Endogenous Ethylene Production Is a Potential Problem in the Measurement of Nitrogenase Activity Associated with Excised Corn and Sorghum Roots

Abstract

Endogenous ethylene production was evaluated as a source of ethylene during acetylene reduction assays with freshly collected roots of fieldgrown corn, Zea mays L. cv Funks G4646, and sorghum, Sorghum bicolor (L.) Moench. cv CK-60A. Ethylene production was not detected when roots were incubated in air without acetylene. The presence of endogenous ethylene production was confirmed when roots were incubated anaerobically and in the presence of 40 millimolar sodium hydrosulfite. Ethylene oxidase activity was also associated with excised roots. The rate of ethylene oxidation was higher than the rates of ethylene accumulation during either acetylene reduction assays or anaerobic incubations. These results indicate that the procedure of incubating roots of grasses in air to monitor endogenous ethylene production is not a valid control in acetylene reduction studies with grasses. The presence of endogenous ethylene production during acetylene reduction assays was demonstrated by using either CO to inhibit nitrogenase activity or chloramphenicol to inhibit nitrogenase synthesis in freshly excised roots. problem in measuring acetylene reduction with soil cores having low nitrogenase activity (3,17). This has not been considered as a problem with excised roots because endogenous ethylene production has not been detected with samples incubated in air without acetylene (4,5,7,12). Ethylene production in soils is not detected during aerobic incubations because the rate of ethylene oxidation is greater than ethylene production (1). It is unknown whether ethylene oxidase activity is associated with excised roots.The objective of this investigation was to examine whether endogenous ethylene production is a source of ethylene during acetylene reduction assays with freshly excised roots of fieldgrown corn and sorghum. This was accomplished by assessing the impact of inhibitors of nitrogenase activity and synthesis on the accumulation of ethylene during acetylene reduction assays. Endogenous ethylene production associated with excised roots was detected when roots were incubated under anaerobic conditions in the presence of sodium hydrosulfite, a reducing agent. A preliminary report of the work has appeared (8).Most of the evidence for nitrogen fixation by bacteria associated with roots of grasses has been obtained by the measurement of nitrogenase activity using the indirect acetylene reduction method in which nitrogenase catalyzes the reduction ofacetylene to ethylene. The use of a prolonged microaerophyllic preincubation period or a long-term acetylene reduction assay has lead to a problem of falsely identifying the presence of nitrogenase activity in grasses or overestimation of rates of N2 fixation (1 1). Measurements of immediate nitrogenase activity by freshly collected grass roots are desirable because they may more closely reflect the presence of and the rate of activity associated with the roots before sampling. Low rates of acetylene reduction by corn and wheat roots have been reported using measurements made...

Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer

“…While our method cannot rule out the possibility of acetylene inhibiting bacterial oxidation of endogenous ethylene that might have been produced by the plant (Debont, 1976), ARA has been successfully used as an indicator of nitrogenase activity in many studies of both above-and belowground tissues and in bacterial isolates from plant tissues (DeLuca et al, 2002;Doty et al, 2009;Bal & Chanway, 2012;Jean et al, 2012;Rout et al, 2013). Endogenous ethylene oxidation, which could potentially be inhibited by acetylene, and therefore confound ARA, has been demonstrated in soil, but only after 8 d of incubation (Debont, 1976), and in wheat (Triticum aestivum) roots, but only in samples with soil attached, not in surface-sterilized roots (Sloger & van Berkum, 1988). Given the short incubation time used here (2 h), the lack of soil in our samples, and the complimentary sequencing data, we think that there is strong evidence for the presence of N 2 -fixing endophytes in P. flexilis.…”

Screening for crop response to Diazotrophic bacteria isolated from potato rhizosphere

“…Further, it has been reported by several other studies that the nitrogenase activities shown by PGPRs vary between 0.25 nmoles/hr to 15.8 nmoles/hr (Sloger and Berkum, 1988;Khan and Doty, 2009). However, most of the bacterial isolates in the current study showed moderate nitrogenase activity (2.5 -2.9 nmol/hr) compared to the previous studies.…”