GENETICS OF LEGUME-RHIZOBIUM SYMBIOSIS

Debarshi DasguptaPALB-4137

• Farmers have known, since the time of the Egyptians, that legumes such as pea, lentil, and clover are important for soil fertility.

• Such practices as green manuring, crop rotation, and intercropping have been known for millennia and were extensively described by the Romans.

• In the 19th century, agriculture in Europe had progressed to the point that both green manuring and intercropping using legume crops was standard practice.

• People observed “bumps” on legume roots as early as the 17th century as evidenced by a drawing published in 1679 by Malpighi (who thought they were insect galls).

Marcello Malpighi (March 10, 1628 – November 29, 1694)

• It took a German scientist, Hermann Hellriegel, in collaboration with Hermann Wilfarth, to recognize that the legume root nodules themselves were responsible for the conversion of atmospheric nitrogen to ammonia (1888).

• The microorganisms were first isolated and cultured from nodules of a number of different legume species by Martinus Beijerinck (1888) of Holland.

Rhizobium (rhiza = root; bios = life).

Haber–Bosch process, is an artificial nitrogen fixation process and is the main industrial procedure for the production of ammonia today.

N2 + 3 H2 → 2 NH3   (ΔH = −92.4 kJ·mol−1)

The BNF symbiosis consists of complex processes of infection of roots by rhizobia,

nodule development, nodule function, and nodule senescence.

The amount of nitrogen fixed by a legume depends on several factors, most

importantly the level of nitrogen already available in the soil: BNF is most active

when soil nitrogen is minimal.

In addition to producing valuable food and animal feed, legumes are beneficial as

rotational crops, green manure, cover crops, forage, and fuelwood.

If a way could be found to mimic nitrogenase catalysis

(a reaction conducted at 0.78 atmospheres N2 pressure and ambient temperatures), huge amounts of energy

(and money) could be saved in industrial ammonia production.

If a way could be found to transfer the capacity to form N-fixing symbioses

from a typical legume host to an important non-host crop species such as corn or

wheat, far less fertilizer

would be needed to be produced and applied in order to sustain crop yields

PHYLOGENY OF Rhizobium RELATED GENERA BASED ON 16s rRNA SEQUENCING

  (12 GENERA CONSISTING OF MORE THAN 70 SPECIES SPANNING α-PROTEOBACTERIA AND β-PROTEOBACTERIA.)

• In the absence of its bacterial symbiont, a legume cannot fix N2.

• Rhizobia can fix N2 when grown in pure culture under microaerophilic conditions (a low-oxygen environment is necessary because nitrogenases are inactivated by high levels of O2).

• In the nodule O2 levels are precisely controlled by the O2-binding protein leghemoglobin.

• Production of this iron-containing protein in healthy N2-fixing nodules is induced through the interaction of the plant and bacterial partners.

• The apoprotein is produced by the plant and the heme (an iron atom bound in a porphyrin ring) is produced by the bacterium.

• Leghemoglobin functions as an “oxygen buffer,” cycling between the oxidized (Fe3+) and reduced (Fe2+) forms of iron to keep unbound O2 within the nodule low.

• The ratio of leghemoglobin-bound O2 to free O2 in the root nodule is on the order of 10,000:1.

• A particular rhizobial species is able to infect certain species of legumes but not others.

• A group of related legumes that can be infected by a particular rhizobial species is called a cross-inoculation group.

• If legumes are inoculated with the correct rhizobial strain, leghemoglobin-rich, N2-fixing nodules develop on their roots.

(a) infection thread cells in this zone multiply

actively , contaminated with

Rhizobium(b) meristem made of small cells that are not

contaminated andthat provide for the growth

of the nodule(c) fixation zone, made up of bacteroids, plant cells filled with rhizobia. Has

nitrogenase.(d) degeneration zone. No

fixation takes place in degenerating cells.(e) vascular system. irrigates the nodule,

bringing in carbohydrates, transporting nitrogen

compounds to the leaves

Fast-growing Rhizobium spp. whose nodulation functions (nif, fix) are encoded on their symbiotic megaplasmids (pSym)Slow-growing Bradyrhizobium spp. whose N-fixation and nodulation functions are encoded on their chromosome.

Process of Nodule Formation1. Recognition of the correct partner by both plant and bacterium and attachment of the bacterium to the root hairs2. Secretion of oligosaccharide signaling molecules (nod factors) by the bacterium3. Bacterial invasion of the root hair4.Movement of bacteria to the main root by way of the infection thread5. Formation of modified bacterial cells (bacteroids) within the plant cells and development of the N2-fixing state6. Continued plant and bacterial cell division, forming the mature root nodule

The roots of legumes secrete organic compounds that stimulate the growth of a diverse rhizosphere microbial community.

If rhizobia of the correct cross-inoculation group are in soil, they form large populations and attach to the root hairs extending from the roots of the plant.

An adhesion protein called rhicadhesin is present on the cell surfaces of rhizobia.

Other substances, such as carbohydrate-containing proteins called lectins and specific receptors in the plant cytoplasmic membrane, also play roles in plant–bacterium attachment.

After attaching, a cell penetrates into the root hair, which curls in response to substances excreted by the bacterium.

The bacterium then induces formation of a cellulosic tube, called the infection thread, by the plant.

Root cells adjacent to the root hairs subsequently become infected by rhizobia, and plant cells divide.

Continued plant cell division forms the tumor -like nodule.

Nodule Development• As these and other tissues develop, the root begins to swell and

the nodule becomes visible.

• In the field, nodules are visible within 21 to 28 days from emergence of the plant.

• The time from planting to the appearance of nodules varies depending on plant growth and availability of mineral nitrogen in the soil.

• Nodules differ in shape, size, color, texture, and location. Their shape and location depend

largely on the host legume.

Nodule FunctionThe rhizobia multiply rapidly within the plant cells and become transformed into swollen, misshapen, and branched cells called bacteroids.A microcolony of bacteroids becomes surrounded by parts of the plant cytoplasmic membrane to form a structure called the symbiosome , and only after it forms does N2 fixation begin.The bacteroids produce the enzyme nitrogenase.The ammonia attaches to a compound provided by the plant, forming amino acids. These amino acids move out of the nodule to other parts of the plant where they undergo further changes.

• It is estimated that the legume-rhizobia symbiosis requires about 10 kg of carbohydrates for each kg of N2 fixed.

• Energy-expensive.

ROOT-NODULE

BACTEROID

Synthesis Of Amino AcidIt was thought that bacteria and higher plants assimilate ammonia into glutamate via the GDH pathway, as in certain fungi and yeasts. NH3 + 2-oxoglutarate + NADPH + H+ <---> glutamate + NADP+

An alternative pathway of ammonia assimilation [involving glutamine synthetase (GS) [ and an NADPH-dependent glutamine:2-oxoglutarate amidotransferase (GOGAT), must be operating when ammonia is present in the growth medium at low levels.

NH3 + glutamate + ATP ---> glutamine + ADP + Pi glutamine + 2-oxoglutarate + NADPH + H+ ---> 2 glutamate + NADP+

THE INFECTION THREAD AND FORMATION OF ROOT

NODULES

Symbiosome

Nodule Senescence• Eventually nodules age and decay.

• Their life span is largely determined by four factors: the physiological condition of the legume, the moisture content of the soil, the presence of any parasites, and the strain of rhizobia forming the nodule.

• As the plant puts more energy into seed production, the nitrogen-fixing activity of the bacteroids

• decreases.

• Eventually the nodules stop functioning and disintegrate, releasing bacteroids into the soil.

• These rhizobia may survive and infect new plants during the next cropping season.

Nodules on Groundnut Soyabean Bengal gram

Clover Guar Soybean EN and IN

Determinate NodulesFormed on tropical legumes by 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 arise just below epidermis; largely internal vascular system.Uninfected cells dispersed throughout nodule; quipped to assimilate NH4+ as ureides (allantoin and allantoic acid)

Formed on temperate legumes (pea, clover, alfalfa);typically by Rhizobium spp.

Cylindrical nodules with a persistent meristem;nodule growth creates zones of different developmentalStages

Nodule arises near endodermis, and nodule vasculature clearly connected with root vascular system

Uninfected cells of indeterminate nodules assimilate NH4+ as amides (asparagine, glutamine)

Indeterminate Nodules

Nitrogenase actually consists of two proteins that work in tandem: the iron (Fe) protein and the molybdenum-iron (MoFe) protein. During the catalytic reduction of dinitrogen, the electrons are transferred from the Fe-protein to the MoFe-protein.

Nitrogenase is extremely sensitive to oxygen. Root nodules of nitrogen‐fixing plants contain the oxygen‐binding protein, leghemoglobin, which protects nitrogenase by binding molecular oxygen.

Nod Genes, Nod Proteins and Nod FactorsRhizobial genes that direct the steps in nodulation of a legume are called nod genes.

Horizontal transfer of such genes as nod and nif that are located on plasmids or transferable regions of chromosomal DNA has taken place.

The nodABC genes encode proteins that produce oligosaccharides called nod factors; these induce root hair curling and trigger cell division in the pea plant, eventually leading to formation of the nodule.

Nod factors consist of a backbone of N-acetylglucosamine to which various substituents are bonded.

The structure of the nod factor determines which plants a given rhizobial species can infect.

Each cross-inoculation group contains nod genes that encode proteins that chemically modify the nod factor backbone to form its species-specific molecule.

nodD encodes the regulatory protein NodD, which controls transcription of other nod genes.

After interacting with inducer molecules, NodD promotes transcription and is thus a positive regulatory protein.

NodD inducers are plant flavonoids, organic molecules that are widely excreted by plants

Luteolin(nodD gene expression inducer)

Genistein(nodD gene

expression

inhibitor)Flavonoids

Discovered that a flavone (luteolin) derived from alfalfa is necessary for activation of nodulation genes (nod ABC) in Sinorhizobium meliloti.

Investigated bacterial nitrogen fixation genes

Gary Ruvkun Sharon R. Long

Medicago truncatulaModel legume plant• Small diploid

genome• Self-fertile• Prolific seeding• Rapid generation

time

F G H I N D1 A B C I J Q P G E F H D3 E K D H A B C(nol) (nod) (nif) (fix)

Gene clusters on R. meliloti pSym plasmid

N M L R E F D A B C I J T C B A H D K E N

Gene clusters on R. leguminosarum bv trifolii pSym plasmid

- - - D2 D1 Y A B C S U I J - - -

Gene cluster on Bradyrhizobium japonicum chromosome

nod (NODULATION) AND nol (nod LOCUS) GENES• Mutations in these genes block nodule formation or alter host

range• Most have been identified by transposon mutagenesis, DNA

sequencing and protein analysis.• four classes: nodD

nodA, B and C (common nodgenes)hsn (host-specific nod genes)other nod genes

Nod D (the sensor)The nod D gene product recognizes molecules (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 nodD alleles recognize various flavonoidstructures with different affinities, and respond withdifferential patterns of nod gene activation

Mutations in nodA,B or C completely abolish the ability of the bacteria to nodulate the host plant; they are found as part of the nod gene “regulon” in all Rhizobia

Products of these genes are required for bacterial induction of root cell hair deformation and root cortical cell division

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

Host-specific nod genesMutations in these genes elicit abnormal root reactions on their usual hosts, and sometimes elicit root hair deformation reactions on plants that are not usually hosts.

loss of nodH function in R. meliloti results in synthesis of a nod factor that is no longer effective on alfalfa but has gained activity on vetch.

The role of the nodH gene product is therefore to add a specific sulfate group, and thereby change host specificity.

May be involved in the attachment of the bacteria to the plant surface, or in export of signal molecules.

exo (EXOPOLYSACCHARIDE) GENES• Exopolysaccharides may provide substrate for signal

production, osmotic matrix needed during invasion, and/or a recognition or masking function during invasion.

• In Rhizobium-legume interactions leading to indeterminate nodules, exo mutants cannot invade the plant properly. They do provoke the typical plant cell division pattern and root deformation, leading to nodule formation, although these are often empty (no bacteroids).

• In interactions that usually produce determinate nodules, exo mutations tend to have no effect on the process.

nif (NITROGEN FIXATION) GENES• Gene products required for symbiotic nitrogen fixation,

and for nitrogen fixation in free-living N-fixing species.

• Example: subunits of Nitrogenase

fix (FIXATION) GENESGene products required to successfully establish a functional N-fixing nodule. Regulatory proteins that monitor and control oxygen levels within the bacteroids.

EXAMPLE: FixL senses the oxygen level; at low oxygen tensions, it acts as a kinase on FixJ, which regulates expression of two more transcriptional regulators:

• NifA, the upstream activator of nif and some fix genes; • FixK, the regulator of fixN Low oxygen tension activates nif gene transcription

and permits the oxygen-sensitive nitrogenase to function.

Stem Nodulating BacteriaWidespread in tropical regions where soils are often nitrogen deficient because of leaching and intense biological activity.

The best-studied system is the tropical aquatic legume Sesbania, which is nodulated by the bacterium Azorhizobium caulinodans.

Some stem-nodulating rhizobia produce bacteriochlorophyll a and thus have the potential to carry out anoxygenic photosynthesis.

Bacteriochlorophyll-containing rhizobia,called photosynthetic Bradyrhizobium, are widespread in nature,particularly in association with tropical legumes.

Future DirectionsEnhancing survival of nodule forming bacterium by improving competitiveness of inoculant strains

Extend host range of crops, which can benefit from biological nitrogen fixation

Engineer microbes with high nitrogen fixing capacity

Enhanced competitive ability in an inoculant strain is a key requirement

Management and manipulation of rhizobial competition as well as genes that influence competition in the rhizosphere

May exhibit several plant growth-promoting effects, such as hormone production, phosphate solubilization, and the suppression of pathogens

CONCLUSION• Recent advances in the understanding of endosymbiotic

and endophytic nitrogen fixation with nonlegume plants may represent original and alternative new avenues for engineering non-legume nitrogen-fixing crops.

• Understanding the molecular mechanism of BNF outside the legumerhizobium symbiosis could have important agronomic implications and enable the use of N-fertilizers to be reduced or even avoided.

• Indeed, in the short term, improved understanding could lead to more sustainable exploitation of the biodiversity of nitrogen-fixing organisms and, in the longer term, to the transfer of endosymbiotic nitrogen-fixation capacities to major non-legume crops.

• The task of engineering the non legumes is a complex method but a concerted effort is required to fill the gaps of our knowledge.