Measurement of nitrogenase activity of intact legume symbionts in situ using the acetylene reduction assay
Measurement of nitrogenase activity of intact legume symbionts in situ using the acetylene reduction assay
复制标题
使用乙炔还原测定原位测量完整豆类共生体的固氮酶活性
DOI：
10.2134/agronj1973.00021962006500030022x
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发表时间：
1973
期刊：
影响因子：
2.1
通讯作者：
L. Boersma
中科院分区：
文献类型：
--
作者：
K. Fishbeck;H. Evans;L. Boersma
乙炔还原测定已广泛用于检测和测量自由生活的微生物、切除的豆科植物和非豆科植物根瘤、根系、土壤核心和从土壤介质中取出的整个结瘤豆科植物的固氮酶活性。进行这项研究是为了确定乙炔还原测定是否可用于测量原位结瘤大豆（Glycine max.）植物培养物的固氮酶活性，并将这些培养物的固氮酶活性与来自可比培养物的分离根瘤和切除的根系统的固氮酶活性进行比较。将在珍珠岩或溶胶中生长的整个培养物置于聚乙烯容器中，盖上有机玻璃盖，暴露于乙炔，并测定乙烯产量。时程实验表明，在 15 分钟的初始滞后期之后，在珍珠岩和土壤中生长的植物的乙炔还原率在至少 90 分钟内呈线性。完整培养物中固氮酶的饱和度是在 pC2H2 为 0.1 atm 时获得的。固氮酶的表观 Km 为 0.05 atm。为了方便起见，在大容器中的测定在 0.025 atm 的 pC2H2 下进行。 Michaelis-Menten 方程用于计算乙炔在饱和水平下的乙炔还原率。乙炔还原率与根瘤鲜重的相关系数，同龄植株为0.99，不同龄植株为0.79。珍珠岩中完整的结节植物或从珍珠岩中取出的结节根系的乙炔减少率显着高于来自可比培养物的分离根瘤的乙炔减少率。在受控环境条件下生长的盆栽植物固氮酶活性的昼夜变化相对较小。该方法可用于评估标准化条件下多孔介质中豆类培养物的固氮酶活性，但事实证明，由于土壤含水量影响培养基内气体扩散速率，该方法在土壤中豆科植物的应用变得复杂
The acetylene reduction assay has been used extensively to detect and measure nitrogenase activity of free-living microorganisms, excised legume and non-legume nodules, and root systems, soil cores, and entire nodulated legume plants removed from their soil medium. This investigation was undertaken to determine whether or not the acetylene reduction assay can be used to measure nitrogenase activity of cultures of nodulated soybean (Glycine max.) plants in situ and to compare the nitrogenase activity of these cultures with the nitrogenase activity of detached nodules and excised root systems from comparable cultures. Entire cultures grown in Perlite or in soll were placed in polyethylene containers, covered with Plexiglas lids, exposed to acetylene, and assayed for ethylene production. Time course experiments showed that rates of acetylene reduction of plants growing in Perlite and in soil were linear for at least 90 min following an initial lag period of 15 min. Saturation of nitrogenase in intact cultures was obtained at a pC2H2 of 0.1 atm. The apparent Km for nitrogenase was 0.05 atm. For convemence, assays in the large containers were conducted at a pC2H2 of 0.025 atm. The Michaelis-Menten equation was used to calculate rates of acetylene reduction at saturating levels of acetylene. The correlation coefficient between rates of acetylene reduction and nodule fresh weight was 0.99 for plants of the same age and 0.79 for plants of different ages. Acetylene reduction rates of either intact nodulated plants in Perlite or nodulated root systems removed from Perlite were significantly greater than acetylene reduction rates of detached nodules from comparable cultures. Relatively little diurnal variation was observed in nitrogenase activity of potted plants grown under controlled environmental conditions. The method is useful for the assessment of nitrogenase activity of legume cultures in a porous medium under standardized conditions, but its application to legumes in soil proved to be complicated by the water content of the soil which influences rates of gas diffusion within the culture medium