Plant & Nitrogen fixation

Rizkita R E Plant Microbe Interaction BI - 5239

Plants must secure their nitrogen in "fixed" form i form, i.e., e incorporated in compounds such as: nitrate ions (NO3) ammonia (NH3) urea (NH2)2CO

NITROGEN FIXATION

Three processes are responsible for most of the nitrogen fixation in the biosphere: 1. atmospheric fixation by lightning 2. biological fixation by certain microbes alone or in a symbiotic relationship with plants p 3. industrial fixation

Biological g Fixation The ability to fix nitrogen is found only in certain bacteria. Some live in a symbiotic relationship with plants of the legume family (e.g., soybeans, alfalfa). Some establish symbiotic relationships with plants other than legumes (e.g., alders). Some nitrogen-fixing bacteria live free in the soil.

Nitrogen-fixing cyanobacteria are essential to maintaining the fertility of semi-aquatic environments i t lik like rice i paddies. ddi

 Biological g nitrogen g fixation is the reduction of atmospheric nitrogen gas (N 2 ) to ammonium ions (NH4+ ) by the oxygen-sensitive enzyme, nitrogenase. Reducing R d i power i is provided id d b by NADPH/f NADPH/ferredoxin, d i via i an Fe/Mo centre. Plant genomes lack any genes encoding this enzyme, y in prokaryotes p y (bacteria). which occurs only Even within the bacteria, only certain free-living bacteria ( Klebsiella, Azospirillum, Azotobacter), blue-green bacteria ( Anabaena) and a few symbiotic Rhizobial species are known nitrogen-fixers. Another nitrogen-fixing nitrogen fixing association exists between an Actinomycete ( Frankia spp.) and alder ( Alnus spp.)

Biological nitrogen fixation requires a complex set of enzymes and a huge expenditure of ATP. ATP Although Al h h the h first fi stable bl product d of f the h process is i ammonia, i this hi is i quickly incorporated into protein and other organic nitrogen compounds.

 The enzyme nitrogenase catalyses the conversion of atmospheric, p , gaseous g dinitrogen (N 2 ) and dihydrogen (H 2 ) to ammonia ( (NH 3 ) ), as shown in the chemical equation The above reaction seems simple enough and the atmosphere is 78% N 2 , so why is this enzyme so important?

Fact :

Nitrogen Fixers (see Tables) Despite p the extreme oxygen-sensitivity yg y of nitrogenase, g some microbes that live in aerobic environments can fix nitrogen. Organisms that fix nitrogen include:
(i) Azotobacter spp. - soil bacteria; occur in oxic environments (fix ~ 0.26 lbs N2 per agricultural acre per year); (ii) Klebsiella spp. pp - soil bacteria, can live in the p presence or absence of oxygen; (iii) Cyanobacteria (e.g. Anabeana spp., Nostoc) - water bacteria; occur in oxic environments (fix ~ 22 lbs N2 per agricultural acre per year); iv) Rhizobium spp. - plant symbionts; responsible for significant fixation (fix approx. 220 lbs N2 per agricultural acre per year).
Nitrogen g Fixing Bacteria Azotobacter vinelandii Clostridium pasteurianum None Klebsiella pneumoniae Various Aerobic Soils and Anaerobic Soils; ; Water;Also in association with plants Rhodospirillum rubrum None Surface of polluted ponds p p (a photosynthetic bacterium)

Associated None Organism Natural N t l Habitat

Aerobic Soils

Anaerobic A bi Soils

Table N9. Nitrogen fixing organisms: representative symbiotic bacteria.

Nonlegume Nonlegume Nonlegume Legume Nitrogen Fixing Organism g Associated Organism Frankia alni Nostoc muscorum Gunnera macrophyll a (tropical herb) In stems; a cyanobact erium Anabaena azollae Azolla (aquatic ( q fern) In leaf pores; a cyanobact erium

Legume

Legume

Rhizobiu Bradyrhiz Rhizobium obium m trifolii japonicum j p meliloti

Alder

Soybean y

Clover

Alfalfa

Natural Habitat

Root nodules in the Alder tree

Root nodules of the soybean

Root R t nodules of clover

Root R t nodules of alfafa

Figure 1. A microbial mat at a hot spring in eastern Oregon. Some of the organisms in the mat are cyanobacteria that can fix nitrogen. Cyanobacteria are found in a variety of terrestrial and aquatic habitats. Many, but not all, are capable of nitrogen fixation. Figure 2. The aquatic fern Azolla is the only fern that can fix nitrogen. It does so by virtue of a symbiotic association with a cyanobacterium y (Anabaena azollae) ). Azolla is found worldwide and is sometimes used as a valuable source of nitrogen for agriculture. The plants shown here are each about 2 cm across. The pale yellow plant has been deprived of cobalt (essential for the cyanobacterial symbiont) and thus is showing typical signs of N deficiency. deficiency Figure 3. This is another example of a cyanobacterial nitrogen-fixing g f g association. . In this case the cyanobacteria y Nostoc (visible as small dark colonies in this photomicrograph) has taken up residence on leaves of a common leafy liverwort (Porella navicularis). The scale bar is 1 mm. mm

Nit Nitrogen Fi Fixation ti

Figures 4. 4 Gunnera sp., sp an unusual angiosperm that contains nitrogennitrogen fixing cyanobacteria in pockets at the base of petioles. This is often referred to as the only angiosperm that forms a nitrogen-fixing symbiosis with cyanobacteria cyanobacteria, however however, this is not strictly true since some tropical angiosperms have cyanobacterial films on their leaf surfaces. Gunnera has some of the largest leaves of any plant and is very common in parts of South and Central America where it is called "nalca nalca."

Figure5. Young plants of red alder (Alnus rubra). Alder is a N-fixing plant that forms a symbiotic association with bacteria (more specifically an actinomycete) ) of f the h genus Frankia F k . There h are about b 21 genera of f nonlegumes that fix N. These plants are collectively called actinorhizal plants and are important contributors of N in ecosystems where fixed N is scarce. In thi figure, this fi all ll plants l t are the th same age and d are growing i in i pure sand. d The Th plants on the left were inoculated with Frankia. The plants on the right were not inoculated and are displaying signs of extreme N deficiency.

Fig.6 Snowbrush (Ceanothus velutinus), a common nitrogen-fixing actinorhizal shrub h b throughout th h t the th western t U.S.
Fig.7. Fi 7 R Root t system t m of f soybean b n (Glycine Gl in m max x), ) a typical t pi l pl plant nt in the legume family. Nitrogen fixation occurs in the root nodules that contain bacteria (Bradyrhizobium for soybean, Rhizobium for most other legumes). g ) Almost all legumes g can fix nitrogen. The legume family (Leguminosae or Fabaceae) includes many important crop species such as pea, alfalfa, clover, common bean, peanut, and lentil.

Fig.8. Roots of pea showing numerous N-fixing nodules.

Mimosa

Symbiotic Nitrogen F xat on Fixation

The Rhizobium-legume association Bacterial associations with certain plant families, p , primarily p y legume g species, make the largest single contribution contr but on to biological b olog cal n nitrogen trogen fixation in the biosphere

It is estimated that more than 300 lbs of nitrogen per acre (340 kg per hectare) can be fixed by fields of alfalfa and other legumes Bacteria that live in the roots of legumes are of the genus Rhizobium, first isolated in.1888 by Beijerinck

Symbiotic Rhizobia are classified in 2 groups:

1. 2. Fast-growing Rhizobium spp. whose nodulation functions (nif, fix) are encoded on their symbiotic megaplasmids (pSym) Slow-growing Bradyrhizobium spp. whose Nfi ti and fixation d nodulation d l ti f functions ti are encoded d d on their chromosome.

Determinate Determ nate nodules

Formed on tropical legumes by y Rhizobium and Bradyrhizobium Meristematic activity not persistent - present only during early stage of nodule formation; after that, cells simply expand rather than divide, to form globose nodules. Nodules N d l arise i just j below b l epidermis; id i l largely l not on internal vascular system

 Uninfected cells dispersed throughout nodule; no u ; equipped qu pp to assimilate ass m at NH 4+ as ureides (allantoin and allantoic acid)

Indeterminate ndeterm nate nodules

Formed on temperate legumes (pea, clover, alfalfa); typically by Rhizobium spp. Cylindrical C li d i l nodules d l with i h a persistent i meristem i ; nodule growth creates zones of different developmental stages Nodule arises near endodermis, and nodule vasculature clearly connected with root vascular system

 Uninfected cells of indeterminate nodules (asparagine, p g , glutamine g ) assimilate NH 4+ as amides (

Typical Associations (cross-inoculation (cross inoculation groups)

R.leguminosarum biovar viciae colonizes pea ( Pisum spp.) pp ) (temperate; ( p indeterminate nodules) ) R.l. biovar trifolii colonizes clover ( Trifolium spp.)(temperate; indeterminate nodules) R l biovar phaseoli colonizes bean ( Phaseolus spp.) (tropical; determinate nodules) Bradyrhizobium japonicum colonizes soybean (tropical; determinate nodules) Rhizobium h NGR 234 colonizes l Parasponia and d tropical plants; very broad host range

Infection and Nodulation

The bacteria find clover roots by chemotaxis-'recognizing' their presence by the phenolic compounds p (aromatic ( 6-carbon ring g: eg.flavonoids) secreted from the roots. The bacteria move toward the seedling clover roots.
Lectins, e.g. trifoliin, f l which h h are sugar binding d proteins produced by plant, are involved in the host specific recognition process between bacteria and legume plant

in the soil.

Rhizobium bacteria live

Legume roots secrete growth promoting g p g substances and organic compounds (proteins, amino acids, organic acids vitamins) which stimulates the growth of the nodule-forming b t i bacteria,

lectins on plant roots interact with specific sites on bacteria, these sites contain specific oligosaccharides (sugars) which are unique for each species of nodule-forming bacteria, and allows plant to recognize and permit entrance of correct type of nodule-forming bacteria (analogous to a lock and key mechanism).

Initial Initi l inf infection cti n occurs ccu s on n root th hairs i s -- curling cu lin of f root hairs often occurs prior to bacterial penetration Nitrogen fixation only occurs in nodules containing viable bacteria

Nodule development process

1. Bacteria encounter root; y are chemotactically y attracted toward specific p they plant chemicals (flavonoids) exuding from root tissue, especially in response to nitrogen limitation

2. Bacteria attracted to the root, attach themselves to the root hair surface producing lectins and secrete specific oligosaccharide signal molecules (nod factors). )

3 In response to oligosaccharide signals, 3. signals the root hair becomes deformed and curls at the tip; bacteria become enclosed in small pocket. Cortical l cell ll division d is induced d d within h the h root.

4. Bacteria then invade the root hair cell and move along l an internal, i l plant-derived l d i d infection thread, multiplying, and secreting polysaccharides that fill the channel.

infection thread

Rhizobium cells expressing GFP (green fluorescent protein) invade a host root hair

 5. Infection thread penetrates through several layers of cort cortical cal cells and then ram ramifies f es within w th n the cortex. Cells in advance of the thread divide and organize themselves into a nodule primordium. 6. The branched infection thread enters the nodule primordium zone and penetrates individual primordium cells. 7. 7 Bacteria are released from the infection thread into the cytoplasm of the host cells, but remain surrounded by the peribacteroid membrane. Failure t form to f rm the PBM results in the activati activation n of fh host st defenses and/or the formation of ineffective nodules. 8. Infected root cells swell and cease dividing. Bacteria within the swollen cells change form to b become m endosymbiotic nd s mbi ti bacteroids, b t id which hi h begin b in to t fix nitrogen.

 The nodule provides an oxygen-controlled environment (leghemoglobin = pink nodule interior) structured to facilitate transport of reduced nitrogen it metabolites t b lit f from th the b bacteroids t id t to th the plant vascular system, and of photosynthate from th h the host t plant l t to t the th bacteroids b t id .

Patriarca et al. 2002. Microbiology and Mol. Biol. Rev. 66:202-223

 Types of bacterial gene functions involved in nodulation d l ti and d nitrogen it fi fixation ti : nod (nodulation) and nol (nod locus) genes mutations in these genes block nodule formation or alter host range most have been identified by y transposon p mutagenesis,DNA DN sequencing and d protein analysis, l in R. meliloti,R. leguminosarum bv viciae and trifolii fall into four classes: 1. nodD 2. nodA, , B and C ( common nod g genes) ) 3. hsn ( host-specific nod genes) 4. other nod genes

Nod D (the sensor) the nod D gene product recognizes molecules (phenylpropanoid-derived (phenylpropanoid derived flavonoids) produced by plant roots and becomes activated as a result of that binding activated nodD protein positively controls the expression of the other genes in the nod gene regulon (signal transduction) different diff t nodD dD alleles ll l recognize i various i fl flavonoid id structures with different affinities, and respond with differential patterns of nod gene activation

Common nod genes - nod ABC

nod factors are active on host plants at very low concentrations (10 -8 to 10 -11 M) but have no effect on non-host species mutations in nodA,B or C completely abolish the ability of the bacteria to nodulate the host plant; they are found as part of gene regulon g in all Rhizobia the nod g products of these genes are required for bacterial induction of root cell hair deformation and root cortical cell division The nod A,B,C gene products are enzymes responsible for synthesis of diffusible nod factors, which are sulfated and acylated beta-1,4-oligosaccharides of glucosamine (other gene products, e.g. NodH, may also be needed for special modifications) The role of the nodH gene product is therefore to add a s specific ifi s sulfate lf t group, and d thereby th b change h h host st s specificity ifi it

 nodC gene encodes NodC protein - an enzyme with N-acetylglucosaminyltransferase activity. Synthesis of the N-acetylglucosamine backbone. backbone nodB gene encodes the NodB protein - a deacetylase enzyme that removes an acetyl group from the Nod factor. nodAgene encodes the NodA protein - an acyltransferase that adds a fatty acid chain to the site deacetylated by NodB Sulfation ( nodH, noeE). Sulfotransferases that modify Nod factors (e.g., (e.g. in R. meliloti). Fatty acid addition ( nodEF). Different NodEF proteins from different Rhizobium species can put different fatty acids g specificity. p y onto Nod factors and change Arabinosylation ( noeC). The NoeC protein adds arabinose to Nod factors - can influence number of nodules that are formed on host plants. Fucosylation F l (e.g., ( nodZ). d ) F Fucose addition dd changes h specificity f of nodulation for some symbionts such as Bradyrhizobium japonicum. Acetylation Ac t l ti n ( (e.g., n nodL). dL) Ac Acetlyation tl ti n c can n influ influence nc N Nod d factor f ct r activity for R. leguminosarumand B. japonicumNGR234 Methylation and Carbamoylation (e.g., nodS). Mutation changes host range for R. R tropici tropici, R R. loti loti, R R. frediiand B B. japonicum japonicum.

Nodulation gene : y produce p low molecular weight g plant p growth g NodA and nodB may promoting compounds that stimulate cell division; nodC may help in
moving these bacteria to the plant.

There are many other factors, most poorly understood, that are part of the attachment p process - fimbriae, , cellulose fibrils and Ca2+-binding g proteins.
Infection may be through the root hair or through wounds or cracks between cells. Hair infection is the most common in crop plants. There is digestion of the cell walls of the root hair caused by some interaction between the plant and the rhizobia. It was thought th t this that thi involved i l d pectic ti enzymes and d other th plant produced digestion enzymes, but the real process is much more complex. It is spatially confined within the root hair curled region and closely associated with the hair metabolism as is indicated by the movement of an enlarged nucleus close to the point of rhizobial hi bi l entry. t There Th are strong t protoplasmic streaming movements in the root hair cells.

Other nod genes : May be involved in the attachment of the bacteria to the plant surface or in export of signal molecules surface, molecules, or proteins needed for a successful symbiotic relationship

exo (exopolysaccharide) genes Encode proteins needed for exopolysaccharide synthesis and secret secretion on In Rhizobium-legume interactions that lead to indeterminate nodules, exo mutants cannot invade the plant properly. p p p y However, , they y do provoke p the typical yp plant p cell division pattern and root deformation, and can even lead to nodule formation, although these are often empty (no bacteroids). In interactions that h usually ll produce d determinate d nodules, d l exo mutations tend to have no effect on the process. Exopolysaccharides may provide substrate for signal production, osmotic matrix needed during invasion, and/or a recognition or masking function during invasion

SUMMARY : 1. NodD p protein recognition g of f different ff compounds p in root exudates (flavonoids) contributes to host specificity. 2. The nod genes encode functions that are involved in the regulation of gene expression (e.g., NodD), producing the core Nod N d factor f t (NodABC) (N dABC) and d d decorating ti nod d f factors t (hsn,e.g., NodH, NoDE). 3. Differences in Nod factor decoration (side chain modifications) contribute to specificity. Specificity is a due to a combination of factors: 1 Types 1. T of f flavonoids fl id made d b by h host t plants l t 2. Nod D sensor proteins in the bacterial symbionts 3. Types of Nod factors produced by the bacteria 4. Amounts of Nod factors produced by the bacteria 5. Exopolysaccharide - for indeterminant nodules (role unclear) 6. Plant hydrolases y (e.g. ( g chitinases that may y degrade g Nod factors) ) 7. Plant lectins - carbohydrate binding proteins (possible role in attachment of bacteria to plant cells) p for Nod factors 8. Plant receptors

nodH encodes an enzyme y involved in modification of nodRm-2 to g give nodRm-1. The enzyme transfers activated sulfate to the nod factor. NodH- mutants lose the ability to produce nodRM-1, instead the mutants make nodRm-2 which is a more hydrophobic compound that is active on Vetch(vicia), not on alfalfa. The NodP and NodQ enzymes are involving in activating sulfur for transfer to the nod factor.

nif (nitrogen fixation) genes Gene p products are required q for symbiotic y nitrogen g fixation, and for nitrogen fixation in free-living N-fixing species

nif

Chemistry of Nitrogen Fixation G Gaseous N2 from f the th atmosphere t h i is reduced d d to t ammonia i This reaction is catalyzed by the enzyme Nitrogenase The chemical reaction for nitrogen fixation: N2 + 10 H+ + 8 e- + 16 ATP ====> 2 NH4 + H2 + 16 ADP + 16 Pi ATP and H ions are supplied by the plant from photosynthesis -- this constitutes a considerable expenditure of energy by the plant Leghemoglobin is a compound found in effective nodules, it is thought to protect the nitrogenase enzyme from oxygen Ammonia is excreted by the bacteriods and incorporated into amino acids and amides, and eventually proteins, by the plant

fix (fixation) genes Gene products required to successfully establish a functional N-fixing g nodule. No fix homologues have been identified in free-living N-fixing bacteria. Example: regulatory proteins that monitor and control oxygen levels within the bacteroids 1. 1 2. FixL Fi L senses the th oxygen level; l l at t low l oxygen tensions, t i it acts t as a kinase, which regulates expression of two more transcriptional regulators: NifA, the upstream activator of nif and some fix genes; FixK, the regulator of fixN (another oxgen sensor?) This k Thi key t transducing d i protein, t i Fi FixL, L i is a novel l hemoprotein h t i kinase with a complex structure. It has an N-terminal membrane -anchoring domain, followed by the heme binding section and a C section, C-terminal terminal kinase catalytic domain. domain Result? Low oxygen tension activates nif gene transcription and permits the oxygen oxygen-sensitive sensitive nitrogenase to function. function

Metabolic genes and transporters Dicarboxylic acid (malate) transport and metabolism Genes for other functions yet to be identified. Host plant role in nodulation 1. Production and release of nod g gene inducers flavonoids 2 2. Activation of plant genes specifically required for successful nodule formation nodulins Suppression of genes normally involved in repelling microbial invaders h st defense host d f s genes s

3.

Early nodulins :
At least 20 nodule-specific or nodule-enhanced genes are expressed in plant roots during nodule formation; most of these appear after the initiation of the visible nodule. Five different nodulins are expressed only in cells containing g growing g g infection f threads. These may m y encode proteins that are part of the plasmalemma surrounding the infection thread, or enzymes needed to make or modify other molecules Twelve nodulins are expressed in root hairs and in cortical ti l cells lls th that t contain t i growing i i infection f ti th threads. ds They are also expressed in host cells a few layers ahead of the growing infection thread.

Late nodulins :
Th The best b t studied t di d and d most t abundant b d t late l t nodulin d li i is th the protein t i component of leghemoglobin. The heme m component mp of f leghemoglobin g m g appears pp to be synthesized by the bacteroids. Oth Other late l t nodulins d li are enzymes or subunits b it of f enzyme that th t function in nitrogen metabolism (glutamine synthetase; uricase) or carbon metabolism (sucrose synthase). Others are associated with the peribacteroid membrane, and probably are involved in transport functions. These late nodulin gene products are usually not unique to nodule function, but are found in other parts of the plant as well. This is consistent with the hypothesis that nodule formation evolved as a specialized form of root differentiation. differentiation

There must be many y other host gene g functions that are needed for successful nodule formation. Example: what is the receptor for the nod f factor? These are being sought through genomic and proteomic analyses, and through generation of plant mutants that fail to p p y nodulate properly The full genome sequencing of Medicago truncatula and Lotus japonicus , both currently underway, will greatly speed up this y process. p discovery

roc roa hab and hac inf noi bar bad nif cof nop

multiplication of rhizobia at or near the root surface adhesion of rhizobia to root hair surface root hair branching and root curling formation of an infection thread nodule initiation: formation of nodule m i t m nodule meristem, d l d development l pm t and d differentiation diff ti ti bacteroid release from infection thread bacterial differentiation onset of nitrogen fixation biochemical and physiological functions associated with nitrogen g fixation maintenance (persistence) of nodule function

Factors Affecting Nitrogen Fixation S il pH Soil H -- as soil il pH H decreases d (i.e., (i soil il becomes b acidic) idi ) - infection i f i and nodulation decreases Soil potassium level -- as soil potassium level decreases - number and size of nodules decrease and nitrogenase activity decreases Soil nitrate level -- as soil nitrate level increases, infection, nodulation, nodule growth, and amount nitrogen fixation decreases

Bacteria B t i migrate mi t d down n root th hair i t to root t cortex t x th through u h infection inf ti n th thread d which consists of a stream of bacteria surrounded by host plant cell wall material Cortical cell of root stimulated by bacteria to divide -- developing a nodule Vascular V l tissue ti i in roots t connects t with ith newly l formed f d vascular l tissue ti i in nodule, particularly indeterminate one. Bacterial B t i l cells ll multiply lti l rapidly idl -- transform t f i into t swollen ll f forms called ll d bacteroids enzymes necessary for nitrogen fixation present in bacteroids

Nitrogenase Protection How do organisms living in oxic environments protect their nitrogenase from O2? (i) Free living Azotobacter has an unusually high rate of respiration. It consumes oxygen so fast that the intracellular concentration of oxygen is kept low enough that nitrogenase is not inhibited. (ii) The facultative aerobe Klebsiella fixes nitrogen only when it is in an oxygen-free f environment. i t (iii) Filamentous cyanobacteria have specialized cells called heterocysts that fix nitrogen. Diffusion of oxygen into these cells is limited. (iv) Rhizobia - see discussion Fig. Nif/fix cluster in R.meliloti : pSym megaplasmid
Nif H prpmoter

Nif K

Nif D

Nif H

fixA

fixB

fix C fix X

Nif A

Nif B

Nitrogenase I & II

Regulator

Cofactor

- O2

 The products of the genes fixLJ encode a sensor and a

transcription t i ti f factor t th that t i induces d th the expression i of f th the gene nifA ifA under anaerobic conditions. The product of nifA is a transcriptional activator protein that in turn activates expression of many nif and fix genes. Among the genes induced by y NifA are two operons p - nifNOQP Q and nifHDK, the latter containing the genes encoding dinitrogenase reductase (nifH) and the two subunits of dinitrogenase (nifDK). An important point is that this regulatory cascade that results in the expression of genes involved in nitrogen fixation will not occur in the presence of oxygen oxygen. This makes sense - since nitrogenase is not functional under oxic conditions, there is no point in expressing these genes when oxygen is present.

the nif genes encode the protein necessary for the nitrogenase complex(fix) and nitrogen fixation. fixation nifA expression is controlled by NtrC which is a sigma factor for RNA polymerase. NtrC activates transcription in the absence of ammonium ions. NifA in turn activates the nif structural genes that encode nitrogenase in the absence of oxygen. The Nterm domain of NifA is responsible for NifA's ability bl to sense oxygen

(i) How was this pathway mapped? Transposon insertion mutagenesis. Complementation of mutants to isolate nitrogen fixation genes using gene libraries. (ii) Why is it called a cascade of gene expression? Because one transcriptional activator (phospho-FixJ) activates the expression of yet another transcriptional activator (NifA) that then activates the expression of other genes. The net effect is a large amplification of gene expression. (iii) How is an "anaerobic" environment achieved in nodules? Nodules are structures on plants where nitrogen fixation is done by symbiotic bacteria bacteria. The plant provides the bacteria with fixed carbon produced by photosynthesis, while the bacteria supply fixed nitrogen to the plant. An anaerobic environment is maintained in the nodule via the collection of f free oxygen by b a heme h protein, t i leghemoglobin l h l bi (LHb)

The equilibrium dissociation constant (Kd) for the interaction between LHb and oxygen is 4 x 10-8 M. This means that when the concentration of oxygen is 4 x 10-8 M (which is extremely low) half of the protein is oxygen-bound and h lf of half f it is i f free. This means that even trace amounts of oxygen will be bound. In other words, LHb has a very high affinity for oxygen. The concentration of LHb in nodules is maintained at very high levels. This helps p to ensure an anaerobic environment. In addition, oxygen does not diffuse into the nodules easily - a thick layer of cells acts as a barrier to oxygen diffusion. diffusion As a result of these phenomena, the final concentration of free oxygen in nodules is 10uM O2, a value so small that the environment is essentially anaerobic However, anaerobic. However it should be remembered that there actually is a great deal oxygen present, but it is bound to LHb. This oxygen is still available as a terminal electron acceptor in energy-generating reactions. ti s

 (iv) How is the cascade turned on and off based on the concentration of oxygen? A sensor-kinase system is employed. The oxygen sensor, FixL, is a heme protein that exists in two states - oxy-FixL when oxygen is bound and deoxy-FixL when no oxygen is bound. D Deoxy-FixL Fi L (but (b t not t oxy-FixL) Fi L) can phosphorylate h h l t itself it lf using i ATP as a substrate, producing phospho-FixL and ADP. The phosphate can subsequently be transferred from phosphoFixL to an inactive transcription factor factor, FixJ FixJ, producing phospho-FixJ. Phosphorylation activates FixJ, continuing the cascade. Thus, covalent modification by the phosphate group induces conformational changes in FixJ that makes it a competent t tt transcription i ti f factor. t Dephosphorylation of phospho-FixJ occurs continuously, even when h oxygen is i not t present. t It must t therefore th f be b regenerated constantly. This mechanism ensures that the system does not get "stuck" in the "on" position; a good biological "switch" switch must be readily reversible so that the organism can quickly adapt to a rapidly changing environment.

Nitrogen fixation and agriculture: the way forward

1. After harvest legume roots left in the soil decay, releasing organic nitrogen compounds for uptake by the next g generation of plants p or rotating g a leguminous g crop p with i h a non leguminous l i one.

2. Improving p g the ability y of the more efficient strains of rhizobia to compete with less efficient strains in the soil to ensure the most effective nodulation of legume plants. 3. Researchers are trying to insert the genes that code for the enzymes involved in nitrogen fixation from bacteria to crop plants, in particular cereals such as wheat, maize and rice. i This Thi would ld decrease d the th need df for nitrogen it f fertilizers tili and increase the protein content of the crop.