Photorespiratory Amino Donors, Sucrose Synthesis and the Induction of CO2 Fixation in Barley Deficient in Glutamine Synthetase and/or Glutamate Synthase

Murray, A. J. S., Black well, R. D., Lea, P. J. and Joy, K.W. 1988. Photorespiratory amino donors, sucrose synthesis andthe induction of CO₂ fixation in barley deficient in glutaminesynthetase and/or glutamate synthase.—J. exp. Bot. 39:845–858. A number of mutants of barley have been produced which lackboth chloroplastic glutamine synthetase and ferredoxin-dependentglutamate synthase activities. The plants accumulated ammoniato the same extent as mutants deficient in only glutamine synthetasebut shared the gas-exchange characteristics of the glutamatesynthase deficient parent. These mutants have been used to demonstratedirectly the ability of alanine to ameliorate the dramatic dropin fixation rate normally exhibited by glutamate synthase deficientmutants on transfer to photorespiratory conditions. Immediatelyafter transfer to air, the mutants deficient in glutamate synthaseactivity demonstrated a reduced ability to incorporate ¹⁴C derivedfrom ¹⁴CO₂ into sucrose. This effect was, however, dependenton the previous induction of CO₂ fixation. Use of ¹⁴CO₂ revealedthat the induction phase of CO₂ fixation was altered in allthree mutants. Neither of the parents nor the double mutantaccumulated sucrose in air under conditions which promote sucroseaccumulation by the wild type. The implications of these resultsfor photosynthesis and the control of sucrose synthesis arediscussed. Key words: Photorespiratory barley mutant, amino donors, sucrose, GS, glutamate synthase.     

Barley mutants lacking chloroplast glutamine synthetase-biochemical and genetic analysis

Eight mutants of barley (Hordeum vulgare cv Maris Mink) lacking the chloroplast isozyme of glutamine synthetase (EC 6.3.1.2.) were isolated by their inability to grow under photorespiratory conditions. The cytoplasmic isozyme of glutamine synthetase was present in the leaves of all the mutants, with activities comparable to the wild-type (10-12 nanokatals per gram fresh weight). The mutant plants developed normally and were fully fertile under conditions that minimize photorespiration. In 1% O₂ the rate of CO₂ fixation in leaves of one of the mutants, RPr 83/32, was the same as the wild-type, but in air this rate declined to 60% of the wild-type after 30 minutes. During this time the ammonia concentration in leaves of the mutant rose from 1 to 50 micromoles per gram fresh weight. Such ammonia accumulation in air was found in all the mutant lines. In back-crosses with the parent line, F₁ plants were viable in air. In the F₂ generation, nonviability in air and the lack of chloroplast glutamine synthetase co-segregated, in both the lines tested. These two lines and four others proved to be allelic; we designate them gln 2a-f. The characteristics of these mutants conclusively demonstrate the major role of chloroplast glutamine synthetase in photorespiration and its associated nitrogen recycling.     

Assimilation of Nitrogen in Mutants Lacking Enzymes of the Glutamate Synthase Cycle

¹⁵N labelling was used to investigate the pathway of nitrogenassimilation in photorespiratory mutants of barley (Hordeumvulgare cv. Maris Mink), in which the leaves have low levelsof glutamine synthetase (GS) or glutamate synthase, key enzymesof ammonia assimilation. These plants grew normally when maintainedin high CO₂, but the deletions were lethal when photorespirationwas initiated by transfer to air. Enzyme levels in roots weremuch less affected, compared to leaves, and assimilation oflabelled nitrate into amino acids of the root showed very littledifference between wild type and mutants. Organic nitrogen wasexported from roots in the xylem sap mainly as glutamine, levelsof which were somewhat reduced in the GS-deficient mutant andenhanced in the glutamate synthase deficient mutant. In theleaf, the major effect was seen in the glutamatesynthase mutant,which had an extremely limited capacity to utilize the importedglutamine and amino acid synthesis was greatlyrestricted. Thiswas confirmed by the supply of [¹⁵N]-glutamine directly to leaves.Leaves of the GS-deficient mutant assimilatedammonia at about75% the rate found for the wild type, and this was almost completelyeliminated by addition of the inhibitormethionine sulphoximine.Root enzymes, together with residual levels of the deleted enzymesin the leaves, have sufficient capacityfor ammonia assimilation,through the glutamate synthase cycle, to provide adequate inputof nitrogen for normal growth of themutants, if photorespiratoryammonia production is suppressed. Key words: Hordeum vulgare, ¹⁵N, glutamine synthetase, glutamate synthase, ammonia assimilation     

Effect of Ammonia on Dark CO2 Fixation by Anabaena Cells Treated with Methionine Sulfoximine

Dark CO₂ fixation by Anabaena cylindrica was stimulated aboutthree-fold by the addition of NH₄Cl to the cells. The ¹⁴CO₂incorporation experiments showed that ¹⁴C is most rapidly incorporatedinto aspartate and then glutamine by adding NH4CI. Glutamineaccumulated predominantly after the addition of NH₄Cl showingthat NH₄ is incorporated into glutamine by glutamine synthetase.The stimulating effect of NH4Cl on CO₂ fixation and amino acidsynthesis was suppressed by methionine sulfoximine, an inhibitorof glutamine synthetase. It was suggested that dark CO₂ fixationwas stimulated by the action of glutamine synthesis which isenhanced by ammonia. (Received February 10, 1981; Accepted April 2, 1981)     

Carbon and nitrogen metabolism in a barley (Hordeum vulgare L.) mutant with impaired chloroplast dicarboxylate transport

A mutant line, RPr79/2, of barley (Hordeum vulgare L. cv. Maris Mink) has been isolated that has an apparent defect in photorespiratory nitrogen metabolism. The metabolism of ¹⁴C-labelled glutamine, glutamate and 2-oxoglutarate indicates that the mutant has a greatly reduced ability to synthesise glutamate, especially in air, although in-vitro enzyme analysis indicates the presence of wild-type activities of glutamine synthetase (EC 6.3.1.2) glutamate synthase (EC 1.4.7.1 and EC 1.4.1.14) and glutamate dehydrogenase (EC 1.4.1.2). Several characteristics of RPr79/2 are very similar to those described for glutamate-synthase-deficient barley and Arabidopsis thaliana mutants, including the pattern of labelling following fixation of ¹⁴CO₂, and the rapid rise in glutamine content and fall in glutamate in leaves on transfer to air. The CO₂-fixation rate in RPr79/2 declines much more slowly on transfer from 1% O₂ to air than do the rates in glutamate-synthase-deficient plants, and RPr79/2 plants do not die in air unless the temperature and irradiance are high. Analysis of (glutamine+NH₃+2-oxoglutarate)-dependent O₂ evolution by isolated chloroplasts shows that chloroplasts from RPr79/2 require a fivefold greater concentration of 2-oxoglutarate than does the wild-type for maximum activity. The levels of 2-oxoglutarate in illuminated leaves of RPr79/2 in air are sevenfold higher than in Maris Mink. It is suggested that RPr79/2 is defective in chloroplast dicarboxylate transport.     

A study of photorespiratory ammonia production in the C4 plant Amaranthus edulis, using mutants with altered photosynthetic capacities

Previous studies have indicated that the rate of photorespiration in C₄ plants is low or negligible. In this study, wild-type and mutant leaves of the C₄ plant Amaranthus edulis were treated with the glutamine synthetase inhibitor, phosphinothricin and the glycine decarboxylase inhibitor, aminoacetonitrile, at different concentrations of CO₂. The time course of ammonia accumulation in leaves of the wild type was compared with a mutant lacking phosphoenolpyruvate carboxylase activity (EC 4.1.1.31), and with three different mutants that accumulated glycine. The increase in the concentration of ammonia in the leaves, stimulated by the treatments was used as a measurement of the rate of photorespiration in C₄ plants. The application of glutamine and glycine maintained the rate of photorespiratory ammonia production for a longer period in the wild type, and increased the rate in a mutant lacking phosphoenolpyruvate carboxylase suggesting that there was a lack of amino donors in these plants. The calculated rate of photorespiration in Amaranthus edulis wild-type leaves when the supply of amino donors was enough to maintain the photorespiratory nitrogen flow, accounted for approximately 6% of the total net photosynthetic CO₂ assimilation rate. In a mutant lacking phosphoenolpyruvate carboxylase, however, this rate increased to 48%, when glutamine was fed to the leaf, a value higher than that found in some C₃ plants. In mutants of Amaranthus edulis that accumulated glycine, the rate of photorespiration was reduced to 3% of the total net CO₂ assimilation rate. The rate of ammonia produced during photorespiration was 60% of the total produced by all metabolic reactions in the leaves. The data suggests that photorespiration is an active process in C₄ plants, which can play an important role in photosynthetic metabolism in these plants.     

Effect of L-Methionine Sulphoximine on the Products of Photosynthesis in Wheat (Triticum aestivum) Leaves

Methionine sulphoximine, an inhibitor of glutamine synthetase,caused ammonia accumulation in detached wheat leaves. The ratewas increased by increased oxygen in the atmosphere and by simultaneouslysupplying glycine or giving extra nitrate; it was decreasedby isonicotinyl hydrazide. Ammonia production was light-dependentand continued at a constant rate in air for at least 2 h. Photosynthesiswas progressively inhibited after the first hour; this inhibitionwas not because of increased stomatal resistance. Leaves suppliedwith 30 mol m^–3 ammonium chloride, without methioninesulphoximine, accumulated more ammonia than leaves treated withthe inhibitor but showed less inhibition of photosynthesis.The inhibitor decreased synthesis of [¹⁴C] amino acids from¹⁴CO₂ in the light but increased the synthesis of [¹⁴C] malateand, relatively, the incorporation of ¹⁴C into sugar phosphates.In the absence of inhibitor, nitrate increased and ammoniumion decreased synthesis of malate. Methionine sulphoximine,by causing a shortage of amino acids, probably inhibited photosynthesisin part by decreasing the recycling of carbon from the photorespiratorycycle back to the Calvin cycle. Key words: Photosynthetic ¹⁴CO₂ assimilation, Methionine sulphoximine, Detached wheat leaves     

The value of mutants unable to carry out photorespiration

Manipulation of the CO₂ concentration of the atmosphere allows the selection of photorespiratory mutants from populations of seeds treated with powerful mutagens such as sodium azide. So far, barley lines deficient in activity of phosphoglycolate phosphatase, catalase, the glycine to serine conversion, glutamine synthetase, glutamate synthase, 2-oxoglutarate uptake and serine: glyoxylate aminotransferase have been isolated. In addition one line of pea lacking glutamate synthase activity and one barley line containing reduced levels of Rubisco are available. The characteristics of these mutations are described and compared with similar mutants isolated from populations of Arabidopsis. As yet, no mutant lacking glutamine synthetase activity has been isolated from Arabidopsis and possible reasons for this difference between barley and Arabidopsis are discussed. The value of these mutant plants in the elucidation of the mechanism of photorespiration and its relationships with CO₂ fixation and amino acid metabolism are highlighted.Abbreviations GS cytoplasmic glutamine synthetase - GS₂ chloroplastic glutamine synthetase - PFR Photon fluence rate - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP Ribulose-1,5-bisphosphate - SGAT serine:glyoxylate aminotransferase     

Development of Photorespiration during Chloroplast Biogenesis in Wheat Leaves

Tobin, A. K., Sumar, N., Patel, M., Moore, A. L. and Stewart,G. R. 1988. Development of photorespiration during chloroplastbiogenesis in wheat leaves.—J. exp. Bot. 39: 833–843. The rate of light-dependent ammonia accumulation in L-methioninesulphoximine (MSO: glutamine synthetase inhibitor)-treated wheat(Triticum aestivum L. cv. Maris Huntsman) primary leaf sectionsincreased with mesophyll cell maturity. Ammonia production inthe more mature sections (beyond 2.0 cm from the basal meristem)was inhibited by elevated CO₂ concentrations and by incubationwith 10 mol m^–3 pyrid-2-yl hydroxymethane sulphonate (HPMS).In contrast, the low levels of ammonia which accumulated inthe immature sections (0 to 2.0 cm from the base) were unaffectedby such treatments. This indicates that the ammonia producedin mature wheat leaf sections is of photorespiratory originand that the capacity of this pathway increases with mesophyllcell and chloroplast development. Rates of CO₂-dependent oxygenevolution by leaf sections (under saturating CO₂) increasedin parallel with ammonia production. Levels of endogenous nitratewere relatively high and increased from 5.15 mol x 10^–13mesophyll cell^–1 in meristematic cells to 6.6 mol x 10^–12mesophyll cell^–1 in mature tissue. There was no significantchange in leaf nitrate level during 30 min light incubationof the wheat leaf sections, indicating that the majority ofthe nitrate was metabolically inactive and stored in the vacuole.Activities of key enzymes of photorespiration (glutamine synthetase,glycollate oxidase), nitrogen metabolism (nitrate reductase,glutamate dehydrogenase, glutamine synthetase) and mitochondrialrespiration (cytochrome oxidase), showed specific and distinctpatterns of development during leaf growth. Chloroplast glutaminesynthetase (GS₂) and peroxisomal glycollate oxidase developedin apparent synchrony with the major increase in activity occurringin regions beyond4.0 cm from the leaf base, i.e. where photorespirationwas developing. Cytosolic glutamine synthetase (GS₁) and nitratereductase (in vivo) activities were identical throughout leafgrowth, reaching maximum rates at 4.0 cm from the base and thenremaining constant. Activities of the mitochondrial enzymesglutamate dehydrogenase (GDH) and cytochrome oxidase were highin meristematic cells and increased in parallel, attaining amaximum towards the leaf tip. This indicated a respiratory,as opposed to a photorespiratory, role for GDH in wheat leafmetabolism. The evidence for controlled, co-ordinated synthesisof pathway enzymes at specific stages of organelle biogenesisis discussed. Key words: Photorespiration, organelle biogenesis     

Carbon Isotope Discrimination by Tissues5

The carbon isotope discrimination ratio of the floral parts,leaves, and stems of barley and oat plants were measured todetermine if net CO₂ fixation by PEP carboxylase (describedin these tissues by other workers) makes a significant contributionto total carbon fixation in these tissues. The ¹³C values rangedfrom –26.6 to –29.6% and are within the range normallyexpected for plants with the C₃ pathway of photosynthesis inwhich autotrophic CO₂ fixation proceeds via RuBP carboxylase.We conclude that PEP carboxylase does not make a substantialcontribution to autotrophic CO₂ fixation in the floral partsof these C₃ plants.     

Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase

Five mutant lines of barley (Hordeum vulgare L.), which are only able to grow at elevated levels of CO₂, contain less than 5% of the wild-type activity of ferredoxin-dependent glutamate synthase (EC 1.4.7.1). Two of these lines (RPr 82/1 and RPr 82/9) have been studied in detail. Leaves and roots of both lines contain normal activities of NADH-dependent glutamate synthase (EC 1.4.1.14) and the other enzymes of ammonia assimilation. Under conditions that minimise photorespiration, both mutants fix CO₂ at normal rates; on transfer to air, the rates drop rapidly to 15% of the wild-type. Incorporation of ¹⁴CO₂ into sugar phosphates and glycollate is increased under such conditions, whilst incorporation of radioactivity into serine, glycine, glycerate and sucrose is decreased; continuous exposure to air leads to an accumulation of ¹⁴C in malate. The concentrations of malate, glutamine, asparagine and ammonia are all high in air, whilst aspartate, alanine, glutamate, glycine and serine are low, by comparison with the wild-type parent line (cv. Maris Mink), under the same conditions. The metabolism of [¹⁴C]glutamate and [¹⁴C]glutamine by leaves of the mutants indicates a very much reduced ability to convert glutamine to glutamate. Genetic analysis has shown that the mutation in RPr 82/9 segregates as a single recessive nuclear gene.Abbreviations GDH glutamate dehydrogenase (EC 1.4.1.2) - GS glutamine synthetase (EC 6.3.1.2) - RuBP ribulose 1,5-bisphosphate     

Regulation of Nodule Glutamine Synthetase by CO(2) Levels in Bean (Phaseolus vulgaris L.)

Nodulated bean (Phaseolus vulgaris) plants were grown for 17 days after infection in normal (0.02%) CO₂ and from day 8 to 17 in high (0.1%) CO₂ in order to increase nitrogen fixation and define how nodule glutamine synthetase (GS) isoforms are regulated by the ammonia derived from the bacteroid. Nitrogenase activity was detected by day 10, and by day 17 activity was over twofold higher in 0.1% of CO₂ compared with plants grown in 0.02% CO₂ and inoculated with Rhizobium wild-type strain CE3. Likewise, plant fresh weight increased in response to increased CO₂, particularly in plants inoculated with the Rhizobium phaseoli mutant strain CFN037. Glutamine synthetase specific activity increased 2.5- to 6.5-fold from day 11 to 17. However, increased CO₂ did not appear to have an effect on GS specific activity. Analysis of the nodule GS polypeptide composition revealed that the γ polypeptide was significantly reduced in response to high CO₂, whereas the β polypeptide was not affected. The significance of this result in relation to the regulation of GS isoforms and their role in the assimilation of ammonia in the nodule is discussed in this paper.     

Effect of altitude on the primary products of photosynthesis and the associated enzymes in barley and wheat

There is little information available on the primary products of photosynthesis and the change in the activity of the associated enzymes with altitude. We studied the same in varieties of barley and wheat grown at 1300 (low altitude, LA) and 4200 m (high altitude, HA) elevations above mean sea level in the western Himalayas. Plants at both the locations had similar photosynthetic rates, leaf water potential and the chlorophyll fluorescence kinetics. The short-term radio-labelling experiments in leaves showed appearance of ¹⁴CO₂ in phosphoglyceric acid and sugar phosphates in plants at both the LA and HA, suggesting a major role of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in CO₂ fixation in the plants at two altitudes, whereas the appearance of labelled carbon in aspartate (Asp) and glutamate (Glu) at HA suggested a role of phosphoenolpyruvate carboxylase (PEPCase) in photosynthesis metabolism. Plants at HA had significantly higher activities of PEPCase, carboxylase and oxygenase activity of Rubisco, aspartate aminotransferase (AspAT), and glutamine synthetase (GS). However, the activities of malate dehydrogenase, NAD-malic enzyme and citrate synthase were similar at the two locations. Such an altered metabolism at HA suggested that PEPCase probably captured CO₂ directly from the atmosphere and/or that generated metabolically e.g. from photorespiration at HA. Higher oxygenase activity at HA suggests high photorespiratory activity. OAA thus produced could be additionally channelised for Asp synthesis using Glu as a source of ammonia. Higher GS activity ensures higher assimilation rate of NH₃ and the synthesis of Glu through GS-GOGAT (glutamine:2-oxoglutarate aminotransferase) pathway, also as supported by the appearance of radiolabel in Glu at HA. Enhanced PEPCase activity coupled with higher activities of AspAT and GS suggests a role in conserving C and N in the HA environment.     

Diurnal fluctuation of light and dark CO2 fixation in peeled and unpeeled leaves of Bryophyllum daigremontiana

Diurnal fluctuation of light and dark CO₂ fixation in peeledand unpeeled leaves of Bryophyllum daigremontiana was examined.A distinct difference in light CO₂ fixation was observed inunpeeled leaves but not in peeled ones. No measurable differencein dark CO₂ fixation was observed in either type. These resultsindicate that the leaves of CAM plants have a high capacityfor CO₂ fixation in the daytime, but it is suppressed by theclosing of the stomata. Also, the rapid depression of CO₂ uptakewhen the illumination was directed at on dark acidified leavescould be prevented by peeling off the epidermis. The net photosyntheticCO₂ uptake in peeled leaves was 77 µmoles/mg chllrophyll/hrin the 3rd leaf and 62 in the 4th leaf. (Received August 7, 1978; )     

Effect of Methionine Sulfoximine on Photosynthetic Carbon Metabolism in Wheat Leaves

Methionine sulfoximine (MSO) greatly reduced the carbon dioxideexchange rate (CER) of detached wheat (Triticum aestivvm L.cv Roland) leaves in 21% O₂, but only slightly reduced it in2% O₂. A supply of 50 mM NH₄Cl had little effect on the CERirrespective of the O₂ concentration. A simultaneous additionof glutamine and MSO protected against the inhibition of photosynthesisto a considerable extent and caused the accumulation of moreNH₃ than did the addition of MSO alone. Fixation of ¹⁴CO₂ in wheat leaves was inhibited by MSO treatmentin 22% O₂, and there was decreased incorporation of ¹⁴G intoamino acids and sugars and increased label into acid fractions.The addition of MSO and glutamine together eliminated the effectof MSO on the photosynthetic ¹⁴CO₂ fixation pattern. NH₄Cl stimulatedthe synthesis of amino acids from ¹⁴CO₂, especially the synthesisof serine in 22% O₂. Our observations show that factors other than the uncouplingof photophosphorylation by accumulated NH₃ may be responsiblefor the early stage of photosynthesis inhibition by MSO underphotorespiratory conditions. ¹Present address: Department of Agricultural Chemistry, KyushuUniversity, Fukuoka 812 Japan. ²Also at U.S. Department of Agriculture, Agricultural ResearchService, Urbana, Illionois 61801, U.S.A. (Received September 13, 1983; Accepted February 2, 1984)     

Regulation of Ribulose Bisphosphate Carboxylase Activity in Intact Wheat Leaves by Light, CO2, and Temperature

The activity of the enzyme ribulose bisphosphate carboxylase(RuBPCase) was estimated after rapidly extracting it from intactwheat leaves pretreated under different light and CO₂ levels.No HCO₃^– was added to the extraction buffer since it isshown to inhibit RuBPCase. The activity increased as light intensityor CO₂ concentration during pretreatment was increased. Enzymeactivity increased as temperature during pretreatment was decreased.Light activation did not affect the affinity of RuBPCase forCO₂. A K_m of 30 µM CO₂ under air level O₂ was determined.CO₂, light and temperature are three main limiting factors ofphotosynthesis. It seems that the activity of RuBPCase is regulatedby these factors according to the requirements for CO₂ fixation.     

Oats Tolerant of Pseudomonas syringae pv. tabaci Contain Tabtoxinine-beta-Lactam-Insensitive Leaf Glutamine Synthetases

Pseudomonas syringae pv. tabaci, a commonly recognized leaf pathogen of tobacco, can infest the rhizosphere of many plants, including oats. Normal oat plants do not survive this infestation as a consequence of the complete and irreversible inactivation of all of their glutamine synthetases by tabtoxinine-β-lactam (TβL), a toxin released by pv. tabaci. We have identified a population of oat (Avena sativa L. var Lodi) plants that are tolerant of pv. tabaci. The tolerant plants had no detectable TβL-detoxification mechanisms. Pathogen growth on these plant roots was not inhibited. These plants contain leaf glutamine synthetases (GS₁ and GS₂) that were less sensitive to inactivation by TβL in vitro; these GSs have normal K_m values for glutamate and ATP when compared with those of GS in control plants. Root glutamine synthetase of the tolerant plants was inactivated in vivo during infestation by the pathogen or by TβL in vitro. When growing without pv. tabaci, the tolerant plants contained normal levels of glutamine synthetase in their roots and leaves and normal levels of protein, ammonia, glutamate, and glutamine in their leaves. However, when the tolerant plants' rhizosphere was infested with pv. tabaci, the plant leaves contained elevated levels of glutamine synthetase activity, protein, ammonia, glutamate, and glutamine. No changes in glutamate dehydrogenase activity were detected in leaves and roots of pathogen-infested tolerant plants.     

Transformation of atmospheric NO2 absorbed in spinach leaves

NO₂ fumigation at 8 ppm of spinach plants resulted in nitriteaccumulation in the leaves in the dark but not in the light.When spinach plants were fumigated with ₁₅N-labeled NO₂ in thelight, amide nitrogen of glutamine, glutamic acid, -amino butyricacid and aspartic acid, in this order, were highly labeled with¹⁵N and nitrate was also labeled. These results suggest thatNO₂-nitrogen (at least some of it) is converted into nitriteand nitrate, and then actively assimilated into amino acidsthrough the glutamine synthetase/glutamate synthase pathwayin spinach leaves. ¹This work was conducted as a part of the special research project"Studies on evaluation and amelioration of air pollution byplants" (1976–1978) at the National Institute for EnvironmentalStudies. (Received July 24, 1978; )     

Effects of elevated CO2 concentration and climate-warming on photosynthesis during winter in Lolium perenne

Long-term effects of atmospheric carbon dioxide concentration(ambient or 700 µmol mol^–1) and air temperature(simulation of field conditions or + 4 C) on leaf photosyntheticrate were examined in Lolium perenne L. cv. Vigor, exposed tonatural illumination during winter. Photosynthetic capacitywas compared over a range of air temperatures and photon fluxdensities of photosynthetically active radiation which wererepresentative of winter climate (5–15 C and 0–500µmol m^–2s^–1), with CO₂ level during measurementsimilar to that during the experimental period. Long-term exposureto increased air temperature reduced leaf CO₂ fixation capacityby 23% (averaged over all measurement conditions), resultingfrom a decline in lightsaturated uptake rate, but not in incident-lightquantum efficiency. CO₂- stimulation was largely absent in plantsgrown in ambient temperature, but pronounced in plants grownunder +4 C, where it compensated for two-thirds of the 23%drop. This enhancing effect of elevated CO₂ level on leaf CO₂uptake rate observed in the warmer treatment, was strongly dependenton measurement temperature, increasing from 5% at 5 C, to upto 32% at 15 C. Measurements of chlorophyll fluorescence anddry matter corresponded with the observed changes in assimilationcapacity, which could not be attributed to a deteriorated nitrogenstatus of the leaves as there was a similar N content on anarea basis. Several hypotheses are considered to explain theobserved CO₂-temperature interactions. Key words: Acclimation, chlorophyll fluorescence, elevated CO₂ level, global warming, low temperature     

Effects of tabtoxin on nitrogen metabolism

Tabtoxin is a chlorosis-inducing toxin produced by the plant pathogenic bacterium Pseudomonas syringae pv. tabaci. Previous studies have indicated that tabtoxin inhibits glutamine synthetase (EC 6.3.1.2) in vitro. We report here that tabtoxin also inhibits glutamine synthetase in vivo. The main evidence was that assimilation of exogenous ¹⁵NH₃ into Asparagus sprengeri protein was rapidly inhibited in isolated cells exposed to tabtoxin. This was associated with an equivalent decline in glutamine synthetase activity in extracts of these cells and the accumulation of extracellular ammonia. Glutamine synthetase was also inhibited in leaves of Nicotiana tabacum L. cv. White Burley treated with tabtoxin and the affected tissue accumulated ammonia and became chlorotic. However, the development of symptoms and accumulation of ammonia was suppressed when the leaves were held in air containing 1% CO₂ to reduce photorespiration. This indicates that the chlorotic symptom did not result from the inhibition of nitrogen assimilation but was a consequence of the interruption of the photorespiratory nitrogen cycle.