A new multigene family inducible by tobacco mosaic virus or salicylic acid in tobacco.

A previously undescribed cDNA family was isolated from tobacco challenged with tobacco mosaic virus (TMV). A cDNA library was constructed with mRNA from upper leaves of Xanthi nc tobacco plants that had been inoculated with TMV on the lower leaves 11 days previously. The library was screened differentially with radiolabeled cDNA synthesized with mRNA from upper, uninoculated leaves of either TMV-inoculated or mock-inoculated tobacco plants. The new cDNA family, designated SAR8.2, had at least five expressed members, one or more of which were inducible by TMV inoculation and by salicylic acid treatment. The cDNAs encoded small, highly basic proteins containing N-terminal hydrophobic signal peptides and highly conserved cysteine-rich C-terminal domains. One of the SAR8.2 family members contained a direct repeat of the C-terminal domain in tandem. Hybridization of SAR8.2 cDNA to tobacco genomic DNAs indicated a gene family of 10-12 members.     

Short-term polyamine response in TMV-inoculated hypersensitive and susceptible tobacco plants

The short-term polyamine response to inoculation, with tobacco mosaic virus (TMV), of TMV-inoculated NN (hypersensitive) and nn (susceptible) plants of Nicotiana tabacum (L.) cv. Samsun was investigated. Free and conjugated polyamine concentrations, putrescine biosynthesis, evaluated through arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activities, and putrescine oxidation, via diamine oxidase (DAO) activity, were analysed during the first 24 h from inoculation. Results were compared with those of mock-inoculated control plants. In NN TMV-inoculated plants undergoing the hypersensitive response (HR), free putrescine and spermidine concentrations had increased after 5 h compared with controls; polyamine conjugates also tended to increase compared with controls. In both virus- and mock-inoculated plants, ADC and ODC activities generally increased whereas DAO activity, which was present in controls, was detectable only in traces in inoculated tissues.
In TMV-infected susceptible plants, free putrescine and spermidine concentrations were lower at 5 h relative to controls, as were polyamine conjugates. No differences were revealed in ADC and ODC activities whereas DAO activity was not detectable. These results further support the hypothesis that polyamines are involved in the response of tobacco to TMV and that, only a few hours after inoculation, the response of hypersensitive plants is distinct from that of susceptible ones.     

Induction of UDP-Glucose:Salicylic Acid Glucosyltransferase Activity in Tobacco Mosaic Virus-Inoculated Tobacco (Nicotiana tabacum) Leaves

Salicylic acid (SA) is a putative signal that activates plant resistance to pathogens. SA levels increase systemically following the hypersensitive response produced by tobacco mosaic virus (TMV) inoculation of tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. The SA increase in the inoculated leaf coincided with the appearance of a [beta]-glucosidase-hydrolyzable SA conjugate identified as [beta]-O-D-glucosylsalicylic acid (GSA). SA and GSA accumulation in the TMV-inoculated leaf paralleled the increase in the activity of a UDP-glucose:salicylic acid 3-O-glucosyltransferase (EC 2.4.1.35) ([beta]-GTase) capable of converting SA to GSA. Healthy tissues had constitutive [beta]-GTase activity of 0.076 milliunits g-1 fresh weight. This activity started to increase 48 h after TMV inoculation, reaching its maximum (6.7-fold induction over the basal levels) 72 h after TMV inoculation. No significant GSA or elevated [beta]-Gtase activity could be detected in the healthy leaf immediately above the TMV-inoculated leaf. The effect of TMV inoculation on the [beta]-GTase and GSA accumulation could be duplicated by infiltrating tobacco leaf discs with SA at the levels naturally produced in TMV-inoculated leaves (2.7-27.0 [mu]g g-1 fresh weight). Pretreatment of leaf discs with the protein synthesis inhibitor cycloheximide inhibited the induction of [beta]-GTase by SA and prevented the formation of GSA. Of 12 analogs of SA tested, only 2,6-dihydroxybenzoic acid induced [beta]-GTase activity.     

Pathway of Salicylic Acid Biosynthesis in Healthy and Virus-Inoculated Tobacco

Salicylic acid (SA) is a likely endogenous regulator of localized and systemic disease resistance in plants. During the hypersensitive response of Nicotiana tabacum L. cv Xanthi-nc to tobacco mosaic virus (TMV), SA levels rise dramatically. We studied SA biosynthesis in healthy and TMV-inoculated tobacco by monitoring the levels of SA and its likely precursors in extracts of leaves and cell suspensions. In TMV-inoculated leaves, stimulation of SA accumulation is accompanied by a corresponding increase in the levels of benzoic acid. 14C-Tracer studies with cell suspensions and mock-or TMV-inoculated leaves indicate that the label moves from trans-cinnamic acid to SA via benzoic acid. In healthy and TMV-inoculated tobacco leaves, benzoic acid induced SA accumulation. o-Coumaric acid, which was previously reported as a possible precursor of SA in other species, did not increase SA levels in tobacco. In healthy tobacco tissue, the specific activity of newly formed SA was equal to that of the supplied [14C]benzoic acid, whereas in TMV-inoculated leaves some isotope dilution was observed, presumably because of the increase in the pool of endogenous benzoic acid. We observed accumulation of pathogen-esis-related-1 proteins and increased resistance to TMV in benzoic acid- but not in o-coumaric acid-treated tobacco leaves. This is consistent with benzoic acid being the immediate precursor of SA. We conclude that in healthy and virus-inoculated tobacco, SA is formed from cinnamic acid via benzoic acid.     

Spermine delays leaf senescence in Lactuca sativa and prevents the decay of chloroplast photosystems

Aliphatic polyamines (PAs) are involved in the delay or prevention of plant senescence, but the molecular mechanism is not clarified. The hypothesis is put forward that one of the mechanisms by which PAs modulate leaf senescence and chlorophyll stabilisation could be due to their modification of chlorophyll-bound proteins, catalysed by transglutaminase (TGase, R-glutaminylpeptide-amine γ-glutamyltransferase; E.C. 2.3.2.13). The retardation of leaf senescence of Lactuca sativa L. by spermine (Spm) was examined during induced cell death using leaf discs, or during the normal developmental senescence of leaves. Over 3 days, in leaf discs, Spm caused a delay of chlorophyll (Chl) decay, an increase of endogenous TGase activity, and a three-fold increase in chlorophyll content when supplied together with exogenous TGase. Spm was conjugated, via TGase, mainly to 22–30 kDa proteins. Long-term experiments over 5 days showed a general decrease in all three parameters with or without Spm. When leaves remained on the plants, Spm-sprayed leaves showed an increase in free Spm 1 h after spraying, mainly in the young leaves, whereas over longer periods (15 days) there was an increase in perchloric acid-soluble and -insoluble Spm metabolites. In senescing leaves, Spm prevented degradation of chlorophyll b and some proteins, and increased TGase activity, producing more PA-protein conjugates. Spm was translocated to chloroplasts and bound mainly onto fractions enriched in PSII, but also those enriched in PSI, whose light-harvesting complexes (LHC) sub-fractions contained TGase. Spm was conjugated by TGase mainly to LHCII, more markedly in the light. Immunodetection of TGase revealed multiple proteins in young leaves, possibly representing different TGase isoforms when TGase activity was high, whereas in already senescent leaves, when its activity decreased, one high-molecular-mass band was found, possibly because of enzyme polymerisation. Spm thus protected senescing Lactuca leaves from the decay of their chloroplast photosystem complexes. The senescence-delaying effects of Spm could be mediated by TGase, as TGase was re-activated to the level in young leaves following Spm treatment.     

Is Salicylic Acid a Translocated Signal of Systemic Acquired Resistance in Tobacco?

Salicylic acid (SA) is a likely endogenous signal in the development of systemic acquired resistance (SAR) in some dicotyledonous plants. In tobacco mosaic virus (TMV)-resistant Xanthi-nc tobacco, SA levels increase systemically following the inoculation of a single leaf with TMV. To determine the extent to which systemic increases in SA result from SA export from the inoculated leaf, SA produced in TMV-inoculated or healthy leaves was noninvasively labeled with 18O2. Spatial and temporal distribution of 18O-SA indicated that most of the SA detected in the healthy tissues was synthesized in the inoculated leaf. No significant increase in the activity of benzoic acid 2-hydroxylase, the last enzyme involved in SA biosynthesis, was detected in upper uninoculated leaves, although the basal level of enzyme activity was relatively high. No increases in SA level, pathogenesis-related PR-1 gene expression, or TMV resistance in the upper uninoculated leaf were observed if the TMV-inoculated leaf was detached up to 60 hr after inoculation. Apart from the inoculated tissues, the highest increase in SA was observed in the leaf located directly above the inoculated leaf. The systemic SA increase observed during SAR may be explained by phloem transport of SA from the inoculation sites.     

Endogenous Methyl Salicylate in Pathogen-Inoculated Tobacco Plants

The tobacco (Nicotiana tabacum) cultivar Xanthi-nc (genotype NN) produces high levels of salicylic acid (SA) after inoculation with the tobacco mosaic virus (TMV). Gaseous methyl salicylate (MeSA), a major volatile produced in TMV-inoculated tobacco plants, was recently shown to be an airborne defense signal. Using an assay developed to measure the MeSA present in tissue, we have shown that in TMV-inoculated tobacco plants the level of MeSA increases dramatically, paralleling increases in SA. MeSA accumulation was also observed in upper, noninoculated leaves. In TMV-inoculated tobacco shifted from 32 to 24°C, the MeSA concentration increased from nondetectable levels to 2318 ng/g fresh weight 12 h after the temperature shift, but subsequently decreased with the onset of the hypersensitive response. Similar results were observed in plants inoculated with Pseudomonas syringae pathovar phaseolicola, in which MeSA levels were highest just before the hypersensitive response-induced tissue desiccation. Transgenic NahG plants unable to accumulate SA also did not accumulate MeSA after TMV inoculation, and did not show increased resistance to TMV following MeSA treatment. Based on the spatial and temporal kinetics of its accumulation, we conclude that tissue MeSA may play a role similar to that of volatile MeSA in the pathogen-induced defense response.     

Serological Comparison of Antigens Related to Local Virus and Bacterial Infections and Aspecific Stresses in Tobacco Leaves

Two “new” precipitin bands (antigens) detected by the immunodiffusion test were demon strated in leaf extracts of tobacco inoculated with tobacco mosaic virus (TMV), Pseudomonas tabaci or treated with mercuric chloride, sodium azide or sodium hypochlorite. One of the precipitin bands was stronger, than the other, These antigens were also detected in the upper, non-infected leaves of tobacco plants when the lower leaves were locally stressed (necrotized) either by TMV or by chemical injury. The “new” antigens formed in the upper leaves were detected even if the TMV-inoculated lower leaves were removed one day after inoculation. The “new” antigens were identical both in the lower and upper leaves and their induction was independent from the stress whether pathogenic or chemical. A coincidence exists between the appearance of “new” antigens and acquired resistance, but this does not mean necessarily a cause-and-effect relationship between the two phenomena. Our experiments indicate that the induction of the synthesis of “new” stress proteins in tobacco is aspecific and the proteins formed are related to the aspecific stress itself rather than to pathogenesis.     

Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco.

Systemic induction of pathogenesis-related (PR) proteins in tobacco, which occurs during the hypersensitive response to tobacco mosaic virus (TMV), may be caused by a minimum 10-fold systemic increase in endogenous levels of salicylic acid (SA). This rise in SA parallels PR-1 protein induction and occurs in TMV-resistant Xanthi-nc tobacco carrying the N gene, but not in TMV-susceptible (nn) tobacco. By feeding SA to excised leaves of Xanthi-nc (NN) tobacco, we have shown that the observed increase in endogenous SA levels is sufficient for the systemic induction of PR-1 proteins. TMV infection became systemic and Xanthi-nc plants failed to accumulate PR-1 proteins at 32 degrees C. This loss of hypersensitive response at high temperature was associated with an inability to accumulate SA. However, spraying leaves with SA induced PR-1 proteins at both 24 and 32 degrees C. SA is most likely exported from the primary site of infection to the uninfected tissues. A computer model predicts that SA should move rapidly in phloem. When leaves of Xanthi-nc tobacco were excised 24 hr after TMV inoculation and exudates from the cut petioles were collected, the increase in endogenous SA in TMV-inoculated leaves paralleled SA levels in exudates. Exudation and leaf accumulation of SA were proportional to TMV concentration and were higher in light than in darkness. Different components of TMV were compared for their ability to induce SA accumulation and exudation: three different aggregation states of coat protein failed to induce SA, but unencapsidated viral RNA elicited SA accumulation in leaves and phloem. These results further support the hypothesis that SA acts as an endogenous signal that triggers local and systemic induction of PR-1 proteins and, possibly, some components of systemic acquired resistance in NN tobacco.     

Tobacco mosaic virus inoculation inhibits wound-induced jasmonic acid-mediated responses within but not between plants

Jasmonic acid (JA) and salicylic acid (SA) have both been implicated as important signal molecules mediating induced defenses of Nicotiana tabacum L. against herbivores and pathogens. Since the application of SA to a wound site can inhibit both wound-induced JA and a defense response that it elicits, namely nicotine production, we determined if tobacco mosaic virus (TMV) inoculation, with its associated endogenous systemic increase in SA, reduces a plant's ability to increase JA and nicotine levels in response to mechanical damage, and evaluated the consequences of these interactions for the amount of tissue removed by a nicotine-tolerant herbivore, Manduca sexta. Additionally, we determined whether the release of volatile methyl salicylic acid (MeSA) from inoculated plants can reduce wound-induced JA and nicotine responses in uninoculated plants sharing the same chamber. The TMV-inoculated plants, though capable of inducing nicotine normally in response to methyl jasmonate applications, had attenuated wound-induced JA and nicotine responses. Moreover, larvae consumed 1.7- to 2.7-times more leaf tissue from TMV-inoculated plants than from mock-inoculated plants. Uninoculated plants growing in chambers downwind of either TMV-inoculated plants or vials releasing MeSA at 83- to 643-times the amount TMV-inoculated plants release, exhibited normal wound-induced responses. We conclude that tobacco plants, when inoculated with TMV, are unable to elicit normal wound responses, due likely to the inhibition of JA production by the systemic increase in SA induced by virus-inoculation. The release of volatile MeSA from inoculated plants is not sufficient to influence the wound-induced responses of neighboring plants. Received: 6 January 1999 / Accepted: 11 January 1999     

A<Emphasis Type="Italic"> Zea mays</Emphasis> 39-kDa thylakoid transglutaminase catalyses the modification by polyamines of light-harvesting complex II in a light-dependent way

A transglutaminase (TGase; EC 2.3.2.13) activity, which shared many properties with the TGase activity of the Helianthus tuberosus chloroplast, was observed in the Zea mays L. chloroplast and in its fractions. This activity was found to be prevalent in thylakoids; bis-(glutamyl) spermidine and bis-(glutamyl) putrescine were the main polyamine conjugates formed. Light stimulated the endogenous thylakoid activity. Putrescine, spermidine and spermine were conjugated to the isolated light-harvesting complex of photosystem II (LHCII) with different degrees of efficiency, spermine being the polyamine most efficiently conjugated. A TGase with a light-sensitive activity was identified in the photosystem II-enriched fraction. Its partial purification on a sucrose gradient allowed the separation of a 39-kDa band, which was immunorecognised by two anti-TGase antibodies (Ab-3 and rat prostatic gland-TGase). Both a colorimetric and a radiometric assay for TGase activity, the former carried out in the presence of biotinylated cadaverine and the latter in the presence of polyamines labelled with radioactive isotopes and resulting in the isolation of glutamyl-polyamines, further confirmed that the thylakoid enzyme is indeed a calcium-dependent transglutaminase (Thyl-TGase). At variance with guinea pig liver and erythrocyte TGases, which are insensitive to light, the activity of the thylakoid transglutaminase is affected by light. Moreover, this enzyme, when tested with purified LHCII as substrate, catalysed the production of mono- and bis-glutamyl-polyamines in equal amounts, whereas the animal enzymes produced mainly mono-derivatives. Herein, it is discussed whether this light sensitivity is due to the enzyme or the substrate.Abbreviations bis-PU Bis-(-glutamyl) putrescine - bis-SD Bis-(-glutamyl) spermidine - bis-SM Bis-(-glutamyl) spermine - Er-TGase Erythrocyte transglutaminase - Gpl-TGase Guinea pig liver transglutaminase - LHCII Light-harvesting complex II - mono-PU Mono-(-glutamyl) putrescine - mono-SD Mono-(-glutamyl) spermidine - mono-SM Mono-(-glutamyl) spermine - PA Polyamine - PU Putrescine - SD Spermidine - SM Spermine - Rpg-TGase Rat prostatic gland transglutaminase - Thyl-TGase Thylakoid transglutaminase     

Role of polyisoprenoids in tobacco resistance against biotic stresses

Infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols, solanesol and a family of polyprenols [from polyprenol composed of 14 isoprene units (Pren-14) to -18, with Pren-16 dominating] in the leaves of resistant tobacco plants Nicotiana tabacum cv. Samsun NN. Upon TMV infection, solanesol content was increased seven- and eight-fold in the inoculated and upper leaves, respectively, while polyprenol content was increased 2.5- and 2-fold in the inoculated and upper leaves, respectively, on the seventh day post-infection. Accumulation of polyisoprenoid alcohols was also stimulated by exogenously applied hydrogen peroxide but not by exogenous salicylic acid (SA). On the contrary, neither inoculation of the leaves of susceptible tobacco plants nor wounding of tobacco leaves caused an increase in polyisoprenoid content. Taken together, these results indicate that polyisoprenoid alcohols might be involved in plant resistance against pathogens. A putative role of accumulated polyisoprenoids in plant response to pathogen attack is discussed. Similarly, the content of plastoquinone (PQ) was increased two-fold in TMV-inoculated and upper leaves of resistant plants. Accumulation of PQ was also stimulated by hydrogen peroxide, bacteria ( P.  syringae ) and SA. The role of PQ in antioxidant defense in cellular membranous compartments is discussed in the context of the enzymatic antioxidant machinery activated in tobacco leaves subjected to viral infection. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase was found in inoculated leaves of resistant tobacco plants but not in susceptible plants.     

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.     

Molecular characterization of messenger RNAs for 'pathogenesis related' proteins la, lb and lc, induced by TMV infection of tobacco

A cDNA library was made to poly(A)-containing RNA from tobacco mosaic virus (TMV)-infected Samsun NN tobacco plants and clones corresponding to mRNAs for the `pathogenesis-related' (PR) proteins 1a, 1b and 1c were identified. One clone was found to contain a complete copy of PR-1b mRNA. The structural organization of this RNA is: a leader sequence of 29 nucleotides, an open reading frame of 504 nucleotides encoding a 30 amino acid long signal peptide and a 138 amino acid long mature protein, and a 3'-non-coding region of 235 nucleotides. Two other clones were found to contain partial copies of PR-1a and PR-1c mRNAs. The data indicate an ~90% homology between the amino acid sequences of PR-1a, -1b and -1c. Using one of the clones as probe it was shown that in the TMV-inoculated lower leaves and the non-inoculated upper leaves of a tobacco plant, the PR-1 mRNAs become detectable from 2 and 8 days after inoculation, respectively.     

Increase in transglutaminase and its extracellular products in response to an inflammatory stimulus by lipopolysaccharide

Transglutaminase (TGase) activities were measured in rat tissues 1-7 days after intraperitoneal injection of saline or lipopolysaccharide (LPS) and in the cells and media from pre-confluent human fibroblasts cultured for two days in the presence or absence of LPS. (-glutamyl)lysine and [3H]putrescine-labelled -glutamyl derivatives in extracellular and cellular fibroblast proteins were also measured. Three effects of LPS were observed. Firstly, total TGase activity is greater in the tissues from the LPS-injected animals, with the maximum increase occurring at 1 day in dermis, epidermis and liver, at 5 days in the aorta and, after a decrease at 2-5 days, at 7 days in the panniculus muscle. Secondly, the fraction of the total activity which is buffer-extractable is greater on days 1 and/or 2 in all the tissues from the LPS-injected rats. Thirdly, in cultures of human fibroblasts, LPS increases that fraction of bound [3H]putrescine and of TGase and its -glutamylamine products which occurs in the extracellular medium. In addition, a higher concentration of TGase-derived crosslinks was found in extracellular as opposed to intracellular proteins. In conjunction with previous findings in skin wound healing and in atherosclerosis these results support the concept of an extracellular function for tissue TGase and indicate that there is a widespread association of increases in TGase and its extracellular products with inflammation and the healing or fibrotic processes which follow it.     

Possible mechanism of action of antiviral proteins from the leaves of Chenopodium album L

Antiviral proteins (AVPs) named CAP-I and CAP-II purified from the leaves of Chenopodium album cv Pusa Bathua-1 induced systemic resistance against tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) in both hypersensitive as well as systemic hosts. An increased accumulation of two polypeptides (approximately 17 kDa and approximately 26 kDa) was observed in untreated upper leaves of Cyamopsis tetragonoloba plants whose basal leaves were treated with CAP-I/CAP-II. Both AVPs exhibited ribosomal RNA N-glycosidase activity on 28S rRNA of tobacco leaves and also caused in vitro degradation of TMV RNA. It is suggested that the CAP-I and -II are multi-functional and may be acting at multiple levels to ensure maximum possible inhibition of viral infection.     

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.     

γ-Glutamylcyclotransferase in tobacco suspension cultures: Catalytic properties and subcellular localization

Steinkamp, R., Schweihofen, B. and Rennenberg, H. 1987. γ-Glutamylcyclotransfer-ase in tobacco suspension cultures: Catalytic properties and subcellular localization.
γ-Glutamylcyclotransferase activity (EC 2.3.2.4) in ammonium sulfate precipitates (40–70% saturation) of extracts from cultured tobacco cells ( Nicoliana tabacum L. cv. Samsun) was analyzed as liberation of 5-oxo-proline from L-γ-glutamyl dipeptides. The enzyme was highly specific for the sulfur containing γ-glutamyl dipeptides γ-glutamyl-L-methionine and γ-glutamyl-i.-cysteine. Maximum activity was obtained at pH 8.7 and 35°C. As also observed with animal γ-glutamylcyclotransferase, the tobacco enzyme exhibited a relatively low substrate affinity (γ-glutamyl-i.-methionine: K_m 2.2 ± 0.4 mM ). In contrast to animal γ-glutamylcyclotransferase, the tobacco enzyme was not inhibited by the D-isomerof the substrate D-γ-glutamyl-i.-methionine; it also did not use the D isomer as a substrate. γ-Glutamylcyclotransferase of tobacco cells was shown to be a soluble enzyme entirely localized in the cytoplasm.     

Two Apoplastic alpha-Amylases Are Induced in Tobacco by Virus Infection

α-Amylase activity (EC 3.2. 1.1) is greatly increased in leaves of tobacco (Nicotiana tabacum L. cv Samsun NN) infected with tobacco mosaic virus (TMV). The kinetics of enzyme induction during the hypersensitive reaction resemble those of other hydrolases known to be pathogenesis-related proteins of tobacco. Two α-amylases were purified from TMV-infected leaves and shown to have features in common with well-characterized pathogenesis-related proteins: they are acidic monomers that can be separated upon electrophoresis on basic native gels, and they are found in the apoplastic compartment of the cell. This extra-cellular localization was demonstrated by comparing the α-amylase partition between the intercellular wash fluid and the cell extract with that of proteins of known cellular compartmentalization. These data indicate an active secretion of both α-amylases produced in tobacco upon TMV infection.