Extremely low frequency weak magnetic fields enhance resistance of NN tobacco plants to tobacco mosaic virus and elicit stress-related biochemical activities

Increasing evidence has accumulated concerning the biological effects of extremely low frequency magnetic fields (ELF-MFs) in different plant models. In the present study, effects of ELF-MFs in tobacco plants reacting to tobacco mosaic virus (TMV) with a hypersensitive response (HR) were evaluated. Plants were exposed for 8 or 24 h (either before or after TMV inoculation) to a static MF, at either -17 or 13 microT, combined with a 10 Hz sinusoidal MF with different intensities (25.6 or 28.9 microT). The working variables were the area and number of hypersensitive lesions in leaves. Following ELF-MFs exposure, an increased resistance was detected, particularly after an 8-h treatment, as shown by the decrease in lesion area and number. Moreover, two enzyme activities involved in resistance mechanisms were analyzed: ornithine decarboxylase (ODC) and phenylalanine ammonia-lyase (PAL). Uninoculated leaves previously exposed to ELF-MFs in general showed a significant increase relative to controls in ODC and PAL activities, in particular for 13 microT static MF plus 28.9 microT, 10 Hz sinusoidal MF (24 h) treatment. In conclusion, ELF-MFs seem to influence the HR of tobacco to TMV, as shown by the increased resistance and changes in ODC and PAL activities, indicating the reliability of the present plant model in the study of bioelectromagnetic interactions.     

Phenylalanine ammonia-lyase in tobacco mosaic virus-infected hypersensitive tobacco. Density-labelling evidence of de novo synthesis.

A strong increase in phenylalanine ammonia-lyase (EC 4.3.1.5) activity occurs in tobacco mosaic virus-infected tobacco leaves developing necrotic local lesions. Comparison of physicochemical properties of the partially purified enzymes extracted from healthy and infected leaves showed that the hypersensitive reaction leads to an increase in the pool size of the same active enzyme molecules as those present in non-infected material. The molecular mechanism of enzyme formation was investigated by radiolabelling with [3H]leucine and by density labelling with 2H2O. Abnormal patterns of incorporation of radioactivity into all soluble proteins were found in infected leaves carrying local lesions. In contrast, uptake of deuterium into the amino acid pool was the same in healthy and infected leaves. Unstimulated phenylalanine ammonia-lyase was shown to be a long-lived enzyme (half-life: 25-35 h). Results of comparative density labelling experiments unequivocally demonstrated that the increased enzyme pool size arose from an increased rate of synthesis mediated by the hypersensitive reaction.     

Plant disease and the regulation of enzymes involved in lignification

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.     

Structurally unrelated algal oligosaccharides differentially stimulate growth and defense against tobacco mosaic virus in tobacco plants

Tobacco plants were treated with structurally unrelated oligosaccharides obtained from Chilean marine macroalgae. These oligosaccharides were prepared by chemical depolymerization of native polysaccharides extracted from brown and red algae and correspond to pure polymers of around 20 units of guluronic acid (Poly-Gu), mannuronic acid (Poly-Ma) and sulphated galactan (Poly-Ga). These oligosaccharides were solubilized in water, at a final concentration of 500 μg mL⁻¹, and sprayed on tobacco leaves, once a week for a month. Their effects on the stimulation of growth and defense against tobacco mosaic virus (TMV) were determined 7 and 15 days after the final spraying, respectively. The activities of several defense and antioxidant enzymes and the levels of water-soluble antioxidant compounds were determined. Plants treated with Poly-Ga and Poly-Ma showed an increase in height of 23% and 49%, respectively, whereas Poly-Gu did not stimulate growth. Plants treated with Poly-Gu, Poly-Ma and Poly-Ga showed an increase in defense against TMV corresponding to decreases in the number of necrotic lesions of 9%, 22% and 74%, respectively. The stimulation of plant growth correlates with activation of the antioxidant enzyme ascorbate peroxidase (AP) and with a decrease in ascorbate level. On the other hand, the stimulation of defense against TMV is correlated with the activation of the defense enzyme phenylalanine ammonia-lyase (PAL). These results indicate that algal oligosaccharides differentially stimulate growth and defense against TMV in tobacco plants and that these processes involve the activation of the enzymes AP and PAL, respectively.     

The symptom difference induced by <Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> in tobacco plants containing the <Emphasis Type="Italic">N</Emphasis> gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was replaced by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in tobacco plants containing the N gene results from the functional difference of their MP genes.     

The symptom difference induced by Tobacco mosaic virus and Tomato mosaic virus in tobacco plants containing the N gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaicvirus (ToMV) are members of the genus Tobamoviruswith a world-wide distribution, and cause severe dis-eases on many economically important crops. TMVand ToMV have very close relationship and both havessRNA genome with a length of about 6400 nucleo-tides, encoding at least three nonstructural proteinsand a 17.6 kD coat protein (CP). Both 126 kD and 183kD proteins function as components of replicase, andthe 30 kD protein is involved in viral ce…     

Tobacco plants epigenetically suppressed in phenylalanine ammonia-lyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus

The response of tobacco (Nicotiana tabacum L. cv. Xanthinc) plants, epigenetically suppressed for phenylalanine ammonia-lyase (PAL) activity, was studied following infection by tobacco mosaic virus (TMV). These plants contain a bean PAL2 transgene in the sense orientation, and have reduced endogenous tobacco PAL mRNA and suppressed production of phenylpropanoid products. Lesions induced by TMV infection of PAL-suppressed plants are markedly different in appearance from those induced on control plants that have lost the bean transgene through segregation, with a reduced deposition of phenofics. However, they develop at the same rate as on control tobacco, and pathogenesis-related (PR) proteins are induced normally upon primary infection. The levels of free salicylic acid (SA) produced in primary inoculated leaves of PAL-suppressed plants are approximately fourfold lower than in control plants after 84 h, and a similar reduction is observed in systemic leaves. PR proteins are not induced in systemic leaves of PAL-suppressed plants, and secondary infection with TMV does not result in the restriction of lesion size and number seen in control plants undergoing systemic acquired resistance (SAR). In grafting experiments between wild-type and PAL-suppressed tobacco, the SAR response can be transmitted from a PAL-suppressed root-stock, but SAR is not observed if the scion is PAL-suppressed. This indicates that, even if SA is the systemic signal for establishment of SAR, the amount of pre-existing phenylpropanoid compounds in systemic leaves, or the ability to synthesize further phenylpropanoids in response to the systemic signal, may be important for the establishment of SAR. Treatment of PAL-suppressed plants with dichloro-isonicotinic acid (INA) induces PR protein expression and SAR against subsequent TMV infection. However, treatment with SA, while inducing PR proteins, only partially restores SAR, further suggesting that de novo synthesis of SA, and/or the presence or synthesis of other phenylpropanoids, is required for expression of resistance in systemic leaves.     

Ornithine decarboxylase activity and the hypersensitive reaction to tobacco mosaic virus in Nicotiana tabacum

The activity of ornithine decarboxylase (ODC) is increased 20 fold in leaves of Nicotiana tabacum cv. Xanthi n.c. following infection with tobacco mosaic virus at 20°. The activity reaches its maximum when localized necrotic lesions appear. There is little or no increase in plants kept at 32° when infection is systemic. However, if the infected plants are transferred to 20°, a marked and rapid increase in ODC activity occurs in the upper leaves, which collapse seven to nine hours after the transfer. ODC activity therefore parallels the activity of phenylalanine ammonia lyase during the hypersensitive reaction. Tyrosine decarboxylase was found to be activated in the same conditions. By contrast no increase in arginine decarboxylase activity could be detected. Temperature has a much greater effect on the polyamine and tyramine content of Xanthi n.c. leaves than does infection with TMV.     

Xanthine Oxidase Activity in the Susceptible and Hypersensitive Responses of Tobacco Leaves to Tobacco Mosaic Virus Infection

A superoxide-producing xanthine oxidoreductase was isolated and quantified after polyacrylamide disc gel electrophoresis of tobacco leaf extracts. The results obtained indicate that, like uricase activity, a slight increase in tobacco xanthine oxidase activity takes place in the susceptible interaction with tobacco mosaic virus (TMV). In contrast, out of three hypersensitive tobacco cultivars tested, only two showed the same slight increase m activity during the late stage of hypersensitive response.
Allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] a specific and potent in vitro and in vivo inhibitor of xanthine oxidoreductase, applied to tobacco plants by root absorption, starting about 8 days before the inoculation, did not affect the hypersensitive response but weakened the hypersensitivity-linked virus localization and promoted the movement of a certain amount of TMV particles and/or virus related material from necrotic lesions which induced systemic necrotic symptoms in uninoculated leaves. However, due to the inefficacy of allopurinol in preventing necrotic lesion development, all results are consistent with the hypothesis that xanthine oxidoreductase, the first enzyme in purine oxidative degradation, plays only a secondary role during induction of primary hypersensitive cell death in TMV infected tobacco leaves.     

Regulation of Ethylene Biosynthesis in Virus-Infected Tobacco Leaves : I. DETERMINATION OF THE ROLE OF METHIONINE AS THE PRECURSOR OF ETHYLENE

The hypersensitive reaction of Samsun NN tobacco leaves to tobacco mosaic virus (TMV) was accompanied by a large increase in ethylene production, just before necrotic local lesions became visible. Normal and virus-induced ethylene production were both largely inhibited by 0.1 millimolar aminoethoxyvinylglycine indicating that methionine is a main ethylene precursor.     

Effect of yeast CTA1 gene expression on response of tobacco plants to tobacco mosaic virus infection

The response of tobacco (Nicotiana tabacum L. cv Xanthi-nc) plants with elevated catalase activity was studied after infection by tobacco mosaic virus (TMV). These plants contain the yeast (Saccharomyces cerevisiae) peroxisomal catalase gene CTA1 under the control of the cauliflower mosaic virus 35S promoter. The transgenic lines exhibited 2- to 4-fold higher total in vitro catalase activity than untransformed control plants under normal growth conditions. Cellular localization of the CTA1 protein was established using immunocytochemical analysis. Gold particles were detected mainly inside peroxisomes, whereas no significant labeling was detected in other cellular compartments or in the intercellular space. The physiological state of the transgenic plants was evaluated in respect to growth rate, general appearance, carbohydrate content, and dry weight. No significant differences were recorded in comparison with non-transgenic tobacco plants. The 3,3'-diaminobenzidine-stain method was applied to visualize hydrogen peroxide (H(2)O(2)) in the TMV infected tissue. Presence of H(2)O(2) could be detected around necrotic lesions caused by TMV infection in non-transgenic plants but to a much lesser extent in the CTA1 transgenic plants. In addition, the size of necrotic lesions was significantly bigger in the infected leaves of the transgenic plants. Changes in the distribution of H(2)O(2) and in lesion formation were not reflected by changes in salicylic acid production. In contrast to the local response, the systemic response in upper noninoculated leaves of both CTA1 transgenic and control plants was similar. This suggests that increased cellular catalase activity influences local but not systemic response to TMV infection.     

Synthesis and localization of pathogenesis-related proteins in tobacco.

The PR1 family of pathogenesis-related proteins from tobacco (Nicotiana tabacum L.) leaves is induced by a variety of pathogenic and chemical agents and is associated with resistance to tobacco mosaic virus. The majority of the PR1 proteins did not copurify with mesophyll protoplasts (the major cell type of the leaf) isolated from tobacco mosaic virus-infected N. tabacum cv. Xanthi-nc leaves. However, these isolated protoplasts were capable of synthesizing and selectively secreting the PR1 proteins. Using monoclonal antibodies for immunofluorescence microscopy, we localized these proteins to the extracellular spaces predominantly in regions adjacent to viral lesions as well as in xylem elements of infected leaves.     

Isolation of tobacco mesophyll cells in intact and active state

A procedure using a fungal pectinase was developed to rapidlyrelease mesophyll cells from tobacco leaves. Fifty to ninetyper cent of the released cells were morphologically intact andwere converted into spherical protoplasts by cellulase treatment.Cells isolated from tobacco mosaic virus-inoculated leaves supportedmultiplication of the virus during subsequent incubation. (Received December 14, 1967; )     

Response of Membrane-bound Mg-activated ATPase of Tobacco Leaves to Tobacco Mosaic Virus

Infectious material was formed at an early stage, and migrated into the mesophyll from the epidermis of tobacco leaves (Nicotiana tabacum cv. Samsun NN) during the period of 1 to 3 hours after inoculation with tobacco mosaic virus (TMV). The activity of membrane-bound Mg²⁺-activated ATPase from the mesophyll was stimulated two to four times within 30 minutes after inoculation with 1.0 microgram per milliliter of TMV. Maximum TMV stimulation of membrane-bound Mg²⁺-activated ATPase activity in epidermis and mesophyll was observed at 0.5 and 3.0 hours after inoculation, respectively. This stimulation was also observed with ultraviolet irradiated TMV (only RNA was destroyed), whereas, the stimulation was not observed with heat-irradiated TMV (both coat and RNA were destroyed). Stimulation equal to that of TMV was observed by inoculation with cucumber green mottle mosaic virus and to a lesser extent with cucumber mosaic virus.

These results illustrate that the stimulus resulting from inoculation with TMV transfers to underlying cells faster than the migration of TMV particles. This stimulus might be closely correlated to the structure of virus, but not to the infectivity of virus.

     

Phenylpropanoid compounds and disease resistance in transgenic tobacco with altered expression of L-phenylalanine ammonia-lyase

Tobacco plants over-expressing L-phenylalanine ammonia-lyase (PAL(+)) produce high levels of chlorogenic acid (CGA) and exhibit markedly reduced susceptibility to infection with the fungal pathogen Cercospora nicotianae, although their resistance to tobacco mosaic virus (TMV) is unchanged. Levels of the signal molecule salicylic acid (SA) were similar in uninfected PAL(+) and control plants and also following TMV infection. In crosses of PAL(+) tobacco with tobacco harboring the bacterial NahG salicylate hydroxylase gene, progeny harboring both transgenes lost resistance to TMV, indicating that SA is critical for resistance to TMV and that increased production of phenylpropanoid compounds such as CGA cannot substitute for the reduction in SA levels. In contrast, PAL(+)/NahG plants showed strongly reduced susceptibility to Cercospora nicotianae compared to the NahG parent line. These results are consistent with a recent report questioning the role of PAL in SA biosynthesis in Arabidopsis, and highlight the importance of phenylpropanoid compounds such as CGA in plant disease resistance.     

Effect of Tobacco Mosaic Virus on Phospholipid Content and Phospholipase D Activity in Tobacco Leaves

The phospholipid content and phospholipase D activity in the leaves of two tobacco (Nicotiana tabacum L.) cultivars were investigated. These cultivars are characterized by different response to the infection with tobacco mosaic virus (TMV). In the infected leaves of a susceptible cv. Samsun, phospholipid content and phospholipase D activity did not change within seven days after TMV infection. The development of a hypersensitive response in the leaves of a resistant cv. Xanthy necrotic was not accompanied by a change in the total phospholipid content as compared to the noninfected leaves. However, the appearance of necrotic lesions and their subsequent expansion resulted in a steady decrease in the level of phosphatidylglycerol in infected leaves. At the same time, phosphatidic acid and diphosphatidylglycerol contents increased. Leaf zones remote from the regions of necrosis development were also characterized by an increased level of phosphatidic acid. There was a tendency for an increase in phospholipase D activity in both the sites of necrosis development and in the leaf regions remote from these sites. The changes in phosphatidic acid content were of similar nature, and therefore a relative increase in phosphatidic acid could result from the phospholipase D activity. This fact suggests a possible involvement of phospholipase D in the development of the hypersensitive response, and this suggestion is supported by a higher enzyme activity in the leaves of healthy plants of the resistant cultivar as compared to the susceptible one. Causes for the changes in the content of some phospholipids, as well as the physiological role of phospholipase D in the hypersensitive response are discussed.     

Somatic hybridization between male sterile Nicotiana tabacum and N. glutinosa through protoplast fusion

Summary Protoplasts derived from suspension cultured cells of cytoplasmic male sterile Nicotiana tabacum (N. debneyi cytoplasm) and of fertile N. glutinosa were fused with the aid of polyethylene glycol (PEG). Out of 1,089 colonies developed from PEG-treated protoplasts, 29 restored whole plants.A somatic hybrid plant was selected on the basis of isoelectrofocusing analysis of Fraction I protein in leaves of regenerated plants. A newly created hybrid contained small subunits of both parents but only a N. glutinosa type large subunit.Male sterile character was conserved in a hybrid plant while leaf morphology was intermediate between the parents. By tobacco mosaic virus infection tests, the hybrid's leaves showed resistant symptoms, hypersensitive local lesions, which were due to N. glutinosa nuclear genome expression.Abbreviations PEG Polyethylene glycol - TMV Tobacco mosaic virus     

Influence du piclorame sur l'activité phénylalanine-ammoniac lyase de Nicotiana tabacum induite par la réaction d'hypersensibilité au virus de la mosaïque du tabac

In Nicotiana tabacum var. Xanthi n.c. phenylalanine ammonia lyase (PAL) activity, which increases significantly during the hypersensitivity reaction to tobacco mosaic virus (TMV), is only partially affected when plants are treated with piclorame, which is known to be an inhibitor of PAL in vivo. Using piclorame together with TMV, it has been possible to distinguish between that increase in PAL activity due to the virus and that dependent on the photoperiod.