A Multichannel System for Steady–State and Continuous Measurements of Gas Exchanges from Legume Roots and Nodules

The objectives of this study were to determine whether attached nodules of soybean (Glycine max L. Merr.) could adjust to gradual increases in rhizosphere PO2 without nitrogenase inhibition and to determine whether the nitrogenase activity of the nodules is limited by P02 under ambient conditions. A computercontrolled gas blending apparatus was used to produce linear increases (ramps) in PO2 around attached nodulated roots of soybean plants in an open gas exchange system. Nitrogenase activity (H2 production in N2:02 and Ar:02) and respiration (CO2 evolution) were monitored continuously as PO2 was ramped from 20 to 30 kilopascals over periods of 0, 5, 10, 15, and 30 minutes. The 0, 5, and 10 minute ramps caused inhibitions of nitrogenase and respiration rates followed by recoveries of these rates to their initial values within 30 minutes. Distinct oscillations in nitrogenase activity and respiration were observed during the recovery period, and the possible basis for these oscillations is discussed. The 15 and 30 minute ramps did not inhibit nitrogenase activity, suggesting that such inhibition is not a factor in the regulation of nodule diffusion resistance. During the 30 minute ramp, a stimulation of nitrogenase activity was observed, indicating that an 02-based limitation to nitrogenase activity occurs in soybean nodules under ambient conditions.

Section: Materials and Methods Plant Culture And Experimental Conditionsmentioning

confidence: 99%

Effects of Gradual Increases in O₂ Concentration on Nodule Activity in Soybean

Hunt¹,

King²,

Layzell³

1989

“…Measurements of CO2 production and H2 evolution from intact attached nodulated roots were made with the computercontrolled gas exchange system described previously (13). Control and experimental plants were sealed in their growth pots and attached to the gas exchange system.…”

Section: Gas Exchange Measurementsmentioning

confidence: 99%

Mechanism of Nitrogenase Inhibition in Soybean Nodules

Layzell

Hunt²,

Palmer³

1990

A novel, pulse-modulated spectroscopic system for measuring fractional leghemoglobin oxygenation and infected cell 02 concentration (0) in intact attached nodules of soybean (Glycine max) is described. The system is noninvasive and uses a pulsed (1000 Hertz) light-emitting diode coupled to an optical fiber to illuminate the nodule with light at 660 nanometer. A second optical fiber receives a portion of the light reflected from the nodule and directs this to a photodiode. A lock-in amplifier measures only the signal from the photodiode which is in phase with the pulsed light from the light-emitting diode, and the voltage output from the amplifier, proportional to reflectance, is used to calculate fractional leghemoglobin oxygenation and the nanomolar concentration of free 02 in the infected cells of the nodule (0 Studies with a variety of legumes have suggested that the diffusion barrier in the nodule inner cortex is variable (11,12,22,27) perhaps through regulation of the relative proportion of water and gas which occupies the intercellular spaces in this tissue layer (12). In many treatments in which nitrogenase activity is inhibited, the resistance to gas diffusion into the nodule is greater than that in nodules of untreated plants (23,26), suggesting that treatments inhibitory to nodule metabolism and nitrogenase activity act by increasing diffusion resistance and thereby decrease the availability of 02 within the infected cells. In this paper, we test this hypothesis using a novel pulse-modulated spectroscopic technique to measure the infected cell 02 concentration ofsoybean nodules in which nitrogenase activity has been inhibited by different treatments.N2 fixation in legume nodules is adversely affected by a wide range of environmental and physiological conditions. For example, drought ( 14,18,25),23), low temperature (14,25), and various treatments which disrupt phloem supply to nodules (9,23,24) are all known to inhibit nitrogenase activity. In treatments which reduce phloem supply to nodules, the mechanism of nitrogenase inhibition has been attributed to a reduction in the availability of carbohydrate, which would presumably reduce the pool size of reductant required by nitrogenase. However, studies have shown that these inhibitions of nitrogenase activity can be partially or fully recovered by increases in rhizosphere PO2 (9,23), indicating that nitrogenase is limited by 02 supply (and thereby ATP availability) rather than reductant availability.

“…The design of the growth pots (8) permitted sealing for open circuit gas exchange measurements of H2 and CO2 evolution from the root systems. The gas exchange system contained computer-controlled mass flow controllers (model FMA-100 series, Omega Engineering, Stamford, CT) which allow linear changes in gas concentrations at a constant total flow rate.…”

mentioning

confidence: 99%

“…CO2 evolution was measured by infrared gas analyzers (model 225, Mark III, Analytical Development Corp., Hoddeson, UK), 02 evolution with an O2 electrode (model CB 1 D, Hansatech, King's Lynn, UK), and H2 evolution with custom-built H2 analyzers (11). The complete system has been described previously (6,8). At a gas flow rate of 500 mL min-' past the root systems, the time constant for the entire system was approximately 30 s. Immediately following the final anaerobic treatment, the root system was reexposed to 100% N2 for approximately 5 min to ensure that leghemoglobin was completely deoxygenated during the 45-s treatments.…”

mentioning

confidence: 99%

Effect of Increases in Oxygen Concentration during the Argon-Induced Decline in Nitrogenase Activity in Root Nodules of Soybean

King

Layzell²

1991

When intact nodulated roots of soybean (Glycine max L. Merr. nodulated with Bradyrhizobium japonicum strain USDA 16) were exposed to an atmosphere lacking N2 gas (Ar:02 80:20), total nitrogenase activity (measured as H2 evolution) and respiration (CO2 evolution) declined with time of exposure. In Ar-inhibited nodules, when the 02 concentration in the rhizosphere was increased in a linear 'ramp' of 2.7% per minute, 93% of the original H2 evolution and 99% of the CO2 evolution could be recovered. The internal nodule 02 concentration (estimated from leghemoglobin oxygenation) declined to 56% of its initial value after 60 minutes of Ar:02 exposure and could be partially recovered by the linear increases in 02 concentration. Nodule gas permeability, as estimated from the lag in ethylene production following exposure of nodules to acetylene, decreased to 26% of its initial value during the Ar-induced decline. Collectively, the results provide direct evidence that the Ar-induced decline results from decreased nodule gas permeability and indicate that the decline in permeability, rather than being immediate, occurs gradually over the period of Ar:02 exposure.The nodules of various legumes undergo a distinct decline in TNA3 and C02 evolution following exposure to 10% acetylene (2, 3, 12-14, 22, 23). A similar decline occurs when N2 in the gas phase is replaced by an inert gas such as Ar (5,13 3 Abbreviations: TNA, total nitrogenase activity as estimated from H2 production in Ar:02 or from acetylene reduction; NR, nodulated root; nod, nodules; Oi, 02 concentration in the cytosol of the infected cells of the nodule; P, nodule permeability to gas diffusion; t, time constant for ethylene production.clines (5, 23), and the ability of 02 at least to partially overcome the acetylene-induced decline (22), suggest that both declines are caused by a decrease in the nodule gas permeability (P), which restricts the entry of 02 for respiration in support of nitrogenase activity (23). However, this suggestion is based on theoretical considerations of the relationship between 02 concentration gradients, 02 uptake rates, and P.To date, neither P nor the infected cell 02 concentration (Os) have been measured in acetylene-or Ar-inhibited nodules, nor has it been demonstrated whether increases in 02 concentration can overcome the Ar-induced decline.In addition, various actinomycete symbioses also exhibit acetylene-induced declines or transient fluctuations of TNA (16,17,19), which are superficially similar to those of legumes. However, the anatomy of the nodules formed in these symbioses differs considerably from that of legume nodules, and the main barrier to gas diffusion is thought to be associated with the Frankia endosymbiont itself (18) rather than with the uninfected nodule cortex, as appears to be the case in legumes (20,24). Silvester and Winship (16) have provided evidence that the acetylene-and 02-induced transient responses in several actinomycete symbioses are associated with the Frankia vesicles and are not due to cha...