Incorporation of C14-amino acids into various cell fractions of tobacco leaves infected with tobacco mosaic virus

VIROLOGY

21,

226-231 (1963)

Incorporation Tobacco

of C14-Amino Acids into Various Cell Fractions Leaves Infected with Tobacco Mosaic Virus’ YUKIMASA

Plant Pathology

Laboratory,

of

HAYASHI

Faculty of Agriculture, Aichi-Ken, Japan Accepted

Nagoya

University,

Anjo,

May 27,1963

F-Amino acid incorporation into various cell fractions of TMV-infected tobacco leaf disks at different times after infection was investigated. The highest rate of incorporation into cytoplasmic protein was observed 6 hours after infection, but no virus was found at this time by serological test or by biological assay on Nicotiana glutinosa. In later stages, the rate of incorporation increased in the nuclear and the chloroplast fractions. The reason for this rise in the protein synthesis in the initial stage of infection is discussed.

the protein synthesis or only of virus accumulation. This paper reports the investigation of the site of virus protein synthesis by means of C14-amino acid incorporation into various cell fractions and of the time-course analysis of the protein synthesis.

INTRODUCTION

It is well known that protein metabolism of tobacco leaves is altered when the leaves are infected with tobacco mosaic virus (TMV) (Commoner and Yamada, 1955 ; Commoner and Rodenberg, 1955; Commoner et al., 1959; Takahashi, 1959). Amino acid activation in infected tobacco leaves was found to increase about 50% as compared to that in uninfected leaves (Hayashi, 1962). This increase suggested that protein biosynthesis in TMV-infected leaves was more active than in normal leaves and that TMV protein was synthesized de novo from free amino acids in the cell. It is important to know in which cellular component virus protein is formed from the activated amino acids and the time after inoculation when protein metabolism in the leaves is altered. Schramm and Riittger (1959) reported that TMV protein was formed in the cytoplasm surrounding the nucleus. Since they first observed virus protein by fluorescent antibody staining 45 hours after infection, it is not known whether the cytoplasm is the actual site of 1 Contribution No. 78, Plant tory, Nagoya University.

Pathology

Labora-

MATERIALS

AND

METHODS

Tobacco leaves were detached from young of Nicotiana tabacum ‘Bright plants Yellow’ grown in a green house, and their petioles were dipped into water at 25” under continuous light for 48 hours. This treatment gave detached leaves with metabolism in a reproducible steady state (Bonner, 1950). After the dipping, leaves were cut along the midrib. One half of each leaf was inoculated with purified TMV solution (0.1 mg/ml) with suspended Carborundum (5% w/w), and the other half was rubbed with Carborundum suspension without TMV. Leaf disks 12 mm in diameter were punched from each of the halfleaves and floated on distilled water in 6-cm petri dishes. These disks were incubated at 25” under cont.inuous illumination from fluorescent lamps (3000 lux). Isotope treatment. After various time intervals, leaf disks were transferred to

226

Cl’-AMINO

227

SCIDS INCORPORATION

petri dishes containing C14-amino acid solutions and incubated for 1-2 hours. nn-CY4-alanine or C14-glycine were usually used, and also Ci4-protein hydrolyzate from Chlorella vulgaris. Then the disks were washed quickly in the cold with corresponding nonlabeled amino acid solution (0.2 mM) to remove external C14-amino acid. The disks were blotted with filter papers and frozen at -18” until fractionation. Fractionation procedure. Fifty frozen leaf disks were homogenized in the cold in a mortar with 0.0125 M trisaminomethane (Tris)-HCl buffer (pH 7.5) containing 0.25 M sucrose, 0.05 M KCl, and 0.01 M MgS04. The homogenate was made to 10 ml, filtered through 4 layers of gauze, and fractionated by centrifugation. The temperature was kept near 0” during the procedure. The nuclear fraction was in the pellet after centrifugation at 500 g for 5 minutes, although this fraction contained considerable amounts of chloroplasts and cell wall fragments as shown by its color and a observation. The resultant microscopic supernatant was centrifuged at 1500 g for 15 minutes to obtain chloroplasts. The mitochondria were in the pellet after 20 minutes’ centrifugation at 15,000 g. This fraction also contained broken chloroplasts. The 15,000 g supernatant was called cytoplasm. In some experiments a microsomal fraction was obtained by 60 minutes’ centrifugation at 104,000 g. The 104,000 g supernatant was called the soluble protein fraction. After fractionation, protein was precipitated with 10% trichloroacetic acid, washed by the modified method of Siekevitz (1952)) and finally suspended in methyl alcohol. An aliquot of this suspension was dried in stainless-steel planchets, and the radioactivity was measured by using a windowless gas-flow Nuclear Chicago suspension was counter. The residual assayed for protein by Nessler’s reagent after digestion with sulfuric acid. Gel filtration technique. Sephadex gel filtration was used to ascertain the incorporation of C14-alanine into the protein

of the cytoplasm (15,000 g supernatant). Sephadex G-25 gel was placed in a 1.2 x 30 cm column and equilibrated with 0.0125 M Tris-HCl buffer (pH 7.5). Two milliliters of the cytoplasm solution was placed on the gel surface and eluted with the same buffer. The effluent was collected in l-ml fractions and assayed for radioactivity and optical density at 280 rnp. TMV infectivity test and serological assay. Leaf disks were inoculated as for the incorporation experiments and homogenized with 5 ml of 0.01 M acetate buffer (pH 6.0) containing 2% bentonite per 25 disks. Leaves of N. glutinosa were prerubbed with acetate buffer (pH 6.0) containing 0.08% bentonite and 5% Carborundum. Immediately after the rubbing, the leaves were inoculated with the above homogenate in a half-leaf experimental design. This inoculating method protects free TMV-RNA from the attack of RNase, as shown by Singer and Fraenkel-Conrat (1961). The precipitin ring test was used to detect virus antigen formed in tobacco leaf disks during the incubation. RESULTS

Incorporation

AND DISCUSSION

into the Whole Cell

In preliminary experiments the rate of incorporation of C14-amino acids into the protein of leaf homogenates increased linearly with time during 4 hours’ incubation on the isotope solution. In subsequent incorporation during l-2 experiments, hours’ incubation was investigated. Data in Table 1 show typical results of the experiments with C14-alanine. Other experiments using C14-glycine or C14-protein hydrolyzate indicated almost the same trend. In uninfected leaves, the incorporation rate into the whole-cell protein increased concomitantly with the time of incubation up to 2448 hours, and later decreased gradually (Table 1). This decrease may be caused by the degradation of leaf disks due to detaching and incubation in distilled water containing no nutrient. The increase in the earlier period seems to reflect the starvation of the disks, though the metabolic activity has been maintained.

HAYASHI

228 TABLE

1

Cl4-ALANINE INCORPORATION INTO VARIOUS CELL FRACTIONS OF TOBACCO LEAVES INFECTED WITH TOBACCO MOSAIC VIRUS T

Incorporation tions

into the Various Cell Frac-

The rate of incorporation into the protein of various cell fractions was measured to determine the site of virus protein synthesis. Hours Cpm in the fractionsa after 5iample In uninfected leaves, the highest incorporaincculation rate was observed in chloroplasts and tion Mit Cyt W.C. Chl Nu ~__cytoplasm, although mitochondria had the highest incorporation rate per milligram 7824e 15320 5560 15102 43806 (165) (870) (1152) (449) (417) protein, especially soon after infection (at 6b 8037 19500 7120 19362 54019 24 hours) (Table 1). This suggests that the (170) (938) (1160) (597) (519) chloroplast is the most active site of pro7833 20416 4820 16320 49389 tein synthesis, although some synt,hetic (145) (911) (1819) (504) (445) activity 126 seems to be located in all cell 8445 20232 7010 18471 54158 fractions (Stephenson et aZ., 1956; Sissakian (138) (1053) (2108) (570) (467) and Filippovich, 1957; Sissakian, 1958). 4394 43688 7560 32682 98324 When cells were infected with TMV, the (235) (2275) (2761) (1089) (878) 18b rate was markedly changed 6725 51084 10340 42969 121118 incorporation (328) (2660) (3240) (1432) (1124) and its rapid increase in the cytoplasmic 4388 9810 2874 5448 22530 fraction was conspicuous 6 hours after the (744) (1645) (710) (1133) (1048) infection. However, its activity 24c decreased 6268 11243 2280 6096 25887 at a later stage. On the other hand, the 1062) (1928) (1056) (1459) (1313) nuclear and chloroplast fractions showed 4538 7313 2940 6180 21021 prominent activity at a later stage (Table (686) (1274) (1054) (1403) (994) 48” 1 and Fig. 2). In this early period (about 4530 4643 1182 4523 14878 (705) (974) (961) (1264) (809) 24 hours) of the infection, the virus antigen was not detected in the cell by either 2385 4890 2076 4769 14120 (396) (852) (995) (1131) (706) serological or biological assay. 72c 2273 3720 1296 3396 10685 The supernatant of 15,000 g centrifuga(392) (653) (635) (911) (562) tion at 6 hours after the infection was examined by Sephadex sieving (Fig. 3)) and a Nu: Nucleus (500 g precipitate) ; Chl: chloroby separation into microsomal and soluble plast (1500 g precipitate); Mit: mitochondria protein fractions (Table 2). The result of (15,000 g precipitate); Cyt: cytoplasm (15,000 g Sephadex treatment indicated that C14supernatant) ; W.C.: whole cell. amino acid was incorporated into the pro6 Leaf disks were incubated on Ci4-nn-alanine tein molecule, since the curve of radioac(0.2 &0.033 rmole/ml) solution for 1 hour beginning 6, 12, or 18 hours after inoculation. tivity corresponded closely wit,h the optical c Leaf disks were incubated on C%x-alanine density at 280 rnp, which shows the amount (0.1 &0.017 pmole/ml) solution for 2 hours be- of protein (Fig. 3). However, Sephadex sievginning 24, 48, or 72 hours after inoculation. ing was unable to show any significant d U: Uninoculated, I: inoculated. difference in the pattern between TMV6 Figures show counts per minute per 100 disks infected cytoplasm and uninfected. The and figures in parentheses show counts per minactivities of microsomal and soluble protein ute per milligram protein. fractions were investigated and a higher TMV infection stimulated the incorincorporation was found in microsomes. Therefore, the rapid incorporation into the poration rate even at 6 hours after inoculation, and the rate of incorporation in cytoplasm at 6 hours after infection was infected cells was larger than that in unmainly caused by the incorporation into t,he infected cells until about 2448 hours microsomes (Table 2). (Table 1 and Fig. 1). In some experiments, These data indicate that the distinct rise the higher rate was seen throughout 48 in the incorporation rate into the cytoplasm hours or more after the inoculation. (15,000 g supernatant) at 6 hours after in-

Cl’-AMINO

229

ACIDS INCORPORATION

Cell degradation

12

24 Hours

36 after

-

48

inoculation

FIG. 1. The ratio of P-amino acid incorporation (cpm/mg protein) into the protein of infected leaf homogenate (I) to that into the protein of uninfected leaf homogenate (U). Each value is the mean of three independent, experiments with CY-alanine and of two independent experiments with P-glycine.

1.8 1.6

0.8 I

12

24 Hours after

36 inoculation

48

FIG. 2. The ratio of P-amino acid incorporation (cpm/mg protein) into the protein of various cell fractions of infected leaf homogenate (I) to that into the protein of comparable cell fractions of uninfected leaf homogenate (U). Each value is the mean of three independent experiments using C”-DL-alanine and of two independent experiments using CY-glycine. Open circles: nuclear fraction (500 g precipitate); filled circles: chloroplast fraction (1500 g precipitate) ; open triangles: mitochondrial fraction (15,000 g precipitate) ; filled triangles : cytoplasmic fraction (15,000 g supernatant).

fection did not directly involve virus protein synthesis, since no virus antigen could be detected at this time, but the active protein synthesis was caused by virus infection. Considering that several new enzymatic activities appear in phage-infected Escherichia coli during the first few minutes after infection (Flaks and Cohen, 1959;

Kornberg et al., 1959), a high activity of protein synthesis in the cytoplasm at, an early stage of infection is very possible. If the high incorporation into the cytoplasm reflects newly formed enzymes induced by virus infection, the transfer of these enzymes from the cytoplasm onto the surface of plastids (nuclear and chloroplast fractions) might be involved in the increase in

HAYASHI

k 2 10000” 8 d z ,’ 500 uE

20 Tube number

REFERENCES

2

Cl4-ALANINE INCORPORATION INTO MICROSOMAL AND SOLUBLE PROTEIN FRACTIONS” Cpm/mg protein in the fraction@

Hours after inoculation

Sample

6 6 6 7 7 7

ACKNOWLEDGMENTS The author wishes to express his gratitude to Professor T. Hirai for faithful advice throughout this investigation and to Professor I. Uritani for reading this manuscript. Thanks are also due to Mr. T. Takahashi for kindly supplying TMV-antiserum.

30

FIG. 3. Analyses of effluent from Sephadex column on which was placed 2 ml of the 15,000 g supernatant from disks incubated 1 hour in Clalanine at 6 hours after inoculation. The radioactivity and protein (responsible for absorbance at 280 rnp) came off in the same fractions. The radioactivity of free C?-amino acid (not incorporated into the protein) came off just after 30 ml was eluted and is not illustrated. Filled circles : cpm/ml of 15,000 g supernatant; open circles: optical density at 280 mp.

TABLE

the incorporation rate in the plastid at the next &age. This suggests the possibility that virus protein is synthesized on the surface of nuclei or/and chloroplasts. Some experiments are now being carried out to clarify the nature of the proteins formed at the initial stage by using DEAE column chromatography and other methods.

Microsome

Soluble protein

UC IC I/U

3975 6363 1.60

1958 2664 1.3G

U I I/U

3150 4262 1.35

1811 2030 1.12

a Leaf disks were incubated on Ci4-nn-alanine (0.2 &c/0.033 pmole/ml) solution for 1 hour beginning at 6 and 7 hours after inoculation. b The 15,000 g supernatant was fractionated into microsomes and supernatant by ultracentrifugation at 104,000 g for 1 hour. c U: Uninoculated; I: inoculated.

BONNER, J. (1950). “Plant Biochemistry,” pp. 299318. Academic Press, New York. COMMONER,B., and RODENBERG,S. D. (1955). Relationship between tobacco mosaic virus and the non-virus proteins. J. Gen. Physiol. 38, 475-492. COMMONER, B., and YAMADA, M. (1955). The nonvirus proteins associated with tobacco mosaic virus. J. Gen. Physiol. 38,459-473. COMMONER,B., LIPPINCOTT, J. A., and SYMINGTON, J. (1959). Kinetics of virus protein and ribonucleic acid biosynthesis, Nature 184, 1992-1998. FLAKS, J. G., and COHEN, S. S. (1959). Virus-induced acquisition of metabolic function. II. Studies on the origin of the deoxycytidylate hydroxymethylase of bacteriophage-induced E. coli. J. &al. Chem. 234, 1507-1511. HAYASHI, Y. (1962). Amino acid activation in tobacco leaves infected with tobacco mosaic virus. Virology 18, 140-141. KORNBERG, A., ZIMMERMAN, S. B., KORNBERG, S. R., and JOSSE, J. (1959). Enzymatic synthesis of deoxyribonucleic acid. IV. Influence of bacteriophage T2 on the synthetic pathway in host cells. Proc. Natl. Acad. Sci. U. S. 45, 772-785. SCHRAMM, V. G., and R~TTGER, B. (1959). Untersuchungen iiber das Tabakmosaikvirus mit fluoreszierenden Antikorpern. 2. Naturforsch. 14, 510-515. SIEKEVITZ, P. (1952). Uptake of radioactive alanine in vitro into the proteins of rat liver fractions. J. Biol. Chem. 195,549-565. SINGER, B., and FRAENKEL-CONRAT, H. (1961). Effects of bentonite on infectivity and stability of TMV-RNA. Virology 14,5965. SISSAKIAN, N. M. (1958). Enzymology of the plastids. Advan. Enzymol. 20,201-236. SISSAKIAN, N. M., and FILIPPOVICH, I. I. (1957).

C4-AMINO

ACIDS

Protein synthesis in isolated structures of the plant cell. Biokhimiya 22, 375-383. STEPHENSON, M. L., THIMANN, K. V., and ZAMECNIK, P. C. (1956). Incorporation of C’“-amino acids into proteins of leaf disks and cell-free

INCORPORATION

231

fractions of tobacco leaves. Arch. Biochem. Biophys. 65, 194-209. TAKAHASHI, W. N. (1959). The role of anomalous noninfectious protein in virus synthesis. Virology 9,437-445.