Rhizospheric NH3 in Soybean

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 objective of this study was to test the hypothesis that NH3 regulates N2 fixation through changes in nodule gas permeability and that decreasing the availability of carbohydrates in the nodule would increase the sensitivity to NH3 toxicity by decreasing the carbon skeletons needed for the initial assimilation of NH3. MATERIALS AND METHODS Plant Growth Conditions Seeds of soybean (Glycine max L. Merr., cv Biloxi) were inoculated with a commercial preparation ofBradyrhizobium japonicum (The Nitragin Co., Milwaukee, WI2) and germinated in artificial soil medium. Seedlings were removed from the soil mixture 3 to 4 d after sowing, the roots were rinsed with tap water, and the seedlings were transferred to a bored No. 3 stopper. The seedling and stopper combination was placed in the lid of a 1.5 L aeroponic chamber (15) that contained half-strength Hoagland solution and was aerated with compressed air supplied to the bottom of each aeroponic chamber through an aquarium glass bead bubbler. Using this system, two plants per chamber were grown in the greenhouse under natural illumination with the photoperiod extended to 16 h by incandescent lamps. When plants had developed three to four fully expanded trifoliolates, they were transferred to the laboratory and placed under a combination of sodium and metal halide lamps on a 16-h photoperiod from 0500 to 2100 h. PPFD at the top of the plant was 2300 ,umol m-2 sfor the high-light treatment or was reduced by a layer of shade cloth to 800 ,umol m-2 s-' 2 Mention of company names or commercial products does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned. 268 www.plantphysiol.org on December 30, 2017 Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved. SOYBEAN ROOT NODULE RESPONSE TO NH3 for the low-light treatment. Plants were maintained under laboratory conditions from 2 to 4 d prior to treatment initiation. The aeroponic chambers in the laboratory were immersed in a water bath that maintained root and nodule temperature at 24°C. The air temperature around the shoots varied with room temperature and was approximately 22°C. Treatment Implementation 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. The plant and assay chamber were placed on an aeroponic chamber so the lower root system was in continuously aerated nutrient solution. The following morning (0730 EST) an initial measurement of nitrogenase activity and nodule gas permeability was made using the "lag-phase" technique (17). The basis of this technique is that the time required to reach steady state C2H4 production rates following the introduction of a saturating concentration of C2H2 into a small assay chamber at a high volumetric flow rate is dependent upon nodule gas permeability. Experimentally, a mixture of 10% (v/v) C2H2 in air was introduced into the assay chamber at a flow rate of 10 mL s-' for a 2-min period. One-mL gas samples were collected from the exit port of the assay chamber every 6 s for the 2 min C2H2 exposure period. C2H2 was removed from the gas supply, and the chamber was flushed with air for 1 min before reducing the air flow rate in the assay chamber to 0.33 mL s-'. The amount of C2H4 in the gas samples was determined by gas chromatographic procedures. Following the initial measurement ofARA and P, NH3 was added to the humidified air supply passed through the assay chamber for an 8-h exposure period beginning at 0800. ARA and P were periodically assayed during the NH3 treatment and over a subsequent 15.5-h recovery period. Known NH3 concentrations were prepared by mixing anhydrous NH3 with humidified air in a gas dosing device (Calibrated Instruments, Inc.). For both the high-light and low-light treatments, three plants were subjected to NH3 concentrations of 0, 274, 41 1, 547, or 684 ,uL L-'; an additional three plants were subjected to an NH3 concentration of 821 tsL L' for the high-light treatment. At the end of the experimental period, nodules in the assay chamber were excised, and nodule number, nodule fresh weight, and nodule dry weight were determined. Mean nodule diameter was estimated from a photocopy ofthe fresh nodules and used in the calculation of P ( 17). RESULTS AND DISCUSSION Prevailing weather conditions and daylength determined the irradiance on plants during their growth in the greenhouse. Plants used for the high-light treatment in the laboratory were grown in the greenhouse from September 14, 1988, to December 20, 1988, and plants used for the low-light treatment were grown from December 10, 1988, to February 20, 1989. The higher irradiance during the growth ofthe high-light treatment plants resulted in increased nodule dry weights and nodule numbers as compared to the low-light treatment plants (Table I). However, there was little difference between the initial ARA and initial P of nodules from plants exposed to the high or low-light treatment in the laboratory (Table I). These results agree with previous reports that increasing photosynthate supply to nodules increases nodule mass but has little effect on specific nitrogenase activity ( 19). Nitrogenase activity of the control treatment (0 qL NH3 L-') was stable over the 24-h experimental period (Fig. 1) and confirms the absence of diurnal oscillations in nitrogenase activity under conditions of constant rhizosphere temperature (18). Exposure of nodules to 274 ,L NH3 L-' decreased ARA of the low-light treatment to approximately 70% of the pretreatment value while having no effect in the high-light treatment. At 411 gL NH3 L', ARA of both the lowand the high-light treatments decreased to around 60% ofthe pretreatment value, and at the end of the experiment, both light treatments had recovered to approximately 90% of the pretreatment value. An increase in the NH3 concentration to 547 ,uL L-' resulted in a further decrease in ARA but a difference existed between light treatments. When subjected to 547 uL NH3 L-', ARA ofthe low-light treatment was decreased more, and showed less recovery, than ARA of the high-light treatment. Similarly, at 684 ,L NH3 L-', ARA of the low-light treatment was decreased more than the high-light treatment. However, neither the high-light nor the low-light treatment recovered ARA after 684,uL NH3 L-' was removed from the system. When nodules were exposed to 821 gL NH3 L', ARA was rapidly and completely inhibited and showed no signs of recovery following the NH3 exposure period. Nodule P was virtually unaffected at NH3 concentrations below 547 ,uL L-1 (Fig. 2). At 547 uL NH3 L-', P for both light treatments was reduced to 70 to 80% ofthe pretreatment value and recovered to approximately 90% of the pretreatment value by the end of the experiment. At NH3 concentrations of 684 and 821 uL L', P rapidly declined during the first 5 h ofNH3 exposure. The determination ofP by the 'lagphase' technique depends upon measurement of time course changes in C2H4 production, and in some cases, ARA was undetectable after 7 h of NH3 treatment at 684 and 821 ,uL L' (Fig. 1). Under the conditions of rapidly decreasing ARA and P during the first 5 h of NH3 exposure and 0 ARA thereafter, P was extrapolated to 0. This may have led to an underestimation of P and partially account for the large error bars at an NH3 concentration of 684 ,L L' (Fig. 2). There was little recovery of P following the removal of NH3 at concentrations of 684 or 821 uL L'. A comparison ofFigures 1 and 2 illustrates that the response ofARA and P to exogenous NH3, was not tightly coupled. A decrease in ARA was observed before there was any effect on P, and the relationship between ARA and P of the high-light versus the low-light treatment at 547 gL NH3 L' indicates that recovery ofP following NH3 exposure was not associated with recovery of ARA. From these results, it appears that although P is affected by NH3, NH3 does not exert a controlling influence over nitrogenase activity by alterations in nodule permeability. Excluding a direct effect of NH3 on nodule permeability, NH3 may decrease nitrogenase activity in at least three other 269 www.plantphysiol.org on December 30, 2017 Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved. PURCELL AND SINCLAIR Table I. Plant Nodule Characteristics, Initial ARA, and Initial P of NH3 Treatments Values reported are the mean ± SEM (n = 3 except where indicated). Light NH3 Nodule Nodule Nodule Initial Initial Regime Concn. Dry Weight Number Diameter ARA P uL L-' g mm mm3s-1 g-1 mms-1 x 1O3 High 0 0.318 ± 0.036 73 ± 16 2.55 ± 0.16 0.84 ± 0.01 19.0 ± 1.5 274 0.258 ± 0.025 42 ± 9 2.81 ± 0.16 0.89 ± 0.09 23.7 ± 3.8 411 0.222 ± 0.048 51 ± 13 2.53 ± 0.07 0.90 ± 0.17 20.3 ± 3.9 547 0.216 ± 0.074 44 ± 12 2.56 ± 0.11 0.85 ± 0.10 16.3 ± 2.3 684 0.209 ± 0.027 38 ± 4 2.74 ± 0.03 0.79 ± 0.05 20.0 ± 2.3 821 0.142 ± 0.027 32 ± 7 2.71 ± 0.05 1.26 ± 0.14 23.3 ± 3.3 Meana 0.228 ± 0.020 47 ± 5 2.65 ± 0.05 0.92 ± 0.05 20.4 ± 1.2 Low 0 0.179 ± 0.022 34 ± 5 2.78 ± 0.10 1.15 ± 0.08 23.0 ± 2.5 274 0.137 ± 0.023 23 ± 1 2.83 ± 0.21 1.15 ± 0.08 25.0 ± 1.2 411 0.149 ± 0.022 31 ± 8 2.76 ± 0.21 0.94 ± 0.08 21.3 ± 3.5 547 0.142 ± 0.011 31 ± 5 2.69 ± 0.10 1.18 ± 0.06 22.0 ± 1.7 684 0.149 ± 0.013 38 ± 9 2.70 ± 0.25 1.06 ± 0.09 19.3 ± 1.9 Meanb 0.149 ± 0.007 32 ± 2 2.71 ± 0.07 1.07 ± 0.03 22.2 ± 1.0