Download Pods and Nods: a new look at symbiotic nitrogen fixing

Pods and Nods: a new look at
symbiotic nitrogen fixing
Nicholas J Brewin
John Innes Centre, Norwich, UK
How can growing a crop plant make fields more fertile? With legumes, this is precisely what
happens. Working in partnership with symbiotic bacteria that create root nodules on their
chosen host, legumes can fix atmospheric nitrogen and enhance the nitrogen status of soils.
How does this symbiosis develop? And how did it evolve? Bacterial and plant genomics are
beginning to provide the answers.
It has been known since ancient times that legumes
contribute significantly to soil fertility. Pliny the Elder
(circa 70 AD) wrote: ‘It is universally agreed that no
manure is more beneficial than a crop of lupins turned in
by the plough or with forks before the plants form pods.’
The important connection between legumes, root nodules
and nitrogen fixation was established in 1888 by two
German soil chemists, Hellriegel and Wilfarth. They
demonstrated how the unique capacity of leguminous
plants to ‘fix’ atmospheric nitrogen for plant growth was
related to the development of root nodules following
infection by soil microorganisms (reviewed by Quispels,
1988). In the same year, Beyerinck (following in the footsteps of Koch and Pasteur) successfully cultured the
microorganisms from lupin and pea nodules. It was
demonstrated that the strain of ‘Bacillus radicicola’
isolated from pea nodules (title image) was capable of
forming nodules on peas but not on lupin, whereas the
converse was true for the lupin-derived strain. However,
another German microbiologist, named Frank, was not
convinced by Beyerinck’s identification. Because he
suspected that fungi or myxomycetes might be the causal
Title image. Pea nodules, the site of biological nitrogen fixation.
The physiology of a legume root nodule is adapted to promote the
activity of the oxygen-sensitive nitrogenase system of rhizobium
bacteria by reducing oxygen concentrations in the central tissues.
Biologist (2002) 49 (3)
agents of nodulation, Frank proposed the neutral name
Rhizobium leguminosarum, and this name has endured
from 1889 to the present day.
Although there were some minor refinements in
nomenclature, not much changed over the next 100
years. Rhizobium genera and species were defined in
the context of cross-inoculation groups. A rhizobial
species was observed to be co-adapted to a particular
range of host plants. Some strains showed a high
degree of host specificity, while others had very broad
host ranges (particularly with tropical legumes). This
anecdotal information was used as the basis for an
inoculum industry that underpinned the globalisation
of agriculture. As legume crops were introduced from
one region of the world to another (soybeans and
lucerne to North America, clovers to Australia), it was
recognised that they needed to be inoculated with an
appropriate rhizobium strain in order to reap the benefits of biological nitrogen fixation. However, there was
no real understanding of the underlying scientific
issues. Why is a particular strain of rhizobium capable
of nodulating one legume host but not another? What
do rhizobial strains have in common and how do they
differ? Finally, what unique feature of legumes allows
them to develop this unique symbiosis with rhizobia?
Over the past two decades, some fascinating answers
have begun to emerge.
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Figure 1. Colonisation of host cells by rhizobia (courtesy of Simon
Walker and Allan Downie). Rhizobium-derived Nod-factor acts as
a signal for root hair deformation (left-hand micrograph). The
infection thread orginates as an intrusion of the host cell wall
(arrows) and propagates from cell to cell as a transcellular tunnel
in the root cortex (right-hand micrograph).
nod genes and Nod-factors
With the onset of bacterial genetics, it has been possible to
define a number of key genes (nod genes) that are critical for
the initiation of nodulation on legumes, but are not necessary for survival as free-living soil bacteria. Analysis of the
functions of nod genes showed that they arecomponents of a
molecular dialogue that results in the initiation of nodule
development on the appropriate host legume.
Apparently, all legumes exude flavonoid compounds
from their roots. Therefore, each legume species has a
distinctive ‘odour’ that is sensed specifically by the nodD
gene product(s) of an infective rhizobium strain. Following
this recognition event, transcription of a set of nod genes
takes place, which culminates in the synthesis and
secretion of specific signal molecules, termed Nod-factors.
Structurally, Nod-factors are decorated oligomers of
N-acetyl glucosamine (the constituent of chitin) and,
characteristically, they each carry an unsaturated acyl
chain N-linked onto the terminal non-reducing sugar.
Nod-factors specifically activate host cells of the appropriate legume (Stougaard, 2001). By a process that is
still not understood at the molecular level, Nod-factors
initiate the twin processes of nodule initiation (by inducing
cortical cell divisions) and cell colonisation (by provoking a
re-organisation of cell wall growth in epidermal cells and
root hairs).
Rhizobial diversity
From the above analysis, an operational definition of
rhizobia emerges. They are soil bacteria carrying a set of
nod genes that allow them to nodulate one or more
legume hosts. There is a presumption that rhizobia also
carry nitrogenase (or nif) genes that allow them to
develop the capacity for biological nitrogen fixation as
part of the root nodule symbiosis. Generally, it is found
that the nod and nif genes are closely linked, but this is
where the taxonomy begins to get interesting. When
rhizobial strains are analysed by standard taxonomic
criteria based on gene sequence comparisons, it is clear
that these bacteria do not form part of a homogeneous
evolutionary clade. The Rhizobiaceae as a distinct taxonomic group does not exist (Spaink et al., 1998). The
clear implication is that nod, nif and associated symbiosis genes have been transferred ‘horizontally’ between
various groups of soil bacteria to create new genetic
combinations that are optimally adapted, both for
survival in the soil and for the nodulation of some particular legume host.
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Until recently, most of the characterised rhizobial
strains had been derived from the standard but limited
range of cultivated legume species. All these bacterial
isolates belong to three distinct branches within the
alpha-2 subgroup of Proteobacteria. In each case, rhizobia
are phylogenetically intertwined with non-symbiotic bacteria (Moulin et al., 2001). The largest branch includes the
genus Rhizobium, which nodulates peas and clovers, and
Sinorhizobium, which nodulates alfalfa (lucerne); however,
Sinorhizobium is much more closely related to the plant
pathogen Agrobacterium and to Brucella (an intracellular
animal pathogen) than it is to Rhizobium. A second branch
includes the genus Bradyrhizobium, with species that
nodulate soybean, lupin and many tropical legumes;
however, closely related species include Rhodopseudomonas (a photosynthetic free-living bacterium). The
third group includes Azorhizobium, which is closely related
to the chemiautotroph Xanthobacter.
This diversity and heterogeneity may represent only the
tip of the iceberg for rhizobial strains. There are about
18 000 species in the family Leguminosae and, so far,
symbionts from only 50 of the 750 genera have been
sampled. In the last year alone, two completely new rhizobial groupings have been identified. Methylobacterium
nodulans, isolated from Crotalaria nodules, represents a
fourth class of alpha-2 subgroup Proteobacteria (the first
rhizobium strain reported to grow on methanol), while
bacteria isolated from Aspalathus nodules were found to
belong to the genus Burkholderia, a member of the
phylogenetically distant beta-subclass of Proteobacteria
(Moulin et al., 2001). Although nitrogen-fixing bacteria
exist in other proteobacterial subclasses, e.g.,
Herbaspirillum (which colonises sugar cane tissues) and
Azoarcus (which colonises rice roots), none has previously
been found to harbour the nod genes essential for establishing legume symbiosis.
Horizontal gene transfer
How does horizontal transfer of nod and nif genes occur?
The clues are to be found in the genomic structures of those
rhizobia that have been examined so far. In the case of
Rhizobium leguminosarum, the cassette of symbiosis
genes is carried on a self-transmissible plasmid (c. 250
kilobases): biovar viciae (which infects peas and vetches)
and biovar trifolii (which nodulates clover) harbour plasmids that confer different host specificities. Depending on
the ecological niche (e.g., a sward of vetch or clover), it is
easy to see how the symbiotic plasmid with a selective
advantage is likely to become established as the dominant
biovar in the soil population.
In the case of Sinorhizobium strain NGR234, there is a
very large symbiotic plasmid (Freiberg et al., 1997). The
extremely broad host range of this strain is apparently the
consequence of the long-term accumulation of a diversity of
nodD variants and a corresponding range of nod genes,
which synthesise a diversity of Nod-factors. Similarly in
the case of Mesorhizobium loti, a ‘symbiosis island’ allows
non-symbiotic bacteria to evolve into symbionts in a single
quantum leap (Sullivan and Ronson, 1998). The 500 kb
cassette of symbiosis genes has become inserted by integration into the chromosome at the phe-tRNA locus, in a
process mediated by a P4 integrase encoded within the
island. Apparently, the symbiosis island is one of a number
of fitness islands that can be acquired by related bacteria.
These mechanisms of horizontal gene transfer contribute
to the ecological adaptation of soil bacteria. In the last
year, the complete genomes of Sinorhizobium meliloti and
Biologist (2002) 49 (3)
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Figure 2. Electron micrograph showing transverse section of a
tubular infection thread (IT) bounded by a primary cell wall
containing pectin and cellulose. Immunogold staining with monoclonal antibody MAC265 highlights root nodule extensin, a
legume-derived glycoprotein that is secreted through the plasma
membrane (arrowheads) and surrounds the invading rhizobia in
the infection thread lumen. Scale bar = 0.5 microns
Mesorhizobium loti have been determined. Thus,
comparative analysis of genome structure and function
will help to define the minimum set of functions associated
with a root-nodulating (rhizobium) strain.
Adaptations for tissue and cell colonisation
It is clear that the nod and nif genes do not represent the
whole of the story as far as nodulation is concerned. For
example, when these genes are introduced into E. coli
nothing happens. What other genes are required for
symbiosis and what attributes of non-symbiotic soil bacteria predispose them to become rhizobia? Once again, the
clues are to be found in comparative genome analysis
among the non-symbiotic relatives of rhizobia. The
answers may be related to the ability of rhizobia and potential rhizobial strains to colonise host tissues (Figures 1
and 2) and to survive under the special conditions found
inside the tissues and cells of the host plant (Figure 3). For
example, the ability of rhizobia to survive in a hypo- or
hyper-osmotic environment may be an adaptive characteristic. The nature of bacterial cell surfaces (e.g., lipopolysaccharide and exopolysaccharide) has also been shown to
affect the ability of rhizobia to colonise host tissues and
cells (Pellock et al., 2000).
Intracellular symbionts and pathogens
Fitness for host cell colonisation is not a trait that is
unique to rhizobia. It is interesting to note that
Sinorhizobium meliloti (a symbiont of alfalfa) is phylogenetically related to Brucella abortus (a mammalian
pathogen). Both strains establish chronic intracellular
infections in their respective hosts, without the induction
of host defence responses. In these two very different relationships, a similar gene encoding a putative membrane
transport protein (bacA) was found to be of critical importance for the maintenance of bacteria within the host cell
(LeVier et al., 2000).
An even more curious coincidence has recently emerged,
with the discovery that endophytic arbuscular mycorrhizae
Biologist (2002) 49 (3)
Figure 3. Endosymbiotic rhizobia are morphologically differentiated
as bacteroids. In central tissues of the root nodule, host cells harbour
thousands of nitrogen-fixing bacteroids. Host cells have large central
nuclei and the vacuole is reduced or absent (upper micrograph). The
electron micrograph (lower micrograph) shows that each bacteroid is
bounded by a plant-derived peribacteroid membrane, forming an
organelle-like structure termed a symbiosome.
(which colonise 90% of all land plants, not just legumes)
sometimes harbour intracellular bacteria that are related
to Burkholderia (Ruiz-Lozano and Bonfante, 2000).
Because Burkholderia has recently been characterised as a
rhizobial genus, this raises some interesting questions
about the direction of evolutionary change. Have intracellular symbiotic microorganisms from mycorrhizae subsequently acquired the ability to colonise legume cells or
have Burkolderia-type rhizobia developed the capacity to
survive as endosymbionts of mycorrhizal hyphae?
The joint occurrence of Burkolderia spp as endosymbionts
of mycorrhizal fungi and as endosymbionts of legume nodule
cells is particularly intriguing. Genetic analysis of symbiotically defective legumes has revealed that about half of the
plant mutants that fail to establish the early stages of
nodule symbiosis are also impaired in the establishment of
the mycorrhizal symbiosis (Bonfante et al., 2000). Yet, the
mycorrhizal fungi evolved the capacity to colonise host plant
cells over 400 million years ago, while the legume symbiosis
is only about 60 million years old. Apparently, rhizobia have
exploited this more ancient host-symbiont signalling system
as part of the signal transduction cascade for Nod-factor. As
an added twist to this curious paradox, it should be pointed
out that rhizobium Nod-factors are simply decorated fragments of oligochitin, and chitins are, of course, the key
component of fungal cell walls.
A fresh look at legume taxonomy
Let us now examine the rhizobium-legume symbiosis from
the viewpoint of the host plant. Can we identify what
unique features of legumes enable them to establish an
interaction with rhizobia? Once again, a taxonomic and
evolutionary perspective may be helpful. Among the 18 000
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Pisium
Medicago
Lotus
Glycine
Vigna
Papilionoideae
Parasponia has been shown to be dependent on the presence
of functional nod genes (Marvel et al., 1987). These observations suggest two important conclusions. First, the predisposition to form nodules is not restricted to the
Leguminosae, but rather to the Rosid clade I. Second, at
least one non-leguminous member of the clade is capable of
recognising the products of rhizobial nod genes.
Swartzia
New excitement for a new century
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Leucaena
Acacia
Mimoisoideeae
Ceratonia
3
Chaemaecrista
Caesalpiniodeae
Bauhinia
Dialium
Figure 4. Simplified evolutionary tree for the Leguminosae
(Fabaceae), showing the nodulating sectors in red and non-nodulating groups in blue. The three putative origins for nodulation
capability are indicated. (Adapted from Doyle, 1998.)
legume species, there are a number of groups that do not
nodulate. In some cases, the symbiotic character may have
been lost during evolution. However, in the Caesalpinioid
sub-family, only 23% of all species are nodulated. Therefore,
it seems probable that nodulation has never developed in
the more primitive legume groups (Doyle, 1998). Plant
taxonomy is now based on comparative sequence analysis
for several well-defined genes (of nuclear, chloroplast and
mitochondrial origin). On this basis, and applying the principle of parsimony, it seems that nodulation must have
evolved at least three times during the evolution of legumes
(Figure 4). This implies some form of predisposition to establish a nodulating symbiosis. To put it another way, rhizobia
may originally have been colonisers of host cells and intercellular spaces in legume roots in much the same way that
mycorrhizal fungi colonise root cortical cells. Only subsequently would the rhizobium-legume symbiosis have
evolved the capacity to form a nodule-like structure as a
physiological adaptation that serves to reduce oxygen
damage to the oxygen-sensitive nitrogenase enzyme system.
The legume-nodulation paradigm is further complicated
when we step outside the family Leguminosae (Fabaceae).
Following a reconfiguration of the taxonomy of higher
plants, the Leguminosae now belong to a grouping termed
Rosid clade I (Doyle, 1998). Unlike all other groups of higher
plants, this clade contains probably seven separate
instances of the evolution of non-legume root nodule
symbioses (although nodule anatomy is more reminiscent of
a modified lateral root than is the case with legume
nodules). In six of these symbioses, the nodule endophyte is
not a rhizobial strain (as currently defined) but is instead an
actinomycete, Frankia, a filamentous Gram-positive
bacterium (Table 1). Examples of actinorhizal plants include
Alnus, Caeanothus and Casuarina. The seventh example of
non-legume root nodule symbiosis involves the ulmaceous
shrub Parasponia, but in this case the endophyte is unquestionably a rhizobium strain. Moreover, nodulation of
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With so much exciting new information, how should the
experiments of Hellriegel, Beyerinck and Frank be interpreted today? First, in answer to Hellriegel and Wilfarth,
nitrogen fixation is a property of the bacterial symbiont
and host specificity is related to different chemical structures for rhizobium-derived Nod-factors. These are chemically decorated lipochitin oligosaccharides encoded by nod
genes. Second, in answer to Beijerinck and Frank, it is now
recognised that root-nodulating bacteria are taxonomically
diverse. As a result of horizontal gene transfer, the capacity to nodulate legumes has spread to many bacterial
groups in the Proteobacteria. Furthermore, the relationship between the rhizobium-legume symbiosis and the
Frankia-Rosid symbiosis is extremely curious. It might be
clarified by a genome-sequencing program that establishes
whether Frankia spp carry anything equivalent to the nod
genes of rhizobia. Legumes are a sub-group of Rosid clade I
and there is already one well-documented case of a rhizobium symbiosis with a non-legume.
Let us finish on a speculative note. Perhaps the concept of
‘rhizobial strains’ should be extended to include all bacteria
capable of inducing nitrogen-fixing root nodules in members of
Rosid clade I (including both legumes and non-legumes). After
more than a century of research, Frank and his colleagues
would probably welcome the re-integration of Frankia into an
all-inclusive group of root-nodulating bacteria.
Acknowledgements
Research on legume nodule development at the John Innes
Centre has been sponsored by the Biotechnology and
Biological Research Council.
Table 1. Families in Rosid Clade I known to include
nodulating plants
Actinorhizal nodules
Rhizobial nodules
Rosaceae
Ulmaceae
Ulmaceae
Elaeagnaceae
Rhamnaceae
Fabaceae
Betulaceae
Casuarinaceae
Myricaceae
Coriariaceae
Datiscaceaeae
Table 1. Rosid clade I is the only group of higher plants in which
root nodule symbiosis has sporadically arisen. This table lists the
groups that nodulate with Frankia or rhizobia.
Biologist (2002) 49 (3)
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Websites
www.noble.org/medicago/
Describes Medicago truncatula as a model legume for molecular
genetic analysis
http://wwwifr.toulouse.inra.fr/meliloti.html
Describes the complete sequence of Sinorhizobium meliloti, the
symbiont of Medicago truncatula
http://helios.bto.ed.ac.uk/bto/microbes/microbes.htm
A good educational site for plant-microbe interactions
www.jic.bbsrc.ac.uk/staff/nick-brewin/index.htm
Includes author’s lecture notes on the rhizobium-legume symbiosis
Nick Brewin DSc is honorary professor at the University of
East Anglia and tutor for graduate studies at the John Innes
Centre. He has studied both bacterial and plant aspects of the
rhizobium-legume symbiosis, particularly in relation to the
mechanisms of tissue and cell invasion.
John Innes Centre
Norwich, NR4 7UH, UK
[email protected]
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