Effects of ribavirin and adenine arabinoside on tobacco mosaic virus in Nicotiana tabacum L. var Xanthi tissue cultures

Although both ribavirin (1--ribofuranosyl-1,2,4-triazole-3carboxamide) and adenine arabinoside inhibited the multiplication of tobacco mosaic virus (TMV) in mechanically inoculated leaf tissues, neither chemical inhibited virus multiplication in unorganized tobacco callus after in vitro inoculation. The adenine deaminase inhibitor, pentostatin, did not increase the activity of adenine arabinoside in cultured cells. Several different developmental conditions and media did not increase the ability of either chemical to eradicate the virus from tobacco tissue cultures. However, the virus was eradicated from TMV-infected callus when grown in the presence of combinations of ribavirin and adenine arabinoside in shoot inducing medium.     

The Mode of Inhibition of TMV- and PVX-Induced RNA-Dependent RNA Polymerases by some Antiphytoviral Drugs

Six nucleobase and nucleoside analogues (6-aminouracil [6-AU], 6-aminothymin [6-AT], 6-fluorothymin [6-FT], 2,4-dioxohexahydro-1,3,5-triazine [DHT], 9-(2,3-dihydroxypropyl) adenine [DHPA] and 1-β-D-ribofuranosyl-1,2,4-triazol-3-carboxamid [ribavirin]) with potential antiphytoviral activity were tested in an in vitro replication system for their effect on tobacco mosaic virus (TMV)-and potato virus X(PVX)-induced RNA dependent RNA polymerase. 6-AU, 6-AT, 6-FT, and DHPA inhibited both polymerases which may be a target of these compounds. A Putative noncompetitive mode of inhibition is discussed. Whereas ribavirin did not affect the in vitr RNA synthesis in the established systems, the uracil-derivative DHT inhibited only the PVX-specific enzyme.     

Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus.

Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells.     

Adenine phosphoribosyltransferase in plant tissues: some effects of kinetin on enzymic activity

Adenine phosphoribosyltransferase activity was measured in extracts of soybean (Glycine max var. Acme) callus and of senescing barley leaves (Hordeum distichon c.v. Prior). The enzyme from soybean callus had Michaelis constants for adenine and 5-phosphoribosyl pyrophosphate of 1.5 and 7.5 μm respectively and was inhibited by AMP and stimulated by ATP. The presence of kinetin was found to considerably increase the activity of adenine phosphoribosyltransferase in extracts of soybean callus and senescing barley leaves.     

Effects of Salicylate on Virus-Infected Tobacco Plants

Salicylate watered onto the soil of tobacco plants in pots reduced the antigen accumulation and local lesion growth of tobacco necrosis virus mechanically inoculated on the leaves. It also retarded the growth of the necrotic centres of lesions and, in parallel, inhibited ethylene production from infected leaves. However, the therapeutic index of salicylate was very small and the chemical had to be applied in advance of, or at the same time as virus inoculation to give good levels of resistance. The number of lesions and their rate of appearance were not affected by salicylate. In addition, it did not induce resistance against multiplication, systemic spread or symptom severity in tobacco plants inoculated with a necrotic strain of potato virus Y. These findings suggest that salicylate is not likely to prove useful as polyvalent chemotherapeutic agent.     

Significance of treatment interval and DNA repair in the enhancement of viral transformation by chemical carcinogens and mutagens

Treatment of Syrian hamster embryo cells with diverse classes of chemical carcinogens enhanced transformation by a carcinogenic simian adenovirus, SA7. Optimal enhancement was a function of time of chemical addition in relation to time of virus addition and cell transfer. Aflatoxin B₁ (AFB₁) and the polycyclic hydrocarbons, benzo(a)pyrene (B(a)P), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz(a)anthracene (DMBA) enhanced SA7 transformation when added prior to virus, but inhibited transformation when added after virus adsorption and cell transfer. The enhancement of SA7 transformation was maximal when cytosine arabinoside, caffeine and 6-acetoxy-benzo(a)pyrene (6-ac-B(a)P) were added after virus, but minimal when added before virus. A third class of chemicals, including β-propiolactone (β-PL), methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (Ac-AAF), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and methylazoxymethanol acetate (MAM-ac), enhanced SA7 transformation added before, or after, virus inoculation and cell transfer. All chemicals, which induced changes in DNA sedimentation in alkaline sucrose gradients and unscheduled DNA (repair) synthesis in hamster cells, increased the frequency of SA7 transformation. However, several chemicals such as dibenz(a,h)anthracene (DB(a,h)A), benzo(e)pyrene (B(e)P), cytosine arabinoside, and caffeine enhanced SA7 transformation but did not induce DNA sedimentation changes or repair. Chemicals that cause DNA damage, which can be repaired by hamster cells, may enhance viral transformation by providing additional sites for integration of viral DNA during the repair process. Chemicals that apparently do not induce DNA repair synthesis may enhance viral transformation by incorporation of viral DNA into gaps in cell DNA at sites of unrepaired damage during scheduled DNA synthesis.     

Two TOBAMOVIRUS MULTIPLICATION 2A homologs in tobacco control asymptomatic response to tobacco mosaic virus

The most common response of a host to pathogens is arguably the asymptomatic response. However, the genetic and molecular mechanisms responsible for asymptomatic responses to pathogens are poorly understood. Here we report on the genetic cloning of two genes controlling the asymptomatic response to tobacco mosaic virus (TMV) in cultivated tobacco (Nicotiana tabacum). These two genes are homologous to tobamovirus multiplication 2A (TOM2A) from Arabidopsis, which was shown to be critical for the accumulation of TMV. Expression analysis indicates that the TOM2A genes might play fundamental roles in plant development or in responses to stresses. Consistent with this hypothesis, a null allele of the TOM2A ortholog in tomato (Solanum lycopersicum) led to the development of bent branches and a high tolerance to both TMV and tomato mosaic virus (ToMV). However, the TOM2A ortholog in Nicotiana glauca did not account for the asymptomatic response to TMV in N. glauca. We showed that TOM2A family is plant-specific and originated from Chlorophyte, and the biological functions of TOM2A orthologs to promote TMV accumulation are highly conserved in the plant kingdom—in both TMV host and nonhost species. In addition, we showed that the interaction between tobacco TOM1 and TOM2A orthologs in plant species is conserved, suggesting a conserved nature of TOM1–TOM2A module in promoting TMV multiplication in plants. The tradeoff between host development, the resistance of hosts to pathogens, and their influence on gene evolution are discussed. Our results shed light on mechanisms that contribute to asymptomatic responses to viruses in plants and provide approaches for developing TMV/ToMV-resistant crops.

Tobacco TOBAMOVIRUS MULTIPLICATION 2A homologs control the asymptomatic response to tobacco mosaic virus and have highly conserved biological functions related to virus multiplication.     

Somatic embryogenesis and plant regeneration in garden leek

High frequency plant regeneration via somatic embryogenesis has been induced from in vitro shoot-base cultures of seedlings of garden leek (Allium porrum L.). Four main steps are involved in the procedure using BDS medium:

- shoot multiplication with 17.6 mM benzyladenine;

- induction of nodular callus from the in vitro shoot base with 9 mM 2,4-dichlorophenoxyacetic acid;

- initiation of embryogenic callus from nodular callus with 9 mM 2,4-dichlorophenoxyacetic acid +7.6 mM abscisic acid;

- plant regeneration from embryogenic callus with 9.8 mM N⁶-(2-isopentenyl)adenine.

The presence of 2,4-dichlorophenoxyacetic acid in the medium and light conditions were shown to be essential for nodular callus induction and somatic embryogenesis. Abscisic acid was not a prerequiste for somatic embryogenesis, but it significantly increased the frequency.     

Transmembrane ferricyanide reduction in tobacco callus cells

Transmembrane ferricyanide reduction in whole cells of normal and of transformed tobacco (Nicotiana tabacum) callus tissue was compared. It was found that low concentrations of indoleacetic acid (IAA, 0.1 μM), gibberellic acid (GA, 0.3 μM), and benzyl adenine (BA, 0.03 μM) stimulate external ferricyanide reduction in normal tobacco callus cells, but inhibit this reaction up to 67% in transformed cells when hormones are applied to cells 10 min prior to assay. Higher concentrations of these growth regulators (1 μM or greater) inhibit transmembrane ferricyanide reduction in both types of cells, with the exception of IAA, giving an initial stimulation of the rate (12%), followed by 24% inhibition after 2 min. The observed external ferricyanide reduction by whole tobacco callus cells may be explained on the basis of a transplasmalemma redox system, which may be associated with the iron metabolism of these cells.     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 μM PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 μM PAA) and with a 16-h light/8-h dark photoperiod (500 μM PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 μM PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 μM as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 μM PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 μM.     

SOME EFFECTS OF CHANGING TEMPERATURE AND OF VIRUS INHIBITORS ON INFECTION BY CUCUMBER MOSAIC VIRUS

Whereas the spinach strain of cucumber mosaic virus fails to multiply and cause symptoms in tobacco plants kept above 30° C., the yellow strain infects at 36° C. and causes more severe symptoms than at 20° C. Increasing the temperature up to 28° C. increases the initial rate at which the spinach strain multiplies, but the virus later reaches much higher concentrations in leaves at lower temperatures, presumably because it is rapidly inactivated at 28° C. Exposing inoculated plants to 36° C. for 6 hr. decreases the number of infections by the spinach strain when the exposure starts within 6 hr. of inoculation, but not afterwards.
Pancreatic ribonuclease inhibits infections by strains of cucumber mosaic virus; inhibition is greatest when the enzyme is present in the inoculum, and when applied to inoculated leaves its effect decreases rapidly with increasing time after inoculation.
Infection by and the multiplication of strains of cucumber mosaic virus in tobacco are only slightly affected by thiouracil and greatly by azaguanine, whereas strains of tobacco mosaic virus are inhibited much more by thiouracil than by azaguanine. Like tobacco mosaic virus, cucumber mosaic virus multiplies more when inoculated leaves are floated in nutrient solutions than in water, but unlike tobacco mosaic virus, its multiplication is not inhibited by thiouracil more in nutrient solutions than in water.     

A Comparative Study of CN-Resistant Respiration in Different Cultures of Tobacco Callus

The callus of Nicotiana rustica cv Gansu yellow flower and N. tabacum cv willow leaf were cultured on ordinary subculture medium (M-1) and on regeneration medium (M-2), respectively. No differentiation was observed in Gansu yellow flower tobacco callus cultures grown on both M-1 and M-2 medium. The respiration of both cultures was partially resistant to cyanide and markedly inhibited by m-chlorobenzhydroxamic acid. The relative contributions of alternative and cytochrome pathway were 31% and 47% of the total respiration, respectively, in M-1 callus cultures. The relative O₂ uptake of the two pathways was not changed significantly in M-2 callus cultures. In subcultured M-1 callus cultures of Willow leaf tobacco, the respiration mediated via alternative pathway was about 29 to 38% of the total respiration, and the cytochrome pathway still was the major respiratory pathway. In M-2 callus cultures in which differentiation occurred, the relative contribution of alternative pathway increased to 41 to 47% of the total respiration, and the cytochrome pathway decreased considerably. These results suggested that the change of respiratory electron transport pathway was probably related to the differentiation of tobacco callus cultures.     

Regulation of Cytokinin Oxidase Activity in Callus Tissues of Phaseolus vulgaris L. cv Great Northern

The regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern has been examined using an assay based on the oxidation of N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C (i⁶ Ade-8-¹⁴C) to adenine. Solutions of exogenous cytokinins applied directly to the surface of the callus tissues induced relatively rapid increases in cytokinin oxidase activity. The increase in activity was detectable after 1 hour and continued for about 8 hours, reaching values two- to three-fold higher than the controls. The cytokinin-induced increase in cytokinin oxidase activity was inhibited in tissues pretreated with cordycepin or cycloheximide, suggesting that RNA and protein synthesis may be required for the response. Rifampicin and chloramphenicol, at concentrations that inhibited the growth of Great Northern callus tissues, were ineffective in inhibiting the increase in activity. All cytokinin-active compounds tested, including both substrates and nonsubstrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokinin-active urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Thidiazuron to the reaction volumes used to assay cytokinin oxidase activity resulted in a marked inhibition of the degradation of the labeled i⁶ Ade-8-¹⁴C substrate. On the basis of this result, it is possible that Thidiazuron may serve as a substrate for cytokinin oxidase, but other mechanisms of inhibition have not yet been excluded.     

Synthesis and Comparative Cytokinin Activities of N-(Purin-6-yl)-d- and -l-amino Acid Methyl Esters

Six pairs of enantiomeric N-(purin-6-yl)amino acid methyl esters were synthesized and tested for their cytokinin activities by three bioassay systems, the growth of tobacco callus, the seed germination of lettuce and the fresh weight increase of excised radish cotyledons.l-(—)-Antipodes were as a whole more active than the corresponding d-(+)-isomers in the tobacco callus and seed germination tests, whereas an uniform tendency was not observed in the radish cotyledon expansion. The discussions were focused on the effects on the biological response of configurational arrangements around the asymmetric center at α-position to the adenine ring and on species difference of cytokinin receptor molecules.     

In Vitro Flowering and High Xanthotoxin in Ammi majus L.

Ammi majus L. (Apiaceae), an important medicinal herb, constitutes the principal commercial source of xanthotoxin that is commonly used in leucoderma. Since the seed set and germination is poor, the present investigation was undertaken to propagate A. majus through tissue culture and monitor the yield of xanthotoxin (furanocoumarin) in the resultant callus and plantlets. The callus obtained from the cotyledonary leaves on MS medium supplemented with indoleacetic acid (IAA) + kinetin (Kn) + casein hydrolysate (CH), differentiated shoot buds on a medium additionally enriched with adenine. Plantlets 3.5 cm tall, resulted upon transfer of shoots to MS medium with indolebutyric acid (ISA) and glutamine. These plantlets flowered in vitro. The yield of xanthotoxin detected in regenerating cultures is more than hitherto reported from any other tissue including the seeds. lsozymes profile for leucineaminopeptidase (LAP), esterase and peroxidase could be used as reliable markers for the characterization of various stages of growth of callus and regenerants. The present report would prove advantageous in multiplication of Ammi majus and also in inducing enhanced levels of xanthotoxin.     

Systemic Acquired Resistance Induced in Tobacco Plants by Localized Virus Infection Does Not Operate Against Challenging Viruses That Infect Systemically

Localized infections produced by tobacco necrosis virus (TNV) or tomato mosaic virus (ToMV) in White Burley tobacco induced a systemic acquired resistance in upper, uninoculated leaves. This resistance was effective against challenge infection by TNV or ToMV but not by potato virus Y, necrotic strain (PVYⁿ), tobacco mosaic virus (TMV) or tobacco rattle virus (TRV), viruses giving systemic infections. Systemic acquired resistance against TNV or ToMV was expressed as a reduction in lesion size but not in viral antigen content of the resulting necrotic local lesions. The acquisition of resistance was concurrent with an increased capacity of the resistant leaves to convert 1-aminocyclopropane-1-carboxylic acid into ethylene. Systemic acquired resistance was ineffective to contrast or minimize in whatever way the systemic challenge infection produced by PVY^N, TMV or TRV. Severity of symptoms and virus multiplication did not differ in resistant leaves from controls. This result does not allow any optimistic promise on possible application of the systemic acquired resistance against severe viral diseases of crops.     

Light cytokinin interactions in shoot formation in epicotyl cuttings of Troyer citrange cultured in vitro

Ilumination did not affect the pathway of shoot regeneration at the cut edges of epicotyl explants of Troyer citrange (Moreira-Dias et al. 2000, 2001), but signigficantly affected the number of developed shoots and the response to exogenous cytokinins. Shoot regeneration at the apical end occurred through a direct organogenic pathway without callus formation. For explants incubated in the light, this regeneration did not require cytokinin addendum, but the number of shoots formed was significantly increased by benzyl adenine, but not by zeatin or kinetin. Incubation in the dark almost suppressed shoot formation at the apical end. The addition of benzyl adenine or kinetin, but not of zeatin, restored shoot formation in the dark to the value obtained in the light. At the basal end of the explants shoot regeneration occurred through an indirect organogenic pathway after the formation of a primary callus. In explants incubated in the light, callus formation and shoot growth was supported by a low (0.5–1 mg l⁻¹) benzyl adenine concentration and by zeatin. Kinetin did not support callus growth. Shoot formation was higher in the presence of benzyl adenine (0.5–1 mg l⁻¹) than of zeatin, but was inhibited by a high (5 mg l⁻¹) benzyl adenine concentration. Incubation in the dark increased callus growth and shoot formation at the basal cut as compared to explants incubated in the light. The three cytokinins tested supported callus growth and shoot formation in the dark, zeatin being the most effective and kinetin the least. In terms of number of shoots developed, the optimum cytokinin addendum depended on the pathway of organogenesis and the conditions of incubation. The maximum number of shoots developed at the apical end was obtained when the incubation was performed in the light in the presence of benzyl adenine. At the basal end, the optimal conditions were incubation in the dark in the presence of zeatin. It was not always possible to define an optimal cytokinin concentration as the curve concentration/response varied from experiment to experiment, which seemed unrelated to the endogenous cytokinin concentration in the explants.     

Durch TMV-infection hervorgerufene veränderung der intensität der glycolyse und des pentosephosphatcyclus bei tabakpflanzen

During the period of the highest multiplication of TMV in tobacco tissues (on the 6th to l0th daya after inoeulation) the activities of NADP-glyceraldehyde-3-phosphate dehydrogenase, fructose diphosphatase, and phosphoglucoisomerase increase,i.e. the 3-phospho-glycerate is broken down more intensely, being the primer of photosynthesis via glycolysis. Products of this metabolic pathway, which are not employed directly for virus synthesis, are stored during this period as free saccharides and starch. The breaking-down rate of these substances increases during this period as well, as shown by increased activities of amylophosphorylase, phosphoglucomutase and saccharase. The increased aetivity of glucose-6-phosphate dehydrogenase is proportional to the rate of TMV multiplication; its high value (more than twice as with the control) insinuates that the virus reproduction deereases contents of some intermediates of the Calvin cycle (especiaily NADPH and ribulose-l,5-diphosphate) which act as inhibitors of the photosynthetic regulative system.