This week I'll discuss a recent paper by Ryoko Oono, one of five from her PhD work. (If you want to hire her as a postdoc, better hurry!) Comparing Symbiotic Efficiency between Swollen versus Nonswollen Rhizobial Bacteroids, published in Plant Physiology, is available for anyone to read on-line.
Here's a little background. Rhizobia are soil bacteria, best known for infecting the roots of legume plants (clover, lupine, bean, pea, alfalfa, and many others) and multiplying inside root nodules, where they "fix" nitrogen gas from the atmosphere, converting it into forms their plant hosts can use, instead of relying on fertilizer.
Left: Alfalfa nodules; copyright Inga Spence, used by permission. Below left: nonswollen bacteroids. Below right: swollen bacteroids.
Apparently, rhizobia in the soil never fix nitrogen. Inside root nodules, however, some of them develop into bacteroids, which use some of the plant-supplied carbon they consume to power nitrogen fixation. They may also hoard some carbon for their own future survival and reproduction, a possible source of conflict with their host.
In nodules of some legume hosts, including pea and alfalfa, bacteroids are swollen (above right) and have lost the ability to reproduce. In other hosts, bacteroids don't look that different from the free-living rhizobia, and retain the ability to reproduce. Why this difference?
"Why" questions in biology can have two different kinds of answers. A "proximate" (as in "nearby") explanation for bacteroid swelling is that it is caused by certain peptides (like proteins, only smaller), which are produced by some legume species but not others, as shown by recent research by Van de Velde, Kondorosi, Mergaert, and collaborators.
"Ultimate" answers (aside from "42") are evolutionary explanations. Why did some legumes evolve the ability to make peptides that cause bacteroids to swell? Why have rhizobia not evolved resistance to this manipulation, particularly since swollen bacteroids have apparently lost the ability to reproduce?
Earlier, Dr. Oono showed that this plant trait (causing bacteroid swelling) has evolved at least five times. This repeated evolution made us suspect that swollen bacteroids might somehow be more beneficial to their plant host, relative to nonswollen ones.
What would "more beneficial" mean? Fixing twice as much nitrogen would be good, but not if that cost the plant three times as much carbon. In that case, it would have been cheaper for the plant to make twice as many nodules. So "more beneficial" has to mean "more efficient", that is, "fixing more nitrogen, relative to their carbon cost."
How could we test the hypothesis that, by making bacteroids swell up, plants make the bacteroids more efficient? Ideally, we wanted two things. First, some way of controlling bacteroid swelling, without changing anything else. Second, some method to measure the ratio of nitrogen fixation by bacteroids to their carbon consumption.
We didn't quite have either of those, but we came close. We could have measured the efficiency of swollen bacteroids of species X in nodules of host species A, and compared that with nonswollen bacteroids of species Y in host B. But how much of that difference would be due to bacteroid swelling, and how much due to other differences between species? Fortunately, there are a few rhizobia what make swollen bacteroids in one host and nonswollen bacteroids in another. We used two rhizobial strains with this useful property. One made swollen bacteroids in pea and nonswollen ones in bean. The other made swollen bacteroids in peanut and nonswollen ones in cowpea.
To estimate nitrogen fixation, we made use of the fact that, in the absence of nitrogen gas -- we used 20% oxygen and 80% argon -- the nitrogen-fixing enzyme produces hydrogen gas, at a rate proportional to what its nitrogen fixation would have been, if nitrogen were available. To measure total carbon consumption, we would have to measure both the carbon stored by bacteroids and the carbon they respired away as carbon dioxide. We assumed that, once a bacteroid has been around for a while, it's probably not accumulating too much carbon, at least relative to the amount it respires. So we measured nitrogenase activity (as hydrogen production) as a function of respiration rate (carbon dioxide production).
One problem is that not all of the carbon dioxide released by a nodule is directly linked to nitrogen fixation. One could argue that the ratio of total hydrogen production to total carbon dioxide production is an overall measure of efficiency at the nodule level, but differences between swollen bacteroids in one host and nonswollen bacteroids in another host could easily reflect differences in nodule structure between legume species. To tie efficiency differences more closely to bacteroid swelling, we measured the increase in respiration with an increase in hydrogen production by the nitrogen-fixing enzyme. That way, baseline differences in nodule respiration between species were left out of the ratio. To increase these rates, we simply increased oxygen concentration slightly, which had no effect on respiration in the oxygen-saturated parts of the roots and nodules, but increased activity by the oxygen-limited bacteroids. This approach was developed by the late John Witty, with whom I spent an enjoyable and productive month at the Welsh Plant Breeding Station, in 1989.
In both cases, swollen bacteroids had more nitrogen-fixing enzyme activity, relative to their respiration cost, when compared to the nonswollen bacteroids of the same rhizobial strain. Consistent with this apparent difference in intrinsic efficiency, each rhizobial strain gave more plant growth, per gram of root nodule, in the host where its bacteroids became swollen.
We think that's why legumes have repeatedly evolved the ability to make rhizobial bacteroids swell up inside their nodules: it costs them less carbon to get the nitrogen they need.
That's fine for the plants, but what about the rhizobia? I mentioned that swollen bacteroids have apparently lost the ability to reproduce. Failure to reproduce is unlikely to fare well under natural selection. So I wouldn't be surprised if further research uncovers some attempts by rhizobia to resist this manipulation by their legume-plant hosts.
But should these rhizobia give up on symbiosis altogether? After all, rhizobia can survive and reproduce in the soil, without nodulating plants. However, each nodule containing swollen, nonreproductive bacteroids also contains many (a million or so) genetically identical rhizobia that haven't yet turned into bacteroids. These nonswollen clonemates of the swollen nonreproductive bacteroids can still reproduce. Many of them are thought to escape alive back into the soil, when the nodule senesces. That's apparently enough of an inclusive-fitness benefit to keep rhizobia coming back to legume roots, through a process similar to kin selection.
I will be discussing some practical implications of our research on legume-rhizobium coevolution next month, at the Soybean Breeders' Workshop in Saint Louis. For example, would legume crops that don't make bacteroids swell up have higher yield if they did? Or does this benefit depend on the environment or on other aspects of legume physiology, so that it's already evolved in every case where it would be beneficial?
This material is based upon work supported by the National Science Foundation under Grant No. NSF/IOS-0918986.