Intracellular location of nitrate reductase and nitrite reductase in spinach and sunflower leaves

Lipid and fatty acid compositions were determined for chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.), sunflower (flelianthus annuus L.), and maize (Zea mays L.) leaves. The lipid composition was simiar in sunflower, spinach, and undifferentiated maize chloroplast envelope membranes and different in maize mesophyll chloroplast envelope membranes. The predominant lipid constituents in all envelope membranes were monogalactosyldiglyceride (27 to 46%), digalactosyldiglyceride (18 to 33%), and phosphatidylcholine (7 to 30%). The fatty acid composition was also similar in sunflower and spinach chloroplast envelope membranes in comparison to those from maize. The major acyl fatty acids of the chloroplast envelope membrane were palmitic (C16:0, 41 and 36%) and linolenic (C18:3, 29 and 40%) adds for spinach and sunflower; palmitic (77%) and steanc (C,8:0, 12%) acids for young maize; and palmitic (61%), stearic (14%), and linolenic (13%) acids for mature maize. The differences in lipid and acyl fatty acid compositions among these plants which vary in their rates of net photosynthesis were largely quantitative rather than qualitative.Methods for isolating chloroplast envelope membranes from plant species with slow rates of net photosynthesis have been developed in several laboratories (4,11,17,20). Recently I have also described the isolation of such membranes from plant species with fast rates of net photosynthesis (21). I believe that differences in the biochemical properties of these envelope membranes might be related to variations in chloroplastic permeability and metabolite transport and hence these variations in biochemical properties could effect photosynthetic efficiency (21).Characterization of these membranes has shown some differences in their transport abilities (18,19,21), enzymic levels (21), and hexosamine contents (22). The lipid compositions were previously determined only with chloroplast envelope membranes from plant species with slow rates of net photosynthesis (4, 12, 17), hence comparative studies are incomplete. This paper describes differences among the lipid and acyl fatty acid compositions of spinach, sunflower, and maize chloroplast envelope membranes. These plant species were selected for investigation because of their known differences in rates of net photosynthesis at high irradiance in normal air. Chloroplasts. Intact mesophyll chloroplasts were isolated from 10-g batches of freshly harvested, rinsed leaves 4 to 6 weeks of age for preparation from spinach, sunflower, and mature maize (5,8, 25). Undifferentiated intact chloroplasts were prepared from 4-to 6-day-old leaves of young maize (15). MATERIALS AND METHODS PlantChloroplast Envelope Membranes. Complete envelope membranes were removed from intact spinach, sunflower, and maize chloroplasts by osmotic shock and purified on a three phase discontinuous sucrose gradient. Details of the isolation procedure were described previously (21). Complete envelope membrane fractions were pooled and diluted 2-fold w...

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confidence: 99%

Lipid and Fatty Acid Composition of Chloroplast Envelope Membranes from Species with Differing Net Photosynthesis

Poincelot¹

1976

“…The plastids were found to contain only part of the total glutamine synthetase, aspartate aminotransferase, and triosephosphate dehydrogenase activity in the cell. Some evidence was obtained for low levels of glutamate dehydrogenase activity in chloroplasts.A proportion of the nitrite reductase extracted from plant cells is associated with a particulate fraction in both leaves and roots (10,21). While most work has suggested that nitrite reductase is localized in the chloroplast, Lips and Avissar (17) have suggested that the enzyme is present in the peroxisome.…”

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confidence: 99%

“…A proportion of the nitrite reductase extracted from plant cells is associated with a particulate fraction in both leaves and roots (10,21). While most work has suggested that nitrite reductase is localized in the chloroplast, Lips and Avissar (17) have suggested that the enzyme is present in the peroxisome.…”

mentioning

confidence: 99%

The Location of Nitrite Reductase and Other Enzymes Related to Amino Acid Biosynthesis in the Plastids of Root and Leaves

Miflin¹

1974

203

Density gradient separation of plastids from leaf and root tissue was carried out. The distribution in the gradients of the activity of the following enzymes was determined: nitrite reductase, glutamine synthetase, acetolactate synthetase, aspartate amninotransferase, catalase, cytochrome oxidase, and triosephosphate isomerase. The distribution of chlorophyll was followed in gradients from leaf tissue. The presence of plastids that have retained their stroma enzymes was denoted by a peak of triosephosphate isomerase activitv. Coincidental with this peak were bands of nitrite reductase, acetolactate synthetase, glutamine synthetase, and aspartate aminotransferase activity. The results suggest that most, if not all, the nitrite reductase and acetolactate synthetase activity of the cell is in the plastids. The plastids were found to contain only part of the total glutamine synthetase, aspartate aminotransferase, and triosephosphate dehydrogenase activity in the cell. Some evidence was obtained for low levels of glutamate dehydrogenase activity in chloroplasts.A proportion of the nitrite reductase extracted from plant cells is associated with a particulate fraction in both leaves and roots (10,21). While most work has suggested that nitrite reductase is localized in the chloroplast, Lips and Avissar (17) have suggested that the enzyme is present in the peroxisome. Miflin (21) found that the enzyme from barley roots banded in sucrose density gradients below the mitochondria and microbodies but he did not assay for any plastid markers and was unable to identify the organelle in question. Subsequently, Dalling et al. (7) found that nitrite reductase from wheat roots, under isopycnic density gradient centrifugation, was distributed coincidently with only one of two peaks of plastid marker enzymes. Because of the unusual distribution of the plastid markers in the gradient and the lack of complete coincidence between them and nitrite reductase, it is not possible from these results to state unequivocally that nitrite reductase is located in root plastids.If it is accepted that nitrite reductase is present in the plas- (19,20,23). Leech and co-workers (9, 16) and Lea and Thurman (15) have shown that chloroplasts have a light-stimulated glutamate dehydrogenase, which appears to be NADPH-dependent and bound tightly to the chloroplast lamellae. There is also evidence that chloroplasts can incorporate ammonia into glutamine (12,25,27). Further, chloroplasts contain a wide range of transaminases (13). In root tissues, glutamate dehydrogenase seems to be solely in the mitochondria (7,21), but, apart from these observations, little seems to have been done on the location of these enzymes in roots.Studies on "4CO2 fixation in chloroplasts indicate that "4C does not readily appear in amino acids, which suggests that the carbon skeletons for the amino acids are derived from outside the chloroplasts. In agreement with this a-ketoglutarate, pyruvate, and P-enolpyruvate stimulate the formation of a-amino nitrogen from nitrite...

“…Most (7,14,21) but not all (9,13) studies have indicated that NiR3 in leaf tissue is localized in the chloroplasts. Recently, additional confirmation of the chloroplastic location has been provided (15,17).…”

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confidence: 99%

Dependence of Nitrite Reduction on Electron Transport Chloroplasts

Neyra¹,

Hageman²

1974

Methyl viologen and phenazine methosulfate (photosystem I electron acceptors), 3-(3,4-dichlorophenyl)-1, l-dimethylurea (DCMU, electron-transport inhibitor), and methylamine (photophosphorylation uncoupler) were used to study the dependence of nitrite reduction on electron transport in chloroplasts.DCMU, methyl viologen, and phenazine methosulfate markedly inhibited, whereas methylamine stimulated NO2-reduction in isolated, intact spinach (Spinacia oleracea L.) chloroplasts. The objective of this study was to use isolated, intact chloroplasts and leaf sections to: (a) show that NO2 reduction is functionally associated with the electron-transport system of the chloroplasts; (b) seek additional evidence that Fd is the electron donor for NO2-reduction in leaves; and (c) evaluate the concept that ATP is not an obligatory metabolite in NO2-reduction. Chloroplast Preparation. The procedures for chloroplast preparation were as described (15), with the following exceptions. Isoascorbate was omitted from the grinding medium A as well as the resuspension medium B. After the homogenate was filtered to remove cell debris, the filtrate was centrifuged at 1SOOg for S min. The pellet was resuspended with 5 ml of medium B, and centrifuged as before. The pellet was again resuspended in 5 ml of medium B and used as a source of intact chloroplasts. Previous work (15) has shown that similar preparations contained approximately 50% intact (Class I) chloroplasts. MATERIALS AND METHODSNitrite Assay. A 2-ml aliquot of the chloroplast preparation was combined with 0.25 ml of 5 mm KNO2 and H20 plus additives (photosynthetic inhibitor, electron acceptors, or uncoupler) to make a final volume of 3.0 ml. The reaction mixtures were incubated in a water bath at 30 C with continuous shaking. Light was provided by a 500-w flood lamp which gave an incident energy of approximately 1.5 X 105 ergs cm-2 sec-1. Nitrite loss was followed by removing 0.2-ml aliquots of the reaction mixture at zero and subsequent time intervals. Nitrite 480 www.plantphysiol.org on May 11, 2018 -Published by Downloaded from