Amino Acid Biosynthesis in the Plastids of Root and Leaves1

Plant Physiol. (1974) 54, 550-555 The Location of Nitrite Reductase and Other Enzymes Related to Amino Acid Biosynthesis in the Plastids of Root and Leaves1 Received for publication January 17, 1974 and in revised form April 1, 1974 BENJAMIN J. MIFLIN2 Division of Natural Sciences, University of California, Santa Cruz, California 95064 ABSTRACT tids, then the question arises as to how many more of the enzymes involved in the synthesis of amino acids are also present Density gradient separation of plastids from leaf and root in the plastid. Studies with isolated chloroplasts have shown tissue was carried out. The distribution in the gradients of the that they are capable of a light-dependent reduction of nitrite activity of the following enzymes was determined: nitrite reduc- and the incorporation of the product into a-amino nitrogen tase, glutamine synthetase, acetolactate synthetase, aspartate (19, 20, 23). Leech and co-workers (9, 16) and Lea and Thur- amninotransferase, catalase, cytochrome oxidase, and triose- man (15) have shown that chloroplasts have a light-stimulated phosphate isomerase. The distribution of chlorophyll was fol- glutamate dehydrogenase, which appears to be NADPH-de- lowed in gradients from leaf tissue. The presence of plastids pendent and bound tightly to the chloroplast lamellae. There is that have retained their stroma enzymes was denoted by a peak also evidence that chloroplasts can incorporate ammonia into of triosephosphate isomerase activitv. Coincidental with this glutamine (12, 25, 27). Further, chloroplasts contain a wide peak were bands of nitrite reductase, acetolactate synthetase, range of transaminases (13). In root tissues, glutamate dehy- glutamine synthetase, and aspartate aminotransferase activity. drogenase seems to be solely in the mitochondria (7, 21), but, The results suggest that most, if not all, the nitrite reductase apart from these observations, little seems to have been done and acetolactate synthetase activity of the cell is in the plastids. on the location of these enzymes in roots. The plastids were found to contain only part of the total glutamine synthetase, aspartate aminotransferase, and triosephos- Studies on "4CO2 fixation in chloroplasts indicate that "4C cell. Some evidence was ob- does not readily appear in amino acids, which suggests that the phate dehydrogenase activity in the carbon skeletons for the amino acids are derived from outside tained for low levels of glutamate dehydrogenase activity in the chloroplasts. In agreement with this a-ketoglutarate, pyru- chloroplasts. vate, and P-enolpyruvate stimulate the formation of a-amino nitrogen from nitrite by isolated chloroplasts (23); these carbon compounds are sufficient for the formation of amino acids re- garded as the heads of the amino acid families (e.g., aspartate and glutamate). Little is known of the location of enzymes responsible for the conversion of these carbon precursors to the a-keto acids required for the formation of amino acids at the A proportion of the nitrite reductase extracted from plant end of their family tree (e.g., leucine, isoleucine, valine, phen- cells is associated with a particulate fraction in both leaves and ylalanine, and tyrosine). One enzyme in this category is roots (10, 21). While most work has suggested that nitrite re- acetolactate synthetase, the first enzyme unique to isoleucine, ductase is localized in the chloroplast, Lips and Avissar (17) leucine, and valine biosynthesis. This enzyme has been ascribed have suggested that the enzyme is present in the peroxisome. to the mitochondria of Neurospora (31) but no work on its sub- Miflin (21) found that the enzyme from barley roots banded in cellular location has been done with green plants. sucrose density gradients below the mitochondria and micro- Recent studies by Miflin and Beevers (24) have produced a bodies but he did not assay for any plastid markers and was technique for the isolation of intact plastids in a relatively pure unable to identify the organelle in question. Subsequently, state. This technique has been used with leaf and root tissues Dalling et al. (7) found that nitrite reductase from wheat roots, and various enzymes of nitrogen metabolism assayed in order under isopycnic density gradient centrifugation, was distributed to answer some of the questions raised above. This paper pre- coincidently with only one of two peaks of plastid marker sents the results of these experiments. enzymes. Because of the unusual distribution of the plastid markers in the gradient and the lack of complete coincidence MATERIALS AND METHODS between them and nitrite reductase, it is not possible from these results to state unequivocally that nitrite reductase is located in Plant Material. Plants were grown as described previously root plastids. (21, 24). If it is accepted that nitrite reductase is present in the plas- Tissue Extraction. Spinach (Spinacia oleracea) leaves were homogenized in 2 volumes/g of tissue of an isolation medium, 10 1 1% Dextran 1 This research was supported by the National Science Founda- consisting of mm KCI, mM MgCl2, w/v T40, tion Grant GB-35376 and the Science Research Council of Great 1% w/v Ficoll, 0.1% w/v bovine serum albumin, made to Britain. volume with 30% (w/v) sucrose containing 79 mM Tricine 2Permanent address: Biochemistry Department, Rothamsted Ex- buffer, pH 7.5, for 5 X 1 sec bursts in an Atomix blender. Pea perimental Station, Harpenden, Herts AL5 2JQ, England. and barley roots were ground in a chilled pestle and mortar in 550 Downloaded from on May 5, 2020 - Published by www.plantphysiol.org Copyright © 1974 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 54, 1974J w LNITROGEN METABOLISM ENZYMES IN PLASTIDS 551 2 volumes of the same medium except that it also contained 1 mM glutathione and the sucrose concentration was 25% w/w. All homogenates were filtered through eight layers of fine it nylon gauze and the filtrate layered on the density gradient. Density Gradients were of w/w sucrose solutions made up to 2 100% with 0.1 M Tricine buffer pH 7.5. Gradients used for 0 spinach leaves were 4 ml of a 60% sucrose cushion followed by 6 ml of a linear gradient from 60 to 42%, followed by 5 ml of 42%, followed by 10 ml of a linear gradient from 42 to 30% with a final 3 ml of 30% sucrose. Eight milliliters of filtered homogenate were placed on top of the gradient. The gradient used for short time centrifugation of the pea root tissue con- sisted of a 4-ml cushion of 60% sucrose, a linear gradient of 18 ml of 50 to 25%, followed by a final 3 ml of 25% sucrose with 10 ml of homogenate layered on top. The gradients were usually prepared about 2 hr before use. Immediately after layering on the filtered homogenate the tubes were balanced and placed in a SW27 rotor in a Beckman Spinco L.2-65 centrifuge and, as soon as vacuum permitted, centrifuged to 4,000 rpm for 5 min and then at 10,000 rpm for a further 10 min. The rotor was allowed to decelerate to 5,000 rpm before turning on the brake. The gradients and ) 2 centrifugation techniques for the longer spins are given in c ) legends to the figures. After centrifugation all gradients were 0t.i fractionated into samples of 1.2 ml on an ISCO density gradient fractionator, run at 2.0 ml/min. The sucrose concentrations 9 of fractions were determined by refractrometry. Enzyme Assays. Catalase was determined by the method of Luck (18), triosephosphate isomerase by the method of Gibbs and Turner (8). Chlorophyll was determined by the method of Arnon (2), nitrite reductase by the method of Bourne and Miflin (4), acetolactate synthetase by the method of Miflin (22), glutamine synthetase by the method of Shapiro and Stadtman oD - (29). Glutamate dehydrogenase was measured by incubating ._.E 100 ul of gradient fraction in 0.2 mM NADPH, 1 mm CaCl2, 0.1% w/v Triton X-100, 10 mm (NH4)2-SO, 10 mm Tricine 4 buffer pH 7.8 in a final volume of 1 ml; the reaction was started by the addition of 100 ,d of 0.1 M a-ketoglutarate, pH 7.5, in phosphate buffer and the absorption of NADPH at 340 nm followed on a recording spectrophotometer. Cytochrome oxidase was measured by the method of Hackett (11) after incubating aliquots of the fraction in 0.1% digitonin for 5 min. Aspartate aminotransferase was measured by the method of Sizer and Jenkins (30). RESULTS Leaf Tissue. The results of a rapid gradient separation of a 10 20 spinach leaf homogenate are shown in Figure 1. The intact chloroplasts are identified by the marker enzyme triose-P Gradient volume(mJ) isomerase (14). Coincident with this band of triose-P isomerase FIG. 1. Distribution of various enzyme activities in a sucrose are bands of glutamine synthetase, acetolactase synthetase, and density gradient centrifugation of a homogenate of spinach leaves. nitrite reductase. The intact chloroplast band was uncon- The arrows denote the region of the peak of activity for catalase taminated by microbody marker enzymes in that less than 2% (CA) and Cyt oxidase (CO). of the total catalase activity was found under the plastid peak. Mitochondrial contamination, as measured by the Cyt oxidase but, as discussed elsewhere (24), it is probably due to the trap- recovered in the peak, was also less than 2%. Using this tech- ping of microbodies by the mitochondria. The results with bar- nique no evidence was obtained for the coincidental banding of ley (Fig. 2) and with other root tissues indicate that roots con- nitrite reductase and catalase. tain microbodies. Root Tissue. The result of an isopycnic gradient centrifuga- In further studies with root tissues the brief centrifugation tion of barley roots (Fig.

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