Enzymology of Glutammne Metabolism Related to Senescence and Seed Development in the Pea ( Pisum sativum

The metabolism of glutamme in the leaf and subtended fruit of the aging pea (Pisum sativum L. cv. Burpeeana) has been studied in relation to changes in the protein, chlorophyll, and free amino acid content of each organ during ontogenesis. Glutamine synthetase IEC 6.3.1.21 acthvity was measured during development and senescence in each organ. Glutamate synthetase IEC 2.6.1.531 activity was followed in the pod and cotyledon during develpment and maturation. Maximal glutamine synthetase activity and free amino acid accmulation occurred together in the young leaf. Glutamine synthetase (in vitro) in leaf extracts greatly exceeded the requirement (in vivo) for reduced N in the organ. Glutamine synthetase activity, although declinig in the senescing leaf, was sufficient (in vitro) to produce glutamine from all of the N released during protein hydrolysis (in vivo). Maximal glutamine synthetase activity in the -d was recorded 6 days after the peak accumulation of the free amino acids in this organ. In the young pod, free amino acids accumulated as glutamate synthetase activity increased. Maximal pod glutamate synthetase actvity occurred shnultaneously with maxhnal leaf glutamine synthetase activity, but 6 days prior to the corresponding maximu of glutamine synthetase in the pod. Cotyledonary glutamate synthetase activity increased during the asshnilatory phase of embryo growth which coincided with the loss of protein and free amino acids from the leaf and pod; maximal activity was recorded simultaneously with maximal pod glutamine synthetase. We suggest that the actity of glutamine synthetase in the supply organs (leaf, pod) funishes the translocated amide necessary for the N nutrition of the cotyledon. The subsequent activity of glutamate synthetase could provide a mechanism for the transfer of hiported amide N to aIpha amino N subsequenty used in protein syntbesis. In vito measurements of enzyme actii Indicate there was sufficient catalytic potential in vivo to accomplsh these proposed roles. During the course of fructification in pisum sativum, substantial amounts of reserve proteins are deposited over a brief period of time in the developing cotyledons (3, 5, 25). The synthesis of protein reserves creates a demand for the necessary amino acid precursors. This demand is met mainly by amino acids synthesized de novo utilizing reduced C and N imported by the seed (22, 37). Most of the N translocated to the ripening fruit is in the amides, glutamine and asparagine (2, 34, 37) and they are the main N donors for in situ synthesis of protein amino acids in the seed (19, 22). The importance of amides in the N nutrition of the ripening ovule is substantiated by the findings that asparagine and (especially) glutamine stimulate growth and protein synthesis in cul'This research was supported in part by National Science Foundation Grant PCM 75-95722 AOl. 2 Present address: C. F. Kettering Research Laboratory, 150 East College Street, Yellow Spring, Ohio 45387. tures (in vitro) of legume cotyledons and various plant embryos (26, 48). The subtending leaf (leaflets plus stipule) in the reproductive node and pod (carpel wall) surrounding the ripening ovules are the most important supply organs contributing the bulk of the reduced N imported by the developing cotyledons (19, 21, 35). Pate et aL (38) have stated that the role of the leaf in cycling solutes to its developing fruit is second only in importance to its role in photosynthetic C fixation. The pod is almost totally committed to the N nutrition of its developing ovules (16, 27, 36, 40). In a previous communication, we demonstrated the presence of proteolytic activity capable of releasing amino acids from the protein of agini leaves and pods (46). In this paper we report the presence of GS (EC 6.3.1.2) and GOGAT (EC 2.6.1.53) activity in aging supply organs and developing recipient organs of the pea. The respective catalytic potentials of these enzymes to incorporate N into translocated glutamine and subsequently utilize this N for assimilatory growth in the cotyledons are crucial to theN economy of the plant. MATERIALS AND METHODS Plants (P. sativum L. cv. Burpeeana) were grown under controlled environmental conditions and the age (days postanthesis) of individual organs was followed as before (46). All studies were conducted with the leaf (leaflets plus stipule) and subtended fruit (pod and cotyledons) of the lowest reproductive node of each plant. Organs were harvested, dissected and combined, and determinations of fresh weight, protein, Chl, and soluble a-amino N were made as described previously (46). Values given for these determinations were confirmed by triplicate analysis of six separately prepared samples. Glutamine Synthetase. Extracts were prepared by a modification of the method of O'Neal and Joy (29). All steps were carried out at 5 C or less. The extraction medium was 50 mm PIPES buffer (pH 6.8) containing 0.33 M sorbitol, 2 mm NaNO3, 10 mM 2-mercaptoethanol, 1 mm MnCl2, 2 mm EDTA, 2 mm sodium arsenate, 4 mM sodium ascorbate, and 0.1% (w/v) BSA. One g of tissue was added to 4 ml ofextraction medium and then completely macerated with a mechanical razor blade chopper (4). The resulting mixture was filtered through Miracloth (Calbiochem) into a conical test tube and centrifuged at 600g for 10 min. The post600g supernatant was collected and the chloroplast-enriched pellet was resuspended in the original extraction medium minus sorbitol, plus 0.1% Triton X. The supernatant and chloroplast fractions were filtered through a Sephadex G-50 gel column (1.5 x 20 cm) as described previously (46). In some cases, the Miracloth filtrate was applied directly to the G-50 column. Samples were eluted from the column with the extraction medium minus sorbitol and 3Abbreviations: GS: glutamine synthetase; GOGAT: glutamate synthetase; PIPES: piperazine-N,N'bis(2-ethane sulfonic acid) monosodium salt, monohydrate; GHA: gamma glutamyl hydroxamate. 494 www.plantphysiol.org on October 31, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. GLUTAMINE METABOLISM IN THE PEA BSA. The protein-rich fractions, which eluted with the void volume, were pooled, analyzed for soluble amino acids (see ref. 46), and assayed for GS activity. GS, unless stated otherwise, was measured as glutamyl transfer activity by a modification of the methods of O'Neal and Joy (30) and Varner and Webster (49). The standard transfer assay mixture contained 30 mM L-glutamine, 0.25 mm ADP, 1.33 mm EDTA, 25 mM hydroxylamine (NH20H, prepared fresh daily and adjusted to neutrality before use), 12 mm sodium arsenate, and 1.5 mm MnCl2 in 40 mm imidazole buffer (pH 6.8). The reaction was initiated by addition of 50 to 100 ,ul of enzyme extract (to a final volume of 3 ml) and incubated under standard conditions at 30 C, pH 6.8, for 5 min. Catalysis was terminated by addition of 0.5 ml of cold 24% (w/v) trichloroacetic acid and 10% (w/v) FeCl2 in 2.5 N HCI. Precipitated protein was immediately removed from the mixture by centrifugation at 5,000g for 10 min. The clarified supernatant was analyzed for the presence of GHA by spectrophotometric determination (540 nm) of its ferric chelate (8, 43). Control reactions of active or denatured (100 C, 15 min) enzyme only, or of substrate only, were similarly incubated and analyzed. A standard curve was prepared from authentic GHA (Sigma) added to a control reaction mixture and treated as the experimental. Extinction values obtained were in agreement with those reported by others (11, 43). Synthesis of GHA during the course of the reaction (as determined from the standard curve) was taken to indicate GS activity. One unit of activity is defined as the amount of enzyme which catalyzed the formation of 1 ttmol of GHA * min-' under the standard conditions described above. Specific activity is units * mgi' of protein in the enzyme extract. The pH optimum for glutamyl transfer activity by GS was determined by adjusting the initial pH of the incubation mixture and assaying under otherwise standard conditions. The reaction mixture was 40 mm sodium citrate-phosphate (pH 3-5) or 40 mm Tris-MES (pH 5-6, 8-9) or 40 mm imidazole (pH 6-8) buffer containing saturating levels of substrate as described above. Control assays containing only enzyme or substrate were also conducted over the pH range of 3 to 9. Extracts from pods were also assayed for GS activity by the biosynthetic method described by O'Neal and Joy (28) except diethylenetriamine pentaacetate (DTPA) was replaced by EDTA in the reaction mixture. Direct evidence for the synthesis of glutamine by leaf and pod extracts was obtained by a modification of the method described by Webster (52) using a reaction mix similar to that of the biosynthetic assay (28). Enzyme extracts from the G-50 column were incubated in 0.1 M Tricine-KOH, 20 mM MgSO4, 1 mM EDTA, 20 mm L-glutamate, 20 mm NH4CI, 10 mm ATP, and 8 mM 2-mercaptoethanol (final vol, 1 ml) at pH 7.8, 30 C, for 20 min. Similar incubations were made with reaction mixtures minus glutamate, NH4CI, or ATP. The synthesis reaction was terminated by addition of4 ml of ice cold acetone and the precipitated protein was removed by centrifugation. An aliquot of the resulting supernatant was applied to Whatman 3MM filter paper and the components were separated by descending flow of 80% phenol in water (v/v). The chromatograms were developed for qualitative evidence of enzyme activity as described previously (6). Protein was determined by the method of Lowry et al. (23). Chl was measured in 80%1o (v/v) acetone by the method of Amnon (1). Glutamate Synthetase. Enzyme extracts were obtained by procedure B described previously (6) except tissue was homogenized in a Polytron homogenizer (setting 3, 10 sec) and the grinding medium was 100 mM HEPES (pH 7.5) containing 0.1% BSA, 2 mM 2-mercaptoethanol, 2 mm EDTA, and 0.4 M sucrose. Enzyme activity was measured as before (6), except 50 to 100 ,ul of 100,000g supernatant was assayed. One unit ofGS activity is the amount of enzyme which catalyzes the oxidation of 1 nmol ofNADH min-' at pH 7.5, 30 C. Specific activity is units mg of proteinm in the enzyme extract. Protein was determined on trichloroacetic acid precipitates by the method of Lowry et al. (23). Purity of substrate amino acids used in all enzyme assays was determined by paper chromatography. Developmental Changes of Enzyme Activities. Catalytic activities reported for each developmental stage of individual organs were measured under experimentally determined optimum conditions. Reaction velocities were taken from the initial linear portion of reaction curves where catalysis was zero order with respect to substrate and first order with respect to time and amount ofenzyme extract in the reaction mixture. Under these conditions, the concentration ofenzyme in the crude tissue extract was assayed quantitatively in terms of catalytic effects and results were confirmed by triplicate assays on each of three separately prepared samples. Data quoted are the mean of these nine determinations. Mixing Experiments. Two enzyme extracts (prepared as described above for GS or GOGAT) containing different levels of enzyme activity and from separate developmental stages, were brought to equal protein concentrations (protein determined by the method of Warburg and Christian 15 1J) then mixed in varying proportions. Samples containing 0, 25, 50, 75, and 100% of each extract were assayed for enzyme activity as described above.