John F. Witty

Respiratory/Carbon Costs of Symbiotic Nitrogen Fixation in Legumes

This chapter presents an overview of the respiratory/carbon costs of symbiotic nitrogen fixation. The various theoretical costings for nitrogen fixation suggest that respiration directly associated with nitrogenase activity will require between 1.77 and 3.01 g C g−1-N (4.35–7.00 mol CO2 mol−1 N2), while respiration of the entire nitrogen-fixing nodules will require between 2.78 and 4.81 g C g−1-N (6.51–11.19 mol CO2 mol−1 N2). Early attempts to measure these costs were beset by methodological problems, but some reliable approaches were developed. Measured values based on root respiration during the period of active nitrogen fixation are in the range of 5–10 g C g−1-N (11.6–23.4 mol CO2 mol−1 N2), with an average value of 6.5 g C g−1-N (15.1 mol CO2 mol−1 N2). On a nodule basis, values in the range of 3–5 g C g−1-N (7–12 mol CO2 mol−1 N2) appear to represent the ‘normal’ for legume nodules, while values below about 2.5 g C g−1-N are likely to be erroneous. The implications of these costings are considered in terms of the need for legumes to carefully regulate nitrogen fixation and the requirement for such regulation systems to be operational in any novel nitrogen-fixing plants.

Reply to ‘Measurement of nitrogenase activity in legume root nodules: In defense of the acetylene reduction assay’ by J.K. Vessey

This article is in response to that of Vessey (1994) who argues that the traditional, closed acetylene reduction assay can still be a valuable tool for measuring relative differences in nitrogenase activity of legumes. To counter this assertion we consider the practical uses of the traditional assay procedure in relation to real research situations. This requires the use of the assay to be considered separately in the different circumstances of pot-grown and field-grown plants. We conclude that for pot-grown legumes there are a few practical applications where the use of the traditional, closed assay procedure is valid and we accept that these can be extended by the careful use of calibrations against open, flow-through systems. However, we doubt that there are many situations where such a calibration approach would have practical advantages over using the flow-through system to obtain the actual measurements. We cannot recommend any form of the uncalibrated acetylene reduction assay for field-based studies and suggest that researchers consider the merits of simple, alternative measurements such as dry weight, yield and total nitrogen.

Factors Limiting N2 Fixation by the Legume-Rhizobium Symbiosis

It is not our intention to produce yet another review of the numerous publications concerning factors limiting N2 fixation but rather to recommend novel directions for the future. The need for a new impetus was perceived in a review of environmental factors by Sprent et al (1983) where it was noted that many alternative hypotheses were available which postulated either a direct effect of these factors on the nodule system or an indirect effect acting via the host plant. Such conflicts must be resolved if research into limiting factors is to contribute to the improvement of nitrogen fixation by legume crops. However, before progress can be made it is necessary to improve the accuracy of techniques which measure nitrogen fixation and related processes, and to focus greater attention on the role of oxygen supply.

Limitations and Errors in Gas Exchange Measurements with Legume Nodules

Physiological studies of nitrogen fixation in legumes have often involved gas exchange measurements of nodules and nodulated roots. Respiration has been studied since the early 1940’s (Allison et al. 1940, Bond 1941), 15N2 fixation since 1952 (Aprison and Burris 1952), H2 evolution since 1957 (Hoch et al. 1957), and acetylene reduction since 1966 (Koch and Evans 1966). However, only recently with the advent of continuous flow-through gas systems has it been possible to demonstrate the errors which can occur when making these measurements. This review will concentrate on errors and limitations associated with the acetylene-induced decline in nodule activity and the effect of disturbance on oxygen diffusion into nodules.

Simple Apparatus for Growth of Nodulated Plants and for Continuous Nitrogenase Assay Under Defined Gas Phase

The central position of oxygen in the control of the nitrogenase activity in both symbiotic and asymbiotic diazotrophs is now well established (Robson and Postgate 1980, Witty et al. 1986). While considerable attention has been given to growing and assaying asymbiotic organisms at a variety of O2 levels and testing for adaptation and response, relatively few experiments have been directed at altering the gas phase of nodulated plants. The early experiments of McConnell (1959) and Crisswell et al. (1976) on alder and soybean, respectively, showed that both groups of plants, when grown with root systems at various pO2 levels, could adapt their growth and nitrogenase activity to a wide range of ambient O2. The mechanisms behind this adaptation were not elucidated, although Crisswell et al. (1976) did include a change in nodule cortical diffusion resistance among their speculations.

Carbon and Nitrogen Metabolism in Plant-Derived Ineffective Nodules of Pea (Pisum sativum L.)

Organogenesis of legume root nodules and the construction of the nitrogen fixation system require exchanges of molecular signals between rhizobia and the host plant to activate the expression of all the necessary genes. Plant mutants with ineffective nodules are potentially useful for studies of the host-plant control and regulation of symbiotic nitrogen fixation. In this paper the nodules of three different Nod+ Fix- pea mutants (Sym 13, Sym 31 and FN1) as well as their parent lines (Sparkle, Sprint 2 and Rondo) formed on plants by the same rhizobial strain and grown in the same controlled environment cabinet, were compared for selected physiological and biochemical parameters. All mutants are monogenic, recessive and non-allelic. The Sprint 2 Fix- (Sym 31) mutant is characterized by a block in bacteroid differentiation and abnormal symbiosome structure (Borisov et al., 1993). By contrast, mutants El 35 (Sym 13) and FN1 were characterized by early senescence of the symbiosomes and nodules as a whole (Kneen et al., 1990; Postma et al., 1990).

Dynamic Control of Oxygen Diffusion Resistance in Nodules

Excess inputs of O2 to infected cells of legume nodules will lead to loss of nitrogenase activity (Witty et al., 1986), whilst insufficient O2 initially curtails N2 fixation and eventually causes oxidant-induced nodule senescence (Escuredo et al., 1996). There is now a considerable weight of evidence showing that the O2 supply to N2-fixing bacteroids is controlled by a variable physical barrier to diffusion which balances O2 influx with its respiratory consumption. However, some workers have suggested biochemical/metabolic alternatives which might substitute for a physical barrier.