Nitrogenase in Soybean Nodules

Soybean (Glycine max cv Hodgson) nitrogenase activity (C2H2 reduction) in the presence or absence of nitrate was studied at various external 02 tensions. Nitrogenase activity increased with oxygen partial pressure up to 30 kilopascals, which appeared to be the optimum. A parallel increase in ATP/ADP ratios indicated a limitation of respiration rate by low 02 tensions in the nodule, and the values found for adenine nucleotide ratios suggested that the nitrogenase activity was limited by the rate of ATP regeneration. In the presence of nitrate, the nitrogenase activity was low and less stimulated by increased P02, although the nitrite content per gram of nodules decreased from 0.05 to 0.02 micromole when P02 increased from 10 to 30 kilopascals. Therefore, the accumulation of nitrite inside the nodule was probably not the major cause of the inhibition. Instead, inhibition by nitrate could be due to competition for reducing power between nitrate reduction and bacteroid or mitochondrial respiration inside the nodule. This is supported by the observation of decrease in ATP/ADP ratios from 1.65, in absence of nitrate, to 0.93 in the presence of this anion at 30 kilopascals 02. Furthermore, the inhibition was suppressed by the addition, to the plant nutrient solution, of 15 millimolar L-malate, a carbon substrate that is considered to be the major source of reductant for the bacteroids in the symbiosis. Soybean in symbiosis with Bradyrhizobium japonicum has two sources of nitrogen nutrition: fixation of atmospheric molecular nitrogen and assimilation of soil nitrogen, found mostly in the form of nitrate. Nitrite production by NR2 activity of the cytosol and the bacteroids in the soybean nodules causes accumulation ofNO2that is probably related to the low PO2 inside the nodules, because raising the external partial pressure of oxygen causes the internal NO2content to decrease (8). This accumulation of nitrite could be the cause of the inhibition of N2 fixation, since NO2is an inhibitor of nitrogenase (27) and ofleghemoglobin (20). However, this hypothesis has been recently questioned (26). Another hypothesis is that nitrate reduction would limit Present address: Station Agronomique de Laon, Institut National de la Recherche Agronomique, Rue F. Christ, BP 101, 02004 Laon Cedex, France. 2 Abbreviations: NR, nitrate reductase; ARA, acetylene reducing activity; MDH, malate dehydrogenase; PEP, phosphoenolpyruvate; PEPcase, phosphoenolpyruvate carboxylase; YEM, yeast extract mannitol; EC, energy charge. the amount of reductants for molecular nitrogen fixation. The reduction of dinitrogen to ammonia is an energy intensive process; it depends upon the photosynthate supply by the host plant (7) and the oxygen diffusion to the bacteroids inside the root nodules (1, 4, 19). Dicarboxylic acids are considered to be the major sources of reducing power for bacteroids (22) although, in vitro, glucose can also be utilized (28). The purpose of the present research was to investigate the short term inhibition of nitrogenase activity by nitrate. The role of nitrite was tested by comparing the effect of external 02 tensions on the nodule nitrite contents and ARA of N03-preincubated soybeans and control plants receiving no nitrate. The hypothesis of competition between NO3reduction and N2 fixation for reducing-power inside the nodules was tested by measuring the effect of an external supply of malate on nodule C2H2 reduction inhibition by nitrate under increasing external 02 tensions. MATERIALS AND METHODS Biological Material and Growth Conditions Soybeans plants, Glycine max (L.) Merr. cv Hodgson, were grown in an aerated liquid nutrient medium described previously (12). Bradyrhizobium japonicum strain PJ 17 (13) inoculant was prepared in the YEM liquid medium. Sterile seeds, pregerminated on YEM agar (1 %), were inoculated by soaking for 30 min in the liquid inoculant. Plants were then transferred into individual culture vessels that consisted of a 1 L serum flask containing 0.9 L of nutrient solution. They were grown in a greenhouse where supplemental light (200 ,umol photons m-2.s-) provided a photoperiod of 16 h. The temperature was 28°C during the day and 22°C during the night. The pH of the nutrient solution was maintained at neutrality by CaCO3 in excess (12). The medium was supplemented with 2 mm urea until 30 d after sowing. Then, plants received no mineral nitrogen except during-the experiments that began-40 d after sowing. At this age of the plants, the nitrogenase -activity per g of nodule fresh weight was maximum (12). Influence of Oxygen on Nitrogenase Activity (C2H2 Reduction) Nitrogenase activity was estimated by the ARA of in situ root nodules incubated in an open flow-through gassing system described previously (6). Plants were transferred to the