Biological nitrogen fixation by legumes

Nitrogen is the main limiting nutrient in both natural and agricultural systems. In much present day agriculture, it is supplied in a form available to plants as fertiliser, usually produced through a chemical process (e.g. the Haber-Bosch process that converts atmospheric nitrogen into ammonia using very high quantities of energy) or from mining mineral deposits. Regardless of whether the fertilisers are produced chemically or by mining, the energetic and environmental costs of their production and use are prodigious.

An alternative way to add nitrogen to the soil, used since the earliest days of agriculture and still the basis of some cropping systems, is biological nitrogen fixation (BNF).  In this, a plant, called a legume (e.g. beans, peas, the whin shown to the right) and bacteria form a symbiosis in which nitrogen from the air is fixed into plant tissue and later released to the soil. This article describes the mechanism of BNF and the symbiosis of legume and bacteria.

 

Advanced Higher practical and data handling activity: Nitrogen fixation in legumes (Word file 401 kb)

The photograph above shows legumes in a meadow - red clover Trifolium pratense and the yellow flowered Lotus pedunculatus (Living Field collection).

Biological nitrogen fixation

In most undisturbed non-agricultural ecosystems - as well as some agricultural ones - soluble nitrogen (ammonium) is made available to plants via a process known as biological nitrogen fixation (BNF), in which bacteria that contain the enzyme complex called nitrogenase (termed “diazotrophs”) can fix atmospheric N2 into ammonia (NH3) using energy from ATP and reductant (electrons) supplied by the metabolism of carbohydrates, such as sugars.

This ammonia, which is potentially toxic to the organism, is usually then immediately converted into amino acids or amides for use by the diazotrophic bacterium in the production of proteins and peptides to facilitate its growth. The fixed N incorporated into diazotrophic bacteria is then released into the environment when they die, usually in the form of amino acids that then become mineralized and available for uptake by other bacteria and by plants.

Symbiosis

Many plants, particularly legumes, but also some other higher plants (e.g. Gunnera, and “actinorhizal” plants, such as Alnus, Casuarina and Myrica), as well as some cycads and the fern Azolla, form mutualistic symbiotic relationships with nitrogen-fixing bacteria. In these systems, soil bacteria located in specialized organs take nitrogen from the atmosphere and convert it to soluble nitrogen which is then available for plant growth. Nitrogen-fixing legumes are one of the main sources of nitrogen in both natural environments and low-input agricultural systems.

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N-fixing legumes and their bacterial symbionts

The family Leguminosae (or Fabaceae) is one of the three largest families of flowering plants, and consists of more than 640 genera with 18,000 plus species. It has been divided taxonomically into three sub-families, the Papilionoideae (468 genera), Mimosoideae (78 genera) and Caesalpinioideae (157 genera), with only the Papilionoideae being represented in temperate regions. The vast majority of legume species of all three sub-families are confined to the tropics and sub-tropics, where they are particularly abundant and diverse in forests, wetlands and savannahs. Although it is often assumed that all legumes can nodulate and fix N, only 20% of legume genera have been tested for their ability to nodulate and fix. Of those tested, few genera (5%) in the Caesalpinioideae are known to nodulate, whereas most genera  nodulate in the Papilionoideae (93%) and Mimosoideae (88%). These proportions will inevitably change as more legumes species are examined.

Examples of nodules formed on members of all three sub-families are shown in Figures 1 - 4. Briefly, they consist of a central zone that contains the N-fixing bacteria, and this is surrounded by an uninfected “cortex”, which may be surmounted by an epidermis; this cortex/epidermis serves to keep out oxygen, to deter pathogens and to prevent desiccation (Figure 2). The bacterium’s nitrogenase enzyme is highly sensitive to oxygen (which can destroy the enzyme if it is exposed to it for anything but a very short time), but the N-fixing bacteria require it for aerobic respiration to supply the large amounts of energy necessary for high rates of BNF. To get round this, legume nodules contain a high concentration of an oxygen-carrying protein called leghaemoglobin, which surrounds the N-fixing bacteria in the infected cells. This leghaemoglobin transfers very rapidly a low concentration of oxygen to the bacteria, and its presence in nodules is essential for their functioning. There is a striking similarity between the oxygen-carrying function of leghaemoglobin in legume nodules and that of animal haemoglobin in blood cells, and it illustrates just what a unique organ the legume nodule is in its adaptation to house bacteria and to encourage them to fix nitrogen as efficiently as possible.

Figure 1. Internal structure of a pea nodule (Papilionoideae) infected by Rhizobium leguminosarum showing the apical meristem (m), blue-stained N-fixing cells within the infected zone (iz), and the peripheral uninfected cortex (c).

Figure 2. Internal structure of a nodule on the tree Erythrophleum ivorense (Caesalpinioideae). Labels are as in Figure 1.

Figure 3. Nodules (arrow) on Piptadenia viridiflora (Mimosoideae) inoculated with Burkholderia phymatum.

Figure 4. Nodules (arrow) on Acacia schaffneri (Mimosoideae).

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Infection and nodule development

In most legumes so far studied, the soil-based bacteria (“rhizobia”; Figures 5 - 10) are attracted to the roots of compatible plants via chemicals released from the roots called flavonoids. These flavonoids can “switch on” the nodulation genes of the rhizobia, which results in the release by the bacteria of chemicals called “Nod Factors” that stimulate the plant to allow the bacteria to enter root hairs and, simultaneously, stimulate the formation within the root cortex (or epidermis) of a nodule meristem.

The bacteria within the root hairs then divide repeatedly and progress into the root cortex via a tube made of by host plant cell wall material called an infection thread (which both directs the bacteria and prevents them from randomly entering the plant cells that the infection thread crosses) until the infection thread containing the bacteria encounters the nodule meristem that is growing outwards to meet it. The infection thread(s) then enters the newly-divided nodule cells and rhizobia are released from the tip of the thread into the cytoplasm. At this point the host cell encloses the newly-released bacteria within a cell membrane so that it is not actually in contact with the plant cytoplasm. These bacteria then differentiate into their symbiotic N-fixing state, and effectively become part of (and dependent upon) the host plant.

This process occurs repeatedly and in most legumes the nodule meristem continues to grow outwards from the root, and the resultant mature nodules (Figures 1 - 4) are usually elongated structures with a meristem at the tip, a region of cells immediately behind the meristem that are being invaded by infection threads, and behind this (and closest to the root) a large infected zone of N-fixing cells. In older nodules, the region immediately adjacent to the connection to the root contains cells that are senescing, and these will eventually become dominant as the nodule ages and finally degrades, thereby releasing rhizobia into the soil to start the nodulation cycle anew.

Figure 5. Bacterial colonies (*) growing on agar supplemented with a mixture of nutrients that favour the growth of symbiotic rhizobia from legume nodules. A nodule has been crushed and the contents streaked onto the agar.

Figure 6. Same as Figure 5, except that in this case the bacteria have been put through a purification process, so that all the colonies (*) have been formed by the same strain.

Figure 7. Mimosa debilis (Mimosoideae) plants after inoculation with different strains of rhizobia. Both plants are nodulated, but the green plant on the left is nodulated (arrow) by an effective N-fixing strain, whereas the yellow plant on the right is nodulated by an ineffective, non-symbiotic strain.

Figure 8. Comparison of the ability of 2 rhizobial strains to nodulate Mimosa setosissima. Strain JPY164 was isolated from nodules on this plant, and is therefore more compatible with it as a symbiont than strain JPY461 which was isolated from another Mimosa species. This is shown by the green and healthy plant on the left (JPY164) and the small yellow plant on the right (JPY461). Interestingly, when it is inoculated with both strains together, M. setosissima is not as healthy as when it is inoculated only with JPY164.

Figure 9. Nodules on the roots of Cyclopia genistoides (Papilionoideae) that have been cut open to reveal the pink-coloured infected tissue in the centre of the nodules. The pink colour (*) is due to the oxygen-carrying protein leghaemoglobin which is essential for the functioning of the N-fixing symbiosis.

Figure 10. The same nodules as in Figure 9 after they have been viewed under a fluorescent microscope. The green fluorescent protein (GFP) that has been “tagged” onto the N-fixing bacteria within the nodules has been excited by low wavelength light, and has consequently emitted light of a longer wavelength (ie. it has fluoresced green light). As the GFP was incorporated into the DNA of the bacteria before they were inoculated on to the roots, this technique shows how GFP can be used to prove that the inoculated strain really is a symbiont.

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Types of root nodule

The aforementioned process seems to be generally true for most legumes, and the elongated nodule shape with a persistent meristem is found in all three sub-families (Figure 1 – 4), and these nodules are termed “indeterminate”. It is not surprising, therefore, that all the perennial woody legumes that are encountered in tropical forests, have nodules of the persistent indeterminate type. There are a few Papilionoid legumes, however, that form nodules with a transient meristem, such as those on soybean (Glycine max) and common bean (Phaseolus vulgaris). These “determinate” nodules are usually spherical, as the centrally-located meristem ceases early in the developmental process, and the cells containing the newly-released rhizobia then divide to form the functional N-fixing nodule. As they don’t have a persistent meristem these nodules tend to have a shorter life than indeterminate ones, but this is sufficient to encompass the relatively short period over which an annual plant (such as soybean) requires symbiotic BNF to supply it with N for its vegetative growth before it flowers and sets seed.

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Stem nodules

Although most legumes that can nodulate usually do so only on their roots, there are a few genera (most in the Papilionoideae) that can also nodulate their stems. This nodulation can be either on the roots that arise from the flooded stems (ie. the adventitious roots, such as those arising from the floating stems on Neptunia; Figure 11), or directly on the stems themselves, and these “true” stem nodules are so far reported only on species of Aeschynomene, Discolobium and Sesbania (Figures 12 - 14). These all grow in tropical wetlands such as the Pantanal in Brazil, as well as in other seasonally-flooded regions of the tropics (eg. Amazon and Orinoco basins). Nodulation of their stems in a flooded environment allows these legumes to continue symbiotic N-fixation in wet conditions that would normally prevent it in non-hydrophytic legumes, as both nodulation and nodule functioning (ie. symbiotic BNF) are very much dependent on a continued supply of oxygen to support the aerobic respiration required by these highly energetically costly processes.

Interestingly, the nodules formed on the aerial stems of these plants are slightly unusual in developmental and structural terms and don’t fit easily into the determinate and indeterminate categories (see above). Another unusual feature is that they often contain fully functional photosynthetic chloroplasts, and hence are able to supply the N-fixing bacteroids with both oxygen and possibly also a more direct supply of carbohydrate. The symbiotic bacteria within stem nodules also tend to be different from the norm eg. in the case of Sesbania rostrata, the main symbiont is Azorhizobium, a bacterium that is not closely related to Rhizobium, and which has the unusual (for rhizobia) capability of fixing N in a free-living (ie. non-symbiotic) state.

Similarly, the symbionts of Aeschynomene spp, although they are usually Bradyrhizobium and thus related to soybean symbionts, the strains that form stem nodules contain bacteriochlorophyll and can photosynthesise, which is not a characteristic of bradyrhizobia from the non-flooding tolerant plant soybean. The photosynthetic ability of stem nodules by both the plant partner (via chloroplasts) and the microsymbiont (via bacteriochlorophyll) has been linked to the very high rates of BNF recorded by these legumes, and is the reason for their promotion as “green manures” in the cultivation of paddy rice in tropical countries (Figures 13 - 14). This use is likely to become more widespread as the costs of mineral N fertilizers rise owing to the inexorable increase in fossil fuel prices that are projected over the next 20 years or so.

Figure 11. Neptunia oleracea (Mimosoideae) plants floating on a pond in the Brazilian Pantanal wetlands. Note the thick, spongy stems that allow the plants to float (arrows); the adventitious roots that arise from these are nodulated.

Figure 12. Nodules (arrows) on the stem of the hydrophytic legume, Discolobium pulchellum (Papilionoideae), which is very abundant in South American wetlands, such as the Pantanal.

Figure 13. in A rice paddy field filled with the semi-aquatic legume Sesbania rostrata (Papilionoideae) as a “green manure”. This plant, which nodulates profusely both on its roots and stems (see Figure 14), fixes large quantities of N, and it can be ploughed into the paddy field prior to the planting of the rice crop. The fixed N which is released from the green manure can then be taken up by the growing rice and hence is a very good substitute for mineral fertilizer.

Figure 14. Photosynthetic stem nodules on S. rostrata (arrows). These unusual nodules form on the stem above the floodwater and allow this plant to fix N in paddy fields, and their photosynthetic ability greatly increases their N-fixing efficiency.

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Glossary

Symbiosis Narrowly defined as a mutually beneficial relationship between two different organisms.

Legume Organism in the higher plant dicotyledonous family Leguminosae (Fabaceae). This family is now divided into 3 sub-families, the Caesalpinioideae, Mimosoideae, Papilionoideae. One of the main distinguishing features of legumes is that many of them (but not all) can form nodules on their roots (and sometimes stems) that house nitrogen-fixing (N-fixing) symbioses with bacteria called rhizobia. They are very widely distributed and are present in all continents and terrestrial ecosystems, except for Antarctica. They range in growth habit from herbs through to large trees (mainly in the tropics and sub-tropics).

Nodule A root (and sometimes stem)-borne symbiotic organ on the roots of legumes and Actinorhizal plants that allow N-fixing bacteria to fix atmospheric dinitrogen (N2) into soluble, ammonium-based compounds that can be used by the host plant for all its nutritional N-requirements. The nodule acts as an interface between the 2 symbionts and allows for the supply of sugars from the plant to the bacteria in exchange for fixed N (ammonium). It also provides a low oxygen environment, which is essential for the expression and function of the bacterial enzyme nitrogenase.

Rhizobia tTrm which used to refer only to bacteria in the genus Rhizobium, but is now a generic term for all soil bacteria that form symbiotic N-fixing nodules on legumes (and the non-legume Parasponia).

ATP adenosine tri-phosphate.

Leghaemoglobin An oxygen-carrying haemo-protein found in legume nodules. It is essential for the functioning of the symbiosis as it rapidly conveys low concentrations of oxygen to support the high respiratory needs of the N-fixing bacteria, but at a concentration sufficiently low not to inactivate the oxygen-sensitive enzyme, nitrogenase.

Nitrogenase Enzyme complex responsible for “fixing” atmospheric nitrogen (dinitrogen) consisting of a molybdenum-iron (MoFe) protein and an iron (Fe) protein. It uses several molecules of ATP (typically 16) to reduce just one molecule of dinitrogen, so it is highly expensive energetically. It is also highly sensistive to oxygen and can be easily denatured unless the organism (free-living or symbiotic) takes steps to protect it (eg. by living inside a root nodule).

Actinorhizal plants Dicotyledonous plants that form a nodulating N-fixing symbiosis with a filamentous actinomycete called Frankia. They are in 8 families (Betulaceae, Casuarinaceae, Coriariaceae, Datiscaceae, Elaeagnaceae, Myricaceae, Rhamnaceae, Rosaceae), and most are woody temperate species.

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Contacts and further information

The main author and contact for this page is Euan James euan.james@scri.ac.uk

Pietro Iannetta, Janet Sprent and Geoff Squire join Euan James in researching the roles of legumes and biological nitrogen fixation in managed and semi-natural ecosystems.

For arguments on expanding the role of legumes in local cropping systems, see the Knowledgescotland policy briefing Scottish crop production should rely on natural and renewable sources of nitrogen by Pietro Iannetta, Geoff Squire and Janet Sprent. Published online 26 March 2010.

The page on Legumes in the 5000 years project describes wild and cultivated legumes in Scotland with emphasis on their ecological functions, uses since the stone age, present day crops and the reliance on imports.

 

[This page began 13 July 2010. Updated 23 September 2010 with Advanced higher activity and minor edits.]