Fixation of atmospheric nitrogen in mutualistic plants

The inability of most plants and animals to fix atmospheric nitrogen is one of the great puzzles in the process of evolution, since nitrogen is in limiting supply in many habitats. However, the ability to fix nitrogen is widely though irregularly distributed amongst both the eubacteria ('true' bacteria) and the archaea (archaebacteria), and many of these have been caught up in tight mutualisms with systematically quite different groups of eukaryotes. Presumably such symbioses have evolved a number of times independently. They are of enormous ecological importance because of nitrogen's frequent importance (Sprent & Sprent, 1990).

The nitrogen-fixing bacteria that have been found in symbioses (not necessarily mutualistic) are members of the following taxa.

parallels with higher plants remarkable morphological responses on the fungi the range of nitrogen-fixing bacteria

1 Rhizobia, which fix nitrogen in the root nodules of most leguminous plants and just one nonlegume, Parasponia (a member of the family Ulmaceae, the elms). At least three genera are recognized: Rhizobium, Bradyrhizobium and Azorhizobium, which are so distinct that they should perhaps be in different families (Sprent & Sprent, 1990), and between them they may comprise 104 or more species.

2 Actinomycetes of the genus Frankia, which fix nitrogen in the nodules (actinorhiza) of a number of nonleguminous and mainly woody plants, such as alder (Alnus) and sweet gale (Myrica).

3 Azotobacteriaceae, which can fix nitrogen aerobically and are commonly found on leaf and root surfaces.

4 Bacillaceae, such as Clostridium spp., which occur in ruminant feces, and Desulfotomaculum spp., which fix nitrogen in mammalian guts.

5 Enterobacteriaceae, such as Enterobacter and Citrobacter, which occur regularly in intestinal floras (e.g. of termites) and occasionally on leaf surfaces and on root nodules.

6 Spirillaceae, such as Spirillum lipiferum, which is an obligate aerobe found on grass roots.

7 Cyanobacteria of the family Nostocaceae, which are found in association with a remarkable range (though rather few species) of flowering and nonflowering plants (see Section 13.10.3), and which we recently met as photobionts in lichens.

Of these, the association of the rhizobia with legumes is the most thoroughly studied, because of the huge agricultural importance of legume crops.

13.10.1 Mutualisms of rhizobia and leguminous plants

The establishment of a liaison between rhizobia and legume plants proceeds by a series of reciprocating steps. The bacteria occur in a free-living state in the soil and are stimulated to multiply by root exudates and cells that have been sloughed from roots as they develop. These exudates are also responsible for switching on a complex set of genes in the rhizobia (nod genes) that control the process that induces nodulation in the roots of the host. In a typical case, a bacterial colony develops on the root hair, which then begins to curl and is penetrated by the bacteria. The host responds by laying down a wall that encloses the bacteria and forms an 'infection thread', within which the rhizobia proliferate extracellularly. This grows within the host root cortex, and the host cells divide in advance of it, beginning to form a nodule. Rhizobia in the infection thread cannot fix nitrogen, but some are released into the host meristem cells. There, surrounded by a host-derived peribacteroid membrane, they differentiate into 'bacteroids' that can fix nitrogen. In some species, those with 'indeterminate' growth like the rhizobia of the pea (Pisum sativum), the bacteroids themselves are unable to reproduce further. Only undifferentiated rhizobia are released back into the soil to associate with another root when the original root senesces. By contrast, in species with 'determinate' growth like those of the soybean (Glycine max), bacteroids survive root senescence and can then invade other roots (Kiers et al., 2003).

A special vascular system develops in the host, supplying the products of photosynthesis to the nodule tissue and carrying away fixed nitrogen compounds (very often the amino acid asparagine) to other parts of the plant (Figure 13.19). The nitrogen-fixing nitrogenase enzyme accounts for up to 40% of the protein in the nodules and depends for its activity on a very low oxygen tension. A boundary layer of tightly packed cells within the nodule serves as a barrier to oxygen diffusion. A hemoglobin (leghemoglobin) is formed within the nodules, giving the active nodules a pink color. It has a high affinity for oxygen and allows the symbiotic bacteria to respire aerobically in the virtually anaerobic environment of the nodule. Indeed, wherever nitrogen-fixing symbioses occur, at least one of the partners has special structural (and usually also biochemical) properties that protect the anaerobic nitrogenase enzyme from oxygen, yet allow normal aerobic respiration to occur around it.

13.10.2 Costs and benefits of rhizobial mutualisms

The costs and benefits of this mutualism need to be considered carefully. From the plant's point of view, we need to compare the energetic costs of alternative processes by which supplies of fixed nitrogen might be obtained. The route for most plants is direct from the soil as nitrate or ammonium ions. The metabol-ically cheapest route is the use of ammonium ions, but in most soils ammonium ions are rapidly converted to nitrates by micro-bial activity (nitrification). The energetic cost of reducing nitrate from the soil to ammonia is about 12 mol of adenosine triphosphate (ATP) per mol of ammonia formed. The mutualistic process (including the maintenance costs of the bacteroids) is energetically slightly more expensive to the plant: about 13.5 mol of ATP. However, to the costs of nitrogen fixation itself we must also add the costs of forming and maintaining the nodules, which may be about 12% of the plant's total photosynthetic output. It is this that makes nitrogen fixation energetically inefficient. Energy, though, may be much more readily available for green plants than nitrogen. A rare and valuable commodity (fixed nitrogen) bought with a cheap currency (energy) may be no bad bargain. On the other hand, when a nodulated legume is provided with nitrates (i.e. when nitrate is not a rare commodity) nitrogen fixation declines rapidly.

The benefits to the rhizobia are more problematic from an evolutionary point of view, especially for those with indeterminate growth, where the rhizobia that have become bacteroids can several steps to a liaison

Endodermis

Cortex

Vascular system

Point of emergence of nodule from root Nodule meristem

Developing bacteroid region Infection thread

Nodule meristem forming from cortical cells

Cells of nodule primordium, now infected and differentiating

Infection thread Inner cortical cells stimulated to divide

Newly infected region - Nodule meristem

Endodermis

Cortex

Vascular system

Point of emergence of nodule from root Nodule meristem

Developing bacteroid region Infection thread

Nodule meristem forming from cortical cells

Nodule Meristem Cells Plants

Nodule meristem

Newly infected region - Nodule meristem

Developing bacteroid region

Root hairs

Nodule meristem

Developing bacteroid region

Root hairs

Figure 13.19 The development of the root nodule structure during the course of development of infection of a legume root by Rhizobium. (After Sprent, 1979.)

fix nitrogen but cannot reproduce. Hence, they cannot themselves benefit from the symbiosis, since 'benefit' must express itself, ultimately, as an increased reproductive rate (fitness). The rhizobia in the infection thread are capable of reproduction (and are therefore able to benefit), but they cannot fix nitrogen and are therefore not themselves involved in a mutualistic interaction. However, since the rhizobia are clonal, the bacteroids and the cells in the infection thread are all part of the same, single genetic entity. The bacteroids, therefore, by supporting the plant and generating a flow of photosynthates, can benefit the cells of the infection thread, and hence benefit the clone as a whole, in much the same way as the cells in a bird's wing can bring benefit, ultimately, to the cells that produce its eggs - and hence to the bird as a whole.

One puzzle, though, since the rhi-why no cheating? zobia associated with a particular plant are typically a mixture of clones, is why individual clones do not 'cheat': that is, derive benefits from the plant, which itself derives benefit from the rhizobia in general, without themselves entering fully into the costly enterprise of fixing nitrogen. Indeed, we can see that this question of cheating applies to many mutualisms, once we recognize that they are, in essence, cases of mutual exploitation. There would be evolutionary advantage in exploiting without being exploited. Perhaps the most obvious answer is for the plant (in this case) to monitor the performance of the rhizobia and apply 'sanctions' if they cheat. This, clearly, will provide evolutionary stability to the mutualism by preventing cheats from escaping the interaction, and evidence for such sanctioning has indeed been found for a legume-rhizobium mutualism (Kiers et al., 2003). A normally mutualistic rhizobium strain was prevented from cooperating (fixing nitrogen) by growing its soybean host in an atmosphere in which air (80% nitrogen, 20% oxygen) was replaced with approximately 80% argon, 20% oxygen and only around 0.03% nitrogen, reducing the rate of nitrogen fixation to around 1% of normal levels. Thus, the rhi-zobium strain was forced to cheat. In experiments at the whole plant, the part-root and the individual nodule level, the reproductive success of the noncooperating rhizobia was decreased by around 50% (Figure 13.20). Noninvasive monitoring of the plants indicated that they were applying sanctions by withholding oxygen from the rhizobia. Cheating did not pay.

13.10.3 Nitrogen-fixing mutualisms in nonleguminous plants

The distribution of nitrogen-fixing symbionts in nonleguminous higher plants is patchy. A genus of actinomycete, Frankia, forms symbioses (actinorhiza) with members of at least eight families of flowering plants, almost all of which are shrubs or trees. The nodules are usually hard and woody. The best known hosts are the alder (Alnus), sea buckthorn (Hippophae), sweet gale (Myrica), she-oak (Casuarina) and the arctic/alpine shrubs Arctostaphylos and Dryas. Ceonothus, which forms extensive stands in Californian chaparral, also develops Frankia nodules. Unlike rhizobia, the species of Frankia are filamentous and produce specialized vesicles and sporangia that release spores. Whilst the rhizobia rely on their host plant to protect their nitrogenase from oxygen, Frankia provides its own protection in the walls of the vesicles, which are massively thickened with as many as 50 monolayers of lipids.

Figure 13.20 The number of rhizobia grew to much larger numbers when allowed to fix nitrogen in normal air (N2 : O2) than when prevented from doing so by manipulation of the atmosphere (Ar : O2). (a) When the different treatments were applied at the whole plant level, there were greater numbers within the nodules (left; P < 0.005) and on the root surface (right; both P < 0.01) and in the surrounding sand (P < 0.01). n = 11 pairs; bars are standard errors. (b) When the different treatments were applied to different parts of the same root system, there were greater numbers within the nodules (left; P < 0.001) and for those in the surrounding water (right; P < 0.01), but not significantly so for those on the root surface. n = 12 plants; bars are standard errors. (c) When the different treatments were applied to individual nodules from the same root system, there were greater numbers on a per nodule basis (P < 0.05) and a per nodule mass basis (P < 0.01). n = 6 experiments; bars are standard errors. (After Keirs et al., 2003.)

(a) Whole-plant experiment 0.6

Nodules

(b) Split-root experiment 8000 r

o 2000

Roots Sand

Roots Sand

Roots Water x 10

N2:O2

AR:O2

(c) Single-nodule experiment

Rh 4

Per nodule

Nodules

Roots Water x 10

Per nodule

Per nodule mass

ha 4000

Cyanobacteria form symbioses with three genera of liverwort (Anthoceros, Blasia and Clavicularía), with one fern (the freefloating aquatic Azolla), with many cycads (e.g. Encephalartos) and with all 40 species of the flowering plant genus Gunnera, but with no other flowering plants. In the liverworts, the cyanobacteria Nostoc live in mucilaginous cavities and the plant reacts to their presence by developing fine filaments that maximize contact with it. Nostoc is found at the base of the leaves of Gunnera, in the lateral roots of many cycads, and in pouches in the leaves of Azolla.

13.10.4 Interspecific competition

The mutualisms of rhizobia and legumes (and other nitrogen-fixing mutualisms) must not be seen as isolated interactions between bacteria and their own host plants. In nature, legumes normally form mixed stands in association with nonlegumes. These are potential competitors with the legumes for fixed nitrogen (nitrates or ammonium ions in the soil). The nodulated legume sidesteps this competition by its access to a unique source of nitrogen. It is in this ecological context that nitrogen-fixing mutualisms gain their main advantage. Where nitrogen is plentiful, however, the energetic costs of nitrogen fixation often put the plants at a competitive disadvantage.

Figure 13.21, for example, shows the results of a classic experiment in which soybeans (Glycine soja, a legume) were grown in mixtures with Paspalum, a grass. The mixtures either received mineral nitrogen, or were inoculated with Rhizobium, or received both. The experiment a classic 'replacement series'

Figure 13.21 The growth of soybeans (Glycine soja, G, o) and a grass (Paspalum, P, •) grown alone and in mixtures with and without nitrogen fertilizer and with and without inoculation with nitrogen-fixing Rhizobium. The plants were grown in pots containing 0-4 plants of the grass and 0-8 plants of Glycine. The horizontal scale on each figure shows the mass of plants of the two species in each container. —R —N, no Rhizobium, no fertilizer; +R —N, inoculated with Rhizobium but no fertilizer; —R +N, no Rhizobium but nitrate fertilizer was applied; +R +N, inoculated with Rhizobium and nitrate fertilizer was supplied. (After de Wit et al., 1966.)

Species Richness Soil Biont

was designed as a 'replacement series' (see Section 8.7.2), which allows us to compare the growth of pure populations of the grass and legume with their performances in the presence of each other. In the pure stands of soybean, yield was increased very substantially either by inoculation with Rhizobium or by application of fertilizer nitrogen, or by receiving both. The legumes can use either source of nitrogen as a substitute for the other. The grass, however, responded only to the fertilizer. Hence, when the species competed in the presence of Rhizobium alone, the legume contributed far more to the overall yield than did the grass: over a succession of generations, the legume would have outcompeted the grass. When they competed in soils supplemented with fertilizer nitrogen, however, whether or not Rhizobium was also present, it was the grass that made the major contribution: long term, it would have outcompeted the legume.

Quite clearly, then, it is in environments deficient in nitrogen that nodulated legumes have a great advantage over other species. But their activity raises the level of fixed nitrogen in the environment. After death, legumes augment the level of soil nitrogen on a very local scale with a 6-12-month delay as they decompose. Thus, their advantage is lost - they have improved the environment of their competitors, and the growth of associated grasses will be favored in these local patches. Hence, organisms that can fix atmospheric nitrogen can be thought of as locally suicidal. This is one reason why it is very difficult to grow repeated crops of pure legumes in agricultural practice without aggressive grass weeds invading the nitrogen-enriched environment. It may also explain why leguminous herbs or trees usually fail to form dominant stands in nature.

Grazing animals, on the other hand, continually remove grass foliage, and the nitrogen status of a grass patch may again decline to a level at which the legume may once more be at a competitive advantage. In a stoloniferous legume, such as white clover, the plant is continually 'wandering' through the sward, leaving behind it local grass-dominated patches, whilst invading and enriching with nitrogen new patches where the nitrogen status has become low. The symbiotic legume in such a community not only drives its nitrogen economy but also some of the cycles that occur within its patchwork (Cain et al., 1995).

13.10.5 Nitrogen-fixing plants and succession

An ecological succession (treated in much more detail in Chapter 17) is the directional replacement of species by other species at a site. A shortage of fixed nitrogen commonly hinders the earliest stages of the colonization of land by vegetation: the initial stages of a succession on open land. Some fixed nitrogen will be contributed in rain after thunderstorms, and some may be blown in from other more established areas, but nitrogen-fixing organisms such as bacteria, cyanobacteria and lichens are important pioneer colonizers. Higher plants with nitrogen-fixing sym-bionts, however, are rarely pioneers. The reason appears to be that open land is usually colonized first by plants with light, dispersible seeds. A legume seedling, however, depends on fixed nitrogen in its seed reserves and the soil before it can grow to a stage where it can nodulate and fix nitrogen for itself. It is likely, therefore, that only large-seeded legumes carry enough fixed nitrogen to carry them through the establishment phase, and species with such large seeds will not have the dispersibility needed to be pioneers (Grubb, 1986; see also Sprent & Sprent, 1990).

Finally, note that since symbiotic nitrogen fixation is energetically demanding, it is not surprising that most of the higher plant species that support nitrogen-fixing mutualists are intolerant of the shade that is characteristic of the late stages of successions. Higher plants with nitrogen-fixing mutualists are seldom in at the beginning of a succession and they seldom persist to the end.

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  • renato angelo
    How many species of flowering plants fix nitrogen?
    7 years ago

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