Decrease in ribosome levels in tobacco infected with a chlorotic strain of cucumber mosaic virus

Physiological Plant Pathology (1981) 19, 99-111

Decrease in ribosome levels in tobacco infected with a chlorotic strain of cucumber mosaic virus P . L. ROBERTst and K. R. WOOD Department of Microbiology, University of Birmingham, Birmingham B 15 2TT, U.K. (AcceptedjiJr publication April 1981)

Although infection of tobacco (cv. Xanthi nc.) with the P6 strain of cucumber mosaic virus (aMV) causes a severe chlorosis in contrast to the mild symptoms induced by the W strain, the time course and extent of accumulation of infectivity was essentially similar for both strains. Infection by CMV-P6, but not by CMV-''''', caused a mo re rapid decrease in cytoplasmic and chloroplas t ribosome con centrations than that which normally occurred in the expanding leaves of healthy tobacco plant s. The decrease was m ost pronounced in systemicall y infected leaves, which showed severe symptoms, and was first detected at about the time symptoms became evident. The rapid decrease in DNA-dependent RNA polymera se acti vity measured in isolated chloroplasts, which was found as leaves exp anded, may explain the decrease in chlo roplast ribosome concentration occu rring in th e exp anding le aves of healthy plants. Ho wever, th is enzyme seems unlikely to be involved in enh anced ribosome loss or symptom formation following infection, since infection by CMV-P6 did not induce a signifi cant ch ange in activity. D egradative activity du e to protease and rib onuclease inc reased slightly after infection with CMV-W, but did not change after infection wit h CM V- P6; increases in degradative enzymes do not, th erefore, ap pea r to be involved in symp tom expr ession.

INTRODUCTION

Infecti on of tobacco (NicoticmQ tabacum cv, Xanthi nc.) with the Price's No.6 (P6) strain of cucumber mosaic virus (CMV) results in a severe chlorosis and abnormal chloroplast development, in contrast to the very mild smptoms induced by ClvfV-l-V [23,34]. As part of an investigation into the determinants of symptom expression in this system, changes in the concentration ofleafribosomes have been studied as a possible intermediate effect leading to the development of severe chlorosis. Although ribosome decreases after virus infection have previously been reported for several viruses, there appeared to be no correlation with symptom severity [16, 20]. In contrast, in the present study, ribosome levels were only found to decrease after infection by the severe P6 strain of ONlV, but not by the mild W strain. Beca use changes in activity ofleafRNA synthesis [8, 9, 10, 16, 18,20], ribonuclease (R Nase) [5, 19,22,35] and protease [11] have been reported to occur after virus infection, the activity of these enzymes was studied in CMV-infected tissue in an effort to elucidate the mechanism of ribosome loss. Part of this work w as presented at the Fourth International Congress for Virology, The Hague, the Netherlands [24] .

t Prese nt address: Institut fUr Virolog ic und Immunbiologie del' U niversi tlit Wilrzburg , 870{) Wiirzburg, West Germany. 0048-4059/81 /040099+ 13 $02.00/0 © 1981 Academic Press Inc. (London) Limited

100

P. L. Roberts and K. R. Wood

MATERIALS AND METHODS

Virus and plants Plants were grown in loamless compost and maintained in greenhouse conditions at 23°C with supplementary lighting provided by mercury and sodium lights to give a 14 h photoperiod. The P6 [17] and W [28] strains of CMV were partially purified from tobacco (cv. Xanthi nc.) using the method of Scott [25] with one cycle of low and high speed centrifugation. For studies on symptom expression, small tobacco plants, with 2 fully expanded leaves and a small rapidly expanding leaf of c. 5 em, were infected with partially purified virus (dilution end-point c. 10 -4) in 0·05 M phosphate buffer, pH 7·5. Plants were subsequently maintained in a Fisons 2640G-E controlled environment cabinet at 23 °0 (15000 lx, 14 h photoperiod). Virus multiplication Samples of 2 disks (6 rom) per leaf were taken from 15 infected plants at various days post infection. Both inoculated and systemically infected leaves were sampled separately and the disks stored at - 20 °0 or - 70 °0 for subsequent assay. Virus was extracted by homogenization of tissue (1 +10; w+v) in 0·05 M phosphate buffer pH 7·5. Samples were assayed on cowpea (Vigna sinensis Endl. cv. Blackeye), using 10 half-leaves, and titres expressed as average lesion number per half-leaf. Ribosome assay Duplicate samples of one disk (6 mm) per leaf were taken from 15 infected plants and from the corresponding leaves of 15 mock-infected plants at various times post infection, and stored at -70 "0. Preliminary experiments had shown ribosome levels to be similar in fresh tissue samples and those briefly stored (1 to 2 h) at - 70 00. Longer periods of storage (1 week), however, caused a slight decrease in ribosomes to levels of 85±20% (95%). Extraction and purification of ribosomes on sucrose gradients were carried out essentially by the method of Randles and Ooleman [20J, which included using RNase during tissue homogenization to convert polysomes to ribosomes. Gradients were analysed at 260 nm with an ISCO absorbance monitor. The area under the A 26 0 peaks was used to estimate ribosome concentration [20]. For each time point, duplicate samples were extracted and each was run on 3 density gradients. Ribosome levels in corresponding samples of healthy and infected tissue were always determined in parallel. Chloroplast DNA-dependent RNA po0Jmcrase Samples ofleafdisks were taken from the systemically infected leaves of 10 plants and from the corresponding leaves of 10 mock-infected plants 5 to 6 days after inoculation, when severe symptoms had developed. Chloroplasts were extracted and purified as described by WoUgiehn [32]. Fresh tissue disks were ground in 0·05 M Tris-HCI, pH 7,3, containing 0·5 M sucrose, 4 mM mercaptoethanol, 10 Il1.M MgCl 2 and 10 msr KOl. The extract was filtered through a 77 J.lrn nylon mesh and centrifuged at 200 g for 5 min to remove starch and nuclear components and then at 2000 g for 10 min to pellet the chloroplasts. In some experiments chloroplasts were further purified by centrifugation (30 000 g, 1 h) on

Ribosome loss in CMV-infected tobacco

101

discontinuous sucrose gradients made by layering equal volumes of 35% (wrv) sucrose on to 60% (wjv) sucrose, both in extraction buffer. When assays were performed on crude tissue extracts, they were prepared by grinding leaf disks in assay buffer directly. Electron microscopy of chloroplast preparations revealed predominantly stripped chloroplasts and membrane fragments. However, the preservation of the integrity of the chloroplast envelope was not considered necessary for studying the polymerase as this is firmly bound to the thylakoid membranes [27, 32]. Polymerase activity was assayed by measuring the incorporation of [3H]uridine 5'-triphosphate (UTP) into TCA insoluble products [27, 32]. Assays of each extract were performed in duplicate. The reaction mixture contained 0·5 M nucleoside triphosphates (ATP, Sigma grade; GTP, type IIS; CTP, type III; Sigma), 0·5 llCi [3H]UTP (sodium salt, 2'0 Ci mmol r>, Radiochemical Centre, Amersham) and chloroplast extract in assay buffer (0'05 MTris-HOI, 4 mM mercaptoethanol, 0·01 M MgCI 2 , pH 8'0) to give a total volume of 0·25 ml, In some experiments 500 ug' phosphoenolpyruvate (monocyclohexamine salt, Sigma) and 10 J.lg pyruvate kinase (type III, Sigma) were also included. The effects of the inhibitors actinomycin D (Merck, Sharpe and Dohme Ltd.), DNase (bovine pancrease, type I, Sigma) or RNase (bovine pancrease, type IIA, Sigma) were tested in some assays. The reaction mixture was incubated at 35 °0 for 20 min before precipitation with 3 ml ice-cold 10% (w/v) TCA containing 0·01 M sodium pyrophosphate. Precipitates were collected on Whatman glass fibre filters and washed with 10% (x/v) TeA and ethanol. Radioactivity was determined in a Philip's PW4510jOI liquid scintillation analyser.

Degradative enzyme assays Samples of leaf disks were taken from inoculated and systemically infected leaves of 5 plants and from the corresponding leaves of 5 mock-infected plants at various times post infection and stored at - 20°C. Each set of plants was sampled only on one occasion to avoid possible enzyme increases induced by leaf sampling.

(a) Ribonuclease. Disks were ground (l +20 to 1+100; w-j-v) in 0·1 M sodium acetate buffer, pH 6·0. In some experiments, 0·5 M KC1 was included to increase the ionic strength and thus possibly increase the solubilization of the RNase [31]. The extract was filtered through 2 layers of gauze before centrifugation (20000 g, 15 min). Assays were performed on crude tissue extracts and on supernatant fractions and activity determined by measuring the increase in low molecular weight 260 nm absorbing material produced by enzyme action on an RNA substrate [30]. Samples of 0·5 ml were added to 0·5 ml of 0·5% (w/v) yeast RNA (B.D.H. Chemicals Ltd.) in 0'1 M acetate buffer, pH 6,0, which resulted in a final pH of 5·5. In some experiments final pHs of 4·0 or 7·0 were used. The mixture was incubated at 37°C for 30 min before termination with 0·2 ml of 0'75% (wjv) uranyl acetate in 25% (vjv) perchloric acid. The precipitate was removed by centrifugation (4000 g, 20 min) and the supernatant diluted with water (I +7) before reading the absorbance at 260 nm against a zero time blank.

P. L. Roberts and K. R. Wood

102

(b) Protease. Disks were ground (1 +15 to 1+50; w+v) in McIlvaine's buffer (0'2 M sodium phosphate adjusted to pH 7·0 with 0·1 M citric acid) diluted to onetenth. The extract was filtered through 2 layers of gauze before centrifugation (20000 g, 15 min) and assays performed on supernatant fractions. Activity was determined by measuring the increase in low molecular weight 280 nm absorbing material produced by action on a protein substrate. Samples of 0·5 rnl were added to 1 ml of 1 % (wjv) casein (low vitamin, Fisons) made up in McIlvaine's buffer, pH 7·0. In some experiments pHs of 5·5 or 8·0 were used. The mixture was incubated at 37 °0 for 30 min before termination with 0·25m! of 40% (wIv) TOA. The precipitate was removed by centrifugation (4000 g, 5 min) and the absorbance of the supernatant read at 280 nm against a zero time blank.

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103

Ribosome loss in CMV-infected tobacco RESULTS

Virus multiplication and nature of symptoms CMV-P6 caused a severe leaf chlorosis, greatest in systemically infected leaves, which was enhanced by infecting small plants and keeping them in controlled environment cabinet conditions. In contrast, CMV-W induced only a very mild leaf chlorosis. For example, a decrease in chlorophyll content of 30% in CMV-P6 and of 10% in CMV-W inoculated leaves and of 62% in CMV-P6 and of 13% in CMV-W systemically infected leaves was observed at a time when symptoms were maximal

[23J. However, in contrast to the great difference in symptom severity induced by the 2 virus strains, the time courses and levels of infectivity development were essentially similar (Fig. 1). The exact times of virus maxima and symptom appearance varied slightly between individual experiments, with a cyclic phenomenon sometimes being more pronounced. In some experiments where the smallest systemically infected leaves showed symptoms before expansion, no infectivity could be detected by the time the leaves were large enough for sampling. Ribosome levels Sucrose density gradient centrifugation provided a good separation of ribosomes from other plant material (Fig. 2) with 2 clear peaks sedimenting as would be expected for 0·8

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P. L. Roberts and K. R. Wood

104

chloroplast (70S) and cytoplasmic (80S) ribosomes [16J. However, the level of ribosomes was significantly lower in severely chlorotic leaves systemically infected with CMV-P6 (Fig. 2). To further investigate this effect, time course studies on ribosome levels were carried out. For both types of ribosome, a rapid decline occurred as the healthy leaves expanded (Figs 3 and 4).

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systernically infected leaves. The cytoplasmic ribosome level in infected (.) or mock-infected (0) and the chloroplast ribosome level in infected (.) or mock-infected (0) plants was determined. Significant differences between infected and mock-infected plants (P=O·05) are indicated (*); the time over which symptoms first appeared is also indicated (--).

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FIG. 4. Ribosome levels in CMV-W inoculated leaves (a) and the first systemically infected leaves (b}, For details of symbols see Fig. 3.

Significantly lower levels of both chloroplast and cytoplasmic ribosomes were found in inoculated and systemically infected leaves of OMV-P6 infected plants and were first detected at about the time when symptoms first appeared (Fig. 3). The effect of P6 was most pronounced in the severely chlorotic second systemically infected leaves in which a decrease in ribosome concentration and symptoms were evident at the earliest time the leaves were large enough for sampling. In contrast, infection by CMV-W had no effect on leaf ribosome levels, even at late times post infection (Fig. 4).

Chloroplast DNA-dependent RNA polymerase The optimal conditions for enzyme assay and the effect of inhibitors on activity were determined on partially purified preparations. Preliminary experiments on crude extracts or sucrose density-purified chloroplasts gave essentially similar results. Activity was optimal at 35 °0, pH 8'0, and in these conditions incorporation of [3H]UTP was linear for approximately 20 min. The presence ofphosphoenolpyruvate and/or pyruvate kinase did not increase activity and so was omitted from subsequent assays. As would be expected for a DNA-dependent RNA polymerase reaction, actinomycin D (100 llg ml r-), DNase (50 llg ml- l ) or RNase (50 ug ml-1) were inhibitory, with the activity of crude extracts being reduced to to, 9 and 2%respectively of that found in controls. Although fresh tissue disks were routinely used, in some later experiments it was found that the activity in crude extracts of tissue frozen at - 70°C was 450% of that of fresh tissue. There was a rapid decrease in polymerase activity as the leaf expanded. For instance, in one representative

P. L. Roberts and K. R. Wood

106

experiment, using crude tissue extracts, the activity in leaves of 7, 11 and 14 em was 44, 27 and 21 % respectively of that in leaves of 4 ern. In preliminary exp eriments, using gradien t-purified chloroplasts, the enz yme activity in CMV-P6 systemically infected leaves was found to be 240 % of that in control m ock-inoculated plants. However, because the control activity in these prepara tions was only c. 10% of that routinely found in partially purified chloroplast preparations, th e significance of this large increase is not clear. The chloroplasts from infected leaves ap peared to be selectively isolated, as the original chlorophyll level of the infected leaf extract was 52% of the control which increased to 81 % in the final gradient purified chloroplast preparation. Ho wever, this effect could only partially explain the large increases in polymerase found after infection. Due to concern over the possible introduction of artifacts by the multi-step procedure required to produce gradient-purified chloroplasts, the effects of CMV-P6 infection were studied on partially purified chloroplasts and crude tissue extracts. Partially purified chloroplast preparations of P6 infected tissue had an activity level of 131 % of that of the controls (Table 1). The possibility that this increase TABLE

I

Chloroplast D NA-dependent RNA polYmeraseactiuiv in CMV-P6 sJstemical{Y infected leaves' Act ivity"

Preparation Partially purified chloroplasts Crude tissue extract

Control

Infec ted

131±IS" l1S±16

• Comparisons were carried out on leaves 5 to 6 da ys post inoculation, when symptoms were maximal ; the results are mean values from several experiments. b Activities are expressed as a percentage of that in mock-infected controls with (±) stan dard error. D ifferences significant at P=0'05 are indicated (" ). c. d 100% activity is 3200 or 1400 resp ectively d min " ! [sH)UTP incorp orated pe r 100 rng leaf tissue in 20 min at 35 °a.

represents the measurement of significant levels of viral replicase (RNA-dependent RNA polymerase) was tested by including inhibitors of host DNA-dependent RNA polymerase in the assay. The results, averaged from several experiments, showed activity to be reduced by actinomycin D to 11% and 10% and by DNase to 8% and 7% of the activity in the control and infected extracts respectively. This would suggest that the increase found after infection was not viral replicase. However, the small increase induced by P6 infection was only just statistically significant and could largely be explained by the selective isolation of the infected leaf chloroplasts as the average chlorophyll level of the infected leaves was 50% of the control, which increased to 58% in the partially purified preparations. Furthermore, studies using crude tissue extracts showed no significant change in polymerase activity after P6 infection (Table 1).

Degradatiue enzymes (a) Ribonuclease. Preliminary experiments revealed that at least 80% of the total ribonuclease activity was located in the soluble supernatant fraction obtained after

Ribosome loss in CMV-infected tobacco

107

centrifugation at 20 000 g. The low activity remaining in the pellet fraction was more variable and could not be released by more severe grinding of the tissue, a higher salt concentration in the extraction buffer or by reducing the centrifugation speed. Studies on the ribonuclease activity were carried out using crude tissue extracts and soluble supernatant fractions. The activity was optimal at pH 5·5. A temperature of 37 °0 was used for assay, although the enzyme was extremely heat stable and activity increased up to at least 60 °e. Mock inoculation of plants caused an increase in ribonuclease activity in abraded leaves only, with activity reaching 260 and 150% of that found in untreated leaves after 1 and 5 days respectively. Leaf size had little effect on activity; the results from one experiment showed activity in leaves of 8, 10 and 12 ern to be 98, 92 and 84% respectively of that in leaves of 5 em. Infection with CMV-P6 had no effect on leaf ribonuclease activity (Table 2). Infection with OMV-W did increase activity in inoculated leaves, although the small increase also found in older (9 day) systemically infected leaves was only just statistically significant. Due to the possible existence of different isozyrnes operative at different pHs, experiments using pHs of 4·0 and 7·0 were carried out; these gave essentially similar results. For instance, at pH 4·0 the activity in P6 systemically infected tissue was 96% and at pH 7·0 the activity in both P6 and W systemically infected tissue was 89% of that in control leaves. TABLE

2

Ribonuclease activiry in leaves irifectetl with P6 or W'

Leaf Inoculated Inoculated Systemically infected Systemically infected

Day 4 7 5 9

Activity" in crude extracts Control P6 100< 100' 100' 1001

105±4 l1l±6 103±4 95±3

W 133±4'" 126±6* 113±6 106±2*

Activity" in supernatant fractions Control P6 W lOad lOaf 100h 100)

117±7 l24±8 95±8 108±6

l36±6'" l36±9 85±9 108±3·

Comparisons were carried out on leaves, when symptoms were present, at various days post Inoculation and the results from several experiments averaged. b Activities are expressed as a percentage of mock-infected controls with (±) standard error. Differences significant at P=O·05 are indicated (oil). c. d••• r• •. h, I, J 100% activity is 272, 216, 296, 200, 280, 336, 352, 336, respectively, units released per g leaf tissue in 30 min at 37 0 0 . One unit is defined as that which in 1 ml has an A2 80 of 1 in a I em light path. a

(b) Protease. Preliminary experiments revealed that at least 75% of the total protease activity was located in the soluble supernatant fraction obtained after centrifugation at 20 000 g. Studies on protease activity were therefore conducted on supernatant fractions. Enzyme activity was optimal at pH 5·5 to 6·5; activity at lower pHs could not be measured due to precipitation of the substrate, and at higher pHs the activity gradually decreased. Assays were carried out at pH 5·5 and 7·0 because of the possible existence of different isozymes operative at different pHs. A temperature of 37 °0 was used, at which activity was nearly maximal, although activity increased slightly beyond this temperature. The activity in leaves 8 h after mock inoculation had increased to 130% of that in the controls, as measured at pH 5·5 and 7·0. At

P. L. Roberts and K, R. Wood

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later times no significant increases were found. Enzyme activity decreased rapidly as the leaf expanded. Results from one experiment in which leaves expanded from 5 cm to 12 em showed that the activity decreased to 26% or 30% when assayed at pH 5·5 or 7·0 respectively. Infection with CMV-P6 had no effect on leaf protease activity in inoculated or systemically infected leaves when assayed at either pH 5·5 or 7·0 (Table 3). Infection with CMV- W did significantly increase leaf protease activity in inoculated leaves, but not in systemically infected leaves (Table 3). TABLE

3

Protease activiry in leave.infected with P6 or we Leaf

Day

Inoculated

5

Systemically infected

5

Systemically infected

8

pRof assay

5·5 7·0 5·5 7'0 5·5 7·0

Activity" Control

P6

W

lOoa 100· lOoe 100"

98±5 94±7 101±4 93±8 88±5 90±3

132±4" 140±4" I03±3 107±7 116±6 124±6

lOOg

ioo«

• Comparisons were carried out 5 days post inoculation when symptoms were present. Supernatant fractions were used for assay. b Activities are expressed as a percentage of mock-infected controls with (±) standard error. Differences significant at P=0'05 are indicated ("). a, d, e. r, " h 100% activity is 14, 10,42,28,40, 26, respectively, units released per g leaf tissue in 30 min at 37 ea. One unit is defined as that which in I m1 has an A sso of 1 in a 1 em light path.

DISCUSSION

Although CMV-P6 and CMV-W differed markedly in the severity of chlorosis they induced, the time course and extent of accumulation of infectivity were essentially similar. There was no relationship, therefore, between virus replication and symptom expression. Infection by CMV-P6, but not by CMV-W, caused an accelerated decrease in extractable chloroplast and cytoplasmic ribosomes compared with that normally occurring in healthy expanding leaves. Decreases in ribosomes on leaf ageing have also been reported by others in plants [2, 4, 16, 20] or plant cell cultures [3J. The accelerated decrease induced by CMV-P6 was most pronounced in systemically infected leaves showing severe symptoms, and was first detected at about the time symptoms became evident. In N. glutinosa infected with lettuce necrotic yellows virus (LNYV), chloroplast ribosomes decreased 1 day after symptoms had appeared with cytoplasmic ribosomes decreasing later [20]. In tobacco infected with tomato spotted wilt virus (TSWV) or tobacco mosaic virus (TMV) only the chloroplast ribosomes were found to decrease [16]. In contrast to the results reported here for CMV, the decrease in ribosomes induced by LNYV and TSWV occurred with both mild and severe strains. In some situations, however, increases in ribosome levels have been found after virus infection [15, 29]. It has been suggested that increased levels of cytoplasmic rRNA and ribosomes are needed for TMV replication, though only in

Ribosome loss in CMV-infected tobacco

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older leaves where leaf ribosome levels are low [9]. In the present study with CMV, increased ribosome levels were never observed and do not appear to be necessary for replication, since the decrease in ribosomes detected after P6 infection did not appear to decrease virus accumulation. The decrease in ribosome levels may, however, be important in symptom expression, although since the changes were first observed at the time of appearance of visible symptoms, it is not possible to conclude whether or not the 2 were causally related. It may be that a study of polysomes or rates of ribosome formation following infection [10, 16,21] would be helpful in this respect. In seeking a cause for the CMV induced decrease in ribosome levels, the activity of DNA-dependent RNA polymerase was studied, since a decreased activity of this enzyme could lead to reduced RNA synthesis and hence to reduced ribosome synthesis. The enzyme studied in partially purified chloroplast preparations and crude tissue extracts is believed to be principally that of the chloroplasts, rather than that of the nucleus, as other workers using similar techniques have shown this to be the most significant activity [6, 13, 26, 27, 32]. The conditions needed for optimal enzyme activity and, in preliminary experiments, its insensitivity to a-amanitin (10 pg ml r-) are consistent with this suggestion [6]. The low activity of the nuclear enzyme is thought to be due in part to the association of DNA with histones and also because it is not expressed unless the nuclei are broken [27]. The large increase in polymerase activity found in the present study when frozen leaf tissue was used instead of fresh tissue may be due to the release of the nuclear enzyme. If this is indeed the case, it would seem that under certain conditions the activity of the nuclear enzyme can become very significant. During leaf expansion, activity decreased substantially, a situation similar to that reported by others [33J, so that it is possible that this may lead to the decrease in ribosome levels found on leaf expansion. Decreases in leaf chloroplast rRNA synthesis [8, 9, 10, 16, 18, 20J and, under certain conditions, also that of the cytoplasm [9], have been reported for several viruses, while the DNA-dependent RNA polymerase activity of chloroplasts decreased after TMV infection of tobacco [10]. In the present study, decreases in chloroplast polymerase activity were not found after infection with CMV-P6; the small increase found in some cases after P6 infection could probably be explained by preferential isolation of infected cell chloroplasts. Changes in the activity of this enzyme therefore do not appear to explain symptom severity and ribosome loss, though it cannot be ruled out that the activity measured in vitro does not accurately represent the activity in vivo; factors involved in regulating polymerase activity [6J might be lost during chloroplast isolation. Also, the activity of the nuclear polymerase deserves investigation as the cytoplasmic ribosomes, in addition to those of the chloroplast, decreased after CMV-P6 infection. The activity of the degradative enzymes, RNase and protease, were also investigated in view of the possibility that increases in these could lead to ribosome degradation and/or symptom expression. The properties of the tobacco RNase preparation were essentially similar to those described by others, although the various RNases reported for tobacco vary with respect to their pH optima and heat stability [30]. Damage of tobacco leaves by mock inoculation caused a large increase in RNase

110

P. L. Roberts and K. R. Wood

activity, an observation similar to that reported by others [1, 5, 22, 35]. In some cases, specific isozymes have been reported to increase [19, 35]. RNase activity changed little on leaf expansion, though others have reported large increases [22] or decreases [5, 19]. Infection by CMV caused a small increase in RNase activity, greatest in inoculated leaves, but only with the mild W strain. Others have found activity to increase up to 500% of that in control leaves after virus infection [5, 19, 22, 35J, though in turnip yellow mosaic virus-infected Chinese cabbage, no differences were found in the levels of RNase incluced by strains which differed in the severity of chlorosis they induced [19]. In the present study with CMV, several pH values were used during RNase assay because of the possible induction of specific isozymes [19, 35]. However, all gave essentially similar results. It would thus appear that changes in RNase activity do not appear to be involved in CMV symptom expression and ribosome loss. In contrast to RNase, there have been fewer studies on leaf protease activity [12]. Here, damage of leaves by mock inoculation caused only a small increase in activity, though activity decreased rapidly on leaf expansion. Infection by CMV-W, but not by CMV-P6, induced a small increase in activity, similar to the slight increase previously reported following infection by a yellow strain of CMV [11]. Due to the possible existence of isozymes [12], pHs of 5·5 or 7·0 were employed in the assay, but both gave essentially similar results. It thus seems that neither gross changes in protease or RNase activity are involved in C:MV symptom expression and ribosome loss; however it cannot be ruled out that changes in minor isozymes or enzyme activities in specific subcellular locations are important. There is, therefore, a symptom related reduction in both cytoplasmic and chloroplast ribosomes (both of which are involved in synthesis of chloroplast proteins [7, 14J) following infection of tobacco by CMV. In addition, development of severe chlorosis induced by C1.1V-P6 is accompanied by inhibition of the biosynthesis of several host polypeptides, possible components of the chloroplast, and an inhibition of chloroplast development [23]. However, the loss of ribosomes induced by CMV could not be simply explained by decreases in DNA-dependent RNA polymerase activity or increases in degradative enzymes, sothat other expl anations need to be sought. The authors are grateful to the Royal Society for the provision of controlled environment cabinets, to the Science Research Council for a studentship (to P.L.R.) and to Karin Roberts for help in preparing the manuscript. REFERENCES I. BAGl, G. & FARKAS, G. L. (1967). On the nature of'increase in ribonuclease activity in mechanically

2. 3. 4. 5. 6.

damaged tobacco leaf tissues. Phytochemistry 6, 161-169. J. A., CALLOW, M. E. & WOOLHOUSE, H. W. (1972). In vitro protein synthesis, RNA synthesis and po1yribosomes in senescing leaves of Perilla. Cell Differentiation 1, 79-89. CELLA, R., SALA, F. & STREET, H. E. (1976). Studies on the growth in culture of plant cells. XIX. Changes in the levels of free and membrane-bound polysomes during the growth of Acer Pseudoplatanus cells in batch suspension culture. Joumal if Experimental Botal!Y 27, 263-276. CLARK, M. F., MA'I'TI!EWS, R. E. F. & RALPH, R. K. (1964). Ribosomes and po1yribosomes in Btassiea PekinellSis. Blochlmica et Biophysica Acta 91, 289-304. DIENER, T. O. (1961). Virus infection and other factors affecting ribonuclease activity of plant leaves. Virology 14, 177-189. DUDA, C. T. (1976). PlantRNApolymerases. AllnualReviewifPlantPhysiology27, 119-132. CALLOW,

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