Symbiotic properties of Lotus pedunculitis root nodules induced by Rhizobium loti and Bradyrhizobium sp. (Lotus)

Vance, C. P., Reibach, P. H. and Pankhurst, C. E. 1987. Symbiotic properties of Lotus pedunculatus root nodules induced by Rhizobium loti and Bradyrhizobium sp. ( Lotus ).
Symbiotic properties of root nodules were evaluated in glasshouse-grown Lotus pedunculatus Cav. cv. Maku inoculated with either a fast-growing Rhizobium loti strain NZP2037 or a slow-growing Bradyrhizobium sp. ( Lotus ) strain CC814s. Although the nodule mass of plants inoculated with NZP2037 was twice that of plants inoculated with CC814s, the yield of NZP2037 shoots and roots was 50% that of CC814s shoots and roots. Nodules induced by Bradyrhizobium fixed substantially more N than nodules induced by R. loti. Glucose requirements [mol glucose (mol N₂ fixed)^-1] of nodules induced by CC814s and NZP2037 were 7.1 and 16.6, respectively. Nodule enzymes of carbon and nitrogen assimilation reflected the disparity of the two sym-bioses. Xylem sap of the symbiosis with the higher yield contained a higher concentration of asparagine [9.86 μmol (ml xylem sap)'] than did the lower yielding symbiosis [5.80 umol (ml xylem sap)"']. Nodule CO₂ fixation was directly linked to nodule N assimilation in both symbioses. The results indicate that the difference between the two symbioses extend to nodule N and C assimilation and whole plant N transport. The data support a role for host plant modulation of bacterial efficiency and assimilation of fixed N.     

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

Summary The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH₄NO₃ (50 to 250 g N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N.Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.     

Symbiotic effectiveness of phage-resistant mutants of two strains of Lotus rhizobia

Summary Phage-resistant mutants were obtained from a fast-growing (NZP2037) and a slow-growing (CC814s) strain of Rhizobium nodulating Lotus. All the mutants were stable and did not differ from the original parent strain in their cultural characteristics. OnLotus pedunculatus the mutants of NZP2037 were as effective in N-fixation as the parent strain but most mutants of CC814s were less effective. These mutants of CC814s were also less effective than the parent strain on several other host plants.     

THE EFFECT OF CO2 ENRICHMENT ON ROOT NODULE DEVELOPMENT AND SYMBIOTIC N2 REDUCTION IN PISUM SATIVUM L.

A very significant increase in N₂(C₂H₂) reduction by Visum sativum L. infected with Rhizobium leguminosarum occurred when plants were grown in the light with 6 hr of CO₂ enrichment (0.00120 atm). Plants grown for 4 wk under 0.00120 atm CO₂ showed significant increases over control plants at 0.00032 atm CO₂ in plant dry weight, N content, root nodule mass, number of nodules, and mean nodule dry weight. Acetylene-reduction assays, however, revealed no reproducible increase in nitrogenase activity/mg nodule in plants subjected to long-term CO₂ enrichment. Both control and CO₂-enriched plants optimized the sink/source ratio between the mass of nodules and the extranodular plant mass. The optimum ratio for N₂ reduction by 4-week-old peas was 0.05. Long-term CO₂ enrichment did not promote root nodule formation to a greater degree than total plant development, and increases in N content were directly proportional to increases in nodule mass. Morphological data revealed significantly greater deposits of starch in root nodules of plants grown under CO₂-enriched conditions. The results are interpreted as showing that short-term increases in CO₂ levels promote N₂ reduction by affecting root nodule functioning, whereas long-term CO₂ enrichment promotes N₂ reduction by increasing total plant and root nodule development.     

Effects of elevated temperature and carbon dioxide on seed-set and yield of kidney bean (Phaseolus vulgaris L.)

It is important to quantify and understand the consequences of elevated temperature and carbon dioxide (CO₂) on reproductive processes and yield to develop suitable agronomic or genetic management for future climates. The objectives of this research work were (a) to quantify the effects of elevated temperature and CO₂ on photosynthesis, pollen production, pollen viability, seed‐set, seed number, seeds per pod, seed size, seed yield and dry matter production of kidney bean and (b) to determine if deleterious effects of high temperature on reproductive processes and yield could be compensated by enhanced photosynthesis at elevated CO₂ levels. Red kidney bean cv. Montcalm was grown in controlled environments at day/night temperatures ranging from 28/18 to 40/30 °C under ambient (350 µmol mol^?1) or elevated (700 µmol mol^?1) CO₂ levels. There were strong negative relations between temperature over a range of 28/18–40/30 °C and seed‐set (slope, ? 6.5% °C^?1) and seed number per pod (? 0.34 °C^?1) under both ambient and elevated CO₂ levels. Exposure to temperature > 28/18 °C also reduced photosynthesis (? 0.3 and ? 0.9 µmol m^?2 s^?1 °C^?1), seed number (? 2.3 and ? 3.3 °C^?1) and seed yield (? 1.1 and ? 1.5 g plant^?1 °C^?1), at both the CO₂ levels (ambient and elevated, respectively). Reduced seed‐set and seed number at high temperatures was primarily owing to decreased pollen production and pollen viability. Elevated CO₂ did not affect seed size but temperature > 31/21 °C linearly reduced seed size by 0.07 g °C^?1. Elevated CO₂ increased photosynthesis and seed yield by approximately 50 and 24%, respectively. There was no beneficial interaction of CO₂ and temperature, and CO₂ enrichment did not offset the negative effects of high temperatures on reproductive processes and yield. In conclusion, even with beneficial effects of CO₂ enrichment, yield losses owing to high temperature (> 34/24 °C) are likely to occur, particularly if high temperatures coincide with sensitive stages of reproductive development.     

Reversal of ethylene action on cocklebur seed germination in relation to duration of pre-treatment soaking and temperature

Abstract At 23°C, both C₂H₄ and CO₂ stimulated the germination of freshly imbibed upper cocklebur (Xanthium pennsylvanicum Wallr.) seeds, but C₂H₄, unlike CO₂, changed to an inhibitor of germination under some soaking conditions. However, when seeds were pre-soaked for more than several hours at 23 °C prior to treatment, C₂H₄ strongly inhibited their germination at 33 °C, the degree of inhibition increasing with the duration of pre-soaking. Maximum inhibition occurred at 1–3 cm³ m^?3 C₂H₄ when seeds were pre-soaked for 1 week; further increases of C₂H₄ concentration and pre-soaking period decreased the inhibitory effect. C₂H₄ was synergistic with CO₂ when C₂H₄ promoted germination, whereas it was antagonistic when inhibitory. Such a transition of the C₂H₄ action occurred at ca. 27 °C. Also 1-andnocyclopropane-1-carboxylic acid, a C₂H₄ precursor, inhibited the germination of pre-soaked seeds at 33 °C, although it promoted the germination at 23 °C. When pre-soaked seeds were prepared for germination by chilling at 8 °C for 3 d, the inhibitory effect of C₂H₄ on the subsequent germination was manifested even at 23 °C. The reversal of the C₂H₄ action from promotion to inhibition in cocklebur seed germination is discussed in relation to the engagement of two respiratory pathways in the imbibed seeds.     

Interactive effects of low atmospheric CO2 and elevated temperature on growth, photosynthesis and respiration in Phaseolus vulgaris

For most of the past 250 000 years, atmospheric CO₂ has been 30–50% lower than the current level of 360 μmol CO₂ mol^–1 air. Although the effects of CO₂ on plant performance are well recognized, the effects of low CO₂ in combination with abiotic stress remain poorly understood. In this study, a growth chamber experiment using a two-by-two factorial design of CO₂ (380 μmol mol^–1, 200 μmol mol^–1) and temperature (25/20 °C day/night, 36/29 °C) was conducted to evaluate the interactive effects of CO₂ and temperature variation on growth, tissue chemistry and leaf gas exchange of Phaseolus vulgaris. Relative to plants grown at 380 μmol mol^–1 and 25/20 °C, whole plant biomass was 36% less at 380 μmol mol^–1× 36/29 °C, and 37% less at 200 μmol mol^–1× 25/20 °C. Most significantly, growth at 200 μmol mol^–1× 36/29 °C resulted in 77% less biomass relative to plants grown at 380 μmol mol^–1× 25/20 °C. The net CO₂ assimilation rate of leaves grown in 200 μmol mol^–1× 25/20 °C was 40% lower than in leaves from 380 μmol mol^–1× 25/20 °C, but similar to leaves in 200 μmol mol^–1× 36/29 °C. The leaves produced in low CO₂ and high temperature respired at a rate that was double that of leaves from the 380μmol mol^–1× 25/20 °C treatment. Despite this, there was little evidence that leaves at low CO₂ and high temperature were carbohydrate deficient, because soluble sugars, starch and total non-structural carbohydrates of leaves from the 200μmol mol^–1× 36/29 °C treatment were not significantly different in leaves from the 380μmol mol^–1× 25/20 °C treatment. Similarly, there was no significant difference in percentage root carbon, leaf chlorophyll and leaf/root nitrogen between the low CO₂× high temperature treatment and ambient CO₂ controls. Decreased plant growth was correlated with neither leaf gas exchange nor tissue chemistry. Rather, leaf and root growth were the most affected responses, declining in equivalent proportions as total biomass production. Because of this close association, the mechanisms controlling leaf and root growth appear to have the greatest control over the response to heat stress and CO₂ reduction in P. vulgaris.     

Photosynthetic characteristics of the shade-adapted C4 grass Muhlenbergia sobolifera (Muhl.) Trin.: control of development of photorespiration by growth temperature

Muhlenbergia sobolifera (Muhl.) Trin., a C₄ grass, occurs in understory habitats in the northeastern United States. Plants of M. sobolifera were grown at 23 and 30°C at 150 and 700 μmol photons m⁻² s⁻¹. The photosynthetic CO₂ compensation point, maximum CO₂ assimilation, dark respiration and the absorbed quantum use efficiency (QUE) were measured at 23 and 30°C at 2 and 20% O₂. Photosynthetic CO₂ compensation points ranged from 4 to 14mm³ dm⁻³ CO₂ and showed limited O₂ sensitivity. The mean photosynthetic CO₂ compensation point of plants grown at 30°C (4·5 mm³ dm⁻³) was 57% lower and 80% less inhibited by O₂ than that of plants grown at 23°C. Photosynthesis was similarly affected by growth temperature, with 70% more O₂ inhibition in plants grown at 23°C; suppression over all treatments ranging from 2 to 11%. Unlike typical C₄ species, plants of M. sobolifera from both temperature regimes exhibited higher CO₂ assimilation rates when grown at low light. Growth temperature and light also affected QUE; plants grown at low light and 23°C had the highest value (0·068 mol CO₂/mol quanta). Measurement temperature and growth light regime significantly affected dark respiration; however, O² did not affect QUE or dark respiration under any growth or measurement conditions. The results indicate that M. sobolifera is adapted to low PPFD, and that complete suppression of photorespiration is dependent upon high growth temperature.     

Characterization and comparisons of five N2-fixing Azolla-Anabaena associations,I. Optimization of growth conditions for biomass increase and N content in a controlled environment*

Abstract Biomass increase, C and N content, C₂H₂ reduction, percentage dry weight and chlorophyll a/b ratios were determined for clones of Azolla caroliniana Willd., A. filiculoides Lam., A. mexicana Presl., and A. pinnata R. Br. as a function of nutrient solution, pH, temperature, photoperiod, and light intensity in controlled environment studies. These studies were supplemented by a glasshouse study. Under a 16 h, 26°C day at a light intensity of 200 μmol m^?2 s^?1 and an 8 h, 19° C dark period, there was no significant difference in the growth rates of the individual species on the five nutrient solutions employed. Growth was comparable from pH 5 to pH 8, but decreased at pH 9. Using the same photoperiod and light intensity but constant growth temperatures of 15–40°C, at 5°C intervals, the individual species exhibited maximum growth, nitro-genase (N²ase) activity and N content at either 25° or 30°C. There was no difference in the temperature optima at pH 6 and pH 8. The tolerance of the individual species to elevated temperature was indicated to be A. mexicana> A. pinnata> A. caroliniana> A.filiculoides. At the optimum temperature, growth rates increased with increasing photoperiod at both pH 6 and pH 8 but N₂ase activity was usually highest at a 16 h light period. At photon flux densities of 100, 200, 400 and 600 μmol m^?2 s^?1, during a 16 h light period and optimum growth temperature of the individual species, N₂ase activity was saturated at less than 200 μmol m^?2 s^?1 and growth at 400 μmol m^?2 s^?1.No interacting effects of light and pH were noted for any species, nor were light intensities up to 1700 μmol m^?2 s^?1 detrimental to the growth rate or N content of any species in a 5 week glasshouse study with a natural 14.5 h light period and a constant temperature of 27.5°C. Using the optimum growth temperature, a 16 h light period, and a photon flux density of at least 400 μmol m^?2 s^?1, the Azolla species all doubled their biomass in 2 days or less and contained 5–6% N on a dry weight basis.     

Physiological and Growth Responses of C3 and C4 Plants to Reduced Temperature When Grown at Low CO2 of the Last Ice Age

During the last Ice age, CO2 concentration ([CO2]) was 180-200 μmol/mol compared with the modern value of 380 μmol/mol,and global temperatures were ～8 ℃ cooler. Relatively little is known about the responses of C3 and C4 species to longterm exposure to glacial conditions. Here Abutilon theophrasti Medik. (C3) and Amaranthus retroflexus L. (C4) were grown at 200 μmol/mol CO2 with current (30/24 ℃) and glacial (22/16 ℃) temperatures for 22 d. Overall, the C4 species exhibited a large growth advantage over the C3 species at low [CO2]. However, this advantage was reduced at low temperature, where the C4 species produced 5× the total mass of the C3 species versus 14× at the high temperature.This difference was due to a reduction In C4 growth at low temperature, since the C3 species exhibited similar growth between temperatures. Physiological differences between temperatures were not detected for either species, although photorespirationlnet photosynthesis was reduced in the C3 species grown at low temperature, suggesting evidence of improved carbon balance at this treatment. This system suggests that C4 species had a growth advantage over C3 species during low [CO2] of the last ice age, although concurrent reductions in temperatures may have reduced this advantage.     

Molecular cloning of a nodulation gene from fast- and slow-growing strains of Lotus rhizobia

Summary Cosmids containing a nodulation gene from Rhizobium loti NZP2037 were isolated using a 12.8 kb nod:: Tn5 EcoRI fragment from the Nod^- mutant strain PN233, as a hybridisation probe. A physical map of the nod region was established using the enzymes EcoRI and HindIII and the site of insertion of Tn5 in PN233 determined. Site-specific exchange of the cloned nod:: Tn5 fragment demonstrated that Tn5, and not an indigenous insertion sequence, was responsible for the nod mutation in PN233. The nod cosmids isolated complemented the Nod^- phenotype of strain PN233 but restoration of the Fix phenotype was variable suggesting a need for marker rescue to occur before nitrogen fixation occurred.Corresponding nod cosmids were isolated from a R. loti strain, NZP2213, that forms ineffective tumour-like structures on Lotus pedunculatus and from the slow-growing strain (Bradyrhizobium sp), CC814s, by in planta complementation of PN233. Hybridisation experiments suggested that the nod gene region of R. loti NZP2037 was more homologous to Bradyrhizobium strain CC814s than with a nod gene region of R. trifolii strain PN100. However, transfer of the R. trifolii nod cosmid into the R. loti Nod mutant PN233, restored the ability of this strain to initiate nodules on Lotus pedunculatus.     

Seasonal CO2 exchange patterns of developing peach (Prunus persica) fruits in response to temperature, light and CO2 concentration

CO₂ exchange rates per unit dry weight, measured in the field on attached fruits of the late-maturing Cal Red peach cultivar, at 1200 μmol photons m^?2S^?1 and in dark, and photosynthetic rates, calculated by the difference between the rates of CO₂ evolution in light and dark, declined over the growing season. Calculated photosynthetic rates per fruit increased over the season with increasing fruit dry matter, but declined in maturing fruits apparently coinciding with the loss of chlorophyll. Slight net fruit photosynthetic rates ranging from 0. 087 ± 0. 06 to 0. 003 ± 0. 05 nmol CO₂ (g dry weight)^?1 S^?1 were measured in midseason under optimal temperature (15 and 20°C) and light (1200 μmol photons m^?2 S^?1) conditions. Calculated fruit photosynthetic rates per unit dry weight increased with increasing temperatures and photon flux densities during fruit development. Dark respiration rates per unit dry weight doubled within a temperature interval of 10°C; the mean seasonal O₁₀ value was 2. 03 between 20 and 30°C. The highest photosynthetic rates were measured at 35°C throughout the growing season. Since dark respiration rates increased at high temperatures to a greater extent than CO₂ exchange rates in light, fruit photosynthesis was apparently stimulated by high internal CO₂ concentrations via CO₂ refixation. At 15°C, fruit photosynthetic rates tended to be saturated at about 600 μmol photons m^?2 S^?1. Young peach fruits responded to increasing ambient CO₂ concentrations with decreasing net CO₂ exchange rates in light, but more mature fruits did not respond to increases in ambient CO₂. Fruit CO₂ exchange rates in the dark remained fairly constant, apparently uninfluenced by ambient CO₂ concentrations during the entire growing season. Calculated fruit photosynthetic rates clearly revealed the difference in CO₂ response of young and mature peach fruits. Photosynthetic rates of younger peach fruits apparently approached saturation at 370 μl CO₂1^?2. In CO₂ free air, fruit photosynthesis was dependent on CO₂ refixation since CO₂ uptake by the fruits from the external atmosphere was not possible. The difference in photosynthetic rates between fruits in CO₂-free air and 370 μl CO₂ 1^?1 indicated that young peach fruits were apparently able to take up CO₂ from the external atmosphere. CO₂ uptake by peach fruits contributed between 28 and 16% to the fruit photosynthetic rate early in the season, whereas photosynthesis in maturing fruits was supplied entirely by CO₂ refixation.     

Combined effects of elevated CO2 and air temperature on carbon assimilation of Pinus taeda trees

Branches of 22-year-old loblolly pine (Pinus taeda, L.) trees growing in a plantation were exposed to ambient CO₂, ambient + 165 μmol mol^?1 CO₂ or ambient + 330 μmol mol^?1 CO₂ concentrations in combination with ambient or ambient + 2°C air temperatures for 3 years. Field measurements in the third year indicated that net carbon assimilation was enhanced in the elevated CO₂ treatments in all seasons. On the basis of A/C_i, curves, there was no indication of photosynthetic down-regulation. Branch growth and leaf area also increased significantly in the elevated CO₂ treatments. The imposed 2°C increase in air temperature only had slight effects on net assimilation and growth. Compared with the ambient CO₂ treatment, rates of net assimilation were ～1·6 times greater in the ambient + 165 μmol mol^?1 CO₂ treatment and 2·2 times greater in the ambient + 330 μmol mol^?1 CO₂ treatment. These ratios did not change appreciably in measurements made in all four seasons even though mean ambient air temperatures during the measurement periods ranged from 12·6 to 28·2°C. This indicated that the effect of elevated CO₂ concentrations on net assimilation under field conditions was primarily additive. The results also indicated that the effect of elevated CO₂ (+ 165 or + 330 μmol mol^?1) was much greater than the effect of a 2°C increase in air temperature on net assimilation and growth in this species.     

Acetylene reduction byDaviesia mimosoides under Eucalyptus

Summary Daviesia mimosoides is a common understorey legume in Eucalyptus forests of the Brindabella Range in southeastern Australia, capable of fixing atmospheric nitrogen. Rates of N fixation were measured by the acetylene-reduction technique over a growing season in the field. Pot trials under controlled conditions were also carried out to elucidate effects of soil moisture, temperature, and light. Average rates in the field varied from about 1–5 μ mol C₂H₄/g/h (wet weight of nodule), but rates up to 14 μ mol C₂H₄/g/h were measured in optimum controlled conditions. Annual N-fixation rates approximate 4.5–7.0 kg/ha. In pot trials, rate of acetylene reduction decreased with soil moisture to about−10 MPa tension, with a marked depression at about−6 MPa, but within the normal field range of soil moisture there was little correlation of moisture with average acetylene reduction rate. Rates were similar in the temperature range of 20–30°C, but were depressed by either low or high temperature (<10 or >30°C). Diurnal fluctuations in acetylene reduction rates were not correlated with solar radiation, but rates were limited by high mid-day temperatures.     

Does oxygen limit nitrogenase activity in soybean exposed to elevated CO2?

Soybean (Glycine max L. Merr) plants grown under control (360 µmol mol^?1) or elevated CO₂ concentration (800 µmol mol^?1) from 33 to 42 d after sowing were assayed for various components of in vivo nitrogenase activity to test the hypothesis that increasing carbohydrate supply to nodules would increase the potential (i.e. O₂ saturated) nitrogenase activity and impose a more severe O₂ limitation on both nodule metabolism and total nitrogenase activity. Within 51 h of elevated CO₂ treatment, significant increases relative to control plants were seen in total nitrogenase activity expressed per plant. After 6 d of elevated CO₂, the total nitrogenase activity per plant was 18% higher than that in control. This was attributed to an initial increase in nodule size, and a subsequent increase in nodule number following plant exposure to elevated CO₂. However, after 9 d of elevated CO₂, the potential and total nitrogenase activities per gram nodule dry weight were lower, not higher than corresponding values in plants in the control treatment. These results did not support the hypothesis. It was concluded that the metabolic capacity of the control nodules were not limited by carbohydrate supply, at least at the assay temperatures employed here.     

Economy of Photosynthate Use in Nitrogen-fixing Legume Nodules: Observations on Two Contrasting Symbioses

The economy of C use by root nodules was examined in two symbioses, Vigna unguiculata (L.) Walp. (cv. Caloona):Rhizobium CB756 and Lupinus albus L. (cv. Ultra):Rhizobium WU425 over a 2-week period in early vegetative growth. Plants were grown in minus N water culture with cuvettes attached to the nodulated zone of their primary roots for collection of evolved CO₂ and H₂. Increments in total plant N and in C and N of nodules, and C:N weight ratios of xylem and phloem exudates were studied by periodic sampling from the plant populations. Itemized budgets were constructed for the partitioning and utilization of C in the two species. For each milligram N fixed and assimilated by the cowpea association, 1.54 ± 0.26 (standard error) milligrams C as CO₂ and negligible H₂ were evolved and 3.11 milligrams of translocated C utilized by the nodules. Comparable values for nodules of the lupin association were 3.64 ± 0.28 milligrams C as CO₂, 0.22 ± 0.05 milligrams H₂, and 6.58 milligrams C. More efficient use of C by cowpea nodules was due to a lesser requirement of C for synthesis of exported N compounds, a smaller allocation of C to nodule dry matter, and a lower evolution of CO₂. The activity of phosphoenolpyruvate carboxylase in nodule extracts and the rate of ¹⁴CO₂ fixation by detached nodules were greater for the cowpea symbiosis (0.56 ± 0.06 and 0.22 milligrams C as CO₂ fixed per gram fresh weight per hour, respectively) than for the lupin 0.06 ± 0.02 and 0.01 milligrams C as CO₂ fixed per gram fresh weight per hour. The significance of the data was discussed in relation to current information on theoretical costs of nitrogenase functioning and associated nodule processes.     

Effects of incubation temperature and oxygen tension on nitrogenase activity of legume root nodules

Summary Acetylene reduction and H₂ evolution by legume root nodules from several plant species depended on incubation temperature; some nodules were active from 2 to 40°C. Acetylene reduction rates differed between plant species, with maximum activity at temperatures between 20 and 30°C forVicia faba, V. sativa, Trifolium pratense, T. subterraneum, Medicago truncatula and soybean, at 35°C forM. sativa and at 40°C for cowpea. OnlyM. sativa and cowpea reduced substantial amounts at 37.5°C. Temperatures from 2 to 10°C only slightly lessened activity ofT. subterraneum andV. sativa nodules. Nitrogenase functioned at temperatures which prevent establishment of other aspects of the symbiosis. The rate of acetylene reduction was constant for several hours at temperatures below 15°C, and activity continued for several days at 2°C for some species, but declined with time at warmer temperatures. Some nitrogenase was denatured at warmer temperatures, but the O₂ tension in the assay vial also affected activity. In closed assay vessels nodule respiration decreased the pO₂ and reduced nitrogenase activity. Activity was restored by adding O₂ or regassing assay vials with air or Ar/O₂. When the pO₂ was maintained, acetylene reduction and H₂ evolution by detached soybean nodules continued unchanged for 6 h.     

Carbon cost of nitrogenase activity in Frankia–Alnus incana root nodules

The carbon cost of nitrogenase activity was investigated to determine symbiotic efficiency of the actinorhizal root nodule symbiosis between the woody perennial Alnus incana and the soil bacterium Frankia. Respiration (CO₂ production) and nitrogenase activity (H₂ production) by intact nodulated root systems were continuously recorded in short-term assays in an open-flow gas exchange system. The assays were conducted in N₂:O₂, thus under N₂-fixing conditions, in all experiments except for one. This avoided the declines in nitrogenase activity and respiration due to N₂ deprivation that occur in acetylene reduction assays and during extended Ar:O₂ exposures in H₂ assays. Two approaches were used: (i) direct estimation of root and nodule respiration by removing nodules, and (ii) decreasing the partial pressure of O₂ from 21 to 15% to use the strong relationship between respiration and nitrogenase activity to calculate CO₂/H₂. The electron allocation of nitrogenase was determined to be 0.6 and used to convert the results into moles of CO₂ produced per 2e^– transferred by nitrogenase to reduction of N₂. The results ranged from 2.6 to 3.4mol CO₂ produced per 2e^–. Carbon cost expressed as gC produced per gN reduced ranged from 4.5 to 5.8. The result for this actinorhizal tree symbiosis is in the low range of estimates for N₂-fixing actinorhizal symbioses and crop legumes. Methodology and comparisons of root nodule physiology among actinorhizal and legume plants are discussed.     

Effectiveness of Lotus Root Nodules: I. MORPHOLOGY AND FLAVOLAN CONTENT OF NODULES FORMED ON LOTUS PEDUNCULATUS BY FAST-GROWING LOTUS RHIZOBIA

The morphology of root nodules formed on Lotus pedunculatusby two fast-growing strains of Rhizobium, NZP2037 which formseffective (nitrogen-fixing) nodules and NZP2213 which formsineffective (non-nitrogen-fixing) nodules, has been studied.The nodules formed by NZP2037 contained a central zone of bacteroid-filledplant cells surrounded by a cortex. In contrast the nodulesformed by NZP2213 contained no Rhizoblum-infected plant cells,but rhizobia were found in localized areas on the nodule surfaceand between the outer two or three cell layers of the nodule.Electron-dense osmiophilic deposits identified as flavolans(condensed tannins) were present in the vacuoles of many uninfectedplant cells in the nodules formed by both Rhizobium strains.This is the first time that flavolans have been positively identifiedin legume root nodules. In the NZP2037 nodule flavolans werepresent in the outer cortical and epidermal cells. In the ineffecitveNZP2213 nodule fiavolans were present in many of the centralnodule cells. The concentration of flavolan in the NZP2213 nodulewas 12 times higher than in the NZP2037 nodule.     

Photosynthetic performance at low temperature of Bouteloua gracilis Lag., a high-altitude C4 grass from the Rocky Mountains, USA

The mechanisms controlling the photosynthetic performance of C₄ plants at low temperature were investigated using ecotypes of Bouteloua gracilis Lag. from high (3000 m) and low (1500 m) elevation sites in the Rocky Mountains of Colorado. Plants were grown in controlled‐environment cabinets at a photon flux density of 700 μ mol m^?2 s^?1 and day/night temperatures of 26/16 °C or 14/7 °C. The thermal response of the net CO₂ assimilation rate (A) was evaluated using leaf gas‐exchange analysis and activity assays of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPCase) and pyruvate,orthophosphate dikinase (PPDK). In both ecotypes, a reduction in measurement temperature caused the CO₂‐saturated rate of photosynthesis to decline to a greater degree than the initial slope of A versus the intercellular CO₂ response, thereby reducing the photosynthetic CO₂ saturation point. As a consequence, A in normal air was CO₂‐saturated at sub‐optimal temperatures. Ecotypic variation was low when grown at 26/16 °C, with the major difference between the ecotypes being that the low‐elevation plants had higher A; however, the ecotypes responded differently when grown at cool temperature. At temperatures below the thermal optimum, A in high‐elevation plants grown at 14/7 °C was enhanced relative to plants grown at 26/16 °C, while A in low‐elevation plants grown at 14/7 °C was reduced compared to 26/16 °C‐grown plants. Photoinhibition at low growth temperature was minor in both ecotypes as indicated by small reductions in dark‐adapted F_v/F_m. In both ecotypes, the activity of Rubisco was equivalent to A below 17 °C but well in excess of A above 25 °C. Activities of PEPCase and PPDK responded to temperature in a similar proportion relative to Rubisco, and showed no evidence for dissociation that would cause them to become principal limitations at low temperature. Because of the similar temperature response of Rubisco and A, we propose that Rubisco is a major limitation on C₄ photosynthesis in B. gracilis below 17 °C. Based on these results and for theoretical reasons associated with how C₄ plants use Rubisco, we further suggest that Rubisco capacity may be a widespread limitation upon C₄ photosynthesis at low temperature.