraction had an apparent molecular mass of 762 kDa. Methylation analysis (from the crude and fractions A and E), suggests that the polysaccharide is an arabinan-rich pectin. In all cases, the polysaccharides bear the same type of structural Ara moieties with highly branched arabinan-rich pectic polysaccharides. The average relative proportions of the arabinosyl linkages is 3:2:1:1 for T-Araf:(1-->5)-Araf:(1-->3,5)-Araf:(1-->2,3,5)-Araf. The crude polysaccharide extract and fractions A and E induced a murine lymphocyte stimulatory effect, as evaluated by the in vitro and in vivo expression of lymphocyte activation markers and spleen mononuclear cells culture proliferation. The lymphocyte stimulatory effect was stronger on B- than on T-cells. No evidence of cytotoxic effects induced by the polysaccharide fractions was found.     

Effects of neuroactive substances on the activity of subcommissural organ cells in dispersed cell and explant cultures

The subcommissural organ (SCO), an ependymal (glial) circumventricular organ, releases glycoproteins into the cerebrospinal fluid; however, the regulation of its secretory activity is largely unknown. To identify neuroactive substances that may regulate SCO activity, we investigated immunocytochemically identified bovine SCO cells by means of calcium imaging. This analysis was focused on: (1) serotonin (5HT) and substance P (SP), immunocytochemically shown to be present in axons innervating the bovine SCO; and (2) ATP, known to activate glial cells. 5HT had no effect on the intracellular calcium concentration ([Ca(2+)](i)), and its precise role remains to be clarified. SP elicited rises in [Ca(2+)](i) in approx. 30% and ATP in even 85% of the analyzed SCO cells. These effects were dose-dependent, involved NK(3) and P2Y(2) receptors linked to G protein and phospholipase C (PLC) activation, and could not be mimicked by forskolin or 8-bromo-cAMP. In 50% of the SP-sensitive cells, the increases in [Ca(2+)](i) comprised calcium release from thapsigargin-sensitive intracellular stores and an influx of extracellular calcium via protein kinase C (PKC)-induced opening of L-type voltage-gated calcium channels (VGCCs). In the remaining SP-sensitive cells, the increase in [Ca(2+)](i) was caused exclusively by influx of extracellular calcium via VGCCs of the L-type. In all ATP-sensitive cells the increase in [Ca(2+)](i) involved calcium release from thapsigargin-sensitive intracellular stores and a PKC-mediated influx of extracellular calcium via L-type VGCCs. Our data suggest that SP and ATP are involved in regulation of the activity of SCO cells.     

Getting to the root: The role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots

Agrobacterium tumefaciens and A. rhizogenes are the causative agents of the crown gall and hairy root diseases, respectively. The pathogenicity of both species is caused by an inter-kingdom transfer of DNA from the bacteria to wounded plant cells. This 'transfer-DNA' (T-DNA) contains oncogenes whose expression transforms the plant recipient cell into a rapidly dividing tumour cell. In the case of A. tumefaciens , three of these oncogenes have been shown to encode enzymes catalyzing the biosynthesis of the plant growth hormones auxin and cytokinin. Therefore, the unorganized cell division in the crown gall tumour can be largely explained by an unregulated overproduction of these plant growth regulators. In contrast, the hairy root disease is characterized by a massive growth of adventitious roots at the site of infection. Because of the similarities of the infection processes, and because A. rhizogenes and A. tumefaciens are very closely related, it has been suggested that the most important A. rhizogenes oncogenes, the so called rol genes, are also encoding proteins involved in the regulation of plant hormone metabolism. However, recent data indicate that this is not the case. Thus the rol genes have functions that most likely are different from producing mere alterations of plant hormone concentrations. This review summarizes recent results concerning the expression and function of the rol genes, and presents a model for the role of these genes, especially rolB and rolC , in the A. rhizogenes infection process.     

Effect of calcium and other metal ions on pectic activities from autolysed cultures of Alternaria alternata

Abstract The effect of increasing concentrations of Ca²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Na⁺ and EDTA on the pectic enzymic activities (polymethylgalacturonase, endopectinase and pectin-lyase) present in the autolytic complex from Alternaria alternata has been studied. In all cases the divalent metal ions and EDTA produced an increased inhibition correlated with increasing concentration of each ion. An opposite effect was shown by the Na⁺ ion, which produced an increase in pectic enzymic activities, principally at low concentrations.     

Consistent and stable expression of the nptII, uidA and bar genes in transgenic Pinus radiata after Agrobacterium tumefaciens-mediated transformation using nurse cultures

An Agrobacterium tumefaciens-mediated transformation protocol has been developed for embryogenic cell cultures of Pinus radiata. Transgenic lines were only produced when embryogenic tissue was placed on nurse tissue during the Agrobacterium co-cultivation and recovery stages of the procedure. Plantlets were regenerated via somatic embryogenesis from ten of the 11 transgenic lines tested and at least 20 of each line were planted in a GMO glasshouse. Expression of the nptII, uidA and bar genes in up to ten plants of each individual transgenic line was evaluated by molecular, biochemical and functional analysis. As expected, expression of the nptII gene varied among the ten lines, while within ten replicates of the same line, nptII expression appeared to be consistent, with the exception of one line, K3. Likewise, the level of GUS activity varied among transgenic lines, but was relatively consistent in plants derived from the same tissue, except for two lines, G4 and G5. Moreover, similar absolute values and pattern of gene expression of uidA was observed in the transgenic plants, for two consecutive years. Plantlets from eight lines survived a spray treatment with the equivalent of 2 kg/ha and 4 kg/ha of the commercial formulation Buster, whereas non-transformed controls died. Southern hybridisation analysis of embryogenic tissue and green needle tissue from putative transgenic lines demonstrated a relatively low number of gene insertions (from one to nine) of both the bar and nptII genes in the nine transgenic lines tested.     

Extracellular exo-polygalacturonase secreted from carrot cell cultures. Its purification and involvement in pectic polymer degradation

Exo-polygalacturonase (exo-PGase, EC 3.2.1.67) activity has been detected in a culture filtrate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular exo-PGase was purified to electrophoretic homogeneity using DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The molecular mass of the purified enzyme was calculated to be 48 kDa from Sephadex G-200 gel filtration, and 50 kDa from sodium dodecyl sulfate (SDS)-PAGE after treatment with SDS and 2-mercaptoethanol. The isoelectric point was at pH 6.2. The K_m and V_max values for polygalacturonate (degree of polymerization: 52) were 14.4 μ M and 25.6 μmol (mg protein)⁻¹ h⁻¹, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.6. The enzyme activity was inhibited by Ba²⁺, Cu²⁺, Mn²⁺ and Hg²⁺. The enzyme was involved in ca 15% hydrolysis of the acidic polymer purified from carrot pectic polysaccharides, and connected with the release of galacturonic acid. Even after an exhaustive reaction the enzyme had, however, little or no effect on cell walls from carrot cell cultures.     

Effects associated with insertion of rol genes on morphogenic potential in explants derived from transgenic Bacopa monnieri (L.) Wettst

Plant Cell, Tissue and Organ Culture (PCTOC) - The present study deals with the establishment of rolA-transgenic and rolB-transgenic plants for the first time through Agrobacterium tumefaciens...     

Function of the aux and rol genes of the Ri plasmid in plant cell division in vitro

Auxin-autonomous growth in vitro may be related to the integration and expression of the aux and rol genes from the root-inducing (Ri) plasmid in plant cells infected by agropine-type Agrobacterium rhizogenes. To elucidate the functions of the aux and rol genes in plant cell division, plant cell lines transformed with the aux1 and aux2 genes or with the rolABCD genes were established using tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells. The introduction of the aux1 and aux2 genes enabled the auxin-autonomous growth of BY-2 cells, but the introduction of the rolABCD genes did not affect the auxin requirement of the BY-2 cells. The results clearly show that the aux genes are necessary for auxinautotrophic cell division, and that the rolABCD genes are irrelevant in auxin autotrophy.Key words: Agrobacterium rhizogenes, auxin-autotrophic cell, auxin biosynthesis, hairy root, plant cell division, Ri plasmid, T-DNA, aux, rol, tobacco BY-2 cells     

Abnormal purine and pyrimidine nucleotide content in primary astroglia cultures from hypoxanthine-guanine phosphoribosyltransferase-deficient transgenic mice

Lesch-Nyhan syndrome is a pediatric metabolic-neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The cause of the metabolic consequences of HGPRT deficiency has been clarified, but the connection between the enzyme deficiency and the neurological manifestations is still unknown. In search for this connection, in the present study, we characterized purine nucleotide metabolism in primary astroglia cultures from HGPRT-deficient transgenic mice. The HGPRT-deficient astroglia exhibited the basic abnormalities in purine metabolism reported before in neurons and various other HGPRT-deficient cells. The following abnormalities were found: absence of detectable uptake of guanine and of hypoxanthine into intact cell nucleotides; 27.8% increase in the availability of 5-phosphoribosyl-1-pyrophosphate; 9.4-fold acceleration of the rate of de novo nucleotide synthesis; manyfold increase in the excretion into the culture media of hypoxanthine (but normal excretion of xanthine); enhanced loss of label from prelabeled adenine nucleotides (loss of 71% in 24 h, in comparison with 52.7% in the normal cells), due to 4.2-fold greater excretion into the media of labeled hypoxanthine. In addition, the HGPRT-deficient astroglia were shown to contain lower cellular levels of ADP, ATP, and GTP, indicating that the accelerated de novo purine synthesis does not compensate adequately for the deficiency of salvage nucleotide synthesis, and higher level of UTP, probably due to enhanced de novo synthesis of pyrimidine nucleotides. Altered nucleotide content in the brain may have a role in the pathogenesis of the neurological deficit in Lesch-Nyhan syndrome.     

发根农杆菌Ri质粒rolB基因研究进展（综述）

RiT-DNArolB基因是发根农杆菌转化植物的决定因子,rolB基因表达引起转基因植物形成大量毛状根（hairy root)。本文介绍近年来rolB基因的位点,表达与调控及RolB蛋白结构与功能方面的研究进展。     

Enzymatic degradation and β-elimination of the pectic substances in cherry fruits

Pectic substances from cherry fruits (Prunus avium) were studied after extraction and purification. They were subjected to β-elimination by heat treatment and depolymerized by endopolygalacturonase from Aspergillus niger. The degraded pectins were fractionated by gel permeation chromatography (Sephadex G-100, Bio-gel P2). The results suggest that the pectic substances largely consist of an α-d-galacturonic backbone interspersed with occasional l-rhamnosyl residues. Neutral sugars as side-chains of varying lengths appear to be concentrated along certain regions of the polymer (‘hairy$\text{)}\text{?}$ in contrast to side-chain free (4mooth’) parts.