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  • 1
    Publication Date: 1979-09-01
    Print ISSN: 0032-0935
    Electronic ISSN: 1432-2048
    Topics: Biology
    Published by Springer
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  • 2
    Publication Date: 1983-03-01
    Print ISSN: 0032-0935
    Electronic ISSN: 1432-2048
    Topics: Biology
    Published by Springer
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  • 3
    Publication Date: 1978-01-01
    Print ISSN: 0032-0935
    Electronic ISSN: 1432-2048
    Topics: Biology
    Published by Springer
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Planta 157 (1983), S. 180-189 
    ISSN: 1432-2048
    Keywords: Hypersensitivity ; Nicotiana (enzyme activity and virus infection) ; O-methyltransferase ; Phenylalanine ammonia-lyase ; Plasmolysis ; Tobacco mosaic virus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Tobacco varieties carrying the N gene from Nicotiana glutinosa respond to infection by Tobacco Mosaic Virus (TMV) by forming necrotic local lesions (hypersensitive reaction), thereby localizing the infection. In this study, infected mesophyll leaf tissue of N. tabacum Samsun NN was treated with the non-permeating, non-metabolizable carbohydrate mannitol. The local lesions developed under iso-osmotic conditions (0.28 M mannitol), though with a slight delay and with a reduced rate of growth, as compared to those on attached leaves. At increasing plasmolysing concentrations of mannitol, necrotization was progressively inhibited, but not completely suppressed. The leaf tissue produced tiny translucent zones, with a delay that increased between the virus inoculation and application of the plasmolytica. Activities of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and O-methyltransferase (OMT, EC 2.1.1.6) are strongly stimulated in hypersensitively reacting tobacco and were used as biochemical markers in the present study. This study was done to determine whether the inhibitory effect of plasmolysis on the elicitation of the hypersensitive response is due to a decrease in virus spread, resulting from the rupture of plasmodesmata or, at least in part, to metabolic alterations of the host cell exposed to osmotic stress. Since necrotization is normally preceded by intense virus multiplication, the inhibitory effects found for early applications (i.e., before local lesion appearance) of plasmolytica could easily be related to an inhibition of virus spread which also occurred in similarly treated leaf tissue of the systemically reacting variety Samsun. The most meaningful data were obtained from mannitol treatments performed on leaf tissue already carrying local lesions, i.e., in which the elicitor(s) and/or the factor(s) of necrotization were already operating. Under iso-osmotic conditions, we found the stimulated PAL and OMT activities characteristic of the hypersensitive response. At plasmolysing concentrations of mannitol, we observed the counteracting effects of two different mechanisms controlling the phenylpropanoid enzymes. Floating the leaf material on the liquid medium induced an ageing-like effect with a continuous increase in enzyme activities that was independent on osmotic pressure and sensitive to cycloheximide. At the same time, the stimulated enzyme activities related to hypersensitivity decreased at a rate related to osmotic pressure. Since PAL and OMT of tobacco leaves are long-lived enzymes, it is likely that the increased de novo synthesis of the enzymes was suppressed by plasmolysis while their degradation and/or inactivation was maintained or even increased. From these results it is concluded that the apparent inhibition of the hypersensitive response by plasmolysis is due to both a decrease in virus spead (artificially caused by the rupture of connections between cells) and to drastic metabolic alterations of the host cell exposed to high osmotic pressure.
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  • 5
    ISSN: 1432-2048
    Keywords: Hypersensitivity ; Nicotiana ; o-Diphenols ; O-Methyltransferases ; Tobacco mosaic virus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three distinct o-diphenol O-methyltransferases (OMTs) were found in leaves of Nicotiana tabacum, variety Samsun NN. They could be clearly distinguished by differences in elution pattern upon chromatography on DEAE-cellulose and in specificity towards 16 diphenolic substrates. The phenylpropanoids caffeic acid and 5-hydroxyferulic acid, whose importance as lignin precursors is well known, were the best substrates of OMT I, but they were also efficiently methylated by the two other OMTs that showed a broader substrate specificity. The highest rates of methylation were observed by assaying these latter enzymes with catechol, homocatechol and protocatechuic aldehyde. The flavonoid quercetin, the major o-diphenol of tobacco leaves, was a good substrate for OMTs II and III, but was also methylated significantly by OMT I. The tobacco OMTs showed both para-and meta-directing activities with protocatechuic acid, protocatechuic aldehyde and esculetin as substrates. Para-O-methylation of the former substrate arose almost exclusively from OMT I whereas that of the two latter substrates from all three enzymes. In healthy leaves the total O-methylating activity varied very much with the batch of plants whereas the relative contributions of the three enzymes were rather constant. On an average, OMTs I, II and III acounted towards caffeic acid, respectively. In tobacco mosaic virus-infected leaves carrying local necrotic lesions we found the same three OMTs with the same substrate specificities, but with increased activities. The degree of stimulation of both OMTs II and III was 2–3 times greater than that of OMT I when the leaves had a moderate number of lesions, and 3–5 times greater with large number of lesions. It is very likely that the changes in both the pattern of the O-methylating enzymes and the concentrations of the naturally occuring o-diphenolic substrates are related to an increased biosynthesis of lignins and of lignin-like compounds. These aromatic polymers could be involved in the cell wall thickening associated with the hypersensitive reaction and with the resistance to virus spread that occur in the cells surrounding the local lesions.
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  • 6
    ISSN: 1432-2048
    Keywords: Hypersensitivity ; Necrogenesis ; Nicotiana ; Phenylalanine ammonia-lyase ; Protoplasts ; Tobacco mosaic virus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Leaves of tobacco varieties carrying the N gene for hypersensitiviy react to tobacco mosaic virus (TMV) infection by forming necrotic lesions and by localizing the virus in the vicinity of these lesions. These changes are accompanied in the host by an increased metabolic activity, in particular by an increased production of phenolic compounds derived from phenylalanine. Necrogenesis apparently destroys cells which have become heavily infected despite this strong defense reaction. However, it has been demonstrated previously (Otsuki et al., 1972) that protoplasts derived from leaves which normally respond in vivo to virus inoculation by forming necrotic local lesions, show no such response when inoculated in vitro. In the present study we have investigated the effect of pre-infecting hypersensitive leaves with TMV on the production or the non-production of the factor(s) of necrosis at the level of either protoplasts or mesophyll cells isolated from these preinfected leaves. Phenylalanine ammonia-lyase (PAL), whose rate of synthesis has been shown (Duchesne et al., 1977) to increase in stimulated cells of infected leaves, was used as a biochemical marker in the search for the stimulus preceding necrogenesis. We found that this stimulus concerning PAL activity was never elicited in either protoplasts or mesophyll cells which were prepared just before the appearance of necrotic local lesions. This result did not depend on the conditions of pre-infection or on the methods used to isolate the protoplasts or mesophyll cells. We also assayed samples derived from pre-infected leaves that were already carrying local lesions, i.e., in which the stimulus and necrogenesis were already operating: not only did the isolated protoplasts and mesophyll cells not sustain the stimulus concerning PAL activity, but the stimulated enzyme activity decreased abruptly and, in most of the experiments, had disappeared within the time necessary for maceration. Evidence is presented showing that the non-elicitation or the abrupt decrease of stimulated PAL activity could not result from a selection of unstimulated cells or from a preferential destruction of stimulated cells during maceration of the leaves. Our results support the view that hypertonic osmotic pressure is responsible for the non-occurence of the hypersensitive response by acting according to one or both of the following processes: it suppresses the contacts through plasmodesmata between neighboring cells and, hence, it also suppresses the cell-to-cell diffusion of the factor(s) eliciting the stimulus; and/or since hypertonic osmotic pressure causes striking differences between leaf cells and protoplasts in total RNA and protein synthesis, these differences might include the suppression of synthesis of the elicitor of hypersensitivity.
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  • 7
    ISSN: 1617-4623
    Keywords: Nicotiana tabacum ; Pathogenesis-related protein ; Chitinase ; Gene expression ; Vacuolar targeting
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A new PR (pathogenesis-related) protein was isolated from tobacco leaves (Nicotiana tabacum cv. Samsun NN), reacting hypersensitively to tobacco mosaic virus (TMV), by zinc chelate chromatography and was therefore named Pz. Its reactivity toward several lectins indicated the presence of bound sugar residues. From the amino acid sequence of tryptic peptides, Oligonucleotide primers were derived which allowed the synthesis of Pz cDNA by PCR. Using this cDNA as probe, near full-length clones were isolated from a library made from poly(A)+ RNA purified from TMV-infected leaves. Sequence analysis revealed similarities with chitinases/lysozymes of various origins and the purified protein was, indeed, shown to hydrolyse different N-acetylglucosamine-containing substrates. Comparison of peptide and cDNA sequences indicated that Pz protein is synthesized as a pre-pro-protein, a seven-amino acid C-terminal peptide probably being involved in the vacuolar targeting of the protein. Pz mRNA and protein were demonstrated to accumulate strongly in TMV-infected tobacco leaves. Pz transcripts were also found in various tissues of healthy plants, indicating that Pz gene expression is developmentally regulated.
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  • 8
    ISSN: 1573-5028
    Keywords: pathogenesis-related proteins ; chitinase ; tobacco ; TMV
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Recently, four chitinases have been detected in tobacco mosaic virus (TMV) infected tobacco: two acidic chitinases that were identified as pathogenesis-related (PR) proteins P and Q and two basic chitinases (Legrand et al., Proc.Natl. Acad. Sci. USA, in press). Here, it was shown that P and Q are closely serologically related but not related to other known acidic tobacco PR proteins. Antisera to P and Q were used to characterize translation products of TMV-induced mRNAs that were hybrid-selected with cDNA clones described previously (Hooft van Huijsduijnen et al., EMBO J 5: 2057–2061, 1986). In this way cDNA clones corresponding to the acidic and basic chitinases were identified. The partial amino acid sequences of the acidic and basic tobacco chitinases that were represented in the clones, showed an approximately 70% homology to each other and to the sequence of a bean chitinase. Although the acidic and basic chitinases differ in apparent molecular weight, they were found to have homologous C-termini. Hybridization of cDNA probes to genomic blots indicated that the acidic and basic chitinases are each encoded by two to four genes in the amphidiploid genome of Samsun NN tobacco. A similar complexity was found for the genes encoding the tobacco PR protein that is homologous to the sweet-tasting protein thaumatin and to the bifunctional trypsin/α-amylase inhibitor from maize.
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