Factors Affecting the Acetylene-Induced Decline during Nitrogenase Assays in Root Nodules of Myrica gale L.

Abstract

Our goal was to determine why the rate of acetylene reduction by nodules of actinorhizal plants declines after an initial peak value. The decline was eliminated by pretreatment with argon, indicating that the decline is initiated by cessation of ammonia synthesis. When 02 concentration was decreased during the decline, the rate of acetylene reduction increased. This shows that during the decline there is either 02 toxicity or competition between respiration and nitrogenase for reductant. The decline was not eliminated when uptake hydrogenase was inactivated by pretreatment with acetylene, showing that cessation of H2 oxidation is not the primary cause of the decline. The effects of a variety of other treatments on the decline were also studied. Overall, we conclude that the cessation of ammonia formation is the primary cause of the acetylene-induced decline. We hypothesize that the supply of reductant for nitrogenase depends on amino acids that are depleted following cessation of ammonia formation. We also conclude that the initial peak rate of acetylene reduction provides the best measure of nitrogenase activity.The acetylene reduction assay for nitrogenase activity has a number of desirable characteristics: it is fast and low in cost and allows repeated assays of the same material. Unfortunately, the rate of C2H2 reduction is often not constant as a function of time, with an initial peak rate being followed by a decline. In legume nodules, there is typically little or no recovery from this decline (7, 21), whereas in actinorhizal plants there is a variable recovery of activity, with the recovered rate approaching the initial peak rate in some instances (9,10,13,15,17,20). We found an especially large decline followed by a strong recovery in water-cultured plants of Myrica gale L. and used this as a model system to study the C2H2-induced decline. Our goal was to determine the causes of this phenomenon and the conditions under which the acetylene reduction assay most accurately measures nitrogenase activity. We examined the effects of cessation of ammonia production, temperature, 02 concentration, inactivation of uptake hydrogenase, and light deprivation. Our results show that nitrogenase activity is affected by changes in metabolism caused by the cessation of ammonia production in the presence of C2H2. We also conclude that the initial peak rate of C2H2 reduction is the best measure of nitrogenase activity. ' This work was supported by U.S. Department of Agriculture grants 87-CRCR-1-2431 and 87-CRCR-1-2440. 1451 MATERIALS AND METHODSMyrica gale L. was grown from seeds, inoculated, and grown as described by Monz and Schwintzer (9). Inoculation was with Frankia strain LLR16 1101, and seedlings were grown in individual water cultures (1/4 strength, nitrogen-free Hoagland solution, 250-mL plastic bottles) in a growth chamber with a 16-h photoperiod (450 umol m-2 s-' PPFD, 70 to 80% humidity, 22°C day, 20°C night). The solution level in the bottles was kept below the nodulated portion of the roots, and the ...

Factors Affecting the Acetylene to 15N2 Conversion Ratio in Root Nodules of Myrica gale L

“…In a previous study (Tjepkema and Schwintzer, 1992), we showed that the extent of the decline and the recovery depends on growth conditions prior to the assay and conditions during the assay, including the acetylene concentration, O2 concentration, and temperature. We further showed that the decline can be eliminated by pretreatment with argon, indicating that it is initiated by cessation of ammonia synthesis.…”

“…These treatments were chosen because they substantially decrease the extent of the recovery (Tjepkema and Schwintzer, 1992, and preliminary experiments), thus allowing a clearer determination of whether the peak rate or the recovered rate of acetylene reduction is the best measure of nitrogenase activity. Plants were water stressed by withholding water beginning 6 d prior to the experiment.…”

“…In a previous study (Tjepkema and Schwintzer, 1992), we showed that the extent of the decline and the recovery depends on growth conditions prior to the 'This work was supported by U.S. Department of Agriculture grants 87-CRCR-1-2431 and 87-CRCR-1-2440 and National Research Initiative Competitive Grants Program grant 92-37305-7758.…”

“…This phenomenon has been termed the acetyleneinduced decline. In legume nodules, there is typically little or no recovery from this decline (Minchin et al, 1983;Witty et al, 1986), whereas in actinorhizal plants there is a variable recovery of activity with the recovered rate closely approaching the initial rate in some instances (Rosendahl and HussDanell, 1988;Tjepkema et al, 1988;Monz and Schwintzer, 1989; Silvester and Hams, 1989;Silvester and Winship, 1990;Winship and Tjepkema, 1990;Tjepkema and Schwintzer, 1992).We found an especially large decline followed by a strong recovery in hydroponically grown plants of Myrica gale L.and have used this as a model system to study the acetyleneinduced decline. In a previous study (Tjepkema and Schwintzer, 1992), we showed that the extent of the decline and the recovery depends on growth conditions prior to the assay and conditions during the assay, including the acetylene concentration, O2 concentration, and temperature.…”

“…This phenomenon has been termed the acetyleneinduced decline. In legume nodules, there is typically little or no recovery from this decline (Minchin et al, 1983;Witty et al, 1986), whereas in actinorhizal plants there is a variable recovery of activity with the recovered rate closely approaching the initial rate in some instances (Rosendahl and HussDanell, 1988;Tjepkema et al, 1988;Monz and Schwintzer, 1989; Silvester and Hams, 1989;Silvester and Winship, 1990;Winship and Tjepkema, 1990;Tjepkema and Schwintzer, 1992).…”