Regulation of Soybean Nitrogen Fixation in Response to Rhizosphere Oxygen

Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle for defense against activated forms of oxygen. Nodulated roots of hydroponically grown soybean plants were exposed to atmospheres containing 2, 21, 50, or alternating 21 and 50 kilopascals of 02. The activities of ascorbate (ASC) peroxidase, monodehydroascorbate (MDHA) reductase, dehydroascorbate (DHA) reductase, and glutathione (GSSG) reductase were higher in nodules exposed to high P02.Nodule contents of ascorbate and reduced glutathione were also greater in the high P02 treatments. Treatment of modulated plants with fixed nitrogen (urea) led to concomitant decreases in acetylene reduction activity, in leghemoglobin content, and in activities of ASC peroxidase, DHA reductase, and GSSG reductase. Activity of MDHA reductase and glutathione concentrations in nodules were not affected by treatment with urea. The enzymes of the ascorbate-glutathione cycle were also detected in uninfected soybean roots, although at levels substantially below those in nodules. These observations indicate that the ascorbate-glutathione cycle can adjust to varying physiological conditions in nodules and that there is a key link between N2 fixation and defenses against activated forms of oxygen.The oxygen relations of legume root nodules are extremely important in maintaining normal nodule functions. There are many aspects to the relationship between oxygen and N2 fixation in nodules. The essential role of leghemoglobin as a 02-binding protein and the 02 sensitivity of nitrogenase have long been recognized as fundamentally important factors in nodules. N2 fixation is an energy-intensive process, requiring large amounts of ATP that must be supplied by oxidative phosphorylation. Nodules have a high rate of respiratory 02 consumption, but measurements with 02 microelectrodes indicate that the P02 in nodule interior spaces is very low (24). The inner cortex of nodules presents a major impediment to 02 diffusion and is a primary factor in maintaining the low internal P02. Numerous studies utilizing flow-through gas systems for measurement of acetylene reduction and respiration have provided indirect evidence that this diffusion barrier is variable and capable of responding to gradual changes in external PO2 without damage to nitrogenase (10,

Section: Resultsmentioning

confidence: 99%

Effects of Ambient Oxygen and of Fixed Nitrogen on Concentrations of Glutathione, Ascrobate, and Associated Enzymes in Soybean Root Nodules

Dalton

Post²,

Langeberg³

1991

“…3 stopper. The seedling and stopper combination was placed in the lid of a 1.5 L aeroponic chamber (15) …”

Section: Plant Growth Conditionsmentioning

confidence: 99%

“…The evening before an experiment began, the plant and stopper combination was transferred from the aeroponic chamber to an assay chamber (15). The upper 9 cm of the nodulated root system of the intact plant was sealed into the assay chamber.…”

Section: Treatment Implementationmentioning

confidence: 99%

Nitrogenase Activity and Nodule Gas Permeability Response to Rhizospheric NH₃ in Soybean

Purcell

Sinclair²

1990

Self Cite

This study was conducted on soybean (Glycine max L. Merr.) nodules to determine if exogenous NH3 exerts a controlling influence over nitrogenase activity through changes in nodule gas permeability (P), and if decreasing carbohydrate availability, as a result of low-light treatment, increases the sensitivity of root nodules to NH3. Nodulated root systems of intact plants were exposed to one of several NH3 concentrations ranging from 0 to 821 microliters per liter for an 8-hour period. Treatments were conducted under high-light (2300 micromoles per square meter per second) or low-light (800 micromoles per square meter per second) conditions. Increasing the NH3 concentration and length of exposure of NH3 caused a progressive decline in acetylene reduction activity (ARA). There was generally a greater reduction in ARA under the low-light treatment compared to the high-light treatment at a particular NH3 concentration. The NH3 concentration necessary to decrease P was greater than that needed to decrease ARA, and there was no evidence of a causal relationship between P and ARA in response to NH3.The inability to increase nitrogenase activity in legumes on a nodule weight basis through increased photosynthate availability (14,19) has led researchers to explore other potential limitations to N2 fixation. Recent work has indicated that N2 fixation rates are closely linked quantitatively to (P') for O2 diffusion (7,16). Despite the observations of P adjustments in response to the environment (7,10,12,16), there is a virtual void in understanding how P changes are achieved.Since NH3 is the initial product of N2 fixation, environmental factors which alter P and N2 fixation also affect NH3 production in nodule bacteroids. Treatments that prevent NH3 production, such as prolonged exposure to 1O% C2H2 (10, 12) or replacement of the N2:02 atmosphere surrounding nodules with an Ar:02 atmosphere (7, 10), decrease P. These reports have led to the suggestion that maintenance of steady-state P is dependent upon the continual production of NH3 (10, 12). However, this hypothesis does not account for either the increase in P under conditions of low NH3 production (as indicated by decreased ARA) associated with decreasing the rhizosphere PO2 from 0.21 to 0.10 MPa, or the decrease in P Abbreviations: P, nodule gas permeability; PO2, partial pressure of 02, ARA, acetylene reduction activity.following an increase in rhizosphere PO2 (16). A causal relationship between NH3 production and P remains to be determined.There have been no reports characterizing the effects of exogenous NH3 on nodule permeability. The addition of NH4' salts to the growth media of pea (Pisium sativium, L.) results in a decreased ARA of detached nodules (4, 5), excised nodulated root systems (1), and intact plants (4, 6) but has no short-term effect on the ARA of isolated bacteroids (4, 6). In addition, NH4' toxicity in plant tissues may be overcome by increasing the light intensity (1, 6) or by the addition of organic acids to the culture media (9). The obj...

“…Soybean nodules contain three cortical cell layers (44% of the nodule volume) (28), an outer cortex, an endodermis, and an inner cortex. The inner cortex functions as a diffusion barrier to balance inward oxygen flux with bacteroid respiratory oxygen demand (15,20,31,32). Tjepkema and Yocum (29) and later Witty et al (34) verified that the oxygen concentration decreased across the cortical layer until it reached a minimum at the inner cortex, the presumed major barrier to gas diffusion.…”

mentioning

confidence: 99%

³¹P Relaxation Responses Associated with N₂/O₂ Diffusion in Soybean Nodule Cortical Cells and Excised Cortical Tissue

Pfeffer

Kumosinski²,

MacFall³

et al. 1992

N2-fixing Bradyrhizobium japonicum nodules and cortical tissue derived from these nodules were examined in vivo by 31P nuclear magnetic resonance (NMR) spectroscopy. Perfusion of the viable nodules and excised cortical tissue with 02 followed by N2 or Ar caused a loss of orthophosphate (Pi) resonance magnetization associated with the major portion of acidic Pi (5 0.9 ppm, pH 5.5) residing in the cortical cells. Soybean nodules contain three cortical cell layers (44% of the nodule volume) (28), an outer cortex, an endodermis, and an inner cortex. The inner cortex functions as a diffusion barrier to balance inward oxygen flux with bacteroid respiratory oxygen demand (15,20,31,32). Tjepkema and Yocum (29) and later Witty et al. (34) verified that the oxygen concentration decreased across the cortical layer until it reached a minimum at the inner cortex, the presumed major barrier to gas diffusion. Numerous investigators (2,7,8,11,27,28) have examined the diffusion of 02 through this barrier and have proposed that the sites of entry are air spaces filled with water the depth of which can be varied through expansion or contraction of the adjacent inner cortex cells. On the other hand, Witty and coworkers (26, 34) have speculated that cell expansion into the intercellular space is responsible for restricting the passage of 02 into the inner nodule in response to diminished respiration. VandenBosch et al. (30) and Bradley et al. (3) observed a unique glycoprotein in the infection thread matrix and intercellular space of pea nodules. Further investigation of this material in soybean nodules by James and coworkers (9) demonstrated that its elaboration was greatly enhanced in the presence of oxygen. These authors (9) hypothesized that this 'occlusion protein' is responsible for increasing 02 diffusion resistance in the cortical cell layer by blocking the intercellular air spaces.At present the nature of the oxygen-regulating miechanism(s) associated with the cortical layer is controversial. In this and the accompanying paper (12), we describe relaxation data obtained from 31P NMR spectroscopy and 'H magnetic resonance imaging studies that suggest that increasing viscosity resulting from the production of cortical cell glycoprotein may be associated with enhanced 02 diffusion resistance in soybean (Glycine max) nodules. MATERIALS AND METHODS