Arginine metabolism in developing soybean cotyledons : I. Relationship to nitrogen nutrition

The free and protein amino acid composition of Glycine max (L.) Merrill cotyledons was determined for the entire developmental period using high performance liquid chromatography. Arginine constituted 18% of the total protein nitrogen throughout development, and there was a linear arginine nitrogen accumulation rate of 1212 nanomoles per cotyledon per day between 16 and 58 days after anthesis. Arginine and asparagine were major constituents of the free amino acid pool, constituting 14 to 62% and 2 to 41% of the total free amino acid nitrogen, respectively. The urea cycle intermediates, citrulline, ornithine, and argininosuccinate were also detected in the free pool. A comparison of the amino acid composition of cotyledonary protein and of seedcoat exudate suggested that 72% of the cotyledon's arginine requirement is satisfied by in situ biosynthesis, and that 20% of the transformed nitrogen is incorporated into arginine. Also, [1-¹⁴C]glutamate and [U-¹⁴C]glutamine were fed to excised cotyledons. After 4 hours, ¹⁴C was incorporated into protein and released as ¹⁴CO₂, but none was incorporated into the C-1 and C-6 positions of free and protein arginine, determined using arginine-specific enzyme-linked assays. It is not currently known whether arginine biosynthesis in the cotyledon involves glutamate delivered from the mother plant or glutamate derived in situ.     

Degradation of 2-carboxyarabinitol 1-phosphate by a specific chloroplast phosphatase

The catalytic degradation of 2-carboxyarabinitol 1-phosphate (CA 1-P), a naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), was investigated by chromatographic and spectroscopic analyses of the reaction products. Carboxy-labeled [¹⁴C]CA 1-P was incubated with a partially purified tobacco (Nicotiana rustica) chloroplast protein that has been shown previously to catalyze metabolism of CA 1-P to a form incapable of inhibiting Rubisco (ME Salvucci, GP Holbrook, JC Anderson, and G Bowes [1988] FEBS Lett 231: 197-201). In the presence and absence of NADPH, ion-exchange chromatography showed a progressive conversion of [2′-¹⁴C]CA 1-P to a labeled compound which coeluted with authentic carboxyarabinitol. Parallel assays with unlabeled CA 1-P showed a concomitant decrease in the ability of reaction samples to inhibit Rubisco activity. In separate experiments, a 1:1 stoichiometry was found between the release of inorganic phosphate from [2′-¹⁴C]CA 1-P and accumulation of the ¹⁴C-labeled product. Liberation of inorganic phosphate was not observed when the tobacco enzyme was incubated with ribulose-1,5-bisphosphate, fructose-1,6-bisphosphate, glucose-1-phosphate, glucose-6-phosphate, or 6-phosphogluconate. Proton nuclear magnetic resonance spectroscopy of the labeled CA 1-P reaction product established its identity as carboxyarabinitol. We therefore propose that light-stimulated degradation of CA 1-P is catalyzed in vivo by a specific phosphatase, 2-carboxyarabinitol 1-phosphatase. Carboxyarabinitol 1-phosphatase activity was detected in the absence of NADPH, but increased threefold when 2 millimolar NADPH was present. Thus, while not required for the reaction, NADPH may play an important role in the regulation of CA 1-P degradation.     

Effect of Water Deficits on Seed Development in Soybean : II. Conservation of Seed Growth Rate

Water deficits during seed filling decrease seed size in soybean (Glycine max L.). This may result from a reduction in the supply of assimilates from the maternal plant and/or an inhibition of seed metabolism. To determine whether maternal or zygotic factors limited seed growth, we examined the effects of a plant water deficit on the supply of sucrose to and its utilization by developing embryos. Plants were grown in the greenhouse, and water deficits were imposed by withholding water for a period of 6 days during linear seed fill. When water was withheld, leaf water potential decreased rapidly, inhibiting canopy photosynthesis completely within 3 days. However, seed dry weight (nodes 7-11) continued to increase at or near the control rate. The level of total extractable carbohydrates in leaf, stem, and pericarp tissue decreased by 70, 50, and 45%, respectively, indicating that reserves were mobilized to support seed growth. Cotyledon sucrose content decreased from about 60 milligrams per gram dry weight to 30 milligrams per gram dry weight. Similarly, the concentration of sucrose in the interfacial apoplast of the cotyledons decreased from approximately 100 millimolar to 50 millimolar. However, the rate of sucrose accumulation by excised embryos, measured in a short-term in vitro assay, increased in response to the water deficit. These results indicate that both source and sink activity in soybean are altered by water deficits to maintain the flux of assimilates to the developing embryos. This may explain why seed growth is maintained, albeit for a shorter duration, when soybean is exposed to water deficits during the seed filling period.     

Localization of ferredoxin isoproteins in mesophyll and bundle sheath cells in maize leaf

Four ferredoxin isoproteins were identified in the C₄ plant Zea mays L. by analysis of extracts from leaves, mesocotyls, and roots of the young seedlings. The relative amounts of the isoproteins isolated from the photosynthetic and nonphotosynthetic organs were different. All the isoproteins were present in the leaves of green and etiolated plants, whereas two out of the four isoproteins were not detected in the roots or in the mesocotyls. During the greening of etiolated seedlings, the level of the two isoproteins unique to the leaf increased markedly. Analysis of the cellular and subcellular distribution of the two major leaf isoproteins showed that one isoprotein was present in the chloroplasts of both mesophyll and bundle sheath cells, whereas the other was only found in the chloroplasts of bundle sheath cells. This is the first report of the cell-specific expression of ferredoxin isoproteins in the leaves of a C₄ plant.     

Functional Analysis of the Photosynthetic Apparatus of Prochlorothrix hollandica (Prochlorales), a Chlorophyll b Containing Procaryote

Light-shade adaptation of the chlorophyll a/b containing procaryote Prochlorothrix hollandica was studied in semicontinuous cultures adapted to 8, 80 and 200 μmole quanta per square meter per second. Chlorophyll a contents based on dry weight differed by a factor of 6 and chlorophyll b by a factor of 2.5 between the two extreme light conditions. Light utilization efficiencies determined from photosynthesis response curves were found to decrease in low light grown cultures due to lower light harvesting efficiencies; quantum requirements were constant at limiting and saturating irradiances for growth. At saturating growth irradiances, changes in light saturated oxygen evolution rate originated from changes in chlorophyll a antenna relative to the number of reaction centers II. At light-limiting conditions both the number of reaction centers II and the antenna size changed. The amount of chlorophyll b relative to reaction center II remained constant. As in cyanobacteria, the ratio of reaction center I to reaction center II was modulated during light-shade adaptation. On the other hand, time constants for photosynthetic electron transport (4 milliseconds) were low as observed in green algae and diatoms. The occurrence of state one to two and state two to one transitions is reported here. Another feature linking photosynthetic electron transport in P. hollandica to that in the eucaryotic photosynthetic apparatus was blockage of the state one to two transition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Although chlorophyll b was reported in association with photosystem I, the 630 nanometer light effect does not exclude that chlorophyll b is the photoreceptor for the state one to two transition.     

Regulation of Pea Mitochondrial Pyruvate Dehydrogenase Complex : Does Photorespiratory Ammonium Influence Mitochondrial Carbon Metabolism?

Inactivation of the pyruvate dehydrogenase complex catalyzed by pyruvate dehydrogenase kinase was studied using intact mitochondria purified from green leaf tissue of pea (Pisum sativum L.) and dialyzed mitochondrial extracts. Thiamine pyrophosphate was inhibitory in dialyzed extracts but not in intact mitochondria, except in the presence of high concentrations of Na⁺. NH₄⁺, at concentrations as low as 20 micromolar, markedly stimulated inactivation in dialyzed extracts. K⁺ in the range 1 to 10 millimolar also enhanced inactivation. In contrast, Na⁺ was without affect at lower concentrations but was inhibitory at 10 to 100 millimolar levels. The effect of NH₄⁺ is discussed in relation to a possible regulatory interaction between photorespiratory NH₄⁺ production and the entry of carbon into the tricarboxylic acid cycle by way of the pyruvate dehydrogenase complex.     

Regulation of Protein Synthesis during Photomorphogenesis of Gametophytes of the Fern Onoclea sensibilis

Gametophytes of the fern Onoclea sensibilis grow as filaments in the dark and in red light and become planar in blue light. Pulse-labeling 4-day-old gametophytes with [³⁵S]methionine at different times after transfer to dark, red, and blue light environments revealed higher rates of amino acid uptake and protein synthesis in blue light than in red light or in the dark. Characterization of the extant and newly synthesized soluble proteins by one- and two-dimensional gel electrophoresis showed that the patterns of protein accumulation and synthesis in gametophytes exposed to short periods of red or blue light were qualitatively indistinguishable from those of gametophytes maintained in the dark. However, some striking increases and decreases in the levels of certain polypeptides were noted and these changes were accentuated during continued growth of gametophytes in the different environments. The results show that photomorphogenesis of gametophytes of O. sensibilis is associated with quantitative rather than qualitative changes in the population of mRNAs available for translation.     

Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules

Effective (N₂-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N₂-fixing) alfalfa recessive for the in₁ gene were compared to determine the effects of the in₁ gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in₁ effective nodules.     

Characterization of a meta-Fluorotyrosine-Tolerant Cell Culture of Eschscholtzia californica Cham

A cell line of Eschscholtzia californica selected for meta-fluorotyrosine (MFT) tolerance was found to have 10-fold increased levels of phenylalanine and tyrosine compared to the parent line, while most other amino acids were only increased 2-fold. Tracer experiments with shikimic acid in the presence of MFT showed that the biosynthesis of the aromatic amino acids was not impaired in the tolerant line. Feeding experiments with phenylalanine, tyrosine, or shikimic acid also revealed a reduced turnover of the pools of the aromatic amino acids in the variant. Thus undisturbed de novo biosynthesis of the aromatic amino acids and dilution of toxic effects of MFT by the enlarged pool sizes seemed to be the main reason for the acquired tolerance. Despite the enlarged availability of the precursor tyrosine, formation of the benzophenanthridine alkaloids was enhanced neither in the growth nor in the production medium.     

P-Nuclear Magnetic Resonance Determination of Phosphate Compartmentation in Leaves of Reproductive Soybeans (Glycine max L.) as Affected by Phosphate Nutrition

Most leaf phosphorus is remobilized to the seed during reproductive development in soybean. We determined, using ³¹P-NMR, the effect phosphorus remobilization has on vacuolar inorganic phosphate pool size in soybean (Glycine max [L.] Merr.) leaves with respect to phosphorus nutrition and plant development. Phosphate compartmentation between cytoplasmic and vacuolar pools was observed and followed in intact tissue grown hydroponically, at the R2, R4, and R6 growth stages. As phosphorus in the nutrient solution decreased from 0.45 to 0.05 millimolar, the vacuolar phosphate peak became less prominent relative to cytoplasmic phosphate and hexose monophosphate peaks. At a nutrient phosphate concentration of 0.05 millimolar, the vacuolar phosphate peak was not detectable. At higher levels of nutrient phosphate, as plants progressed from the R2 to the R6 growth stage, the vacuolar phosphate peak was the first to disappear, suggesting that storage phosphate was remobilized to a greater extent than metabolic phosphate. Under suboptimal phosphate nutrition (≤ 0.20 millimolar), the hexose monophosphate and cytoplasmic phosphate peaks declined earlier in reproductive development than when phosphate was present in optimal amounts. Under low phosphate concentrations (0.05 millimolar) cytoplasmic phosphate was greatly reduced. Carbon metabolism was coincidently disrupted under low phosphate nutrition as shown by the appearance of large, prominent starch grains in the leaves. Cytoplasmic phosphate, and leaf carbon metabolism dependent on it, are buffered by vacuolar phosphate until late stages of reproductive growth.