This week's paper was published in Science (Beatty and Good 2011). It discusses the prospects for developing cereals, like wheat or rice, that can use ("fix") atmospheric nitrogen. This paper was brought to my attention both by my wife, Cindy, by Andy McGuire. As my first graduate student, at UC Davis, Andy tested the use of legume "green manures", which form symbioses with nitrogen-fixing rhizobia bacteria, to supply nitrogen to a subsequent wheat crop. (McGuire et al. 1998)
This week's paper suggests three approaches. Maybe we could engineer cereals to host rhizobia in root nodules, similar to those found on legumes. Maybe we could encourage looser associations between cereals and bacteria to fix nitrogen in or on the plant, even without nodules. Or, maybe we could engineer the plants themselves to fix nitrogen.
The authors note that the proposed cereal-root nodules would need to provide a low-oxygen environment, to protect the key enzyme, nitrogenase. Just keeping oxygen low isn't that difficult. Legume nodules have a physical barrier to gas diffusion that limits oxygen influx to the nodule interior, and it might not be that hard to do something similar in cereals. But rhizobial nitrogen fixation is powered by respiration, which requires lots of oxygen. So rhizobia need a high oxygen flux, in addition to a low oxygen concentration. Diffusion is slow at the low concentrations beyond the diffusion barrier, so diffusion of free oxygen within rhizobia-infected cells can't meet demand. Much of the oxygen is carried by diffusion of a plant hemoglobin, whose high concentration makes the inside of legume root nodules red.
And that's not the end of the challenges we would need to overcome to put functional root nodules on cereals. Suppose the diffusion barrier were just thick enough that respiratory uptake drops oxygen concentration from atmospheric (21 kPa) to the targeted near-zero concentration (1 kPa, say) across the diffusion barrier. What if the respiration rate drops to 90% of it's current value? (A slight decrease in soil temperature could have this effect, as could a shortage of photosynthate.) The concentration drop across the barrier is proportional to the flux (Fick's Law of Diffusion, similar to Ohms law for electricity), so the concentration drop would now be 18 kPa instead of 20 kPa. So the nodule-interior oxygen would now be 3 kPa instead of 1 kPa. 3 kPa is probably high enough to destroy nitrogenase.
Evolution has solved this problem, too, at least in legumes. As conditions change, legumes adjust the gas permeability of their nodules to keep oxygen low enough to protect nitrogenase, but high enough for respiration to meet their nitrogen-fixation needs. When sheep graze clover plants, the resulting photosynthate shortage decreases nodule respiration, so you might expect nodule-interior oxygen levels to rise, perhaps endangering nitrogenase. But clover plants decrease their nodule gas permeability decreases so much that nodule-interior oxygen actually decreases, rather than increasing (Hartwig et al. 1987, Denison and Okano 2003). Cereal root nodules would need something similar.
If we could engineer cereal crops to allow infection by rhizobia and support their reproduction inside root nodules that actively regulate oxygen supply, maybe the rhizobia would fix nitrogen there... at first.
But, like all living things, rhizobia evolve. Any mutation that reduced rhizobial investment in nitrogen fixation would free resources for additional rhizobial reproduction. If there were only one rhizobial genotype per plant, this "cheating" would be self-defeating, because a nitrogen-starved plant would have less photosynthate to support rhizobia. But, with multiple genotypes per plant, we have a "tragedy of the commons", favoring cheaters.(Denison 2000, West et al. 2002)
Legume evolution has found at least a partial solution to this problem, too, although there's probably room for improvement. Although moderate levels of cheating may be tolerated (Kiers et al. 2006), major diversion of resources from nitrogen fixation to rhizobial reproduction triggers host "sanctions", which reduce the fitness of rhizobial cheaters (Kiers et al. 2003, Oono et al. 2011). If we don't want nitrogen-fixing cereals to waste photosynthate supporting nonfixing rhizobia, they would need to impose sanctions, too. Or maybe growing them in rotation with sanction-imposing legumes hosting the same rhizobia would be enough to keep rhizobial cheaters rare.
What about the second option, cereals without nodules, but associating with nitrogen fixers? To meet a significant fraction of a cereal's nitrogen needs, we would need to regulate oxygen supply, as in nodules. And we would soon face the same problem of cheaters (Kiers and Denison 2008), so we'd also need some form of sanctions. If we're have to duplicate the functions of nodules anyway, why not copy nodules, rather than starting from scratch?
Engineering the cereals themselves to fix nitrogen, without rhizobia, would solve the problem of cheaters. The article suggests that it might be possible to add nitrogen fixation to plant mitochondria, the current site of respiration, or to chloroplasts, responsible for photosynthesis. Photosynthesis generates oxygen, which would tend to destroy nitrogenase. But the authors say that "some cyanobacteria perform photosynthesis and nitrogen ﬁxation in the same space but at separate times" - maybe chloroplasts could somehow shield nitrogenase from oxygen during the day, when they're photosynthesizing, and activate nitrogenase only at night. This seems possible, at least in theory. But could it be done soon enough to help avert looming food shortages?
The article closes with the hope that "if nitrogen supply and carbon metabolism can be closely coupled, excess nitrogen would not be lost to the environment." That is already true of legumes, which shut down nodules when they have as much nitrogen as they need (Denison and Harter 1995). But this feedback control evolved - legumes that wasted photosynthate on "extra" nitrogen fixation were out-competed by more-frugal mutants - it would not be an "automatic" outcome of any of the approaches proposed.
So, again, any attempt to develop nitrogen-fixing cereals would benefit from copying what legume nodules already do. It's too bad that we don't yet know the mechanisms that regulate oxygen supply in legume nodules, impose sanctions on rhizobial cheaters, or adjust nitrogen-fixation rate to match nitrogen needs. Regulation of nodule gas permeability appears to be involved in all three, but we don't know how gas permeability is regulated. It's too bad that nobody wants to fund that kind of research any more.
Beatty P. H., A. G. Good. 2011. Future Prospects for Cereals That Fix Nitrogen. Science 333:416-417.
Denison R. F. 2000. Legume sanctions and the evolution of symbiotic cooperation by rhizobia. American Naturalist 156:567-576.
Denison R. F., Y. Okano. 2003. Leghaemoglobin oxygenation gradients in alfalfa and yellow sweetclover nodules. Journal of Experimental Botany 54:1085-1091.
Denison R. F., B. L. Harter. 1995. Nitrate effects on nodule oxygen permeability and leghemoglobin. Nodule oximetry and computer modeling. Plant Physiology 107:1355-1364.
Hartwig U., B. Boller, and J. Nösberger. 1987. Oxygen supply limits nitrogenase activity of clover nodules after defoliation. Annals of Botany 59:285-291.
Kiers E. T., R. F. Denison. 2008. Sanctions, cooperation, and the stability of plant-rhizosphere mutualisms. Annual Review of Ecology, Evolution, and Systematics 39:215-236.
Kiers E. T., R. A. Rousseau, and R. F. Denison. 2006. Measured sanctions: legume hosts detect quantitative variation in rhizobium cooperation and punish accordingly. Evolutionary Ecology Research 8:1077-1086.
Kiers E. T., R. A. Rousseau, S. A. West, and R. F. Denison. 2003. Host sanctions and the legume-rhizobium mutualism. Nature 425:78-81.
McGuire A. M., D. C. Bryant, and R. F. Denison. 1998. Wheat yields, nitrogen uptake, and soil water content following green manure vs. fallow. Agronomy Journal 90:404-410.
Oono R., C. G. Anderson, and R. F. Denison. 2011. Failure to fix nitrogen by non-reproductive symbiotic rhizobia triggers host sanctions that reduce fitness of their reproductive clonemates. Proceedings of the Royal Society B 278:2698-2703.
West S. A., E. T. Kiers, E. L. Simms, and R. F. Denison. 2002. Sanctions and mutualism stability: why do rhizobia fix nitrogen? Proceedings of the Royal Society B 269:685-694.