Microelectrode Measurements of Hydrogen Concentrations and Gradients in Legume Nodules

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Abstract
This paper describes the construction and operation of H2 specific microelectrodes and their application to measurements of H2 concentrations, H2 gradients and H2 inhibition of N2-fixation in legume root nodules. Electrode construction was similar to that of O2 specific microelectrodes previously reported. They comprise an outer casing drawn to a 2–20 μm tip plugged with silicone rubber and a concentric inner electrode made from glass coated platinum wire. The exposed tip of the Pt wire was placed close to the silicone plug and polarized positively at 0·4 V with respect to an internal Ag reference electrode. With an internal electrolyte of KC1/HC1 current flow through the electrode was proportional to H2 concentration and independent of CO2 and O2. With appropriate amplification and screening the detection limit for this system was 0·0001 atm H2 (4·0 μmol m−3). Within newly detached nodules of Hup−ve symbioses of soyabean, pea and clover H2 concentration varied from 0·009 to 0·014atm compared with 0·021 atm in lupin nodules. In nodules formed by the Hup−ve soyabean/RCR3442 symbiosis internal pH2 increased from 0·012 atm to 0·09 atm when external pO2 was raised to 0·60 atm. Hydrogen could not be detected within nodules of the Hup+ve Clarke/RCR3407 symbiosis even when N2 in the gas phase was replaced with Ar and external pO2 was increased to 0·60 atm. An assessment of H2 inhibition of nitrogen fixation in the soyabean (Clarke/RCR3442) symbioses involved measurements of H2 production at increasing internal H2 levels, induced by stepped increases in gas phase H2 concentration. The initial relative efficiency of 0·66 (calculated from the pH2 of nodules exposed to air and Ar/O2 mixtures) started to decrease at an internal pH2 of 0·02 to 0·03 atm and fell by 80% to 0·18 at an internal pH2 of 0·1 atm. This threshold value for inhibition is above the measured mean H2 concentration for this symbiosis of 0·01 atm. Hydrogen gradients through the nodule showed a sharp increase in the region of the inner cortex, which was reciprocal to a decrease in O2 concentration, and a shallow gradient through the infected zone. These results indicate that the inner air spaces in the nodule are interconnected and confirm that the barrier to O2 diffusion is located in the inner cortex.

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