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Localization of tRNAs and Aminoacyl-tRNA Synthetases in Cytoplasm, Chloroplasts and Mitochondria of Glycine max, L.
Localization of tRNAs and Aminoacyl-tRNA Synthetases in Cytoplasm, Chloroplasts and Mitochondria of Glycine max, L.::-) DOUGLAS SINCLAIR and D. T. N. PILLAY Department of Biology, University of Windsor, Windsor, Ontario N9B 3P4, Canada Received August 19, 1980 . Accepted December 15, 1980
Summary Soybean leucyl-tRNAs and synthetases were localized in cytoplasm, mitochondria and chloroplasts. Totalleucyl-tRNA synthetase (cytoplasmic) resolves into three enzyme peaks when chromatographed on hydroxylapatite columns (HA) while mitochondrial and chloroplastic enzymes result in one peak each. Cytoplasmic enzyme peak 1 preferentially charges cytoplasmic tRNAlell species 5 and 6 and chloroplastic tRNAl," species 5 and 6. Cytoplasmic enzyme peak 2 preferentially charges cytoplasmic or mitochondrial tRNAl," species 1-4. Similarl~, cytoplasmic enzyme peak 3 preferentially charges cytoplasmic or mitochondrial tRNA'" species 1- 4. Chloroplast enzyme acylates only two tRNAl,u species 5 and 6 of chloroplast tRNA and preferentially charges peaks 5 and 6 of total cytoplasmic tRNA. Mitochondrial enzyme acylates four mitochondrial tRNAlell species 1-4 and the same four cytoplasmic tRNAlell species 1-4. However, the acylation of tRNAleu species 3 and 4 using mitochondrial tRNA is considerably increased. E. coli tRNA, charged with homologous synthetase and separated on RPC-5 column yields five tRNAlell species. Cytoplasmic enzyme (crude) acylates all five tRNAleu species of E. coli. Cytoplasmic enzyme 1 from HA column or purified chloroplastic enzymes acylate all five tRNAl," species of E. coli whereas c~toplasmic enzyme 2 and 3 and/or purified mitochondrial enzyme preferentially acylate tRNA '" species 1 and 2 of E. coli.
Key words: Glycine max, tRNA, aminoacyl-tRNA synthetase, soybean cotyledon, chloroplast, mitochondria, cytoplasm, RPC- 5 chromatography.
Introduction When a cell passes from one physiological state to another, both qualitative and quantitative changes in certain isoaccepting tRNAs and aminoacyl-tRNA synthetases have been observed. Previous studies on tRNAs and synthetases in soybean have revealed that the complement of tRNAl,u isoaccepting species change during ageing of the cotyledons (Bick et aI., 1970; Patel and Pillay, 1976; Shridhar and Pillay, 1976). The relative amounts of six tRNAleu species in young and older cotyledons change, with such change being more.pronounced in tRNAleu species 5 and 6. This raised the question as to what brings about the increase in species 5 and 6 and further since most ,C) This work was supported by a Grant A-1984 from the National Sciences and Engineering Council of Canada.
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of these studies were conducted with the total tissue including the cytoplasmic and organellar tRNAs and synthetases it was difficult to assess these changes in terms of their localization. In a plant cell, protein synthesis takes place in three compartments; in the cytoplasm, in the mitochondria and in the chloroplast. Localized within the organelles (mitochondria and chloroplast) are precisely the same categories of informational, transcriptional and translational macromolecules that have been characterized in the cytoplasm of eukaryotes and in prokaryotes. In many respects, protein synthesis in organelles resemble protein synthesis in prokaryotes as seen by the sensitivity of organellular protein synthesis to certain antibiotics such as choramphenicol, or the formylation of organelle initiator methionyl tRNA (Boulter et ai., 1972), rather than that taking place in the plant cell cytoplasm. In order to provide a definitive answer this investigation was undertaken to determine the presence of unique tRNAs and synthetases in the chloroplasts, the mitochondria and in the cytoplasm of Glycine max through the use of homologous and heterologous amino acylation experiments. In addition E. coli tRNAleu and leutRNA synthetases were included in these comparative studies to provide further proof that tRNAs and synthetases from plant organelles resemble those from bacteria and thus add credence to the endosymbiont theory. Materials and Methods Plant Material Soybean seeds (Glycine max var-Harcor) were soaked in distilled water for 3-4 hours and sown in moist vermiculite. Cotyledons and hypocotyls were harvested after 5 days of germination in the dark at 27 - 29°C. Leaves were harvested after 10 days of germination in the light, in a plant growth chamber.
Isolation of Transfer RNA from the cotyledons Transfer RNA was isolated from total RNA, prepared from freshly harvested cotyledons or those stored for several days at -20°C. The total RNA was extracted according to procedures described by Pillay and Cherry, 1974.
Preparation of Leucyl-tRNA Synthetase Extraction and partial purification of the enzyme from freshly harvested tissue was performed according to procedures described (Pillay and Cherry, 1974; Patel and Pillay, 1976).
Hydroxylapatite Column Chromatography Amino acyl-tRNA synthetase preparations from the cotyledons, chloroplasts and mitochondria were separated on hydroxylapatite columns (HA columns) made of a mixture of 10 g of hydroxylapatite and 1 g of cellulose powder (Whatman DF 11) with 0.5 g portions of cellulose at the top and bottom of the packing, and all other manipulations were as described previously (Patel and Pillay, 1976; Shridhar and Pillay, 1976).
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Transfer RNA Aminoacylation Assay The reaction was carried out at 30°C. Unless otherwise stated 1 ml of the reaction mixture contained 100 mM Tris HCI (pH 7.8); 10 mM MgCI 2 ; 1 mM ATP: 0.2 % soluble polyvinylpyrolidone -0.2 mg· ml- 1 of tRNA, 0.2 mg/ml of enzyme and 20 ~l unneutralized solution of L-(4,S-JH leucine solution 60 Ci . mmol- 1}. Reaction was terminated after 20 min by the addition of 5 ml of ice cold 5 % trichloracetic acid. The samples were filtered through glass fiber filters (What man GFIA) and counted in a liquid scintillation counter.
Isolation of Organelles Mitochondria Seeds of Glycine max were grown in the dark and the hypocotyls harvested after five days. Mitochondria were extracted as described by Guillemaut et aI., 1972. Batches of 200 g of hypocotyls were quickly homogenized in a Waring blender at 0° - 4 °C in 200 ml buffer containing 0.7 M mannitol, 0.004 M ATP, 0.001 M EDTA and 1 mg· ml- 1 of bovine serum albumin and the pH was adjusted to 7.2 with triethanolamine. The homogenate was then filtered through several layers of fine nylon cloth and centrifuged at 1000 xg for 5 min. The supernatant was layered onto a sucrose cushion and the pH was adjusted to 7.2 using triethanolamine. The ratio of 5: 2 supernatant to cushion was centrifuged at 8000 xg for 10 min. The mitochondria pellet was then resuspended in the appropriate buffer for either enzyme preparation or tRNA extraction.
Chloroplasts Seeds of Glycine max were grown in the light and the leaves harvested after 10 days. Batches of 100 g of leaves were quickly homogenized in a Waring blender in 350 ml buffer containing 0.05 M Tris-HCI (pH 8.0), 0.003 M EDTA: 0.001 M l3-mercaptoethanol, 0.3 M mannitol and 1 % BSA (Burkard et aI., 1972). The resultant slurry was then passed through several layers of 50 m~ mesh nylon cloth squeezing gently. The filtrate was then passed through several layers of 25 m~ mesh nylon cloth followed by 10 m~ mesh nylon cloth without squeezing. The filtrate was centrifuged for 90 s at 1000 x g. The supernatant was quickly poured off and the pellet resuspended in the extraction buffer (1 ml for 10 g of tissue). This was followed by centrifugation at 1000 xg for 90 s. The supernatant was discarded and the pellet stored in the freezer. All operations were done at 0° - 4 0C.
Measurement of Oxygen Consumption The rate of oxygen consumption at 30°C by mitochondrial suspensions was measured polargraphically using a Clark type oxygen electrode connected through an oxygen monitor (Yellow Springs Instruments; model 53) to a Heath Chart recorder (model SR-201A). The electrode was calibrated with air-saturated distilled water at 30°C and the dissolved oxygen content of the incubation medium was determined according to Beechey and Ribbons, 1972. Rates of oxygen consumption by mitochondrial suspensions were measured according to Estabrook, 1967 in a 3 ml reaction mixture.
Extraction of Chloroplast and Mitochondrial tRNA Generally 60-80 ml extraction medium containing 0.02 M Tris HCI pH 7.4, 0.01 M MgCl 2 and 1 % SDS was used for organelle preparation from a kilogram of tissue. To each tube containing organelle pellet,S ml extraction buffer was added; the pellet quickly dissolved and immediately mixed with water saturated phenol, with the phenol buffer ratio at 1 : 1 by volume with the extraction medium. The mixture was stirred for 30 min and then centrifuged at
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3500 xg for 15 min. The aqueous phase was extracted once again. A 1110 volume of 20 % Potassium acetate (pH 5.0) was added to the aqueous phase, followed by 2 volumes of 95 % ethanol and stirred overnight at -20°C. The pellet was collected by centrifugation at 1000 g and high molecular weight RNA was precipitated with 1 M NaCI and centrifuged at 1000 xg for 15 min. The supernatant was then diluted with Tris pH 7.5 to bring the Tris concentration to 0.01 M, with MgClz concentration at 0.1 M and the final NaCI concentration to 0.2 M. This is the optimum condition for DNAase activity. 10 /.Ig of DNAase/ml was added and thereaction allowed to proceed for 90 min at 4 0C. The solution was then absorbed on a DEAE cellulose column in 0.1 M Tris. Washing continued till the absorbance at 260 was less than 0.03 O.D. The tRNA was eluted with 1 M Tris and fractions showing an O.D. greater than 0.2 were pooled. Two volumes of 95 % ethanol were added to rhe pooled fractions and stored overnight at -20°C. The tRNA pellet was collected by centrifugation and suspended in distilled water and the solution was stored at - 20°C. Aminoacylation
0/ tRNA
Aminoacylation of tRNA was carried out at 30°C for 30 min in a 0.5 ml reaction mixture containing 10 mM Tris HCI pH 7.8 and optimal concentrations of Mg++, ATP and JH aminoacid. After incubation 50 /.II of the reaction mixture was applied to Whatman No.3 filter paper discs which were dried and immersed in cold 5 % TCA. All other procedures were as described previously (Patel and Pillay, 1976).
E. coli tRNA and Synthetases tRNA and aminoacyl-tRNA synthetases from E. coli was prepared according to procedures described by G. S. Swamy (1980). RPC- 5 Chromatography
tRNA samples from cytoplasm, chloroplast and mitochondria or E. coli were aminoacylated with 3H-Leucine using either homologous and heterologous aminoacyl-tRNA synthetases and the charged samples were chromatographed on RPC-S column as described previously (Pearson et al., 1971; Pillay and Gowda, 1980). Results and Discussion Localization of tRNAs and aminoacyl-tRNA synthetases in cytoplasm, chloroplasts and mitochondria of soybeans and aminoacylation of tRNA species by homologous and heterologous enzymes is described here.
Optimum A TP and Mg+2 levels A series of assays were carried out to optimize Mg+2 and ATP concentration in our assay system. Using a pH optimum of 7.8 for chloroplast assay system a range of concentrations for ATP (1- 2.5 mM) and for magnesium (0.05 to 5 mM) were tested. For the mitochondrial assay system concentrations of ATP (0.5 - 2 mM) and magnesium
(0.5-5 mM) were tested. Activity in both organelle systems was inhibited only %) when A TP and Mg+ + levels were varied from 0.5 -10 mM A TP and 0.5 -10 mM ATP and 0.5 -15 mM Mg. Based ort maximum activity at optimal ATP
slightly (10
and Mg+ + concentrations, the standard assay reaction mixture used contained 1 mM
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A TP and 10 mM Mg for all three systems tested. Time course experiments were conducted using cytoplasmic, chloroplastic and mitochondrial preparations in order to determine the time required for maximum acylation.
Leucyl·tRNA synthetases from cytoplasm, chloroplast and mitochondria A crude preparation of total soybean cotyledon leucyl-tRNA synthetase chromatographed on hydroxylapatite column and assayed for enzyme activity using cytoplasmic tRNA as substrate results in three peaks of leucyl-tRNA synthetase activity and the individual peaks are designated as enzyme 1, 2 and 3 (Fig. 1 a). Cytoplasmic enzyme 1 is the one which is chloroplast-specific, since it exclusively acylated tRNAleu species 5 and 6, if the substrate used is either cytoplasmic or chloroplastic tRNA. Cytoplasmic enzyme 2 and 3 are less specific and they both acylate either cytoplasmic or mitochondrial tRNAleu species 1-4. Leucyl-tRNA synthetase preparations from purified chloroplasts or mitochondria chromatographed on HA column and assayed using homologous tRNAs (chloroplastic or mitochondrial) yield single enzyme peaks from each organelle (Fig. 1 b, c). Our results for chloroplast enzyme is at slight variance to the observation in Pha· seolus (Guillemaut et aI., 1975) where two chloroplast enzyme peaks were obtained on the first HA chromatography and later only one peak was obtained on repeat chromatography of the enzyme on HA. Small amounts of mutual contamination of
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organellar or cytoplasmic enzymes is unavoidable due to extreme difficulties of isolating pure preparations from a single cell which is highly compartmentalized. Chloroplastic leucyl-tRNA synthetase chromatographed on HA elutes as a single peak (Fig. 1 b) which is similar to cytoplasmic enzyme 1 in its ability to exclusively charge only leucyl-tRNA species 5 and 6 when we use either chloroplast or cytoplasmic'tRNA. Similarly, mitochondrial synthetase elutes as a single peak (Fig. 1 c) on HA and is capable of acylating tRNA1eu species 1 to 4 from either cytoplasmic or mitochondria. Species 3 and 4 which are believed to be mitochondria-specific increase about 110 %.
Acylation 0/cytoplasmic, chloroplastic and mitochondrialleucyl-tRNAs by homologous and heterologous enzymes Charged leucyl-tRNA preparations from soybean which fractionate into six peaks on RPC-S chromatography (Fig. 5 a) will be used as control in the discussion that follows. Following aminoacylation of chloroplast-tRNA with a total crude cytoplasmic enzyme and chromatography on RPC-S, a three-fold increase in tRNAleu species 5 and 6 is observed compared to control. Although tRNAleu species 3 and 4 (mitochondrial) essentially disappear, small peaks of tRNAleu species 1 and 2 are still present indicative of some cytoplasmic contamination (data not presented). In Figure 2 a comparison of the aminoacylation capabilities of cytoplasmic and chloroplast enzyme is made. Irrespective of the source of tRNA (cytoplasmic or chloroplastic) both enzymes appear similar in their ability to charge leucyl-tRNAs 5 and 6. The activity
Fig, 2: Comparison of leucyl-tRNA profiles on RPC-5 chromatography, after aminoacylation of cytoplasmic and chloroplastic tRNA by (a) cytoplasmic tRNA charged by cytoplasmic enzyme 1. (b) chloroplast tRNA charged by cytoplasmic enzyme 1. (c) chloroplast tRNA charged by chloroplast enzyme for HA column. (d) cytoplasmic tRNA charged by chloroplast enzyme for HA column.
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enzyme, it is clear that no charging occurs for tRNNeu 1 to 4. This is positive proof that the enzyme preparations were highly pure and uncontaminated. Aminoacylation of mitochondrial tRNA with a crude cytoplasmic enzyme and chromatography on RPC-5, only tRNAleu species 1 to 4 are present with a sharp increase in activity of peaks 3 and 4 which have been reported to be mitochondriaspecific (Fig. 3 a). Similar results were obtained when mitochondrial tRNAs were charged by cytoplasmic enzymes 2 and 3 and chromatographed on RPC-5 (Fig. 3 b, c). In Figure 4 a comparison of the aminoacylation capabilities of mitochondrial tRNAs by purified mitochondrial enzyme on HA and cytoplasmic enzyme (total) is shown. Both in the homologous and heterologous aminoacylation it is obvious that total crude cytoplasmic or purified mitochondrial enzyme can acylate only four tRNNeu species (Fig. 4 a, c, d). In Figure 4 b, a crude cytoplasmic enzyme again is capable of acylating only four mitochondrial tRNAs. As in the case of chloroplasts these results are in agreement with the findings in Phaseolus (Guillemaut et a!., 1975), tobacco (Guderian et a!., 1972), and Euglena (Barnett and Brown, 1967; Parthier and Krauspe, 1973, 1974 a, b), and we can safely state that great similarity exists between cytoplasmic and mitochondrial tRNAs and synthetases. This is further confirmed by the results presented in Figure 5 wherein cytoplasmic tRNA acylated by either total crude cytoplasmic enzyme or with cytoplasmic enzymes 2 or 3 chromatography on RPC-5 yields the same four tRNA species as in the case of mitochondria.
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Fig. 6: Comparison of leucyl-tRNA profiles on RPC-5 chromatography after aminoacylation of E. coli by (a) E. coli enzyme, (b) chloroplastic enzyme from HA column, (c) cytoplasmic enzyme 1 and (d) total cytoplasmic enzyme.
Specificity of the organellar synthetase was checked by heterologous aminoacylation experiments with E. coli tRNA and synthetase. E. coli tRNA aminoacylated by E. coli synthetase or total cytoplasmic enzyme and separation on RPC-5 results in five tRNAleu species as shown in Fig. 6 a and d. Similarly, E. coli tRNA charged by either chloroplastic or cytoplasmic enzyme 1 yields five ~RNAleu species upon RPC-5 chromatography (Fig. 6 b, c), which is in agreement with the results in Phaseolus (Guillemaut et aI., 1975 and Jeannin et aI., 1976) for leucine, proline and lysine. However, aminoacylation of E. coli tRNA by cytoplasmic enzyme 2 or 3 or mitochondrial specific enzyme and chromatography on RPC-5 shows only two tRNAleu species (Fig. 7 a, b, c). Our results support the observations in Phaseolus (Guillemaut et aI., 1975), tobacco (Guderian et aI., 1972) and Euglena (Barnett et aI., 1978; Parthier, 1973; Parthier et aI., 1978), that in the soybean leucine system there is a similarity between cytoplasmic and mitochondrial enzymes on the one hand and between chloroplast and E. coli enzyme on the other. In the Phaseolus (Guillemaut et aI., 1975; Jeannin et aI., 1976) system for proline, lysine and methionine there appears to be a similarity between chloroplast, mitochondria and bacterial enzymes. Results presented together with those from Phaseolus (Guillemaut et a!., 1975), tobacco (Guderian et a!., 1972), Euglena (Barnett et a!., 1978), peas (Aliev and Filippovich, 1968) cotton (Merrick and Dure, 1972, 1973), Lupinus (Augustyniak and Pawelkiewicz, 1978) and wheat (Augustyniak et a!., 1979) all confirm the existence of chloroplastic specific and mitochondria specific tRNAs and aminoacyl-tRNA synthetases in plants. Only in the case of Phaseolus (Weil et a!., 1977) that organelle specific tRNAs have been studied for a number of amino acids, while
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other reports have looked at only certain amino acids. It is clear from these studies that the number of tRNA species for a particular amino acid localized in chloroplast is different for different amino acid and further with respect to leucyl-tRNA species in Phaseolus there are three species whereas in soybean, peas and tobacco we have only two isoaccepting species. The difference in the occurrence of an extra chloroplastic tRNA species in the same family of leguminoseae is not clear at this time. We are in the process of investigating other amino acids in the soybean. Since organelle-specific tRNAs have been demonstrated in several plant systems, the origin of these tRNAs, if they are coded by nuclear genes or organellar DNA is a matter of great interest. Although we have not attempted any studies in this aspect, recent work (Steinmetz and Weil, 1976) shows that chloroplastic specific tRNAphe and tRNA leu hybridize with chloroplast DNA whereas the corresponding cytoplasmic species do not. The significant role of organelle specific tRNAs is still a matter for further investigation.
With regard to the results in soybean for aminoacyl-tRNA synthetases, we are inclined to endorse the assumption made in Phaseolus (Guillemaut et aI., 1975) that chloroplast specific leucyl-tRNA synthetase though different from the cytoplasmic enzyme in its location, chromatographic properties and tRNA specificity could still be coded for by the nuclear gene which codes for the cytoplasmic enzyme and further modified as it enters the organelles. It has been shown in Euglena that chloroplast aminoacyl-tRNA synthetases are synthesized on cytoplasmic ribosome (Parthier, 1973 and Hecker et aI., 1974). Additional evidence to support this view is gathered by Z. Pjlanzenphysiol. Bd. 104.
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observations in Neurospora (Weil et aI., 1977) and particularly by the bleached mutant Euglena (Reger et aI., 1970) which lacks chloroplast DNA, but still capable of aminoacylating a light inducible tRNNhe suggesting that this chloroplast enzyme might have been coded by nuclear genes, synthesized on cytoplasmic ribosomes and later transported into chloroplast. Previous reports (Bick et aI., 1970; Shridhar and Pillay, 1976 and Patel and Pillay, 1976) have shown changes in tRNAleu species and synthetases in soybeans and peas, particularly with the onset of germination or during ageing. Since tRNA serves as a functional link between the genetic information encoded in the mRNA and protein synthesis specific changes in tRNA populations have various implications for the regulation of cellular events during differentiation, development and ageing. It was also suggested that cytokinins such as 6-benzyladenine (6BA) and zeatin defer senescence and also enhance the acylation of leucyl-tRNAs 5 and 6 in soybeans (Pillay and Cherry, 1974) and pea (Wright et aI., 1973) systems. The question still remains as to what brings about the increase in species 5 and 6. One of the suggestions made so far includes either differential synthesis or degradation of tRNAleu species. Now that is clearly established that species 5 and 6 are chloroplast specific and with ageing there appears to be degradative process in operation that decreases the activity of these species in yellowing leaves or cotyledons. However, treatment with cytokinins may retard senescence and delay the degradative process by controlling the production of hydrolytic enzymes and maintain the level of RNA and protein. The exact mechanism how cytokinins bring about this is still to be understood. References AUGUSTYNIAK, J., U. KARWOSKA, M RUDZINSKA, and Z. GOZDZICKA-]OSEFIAK: Proc. Int. Biochem. Congo p. 117 {1979}. AUGUSTYNIAK, H. and]. PAWELKIEWICZ: Acta. Biochim. Pol. 25, 91-97 {1978}. BARNETT, W. E.: Proc. Nat!' Acad. Sci. 53(6), 1462-1467 {1967}. BARNETT, W. E. and D. H. BROWN: Proc. Nat!' Acad. Sci. 57, 185 {1967}. BARNETT, W. E., D. H. BROWN, and]. L. EPLER: Proc. Nat!. Acad. Sci. 57, 1775-1781 {1967}. BARNETT, W. E. and]. L. EPLER: Cold Spring Harbor Symp. Quant. Bio!. 31,549 (1966). BARNETT, W. E., S. D. SCHWARTZBACH, and L. I. HECKER: Proc. Nat!. Acad. Sci. 63, 1261 {1978}. BEECHEY, R. B. and D. W. RIBBONS: Methods in Microbiology. 6 b, 25-53 {1973}. BICK, M. D., H. LIEBKE, ]. H. CHERRY, and B. L. STREHLER: Biochem. Biophys. Acta. 204, 175-182 (1970).
BOULTER, D., R. ]. ELLIS, and A. YARWOOD: Bio!. Rev. 47, 113 -175 (1972). BURKARD, G., P. GUILLEMAUT, and J. H. WElL: Biochem. Biophys, Acta. 224, 184 {1970}. BURKARD, G.,J. P. YAULTIER, and]. H. WElL: Phytochemistry. 11,1351-1353 {1972}. ESTABROOK, R. W.: Methods in Enzymology. 10,41-47 {1967}. GUDERIAN, R., R. PULLIMAN, and H. GORDON: Biochem. Biophys. Acta. 262,50-65 {1972}. GUILLEMAUT, P., G. BURKARD, and J. H. WElL: Febs. Lett. 60, 286 {1972}. GUILLEMAUT, P., A. STEINMETZ, G. BURKARD, and J. H. WElL: Biochem. Biophys. Acta. 378, 64-72 {1975}.
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HECKER, L. I., J. EGAN, R. J. REYNOLDS, C. E. NIX, J. A. SCHIFF, and W. E. BARNETT: Proc. Natl. Acad. Sci. USA 71,1910-1914 (1974). ]EANNIN, G., G. BURKARD, andJ. H. WElL: Biochem. Biophys. Acta. 442, 24-31 (1976). MERRICK, W. C. and L. S. DURE: J. BioI. Chern. 247, 7988-7999 (1972). - - Biochemistry. 12, 624-635 (1973). PARTHIER, B. and R. KRAUSPE: Plant Sci. Lett. 1,211 (1973). - - Biochem. Physiol. Pflanzen. 165, 1-17 (1974 a). PARTHIER, B.: FEBS Lett. 38,70-74 (1973). PARTHIER, B., F. MUELLER-URI, and R. KRAUSPE: In Chloroplast Development, ed. G. AKOYUNOGLOU et. aI., 687 -693 (1978). PATEL, H. and D. T. N. PILLAY: Phytochemistry. 15(3), 401-405 (1976). PEARSON, R. L., J. F. WEISS, and A. D. KELMERS: Biochem. Biophys. Acta. 228, 770-774 (1971). PILLAY, D. T. N. andJ. H. CHERRY: Canadian]ournal of Botany. 52, 2499-2504 (1974). PILLAY, D. T. N. and S. GOWDA: Gerontology. 27 (4), 194-204 (1981). REGER, B. J., S. A. FAIRFIELD, J. L. EPLER, and W. E. BARNETT: Proc. Nat!. Acad. Sci. 67, 1207 (1970).
SHRIDAR, V. and D. T. N. PILLAY: Phytochemistry. 15, 1809-1812 (1976). STEINMETZ, A. andJ. H. WElL: Biochim. Biophys. Acta. 454, 429 (1976). SWAMY, G. S.: Ph. D. Thesis, University of Windsor, Windsor, Canada (1980). WElL, ]. H., G. BURKARD, P. GUILLEMAUT, G. ]EANNIN, R. MARTIN, and A. STEINMETZ: Nucleic Acid and Protein Synthesis in Plants. 97 -120 (1977). WRIGHT, R. D., D. T. N. PILLAY, and]. H. CHERRY: Mech. Age. Dev. 1,403-412 (1973).
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Summary Soybean leucyl-tRNAs and synthetases were localized in cytoplasm, mitochondria and chloroplasts. Totalleucyl-tRNA synthetase (cytoplasmic) resolves into three enzyme peaks when chromatographed on hydroxylapatite columns (HA) while mitochondrial and chloroplastic enzymes result in one peak each. Cytoplasmic enzyme peak 1 preferentially charges cytoplasmic tRNAlell species 5 and 6 and chloroplastic tRNAl," species 5 and 6. Cytoplasmic enzyme peak 2 preferentially charges cytoplasmic or mitochondrial tRNAl," species 1-4. Similarl~, cytoplasmic enzyme peak 3 preferentially charges cytoplasmic or mitochondrial tRNA'" species 1- 4. Chloroplast enzyme acylates only two tRNAl,u species 5 and 6 of chloroplast tRNA and preferentially charges peaks 5 and 6 of total cytoplasmic tRNA. Mitochondrial enzyme acylates four mitochondrial tRNAlell species 1-4 and the same four cytoplasmic tRNAlell species 1-4. However, the acylation of tRNAleu species 3 and 4 using mitochondrial tRNA is considerably increased. E. coli tRNA, charged with homologous synthetase and separated on RPC-5 column yields five tRNAlell species. Cytoplasmic enzyme (crude) acylates all five tRNAleu species of E. coli. Cytoplasmic enzyme 1 from HA column or purified chloroplastic enzymes acylate all five tRNAl," species of E. coli whereas c~toplasmic enzyme 2 and 3 and/or purified mitochondrial enzyme preferentially acylate tRNA '" species 1 and 2 of E. coli.
Key words: Glycine max, tRNA, aminoacyl-tRNA synthetase, soybean cotyledon, chloroplast, mitochondria, cytoplasm, RPC- 5 chromatography.
Introduction When a cell passes from one physiological state to another, both qualitative and quantitative changes in certain isoaccepting tRNAs and aminoacyl-tRNA synthetases have been observed. Previous studies on tRNAs and synthetases in soybean have revealed that the complement of tRNAl,u isoaccepting species change during ageing of the cotyledons (Bick et aI., 1970; Patel and Pillay, 1976; Shridhar and Pillay, 1976). The relative amounts of six tRNAleu species in young and older cotyledons change, with such change being more.pronounced in tRNAleu species 5 and 6. This raised the question as to what brings about the increase in species 5 and 6 and further since most ,C) This work was supported by a Grant A-1984 from the National Sciences and Engineering Council of Canada.
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of these studies were conducted with the total tissue including the cytoplasmic and organellar tRNAs and synthetases it was difficult to assess these changes in terms of their localization. In a plant cell, protein synthesis takes place in three compartments; in the cytoplasm, in the mitochondria and in the chloroplast. Localized within the organelles (mitochondria and chloroplast) are precisely the same categories of informational, transcriptional and translational macromolecules that have been characterized in the cytoplasm of eukaryotes and in prokaryotes. In many respects, protein synthesis in organelles resemble protein synthesis in prokaryotes as seen by the sensitivity of organellular protein synthesis to certain antibiotics such as choramphenicol, or the formylation of organelle initiator methionyl tRNA (Boulter et ai., 1972), rather than that taking place in the plant cell cytoplasm. In order to provide a definitive answer this investigation was undertaken to determine the presence of unique tRNAs and synthetases in the chloroplasts, the mitochondria and in the cytoplasm of Glycine max through the use of homologous and heterologous amino acylation experiments. In addition E. coli tRNAleu and leutRNA synthetases were included in these comparative studies to provide further proof that tRNAs and synthetases from plant organelles resemble those from bacteria and thus add credence to the endosymbiont theory. Materials and Methods Plant Material Soybean seeds (Glycine max var-Harcor) were soaked in distilled water for 3-4 hours and sown in moist vermiculite. Cotyledons and hypocotyls were harvested after 5 days of germination in the dark at 27 - 29°C. Leaves were harvested after 10 days of germination in the light, in a plant growth chamber.
Isolation of Transfer RNA from the cotyledons Transfer RNA was isolated from total RNA, prepared from freshly harvested cotyledons or those stored for several days at -20°C. The total RNA was extracted according to procedures described by Pillay and Cherry, 1974.
Preparation of Leucyl-tRNA Synthetase Extraction and partial purification of the enzyme from freshly harvested tissue was performed according to procedures described (Pillay and Cherry, 1974; Patel and Pillay, 1976).
Hydroxylapatite Column Chromatography Amino acyl-tRNA synthetase preparations from the cotyledons, chloroplasts and mitochondria were separated on hydroxylapatite columns (HA columns) made of a mixture of 10 g of hydroxylapatite and 1 g of cellulose powder (Whatman DF 11) with 0.5 g portions of cellulose at the top and bottom of the packing, and all other manipulations were as described previously (Patel and Pillay, 1976; Shridhar and Pillay, 1976).
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Transfer RNA Aminoacylation Assay The reaction was carried out at 30°C. Unless otherwise stated 1 ml of the reaction mixture contained 100 mM Tris HCI (pH 7.8); 10 mM MgCI 2 ; 1 mM ATP: 0.2 % soluble polyvinylpyrolidone -0.2 mg· ml- 1 of tRNA, 0.2 mg/ml of enzyme and 20 ~l unneutralized solution of L-(4,S-JH leucine solution 60 Ci . mmol- 1}. Reaction was terminated after 20 min by the addition of 5 ml of ice cold 5 % trichloracetic acid. The samples were filtered through glass fiber filters (What man GFIA) and counted in a liquid scintillation counter.
Isolation of Organelles Mitochondria Seeds of Glycine max were grown in the dark and the hypocotyls harvested after five days. Mitochondria were extracted as described by Guillemaut et aI., 1972. Batches of 200 g of hypocotyls were quickly homogenized in a Waring blender at 0° - 4 °C in 200 ml buffer containing 0.7 M mannitol, 0.004 M ATP, 0.001 M EDTA and 1 mg· ml- 1 of bovine serum albumin and the pH was adjusted to 7.2 with triethanolamine. The homogenate was then filtered through several layers of fine nylon cloth and centrifuged at 1000 xg for 5 min. The supernatant was layered onto a sucrose cushion and the pH was adjusted to 7.2 using triethanolamine. The ratio of 5: 2 supernatant to cushion was centrifuged at 8000 xg for 10 min. The mitochondria pellet was then resuspended in the appropriate buffer for either enzyme preparation or tRNA extraction.
Chloroplasts Seeds of Glycine max were grown in the light and the leaves harvested after 10 days. Batches of 100 g of leaves were quickly homogenized in a Waring blender in 350 ml buffer containing 0.05 M Tris-HCI (pH 8.0), 0.003 M EDTA: 0.001 M l3-mercaptoethanol, 0.3 M mannitol and 1 % BSA (Burkard et aI., 1972). The resultant slurry was then passed through several layers of 50 m~ mesh nylon cloth squeezing gently. The filtrate was then passed through several layers of 25 m~ mesh nylon cloth followed by 10 m~ mesh nylon cloth without squeezing. The filtrate was centrifuged for 90 s at 1000 x g. The supernatant was quickly poured off and the pellet resuspended in the extraction buffer (1 ml for 10 g of tissue). This was followed by centrifugation at 1000 xg for 90 s. The supernatant was discarded and the pellet stored in the freezer. All operations were done at 0° - 4 0C.
Measurement of Oxygen Consumption The rate of oxygen consumption at 30°C by mitochondrial suspensions was measured polargraphically using a Clark type oxygen electrode connected through an oxygen monitor (Yellow Springs Instruments; model 53) to a Heath Chart recorder (model SR-201A). The electrode was calibrated with air-saturated distilled water at 30°C and the dissolved oxygen content of the incubation medium was determined according to Beechey and Ribbons, 1972. Rates of oxygen consumption by mitochondrial suspensions were measured according to Estabrook, 1967 in a 3 ml reaction mixture.
Extraction of Chloroplast and Mitochondrial tRNA Generally 60-80 ml extraction medium containing 0.02 M Tris HCI pH 7.4, 0.01 M MgCl 2 and 1 % SDS was used for organelle preparation from a kilogram of tissue. To each tube containing organelle pellet,S ml extraction buffer was added; the pellet quickly dissolved and immediately mixed with water saturated phenol, with the phenol buffer ratio at 1 : 1 by volume with the extraction medium. The mixture was stirred for 30 min and then centrifuged at
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3500 xg for 15 min. The aqueous phase was extracted once again. A 1110 volume of 20 % Potassium acetate (pH 5.0) was added to the aqueous phase, followed by 2 volumes of 95 % ethanol and stirred overnight at -20°C. The pellet was collected by centrifugation at 1000 g and high molecular weight RNA was precipitated with 1 M NaCI and centrifuged at 1000 xg for 15 min. The supernatant was then diluted with Tris pH 7.5 to bring the Tris concentration to 0.01 M, with MgClz concentration at 0.1 M and the final NaCI concentration to 0.2 M. This is the optimum condition for DNAase activity. 10 /.Ig of DNAase/ml was added and thereaction allowed to proceed for 90 min at 4 0C. The solution was then absorbed on a DEAE cellulose column in 0.1 M Tris. Washing continued till the absorbance at 260 was less than 0.03 O.D. The tRNA was eluted with 1 M Tris and fractions showing an O.D. greater than 0.2 were pooled. Two volumes of 95 % ethanol were added to rhe pooled fractions and stored overnight at -20°C. The tRNA pellet was collected by centrifugation and suspended in distilled water and the solution was stored at - 20°C. Aminoacylation
0/ tRNA
Aminoacylation of tRNA was carried out at 30°C for 30 min in a 0.5 ml reaction mixture containing 10 mM Tris HCI pH 7.8 and optimal concentrations of Mg++, ATP and JH aminoacid. After incubation 50 /.II of the reaction mixture was applied to Whatman No.3 filter paper discs which were dried and immersed in cold 5 % TCA. All other procedures were as described previously (Patel and Pillay, 1976).
E. coli tRNA and Synthetases tRNA and aminoacyl-tRNA synthetases from E. coli was prepared according to procedures described by G. S. Swamy (1980). RPC- 5 Chromatography
tRNA samples from cytoplasm, chloroplast and mitochondria or E. coli were aminoacylated with 3H-Leucine using either homologous and heterologous aminoacyl-tRNA synthetases and the charged samples were chromatographed on RPC-S column as described previously (Pearson et al., 1971; Pillay and Gowda, 1980). Results and Discussion Localization of tRNAs and aminoacyl-tRNA synthetases in cytoplasm, chloroplasts and mitochondria of soybeans and aminoacylation of tRNA species by homologous and heterologous enzymes is described here.
Optimum A TP and Mg+2 levels A series of assays were carried out to optimize Mg+2 and ATP concentration in our assay system. Using a pH optimum of 7.8 for chloroplast assay system a range of concentrations for ATP (1- 2.5 mM) and for magnesium (0.05 to 5 mM) were tested. For the mitochondrial assay system concentrations of ATP (0.5 - 2 mM) and magnesium
(0.5-5 mM) were tested. Activity in both organelle systems was inhibited only %) when A TP and Mg+ + levels were varied from 0.5 -10 mM A TP and 0.5 -10 mM ATP and 0.5 -15 mM Mg. Based ort maximum activity at optimal ATP
slightly (10
and Mg+ + concentrations, the standard assay reaction mixture used contained 1 mM
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A TP and 10 mM Mg for all three systems tested. Time course experiments were conducted using cytoplasmic, chloroplastic and mitochondrial preparations in order to determine the time required for maximum acylation.
Leucyl·tRNA synthetases from cytoplasm, chloroplast and mitochondria A crude preparation of total soybean cotyledon leucyl-tRNA synthetase chromatographed on hydroxylapatite column and assayed for enzyme activity using cytoplasmic tRNA as substrate results in three peaks of leucyl-tRNA synthetase activity and the individual peaks are designated as enzyme 1, 2 and 3 (Fig. 1 a). Cytoplasmic enzyme 1 is the one which is chloroplast-specific, since it exclusively acylated tRNAleu species 5 and 6, if the substrate used is either cytoplasmic or chloroplastic tRNA. Cytoplasmic enzyme 2 and 3 are less specific and they both acylate either cytoplasmic or mitochondrial tRNAleu species 1-4. Leucyl-tRNA synthetase preparations from purified chloroplasts or mitochondria chromatographed on HA column and assayed using homologous tRNAs (chloroplastic or mitochondrial) yield single enzyme peaks from each organelle (Fig. 1 b, c). Our results for chloroplast enzyme is at slight variance to the observation in Pha· seolus (Guillemaut et aI., 1975) where two chloroplast enzyme peaks were obtained on the first HA chromatography and later only one peak was obtained on repeat chromatography of the enzyme on HA. Small amounts of mutual contamination of
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organellar or cytoplasmic enzymes is unavoidable due to extreme difficulties of isolating pure preparations from a single cell which is highly compartmentalized. Chloroplastic leucyl-tRNA synthetase chromatographed on HA elutes as a single peak (Fig. 1 b) which is similar to cytoplasmic enzyme 1 in its ability to exclusively charge only leucyl-tRNA species 5 and 6 when we use either chloroplast or cytoplasmic'tRNA. Similarly, mitochondrial synthetase elutes as a single peak (Fig. 1 c) on HA and is capable of acylating tRNA1eu species 1 to 4 from either cytoplasmic or mitochondria. Species 3 and 4 which are believed to be mitochondria-specific increase about 110 %.
Acylation 0/cytoplasmic, chloroplastic and mitochondrialleucyl-tRNAs by homologous and heterologous enzymes Charged leucyl-tRNA preparations from soybean which fractionate into six peaks on RPC-S chromatography (Fig. 5 a) will be used as control in the discussion that follows. Following aminoacylation of chloroplast-tRNA with a total crude cytoplasmic enzyme and chromatography on RPC-S, a three-fold increase in tRNAleu species 5 and 6 is observed compared to control. Although tRNAleu species 3 and 4 (mitochondrial) essentially disappear, small peaks of tRNAleu species 1 and 2 are still present indicative of some cytoplasmic contamination (data not presented). In Figure 2 a comparison of the aminoacylation capabilities of cytoplasmic and chloroplast enzyme is made. Irrespective of the source of tRNA (cytoplasmic or chloroplastic) both enzymes appear similar in their ability to charge leucyl-tRNAs 5 and 6. The activity
Fig, 2: Comparison of leucyl-tRNA profiles on RPC-5 chromatography, after aminoacylation of cytoplasmic and chloroplastic tRNA by (a) cytoplasmic tRNA charged by cytoplasmic enzyme 1. (b) chloroplast tRNA charged by cytoplasmic enzyme 1. (c) chloroplast tRNA charged by chloroplast enzyme for HA column. (d) cytoplasmic tRNA charged by chloroplast enzyme for HA column.
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of these peaks have been calculated at 51 % and 49 % respectively. With the exception of Fig. 2 d which indicates two minor peaks possibly due to contamination of the Z. Pjlanzenphysiol. Bd. 104. S. 299-310. 1981.
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enzyme, it is clear that no charging occurs for tRNNeu 1 to 4. This is positive proof that the enzyme preparations were highly pure and uncontaminated. Aminoacylation of mitochondrial tRNA with a crude cytoplasmic enzyme and chromatography on RPC-5, only tRNAleu species 1 to 4 are present with a sharp increase in activity of peaks 3 and 4 which have been reported to be mitochondriaspecific (Fig. 3 a). Similar results were obtained when mitochondrial tRNAs were charged by cytoplasmic enzymes 2 and 3 and chromatographed on RPC-5 (Fig. 3 b, c). In Figure 4 a comparison of the aminoacylation capabilities of mitochondrial tRNAs by purified mitochondrial enzyme on HA and cytoplasmic enzyme (total) is shown. Both in the homologous and heterologous aminoacylation it is obvious that total crude cytoplasmic or purified mitochondrial enzyme can acylate only four tRNNeu species (Fig. 4 a, c, d). In Figure 4 b, a crude cytoplasmic enzyme again is capable of acylating only four mitochondrial tRNAs. As in the case of chloroplasts these results are in agreement with the findings in Phaseolus (Guillemaut et a!., 1975), tobacco (Guderian et a!., 1972), and Euglena (Barnett and Brown, 1967; Parthier and Krauspe, 1973, 1974 a, b), and we can safely state that great similarity exists between cytoplasmic and mitochondrial tRNAs and synthetases. This is further confirmed by the results presented in Figure 5 wherein cytoplasmic tRNA acylated by either total crude cytoplasmic enzyme or with cytoplasmic enzymes 2 or 3 chromatography on RPC-5 yields the same four tRNA species as in the case of mitochondria.
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Fig. 6: Comparison of leucyl-tRNA profiles on RPC-5 chromatography after aminoacylation of E. coli by (a) E. coli enzyme, (b) chloroplastic enzyme from HA column, (c) cytoplasmic enzyme 1 and (d) total cytoplasmic enzyme.
Specificity of the organellar synthetase was checked by heterologous aminoacylation experiments with E. coli tRNA and synthetase. E. coli tRNA aminoacylated by E. coli synthetase or total cytoplasmic enzyme and separation on RPC-5 results in five tRNAleu species as shown in Fig. 6 a and d. Similarly, E. coli tRNA charged by either chloroplastic or cytoplasmic enzyme 1 yields five ~RNAleu species upon RPC-5 chromatography (Fig. 6 b, c), which is in agreement with the results in Phaseolus (Guillemaut et aI., 1975 and Jeannin et aI., 1976) for leucine, proline and lysine. However, aminoacylation of E. coli tRNA by cytoplasmic enzyme 2 or 3 or mitochondrial specific enzyme and chromatography on RPC-5 shows only two tRNAleu species (Fig. 7 a, b, c). Our results support the observations in Phaseolus (Guillemaut et aI., 1975), tobacco (Guderian et aI., 1972) and Euglena (Barnett et aI., 1978; Parthier, 1973; Parthier et aI., 1978), that in the soybean leucine system there is a similarity between cytoplasmic and mitochondrial enzymes on the one hand and between chloroplast and E. coli enzyme on the other. In the Phaseolus (Guillemaut et aI., 1975; Jeannin et aI., 1976) system for proline, lysine and methionine there appears to be a similarity between chloroplast, mitochondria and bacterial enzymes. Results presented together with those from Phaseolus (Guillemaut et a!., 1975), tobacco (Guderian et a!., 1972), Euglena (Barnett et a!., 1978), peas (Aliev and Filippovich, 1968) cotton (Merrick and Dure, 1972, 1973), Lupinus (Augustyniak and Pawelkiewicz, 1978) and wheat (Augustyniak et a!., 1979) all confirm the existence of chloroplastic specific and mitochondria specific tRNAs and aminoacyl-tRNA synthetases in plants. Only in the case of Phaseolus (Weil et a!., 1977) that organelle specific tRNAs have been studied for a number of amino acids, while
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other reports have looked at only certain amino acids. It is clear from these studies that the number of tRNA species for a particular amino acid localized in chloroplast is different for different amino acid and further with respect to leucyl-tRNA species in Phaseolus there are three species whereas in soybean, peas and tobacco we have only two isoaccepting species. The difference in the occurrence of an extra chloroplastic tRNA species in the same family of leguminoseae is not clear at this time. We are in the process of investigating other amino acids in the soybean. Since organelle-specific tRNAs have been demonstrated in several plant systems, the origin of these tRNAs, if they are coded by nuclear genes or organellar DNA is a matter of great interest. Although we have not attempted any studies in this aspect, recent work (Steinmetz and Weil, 1976) shows that chloroplastic specific tRNAphe and tRNA leu hybridize with chloroplast DNA whereas the corresponding cytoplasmic species do not. The significant role of organelle specific tRNAs is still a matter for further investigation.
With regard to the results in soybean for aminoacyl-tRNA synthetases, we are inclined to endorse the assumption made in Phaseolus (Guillemaut et aI., 1975) that chloroplast specific leucyl-tRNA synthetase though different from the cytoplasmic enzyme in its location, chromatographic properties and tRNA specificity could still be coded for by the nuclear gene which codes for the cytoplasmic enzyme and further modified as it enters the organelles. It has been shown in Euglena that chloroplast aminoacyl-tRNA synthetases are synthesized on cytoplasmic ribosome (Parthier, 1973 and Hecker et aI., 1974). Additional evidence to support this view is gathered by Z. Pjlanzenphysiol. Bd. 104.
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observations in Neurospora (Weil et aI., 1977) and particularly by the bleached mutant Euglena (Reger et aI., 1970) which lacks chloroplast DNA, but still capable of aminoacylating a light inducible tRNNhe suggesting that this chloroplast enzyme might have been coded by nuclear genes, synthesized on cytoplasmic ribosomes and later transported into chloroplast. Previous reports (Bick et aI., 1970; Shridhar and Pillay, 1976 and Patel and Pillay, 1976) have shown changes in tRNAleu species and synthetases in soybeans and peas, particularly with the onset of germination or during ageing. Since tRNA serves as a functional link between the genetic information encoded in the mRNA and protein synthesis specific changes in tRNA populations have various implications for the regulation of cellular events during differentiation, development and ageing. It was also suggested that cytokinins such as 6-benzyladenine (6BA) and zeatin defer senescence and also enhance the acylation of leucyl-tRNAs 5 and 6 in soybeans (Pillay and Cherry, 1974) and pea (Wright et aI., 1973) systems. The question still remains as to what brings about the increase in species 5 and 6. One of the suggestions made so far includes either differential synthesis or degradation of tRNAleu species. Now that is clearly established that species 5 and 6 are chloroplast specific and with ageing there appears to be degradative process in operation that decreases the activity of these species in yellowing leaves or cotyledons. However, treatment with cytokinins may retard senescence and delay the degradative process by controlling the production of hydrolytic enzymes and maintain the level of RNA and protein. The exact mechanism how cytokinins bring about this is still to be understood. References AUGUSTYNIAK, J., U. KARWOSKA, M RUDZINSKA, and Z. GOZDZICKA-]OSEFIAK: Proc. Int. Biochem. Congo p. 117 {1979}. AUGUSTYNIAK, H. and]. PAWELKIEWICZ: Acta. Biochim. Pol. 25, 91-97 {1978}. BARNETT, W. E.: Proc. Nat!' Acad. Sci. 53(6), 1462-1467 {1967}. BARNETT, W. E. and D. H. BROWN: Proc. Nat!' Acad. Sci. 57, 185 {1967}. BARNETT, W. E., D. H. BROWN, and]. L. EPLER: Proc. Nat!. Acad. Sci. 57, 1775-1781 {1967}. BARNETT, W. E. and]. L. EPLER: Cold Spring Harbor Symp. Quant. Bio!. 31,549 (1966). BARNETT, W. E., S. D. SCHWARTZBACH, and L. I. HECKER: Proc. Nat!. Acad. Sci. 63, 1261 {1978}. BEECHEY, R. B. and D. W. RIBBONS: Methods in Microbiology. 6 b, 25-53 {1973}. BICK, M. D., H. LIEBKE, ]. H. CHERRY, and B. L. STREHLER: Biochem. Biophys. Acta. 204, 175-182 (1970).
BOULTER, D., R. ]. ELLIS, and A. YARWOOD: Bio!. Rev. 47, 113 -175 (1972). BURKARD, G., P. GUILLEMAUT, and J. H. WElL: Biochem. Biophys, Acta. 224, 184 {1970}. BURKARD, G.,J. P. YAULTIER, and]. H. WElL: Phytochemistry. 11,1351-1353 {1972}. ESTABROOK, R. W.: Methods in Enzymology. 10,41-47 {1967}. GUDERIAN, R., R. PULLIMAN, and H. GORDON: Biochem. Biophys. Acta. 262,50-65 {1972}. GUILLEMAUT, P., G. BURKARD, and J. H. WElL: Febs. Lett. 60, 286 {1972}. GUILLEMAUT, P., A. STEINMETZ, G. BURKARD, and J. H. WElL: Biochem. Biophys. Acta. 378, 64-72 {1975}.
GUILLEMAUT, P. andJ. H. WElL: Biochem. Biophys. Acta. 407, 240-248 {1975}. Z. Pjlanzenphysiol. Bd. 104.
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HECKER, L. I., J. EGAN, R. J. REYNOLDS, C. E. NIX, J. A. SCHIFF, and W. E. BARNETT: Proc. Natl. Acad. Sci. USA 71,1910-1914 (1974). ]EANNIN, G., G. BURKARD, andJ. H. WElL: Biochem. Biophys. Acta. 442, 24-31 (1976). MERRICK, W. C. and L. S. DURE: J. BioI. Chern. 247, 7988-7999 (1972). - - Biochemistry. 12, 624-635 (1973). PARTHIER, B. and R. KRAUSPE: Plant Sci. Lett. 1,211 (1973). - - Biochem. Physiol. Pflanzen. 165, 1-17 (1974 a). PARTHIER, B.: FEBS Lett. 38,70-74 (1973). PARTHIER, B., F. MUELLER-URI, and R. KRAUSPE: In Chloroplast Development, ed. G. AKOYUNOGLOU et. aI., 687 -693 (1978). PATEL, H. and D. T. N. PILLAY: Phytochemistry. 15(3), 401-405 (1976). PEARSON, R. L., J. F. WEISS, and A. D. KELMERS: Biochem. Biophys. Acta. 228, 770-774 (1971). PILLAY, D. T. N. andJ. H. CHERRY: Canadian]ournal of Botany. 52, 2499-2504 (1974). PILLAY, D. T. N. and S. GOWDA: Gerontology. 27 (4), 194-204 (1981). REGER, B. J., S. A. FAIRFIELD, J. L. EPLER, and W. E. BARNETT: Proc. Nat!. Acad. Sci. 67, 1207 (1970).
SHRIDAR, V. and D. T. N. PILLAY: Phytochemistry. 15, 1809-1812 (1976). STEINMETZ, A. andJ. H. WElL: Biochim. Biophys. Acta. 454, 429 (1976). SWAMY, G. S.: Ph. D. Thesis, University of Windsor, Windsor, Canada (1980). WElL, ]. H., G. BURKARD, P. GUILLEMAUT, G. ]EANNIN, R. MARTIN, and A. STEINMETZ: Nucleic Acid and Protein Synthesis in Plants. 97 -120 (1977). WRIGHT, R. D., D. T. N. PILLAY, and]. H. CHERRY: Mech. Age. Dev. 1,403-412 (1973).
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