SOLUBLE PROTEINS AND ENZYMES IN TIM SUBCELLULAR FRACTIONS OF VIRUS.INFECTED TOBACCO LEAF M. A. EL-MELEIGII, S. J. SHEEN1, and R. H. LOWE'z ldepartment of Plant Patholop; and 2AR, SEA, USDA, Department of Agron<tmy, University o.f Kentuclcy, Lexington, Kentucky 40546, U.S.A. Received l2 May 1980, acr:epted 3 Aug. 1980. Er--MplrIct, M. A., SHssN, S. J. rno LowE, R. H. 1981. Soluble proteins and enzymes in the subcellular fractions of virus-infected tobacco leaf. Can. J. Plant Sci. 61: 135-142. Leaves of bright tobacco NC 95 infected with tobacco vein mottling virus (TVMV) and tobacco mosaic virus (TMV) were fractionated into subcellular fractions for protein quantitation and enzyme assay. Both viruses increased soluble proteins in the nuclear, chloroplastic and ribosomal fractions, whereas TMV infection lowered cytoplasmic protein content. Leaves infected with TVMV significantly increased total soluble proteins. Virus infection enhanced peroxidase activity in the cytoplasm but decreased activity in the chloroplastic and mitochondrial fractions. Potyphenoloxidase concentrated in the chloroplast and its activity were not altered by vrrus infection. Ribosomal fraction was rich in protease in both virus-infected leaves. In the chloroplasts, protease increased in activity in the TMV-infected leaves but maintained a level as low as the control in leaves showins TVMV svmdtom. The accumulation of soluble proteins in the TVMV-infecred leavis is possibly.'in part. attribured ro a low concentration of protease. Des feuilles de tabac clair NC 95 infect6es oar les virus de la marbrure des nervures du tabac (TVMV) et de la mosaique du tabac (TMV) ont 6t6 s6par6es en fractions sub-cellulaires puis soumises i I'analyse quantitative des prot6ines et au dosage de I'activit6 enzymatique. Les deux virus ont accru la teneur en prot6ines solubles dans le noyau, les chloroplastes et les ribosomes, alors que le TMV I'abaissait dans le cytoplasme. Les feuilles infect6es par TVMV produisaient significativement plus de prot6ines solubles. Les viroses ont stimul6 I'activit6 de la peroxydase dans le cytoplasme mais l'ont r6duite dans les chloroplastes et les mitochondries. La polyph6noloxydase se concentrait dans les chloroplastes et son activit6 est rest6e insensible h la pr6sence des virus. La fraction ribosomes 6tait riche en prot6ase dans les feuilles infect6es par les deux virus. Dans les chioroplastes, I'activit6 de la prot6ase a augment6 dans les feuilles envahies par TMV mais est rest6e au niveau des t6moins chez les feuilles attaqu6es par TVMV. L'accumulation des prot6ines solubles dans les feuilles infect6es par TVMV serait en partie le r6sultat de la faible concentration de prot6ases dans ces tissus. New proteins are known to be synthesized in the cells of tobacco plants infected with tobacco mosaic virus (TMV); however, the nature and sites of the virus-related orotein synthesis and accumulation in the cellare not yet fully elucidated. Reddi (1968) reported that TMV-related proteins in cytoplasm are of nuclear origin and are synthesized in association with ribosomes in the nucleus. Can. J. Plant Sci. 6l: 135-142 (Jan. 19tl) Zaitlin and Hariharasubramanian (1970) and Zaitlin et al. (1968) presented evidence that cytoplasmic ribosomes are involved in the synthesis of virus coat protein, and TMV particles are assembled neither in the nucleus nor in the chloroplast. In the same virus-plant host system, Singer ( 1971,l9'72) discovered a new low molecular weight protein in all subcellular fractions except the nucleus and identified it as TMV coat 135
136 CANADIAN JOURNAL OF PLANT SCIENCE protein. She concluded that regardless of where the coat protein is synthesized, the chloroplast is the site for a rapid assembly of virus particles. Virus infection affects the concentration of soluble proteins in plants. Protein content was lower in the mottling leaves of TMV-infected tobacco plants than in the leaves of non-infected ones (Kluge et al. 19'71:Yan Loon 1975; Wildman 1959). In contrast. tobacco leaves infected with tobacco vein mottling virus (TVMV) exhibited very mild mottling symptoms which are accompanied with a substantial increase in soluble proteins (Pirone and Davis 19'7'7; Sheen and Lowe 1979). Characterization of soluble proteins in virus-infected plants was limited to qualitative analysis. Quantitative variation of soluble proteins in subcellular fractions of virus-infected tissues has been overlooked in most cases. The objective of the present investigation was, therefore, to determine the soluble protein distribution in the subcellular fractions of tobacco leaves as they are possibly affected by TMV or TVMV infection. In addition, peroxidase, polyphenoloxidase and protease activity in the same subcellular fractions was measured to elucidate whether causal relationships exist between the cellular distribution of the two oxidases and the severity of virusinduced mottling symptoms and between protease activity and soluble protein accumulation in subcellular fractions. MATERIALS AND METHODS Bright tobacco (Nicotiana tabacum L.) cultivar NC 95 was grown in 25-cm pots with a l4-h-day photoperiod in a greenhouse. Six plants in two groups of three were inoculated at the six-leaf stage with either TMV or TVMV. Three plants were rubbed with distilled water as controls. Development of mosaic and monling symptoms indicated infection of the inoculated plants. Leaves above the site of inoculation were harvested on an individual plant basis 4 wk atter inoculation. Samples were taken from each plant by punching leafdiscs, 5 cm in diameter, through the tip, middle and basal portions of leaf panels. Cell fractionation was by the procedures described by Singer (l97 ll. Twenty grams of fresh leaf discs were ground with a cold mortar and pestle in 20 ml of prechilled 25 mm Tris-HCl buffer, ph 7.8, containing 0.5 M sucrose, 10 mm MgCl2 and 5 mm 2-mercaptoethanol. The homogenate was squeezed through four layers of cheesecloth and one layer of Miracloth and was centrifuged at 2500 g for 30 min to pellet the nuclear and chloroplastic fractions. The supernatant was centrifuged at 100003 for 30 min to petlet a crude mitochrondrial fraction. the l0 000 g supernatant was centrifuged at I 05 000 g for 2 h to pellet ribosomes and virus particles. The 105 000 I supematant was regarded as the cytoplasmic fraction. All preparations were carried out at 4"C. Chloroplasts which pelleted-out, along with nuclei, were solubilized by shaking for I h in O.25VoTriton X-100 dissolved in the extractron buffer devoid of sucrose. The mixture was then centrifuged at l00g to pellet nuclei. Nuelci were ruptured by shaking for 30 min in the above buffer containing 4TcTritonX-100 (Bonner 1976). The nuclear and chloroplastic fractions were sonicated for 10 min followed by centrifugation at 21 OOO g for 15 min, and the supernatant was saved. Proteins of the nuclear and chloroplastic fractions were precipitated by lova ammonium sulfate saturation and redissolved in 0'15 M sodium phosphate buffer, ph 7. Mitochondrial and ribosomal fractions were also suspended in the same buffer and sonicated. Final volumes of all fractions except cytoplasmic fraction were 5 ml. Peroxidase and polyphenoloxidase assay was the method of Sheen and Calvert ( I 969). Protease was assayed in a reaction mixture containing 10 mg Azocolle, 2 ml of citrate phosphate buffer, ph 5, and 0.5 ml of enzyme solution. The mixture was incubated at 40"C for 60 min, filtered through a cotton plug and read at 560 nm (Moore 1969). Total soluble proteins were quantified by the Biuret phenol method (Brewer et al. 1974) using bovine serum albumin as a standard. Data between treatments were subjected to t-tests in all cases. REST]LTS Soluble protein content in the subcellular fractions of virus-infected tobacco leaf is Dresented in Table 1. Virus infection iignificantly increased protein content in the
EL-MELEIGI ET AL, - VIRUS-INFECTED TOBACCO LEAF I31 Table 1. Soluble protein quantity in the subcellular fractions of tobacco leaves infected with tobacco vein mottling virus (TVMV) and tobacco mosaic virus (TMV) Virus infected Nuclei Chloroplasts Mitochondria Ribosomes Cytoplasm Total (mg/g fresh wt) 0.28 0.23 0. 15 0.48** 0.20 0.64*+ Control TVMV TMV o.75 1.70+* 1.80x* 1.00 1.44* 1.44+ 8,*xSignificant differences from the control at the 5Vc and lvo probability, respectively. 15. l5 16.40 13.40* ll.41 z0.n* 11.48 nuclear, chloroplastic and ribosomal fractions but did not alter the level in the mitochondrial fraction. Leaves infected with TMV had decreased concentrations of soluble proteins in the cytoplasmic fraction. The TVMV-infected leaves showed the highest amount of total soluble proteins, which supports previous reports (Pirone and Davis 1977; Sheen and Lowe 1979). Specific activity ofpreoxidase in response to virus infection varied among the subcellular fractions (Fig. l). The nuclear and cytoplasmic fractions represented the lowest and highest activities, respectively. Peroxidase in the TMv-infected leaf had a significantly higher activity in the nuclear and cytoplasmic fractions than in the corresponding fractions of the control. In contrast, the control exhibited greater activities in the mitochondrial and ribosomal fractions than the diseased leaf. The distribution pattern of peroxidase activity in the subcellular fractions of the leaf with TVMV infection was similar to that of TMV-infected leaf exceot that the nuclear and ribosomal f,ractions hid levels of activity comparable to that of the healthy leaves. When considering soluble proteins from all subcellular fractions, more than 93Vo of the preoxidase activity appeared in the cytoplasmic fraction. Polyphenoloxidase activity was highest in the chloroplastic fraction, followed in decreasing order by mitochondrial, cytoplasmic and nuclear fractions (Fig. 2). This oxidase was not detectable in the ribosomal fraction. Virus infection lowered the activity in the chloroplastic and mitochondrial fractions, although the difference did not reach statistical significance. The high specific activity in the nuclear fraction of TMV-infected leaves accounted for less than 47o of the total activity. Almost l}vo of the activity was associated with the chloroplastic fraction and about 25Vo was in the cytoplasmic fraction. Specific activity of protease varied with subcellular fractions but generally increased in response to virus infection (Fig. 3). The ribosomal fraction showed the highest specific activity. The virus-infected leaves contained more than double the amount of activity found in the healthy tissues; however, difference between the two viruses was not apparent. On the basis of total soluble proteins, ribosomal protease activity in plants infected with TVMV and TMV or not infected accounted for 45Vo,337o, and 207o of total activities, respectively. Although the chloroplastic fraction of TVMVinfected leaves contained 44Vo more extractable proteins than that of the control, the total protease activity in this fraction ofboth infected and control leaves was similar (M 0.75 x 10-3/mgprotein/min). However, this value accounted for only 16%o of the total protease activity in the TVMV-infected leaf but 36Vo in the control. A changed absorbance value of 2.08 x 10-3/mg protein/min was obtained in the chloroplastic fraction of TMV-infected leaves, and this represented 36Vo of the total protease activity. DISCUSSION Soluble protein content in cellular organel-
l3rl Z z Fq0 O E c I <3n ; F?rn u-- ; OEUo 'ln CANADIAN JOURNAL OF PLANT SCIENCE les, except the mitochondria, increased upon virus infection. Since virus particles are not assembled in the nuclei and chloroplasts of infected leaves (Zaitlin and Hariharasubramanian 1910; Zaitlin et al. 1968), the majority of virus particles should be sedimented in the subcellular fraction of ribosomes which are also the sites of virus coat protein synthesis. lt is therefore possible that virus coat protein contributed to the increase of soluble proteins in this fraction. Furthermore, virus coat protein is present in a conjugated form in virus particles. The weak alkali treatment in the Biuret phenol method for protein quantification may not break down the conjugation of proteins and nucleic acids. Ifthis is the case, the soluble protein content in the ribosomal fraction would be underestimated. On the other hand, the increase in protein content of chloroplastic and nuclear fractions in virus-infected tissue is likely attributable to the induction of host protein synthesis in these organelles in response to vlrus infection. These results are in agreement with previous reports (Reddi 1968; Zaitlin et al.1968). In a number of virus and Plant host systems, peroxidase and polyphenoloxidase increased in activity during the appearance NT}CLEAR CHLOROPLASTIC MITOCFOIIDRIAL RIFSfiAL CYTOPI-A$4IC FMCTION FMCTION FRACTION FRACTION FRACTION Fig. 1. Peroxidaseactivityinthesubcellularfractionsof tobaccoleavesinfectedwithtobaccovein mjttling virus (TVMV) und-tobu..o mosaic virus (TMV). Asterisk indicates significant difference (P < 0.05) from the control.
EL.MELEIGI ET AL, VIRUS-INFECTED TOBACCO LEAF 139 of necrosis but remained at a low level of activity when mottling systems developed (Kopp et al. 1976; Sheen and Diachun 1978). This is substanriated by the low activity of both oxidases in the TMV- and TVMV-infected leaves as calculated on the basis of total soluble Droteins and the specific activity of oxidaies in subcellular fractions (Table 2). Polyphenoloxidase activity was associated mainly with the chloroplastic fraction. The considerable amount of this oxidase in the mitochondrial fraction could be the results of contamination with chloroplast fragments due to the inherent nature of cell fractionation procedures. O --l X z E =F O E L Iz - : F 2o io J 2U E l I 16 12 Table 2. Activities of peroxidase, polyphenoloxidase and protease calculated on the basis of total soluble proteins and their specific activities in subcellular fractions of virus-infected and noninfected tobacco leaves Virus infection Peroxidase Polyphenoloxidase Protease Control 43.63 TVMV 48.09 - TMV 49.61 (A Almg proteinlmial- 1.93x10r 0.13x10-3 1.85x 1O-'z 0.23x 10-3 l.9l x 10-'z 0.46x 10j The protease activity of tobacco leaf increased in parallel with leaf senescence (De Jong 1972;EI-Meleigi et al. 1981). In the present work, a stimulation of protease fli-clem FRACTION CI-LOROH-ASTIC I4ITOCI-ONDRIAL RIBOSCTTAL CYTOPLASI,IIC FRACTIoiI FMcTIOt'l FMCTION FRACTToN Fig. 2. Polyphenoloxidase activity in the subcellular fractions of tobacco leaves infected with tobacco vein mottling virus (TVMV) and tobacco mosaic virus (TVM). Asterisk indicates sienificant difference (P < 0.05) from the control.
140 CANADIAN JOURNAL OF PLANT SCIENCE activity by virus infection is evident (Table 2). Protease activity increased more in the TMV-infected plants than in those developing TVMV symptoms. A distinct difference in protease activity in the leaves infected with these viruses appeared for the chloroplastic fraction. This difference may bear importance in symptom development. It seems likely that an increase of protease inside chloroplasts would exert stress on the function and development of this organelle. Degradation of chloroplasts would result in yellowing of the leaf as displayed in the mosaic symptoms of the TMV-infected leaves. On the other hand, the low protease activity in the chloroplastic fraction of the - >< z = -z u6& L I -t ; ;tr O q u <1 UI F? o TVMV-infected leaves may partially explain the mild mottling symptoms and the accumulation of soluble proteins in the diseased tissue, if the degradation of fraction 1 protein is minimized owing to a low protease activity in the chloroplasts. Of interest is the high protease activity in the ribosomal fraction. Plant cytoplasm (cell sap) contains 80 S ribosomes which are associated with rough endoplasmic reticulum. Endoplasmic reticulum is likely sedimented down along with ribosomes in the process of cellular fractionations. For studies of the GA3-controlled formation and secretion of hydrolases, including protease in cereal grains, there is evidence that rough NrcLEAR FRACTION CHLOROPLASTIC FMCTION MITMrcNDRIAL FRACTION RIBOSOI4AL FMCTION CYTOPLASI'II C FRACTION Fig. 3. Protease activity in the subcellular fractions of tobacco leaves infected with tobacco vetn mottling virus (TVMV) and tobacco mosaic virus (TMV). Asterisk indicates significant difference (P < 0.05) from the control.
EL-MELEIGI ET AL. VIRUS-INFECTED TOBACCO LEAF - t4l endoplasmic reticulum participates in the synthesis and exportation of secreted proteins in eukaryotic ceils (Varner and Ho 1916). The present results, therefore, substantaite the association of protease with endoplasmic recitulum in the ribosomal fraction. Virus coat proteins are supposedly assembled on the ribosomes. Whether the proteases which increased in resdonse to virus infection can degrade the coat protein and/or slow down the assembly of virus particles remains an open question, although it has been demonstrated that TMV proteins are resistant to several proteolytic enzvmes (Matthews lg10). Ai any rate, ui.u, infection may accelerate leaf senescence, which is usually preceded with an increase in protease activity. Proteases deteriorate cell structure and function and eventually lead to cell death. ACKNOWLEDGMENTS This research was supported in part by the U.S. Depaftment of Agriculture Cooperative Agreement No. 12-14-'7001-1041. This paper is published with rhe approval of bii..tor, Kentucky Agricultural Experiment Station as Journal Series Paper No. i8b- I I -3-gg). BONNER, J. 1976. The nucleus. pas.es37-64in J. Bonner and J. E. Varner, eds. plant biochemistry, 3rd ed., Academic press, New York. BREWER, J. M., PEACE, A. J. ANd ASHWORTH, R. L. 1974. Experimenral techniques in biochemistry. prenrice ilall. lnc.. Englewood Clifls. N.J. 3iO pp. DE JONG, D. W. 1972. Detergenr extraction of enzymes tiom tobacco leaves varying in maturity. Plant Physiol. 50: 7j3-737. EL-MELEIGI, M. A., SHEEN, S. J. andlowe. R. H. 1980. Changes in fraction 2 protein and associated enzymes during tobacco growth. Tob. Sci. (in press). KLUGE, W., PAUNOW, S. and SCHUSTER. G. 1977. On the action of some metabolically active substances on the protein content and the multiplication of virus in leaves of Nicotiana tabaccum L. Phytopath. Z.88: ll-l'l. MATTHEWS, R. E. F. 1970. plant virolosv. Academic Press. New york. 778 pp. MOORE, G. L. 1969. Use of Azo-bound colasen to measure reaction velocities of oroteolvtic enzymes. Anal. Biochem. 32: 122-l)7. PIRONE, T. P. and DAVIS, D. L. lgii. Modification of the chemical composition of burley tobacco by infection with tobacco vein moftling virus. Tob. Sci. 2: 83-84. REDDI, K. K. 1968. Biochemical events with the cell followed infection with tobacco mosaic virus. Pages 79-90 int. Hirai, Z. Hidaka, and I. Uritani, eds. Biochemical regulation in diseased plants or injury. Kyonitsu Printing Co. Ltd., Tokyo, Japan. SHEEN, S. J. and CALVERT, J.1969. studies of polyphenol contents, activities and isozymes of polyphenoloxidase and peroridase during air curing in three tobacco types. Plant physiol. 44: 199-204. SHEEN, S. J. and DIACHUN, S. 19i8. Relationship of peroxidase and polyphenoloxidase activity to virus symptoms in red clover. Acta Phytopathol. 13: 243-253. SHEEN, S. J. and LOWE, R. H. 1979. proteins and related nitrogenous compounds in virusinfected tobacco plants. Can. J. Plant Sci. 59: 1099-i 107. SINGER, B. 1971. Protein synrhesis in virus infected plants. I. The number and nature of TMV-directed proteins detected on polyacrylamide gels. Virology 46: 247-255. SINGER, B. 1972. Protein synthesis in virus infected plants. II. The synthesis and accumulation of TMV coat protein in subcellular fractions of TMV infected tobacco. Virology 47: 397-404. VAN LOON, L. C. 1975. Polyacrylamide disk electrophoresis of soluble leaf proteins from Nicotiana tabaccum var. "Samsun" and "Samsun NN" III. Influence of temperature and virus strarn on changes induced by tobacco mosaic virus. Physiol. Plant Pathol. 6:289-300. VARNER, J. E. and HO, D. T. H. 1976. Hormones. PagesT13-7701n J. BonnerandJ. E. Varner, eds. Plant biochemistry, 3rd ed. Academic Press. New York. WILDMAN, S. G. 1959. The process of infection and virus synthesis with tobacco mosaic virusandotherplantviruses. Pages l-31jn F. M. Burnet and W. M. Stanley, eds. The viruses, Vol. 2, Plant and bacterial viruses. Academic press, New York. ZAITLIN, M. and HARIHARASUBRAMA- NIAN, V. 1970. Protein in tobacco mosaic virus-infected plants. Biochem. Biophys. Res. Commun. 39: 103 l- 1036.
t42 CANADIAN JOURNAL OF PLANT SCIENCE ZAITLIN, M., SPENCER, D. an<l WHIT- Biochemical regulation in diseased plants or FIELD. P. R. 1968. Studies on the intefcellular injury. Kyonitsu Printing co Ltd, Tokyo' site of tobacco mosaic virus assembly. Pages Japan' 9l - 103 in T. Hirai. Z. Hidaka and I. Uritani, eds