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     

The fine structure and photosynthetic cost of structural leaf variegation

The leaves of some plants display an optical patchiness on their upper side, displaying light- and dark-green areas with high and low reflectance, respectively. In this investigation, we studied the fine structure of the corresponding sectors and we asked whether the lost reflected light entails a photosynthetic cost to these leaves. Four species, i.e. Arum italicum, Ranunculus ficaria, Cyclamen hederifolium and Cyclamen persicum were investigated. Scanning electron microscope examination revealed that epidermal cells of light-green sectors of all species are more bulgy than corresponding cells of neighboring dark-green leaf sectors. The comparative anatomical study revealed that (i) epidermis thickness of the light-green areas and the number of mesophyll cell layers does not differ from those of the adjacent dark-green leaf sectors and (ii) palisade cells of light-green sectors are slightly larger and more loosely arranged, allowing a much higher percentage of intercellular air spaces. The latter histological feature seems to provide the structural basis for the different optical properties between the two leaf sectors. Contrary to expectations, net photosynthetic rates (expressed on a leaf area basis) were similar in the light-green and the dark-green areas of the two cyclamen species. Yet, in C. persicum net photosynthesis was higher in the light-green areas, if expressed on a dry mass basis. The small size of the light-green spots in the rest of the test plants precluded CO₂ assimilation measurements, yet maximum linear photosynthetic electron transport rates displayed no differences between the two sectors in all plants. Thus, the assumption of a photosynthetic cost in the light-green areas was not confirmed. On the contrary, a higher construction cost was evident in the dark-green areas of three species, displaying a significantly higher specific leaf mass, without any photosynthetic benefit. The results on net photosynthesis were compatible with leaf optical properties and pigment levels. Thus, in spite of the considerably higher reflectance of the light-green areas and their lower (yet normal for a green leaf) chlorophyll levels, corresponding differences in absorptance were slight. In addition, dry mass-based pigment contents in dark-green areas were higher, while chlorophyll a/b (in two species) and carotenoid/chlorophyll ratios (in three species) were lower, pointing to a shade adaptation in these sectors. We conclude that in variegated leaves of this kind, dark-green areas are more costly to build and probably less photosynthetically active. We argue that the high pigment contents of dark-green areas establish steep light gradients in the corresponding mesophyll, rendering deeper chloroplast layers more shade adapted.     

Changes in Composition of Soluble Intercellular Proteins Isolated from Healthy and TMV-Infected Nicotiana tabacum L. cv. Xanthi-nc

Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), polyphenoloxidases, peroxidases and proteases activity and PR-proteins composition in leaf tissue and intercellular fluid (ICF) isolated from leaf tissue of healthy and TMV-infected hypersensitive tobacco (Nicotiana tabacum L. cv. Xanthi-nc) plants (non-inoculated leaves) were studied. The amount of the proteins and the enzymes of intercellular space was less than 3 % of the total amount of proteins and the enzymes found in homogenate of healthy leaves. The TMV infection did not significantly change this observation. The great increase in the activities of the enzymes was observed in homogenates of the infected leaves, especially of the enzymes involved in biosynthesis of precursors needed for virus multiplication (G6P DH, RNase, PME, PDE). This is in contrast with the activities of the enzymes of ICF, which were only partly increased. The ICF proteins of infected plants were separated by means of ion exchange chromatography on DEAE cellulose. The isozymes of peroxidase, polyphenoloxidase, PME and PDE were identified. Using discontinuous nondenaturating polyacrylamide gel electrophoresis of DEAE cellulose fractions, the detection of isozymes of peroxidases and PR-proteins was performed. By means of SDS-PAGE the molecular masses of PR-proteins were identified: 15 – 16 kDa (group 1), 27 – 28 kDa (group 3: chitinases) and 36 – 40 kDa (group 2a: -1,3-glucanases).     

Dark and light green tissues of tobacco leaves systemically infected with tobacco mosaic virus

There are significant changes in the structure of the upper tobacco (Nicotiana tabacum L.) leaves systemically infected with tobacco mosaic virus (TMV) especially in the light green tissue (LGT). Dark green areas (DGI) had intermediate status between healthy tissue and LGT. DGI contained significantly less infectious TMV and viral antigen than the LGT. The DGI, LGT and healthy tissues did not differ in the permeability of cell membranes and in the set of acidic pathogenesis-related (PR) proteins but the total content of PR-proteins in the healthy plants was higher than in the infected ones with the DGI being intermediate between healthy tissue and LGT. The crude leaf extracts from DGI and LGT showed less total ribonuclease activity and ribonuclease isozymes in comparison with control.     

Potato virus-Y multiplication in susceptible tobacco cultivar and transgenic breeding line producing coat protein mRNA

Changes in ribonucleases (RNases) and glucose-6-phosphate dehydrogenase (G6P DH) activities, their content and subcellular localisation were studied in relation to virus multiplication in susceptible (cv. Samsun) or resistant (transgenic breeding line NCTG 83) tobacco plants infected with the potato virus Y^N (necrotic strain of PVY). Activities of RNases and G6P DH from diseased susceptible tobacco plants were markedly increased during the experimental period and significantly correlated with the multiplication curve of the PVY^N. In contrast, the activities of RNases and G6P DH were not changed after PVY inoculation of resistant breeding line NCTG 83 producing the CP mRNA of PVY. Changes in the content and in the subcellular localisation of RNases and G6P DH isozymes were also determined in mesophyll protoplasts isolated from healthy as well as PVY^N infected plants of both cultivars by differential centrifugation of broken protoplasts on day eight post inoculation (the culmination of multiplication curve of PVY and enhanced activity of both enzymes). The chloroplasts fraction from infected protoplasts showed an enhanced content of RNases (192.4% when compared with that from healthy control ones), and of G6P DH (174.4 %). The cytosol fraction from infected protoplasts contained slightly enhanced levels of G6P DH (117.4 %) and considerably enhanced levels of RNases (141.7 %). No significant differences in the activities, contents and subcellular localisation of RNases and/or G6P DH isozymes were observed in the resistant line NCTG 83. This is in accordance with no detectable contents of PVY. This revised version was published online in July 2006 with corrections to the Cover Date.     

Alterations in the activities of the enzymes of proline metabolism in Ragi (Eleusine coracana) leaves during water stress

Free proline content in Ragi (Eleusine coracana) leaves increased markedly (6 to 85 fold) as the degree of water stress, created by polyethylene gylcol treatment, was prolonged There was also a marginal increase in soluble proteins in the stressed leaves as compared to that in the controls. Water stress stimulated the activities of ornithine aminotransferase and pyrroline-5-carboxylate reductase, the enzymes of proline biosynthesis and markedly inhibited the enzymes involved in proline degradation viz., proline oxidase and pyrroline-5-carboxylate dehydrogenase. These results suggest that increase in free proline content of Ragi leaves could be due to enhanced activities of the enzymes synthesizing proline but more importantly due to severe inhibition of the enzymes degrading proline. These observations establish for the first time, the pathway of proline metabolism in plants by way of detection of the activities of all the enzymes involved and also highlight the role of these enzymes in proline accumulation during water stress.     

Virus-induced virus inhibitors from systemically virus-infected plants

After infection ofNicotiana tabacum cv. Samsun with tobacco mosaic virus (TMV) crude extracts from dark-green spots of upper leaves had a more strongly marked inhibitory effect upon TMV addedin vitro than crude extracts from the surrounding light-green tissue. Likewise, crude extracts from leaves ofNicotiana tabacum cv. Samsun showing recovery after infection with tobacco ringspot virus (TRV) were seen to have a marked inhibitory effect on TMV addedin vitro. The results obtained suggest that virus inhibitors are produced after virus infections not only in hypersensitive hosts but also in systemic hosts. Necrotizing processes are not an indispensable prerequisite of the production of virus-induced virus inhibitors.     

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.     

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.     

The content of ATP,ADP, AMP,Pi, the activity of enzymes involved in the glycolytic pathway and some problems of its regulation,and energy balance in tobacco plants infected with potato virus Y

The content of ATP, ADP, AMP, P_i, the activity of the enzymes involved in the glycolytic pathway, some problems of their regulation by adenine nucleotides and some basic problems connected with tissue energy balance were studied in tobacco plants infected with the potato virus Y (PVY). The contents of ATP and ΣAdN were increased in virus-infected tissues when compared with healthy tissues and correlated with the PVY reproduction curve. ADP and AMP contents decreased just after the inoculation and increased at the end of the experimental period, P_i content was not influenced by the infection. The activities of the key enzymes of the glycolytic pathway (6-phosphofructokinase, hexosediphosphatase, and pyruvate kinase), determined both in crude homogenates and after partial purification, did not differ during the entire experimental period from the values found in healthy control tissues, similarly as the activities of glucosephosphate isomerase, glyceraldehydephosphate dehydrogenase, phosphoglyceromutase and enolase observed in crude homogenates. The unchanging AEC value in virus-infected tissues simultaneously indicated that no change in the rate of the glycolytic pathway occurred even under “invivo” conditions at the period of the acute stage of infection.     

Correlation of viral RNA biosynthesis with glucose-6-phosphate dehydrogenase activity and host resistance

Changes in glucose-6-phosphate dehydrogenase (G6P DH; EC 1.1.1.49) activity caused by infection of tobacco ( Nicotiana tabacum L.) leaves with potato virus Y (PVY), cucumber mosaic virus, potato virus X, tobacco rattle virus and turnip mosaic virus, the subcellular localisation of G6P DH isozymes in mesophyll protoplasts derived from healthy and PVY-infected tobacco leaves, as well as G6P DH control and the relationship of its isozymes with the degree of tobacco resistance to PVY multiplication, were studied. The activities of G6P DH were markedly increased in locally and systemically infected leaves and the time courses of the activity linearly correlated with those of virus multiplication. In leaves infected with PVY, the activity time courses of the crude and the partially purified G6P DH were coincident. This probably indicates the involvement of coarse regulation of the enzyme. PVY content linearly correlated with enhanced G6P DH activity in leaf discs derived from susceptible, tolerant and resistant cultivars of tobacco. The increased activity of the enzyme in infected protoplasts and plant tissues was predominantly caused by the increased activity of chloroplastic isozymes. This was confirmed by the specific staining of isozymes after electrophoretic separation of chloroplastic proteins of tobacco leaves. These findings enable the degree of resistance to virus multiplication to be quantified for the use of gene manipulation and breeding.     

Gene expression of ascorbic acid-related enzymes in tobacco

GDP-D-mannose pyrophosphorylase (GMPase) and L-galactono-1, 4-lactone dehydrogenase (GalLDH) are key enzymes in L-ascorbic acid (AsA) biosynthesis of plants, and a full-length cDNA for GMPase was isolated from tobacco using PCR. Additionally, expression of GMPase, GalLDH and other AsA-related enzymes was examined in tobacco tissues and cultured BY-2 cells, and the relationship between their expression patterns and AsA content is discussed. It was found that the expression of GalLDH and GMPase mRNAs was markedly suppressed by loading AsA, suggesting that AsA concentration in the cells may regulate AsA biosynthesis. Moreover, the expression of GMPase and GalLDH mRNAs in tobacco leaf also suggested that AsA biosynthesis may be induced by light.     

Enzymes of starch and sugar phosphate metabolism in achlorophyllous ribosome-deficient plastids from high-temperature-grown rye leaves

Several enzymes of non–photosynthetic sugar phosphate and starch metabolism were measured in gradient–purified chloroplasts from normal rye leaves ( Secale cereale L. cv. Halo) grown at 22°C and in the non-photosynthetic plastids isolated from 70S ribosome-deficient rye leaves grown at a non–permissive elevated temperature of 32°C. Activities of the enzymes phosphoglycerate kinase (EC 2.7.2.3), hexokinase (EC 2.7.1.1), phosphoglucose isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate de-hydrogenase (EC 1.1.1.46), ADPglucose pyrophosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21), and phosphorylase (EC 2.4.1.1) were present in ribosome-deficient plastids from 32°C-grown leaves indicating a cytoplasmic origin of the plastid-specific forms of these enzymes. While the photosynthetic marker enzyme NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) was considerably diminished, both the specific activities and the total activities per leaf of the plastid-specific forms of hexokinase, phosphoglucose isomerase and phosphoglucomutase were markedly increased in the ribosome–deficient plastids, relative to normal chloroplasts. The results demonstrate that after elimination of functional protein synthesis in the chloroplasts the supply of chloroplast–specific enzymes by the cytoplasm is not generally suppressed as observed for many enzymes and proteins involved in photosynthesis, but may even be increased in accord with changed metabolic demands.     

Depletion of the photosystem II core complex in mature tobacco leaves infected by the flavum strain of tobacco mosaic virus

The flavum strain of Tobacco mosaic virus (TMV) differs from the wild-type (wt) virus by causing strong yellow and green mosaic in the systemically infected developing leaves, yellowing in the fully expanded leaves, and distinct malformations of chloroplasts in both types of infected tissues. Analysis of the thylakoid proteins of flavum strain-infected tobacco leaves indicated that the chlorosis in mature leaves was accompanied by depletion of the entire photosystem II (PSII) core complexes and the 33-kDa protein of the oxygen evolving complex. The only change observed in the thylakoid proteins of the corresponding wt TMV-infected leaves was a slight reduction of the alpha and beta subunits of the ATP synthase complex. The coat proteins of different yellowing strains of TMV are known to effectively accumulate inside chloroplasts, but in this work, the viral movement protein also was detected in association with the thylakoid membranes of flavum strain-infected leaves. The mRNAs of different enzymes involved in the chlorophyll biosynthesis pathway were not reduced in the mature chlorotic leaves. These results suggest that the chlorosis was not caused by reduction of pigment biosynthesis, but rather, by reduction of specific proteins of the PSII core complexes and by consequent break-down of the pigments.     

Influence of Antiviral Factor on Tobacco Mosaic Virus RNA Biosynthesis in Tobacco

An antiviral factor (AVF) was separated by removing virus particles from extracts of tobacco mosaic virus (TMV) infected leaves using calcium phosphate gel and by column chromatography on DEAE cellulose. AVF was not found in the extracts from healthy plants. The AVF restricted the virus infectivity in vivo and significantly decreased the activity of key enzymes of metabolic pathways tending to the purine and pyrimidine nucleotides biosynthesis of viral- RNA (glucose-6-phosphate dehydrogenase, ribonucleases, phosphomonoesterase and phosphodiesterase). No inhibition of these enzymes was observed in vitro when the effect of different concentrations of AVF (0.25 – 250 µg cm^–3) was examined.     

Glucose-6-Phosphate Dehydrogenase/6-Phosphogluconate Dehydrogenase Ratio and the Glucose-6-Phosphate, 6-Phosphogluconate and Fructose-6-Phosphate Contents in Tobacco Plants Infected with Potato Virus Y

The ratio of activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (G6P DH/6PG DH), and the contents of glucose-6-phosphate (G6P), 6-phosphogluconate (6PG) and fructose-6-phosphate (F6P) were studied at various stages of potato virus Y (PVY) multiplication in Nicotiana tabacum cv. Samsun. G6P DH/6PG DH increased through the experiment from 0.42 to 0.53 in leaves of healthy tobacco, and up to 0.59 in PVY systemically infected leaves. However, these ratios in the ruptured protoplast preparations, and the chloroplast and cytosol fractions of healthy protoplasts were similar to that from infected ones. The ratio lower than 1, found in the healthy and/or PVY- infected leaf tissues and in the infected protoplasts as well, confirms the assumption that G6P DH is the control enzyme of oxidative pentosephosphate pathway not only in the healthy but also in the infected plants. The contents of G6P, 6PG and F6P in the period of the highest PVY multiplication were strongly decreased (to 30 – 50 % when compared with control healthy leaves) and were negatively correlated with the G6P DH and 6PG DH activities.     

High content of endogenous cytokinins stimulates activity of enzymes and proteins involved in stress response in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

Transgenic Pssu-ipt tobacco with elevated content of endogenous cytokinins grown under in vitro conditions exhibited elevated activities of antioxidant enzymes (i.e. catalase, ascorbate peroxidase, guaiacol and syringaldazine peroxidase, glutathione reductase) and some of enzymes involved in anaplerotic pathways such as glucose-6-phosphate dehydrogenase, glycolate oxidase, NADP-malic enzyme, NADP-isocitrate dehydrogenase, and glutamate dehydrogenase compared to control non-transgenic SR1 tobacco. Higher activities of peroxidases, NADP-malic enzyme, and glutamate dehydrogenase were maintained in transgenic grafts after several weeks of the growth under ex vitro conditions, while transgenic rooted plants showed only the increase in activity of glycolate oxidase compared to control non-transformed tobacco. Total activities of superoxide dismutase were lower in both types of Pssu-ipt tobacco contrary to controls under both growth conditions. The presence of PR-1 protein and proteins with elevated activities of chitinase was proved in the extracellular fluid in both transgenic types under both in vitro and ex vitro conditions.     

Regulation of metabolic pathways PVY-RNA biosynthesis in tobacco: Host’s RNA degradation

Tobacco plants infected with the potato virus Y (PVY) were studied during the acute-infection period. The control enzymes of metabolic pathway of hosts RNA degradation tending to biosynthesis of PVY-RNA, its coarse/fine regulation and content of hosts RNA were monitored. Activities of ribonucleases, phosphomonoesterases and phosphodiesterases in both the crude homogenates and the partially purified enzyme preparations from the diseased leaves were markedly increased when compared to the tissues from healthy plants. The curves of enzyme activities positively correlated with the multiplication curve of the PVY and negatively correlated with the decreased contents of hosts RNA. The enzyme activity in homogenate samples did not significantly differ from the corresponding purified enzyme preparations.     

Photosynthesis in leaves of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. infected with tobacco mosaic virus

In tobacco leaves inoculated with tobacco mosaic virus (TMV), changes in chlorophyll (Chl) and carotenoid contents, parameters of slow Chl fluorescence kinetics, i.e. the maximum quantum yield of photosystem (PS2) photochemistry F_v/F_m, the effective quantum yield of photochemical energy conversion in PS2 Φ₂, ratio of quantum yields of photochemical and concurrent non-photochemical processes in PS2 F_v/F₀, non-photochemical quenching (NPQ), and photochemical activities of isolated chloroplasts from systemically infected tobacco leaves were investigated. We compared two successive stages of infection, the first in the stage of vein clearing at 9^th day post inoculation (dpi) and the second at 22^nd dpi when two different regions, i.e. light- (LGI) or dark-green (DGI) islands in the infected leaf were apparent and symptoms were fully developed. These two different regions were measured separately. The Chl and carotenoid contents in infected leaves decreased with a progression of infection and were lowest in LGI in the second stage. Also the ratio of Chl a/b declined in similar manner. The maximum quantum yield of PS2 photochemistry F_v/F_m, was decreased in the following order: first stage, DGI, and LGI. The same is true for the ratio F_v/F₀. The decrease of Φ₂ in infected leaves declined as compared to their controls. On the contrary, NPQ increased in infected leaves, the highest value was found in the first infection stage. Photochemical activities of the whole electron transport chain in isolated chloroplasts dramatically declined with the progression of symptoms, the lowest value was in LGI. Similarly, but to a lesser extent, the activity of PS2 in isolated chloroplasts decreased in infected leaves. Generally, the most marked impairment of the photosynthetic apparatus was manifested in the LGI of infected leaves.     

Changes in ribosephosphate isomerase and ribosephosphate pyrophosphokinase activities in tobacco infected with PVY

Changes in ribosephosphate isomerase and ribosephosphate pyrophosphokinase activities occurring in tobacco leaf tissues infected with the potato virus Y (PVY) were studied at the stage of acute infection. The results obtained have shown that during the entire experimental period the activities of both enzymes were at the end of a dark phase much higher in virus-infected tissues compared with the values found in healthy control plants. The courses of the activity curves of both enzymes were consistent and correlated with the reproduction curve of PVY. The results obtained suggest a direct involvement of both enzymes inde novo biosynthesis of the virus RNAvia the oxidative pentose phosphate pathway.