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ABSTRACIAn open ps exchange system was used to monitor the nonsteady state and steady state changes in nitrogenase activity (H2 evolution in N2:02 and Ar:02) and respiration (CO2 evolution) in attached, excised, and sliced nodules of soybean (Glycine max L. Menf.) exposed to extenal P02 of 5 to 100%. In attached nodules, increases in external P02 in steps of 10 or 20% resulted in sharp declines in the rates of H2 and CO2 evolution. Recovery been proposed (18). Evidence for a distinct barrier to gas diffusion in the nodule cortex has been obtained experimentally by direct measurements of P02 in the outer and central tissues of soybean nodules (2 1).The exact nature of the diffusion barrier and the manner in which it is regulated are unknown, but it has been suggested that the nodules of many symbiotic associations increase their diffusion resistance to O2 entry when they are exposed to an atmosphere containing 10% C2H2, or one in which N2 is replaced by Ar (25). This increase in diffusion resistance is apparent as a decline in nodule respiration rate, with a concomitant decline in C2H2 reduction or H2 production rate, during the first 30 min of exposure to C2H2 or Ar. The presence or absence of an C2H2-or Ar-induced decline has been correlated with the optimum P02 for N2 fixation within a specific legume-Rhizobium association and, by inference, with the speed with which the diffusion barrier is regulated in that association (25). It has also been suggested that the regulation of the diffusion resistance of the nodule requires physical changes in the diffusion barrier (4), and this may limit the speed with which the nodule responds to changes in its gaseous environment.The aims of this study were (a) to determine whether the gas exchange characteristics of soybean nodules following changes in external P02 are consistent with the presence of a variable diffusion barrier in the nodule cortex, and (b) if so, to identify experimental conditions which could be used to vary this diffusion barrier. An open circuit gas exchange system was used to monitor continuously changes in respiration (CO2 evolution) and nitrogenase activity (H2 evolution in N2:02 and Ar:02) as the P02 surrounding the nodules was varied. Nonsteady state measurements of CO2 production and H2 evolution were used to determine the speed with which the nodules adjust to changes in rhizosphere O2 concentration. Steady

IYXX. Oxygen limitation of N, fixation in stem-girdled and nitrate-treated soybean. -Physiol. Plant. 73: 11S121.The effects of increasing rhizosphere PO, on nitrogenase activity and nodule resistance to O2 diffusion were investigated in soybean plants [Glycine max (L.) Merr. cv. Harosoy 631 in which nitrogenase (EC 1.7.99.2) activities were inhibited by (a) removal of the phloem tissue at the base of the stem (stem girdling), (b) exposure of roots to 10 mM NOj over 5 days (NOj-treated). or (c) partial inactivation of nitrogenase activity by an exposure of nodulated roots to 100 kPa O2 (O?-inhibited). In control plants and in plants which had been treated with 1 0 kPa 02, increasing rhizosphere 0, concentrations in 10 kPa increments from 20 to 70 kPa did not alter the steady-state nitrogenase activity. In contrast. in plants in which nitrogenase activities were depressed by stem girdling or by exposure to NOj. increasing rhizosphere p 0 2 resulted in a recovery of 57 or 67%. respectively. of the initial, depressed rates of nitrogenase activity. This suggests that the nitrogenase activity of stemgirdled and NO;-treated soybeans was O,-limited.O2 at 20 kPa and an apparent insensitivity of diffusion resistance to increases in external PO?.

Communicated by J.H.Weil tant of Bradyrhizobiumjaponicum, the amounts of GS mRNA did not increase over that in roots. These experiments, together with the time course of increase in GS mRNA transcripts, suggest that the genes encoding cytosolic GS are directly induced by the available ammonia.

In many legumes, the nitrogen fixing root nodules produce H2 gas that diffuses into soil. It has been demonstrated that such exposure of soil to H2 can promote plant growth. To assess whether this may be due to H2-oxidizing microorganisms, bacteria were isolated from soil treated with H2 under laboratory conditions and from soils collected adjacent to H2 producing soybean nodules. Nineteen isolates of H2-oxidizing bacteria were obtained and all exhibited a half-saturation coefficient (Ks) for H2 of about 1 ml l(-1). The isolates were identified as Variovorax paradoxus, Flavobacterium johnsoniae and Burkholderia spp. using conventional microbiological tests and 16S rRNA gene sequence analysis. Seventeen of the isolates enhanced (57-254%) root elongation of spring wheat seedlings. Using an Arabidopsis thaliana bioassay, plant biomass was increased by 11-27% when inoculated by one of four isolates of V. paradoxus or one isolate of Burkholderia that were selected for evaluation. The isolates of V. paradoxus found in both H2-treated soil and in soil adjacent to soybean nodules had the greatest impact on plant growth. The results are consistent with the hypothesis that H2-oxidizing bacteria in soils have plant growth promoting properties.

Several investigations of legumes have attempted to measure the cost of symbiotic N2 fixation in terms of respired carbohydrate and to compare such costs with those of nonnodulated plants utilizing NO3. Some studies (6, 12) have reported little difference in C economy with the two forms of N assimilation; in others (11,20,21) a considerably lower output of respired CO2 has been observed for N03-reducing plants than for those fLxing N2. Variation in results might be due to differences in pattern of NO3 assimilation by the various species used, since if NO3 reduction were accomplished in leaves from photosynthetically generated reductant (4) a lower respiratory requirement for NO3 assimilation would be expected than if NO3 were reduced heterotrophically in roots. Interpretation has also proved difficult due to lack of strict physiological comparability between nodulated and N03-reducing plants (3, 6, 12), and to technical difficulties in measuring the separate respiratory outputs of shoots, root, and nodules of intact living plants (1,10,20 Dry Weight and C and N Analyses. Plants were separated into leaflets, stem + petioles, unexpanded apical regions of shoots (including inflorescence, ifpresent), roots, and nodules (ifpresent). Levels of C and N in dry matter were determined as described previously (7,15). Salicylic acid was used in Kjeldahl digests of dry matter ofNO0-grown plants so that N03-N would be measured in determinations of total N(5).Collection and Analysis of Xylem and Phloem Sap. Root bleeding exudate (xylem sap) was sampled over a 15-min period from root stumps of freshly decapitated plants, and phloem sap was collected from shallow incisions in petioles of mature leaves, or from base or top of shoots, at the locations described in an earlier study (16). Collections were made at least three times during the study interval of 10 days. Analyses of sap for sugars, organic acids, amides, and amino acids were as detailed elsewhere (14,(17)(18)(19).

The aim of the present study was to test the hypothesis that the N content or the composition of the phloem sap that supplies nodulated roots may play a role in the feedback regulation of nitrogenase activity by increasing nodule resistance to O, diffusion. High nitrate levels in soils are known to inhibit both nodule formation and nitrogenase activity in legume nodules (Streeter, 1988). The inhibitory effects of nitrate on specific nitrogenase activity have been classified into those that occur within the first 2 or 3 d (Vessey et al., 1988b;Minchin et al., 1989; Escuredo et al., 1996) and those that result from longer term exposure to nitrate (Minchin et al., 1989; Escuredo et al., 1996). There is widespread evidence that the initial effect of nitrate exposure causes the respiration rate and nitrogenase activity of legume nodules to become severely O, limited, as a result of an increase in the nodule resistance to O, diffusion and a decrease in the concentration of O, in the bacteria-infected cells (Schuller et al., 1988;Vessey et al., 1988a;Minchin et al., 1989;Layzell et al., 1990;Vessey and Waterer, 1992; Hunt and Layzell, 1993; Denison and Harter, 1995; Escuredo et al., 1996). In nitrate-inhibited nodules nitrogenase activity and nodule respiration rates can recover partially or sometimes to rates approximating a pre-decline status by increases in externa1 p0, to levels greater than that in air (Minchin et al., 1986(Minchin et al., , 1989Schuller et al., 1988;Vessey et al., 1988a; Faurie and Soussana, 1993; Kaiser et al., 1994; Escuredo et al., 1996).Various hypotheses have been proposed to explain the initial inhibitory effects of nitrate and the role that O, plays in the phenomena. For example, within the first few days of exposure, nitrate accumulates in the nodule cortex where it has been proposed to cause water to move out of cortical cells and into the gas-filled intercellular spaces, thus increasing the resistance of the nodule to O, diffusion (Sprent et al., 1987;Minchin et al., 1989;Serraj et al., 1995). Nitrate could also enter the central zone of nodules, where it may be converted to nitric oxide, which could bind to leghemoglobin to form nitrosylleghemoglobin, a heme protein that is unable to facilitate the diffusion of O, to the bacteroids (Kanayama and Yamamoto, 1990a, 1990b Kanayama et al., 1990). However, the latter hypothesis has been recently challenged by a nodule oximetry study (Denison and Harter, 1995) that showed no decrease in the functional leghemoglobin concentration in nodules that are exposed to nitrate.Other studies have shown that nitrate reduces photosynthate partitioning to nodules (Vessey et al., 1988a, 198813; Faurie and Soussana, 1993), and because treatments that disrupt the phloem sap supply to nodules (e.g. nodule excision, plant defoliation, stem girdling, or stem chilling) also induce severe O, limitation within the infected cells (Minchin et al., 1985;Walsh et al., 1987;Schuller et al., 1988; Vessey et al., 1988a, 198813; Faurie and Soussana, 1993; Hartwig ...