L. Šindelář

Benzothiadiazole as an Inducer of β-1,3-Glucanase and Chitinase Isozymes in Sugar Beet

The effect of benzothiadiazole (BTH) on protein synthesis was studied in sugar beet plants. Extracellular proteins induced by 0.025 % BTH were examined and their pattern was compared with that induced by sodium salicylate, chitosan, paraquat, AgNO3, and by tobacco necrosis virus. BTH induced synthesis of at least 9 acidic and 6 basic proteins; three of them appeared as acidic chitinase isozymes, three as acidic β-1,3-glucanase isozymes, three as basic chitinase isozymes, and one as a basic β-1,3-glucanase isozyme. One of the basic chitinase isozymes was found also in control plants. The most of the newly formed proteins was also induced by the other inducers under study regardless of the necrotic or symptomless reaction of plants. The benzothiadiazole proved to be an efficient inducer of proteins in sugar beet.

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 μM) 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 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: β-1,3-glucanases).

Changes in Glucose, Fructose and Saccharose Metabolism in Tobacco Plants Infected with Potato Virus Y

The content of glucose, fructose and saccharose as well as changes in the activities of enzymes involved in their biosynthesis and degradation were studied in tobacco plants infected with potato virus Y (necrotic strain) during the acute-infection period. Over the first part of this period, accumulation of saccharose, glucose and fructose was observed concurrently with decreased activities of the enzymes metabolizing saccharose, glucose and fructose (saccharases, saccharose synthase and hexokinases) and enhancement in the activities of enzymes synthesizing these carbohydrates (saccharosephosphate synthase, glucose-6-phosphate and/or fructose-6-phosphate phosphatases). The subsequent period was characterised by a reduction in both phosphatases that (together with just slightly raised saccharosephosphate synthase) could hardly produce enough sugars for the highly stimulated enzymes such as saccharases, saccharose synthase, and both kinases. Presumably for this reason, the previously increased content of sugars was considerably reduced to the level of control plants. The activities of glucokinase, fructokinase, saccharases and saccharose synthase were strongly increased at the culmination of virus multiplication and negatively correlated with the content of free glucose, fructose and saccharose.

TMV-RNA Biosynthesis in the Light-Green and Dark-Green Regions of Tobacco Leaves

Changes in the proteins, chlorophyll, virus content and activity of key enzymes of viral RNA biosynthesis were investigated in the light- and dark-green regions of tobacco leaves systemically infected with tobacco mosaic virus. The protein content was increased to 118 % in the dark-green islands in contrast to 60 % in the light-green regions when compared with the control healthy leaves. The comparative analysis of soluble proteins from healthy and light- or dark-green regions of leaves by means of SDS-PAGE revealed that the main soluble proteins are equal in pattern but differ in quantity. The contents of chlorophylls did not differ from healthy tissues in the dark-green islands but were considerably lower in the light-green regions. The content of virus in light-green tissues was about 10 times higher than in the dark-green islands. The activities of key enzymes of oxidative pentosephosphate cycle -- glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase -- did not differ from healthy tissues in the dark-green islands but were considerably higher in the light-green tissues when compared with healthy control. Similar relationships were observed for ribonuclease, phosphomonoesterase and phosphodiesterase activities. The biosynthesis of viral RNA in the dark-green islands is probably restricted by the steady (or reduced) activities of key enzymes of these metabolic pathways.

Hexokinases of tobacco leaves: influence of plant age on particulate and soluble isozyme composition

Changes in hexokinase particulate and soluble isozyme composition and activities in leaves of 65- and 115-d-old tobacco plants were determined by ion exchange chromatography on DEAE cellulose. During plant ageing, the activities of glucose and of fructose phosphorylating isozymes of particulate hexokinase decreased to 9.9 and 9.2 % of initial value, respectively. The activity of soluble hexokinase decreased to a lesser extent: that of glucose phosphorylating isozyme to 49.8 % and of fructose phosphorylating isozyme to 37.8 %. The activity of soluble fructokinase isozyme dropped to 34.8 %. Thus also the ratio of particulate and soluble isozymes was dependent on the age of leaf tissue.

Subcellular localization of ribonuclease isoenzymes in tobacco mesophyll protoplasts and their changes induced by infection of PVY

Changes in the content and subcellular localization of ribonuclease isoenzymes were determined in mesophyll protoplasts prepared fromNicotiana tabacum L. cv. Samsun from healthy and potato virus Y (PVY) infected plants. Intact chloroplasts, mitochondria and soluble cytosolic proteins were obtained after protoplast disintegration by means of differential centrifugation. The 1 000g pellet from healthy protoplasts contained 7.3 %, the 15 000g pellet 13.5 % and 15 000g supernatant 82.1 % of the total activity of ribonucleases. The 1 000g pellet from infected protoplasts contained 10.4%, the 15 000g pellet 10.0% and 15 000g supernatant 89.6 % of the total activity of ribonucleases. The activity of these enzymes in infected protoplasts was enhanced in crude homogenate to 137.0 % (P<0.001), in 1 000g pellet to 194.8 % (P<0.001), in 15 000g pellet to 101.3 % (NS), and in 15 000g supernatant to 149.4 % (P<0.001) of that in healthy noninoculated protoplasts.

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 host’s RNA degradation tending to biosynthesis of PVY-RNA, its coarse/fine regulation and content of host’s 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 host’s RNA. The enzyme activity in homogenate samples did not significantly differ from the corresponding purified enzyme preparations.

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.

Changes in Ribonuclease and Glucose-6-Phosphate Dehydrogenase Activities Induced by Beet Necrotic Yellow Vein Virus in Sugar Beet

Activities of host ribonucleases and glucose-6-phosphate dehydrogenase were studied in three cultivars (Monosvalof, Steffi and Rimini) of sugar beet differing in their resistance to beet necrotic yellow vein virus (BNYVV). No differences were found in the susceptibility of cultivars to BNYVV between mechanically inoculated and Polymyxa betae (a natural fungal vector of the virus) infected plants, but the culmination of reproduction curves of BNYVV in mechanically inoculated plants was observed one week earlier than in plants inoculated by means of P. betae. The activities of ribonucleases corresponded with virus multiplication. In roots, activities of ribonucleases reached a maximum at day 7; in leaves, maximum activity was found at day 21 in cv. Monosvalof, and at day 14 in cv. Steffi. The relatively resistant cultivar Rimini showed much lower activities. The activity of glucose-6-phosphate dehydrogenase was only slightly increased at the time of culmination of the BNYVV reproduction curve in cvs. Monosvalof and Steffi.