Argon-Induced Decline in Nitrogenase Activity in Root Nodules of Soybean1

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). Both the acetyleneand Ar-induced declines can be largely prevented by carrying out experiments with nodules adapted to higher 02 concentrations (5, 23). Furthermore, Witty et al. (22) showed that nodules could recover from the acetylene-induced decline ifthe rhizosphere 02 concentration was increased in stepwise increments. The 02 dependence of the acetyleneand Ar-induced deSupported by an operating grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) and an Advisory Research Council grant (Queen's University) to D. B. L. B. J. K. acknowledges the financial support of NSERC and Queen's University. 2 Present address: Department of Botany, University of British Columbia, Vancouver, B.C., Canada V6T 2B1. 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 of02 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 acetyleneor 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 acetyleneand 02-induced transient responses in several actinomycete symbioses are associated with the Frankia vesicles and are not due to changes in nodule gas permeability. Rather, the authors attribute them to a biochemical 'switch-off' of nitrogenase activity. A similar switchoff has previously been suggested to occur in soybean nodules in response to increases in 02 concentration (5-7) and could possibly be involved in the response of legumes to acetylene and Ar:02 as well. Therefore, on the basis of the studies to date, it may be premature to attribute either the acetyleneor Ar-induced declines to decreased P, either in whole or in part. In light of this, the aims of the present study were two-fold: to characterize the time course of the Ar-induced decline in respiration and H2 evolution in intact, attached soybean nodules; and to determine whether or not the decline results from decreased P and consequent 02 limitation of nitrogenase activity. MATERIALS AND METHODS Plant Culture and Gas Exchange Measurements Seeds of soybean (Glycine max L. Merr. cv Harosoy 63 or Maple Arrow) were inoculated with Bradyrhizobium japonicum USDA 16, a strain which lacks uptake hydrogenase activity (10), and grown in silica sand culture as described previously (5) in a growth chamber with 16-h photoperiod,