Hexokinases of Tobacco Leaves: Changes in the Cytosolic and Non-Cytosolic Isozyme Complexes Induced by Tobacco Mosaic Virus Infection

Changes in the cytosotic (soluble) and the non-cytosolic (particulate) isozyme composition of hexokinases and in their properties were studied by ion exchange chromatography on DEAE cellulose after the subcellular fractionation both in the healthy and the tobacco mosaic virus (TMV) infected tobacco leaves. Three main isozyme complexes were obtained: one particulate fraction (the particulate hexokinase phosphorylating both glucose and fructose, EC 2.7.1.1), and two soluble fractions (the soluble hexokinase phosphorylating both the glucose and the fructose, and the soluble fructokinase, which phosphorylates primarily fructose, EC 2.7.1.4). The total fructokinase activities were nearly twice higher than the total glucokinase activities (188.6 % of glucokinase activity in healthy plants and 181.3 % in infected plants). The total particulate glucokinase activity was increased to 120.6 % and the fructokinase to 118.9 % in TMV infected tissue when compared with healthy control. The similar pattern of activity was observed for soluble hexokinase isozymes - the sum of soluble glucokinase activity was increased to 175.4 % and of fructokinase activity to 131.2 % in TMV infected tissue. The isozymes isolated both from the healthy control and TMV-infected leaves had the similar elution profiles, displayed Michaelis-Menten kinetics, showed the identical profiles of pH optima and were Mg²⁺ dependent with the highest enzyme activity at equimolar Mg²⁺ and ATP concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Tobacco-mosaic-virus-induced increase in abscisic-acid concentration in tobacco leaves:

The concentrations of free and bound abscisic acid (ABA and the presumed ABA glucose ester) increased three- to fourfold in leaves of White Burley tobacco (Nicotiana tabacum L.) systemically infected with tobacco mosaic virus. Infected leaves developed a distinct mosaic of light-green and dark-green areas. The largest increases in both free and bound ABA occurred in dark-green areas. In contrast, virus accumulated to a much higher concentration in light-green tissue. Free ABA in healthy leaves was contained predominantly within the chloroplasts while the majority of bound ABA was present in non-chloroplastic fractions. Chloroplasts from light-green or dark-green tissues were able to increase stromal pH on illumination by an amount similar to chloroplasts from healthy leaf. It is unlikely therefore that any virus-induced diminution of pH gradient is responsible for increased ABA accumulation. Tobacco mosaic virus infection had little effect on free ABA concentration in chloroplasts; the virus-induced increase in free ABA occurred predominantly out-side the chloroplast. The proportional distribution of bound ABA in the cell was not changed by infection. Treatment of healthy plants with ABA or water stress increased chlorophyll concentration by an amount similar to that induced by infection in dark-green areas of leaf. A role for increased ABA concentration in the development of mosaic symptoms is suggested.Abbreviations ABA abscisic acid - TMV tobacco mosaic virus     

Transgenic tobacco plants regenerated from leaf disks can be periclinal chimeras

Amongst rolC transgenic tobacco plants regenerated from leaf disks 6.5% are periclinal chimeras, i.e. plants with genetically different cell populations in different cell layers. The expression of the rolC gene of Agrobacterium rhizogenes causes a reduction in pigment content in leaves. The chimeric composition of the regenerated plants becomes thus apparent as light green leaf tissue in the transgenic region, tissue flanked by dark green wild-type sectors. Southern and northern blot analysis confirmed the chimeric nature of such plants. Investigation of selfed progeny of chimeric plants on selective media indicates that layer invasion in reproductive tissues can occur in tobacco early during the formation of the flower buds. The results show (1) that tobacco plants regenerated from leaf disks and grown on selective media have not necessarily the same clonal origin and (2) that they can give rise to non-transgenic offspring. The chimeric plants provide insight on the effect of rolC gene expression on microsporogenesis.     

Detection of tobacco mosaic virus and tomato mosaic virus in pepper seeds by enzyme linked immunosorbent assay (ELISA)

Pepper seed samples were tested for the infection of tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) by enzyme linked immunosorbent assay (ELISA). Out of 26 pepper seed samples tested, 17 were infected with TMV and ToMV in ELISA. About 34.7% of pepper seed samples were found to be healthy. Infections of TMV or ToMV were recorded to be 61.53% and 11.5%, respectively of the total tested seed samples.     

Biosynthesis of tobacco mosaic virus RNA in tobacco protoplasts

Changes in the number of protoplasts, viability, protein and chlorophyll contents and ribonucleases activity were studied in tobacco mesophyll protoplastsin vitro inoculated with tobacco mosaic virus (TMV). The number of protoplasts slowly increased during the cultivation period and the viability decreased from 95 to 67% in the control noninoculated protoplasts, and to 55% in the infected protoplasts. 30 h after inoculation the protein and chlorophyll contents strongly decreased to 25–30% and 17–19%, respectively, in comparison with contents 3 h after inoculation. The chlorophylla/b ratio decreased from 2.11 and 2.02 to 0.79 and 0.60 in healthy and infected protoplasts, respectively. The activities of ribonucleases in protoplasts quickly decreased during experiment but they were higher in infected than in noninfected protoplasts (between 20 to 30 h after inoculation they were 132 to 146% higher than that in healthy controls). These activities corresponded to the multiplication curve of TMV.     

Analysis ofcis-regulatory elements involved in induction of a tobacco PR-5 gene by virus infection

cis-Regulatory elements involved in tobacco mosaic virus (TMV)-inducible expression were indentified in a tobacco PR-5 gene, encoding an acidic thaumatin-like protein. By fusing upstream sequences of the PR-5 gene to the GUS reporter gene and analysing transgenic plants containing these fusions for local and systemic induction of GUS activity by TMV, it was found that sequences between-1364 and-718 are involved in TMV induction of PR-5 gene expression.     

Stimulation by abscisic acid of RNA synthesis in discs from healthy and tobacco mosaic virus-infected tobacco leaves

Uptake of abscisic acid from the culture medium by discs of healthy and tobacco mosaic virus-infected tobacco leaves was measured. Small (two to five-fold) increases in abscisic acid concentration in discs caused increases in rates of [³H]uridine and [³H]adenine incorporation into total nucleic acid, virus RNA and host ribosomal RNA. Net accumulation of virus RNA was also enhanced by abscisic acid. This evidence for stimulation of RNA synthesis is compared with previous reports showing inhibition of RNA synthesis in other tissues. It is suggested that the increase in endogenous abscisic acid caused by tobacco mosaic virus infection may be at least partly responsible for observed increases in rates of RNA synthesis after infection.Abbreviations ABA abscisic acid - TMV tobacco mosaic virus     

Changes in Glucose-6-Phosphate Dehydrogenase, Ribonucleases, Esterases and Contents of Viruses in Potato Virus Y Infected Tobacco Superinfected with Tobacco Mosaic Virus

Effects of the superinfection with tobacco mosaic virus (TMV) on susceptible tobacco plants infected with potato virus Y (PVY) were determined. Dynamic changes in the TMV and/or PVY contents, the ribonucleases (RNases), the phosphomonoesterase (PME), the phosphodiesterase (PDE) and the glucose-6-phosphate dehydrogenase (G6P DH) activities were studied. The PVY infection caused a substantial reduction in the multiplication of TMV. The content of TMV in the PVY inoculated leaves amounts to 6 and 9 % in the PVY systemically infected leaves when compared with single TMV. Surprisingly, the challenging virus (TMV) enhanced the content of inducing virus (PVY) in the locally inoculated leaves up to 130 – 141 %. In contrast, the reduction of PVY content down to 35 – 40 % by TMV was seen in the PVY systemically infected leaves. The activities of the RNase, the PME, the PDE and the G6P DH were increased (when compared with the healthy plants) during the acute phase of single virus multiplication (PVY or TMV). The increase in the activities of the enzymes in the leaves with mixed infection was at least as high as the sum of the increases of single infections. Moreover, a higher increase than the sum was seen for G6P DH and PDE (by about 20 – 35 %). This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular tagging of the tobacco chromosome carrying the TMV-resistance gene (N gene) by Agrobacterium-mediated transformation

Summary The hypersensitive response of tobacco to inoculation with tobacco mosaic virus (TMV) is controlled by a single dominant gene, the N gene. As a first step in localizing and transferring the N gene, we have prepared a line of tobacco plants in which the kanamycin-resistance (Km^r) gene is closely linked to the N gene. Nicotiana tabacum plants heterozygous for the N gene were transformed to Km^r by Agrobacterium carrying pMON200. Eighty-nine independent transformed clones were regenerated and were backcrossed with nontransformed, TMV-sensitive plants. Progeny from these crosses were screened first for Km^r; then the Km^r progeny were inoculated with TMV and scored for the hypersensitive response. Of the initial 89 clones, 68 appeared to have integrated a single functional Km^r gene. Initial tests for TMV resistance indicated possible linkage between Km^r and the N gene in 11 plants. With further testing, linkage has been established for two of these plant lines. In one of these lines, the two genes were 30–40 map units apart, and evidence of somatic instability in the linkage was obtained. However, in the second line, linkage between Km^r and the N gene was tight, and recombination between the genes in this case was only 5%. Southern hybridization revealed that this plant contained only a single copy of the Km^r gene. Linkage between Km^r and the N gene in this plant line has been verified in each of two additional backcross generations.Abbreviations nptII Neomycin phosphotransferase gene - Km^r kanamycin resistant - Km^s kanamycin sensitive - TMV tobacco mosaic virus - TMV-R TMV resistant - TMV-S TMV sensitive     

Effect of “osmotic stress” on protein and nucleic acid synthesis in isolated tobacco protoplasts

The incorporation of labeled precursors into RNAs and proteins of isolated tobacco (Nicotiana tabacum L.) leaf protoplasts decreases with increasing osmotic pressure in the incubation medium. The incorporation of precursors into RNA and proteins is linear for 15–18 h after the isolation of the protoplasts, irrespective of the osmolarity of the culture media. The uptake of precursors is also affected by the osmolarity of the medium. However, the osmotic stress-induced inhibition of incorporation of precursors into RNA and proteins is also apparent if the differences in uptake are taken into consideration in the calculation. Incorporation of ³²P into TMV-RNA is also inhibited by osmotic stress. As assayed by the double labeling ratio technique, osmotic stress has less unequivocal effect on TMV protein synthesis.Abbreviations PP protoplast - RNase ribonuclease - rRNA ribosomal ribonucleic acid - SDS sodium dodecyl sulfate - SSC 0.1 M Na-acetate in 0.15 M NaCl - TCA trichloroacetic acid - TMV tobacco mosaic virus     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

The type-1 and type-2 ribosome-inactivating proteins from<Emphasis Type="Italic"> Iris</Emphasis> confer transgenic tobacco plants local but not systemic protection against viruses

The antiviral activity of the type-2 ribosome-inactivating protein (RIP) IRAb from Iris was analyzed by expressing IRAb in tobacco (Nicotiana tabacum L. cv. Samsun NN) plants and challenging the transgenic plants with tobacco mosaic virus (TMV). Although constitutive expression of IRAb resulted in an aberrant phenotype, the plants were fertile. Transgenic tobacco lines expressing IRAb showed a dose-dependent enhanced resistance against TMV infection but the level of protection was markedly lower than in plants expressing IRIP, the type-1 RIP from Iris that closely resembles the A-chain of IRAb. To verify whether IRIP or IRAb can also confer systemic protection against viruses, transgenic RIP-expressing scions were grafted onto control rootstocks and leaves of the rootstocks challenged with tobacco etch virus (TEV). In spite of the strong local antiviral effect of IRIP and IRAb the RIPs could not provide systemic protection against TEV. Hence our results demonstrate that expression of the type-1 and type-2 RIPs from Iris confers tobacco plants local protection against two unrelated viruses. The antiviral activity of both RIPs was not accompanied by an induction of pathogenesis-related proteins. It is suggested that the observed antiviral activity of both Iris RIPs relies on their RNA N-glycohydrolase activity towards TMV RNA and plant rRNA.Abbreviations GUS -Glucuronidase - IRAb Iris agglutinin b - IRIP Iris type-1 RIP - PAG Polynucleotide:adenosine glycosylase - PAP Phytolacca americana antiviral protein - PR Pathogenesis-related - RIP Ribosome-inactivating protein - TCS Trichosanthin - TEV Tobacco etch virus - TMV Tobacco mosaic virus     

Characterization of tobacco plants expressing a bacterial salicylate hydroxylase gene

Transgenic tobacco plants that express the bacterial nahG gene encoding salicylate hydroxylase have been shown to accumulate very little salicylic acid and to be defective in their ability to induce systemic acquired resistance (SAR). In recent experiments using transgenic NahG tobacco and Arabidopsis plants, we have also demonstrated that salicylic acid plays a central role in both disease susceptibility and genetic resistance. In this paper, we further characterize tobacco plants that express the salicylate hydroxylase enzyme. We show that tobacco mosaic virus (TMV) inoculation of NahG tobacco leaves induces the accumulation of the nahG mRNA in the pathogen infected leaves, presumably due to enhanced stabilization of the bacterial mRNA. SAR-associated genes are expressed in the TMV-infected leaves, but this is localized to the area surrounding necrotic lesions. Localized acquired resistance (LAR) is not induced in the TMV-inoculated NahG plants suggesting that LAR, like SAR, is dependent on SA accumulation. When SA is applied to nahG-expressing leave's SAR gene expression does not result. We have confirmed earlier reports that the salicylate hydroxylase enzyme has a narrow substrate specificity and we find that catechol, the breakdown product of salicylic acid, neither induces acquired resistance nor prevents the SA-dependent induction of the SAR genes.     

Purification of tobacco O-methyltransferases by affinity chromatography and estimation of the rate of synthesis of the enzymes during hypersensitive reaction to virus infection

The three tobacco (Nicotiana tabacum L.) S-adenosyl-L-methionine: o-diphenol-O-methyltransferases (OMTs; EC 2.1.1.6) were purified to homogeneity by affinity chromatography on adenosine-agarose. Amounts and catalytic actities of the enzymes were measured in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus. The drastic increase in activity of each enzyme upon infection was shown to arise from the accumulation of enzymatic protein with constant specific enzymatic activity. Rates of OMT synthesis were determined from pulse-labeling experiments with L-[¹⁴C]leucine injected into the leaves. The specific radioactivities of the homogenous enzymes were compared in healthy and tobacco mosaic virus-infected tobacco. The results demonstrated that increase in OMT amounts is a consequence of de novo synthesis of the enzymes.Abbreviations DEAE diethylaminoethyl - OMT O-methyltransferase - SAM S-adenosyl-L-methionine - TMV tobacco mosaic virus     

Glucose-6-Phosphate Dehydrogenase, Ribonucleases and Esterases upon Tobacco Mosaic Virus Infection and Benzothiodiazole Treatment in Tobacco

Effect of the benzothiodiazole (BTH) pre-treatment was monitored during the acute infection stage in the susceptible and the hypersensitive tobacco plants infected with the tobacco mosaic virus (TMV). Dynamic changes in the contents of chlorophyll, the total proteins, and the pathogenesis related proteins (PR-proteins), and activities of ribonucleases (RNase), phosphomonoesterase (PME), phosphodiesterase (PDE), and glucose-6-phosphate dehydrogenase (G6P DH) were studied. Neither the protein nor the chlorophyll contents were significantly changed by the TMV infection and/or the BTH treatment. The BTH pre-treatment caused a substantial reduction in the multiplication of TMV in the locally-infected leaves of the hypersensitive cultivar Xanthi-nc (to 15.1%). A lesser decrease (to 50.3%) was observed in the locally-infected leaves of susceptible cultivar Samsun. But in the systemically-infected leaves of this cultivar, only a 4-d delay in the multiplication of TMV was found. In the locally-infected leaves of both cultivars, the activities of the RNase, PME, PDE and G6P DH were sharply increased during the acute phase of TMV multiplication (when compared with the healthy plants) and the curves of these activities correlated with the multiplication curves of TMV. The BTH alone also strongly enhanced the activities of these enzymes early after application. Only low additional increases in some enzymes and even slight declines in the others were observed when the inoculation of leaves of cultivar Xanthi-nc followed the pre-treatment with the BTH. No inhibition of the enzymes was observed when the direct effect of different concentration of the BTH (1 – 1000 M) was examined in vitro during a measurement of the activity. The analysis of intercellular proteins by PAGE under native conditions shows the similar spectrum of the proteins extracted from either the BTH-treated or the TMV-infected tobacco cv. Xanthi-nc.     

In vitro propagation of plant virus using different forms of plant tissue culture and modes of culture operation

Plant virus accumulation was investigated in vitro using three different forms of plant tissue culture. Suspended cells, hairy roots and shooty teratomas of Nicotiana benthamiana were infected with tobacco mosaic virus (TMV) using the same initial virus:biomass ratio. Viral infection did not affect tissue growth or morphology in any of the three culture systems. Average maximum virus concentrations in hairy roots and shooty teratomas were similar and about an order of magnitude higher than in suspended cells. Hairy roots were considered the preferred host because of their morphological stability in liquid medium and relative ease of culture. The average maximum virus concentration in the hairy roots was 0.82 ± 0.14 mg g⁻¹ dry weight; viral coat protein represented a maximum of approximately 6% of total soluble protein in the biomass. Virus accumulation in hairy roots was investigated further using different modes of semi-continuous culture operation aimed at prolonging the root growth phase and providing nutrient supplementation; however, virus concentrations in the roots were not enhanced compared with simple batch culture. The relative infectivity of virus in the biomass declined by 80–90% during all the cultures tested, irrespective of the form of plant tissue used or mode of culture operation. Hairy root cultures inoculated with a transgenic TMV-based vector in batch culture accumulated green fluorescent protein (GFP); however, maximum GFP concentrations in the biomass were relatively low at 39 μg g⁻¹ dry weight, probably due to genetic instability of the vector. This work highlights the advantages of using hairy roots for in vitro propagation of TMV compared with shooty teratomas and suspended plant cells, and demonstrates that batch root culture is more effective than semi-continuous operations for accumulation of high virus concentrations in the biomass.     

Changes in non-protein nitrogen metabolism during tobacco mosaic virus biosynthesis

1. Discs cut from tobacco leaf tissue infected with tobacco mosaic virus and cultured in water contain less non-protein nitrogen than comparable uninfected discs during the time at which TMV is formed. This deficiency disappears when virus formation ceases. Discs cultured in nutrient solution form about twice as much TMV as discs cultured in water. The maximum non-protein nitrogen deficiency is comparable in magnitude to the amount of virus synthesized. 2. The largest difference between injected and uninfected tissue occurs in the ammonia content. Smaller, but significant differences in amide content are found. Infected discs cultured in water show no significant differences from control discs in free amino acid content; infected discs cultured in nutrient solution develop a small deficiency in amino acid nitrogen. 3. The general patterns of change in composition of the pool of soluble nitrogen are similar in both infected and uninfected discs. 4. The data indicate that the bulk of the nitrogen incorporated into virus protein is withdrawn from the leaf's pool of soluble nitrogen; virus is formed de novo from ammonia nitrogen and non-nitrogenous carbon sources. The effect of virus infection on host nitrogen metabolism appears to be due to the formation of virus rather than to its presence.     

Phenylalanine ammonia-lyase levels in protoplasts isolated from hypersensitive tobacco pre-infected with tobacco mosaic virus

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.Abbreviations OMT O-methyltransferase - PAL phenylalanine ammonia-lyase - TMV Tobacco mosaic virus