β-Oxidation enzymes in the mitochondria of Arum and oilseed rape

-Oxidation enzymes were detected both in the mitochondria and microbodies of Arum maculatum L. spadices and Brassica napus L. seeds. It is apparent that the mitochondrial membrane barrier, which remains intact after sucrose-density-gradient centrifugation, prevents rapid access of acyl-GoA substrates to matrix oxidation tes. Thus intact mitochondria showed little -oxidation enzyme activity. Rupturing of the mitochondrial membrane allowed rapid access of acyl CoAs to matrix sites. Consequently, in ruptured mitochondria, high -oxidation enzyme activities were measured.C. Masterson thanks the Science and Engineering Research Council for the award of a postgraduate student maintenance grant. D.R. Thomas and C. Wood thank their relatives for continuing financial support. The authors also thank West Cumberland Farmers Ltd., Hexham, UK for their gift of oilseed rape seeds.     

Effect of cyclopentene ��,�¡�-triketone disodium salt on the activity of hydrolases and the state of tobacco mosaic virus particles in tobacco leaves

The activity of hydrolases (protease, RNase) in uninfected and tobacco mosaic virus-infected tobacco leaves of the Samsun variety, untreated and treated with disodium salt of 2-acetyl-4-hydroxycarbonyl-methylthio-5-chlorocyclopent-4-en-1,3-dione (DS), was determined. It was shown that treatment of leaves with this compound significantly increased the activity of hydrolases in them compared to untreated leaves. In infected leaves treated with DS one day before infection, along with an increased level of hydrolases, one revealed more viral particles exposed to destructive changes in infected, rather than untreated, leaves. It is assumed that the DS-caused activation of hydrolases promotes the destruction of viral particles and is therefore one of the cell defense mechanisms induced by this compound that prevents the intracellular accumulation of virus.     

Expression of a soybean β-conclycinin gene under the control of the Cauliflower Mosaic Virus 35S and 19S promoters in transformed petunia tissues

A gene encoding the -subunit of -conglycinin was ligated to the 19S and 35S promoters of Cauliflower Mosaic Virus and introduced into petunia plants on a disarmed Ti-plasmid using Agrobacterium tumefaciens. Transformed cells were regenerated into whole plants and ummunoreactive polypeptides and hybridizable, polyadenylated mRNA were detected in transformed tissues. Expression from the 35S promoter was 10 to 50 times greater than expression from the 19S promoter. The level of immunodetectable polypeptides was greater in seeds than in leaves or callus tissue. In addition, the pattern of -polypeptide breakdown products was distinctive in seeds and leaves. We conclude that in seeds the higher levels of the -polypeptide reflect enhanced stability of this protein.     

Induction of aphid resistance in tobacco by the cucumber mosaic virus CMV∆2b mutant is jasmonate-dependent

Cucumber mosaic virus (CMV) is vectored by aphids, including Myzus persicae. Tobacco (Nicotiana tabacum ‘Xanthi’) plants infected with a mutant of the Fny strain of CMV (Fny-CMVΔ2b, which cannot express the CMV 2b protein) exhibit strong resistance against M. persicae, which is manifested by decreased survival and reproduction of aphids confined on the plants. Previously, we found that the Fny-CMV 1a replication protein elicits aphid resistance in plants infected with Fny-CMVΔ2b, whereas in plants infected with wild-type Fny-CMV this is counteracted by the CMV 2b protein, a counterdefence protein that, among other things, inhibits jasmonic acid (JA)-dependent immune signalling. We noted that in nontransformed cv. Petit Havana SR1 tobacco plants aphid resistance was not induced by Fny-CMVΔ2b, suggesting that not all tobacco varieties possess the factor(s) with which the 1a protein interacts. To determine if 1a protein-induced aphid resistance is JA-dependent in Xanthi tobacco, transgenic plants were made that expressed an RNA silencing construct to diminish expression of the JA co-receptor CORONATINE-INSENSITIVE 1. Fny-CMVΔ2b did not induce resistance to M. persicae in these transgenic plants. Thus, aphid resistance induction by the 1a protein requires JA-dependent defensive signalling, which is countered by the CMV 2b protein.     

Stability of transgene integration and expression in subsequent generations of doubled haploid oilseed rape transformed with chitinase and β-1,3-glucanase genes in a double-gene construct

A double-gene construct with one chitinase and one β-1,3-glucanase gene from barley, both driven by enhanced 35S promoters, was transformed into oilseed rape. From six primary transformants expressing both transgenes 10 doubled haploid lines were produced and studied for five generations. The number of inserted copies for both the genes was determined by Southern blotting and real-time PCR with full agreement between the two methods. When copy numbers were analysed in different generations, discrepancies were found, indicating that at least part of the inserted sequences were lost in one of the alleles of some doubled haploids. Chitinase and β-1,3-glucanase expression was analysed by Western blotting in all five doubled haploid generations. Despite that both the genes were present on the same T-DNA and directed by the same promoter their expression pattern between generations was different. The β-1,3-glucanase was expressed at high and stable levels in all generations, while the chitinase displayed lower expression that varied between generations. The transgenic plants did not show any major impact on fungal resistance when assayed in greenhouse, although purified β-1,3-glucanase and chitinase caused retardment of fungal growth in vitro.     

Mode of action of N,N′-diphenylurea: The isolation and identification of the cytokinins in the transfer RNA from tobacco callus grown in the presence of N,N′-diphenylurea

Summary The tRNA from cytokinin-dependent tobacco callus (Nicotiana tabacum) grown on mineral medium containing N,N-diphenylurea as the source of cytokinin was found to contain 3 cytokinin-active ribonucleosides. The 2 ribonucleosides present in the largest amounts were identified conclusively by their chromatographic properties, ultra-violet and low-resolution mass spectra as the naturally-occurring cytokinins 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9--D-ribofuranosylpurine and 6-(3-methyl-2-butenylamino)-9--ribofuranosylpurine. A third ribonucleoside, present in smaller amounts, was identified as another naturally-occurring cytokinin 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9--D-ribofuranosylpurine on the basis of its chromatographic behaviour. No evidence was found to associate the mode of action of the non-purine cytokinin, N,N-diphenylurea, with tRNA.Abbreviation DPU N,N-diphenylurea     

Leaf developmental stage and tissue location affect the detection of β-glucuronidase in transgenic tobacco plants

Expression in Nicotiana tabaccum L. plants containing the -glucuronidase (GUS) gene under the control of the 35S (CaMV promoter) was affected by tissue type and ontogenic development of the leaves. GUS activity in ontogenetically younger leaves was 1003–1022 nmol 7-hydroxy-4-methylcoumarin (MU) formed mg^–1 (protein) min^–1 and in ontogenetically older leaves was only 140–198 nmol (MU) mg^–1 (protein) min^–1.     

Effect of chitosan on tobacco mosaic virus（TMV） accumulation,hydrolase activity,and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus（TMV） in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV（2 μg/mL） and chitosan（1 mg/mL） were lower in the early period of infection（3 days after inoculation）, by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases（proteases, RNases） in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid（PTA）-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles（18 nm in diameter, 300 nm long）, these suspensions contained abnormal（swollen, “thin” and “short”） virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that “thin” virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.     

Binding between elongation factor 1A and the 3ʹ-UTR of Chinese wheat mosaic virus is crucial for virus infection

The Chinese wheat mosaic virus (CWMV) genome consists of two positive-strand RNAs that are required for CWMV replication and translation. The eukaryotic translation elongation factor (eEF1A) is crucial for the elongation of protein translation in eukaryotes. Here, we show that silencing eEF1A expression in Nicotiana benthamiana plants by performing virus-induced gene silencing can greatly reduce the accumulation of CWMV genomic RNAs, whereas overexpression of eEF1A in plants increases the accumulation of CWMV genomic RNAs. In vivo and in vitro assays showed that eEF1A does not interact with CWMV RNA-dependent RNA polymerase. Electrophoretic mobility shift assays revealed that eEF1A can specifically bind to the 3ʹ-untranslated region (UTR) of CWMV genomic RNAs. By performing mutational analyses, we determined that the conserved region in the 3ʹ-UTR of CWMV genomic RNAs is necessary for CWMV replication and translation, and that the sixth stem-loop (SL-6) in the 3ʹ-UTR of CWMV genomic RNAs plays a key role in CWMV infection. We conclude that eEF1A is an essential host factor for CWMV infection. This finding should help us to develop new strategies for managing CWMV infections in host plants.     

Involvement of abscisic acid-dependent and — Independent pathways in the upregulation of antioxidant enzyme activity during NaCl stress in cotton callus tissue

The role of abscisic acid (ABA) in the signal transduction pathway associated with NaCl-induced up-regulation of antioxidant enzyme activity was examined in a NaCl-tolerant cotton callus cell line treated with NaCl, ABA, paraquat, or H₂O₂ in the presence and absence or fluridone, an inhibitor of terpene, and therefore, ABA synthesis. Treatment with NaCl resulted in a rapid increase (within 30 minutes) in the ABA levels of the callus tissue, and the NaCl, ABA, and paraquat treatments induced rapid increases in the activities of superoxide dismutase, catalase, peroxidase, and glutathione reductase. Pre-treatment with fluridone significantly suppressed the NaCl-induced increases, but only slightly delayed the increases in tissue subjected to exogenous ABA treatment. This implies that ABA is involved in the signal transduction pathway associated with the NaCl-induced up-regulation of these antioxidant enzymes. Pre-treatment with fluridone had no effect on the paraquat-induced increases, suggesting that these enzymes can also be up-regulated by a pathway other than the one mediated by ABA. Both the NaCl and paraquat treatments produced significant increases in the superoxide levels within the callus, but the increase resulting from the paraquat treatment was significantly higher than the increase resulting from the NaCl treatment. These data suggest that NaCl stress results in the production of reactive oxygen intermediates (ROI) which signals the induction of an ABA-dependent signaling pathway. The production of very high levels of ROI, such as those that occur with paraquat treatment or perhaps during periods of prolonged or extreme stress, may induce an ABA-independent signaling pathway.     

Effects of the cucumber mosaic virus 2a protein on aphid–plant interactions in Arabidopsis thaliana

The cucumber mosaic virus (CMV) 2a RNA-dependent RNA polymerase protein has an additional function in Arabidopsis thaliana, which is to stimulate feeding deterrence (antixenosis) against aphids. Antixenosis is thought to increase the probability that aphids, after acquiring CMV particles from brief probes of an infected plant's epidermal cells, will be discouraged from settling and instead will spread inoculum to neighbouring plants. The amino acid sequences of 2a proteins encoded by a CMV strain that induces antixenosis in A. thaliana (Fny-CMV) and one that does not (LS-CMV) were compared to identify residues that might determine the triggering of antixenosis. These data were used to design reassortant viruses comprising Fny-CMV RNAs 1 and 3, and recombinant CMV RNA 2 molecules encoding chimeric 2a proteins containing sequences derived from LS-CMV and Fny-CMV. Antixenosis induction was detected by measuring the mean relative growth rate and fecundity of aphids (Myzus persicae) confined on infected and on mock-inoculated plants. An amino acid sequence determining antixenosis induction by CMV was found to reside between 2a protein residues 200 and 300. Subsequent mutant analysis delineated this to residue 237. We conjecture that the Fny-CMV 2a protein valine-237 plays some role in 2a protein-induced antixenosis.     

A combined computational and experimental investigation of the [2Fe–2S] cluster in biotin synthase

Biotin synthase was the first example of what is now regarded as a distinctive enzyme class within the radical S-adenosylmethionine superfamily, the members of which use Fe/S clusters as the sulphur source in radical sulphur insertion reactions. The crystal structure showed that this enzyme contains a [2Fe–2S] cluster with a highly unusual arginine ligand, besides three normal cysteine ligands. However, the crystal structure is at such a low resolution that neither the exact coordination mode nor the role of this exceptional ligand has been elucidated yet, although it has been shown that it is not essential for enzyme activity. We have used quantum refinement of the crystal structure and combined quantum mechanical and molecular mechanical calculations to explore possible coordination modes and their influences on cluster properties. The investigations show that the protonation state of the arginine ligand has little influence on cluster geometry, so even a positively charged guanidinium moiety would be in close proximity to the iron atom. Nevertheless, the crystallised enzyme most probably contains a deprotonated (neutral) arginine coordinating via the NH group. Furthermore, the Fe···Fe distance seems to be independent of the coordination mode and is in perfect agreement with distances in other structurally characterised [2Fe–2S] clusters. The exceptionally large Fe···Fe distance found in the crystal structure could not be reproduced.     

Origin of Scm1 and Scm2– two loci conferring resistance to sugarcane mosaic virus (SCMV) in maize

Sugarcane mosaic virus (SCMV) causes serious losses of grain and forage yield of maize (Zea mays L.) in Europe. Two dominant genes, Scm1 and Scm2, have been identified to confer resistance to SCMV. Scm1 is located on the short arm of chromosome 6 and Scm2 near the centromere region of chromosome 3. In the present study,resistant, partially resistant, and susceptible maize inbred lines, together with their ancestral lines, were evaluated with molecular markers to trace back the origin of Scm1 and Scm2. The banding patterns indicated that the Scm1 region, originally identified in resistant European line FAP1360A, was derived from its ancestral line FAP954A. The other two resistant European lines, D21 and D32, most likely carry the same Scm1 region, which originated from their common ancestral line A632. This Scm1 region was also present in three partially resistant lines, D09, FAP1396A and FAP693A, but not in the resistant U.S. inbred Pa405. Apart from FAP954A and A632, none of the remaining ancestral lines and none of the susceptible lines harbored the Scm1 region. The Scm2 region present in FAP1360A was obviously transmitted from its ancestral line Co125. However, the presence of the respective Scm2 region was not confirmed in the other three resistant lines (D21, D32 and Pa405), the remaining ancestral lines, and all partially resistant lines by using closely linked markers. Received: 22 July 1999 / Accepted: 30 July 1999     

Catalytic mechanism of the primary human prostaglandin F2α synthase, aldo-keto reductase 1B1--prostaglandin D2 synthase activity in the absence of NADP(H)

Aldo-keto reductase 1B1 and 1B3 (AKR1B1 and AKR1B3) are the primary human and mouse prostaglandin F(2α) (PGF(2α)) synthases, respectively, which catalyze the NADPH-dependent reduction of PGH(2), a common intermediate of various prostanoids, to form PGF(2α). In this study, we found that AKR1B1 and AKR1B3, but not AKR1B7 and AKR1C3, also catalyzed the isomerization of PGH(2) to PGD(2) in the absence of NADPH or NADP(+). Both PGD(2) and PGF(2α) synthase activities of AKR1B1 and AKR1B3 completely disappeared in the presence of NADP(+) or after heat treatment of these enzymes at 100 °C for 5 min. The K(m), V(max), pK and optimum pH values of the PGD(2) synthase activities of AKR1B1 and AKR1B3 were 23 and 18 μM, 151 and 57 nmol·min(-1)·(mg protein)(-1), 7.9 and 7.6, and pH 8.5 for both AKRs, respectively, and those of PGF(2α) synthase activity were 29 and 33 μM, 169 and 240 nmol·min(-1)·(mg protein)(-1), 6.2 and 5.4, and pH 5.5 and pH 5.0, respectively, in the presence of 0.5 mm NADPH. Site-directed mutagenesis of the catalytic tetrad of AKR1B1, composed of Tyr, Lys, His and Asp, revealed that the triad of Asp43, Lys77 and His110, but not Tyr48, acts as a proton donor in most AKR activities, and is crucial for PGD(2) and PGF(2α) synthase activities. These results, together with molecular docking simulation of PGH(2) to the crystallographic structure of AKR1B1, indicate that His110 acts as a base in concert with Asp43 and Lys77 and as an acid to generate PGD(2) and PGF(2α) in the absence of NADPH or NADP(+) and in the presence of NADPH, respectively.     

Epidemics of Tomato torrado virus,Pepino mosaic virus and Tomato chlorosis virus in tomato crops: do mixed infections contribute to torrado disease epidemiology?

Torrado disease was first observed in protected tomato crops in the Murcia province of Spain in spring 2001, causing serious concern to regional tomato producers. The disease-causing agent was initially identified as a picorna-like bipartite plant RNA virus, now known as Tomato torrado virus (ToTV), but several additional torradoviruses inducing similar disease symptoms have been described more recently. We studied the incidence of torradoviruses between 2005 and 2008 in two parts of Murcia (Spain) where tomato crops are grown commercially. We also analysed the potential association among ToTV, Pepino mosaic virus (PepMV) and Tomato chlorosis virus (ToCV) in samples showing torrado symptoms of varying severity. ToTV was the only torradovirus found in the samples (predominantly as single infections), but double and triple infections comprising ToTV, PepMV and/or ToCV were also detected. There was no evidence of a specific association among the viruses as the frequencies of mixed infections did not deviate from those expected to occur by chance. Statistical analysis of the potential association between torrado symptoms and the type of infection (single or multiple) was inconclusive. To determine whether co-infections with ToTV and PepMV have any marked influence on the torrado disease, we analysed torrado symptom severity and virus accumulation in tomato plants experimentally infected with ToTV-CE, PepMV-Sp13 and PepMV-PS5 in single and mixed infections. The severity of the torrado symptoms was not affected by the presence of PepMV. In single infections, the ToTV titre remained very low, reaching its maximum in the early stages of infection and declining rapidly thereafter, whereas the disease symptoms became more severe over the same timescale. In mixed infections, the accumulation of both ToTV and PepMV was altered with respect to single infections, and the magnitude of this alteration appeared to be virus and strain specific. Therefore, ToTV and PepMV mixed infections may modulate the epidemiology of both viruses in a complex way by altering virus fitness. The impact of our studies on efforts to track and prevent the spread of torrado disease is discussed.     

An integrative methodology to predict dispersal of genetically modified genotypes in oilseed rape at landscape-level—A study for the region of Schleswig-Holstein,Germany

Potential environmental as well as socio-economic effects of the cultivation of genetically modified (GM) oilseed rape (OSR) may be caused by large-scale dispersal of transgenes. We present an up-scaling approach that was based on scenario assumptions concerning the percentage of GM cultivation and took into account natural and anthropogenic variation of involved dispersal processes. The applied methods include computer modelling and spatial analysis. A simulation model (GeneTraMP) was used to calculate the spatio-temporal pattern of the spread of a neutral transgene (without any specific function) in OSR. Basic scenario calculations were carried out for different spatial configurations covering 1 km² each and taking into account information on climate and cultivation systems of the region of the federal state of Schleswig-Holstein, Germany. For the exemplary regional study presented here, we analysed the numbers of flowering plants of GM OSR in different types of locations as predicted by the model. The results confirmed the expectation of a very high variability of GM occurrences at distinguishable intensity levels which were closely related to the proximity of areas of intended GM oilseed rape cultivation and may be described by a combination of management parameters and location type.The up-scaling method included a spatial analysis of the target region. Based on satellite images and digital maps, the structure of the region was analysed resulting in a map of Schleswig-Holstein that represents each single field, also including information on crop rotation, ownership and production systems. Applying GIS queries to this database, we identified the area of relevant location types. Both, the model results and the spatial data were used to predict the total numbers of flowering GM OSR plants for the region of Schleswig-Holstein. As an important feature, the up-scaling of modelling results to a larger scale allows for a comprehensive analysis by also enclosing regional parameters, as, for example the cropping density. The presented methods can support decision making if they are incorporated into the planning of an environmental monitoring of commercial GM crops or into life cycle assessment and cost–benefit analyses of GMO cultivation.