RNA-synthesizing enzymes in healthy and TMV-infected tobacco leaves

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

RNA-Synthesizing

164, 218-223 (1974)

Enzymes

in Healthy

Tobacco Separation

and Properties

Leaves

of Enzymes

Catalyzing

Polymerization

STURE BRISHAMMAR Department

of Plant Pathology

and Entomology,

and TMV-infected

l AND

Agricultural

Received November

Nucleotide

NILS JUNTTI

College of Sweden, S-750 07 Uppsala 7, Sweden 27, 1973

A separation procedure, preparation of the extract and a gel filtration on 8% agarose, has been developed for the separation of enzymes catalyzing nucleotide polymerization in healthy and TMV-infected tobacco leaves. The profiles of the gel filtrations were almost the same from infected and uninfected materials. A number of nucleotide polymerizing and nucleolytic activities were revealed. A DNA-directed RNA polymerase was demonstrated as well as three homopolymerase activities. Incorporations of ATP and CTP, requiring a primer (preferentially tRNA), were found in the same fractions and were probably caused by the same enzyme. An incorporation of GTP using poly(G) as primer likewise was detected. These polymerizations were inhibited in the presence of 10 mM pyrophosphate while 100 mM orthophosphate was not inhibitory. Furthermore, two peaks of nuclease activities have been found. The slow-eluting peak indicates an enzyme (enzymes) of approximately the same size as pancreatic ribonuclease (M, = 13,680). The fast-eluting peak corresponds to molecular weights of the order of 150.000.

The synthesis of viral nucleic acid in paper will treat the enzyme polynucleotide tobacco plants infected with TMV (to- phosphorylase. bacco mosaic virus) has frequently been MATERIALS AND METHODS the center of a manifest interest (1-3). This Plant materials and uirus used. Preparations were is a consequence of the outstanding position of TMV as a model virus. The bio- made from healthy and systemically TMV-infected chemical mechanisms within the tobacco leaves of Nicotianu tabacum L. var. Samsun. The cell, however, are largely unknown and, virus strain Flauum was used at the inoculation and therefore, it has become obvious that it is the sap inoculum was prepared from frozen TMVinfected leaves. The plants were grown in soil in lo-cm necessary to make a survey of the normal pots and were cultivated in a greenhouse. The light RNA-synthesizing enzymes (and the ribo- period was 16 hr, with additional illumination from nucleases) of the cell. This report describes fluorescent lamps (light intensity about 6000 lux) a separation procedure elaborated for the during the dark seasons. At daytime the temperature purpose of revealing the existence of en- was between 22 and 26°C and at night 16-20°C. The zymes which catalyze nucleotide polymeri- greenhouse kept a relative humidity of about 50%. zation. The paper also will describe some The first leaf having a pointed tip (usually number 7 properties of these enzymes. The procedure including the cotyledons) and the leaf next below were has been applied to both healthy and inoculated when the plants had 11-13 visible leaves. TMV-infected material. A subsequent The inoculated leaves were carefully rinsed with tap 1This is paper I in a series. Paper II is the next particle in this issue (Arch. Biochem. Biophys. 164, 224-232, 1974).

water. Under these conditions leaves having a length of 20-45 mm at the time of inoculation, usually number 9-11, showed vein clearing on the major part of the lamina after 4-5 days. This leaf of the infected

218 Copyright All rights

Q 1974 by Academic Press, of reproduction in any furm

Inc. reserved

TOBACCO

NUCLEOTIDE

POLYMERIZING

plant or the corresponding leaf of the healthy plant was used at the preparations. The time for harvest was standardized to 1 day after vein clearing for the systemically infected leaf and to the analogous time for the healthy leaf. Chemicals. Unlabeled riboside triphosphates (sodium salts: ATP, Sigma grade; CTP, type IV; GTP, type II-S; UTP, type IV), yeast RNA (type XI), yeast tRNA (type I), salmon DNA (type III), poly(C), poly(G), phosphoenolpyruvate (trisodium salt), hemoglobin (bovine, type I), and Cordycepin (grade III) were obtained from Sigma Chemical., St. Louis, MO. Pyruvate kinase (rabbit muscle, EC 2.7.1.40), poly(A), and poly(U), were purchased from Boehringer and Soehne GmbH, Mannheim, West Germany. Actinomycin D was a gift from Merck, Sharp, and Dohme, Rahway, NJ. Polyamines were supplied by Fluka AG, Switzerland. The labeled nucleotides were purchased from the Radiochemical Centre, Amersham, England. RNA preparations. Tobacco mosaic and clover yellow vein virus RNAs were prepared by conventional phenol extraction and alcohol precipitation. Escherichia coli (E. coli) RNA was a gift of Dr. B. Gberg, Uppsala. Buffers. Homogenization buffer (H buffer), adjusted to pH 8.4 (at room temperature) with HCI consisted of: 100 mM Tris-HCl, 100 mM NH,Cl, 50 mM mercaptoethanol, 5 mM MgCl,, and 10 mM EDTA. Elution buffer (EB) contained besides Tris-HCl: 106 rnM NH&l, 50 mM mercaptoethanol, 5 mM MgCI,, and 1 mM EDTA. The pH was varied from 7.6 to 8.4 (at room temperature) and the molarity of Tris-HCl from 10 to 50 mM. Separation

of the Enzymes

a. Preparation of enzyme extracts. All operations were performed at 4°C. The homogenates were prepared by grinding healthy or infected leaves, after removing their mid veins, in a prechilled mortar with purified sea sand. For each gram fresh weight of tissue 1.5 ml of H buffer was added before grinding. The homogenates were strained through a double layer of cheesecloth and centrifuged for 10 min at 10,OOOgin the refrigerated (4°C) Sorvall RCS-B centrifuge. The supernatant was gel filtrated on a Sephadex G-50 column (3.2 x 90 cm) to get rid of low molecularweight substances. The void volume material was centrifuged at 100,OOOgfor 1 hr with the Spinco L2-50 preparative ultracentrifuge. The discarded precipitate contained high molecular-weight green material. The supematant was used for the subsequent gel filtration on 8% agarose (after a concentration of the solution). b. Concentration of solutions. To obtain concentrations of about 20-30 times, a special concentration equipment had to be used. Nitrogen gas with a pressure of 5 kg/cm* was conveyed through an Amicon

ENZYMES

Ultrafiltration cell, Model 52, (Amicon Ltd, Massachusetts), with a volume of 65 ml. This cell was without any ultrafiltration membrane. The solution was carried from the cell through a narrow tubing to an Amicon Column Eluate Concentrator, CEC 1, with a membrane (PM-lo) of 10,000 M, retention limit. c. Gel filtration on agarose. Spherical agarose gel (8%), Bio-Gel A-l.5 m, 200-400 mesh (Bio-Rad Laboratories, Richmond, CA), was packed into 3.2 x 160~cm chromatography column (4). The total volume was 1250 ml and the void volume 380 ml. The sample was introduced into the bed by means of a peristaltic pump. The procedure was carried out in a cold room (4°C). Protein determination. Calculations were based on spectrophotometric readings at 280 nm in a Zeiss M4Q III spectrophotometer. The calculations were performed under the assumptions that Beer’s law obtains for the various solutions giving optical density values OD:Fm = 10 (1% solution, l-cm light path). Assays of nucleotide polymerization activities. Enzyme assays were based on the rate of incorporation of [3H]ribonucleoside 5’-triphosphates into acid-insoluble material. The same technique was used in all cases, but the assay mixture differed with respect to enzyme, nucleoside triphosphates, and primers. The basic standard assay medium contained in a final volume of 0.250 ml: 50 mM Tris-HCl, pH 8.2, 5 mM MgSO,, 40 mM NH&l, 2 mM mercaptoethanol, 0.4 mM EDTA, 15 pug pyruvate kinase, and 5 mM phosphoenolpyruvate. Incubations were carried out at 30°C for 20 min, and terminated by placing the reaction mixture in an ice bath and by addition of 3 ml of 10% trichloroacetic acid containing 0.9% sodium pyrophosphate. The precipitate was filtered onto a glass fiber disk (Whatman GF/C, 2.5~cm diam) which was washed with 5 x 5 ml of the same medium and afterward with 5 x 5 ml of ethanol. The filters were dried and counted in 5 ml of toluene scintillator in a Packard Liquid scintillation spectrometer. The ethanol was included in the wash, since it appeared that this treatment removed any remaining colored substances of the plant and lowered the background level to about 35 cpm. This background value was always subtracted. Assa.y mixture of ribohomopolymer synthesis. To the basic standard assay medium was added: enzyme fraction, 1.0 &i [sH]nucleoside triphosphate (sp act about 25 Ci/mmole), 50 pg of a primer, and 5 pg actinomycin D. Poly(A) synthesis was assayed with [$H]ATP and yeast tRNA. Poly(C) synthesis was assayed with [3H]CTP and yeast tRNA. Poly(G) synthesis was assayed with [3H]GTP and poly(G). mixture of DNA-dependent RNA Assay polymerization. To the basic standard assay medium was added: enzyme fraction, 1.0 PCi [3H]UTP (sp act 15 Ci/mmole), 0.12 mM of each of the three other (unlabeled) nucleoside triphosphates, and 25 pg of

220

BRISHAMMAR

AND JUN’ITI

salmon DNA as a template. The pH was in this case the investigations. The gel filtrations with 8.0. healthy and infected materials revealed a Ribonuclease assay. The enzyme activity of plant number of nucleotide polymerizing and ribonucleases was determined according to Reddi (5). nucleolytic activities. Thus, DNA-directed The cleavage of RNA was followed by the formation of mono- and oligonucleotides, which were separated RNA synthesis and three types of ribohofrom the partially digested RNA fragments by acid mopolymerizations were detected. In addiprecipitation. The concentration of acid-soluble nu- tion two peaks of nuclease activity could be cleotides was determined spectrophotometrically at demonstrated. 260 nm. One unit of ribonuclease activity was the amount of ribonuclease needed to cause an increase in The Ribohomopolymerization Activities optical density of 0.005. (It was important to use a Incorporation of ATP was recorded as a very pure grade of RNA.) RESULTS

This elaborated method for separation of fresh materials from healthy and systemically TMV-infected tobacco leaves comprised two steps: the preparation of the extract (including the succeeding concentration of the sample solution) and a gel filtration on 8% agarose gel. The profiles of the protein curves at the gel filtrations of healthy (Fig. 1) and infected (Fig. 3) materials looked roughly like each other. The second sharp peak was in general higher with healthy material while the third peak grew bigger after the infection. These separations were very reproducible throughout

sharp peak in the second part of the gel filtration curve (Fig. 1). This activity was probably due to the presence of a homopolymerase, as the activity did not drop by the omission of the three unlabeled nucleoside triphosphates. The values were always lower when the other three triphosphates were present. In the same way an incorporation of CTR could also be registered (Fig. 1). These activities were apparently not caused by the enzyme polynucleotide phosphorylase since no inhibition was obtained when 100 mM orthophosphate was added to the assay mixture. In the presence of 10 mM pyrophosphate no activity was detected. The enzyme fractions were assayed with

FRACTION NUMBER

FIG. 1. Gel filtration on 8% agarose gel of material from healthy leaves. Eluting buffer: 10 rnM EB, pH 7.6. Flow rate: 39 ml/hr. Fraction volume: 13 ml. Sample: 4 ml. Optical density at 280 nm (0-O). The activities of poly(A) synthesis (O-O) and poly(C) synthesis (A-A) are expressed in cpm incorporated. The activity of ribonuclease (A-A) is expressed in RNase units/ml.

TOBACCO NUCLEOTIDE

POLYMERIZING

TABLE I

cordycepin, an inhibitor of poly(A) polymerase, in the assay medium. This drug was without inhibitory effect at concentrations up to 0.4 mg/ml. Preliminarily, the experiments have shown that the synthesis of poly(A) and poly(C) were of the same magnitude after the TMV-infection. Influence

of incubation

EFFECT OF PRIMERS ON RIBOHOMOPOLYMER SYNTHESIZINGACTIVITY”

Enzyme activity expressed in cpm incorporated

Primer added (50 &assay)

temperature.

The poly(A) synthesis showed a varying pattern at different temperatures (Fig. 2). It appeared that all slopes of the curves for the temperatures 25“C-45 “C were rather steep within the first 20 min, but after 45 min the highest rate of incorporation was obtained at 20°C. A slight decrease of the rate could be noticed after 90 min with all examined temperatures. Effect of pH and divalent ions. The poly(A) and poly( C) polymerizations proceeded maximally at pH 9.0-9.2 and at 0.5-5 mM MgSO, . Increasing Mg2+ concentration above that level decreased gradually the activities; however, complete inhibition of the incorporations was not observed up to 20 mM Mg2+ in the assay medium. No activity was obtained in the absence of Mg2+ or when Mg2+ was replaced by Mn2+. Dependence of primer. The incorporations of ATP and CTP were dependent on the addition of a primer and showed a preference for yeast tRNA (Table I). The other natural primers used, stimulated some activity while the synthetic riboho-

221

ENZYMES

None TMV RNA CYVV RNA Yeast RNA Yeast tRNA E. coli RNA Poly(A) POlY(C) Poly(G) POlY

WI

[3H]ATP

[3H]CTP

160 360 1,900 2,200 24,000 490 250 200 350 220

120 1,300 2,000 2,100 25,000 400 230 500 280 190

“The assays were performed under the standard assay conditions for ribohomopolymer synthesis as described in Materials and Methods. (TMV = tobacco mosaic virus; CYVV = clover yellow vein virus.)

mopolymers caused very low levels of incorporation. These studies indicated that a single enzyme catalyzed the incorporation of ATP and CTP and the optimal assay conditions seemed to coincide. Incorporation of GTP has also been detected. This reaction required Mg2+ and poly(G) as a primer. Mostly the activity was found in the fractions with incorporations of ATP and CTP but could sometimes appear in

25'

0 0

I 10

I 20

I 30

I 40

f 50

I 60

I 70

I 60

I 90

min

Fro 2. The influence of the incubation temperature on the incorporation of [3H]ATP. The assay was performed as described in Materials and Methods.

222

BRISHAMMAR

the end of the curve instead (Fig. 3), and then the peak of activity ended abruptly. DNA-directed RNA synthesis. Incorporation of [3H]UTP was obtained in the presence of the three other unlabeled nucleoside triphosphates when DNA was added as a template. The activity could be recorded in the early part of the gel filtration curve (Fig. 3). Since this activity was missing without DNA, or with DNA in the presence of actinomycin D, it appeared to be likely that the reaction was caused by the DNA-dependent RNA polymerase, or transcriptase, (EC 2.7.7.6) of the tobacco plant cell. The incorporations were rather low but the variables of the assay have not, been studied so the reaction may have proceeded far from optimal conditions. From the position in the gel filtration curve the enzyme(s) may have approximately the same size as the transcriptase found in Escherichia Detection

coli (M, 360,000-450,000). of plant ribonuclease. The es-

tablishment of ribonuclease activities at the purification was considered necessary, since nucleases probably may counteract nucleotide polymerization. The agarose gel filtration (Fig. 1) revealed two separate peaks of ribonuclease activity. The slow-

FRACTION

AND

JUNTTI

eluted one was obtained in the very end of the elution curve. This position indicated that the corresponding enzyme was of the same size as pancreatic ribonuclease. The fast-eluted activity, on the other hand, appeared unexpectedly early within the curve. This peak of ribonuclease activity coincided with the sharp peak of the protein curve (Fig. 1). In exactly the same zone was also the enzyme polynucleotide phosphorylase detected, as is described in a subsequent paper (6). For this reason the nuclease activity was initially thought to be a result of the phosphorolytic cleavage of RNA by the polynucleotide phosphorylase; i.e., the formation of nucleoside diphosphates. In that case this reaction should have been stimulated by the addition of 0.1 M orthophosphate and 5 mM MgSO, together with a raising of the pH to 8.2 (7). Since no increase of the RNAdecomposing activity was obtained under these conditions, the enzyme must have been one of the tobacco leaf ribonucleases reported (8). The two peaks of nuclease activity have not been further studied with respect to substrate specificity and pH optimum. However, the fast-eluted RNase obviously did not degrade poly(A) when

NUMBER

FIG. 3. Gel filtration on 8% agarose gel of material from infected leaves. Eluting buffer: 10 mM EB, pH 7.6. Flow rate: 39 ml/hr. Fraction volume: 13 ml. Sample: 4 ml. Optical density at 280 nm (0-O). The activity of the DNA-dependent RNA polymerase (A-A). The first peak of poly(G) synthesis (I) (O--O). The second peak of poly(G) synthesis (II) (x-x). The activities are expressed in cpm incorporated.

TOBACCO NUCLEOTIDE

POLYMERIZING

this polymer was used as primer in the assay of the coexisting polynucleotide phosphorylase (6). The unexpected fall of the poly(G) synthesis in the last part of the slow-eluted peak of activity (Fig. 3) was probably the result of the degrading action by the small ribonuclease(s) found in the end of the elution curve (Fig. 1). These investigations did not reveal if the levels of nuclease activity change upon virus infection or how many enzymes the two peaks represent. DISCUSSION

Many signs indicate that the incorporation of ATP and CTP are caused by one enzyme which shows characteristics of ATP(CTP): tRNA nucleotidyltransferase (EC 2.7.7.20) (9). Thus, the reaction was primed with tRNA and the pH optimum was at pH 9.0-9.2. A true poly(A) polymerase ought to have used poly(U) as a template or poly(A) as a primer and become inhibited by cordycepin (lo), which was not the case. On the other hand, a poly(A) polymerase of plant cells may be different from those in animals and bacteria. Multiple forms of DNA-dependent RNA polymerase have been demonstrated in cells of higher plants (11). In the nuclei of eukaryotic cells there exist more than one type of this enzyme (12). Transcriptase has also been found in tobacco chloroplasts associated with the chloroplast membrane (13). The double-peak of activity which appears in the figure (Fig. 3) may indicate that the detected transcriptase activity originated from many forms of this enzyme which are derived from different cell organelles. Some of these forms may require tobacco DNA, or DNA from other plants, and manganese instead of magnesium. The nuclease activities have been regarded to be the main hindrance in the detection of RNA polymerases in tobacco leaves (1). Therefore, it is very important to find out their chromatographic positions in relation to polymerases. It is also urgent to possess this knowledge before a further purification of the polymerases can be achieved. The literature does not disclose any de-

ENZYMES

223

terminations of molecular weights of the tobacco leaf ribonucleases. The slow-eluted peak of activity in these experiments reflects the anticipated relatively small size of most ribonucleases known. The fasteluted peak, on the other hand, corresponds to an unexpectedly large size of molecules. This is nevertheless in accord with the report (14) on an easily precipitated fraction with ribonuclease activity at ammonium sulfate precipitation of tobacco leaf extract which also indicates a large molecule. ACKNOWLEDGMENTS The authors are indebted to Professor H. Zech and Dr. P. Oxelfelt for valuable advice, to Professor K. Bjijrling for his support, and to Mr. R. Marciniak for skilful technical assistance. Financial support for this investigation was obtained from the Swedish Natural Science Research Council and The Swedish Council for Forestry and Agricultural Research. REFERENCES 1. KARASEK, M., AND SCHRAMM, G. (1962) Biochem. Biophys. Res. Commun. 9, 63-68. 2. REDDI, K. K. (1969) Biochembtv 62, 604-611. 3. BRADLEY, D. W., AND ZAITLIN, M. (1971) Virology 45, 192-199. 4. PORATH, J., AND BENNICH, H. (1962) Arch. Biothem. Biophys. Suppl. 1, 152-156. 5. REDDI, K. K. (1967) in Procedures in Nucleic Acid

Res. (Cantoni, G. L., and Davies, D. R., eds.), Vol. 1, pp. 71-78, Harper and Row. New York. 6. BRISHAMMAR, S., AND JUNITI, N. (1974) Arch. Biochem. Biophys.

164,224-232.

7. SINGER, M. F. (1967) in Procedures in Nucleic Acid Res. (Cantoni, G. L., and Davies, D. R., eds.), Vol., pp. 245-262, Harper and Row, New York. 8. FRISCH-NIGGEMEYER, W., AND REDDI, K. K. (1957) Biochim. Biophys. Acta 26, 40-46. 9. LI’ITAUER, U. Z., AND DANIEL, V. (1967) in Proce-

dures in Nucleic Acid Res. (Cantoni, G. L., and Davies, D. R., eds.), Vol. 1, pp. 353-361. Harper and Row, New York. 10. DARNELL, J. E., PHILIPSON, L., WALL, R., AND ADESNIK, M. (1971) Science 174, 507-510. II. POLYA, G. M., AND JAGENDORF,A. T. (1971) Arch. Biochem. Biophys. 146,635-648. 12. ROEDER, R. G., AND RULER, W. J. (1970) Proc. Nat. Acad. Sci. USA 65, 675-682. 13. TEWARI, K. K., AND WILDMAN, S. G. (1967) Proc. Nat. Acad. Sci. USA 58, 689-696. 14. PIRIE, N. W. (1968) Rothamsted Report, Part I, 114-115.