The effects of tobacco mosaic virus synthesis on the free amino acid and amide composition of the host

1. Changes in concentrations of free amino acids and amides have been determined in TMV-infected tobacco leaf discs and in comparable uninfected discs during the time of virus formation. 2. During the period of rapid virus formation the infected discs show a transitory deficiency (as compared to uninfected discs) in glutamine, asparagine, aspartic acid, glutamic acid, serine, and to a lesser extent in valine, threonine, and proline. About 100 hours before this time smaller deficiencies in the concentrations of these components also occur. The latter effect is probably associated with the early synthesis of a non-virus protein in infected tissue. 3. Comparison of the above effects with the known amino acid composition of TMV indicates that it is unlikely that the virus protein is synthesized by condensation of appropriate free amino acids. Rather, the deficiencies observed appear to result from removal of ammonia from the nitrogen pool during synthesis of new proteins in infected tissue. Equilibrium shifts resulting from ammonia withdrawal probably account for the observed deficiencies in amides and free amino acids. TMV protein, therefore, appears to be synthesized de novo, from non-protein nitrogen, probably ammonia. 4. It is suggested that the changes in free amino acid concentrations induced by virus formation may account for some of the symptoms observed in infected plants.     

Relationships between tobacco mosaic virus biosynthesis and the nitrogen metabolism of the host

1. Comparisons of the nitrogen content of TMV-infected and uninfected tobacco leaf discs at various times after inoculation show that virus synthesis is associated with a net increase in protein content. This excess protein is due to: (a) TMV, (b) an excess in insoluble protein which develops soon after inoculation and ends about 100 hours before cessation of TMV synthesis, and (c) an excess in soluble non-virus protein, which is variable in size and which only occurs during the time of virus synthesis. A deficiency in non-protein nitrogen occurs during the time when virus appears. 2. Isotope experiments with N¹⁵-labelled nutrient show that: (a) The bulk of TMV nitrogen is derived from the free ammonia of the host tissue. (b) Amino acid residues of TMV protein are not derived from the corresponding free amino acids in the host. (c) The appearance of TMV is preceded by the synthesis of an insoluble precursor of the virus which is then converted into TMV or some soluble intermediate protein. This effect is associated with a cell particulate which represents a small fraction of the total insoluble protein. (d) Infected tissue synthesizes de novo small amounts of soluble non-virus protein, which may represent intermediates in TMV synthesis. (e) Infected tissue fails to synthesize a rapidly turned-over soluble protein which is synthesized in comparable uninfected tissue. (f) TMV synthesis is preceded by a temporary enhancement of the metabolic stability of an insoluble protein component. 3. The results lead to the conclusion that TMV formation is due to diversion of some part of the host's protein-synthesizing apparatus from its normal course.     

Powdery mildew of tobacco (Erysiphe cichoracearum DC.)

Percentage germination, and growth of hyphae from single conidia of Erysiphe cichoracearum DC., were measured on leaf discs from topped and intact tobacco plants, grown in aerated nutrient solutions consisting of basal medium plus large or small amounts of potassium. The effect of supplying sodium was also studied. Discs were incubated on water and on 10% sucrose solution. Changes in free amino nitrogen and carbohydrate in comparable uninfected leaf discs, before and after incubation, were also measured. Potassium deficiency resulted in more free amino nitrogen and soluble carbohydrate and less insoluble carbohydrate, per cm.² of leaf. Spore germination was not greatly affected by treatments, though it was usually less on discs from potassium-deficient leaves. The pathogen grew slower on potassium-deficient leaf discs, whether they were incubated on water or on sucrose. Incubating discs from some leaves on sucrose, compared with water, gave greatly increased sugar content and less fungal growth; discs from other leaves had a much smaller increase in sugar, and hyphal length was similar to that on discs incubated on water. Sodium, when potassium was scarce, increased potassium deficiency symptoms, free amino nitrogen and sugar content, and resistance to powdery mildew.     

Relationships between tobacco mosaic virus and the non-virus proteins

1. The non-virus proteins, A4, B3, and B6, characteristically found in tobacco leaf infected with TMV exhibit specific immunochemical cross-reactions with serum prepared against the virus. The close immunochemical relations which occur among these proteins do not extend to any normal tobacco leaf proteins. 2. The rate of appearance of the non-virus proteins in newly infected cultured leaf tissue at various times after inoculation has been determined by immunochemical techniques and by direct isolation of the proteins. Both methods give comparable results. The non-virus proteins appear abruptly at about 220 hours after inoculation, when the TMV content is about one-third its final value. The amount of A4 rises rapidly and then levels off. The B6 content rises rapidly and continuously over the course of the experiment. B3 appears last, and increases in amount considerably more slowly than A4 and B6. 3. The isotope contents of TMV, B3, and B6 which appear in given intervals after inoculation in newly infected leaf cultured in nutrient containing N¹⁵H₄ have been compared. The isotope levels of concurrent TMV, B3, and B6 are identical within the experimental error. The isotope conditions employed in this experiment lead to the conclusion that this coincidence of N¹⁵ levels means that the virus and non-virus proteins are probably synthesized at about the same time from the same non-protein source of nitrogen. 4. Possible interpretations of the available data on the non-virus proteins are discussed. It is likely that one or more of these proteins represents small protein units which occur in the TMV nucleoprotein.As they exist in the infected leaf, the non-virus proteins are probably no longer available to the biochemical processes which lead to TMV synthesis. They are probably not precursors of TMV protein in a temporal sense.     

The Effect of Light on Tobacco Mosaic Virus (TMV) Formation

Discs were punched from TMV-inoculated tobacco leaves (Nicotiana tabacum L.) and illuminated while floating on half strength Vickery's solution maintained at 24°C. After 48 hours some discs were placed in the dark for 24 hours and the amount of TMV formed in the light and dark compared. Discs from young leaves formed more virus in the light than in the dark. Discs from older leaves produced less virus, but as much in the dark as in the light.     

Powdery mildew of tobacco (Erysiphe cichoracearum DC.): Susceptibility of proximal and distal parts of leaves from different stalk positions on intact and topped field plants in relation to free amino nitrogen and carbohydrate content*

The area covered by visible mycelium of E. cichoracearum on the upper surface of leaves 4, 8, 12 and 16 of tobacco plants in field plots in Rhodesia was expressed as percentages of the proximal and distal halves at weekly intervals. Free amino nitrogen and carbohydrate in discs from proximal and distal halves of the same leaves were analysed when each leaf was expanding rapidly and was not infected, and several weeks later, when the rate of expansion had slowed down and there was slight infection. On two other occasions, similar leaf discs were inoculated with conidia, to measure the percentage germination and hyphal length from individual conidia after incubation for 2–3 days at constant temperature and humidity; duplicate discs were chemically analysed. Leaves were not susceptible until at least 6 weeks after they had emerged from the bud. Soluble carbohydrate increased and free amino nitrogen decreased during the change from resistance to susceptibility. Proximal parts of leaves were usually infected first; they initially contained less amino nitrogen and soluble carbohydrate than distal parts. All parts of the leaf seemed to be equally susceptible later, when there were no differences in their amino nitrogen or soluble carbohydrate. Upper leaves of intact plants had more natural infection than those from corresponding leaves from topped plants. More conidia germinated on discs from them and produced longer hyphae. The discs from intact plants contained less free amino nitrogen and more soluble carbohydrate than those from topped plants. The accuracy of visual assessments of susceptibility was, generally, confirmed by measurements of percentage germination and length of hyphae from individual conidia on leaf discs. Regressions of hyphal length on leaf composition showed that susceptibility was apparently related inversely to free amino nitrogen and water content and directly to insoluble carbohydrate per unit dry matter.     

Acidic and basic class III chitinase mRNA accumulation in response to TMV infection of tobacco

Complementary DNA clones encoding acidic and basic isoforms of the class III chitinase were isolated from Nicotiana tabacum. The clones share ca. 65% identity, are equally homologous to the class III chitinases from cucumber and Arabidopsis, and are members of small gene families in tobacco. An acidic class III chitinase was purified from the intercellular fluid of tobacco leaves infected with tobacco mosaic virus (TMV). Partial amino acid sequencing of the protein confirmed that it was encoded by one of the cDNA clones. The mRNAs of the class III chitinases are coordinately expressed in response to TMV infection, both in infected and uninfected tissue. The acidic and basic class III chitinases constitute previously undescribed pathogenesis-related proteins in tobacco.     

Chemical suppression of the symptoms of two virus diseases

Carbendazim applied at the rate of 2 g per plant to the roots of tobacco (Nicotiana tabacum cv. White Burley) plants before infection with tobacco mosaic virus (TMV) caused very considerable reduction in the severity of disease symptoms in systemically infected leaves but did not affect their virus content. Leaves of untreated, infected plants had a greatly reduced chlorophyll content 100 days after infection whereas the chlorophyll content of leaves of infected plants treated with carbendazim was similar to that of normal uninfected leaves. Carbendazim had no effect on the infectivity of TMV in vitro or on the local lesion reaction of N. glutinosa plants when inoculated with TMV. Carbendazim was applied to lettuce cv. Cobham Green at a total rate of o-i g per plant before and after they were infected with beet western yellows virus and the plants were then grown on in the field. At harvest time (50 days after infection) almost all the treated virus-infected plants were of a normal green appearance, whereas the untreated controls were almost all very severely yellowed and unmarketable.     

Effect of Light on Tobacco Mosaic Virus Formation under Anaerobic Conditions

Tobacco leaf discs, infected with tobacco mosaic virus (TMV), were floated on Vickery's solution and kept under N₂ in the light, conditions where the only source of ATP was assumed to be cyclic photophosphorylation. Usually the virus content was unaltered or decreased during the next 24 hours; occasionally there was some TMV formation, but less than in air and light, and it was abolished by 10^?5 M DCMU. This suggested that ATP produced by cyclic photophosphorylation was not used in TMV formation. Infected discs exposed to N₂ for longer than 2 hours formed less virus when transferred to air and light than discs not exposed to N₂, presumably because some breakdown in the TMV-forming apparatus occurred in ATP deficient conditions.     

SOME EFFECTS OF VIRUS INFECTION ON LEAF WATER CONTENTS OF NICOTIANA SPECIES

Infection with tobacco mosaic virus decreases the water content which detached tobacco leaves attain when kept for 20 hr. in conditions of minimum water stress, and does so more when the plants are kept in light before inoculation than when they are kept in darkness. No such effects of infection during the first day after inoculation were obtained with tobacco leaves infected with either tobacco etch virus or potato virus X , or with Nicotiana glutinosa leaves infected with tobacco mosaic virus. These results, like those showing early effects of TMV on respiration and photosynthesis of tobacco leaves, suggest that inoculation with TMV affects deeper leaf tissues than the epidermis earlier in tobacco leaves than in other leaves, and earlier than other viruses in tobacco leaves.     

Cytochrome Oxidase and Mitochondrial Protein in Extracts of Leaves of Nicotiana glutinosa L. Infected with Tobacco Mosaic Virus

Leaves of N. glutinosa were inoculated with tobacco mosaic virus,and infection allowed to develop in situ or in discs cut fromthe leaves and floated on water or culture solution. Extracts,made from the leaves and discs when their respiration was greaterthan that of comparable uninfected tissue, contained amountsof protein N, chlorophyll, glycollic oxidase, and mitochondrialprotein N, similar to those in extracts from uninfected tissue,but slightly less cytochrome oxidase and considerably more polyphenoloxidase.No evidence was obtained for the view that the virus-inducedrespiration reflects an increase in the amount of mitochondrialmaterial.     

An Analysis of the Effects of Tobacco Mosaic Virus on Growth and the Changes in the Free Amino Compounds in Young Tomato Plants

Tomato plants at the four-leaf stage were inoculated on thefirst leaf with TMV in a growth room and the effects studiedin systemically-mfected leaves with reference to growth, virusmultiplication and changes in water, nitrogen, nitrate and chlorophyllcontents. Parallel changes in the free amino compounds werealso studied in the growth room (incident radiation 152 mwhcm^-2 day^-1) and in two experiments in a glasshouse (352 and226 mwh cm^-2 day^-1) Dry matter accumulation and leaf expansion in leaves 3, 4 and5 were checked by TMV 5–7 days after inoculation but notin leaf 2. In the period 7–25 days after inoculation therelative growth rates of whole plant and leaves 3 and 6 andnet assimilation rate were not affected by TMV. Stem heightand dry weight were not affected by TMV but ‘root’dry weight was reduced from days 5–15. Virus was presentin the stem and in leaves 2 and 6 by days 3, 5 and 15 respectivelyso that infection per se did not always check growth. Chlorophyllcontent of systemically-infected leaves was reduced 10 daysafter inoculation. Total N and ammonia contents were not affectedby TMV but infected leaves contained less nitrate. At the two lower levels of incident radiation the initial effectof TMV was to reduce the content of total free protein aminoacids and amides, which were minimal 5–7 days after inoculation.In the glasshouse experiment a reduction could be measured only1 day after inoculation or before virus was present in the youngerleaves. With high incident radiation there was no initial reductionbut an increase at day 13 when mottling symptoms were visible.Total non-protein amino acids, of which amino butyric acidwas the major constituent, were increased by TMV in all threeexperiments for up to 13 days after inoculation. It is suggested that inoculation of a leaf with TMV temporarilyinterferes with export of photosynthates and import of root-synthesisedamino acids and that the results reported above can be interpretedin this context. Evidence in support of this is adduced froman experiment in which 13 foliar sprays of gibberellic acid(2·5 ppm) were combined with TMV inoculation and thechanges in free amino compounds followed. It is concluded that analyses of the changes in individual freeamino compounds are unlikely to provide useful information concerningthe sources of virus coat protein.     

THE RESPIRATION OF TOBACCO LEAVES AFTER SYSTEMIC INFECTION WITH TOBACCO MOSAIC VIRUS

The rate of CO, production per g. dry matter of the younger leaves of tobacco plants systemically infected with tobacco mosaic virus was about 10 yo less than that of comparable healthy leaves. Older infected leaves, showing well-developed mosaic symptoms, had the same respiration rate as comparable healthy leaves. These results were independent of seasonal change in light conditions during the growth of the plants. Older leaves, but not younger leaves, of infected plants had a lower initial water content, and both absorbed less water during the experimental period, than leaves from healthy plants. The effects of TMV infection on water content were so great that the rate of CO, production per g. fresh weight was sometimes significantly increased by infection. This reversal of the apparent effect of infection on respiration rate, depending on the basis of reference may partly account for contradictory results reported previously by other workers. Other causes for contradictory results are discussed.     

Tomato yellow leaf curl virus DNA in callus cultures derived from infected tomato leaves

Callus cultures were induced from leaves of a tomato plant infected with tomato yellow leaf curl virus (TYLCV) and analyzed for viral DNA presence during successive subcultures. No TYLCV DNA was detected in calli sampled after eight months of culture. Considerable differences in the presence of TYLCV DNA were found within sectors of a callus culture and between different callus cultures, throughout the entire eight months period. Infected calli which were cultured at sub-optimal temperature (15°C) retained the viral DNA longer than at 25 °C. The results suggested that TYLCV disappearance during callus culture was due to a disruption of some of the cell-to-cell connections, resulting in islands of infected cells in the midst of uninfected tissue and/or to the competition between the rate of cell division and that of viral DNA replication.Abbreviations BA benzyladenine - CMV cucumber mosaic virus - NAA naphthaleneacetic acid - TMV tobacco mosaic virus - TYLCV tomato yellow leaf curl virus     

Nitrogen and Carbohydrate Metabolism of Japanese Mint as Affected by Phosphorus Deficiency and Seasonal Variations

The paper presents effects of phosphorus deficiency and seasonal variations on nitrogen and carbohydrate metabolism of Japanese mint (Mentha arvensis L. var. piperascens, Holmes). Mint plants were grown in sand cultures under full nutrition and under phosphorus deficiency conditions during winter and summer. Various nitrogen and sugar fractions were determined in the component parts at specified periods of growth stages. Phosphorus deficiency disturbed the nitrogen metabolism at the stages for amide and amino acid formation, which resulted in an accumulation of carbohydrates. The content of total soluble and total nitrogen was higher and that of insoluble nitrogen was lower in summer as compared with winter plants. Of the soluble fractions, the ammonia, amide and nitrate nitrogen were higher and the ‘rest’ nitrogen lower in winter plants. Nitrate formed the highest bulk of the pool of soluble nitrogen in winter, whereas in summer ‘rest’ nitrogen was most abundant. The content of total sugar in winter plants far exceeded that of summer plants, which was wholly due to increase in sucrose content. Glucose was the predominant reducing sugar in both seasons. However, the summer plants were richer in glucose than those from the winter.     

Effects of Two Strains of Tobacco Mosaic Virus on Photosynthetic Characteristics and Nitrogen Partitioning in Leaves of Nicotiana tabacum cv Xanthi during Photoacclimation under Two Nitrogen Nutrition Regimes

Photoacclimation was studied in tobacco leaves (Nicotiana tabacum cv Xanthi) infected with two strains of tobacco mosaic virus (TMV) and grown under different light and nitrogen nutrition regimes. Photosynthetic acclimation measured by the quantum yield and the maximum rate in saturating light of CO2-saturated photosynthesis was impaired to a greater extent in tobacco leaves infected with TMV strain PV230 than in those infected with TMV strain PV42. Infection with TMV strain PV230 severely impaired photosynthetic acclimation at high light/low nitrogen and during transfer from low to high light. Expanding leaves showing chlorotic-mosaic symptoms had greatly reduced capacity to acclimate to high light compared with controls and with developed leaves without visible symptoms. We conclude that the failure of expanding leaves to acclimate was largely due to the destruction of chloroplasts in yellow areas of the tissue, accompanied by severe reduction in ribulose-1,5-bisphosphate carboxylase/oxygenase levels, and corresponding reduction in photosynthesis on a leaf-area basis. When corrected for areas of healthy green tissue, photoacclimation of infected leaves was the same as that of controls. Visible symptom development was greatest in high light/low nitrogen treatments. In developed leaves without visible symptoms, virus accumulation, which was as extensive as in expanding leaves, accelerated senescence and impaired photoacclimation during transfer from low light to high light. Generally, infection with TMV strain PV42 did not impair photosynthetic acclimation and even enhanced it in some treatments, even though virus accumulated to the same concentration as in PV230-infected leaves. These data show that TMV does not simply impair photoacclimation in tobacco by competing with chloroplasts for leaf nitrogen reserves. Rather, specific properties of severe strains, such as PV230, which lead to visible symptom development and patchy loss of photosynthetic activity in expanding leaves as well as general acceleration of chloroplast senescence in developed leaves, contribute to impaired photoacclimation, which is generally exacerbated by low nitrogen nutrition.     

烟草在PVYN病毒与烟蚜作用下对高CO2浓度的响应

以CO2浓度为主处理因子,研究了加倍CO2浓度和对照大气CO2浓度条件下,烟蚜、马铃薯Y病毒N株(PVYN)以及二者共同作用下烟草各指标的响应。结果表明,在当前CO2浓度条件下,PVYN、烟蚜及两者联合作用对烟草生物量影响不显著;而在未来高CO2浓度条件下,PVYN、烟蚜及两者联合作用对烟草生物量影响很大。CO2浓度升高后,PVYN和蚜虫二者联合作用显著降低烟草产量,危害加重,高CO2的"肥料"作用被极大地削弱。在有烟蚜、PVYN以及两者共同作用时烟草的化学物质及主要的次生代谢物烟碱的含量对CO2浓度升高的响应也发生一定的变化,表现在:高CO2浓度条件下,蚜虫、蚜虫与PVYN共同作用显著增加了烟草的含氮量;显著减少了烟叶含糖量;PVYN及其与蚜虫共同作用显著升高叶片可溶性蛋白含量;当高CO2浓度下,各处理的烟草烟碱含量均显著下降,而且PVYN感染的烟叶烟碱含量无论在哪一种CO2浓度条件下,都比无毒无虫的对照烟叶烟碱含量升高。结果显示,烟蚜和马铃薯Y病毒N株(PVYN)对烟草的产量、营养物质及防御物质都有影响;CO2浓度升高对烟草的生长有促进作用,增加了烟草的产量,但蚜虫的危害和PVYN感染使烟草产量下降,在高CO2浓度条件下,烟蚜和PVYN共同作用相对于目前CO2浓度对烟草产量的危害加重。     

Vergleich des Eiweissanteiles des Wildstammes vom Tabakmosaikvirus mit dem einer Temperaturmutante

Summary The protein of the common strainvulgare of tobacco mosaic virus was compared analytically with the protein of a temperature-mutantthermophilum. The latter was isolated from tobacco plants which were infected with common TMV and cultured at 35°C. Per polypeptid chain of 17 500 molecular weight the protein of the mutation strain contained one threonine residue more and one isoleucine (or leucine) residue less than common TMV protein.     

Studies in the Physiology of Lichens: With two Figures in the Text: The Effects of Starvation and of Ammonia Absorption upon the Nitrogen Content of Peltigera polydactyla

Discs cut from the thallus of the lichen Peltigera polydactylacontain relatively large amounts of ammonia nitrogen and smallamounts of amide nitrogen. During starvation in the dark theamounts of ammonia and amide nitrogen remain unchanged, butthe amount of amino-nitrogen increases. Absorption of ammoniaresults in increases in the amounts of ammonia and amino-nitrogenin the discs, but not in the amount of amide nitrogen. It istherefore concluded that amides do not have an important quantitativerole in the nitrogen metabolism of the discs. Ammonia absorption is stimulated by addition of glucose to themedium, but the very slow rate of protein synthesis is onlyslightly increased by glucose. The rate of nitrate absorptionis much slower than that of ammonia absorption. The ecological implications of the results are discussed, andexisting knowledge of the nitrogen metabolism of lichens isreviewed.     

Susceptibility of Tobacco Leaves to Photoinhibition following Infection with Two Strains of Tobacco Mosaic Virus under Different Light and Nitrogen Nutrition Regimes

Sensitivity to photoinhibition under high light stress (2000 [mu]mol photons m-2 s-1 for 2 h in air) and recovery from this stress were examined in leaves of control, uninfected tobacco (Nicotiana tabacum cv Xanthi) leaves and in leaves in tobacco plants infected with tobacco mosaic virus (TMV) when grown under low light (150-200 [mu]mol photons m-2 s-1) or high light (1200 [mu]mol photons m-2 s-1) with high (8.0 mM) or low (0.5 mM) nitrate supply. Photoinhibition was monitored using the dark-adapted fluorescence parameters variable fluorescence/maximum fluorescence, an indicator of photosynthetic efficiency that correlated well with the quantum yield of photosynthetic oxygen evolution, and initial fluorescence, potentially an indicator of photoinhibitory damage. Susceptibility to photoinhibition was greater in low light- and low nitrogen-grown control plants than in high light- or high nitrogen-treated plants. Compared with uninfected controls, infection with the masked strain PV42 increased susceptibility to photoinhibition only in plants grown under low light/low nitrogen conditions. In expanding leaves, infection with severe strain TMV PV230 markedly accelerated photoinhibition under these conditions and under high light/low nitrogen conditions, even before visible symptoms were evident. High nitrogen levels during growth protected against this accelerated photoinhibitory response to virus infection during light stress and generally promoted recovery, at least prior to symptom development. As symptoms developed, the yellow regions provided evidence for chronic photoinhibitory damage, prior to and during the stress treatment, irrespective of growth conditions. Green regions of leaves showing visible symptoms were generally indistinguishable from control, uninfected plants during photoinhibitory stress and recovery. In developed leaves that remained free of visible symptoms during the experiments, in spite of the accumulation of about the same amounts of virus protein (S. Balachandran, C.B. Osmond, A. Makino [1994] Plant Physiol 104: 1043-1050) infection led to an acceleration of photoinhibition during stress treatments, especially in low light/low nitrogen treatments, in which chronic photoinhibitory damage was evident. These studies suggest a role for photoinhibitory damage in the acceleration of visible symptom development following TMV PV230 infection of expanding leaves, as well as in acceleration of senescence in developed leaves without visible symptoms.