Nitrogen Nutrition of Nodules in Relation to ;N-Hunger' in Cowpea (Vigna unguiculata L. Walp)

Early growth, nodule development, and nitrogen fixation by two cultivars of cowpea (Vigna unguiculata L. Walp), one large-seeded (Vita 3; 146.0 ± 0.9 milligrams seed dry weight, 4.1 ± 0.2 milligrams seed N), the other small-seeded (Caloona; 57.5 ± 2.5 milligrams seed dry weight, 1.8 ± 0.1 milligrams seed N), were compared under conditions of sand culture with nutrient solution free of combined N. The seed stocks used had been obtained from plants uniformly labeled with ¹⁵N, thus enabling changes with time in distribution of cotyledon and fixed N among plant parts to be measured by isotope dilution. Caloona, but not Vita 3, showed physiological symptoms of `N hunger,' i.e. transient loss of chlorophyll (visible yellowing) and N from the first-formed unifoliolate leaves at or around the onset of symbiotic functioning and N<sub>2</sub> fixation. The smaller-seeded Caloona showed higher early nitrogenase activity than the larger-seeded Vita 3 and by 28 days had fixed 6.6 milligrams of N per milligram of seed N [mg N · (mg seed N)<sup>−1</sup>] versus only 3.5 mg N · (mg seed N)<sup>−1</sup> in Vita 3. Both cultivars lost around 30% of their initial seed N at germination, mostly as fallen cotyledons. Abscised cotyledons of Caloona contained 1.21 ± 0.17% N; those of Vita 3 contained 2.61 ± 0.37% N. When compared on the basis of cotyledon N available for seedling growth, Caloona was shown to have fixed 10.6 mg N · (mg seed N)<sup>−1</sup> and Vita 3 only 5.3 mg N · (mg seed N)<sup>−1</sup>. Most of the cotyledon N withdrawn from the unifoliolate leaf pair of Caloona during `N-hunger' was committed to early nodule growth and, in total, 20 to 25% of the cotyledon N resource of this cultivar was ultimately invested in establishment of symbiosis compared with only 7% in Vita 3.     

Diurnal variation in the functioning of cowpea nodules

Nitrogenase (EC 1.7.99.2) activity of nodules of cowpea (Vigna unguiculata [L.] Walp), maintained under conditions of a 12-hour day at 30°C and 800 to 1,000 microeinsteins per square meter per second (photosynthetically active radiation) and a 12-hour night at 20°C, showed a marked diurnal variation with the total electron flux through the enzyme at night being 60% of that in the photoperiod. This diurnal pattern was, however, due to changes in hydrogen evolution. The rate of nitrogen fixation, measured by short-term ¹⁵N₂ assimilation or estimated from the difference in hydrogen evolution in air or Ar:O₂ (80:20; v/v), showed no diurnal variation. Carbon dioxide released from nodules showed a diurnal variation synchronized with that of nitrogenase functioning and, as a consequence, the apparent `respiratory cost' of nitrogen fixation in the photoperiod was almost double that at night (9.74 ± 0.38 versus 5.70 ± 0.90 moles CO₂ evolved per mole N₂ fixed). Separate carbon and nitrogen balances constructed for nodules during the photoperiod and dark period showed that, at night, nodule functioning required up to 40% less carbohydrate to achieve the same level of nitrogen fixation as during the photoperiod (2.4 versus 1.4 moles hexose per mole N₂ fixed).

Stored reserves of nonstructural carbohydrate of the nodule only partly satisfied the requirement for carbon at night, and fixation was dependent on continued import of translocated assimilates at all times. Measurements of the soluble nitrogen pools of the nodule together with ¹⁵N studies indicated that, both during the day and night, nitrogenous products of fixation were effectively translocated to all organs of the host plant despite low rates of transpiration at night. Reduced fluxes of water through the plant at night were apparently counteracted by increased concentration of nitrogen, especially as ureides, in the xylem stream.

     

Experimental determination of the respiration associated with soybean/rhizobium nitrogenase function, nodule maintenance, and total nodule nitrogen fixation

The total metabolic cost of soybean (Glycine max L. Mer Clark) nodule nitrogen fixation was empirically separated into respiration associated with electron flow through nitrogenase and respiration associated with maintenance of nodule function.

Rates of CO₂ evolution and H₂ evolution from intact, nodulated root systems under Ar:O₂ atmospheres decreased in parallel when plants were maintained in an extended dark period. While H₂ evolution approached zero after 36 hours of darkness at 22°C, CO₂ evolution rate remained at 38° of the rate measured in light. Of the remaining CO₂ evolution, 62% was estimated to originate from the nodules and represents a measure of nodule maintenance respiration. The nodule maintenance requirement was temperature dependent and was estimated at 79 and 137 micromoles CO₂ (per gram dry weight nodule) per hour at 22°C and 30°C, respectively.

The cost of N₂ fixation in terms of CO₂ evolved per electron pair utilized by nitrogenase was estimated from the slope of H₂ evolution rate versus CO₂ evolution rate. The cost was 2 moles CO₂ evolved per mole H₂ evolved and was independent of temperature.

In this symbiosis, nodule maintenance consumed 22% of total respiratory energy while the functioning of nitrogenase consumed a further 52%. The remaining respiratory energy was calculated to be associated with ammonia assimilation, transport of reduced N, and H₂ evolution.

     

Photosynthesis and Respiration of Developing Populus tremuloides Internodes

The photosynthetic and respiratory performance of developing internodes of Populus tremuloides was evaluated by infrared gas analysis. Anatomical and morphological transitions were related to metabolic activity. Photosynthetic rates ranged from 6.0 to 10.0 milligrams CO₂ per decimeter squared per hour in the youngest internodes to 2.5 to 3.8 milligrams CO₂ per decimeter squared per hour in internodes with fully developed bark tissues. Respiration exceeded the rate of photosynthesis on the average by a factor of two. Stem photosynthesis increased with temperature up to 40°C and declined steeply between 40 and 50°C. Stem respiration increased nearly linearly to temperatures as high as 50°C.     

Significance of hydrogen evolution in the carbon and nitrogen economy of nodulated cowpea

The carbon and nitrogen economies of a single cultivar of cowpea (Vigna unguiculata (L.) Walp.cv Caloona) nodulated with either a high H₂-evolving strain (176A27) or a low H₂-evolving strain (CB756) of Rhizobium were compared. The two symbioses did not differ in total dry matter production, seed yield, nitrogen fixed, the spectrum of nitrogenous solutes produced by nodules for export, or the partitioning of net photosynthate within the plant throughout the growth cycle. Detailed examination of the carbon and nitrogen economy of the nodules, however, showed a significant difference between the symbioses. Nodules formed with CB756 lost less CO₂ in respiration compared to the higher H₂-evolving symbioses and this could have been largely responsible for a 36% better economy of carbon use in CB756 nodules during the period of maximum H₂ evolution (48-76 days) and over the whole growth period (20-90 days), a 16% economy. In terms of overall net photosynthate generated by the plant, these economies were equivalent to 5% and 2% of the carbon utilized in the two periods, respectively. From the differences in H₂ evolution and CO₂ production by nodules of the two symbioses, the cost of H₂ evolution was found to be 3.83±0.6 millimoles CO₂/millimoles H₂ for plants grown in sand culture and 1.69 ± 0.48 millimoles CO₂/millimoles H₂ for those in water culture. In both symbioses, the ratio of H₂ evolution to N₂ fixed varied markedly during ontogeny, indicating a significant variation in the relative efficiency and thus metabolic cost of N₂ fixation at different stages during development.     

Effects of Glycolate Pathway Intermediates on Glycine Decarboxylation and Serine Synthesis in Pea (Pisum sativum L.)

The study aimed to test the hypothesis that ammonia production by Rhizobium bacteroids provides not only a source of nitrogen for growth but has a central regulatory role in maintaining the metabolic activity and functional integrity of the legume nodule. Production of ammonia in intact, attached nodules was interrupted by short-term (up to 3 days) exposure of the nodulated root systems of cowpea (Vigna unguiculata L. Walp cv Vita 3: Rhizobium CB 756) and lupin (Lupinus albus L. cv Ultra: Rhizobium WU 425) to atmospheres of argon:oxygen (80:20; v/v). Treatment did not affect nodule growth, levels of plant cell and bacteroid protein, leghaemoglobin content, or nitrogenase (EC 1.7.99.2) activity (acetylene reduction) but severely reduced (by 90%) synthesis and export of the major nitrogenous solutes produced by the two symbioses (ureides in cowpea, amides in lupin). Glutamine synthetase (EC 6.3.1.2) and NAD:glutamate oxidoreductase (EC I.4.1.2) were more or less stable to Ar:O₂ treatment, but activities of the glutamine-utilizing enzymes, glutamate synthase (EC 2.6.1.53), asparagine synthetase (EC 6.3.5.4) (lupin only), and de novo purine synthesis (cowpea only), were all markedly reduced. Production and export of nitrogenous solutes by both symbioses resumed within 2 hours after transferring Ar:O₂-treated plants back to air. In each case the major exported product of fixation after transfer was initially glutamine, reflecting the relative stability of glutamine synthetase activity. Subsequently, glutamine declined and products of its assimilation became predominant consistent with resurgence of enzymes for the synthesis of asparagine in lupin and ureides in cowpea. Enzymes not directly involved with either ammonia or glutamine assimilation (purine synthesis, purine oxidation, and carbon metabolism of both bacteroids and plant cells) also showed transient changes in activity following interruption of N₂ supply. These data have been interpreted to indicate a far-reaching effect of the production of ammonia by bacteroids on a wide range of enzymes, possibly through control of protein turnover, rather than a highly specific effect of ammonia, or some product of its assimilation, on a few enzyme species.     

Conservation in Soil of H2 Liberated from N2 Fixation by Hup- Nodules

Pigeon peas (Cajanus cajan) were grown in large soil columns (90-cm length by 30-cm diameter) and inoculated with four different strains of cowpea rhizobia, which varied with respect to hydrogen uptake activity (Hup). Despite the profuse liberation of H₂ from Hup^- nodules in vitro, H₂ gas was not detected in any of the soil columns. When H₂ was injected into the columns, the rates of consumption were highest in the treatments (including control) containing Hup^- nodules (218 and 177 nmol · h⁻¹ · cm⁻²) and lowest in the Hup⁺ treatments (158, 92, and 64 nmoles · h⁻¹ · cm⁻²). In situ H₂ uptake rates in small soil cores at fixed distances from the nodules decreased exponentially with distance from the nodule (R² = 0.99). This decrease in H₂ consumption was associated with a similar decrease in numbers of H₂-oxidizing chemolithotrophic bacteria as determined by the most-probable-number method. On the basis of two equations derived separately upon diffusive theory (Fix's Law) and kinetic theory (Michaelis-Menten), the empirically derived rate constants and coefficients indicated that all of the H₂ emitted from Hup^- nodules would be consumed by H₂-oxidizing bacteria within a 3- to 4.5-cm radius of the nodule surface. It is concluded that H₂ is not lost from the soil-plant ecosystem during N₂ fixation in C. cajan but is conserved by H₂-oxidizing bacteria.     

Effect of temperature on nitrogenase functioning in cowpea nodules

Nitrogenase (EC 1.7.99.2) activity of a cowpea (Vigna unguiculata (L.) Walp cv Caloona) symbiosis formed with a Rhizobium strain (176A27) lacking uptake hydrogenase and maintained under conditions of a 12-hour day at an air temperature of 30°C (800-1000 microeinsteins per square meter per second) and a 12-hour night at an air temperature of 20°C showed a marked diurnal variation in ratio of nitrogen fixed to hydrogen evolved. As little as 0.3 micromole nitrogen was fixed per micromole hydrogen evolved in the photoperiod versus up to 0.6 in the dark period. In plants maintained under the same diurnal illumination regime but at constant (day and night) air temperature (30°C), this difference was abolished and a relatively constant ratio of nitrogen fixed to hydrogen evolved (around 0.3 micromole per micromole) was observed day and night. Exposure of nodulated roots to a range of temperatures maintained for 2 hours in a single photoperiod indicated that, whereas hydrogen evolution increased with increasing temperature from 15°C to a maximum around 35°C, nitrogen fixation was largely unaffected over this temperature range. Both functions of the enzyme declined sharply at temperatures above 38°C. A similar general response of nitrogen fixation to root temperature was observed in glasshouse-grown, sand-cultured plants maintained under a range of temperatures (from 15 to 35°C) for a 14-day period in mid vegetative growth. The effect of temperature on the proportion of electrons allocated to proton reduction compared with nitrogen reduction showed a linearly increasing relationship (correlation coefficient = 0.96) between 15°C and 47°C.     

Carbon Dioxide Fixation by Lupin Root Nodules: I. Characterization, Association with Phosphoenolpyruvate Carboxylase, and Correlation with Nitrogen Fixation during Nodule Development

In vivo CO₂ fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin (Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N₂ fixation. PEP carboxylase activity was predominantly located in the bacteroid-containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO₂ fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO₂ fixation in mature nodules was 7.9 nmol hour⁻¹ mg fresh weight⁻¹, similar to rates of N₂ fixation and reported values for amino acid translocation.     

Nodulation of Pole Bean (Phaseolus vulgaris L.) by Rhizobium Species of Two Cross-Inoculation Groups

Physiology and morphology of pole bean (Phaseolus vulgaris L. cv Kentucky Wonder) root nodules induced by two Rhizobium species of different cross-inoculation groups have been compared. Root nodules induced by Rhizobium sp. 127E15, which is a strain of the cowpea group Rhizobium, were pinkish, had irregular shapes, and were only partially effective. Their peak acetylene reduction activity was 4.36 μmol of C₂H₄ formed per g of fresh nodules per h at 30 days after inoculation. The effective nodules induced by Rhizobium phaseoli 127K14, which is a strain of the bean group Rhizobium, were dark red, spherical, and showed peak acetylene reduction activity of 15.95 μmol of C₂H₄ formed per g of fresh nodules per h at 15 days after inoculation. The partial effectiveness of 127E15-induced nodules was associated with fewer infected cells, a delay in the increase of bacteroid population within the host cells, abundance of cytoplasmic vesicles in the host cells, more bacteroids within a membrane envelope (peribacteroid membrane), and the inability of bacteroids to completely fill up the host cytoplasmic space. The 127K14-induced nodules were fully mature, with host cells filled with bacteroids by 12 days after inoculation. In contrast, the 127E15-induced nodules did not reach a similar developmental stage even 30 days after inoculation.     

Relationship between N(2)-Fixing Efficiency and Natural N Enrichment of Soybean Nodules

Non-nodular tissue of soybean (Glycine max L. Merrill) plants grown hydroponically in the absence of added N have a ¹⁵N abundance close to that of atmospheric N₂. In contrast, nodules are usually enriched in ¹⁵N. In this paper, we report measurements of the ¹⁵N abundance of foliar tissue and nodules of soybeans inoculated with 11 variably efficient strains of Rhizobum japonicum and grown hydroponically with no added N. The efficiency of the 11 symbioses varied over a wide range as judged by a 16-fold difference in N content. The degree of ¹⁵N enrichment of nodules was closely correlated with N₂-fixing efficiency (milligrams N fixed per milligram N in the nodules).

These results confirm prior preliminary data based on six variably efficient R. japonicum strains. The strong correlation between ^NN enrichment of soybean nodules and N₂-fixing efficiency is consistent with the hypothesis that new nodule tissue is synthesized from a pool of recently fixed N within the same nodule.

     

Short-Term Effects of CO(2) on Gas Exchange of Leaves of Bigtooth Aspen (Populus grandidentata) in the Field

The short term effects of increased levels of CO₂ on gas exchange of leaves of bigtooth aspen (Populus grandidentata Michx.) were studied at the University of Michigan Biological Station, Pellston, MI. Leaf gas exchange was measured in situ in the upper half of the canopy, 12 to 14 meters above ground. In 1900 microliters per liter CO₂, maximum CO₂ exchange rate (CER) in saturating light was increased by 151% relative to CER in 320 microliters per liter CO₂. The temperature optimum for CER shifted from 25°C in 320 microliters per liter CO₂ to 37°C in 1900 microliters per liter CO₂. In saturating light, increasing CO₂ level over the range 60 to 1900 microliters per liter increased CER, decreased stomatal conductance, and increased leaf water use efficiency. The initial slope of the CO₂ response curve of CER was not significantly different at 20 and 30°C leaf temperatures, although the slope did decline significantly during leaf senescence. In 1900 microliters per liter CO₂, CER increased with increasing light. The light saturation point and maximum CER were higher in 30°C than in 20°C, although there was little effect of temperature in low light. The experimental results are consistent with patterns seen in laboratory studies of other C₃ species and define the parameters required by some models of aspen CER in the field.     

Calorimetry of nitrogenase-mediated reductions in detached soybean nodules

Heat evolved by isolated soybean (Glycine max cv Clark) nodules was measured to estimate more directly the metabolic cost associated with the symbiotic N₂ fixation system. A calorimeter constructed by modifying standard laboratory equipment allowed measurement on 1 gram of detached nodules under a controlled gas stream. Simultaneous gas balance and heat output determinations were made.

There was major heat output by nodules for all of the nitrogenase substrates tested (H⁺, N₂, N₂O, and C₂H₂) further establishing the in vivo energy inefficiency of biological N₂ fixation. Exposure to a short burst of 100% O₂ partially inactivated nitrogenase to permit calculations of heat evolved per mole of substrate reduced. The specific rate of heat evolution for H⁺ reductions was 171 ± 6 kilocalories per mole H₂ evolved in an Ar-O₂ atmosphere, that for N₂ fixation was 784 ± 26 kilocalories per mole H₂ evolved and N₂ fixed, and that for C₂H₂ reduction was 250 ± 12 kilocalories/mole C₂H₄ formed. When the appropriate thermodynamic parameters are taken into account for the different substrates and products, a ΔH′ of −200 kilocalories per mole 2e⁻ is shown to be associated with active transfer of electrons by the nitrogenase system. These values lead to a calculated N₂ fixation cost of 9.5 grams glucose per gram N₂ fixed or 3.8 grams C per gram N₂, which is in close agreement with earlier calculations based on nodular CO₂ production.

     

Mineral Soils as Carriers for Rhizobium Inoculants

Mineral soil-based inoculants of Rhizobium meliloti and Rhizobium phaseoli survived better at 4°C than at higher temperatures, but ca. 15% of the cells were viable at 37°C after 27 days. Soil-based inoculants of R. meliloti, R. phaseoli, Rhizobium japonicum, and a cowpea Rhizobium sp. applied to seeds of their host legumes also survived better at low temperatures, but the percent survival of such inoculants was higher than peat-based inoculants at 35°C. Survival of R. phaseoli, R. japonicum, and cowpea rhizobia was not markedly improved when the cells were suspended in sugar solutions before drying them in soil. Nodulation was abundant on Phaseolus vulgaris derived from seeds that had been coated with a soil-based inoculant and stored for 165 days at 25°C. The increase in yield and nitrogen content of Phaseolus angularis grown in the greenhouse was the same with soil-and peat-based inoculants. We suggest that certain mineral soils can be useful and readily available carriers for legume inoculants containing desiccation-resistant Rhizobium strains.     

Purification and Characterization of NADH-Glutamate Synthase from Alfalfa Root Nodules

Glutamate synthase (GOGAT), a key enzyme in the pathway for the assimilation of symbiotically fixed dinitrogen (N₂) into amino acids in alfalfa (Medicago sativa L.) root nodules, was purified and used to produce high titer polyclonal antibodies. Purification resulted in a 208-fold increase in specific activity to 13 micromole per minute per milligram of protein and an activity yield of 37%. Further purification to near homogeneity was achieved by fast protein liquid chromatography, but with substantial loss of activity. Enzymic activity was highly labile, losing 3% per hour even when substrates, stabilizers, and reducing agents were included in buffers. However, activity could be partially stabilized for up to 1 month by storing GOGAT at −80°C in 50% glycerol. The subunit molecular weight of GOGAT was estimated at 200 ± 7 kilodaltons with a native molecular weight of 235 ± 16 kilodaltons, which suggested that GOGAT is a monomer of unusually high molecular weight. The pl was estimated to be 6.6. The K_m values for glutamine, α-ketoglutarate, and NADH were 466, 33, and 4.2 micromolar, respectively. Antibodies were produced to NADH-GOGAT. Specificity of the antibodies was shown by immunotitration of GOGAT activity. Alfalfa nodule NADH-GOGAT antibodies cross-reacted with polypeptides of a similar molecular weight in a number of legume species. Western blots probed with anti-GOGAT showed that the high GOGAT activity of nodules as compared to roots was associated with increased levels of GOGAT polypeptides. Nodule NADH-GOGAT appeared to be highly expressed in effective nodules and little if any in other organs.     

Effect of pO(2) during Growth on the Gaseous Diffusional Properties of Nodules of Cowpea (Vigna unguiculata L. Walp.)

Adaptations of nodules of cowpea (Vigna unguiculata L. Walp. cv Vita 3: Bradyrhizobium CB 756) to growth in pO₂ ranging from 1 to 80% O₂ (volume/volume) involved both readily reversible mechanisms of adjustment and more stable alterations which together resulted in nodules with widely ranging resistance to diffusion of gases. Those grown in subambient pO₂ (1-5% O₂ were altered such that rapid diffusional adjustment was unable to prevent irreversible loss of nitrogenase on their transfer to higher levels of O₂. Those cultured in 80% had adapted to over-supply of O₂ such that their transfer to lower levels of O₂ limited both nitrogenase and respiratory CO₂ release. There was also some evidence for `protective respiration.' Measurement of diffusional properties based on gas exchange kinetics indicated that gaseous permeability values for nodules from 5 to 40% O₂ were relatively constant around 20 × 10⁻³ millimeters per second, while those for nodules from 1% O₂ were as high as 67.7 × 10⁻³ millimeter per second and from 80% as low as 6.8 × 10⁻³ millimeters per second. Estimates of the thickness of the diffusion barrier ranged from 7.5 micrometers for nodules from 1% O₂ to 71.9 micrometers in those from 80% O₂.     

Characterization of Rhizobia from Ineffective Alfalfa Nodules: Ability to Nodulate Bean Plants [Phaseolus vulgaris (L.) Savi.]

This study was initiated to characterize Rhizobium isolates obtained from root nodules of ineffectively nodulated, field-grown alfalfa (Medicago sativa L.) plants. The purpose was to determine if these isolates possessed characteristics which would explain either their ineffectiveness in N₂ fixation or their apparent ability to tolerate the moderately acid soil conditions from which they originated. Isolates were characterized by analysis of growth rate, 39°C tolerance, acid production on conventional media, and symbiotic performance. All isolates were ineffective in N₂ fixation on alfalfa, and they contained one or more anomalous characteristics. These included either slow growth rate, lack of 39°C tolerance, or lack of acid production on conventional media. Infectiveness tests on a broad range of legumes revealed that the isolates formed root nodules on M. sativa, Medicago lupulina L., and Phaseolus vulgaris (L.) Savi. (common bean). These results provide evidence that, in some situations, ineffective nodulation of M. sativa in the field may be due to the presence of promiscuous, native Rhizobium species.     

Effects of Temperature and CO(2) Enrichment on Carbon Translocation of Plants of the C(4) Grass Species Echinochloa crus-galli (L.) Beauv. from Cool and Warm Environments

Plants of Echinochloa crus-galli from Québec and Mississippi were grown under two thermoperiods (28°C/22°C, 21°C/15°C) and two atmospheric CO₂ concentrations (350 and 675 microliters per liter) to examine possible differential responses of northern and southern populations of this C₄ grass species. Translocation was monitored using radioactive tracing with short-lived ¹¹C. CO₂ enrichment induced a decrease in the size of the export pool in plants of both populations. Other parameters did not strongly respond to elevated CO₂. Low temperature reduced translocation drastically for plants from Mississippi in normal CO₂ concentration, but this reduction was ameliorated at high CO₂. Overall, plants from Québec had a higher ¹¹C activity in leaf phloem and a higher percentage of ¹¹C exported, whereas these northern plants had lower turnover time and smaller pool size than plants from the southern population.     

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.     

Low growth temperature effects a differential inhibition of photosynthesis in spring and winter wheat

In vivo room temperature chlorophyll a fluorescence coupled with CO₂ and O₂ exchange was measured to determine photosynthetic limitation(s) for spring and winter wheat (Triticum aestivum L.) grown at cold-hardening temperatures (5°C/5°C, day/night). Plants of comparable physiological stage, but grown at nonhardening temperatures (20°C/16°C, day/night) were used in comparison. Winter wheat cultivars grown at 5°C had light-saturated rates of CO₂ exchange and apparent photon yields for CO₂ exchange and O₂ evolution that were equal to or greater than those of winter cultivars grown at 20°C. In contrast, spring wheat cultivars grown at 5°C showed 35% lower apparent photon yields for CO₂ exchange and 25% lower light-saturated rates of CO₂ exchange compared to 20°C grown controls. The lower CO₂ exchange capacity is not associated with a lower efficiency of photosystem II activity measured as either the apparent photon yield for O₂ evolution, the ratio of variable to maximal fluorescence, or the level of reduced primary quinone electron acceptor maintained at steady-state photosynthesis, and is most likely associated with carbon metabolism. The lower CO₂ exchange capacity of the spring cultivars developed following long-term exposure to low temperature and did not occur following over-night exposure of nonhardened plants to 5°C.