Oxygen control of nitrogen fixation in Frankia and Coriaria arborea

Abstract

Oxygen has a central role in regulation of nitrogen fixation activity in both asymbiotic Frankia and Frankia - actinorhizal plant symbioses. Daily addition of fresh medium at a low dilution rate (D=0.125 day⁻¹) allowed Frankia to be maintained in continuous culture with a stable rate of growth and nitrogenase activity for periods of more than 30 days. Use of continuous cultures permitted the effects of oxygen on vesicle morphology and nitrogenase activity to be investigated independent of normal developmental changes occurring in batch culture. Frankia grown at higher oxygen concentrations formed thicker-walled vesicles which allowed adaptation of nitrogenase activity over a pO₂ range of 5 - 40 kPa O₂. Recovery of nitrogenase activity after oxygen shock (2 to 21 kPa O₂) followed formation of thicker-walled vesicles. Vesicle walls also showed a thickening response to pO₂ level under conditions where nitrogenase activity was inhibited (in Ar : O₂ atmospheres) or not detected (in cultures grown on nitrate-containing medium). These results indicated the presence of vesicles and the induction of nitrogenase activity in culture is not as synonymous as was originally believed. GC/MS analysis of lipid extracts from Frankia cultures and actinorhizal nodules confirmed the high hopanoid content of Frankia. Differences in the relative proportions of hopanoids present were also detected between samples and the total amount of hopanoids measured in Frankia cultures was slightly higher at higher oxygen concentrations. Most work on Frankia symbioses focussed on the Coriaria arborea association and highlighted startling similarities between this actinorhizal symbiosis and Rhizobium - legume associations. Coriaria showed a legume-like, non-recoverable decline in nitrogenase activity and nodule respiration following exposure to 10 kPa acetylene or argon. The extent of the acetylene-induced decline was diminished with increasing plant age and adaptation to above atmospheric oxygen concentrations. Lag-phase measurements indicated acetylene-induced declines in Coriaria are due to an apparent increase in nodule diffusion resistance which has also been observed in legumes. Defoliation of Coriaria also caused a decline in nitrogenase and respiratory activity associated with an apparent increase in nodule diffusion resistance although the effect was delayed for approximately two hours. Continuous flow assay of nitrogenase activity in several actinorhizal species and one legume, exposed to alternate gas streams of nitrogen and helium at varying oxygen tensions, indicated a small component of the diffusion pathway in Coriaria arborea and Myrica gale nodules is an air-filled pathway. In Alnus glutinosa, Casuarina cunninghamiana and Medicago sativa nodules the diffusion pathway has some “water-filled” or “solid” diffusion barrier. Speculation is offered on the location and functioning of fixed and variable diffusion resistance in Coriaria nodules based on results from this study and previous work.