Plant Physiol, October 2000, Vol. 124, pp. 531-540
UPDATE ON NODULE DEVELOPMENT
Regulators and Regulation of Legume Root Nodule
Development
Jens
Stougaard*
Laboratory of Gene Expression, Department of Molecular and
Structural Biology, University of Aarhus, Gustav Wieds Vej 10, 8000 C
Aarhus, Denmark
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INTRODUCTION |
Nitrogen is the nutrient plants
require in the highest amount, and in agriculture nitrogen availability
has a major influence on both yield and product quality. In nature
plants acquire nitrogen by assimilation of nitrate and ammonium or from
dinitrogen through association with nitrogen-fixing bacteria. Symbiotic
nitrogen fixation, where the plant supplies the carbon source for the
energy-dependent reduction of dinitrogen and protects the
oxygen-sensitive nitrogenase enzyme, is among the most effective
fixation systems. To establish a symbiosis, the bacterial
microsymbionts gain access to single plant cells and install themselves
in compartments surrounded by a plant membrane. In Gunnera
sp. the cyanobacterium Nostoc sp. invades
pre-existing stem glands and forms nitrogen-fixing heterocysts in
infected cells. In most other symbiotic interactions, a specialized
plant organ, the root nodule, is developed to provide optimal
conditions for the nitrogen-fixing bacteria. Among woody plant species
belonging to eight different families, an interaction with the
gram-positive genus Frankia leads to the development of
actinorhizal root nodules. In legumes, gram-negative soil bacteria belonging to the family Rhizobiaceae (here collectively called Rhizobium) infect root tissue and induce the formation of
the nitrogen fixing nodules. Why certain plants are able to develop root nodules is unclear, but recent phylogenetic studies based on DNA
sequence analysis place all plants involved in rhizobial or
actinorhizal symbiosis in the same lineage and suggest that the
predisposition for nodulation evolved only once (Soltis et al., 1995
;
Doyle, 1998).
The relationship between Rhizobium and legume plants is
selective. Individual species of rhizobia have a distinct host range allowing nodulation of a particular set of legume plants. For example,
Rhizobium leguminosarum bv viciae nodulates pea
and vetch, whereas Bradyrhizobium japonicum nodulates
soybean. At the other extreme, the exceptionally broad host-range
Rhizobium sp. NGR234 nodulates 353 legume species
representing 122 genera (Pueppke and Broughton, 1999). Differences in
both infection processes and organogenic programs are reflected in
variations in root nodule morphology (Doyle, 1998), but overall there
are pronounced developmental similarities as would be expected from a
common ancestry. To cover most aspects of this unusual plant-prokaryote
symbiosis, the study of nodulation is a multifaceted
research area aiming to understand this plant-microbe interaction in a
framework of physiological and developmental processes underlying
infection and organogenesis. With this perspective, this
Update draws on observations from different
Rhizobium-legume interactions. The following sections focus
on plant control of root nodule organ formation and sketch the way
plant genetics and functional genomics are changing our thinking. The
early signal exchange as well as the biosynthesis and properties of the
bacterial Nod factor signal molecules have been reviewed extensively
(Dénarié et al., 1996; Spaink, 1996; Downie and Walker,
1999, and refs. therein) and will be presented only briefly.
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HISTOLOGY AND NODULE DEVELOPMENT |
In the most studied legumes, infection occurs via an infection
thread that takes the bacteria through the root hair into the root
cortex and distributes them to cells, which become the infected cells
of the nitrogen-fixing nodule (Fig. 1).
The root zone susceptible to invasion is located behind the root tip
where root hairs are still growing and competent for invasion. In
response to attached bacteria, root hairs deform and curl setting up a
pocket that provides a site for initiating the infection. The
infection thread is a plant-derived structure originating from plasma
membrane invagination accompanied by external deposition of cell wall
material. In advance of the intracellular "inward"
progressing thread, root cortical cells dedifferentiate and reenter the
cell cycle. Cortical cells prepared for infection thread passage appear
to be arrested in the G2 phase, whereas cells completing the cycle
resume division to establish the nodule primordium. Later in the
process, pattern formation and cell differentiation specify tissue and
cell types. The bacteria are endocytosed into a subset of cells where
they differentiate into nitrogen-fixing bacteroids surrounded by the peribacteroid membrane of the symbiosome. In the mature functional nodule, peripheral vascular bundles are connected to the root vasculature and the main tissues/cell types can be distinguished cytologically and to some extent with molecular markers. In determinate nodules such as soybean the meristematic activity ceases early, and the
nodule grows by expansion giving a spherical shape (Fig. 1). All
developmental stages from root hair curling to nodule senescence are
consequently phased in time. Indeterminate nodules such as pea maintain
an active meristem depositing cells that are subsequently infected.
This results in a cylindrical shape with the organ formative
developmental stages represented along the longitudinal axis. The type
of nodule formed is specified by the plant host.
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Figure 1.
A, Whole mount of a developing Lotus
japonicus root nodule invaded by two infection threads.
Fluorescence microscopy shows the autofluorescent nodule cells and the
Mesorhizobium loti bacteria stained for  -galactosidase
activity expressed from a lacZ reporter gene. The
determinate root nodules of L. japonicus are initiated from
cell division in the outer cortex, whereas indeterminate nodules (of
pea, for example) are initiated from cell divisions in the inner
cortex. Conditions or factors influencing nodule initiation or
development are listed to the right and left of the root nodule. Where
different responses to the factor or condition have been reported in
individual legume species, this is indicated with the following: +,
positive effect;  , negative effect; or 0, lack of response. EPS,
Exopolysaccharides; LPS, lipopolysaccharides. B, Structure of a
bacterial LCO Nod factor. The acetylated fucosyl in blue results from
NodZ and NolL modification of the R. leguminosarum
LCO.
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MUTUAL SIGNAL EXCHANGE STARTS THE PROCESS |
The early plant host signals secreted into the rhizosphere can be
(iso)flavonoids, stachydrines, or aldonic acids. Best studied are the
flavonoids that, in conjunction with the rhizobial NodD transcriptional activator, induce expression of the nod gene
regulon. In turn, nod gene products synthesize and transport
the Nod factor (Fig. 1B), the major early signal molecule perceived by
the host plant. A flexible interaction with the host is enabled by
several mechanisms. Alternative NodD activators recognizing
different plant flavonoids provide an extended host range in some
bacterial strains. B. japonicum even has an alternative two
component regulatory pathway for activating its nod regulon,
and in Sinorhizobium meliloti nod gene expression is
fine-tuned by positive- and negative-control circuits.
Nod factors are low-Mr ,1-4-linked
N-acetyl glucosamine compounds (Lerouge et al., 1990)
typically carrying a fatty acid on the non-reducing sugar and sulfuryl,
fucosyl, mannosyl, or arabinosyl groups at the reducing terminal sugar.
Additional substitutions include carbamoyl, glycerol, and fucosyl
derivatives (Dénarié et al., 1996; Spaink, 1996). When
purified and applied in the absence of bacteria these
lipochitooligosaccharides (LCOs) function as mitogens or
"morphogens" on some legume roots. For example, addition of
nanomolar concentrations induces root hair deformation in most legumes.
In more responsive plants, pre-infection threads (cytoplasmic bridges
in G2 arrested cortical cells), cortical cell divisions, and empty
nodule structures with an anatomy comparable to rhizobial-induced
nodules also develop (Dénarié et al., 1996; Spaink, 1996,
and references therein). These responses show that some legumes encode
all functions necessary to develop the nodule once the process has been
switched on. Spontaneous nodule development on certain alfalfa mutants
grown axenically supports this idea.
Rhizobium strains differing in their repertoire of
nod genes produce LCOs with different structural features.
Biological activity is determined by the length of the chitin backbone,
the structure of the lipid, and a suite of other substitutions on the
oligosaccharidic backbone. Host specificity results at least partly
from a two-way communication where both plant flavonoid activation of
nod genes and plant perception of the resulting bacterial
LCO signal molecules are required for nodulation to occur (Fig.
2A). For example, R. leguminosarum bv viciae can be genetically modified to
produce an acetyl fucosylated LCO under control of a
flavonoid-independent NodD activator (Figs. 1B and 2B). This
modified LCO is now recognized by L. japonicus plants
resulting in nodulation by the nod gene deregulated R. leguminosarum strain (Bras et al., 2000).
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Figure 2.
Schematic representation of the early
communication between legume host and Rhizobium. A,
Symbiotic development is initiated only when the correct plant and
bacterial signal molecules are synthesized, presented, and perceived.
B, Changes of the genetic repertoire of R. leguminosarum bv
viciae leads to the synthesis of different LCOs and
changes of host specificity. Stepwise addition of a
flavonoid-independent NodD activator plus NodZ fucosyl transferase and
NolL acetyl transferase allows R. leguminosarum bv
viciae to nodulate L. japonicus. , No
nodulation; +, slow nodulation; +++, normal nodulation.
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SIGNAL RELAY IN NOD-FACTOR PERCEPTION |
Bacterial LCO signals are first perceived by epidermal cells in
the zone of root hair differentiation but the earliest responses to
Rhizobium or to the addition of LCO has mainly been studied in more developed root hairs protruding from the root surface where
they are easily accessible for microscopical and physiological analysis. Using a combination of electrophysiology and fluorescence microscopy with ion sensitive dyes, it was shown that LCO-induced ion
fluxes precedes root hair deformation (Fig.
3). A rapidly induced
Ca2+ influx and a transient plasma membrane
depolarization associated with Cl
and
K+ effluxes occur within seconds. This is
accompanied by alkalinization of root hair cytoplasm and after some
minutes of Ca2+ oscillation (Ehrhardt et al.,
1996; for mechanism, see Felle et al., 1998). Subsequently
rearrangement of actin filaments and redirection of root hair tip
growth is observed. Although root hairs that remain uninfected (or are
not competent for rhizobial infection) may also respond, it is
conceivable that these rapid physiological changes are transmitted into
a signal transduction pathway leading to activation of genes regulating
nodulation. Absence of Ca2+ oscillation in the
alfalfa MN-NN1008 non-nodulating mutant, and the overlap of LCO
chemical structure requirements needed for activity on the plant and
for eliciting changed root hair physiology favors this
interpretation.
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Figure 3.
Early physiological changes detected in root hairs
following LCO treatment or rhizobial inoculation. The order of events
roughly follows the timing of changes but does not indicate a causal
relationship.
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Pharmacological experiments using agonists and antagonists [e.g.
mastoparan, pertussis toxin, EGTA,
2,5-di(t-butyl)-1,4-benzohydroquinone, and
La3+] of possible signal transduction components
suggested that small trimeric GTPases together with phospholipase C and
phosphoinositides are involved (Pingret et al., 1998). The causal
relation between the various physiological changes now needs to be
established and related to activation of downstream plant genes. For
this work plant symbiotic mutants defining genetically separable steps and root hair expressed genes will be useful tools. It is interesting that a recently cloned LjCbp1 gene encoding a putative
Ca2+-binding protein is expressed in an
LCO-dependent fashion in root epidermal cells (Webb et al., 2000).
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THE ELUSIVE NOD FACTOR RECEPTOR |
The plant preference for particular rhizobial partners as well as
the low operational concentrations and structural specificity of
bacterial LCO signals suggest that signal perception is mediated by a
plant receptor. At present a bona fide receptor for binding the LCO and
amplifying the bacterial signal has not been identified. Two binding
sites (NFBS1 and NFBS2) were described in microsomal fractions from
alfalfa roots and tissue culture cells. NFBS1 has low substrate
affinity, whereas NFBS2 binds LCO with higher affinity (Gressent et
al., 1999). Both sites have specificity for LCO but will also bind
derivatives without the sulfuryl substitution needed for in vivo
activity. This discrepancy could result from a receptor that in vitro
was depleted of a subunit adding specificity.
A novel lectin type protein was recently purified from Dolichos
biflorus and shown to bind LCO with high affinity (Etzler et al.,
1999). This lectin also has apyrase (nucleotide phosphohydrolase) activity, suggesting a function at the start of a signal transduction (phosphorylation) pathway and thus adding a new twist to the study of
lectins. Expression of pea or soybean genes encoding seed lectins with
non-catalytic sugar-binding sites was previously shown to extend the
host range of clover and Lotus corniculatus. This effect was
attributed to the lectin's ability to attach sufficient bacteria at
the root hair tip prior to infection or enhancement of the LCO induced
mitogenic response rather than being mediated by an LCO-binding/receptor function (van Rhijn et al., 1998; Díaz et al., 2000).
The question concerning the nature and localization of the Nod factor
receptor is still open, leaving room for alternative models (Hirsch,
1992; Ardourel et al., 1994; Schultze and Kondorosi, 1998). Intriguing
results from studies with bacterial mutants, non-specific LCOs, and
chitin-oligomers suggest a perception mechanism using two receptors of
different stringency or a receptor with a complex substrate
interaction. Simple LCO derivatives (O-acetylated chitin
oligosaccharides) can, for example, induce cortical cell divisions
after microtargeting into Vicia roots and in soybean, transient induction of the early nodulin Enod40 gene can be
obtained with an unsubstituted chitin pentamer. This points toward a
hierarchy of plant responses requiring different LCO specificity for
execution and a mechanism where LCO transport, localization, and
receptor affinity may be influenced by plant chitinases or glycosyl
hydrolases can be envisaged.
Uncoupling of cell division and infection thread formation seen for
example in sym5 mutants of pea and sym4 of
sweetclover could lend support for a two receptor model, but the
abundance of generally unresponsive plants mutants indicate that common steps or interactive pathways are involved. Future map-based cloning of
loci believed to be involved in LCO perception, for example, Sym2 from pea and Sym5 from L. japonicus are likely to clarify some of the questions and may also
explain why infection threads are only observed in the presence of bacteria.
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DEVELOPMENTAL CROSS TALKING: CHECKS AND BALANCES |
Development of functional root nodules relies on synchronized
activation of particular gene sets in both symbionts. The
stage-characteristic arrest observed with rhizobial as well as plant
mutants indicates that the processes are highly coordinated, but
information on "late" signal exchange is somewhat sparse. However,
continued expression of nod genes in bacteria
contained within infection threads and localization of internalized
immunoreactive LCO in cells of maturing root nodules, indicate a
connection to the early LCO signaling. After endocytosis, the
synthesis of LCOs is down-regulated in bacteroids. Endocytosis and
bacterial differentiation appear therefore to mark a shift in
plant-rhizobial communication.
Bacterial surface polysaccharides are known to be involved in the
infection process. In some symbiotic interactions, rhizobial mutants
that are deficient in exo- or lipo-polysaccharides (EPS I, EPS II, and
LPS) are defective in the infection process and may provoke increased
host defense reactions suggesting that surface polysaccharides shield
the bacteria. However, EPS mutants are partially rescued by exogenous
application of picomolar concentrations of
low-Mr polysaccharide fractions of EPS I or
EPS II, suggesting that these function as signal molecules
(González et al., 1996).
Secreted proteins also contribute to signaling. Strains of R. leguminosarum bv viciae excrete NodO, a protein shown
to form Ca2+-transporting ion channels in vitro.
In vivo a NodO-mediated enhancement of infection thread progression and
nodulation was observed in partially compatible pea hosts. The NGR234
strain has a type-III protein secretion system known from pathogenic
bacteria to export proteinaceous pathogenicity factors. Mutations
preventing secretion of the NGR234 NolX and y4xl proteins (functions
unknown) change the nodulation pattern on some but not all legume
hosts, implying that aspects of protein signaling are shared between
pathogens and symbionts (Viprey et al., 1998). It will be interesting
to determine the targets of these "late" signals and address the possibility of positive roles in nodule development as well as defense avoidance.
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SECONDARY SIGNALING THROUGH PHYTOHORMONES |
Most of the plant architecture is formed by post-embryonic
development, and changes in relative hormone concentrations under the
influence of biotic and abiotic factors strongly influence the
developmental fate of cells and organs. Formation of root nodules is no
exception, and several lines of evidence suggest a role for
phytohormones in secondary signaling. Incubation with auxin transport
inhibitors results in development of empty nodule-like structures on
roots of some legumes and expression of genes such as
Enod12, Enod40, and Enod2, which are
normally expressed during early phases of root nodule organogenesis
(Fang and Hirsch, 1998). Using an auxin sensitive reporter gene, it was
shown that Rhizobium or external addition of LCO leads to a
rapid transient and local inhibition of acropetal auxin transport in
clover roots (Mathesius et al., 1998). This indicates that a changed
hormone balance follows the primary LCO signal at the site of nodule
initiation possibly sensitizing cells for division. Flavonoids could
act as secondary effectors in this process, because the phenylpropanoid
pathway is up-regulated during nodulation, and flavonoid inhibition of auxin transport has been reported.
For alfalfa, externally supplied cytokinin partially mimics the Nod
factor application. Prolonged local exposure by expression of the
cytokinin biosynthesis tzs gene in a
Nod- Rhizobium resulted in the development
of bacteria-free nodule-like structures. In Sesbania and
alfalfa, the LCO responding genes Enod12 and
Enod40 as well as the Enod2 gene are all
up-regulated by cytokinin, and cell divisions are activated in these
roots. Cytokinin and LCO may thus be part of or influence the same
signal transduction during nodulation.
Although there are differences in legume responses, exogenous
application of ethylene generally influences nodulation negatively, and
agents inhibiting ethylene biosynthesis or perception
[L-
-(2-aminoethoxyvinyl)-glycine and
Ag+] increase nodulation. A drastic increase in
persistent infections and numbers of nodules developing in the
Medicago truncatula ethylene-insensitive sickle
mutant shows that ethylene is involved in a local regulation of
infection (Penmetsa and Cook, 1997). However, a comparable ethylene-insensitive mutant of soybean was unaffected in nodulation, illustrating the difficulties of these hormonal investigations without
a firm knowledge of the involved mechanisms (Schmidt et al., 1999).
Alfalfa has indeterminate nodules, whereas soybean has determinate
ones. One could speculate that the ethylene-mediated switch from
indeterminate to determinate nodules observed on Sesbania is
another manifestation of a differential response in the two nodule
types. In pea roots, nodules develop preferentially opposite protoxylem
poles. Localization of ACC oxidase transcripts (and by extrapolation
ethylene) in cells between protoxylem poles in combination with a
gradient of the uridine "stele factor" from the poles may provide
the positional information for this positioning.
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NITROGEN CONTROL AND AUTOREGULATION OF NODULATION |
Successful nodulation occurs under nitrogen-limiting conditions
where a fraction of the invasion events progress into functional nodules. Even under optimal conditions, most infection threads are
arrested in hypodermal root cell layers and the actual nodule numbers
are limited by the plant. An autoregulatory mechanism enables cortical
cell divisions in older nodule primordia to suppress younger cell
division foci systemically (Caetano-Anolles and Gresshoff, 1991).
Hypernodulating mutants developing excess nodules escape autoregulation
and lack the normal nitrate suppression, indicating that nitrate exerts
its mainly local effect via the autoregulatory pathway. One report
suggested that ethylene might be involved in nitrate repression in
alfalfa, but data from pea did not support a role for ethylene. A model
predicting transport of a nodule-derived compound to the shoot and
return of an inhibitor was formulated on the basis of grafting
experiments demonstrating shoot genotype control of the root phenotype
(Caetano-Anolles and Gresshoff, 1991). The identity of these compounds
remains as yet unknown, but both nitrate and autoregulation act
independently of nutritional effects per se. Recent characterization of
the hypernodulation har1 mutant from Lotus
suggests that regulation of nodule numbers is integrated in the
mechanisms controlling lateral root development (Wopereis et al.,
2000).
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NUMEROUS GENES ARE ACTIVATED DURING NODULE DEVELOPMENT |
Formation of a new organ requires temporally and spatially
controlled activity of genes and gene products participating in the
organogenic process. Using biochemical or molecular techniques, many
proteins (called nodulins) were found at elevated levels in root
nodules, and many genes were highly induced in particular cell types or
nodule tissues. The earliest expressed genes such as Enod12,
Rip1, and LjCbp1are active in root hairs, whereas
genes encoding proteins needed in the physiology and biochemistry of the mature active nodule tend to be strongly induced just before onset
of nitrogen fixation. Leghemoglobin, involved in oxygen protection of
nitrogenase, is the classical example of this class of "late"
nodulins. The complexity of gene regulation during nodulation is also
exemplified by the leghemoglobin gene family where some members are
already expressed in root hairs and primordial cells (Cvitanich et al.,
2000). From the studies of induced genes so far only the
Enod40 gene has emerged with a regulatory role in nodule
initiation. The Enod40 was suggested to encode a small 12- to 13-amino acid peptide (Franssen, 1998) and a 3' RNA proposed to control translation efficiency or location. In accordance with a
regulatory role, Enod40 is transcriptionally up-regulated in root pericycle cells within hours after inoculation or LCO application and prior to division of cortical cells.
With the numerous expressed sequence tag (EST) sequences now
available for various legumes, a comprehensive view of the
genes active in root nodules will appear and probably change the
nodulin concept. Already a complete overview of the genes (ESTs) is
only possible with bio-informatics tools, and readers are therefore referred to databases and Web pages for access and mining of this information (www.kazusa.or.jp/en/plant/lotus/EST/,
www.mbio.aau.dk/~chp/, www.bio-SRL8.stanford.edu,
http://212.6.137.235/agowa/, and
www.ncgr.org/research/mgi).
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FUNCTIONAL ANALYSIS OF NODULIN GENES |
The challenge is to assign function to genes of the EST
inventories and to provide a detailed analysis of key genes governing nodule formation and function. Although similarity to known genes will
help, the scope of this task is clear from the present short list
showing nodulins studied using molecular genetics (Table I). Techniques for gene knockout have
unfortunately proven difficult to establish in plants and functional
studies in legumes often rely on sense/antisense studies in the
absence of a null phenotype. For example, some plants overexpressing an
Enod40 cDNA demonstrated accelerated nodulation, whereas
other lines showing cosuppression of the endogenous Enod40
gene, developed modified or fewer nodules (Charon et al., 1999).
Studies with the Enod12 gene were less complicated. In a
progeny segregating null alleles, it was found that absence of
Enod12 did not influence nodule development or function.
Either the gene was dispensable or redundancy compensates for the null
allele. The recent demonstration that double-stranded RNA interference
can selectively reduce gene activity in Arabidopsis (Chuang and
Meyerowitz, 2000) may in the future allow more effective in planta
studies of gene function in legumes.
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Table I.
List of genes and proteins analyzed in planta to
determine a role in root nodule development or function
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LEGUME GENETICS WILL IMPROVE ANALYSIS OF REGULATORY MECHANISMS |
The regulatory mechanisms controlling nodule development has so
far been approached with studies of the early signaling or regulation
of nodulin gene promoters. Conceptually these studies start at either
end of the developmental process, aiming to meet in between. To fill
the gap, a genetic approach offering a more direct identification of
central players and assembly of pathways from epistasis studies is now
gaining momentum. The potential contribution from plant genetics in
gene identification and functional analysis was recently demonstrated
by the isolation of the L. japonicus nodule inception
(Nin) gene from a symbiotic locus tagged with the maize
transposon Ac (Schauser et al., 1999). Symbiotic mutants
have been known in soybean and pea for many years, but Nin
is the first genetically defined symbiotic locus characterized at the
molecular level and an example of the advantages offered by model
legumes. Phenotypically, nin mutants are non-nodulating. Cortical cell divisions, the first step to nodule primordia
formation, appear not to be initiated. Like the wild-type plants,
nin mutants exhibit root hair deformation after
inoculation, indicating that LCO signal perception is functional.
These observations point to a Nin function at the junction
of signal transduction and gene activation. The presence of
putative transcription factor domains in the NIN protein as well as
regional similarity to the minus dominance (Mid) protein controlling
gametogenesis in Chlamydomonas support this idea.
Figure 4 shows the NIN protein structure
and outlines a working model for the role of Nin. Based on
one mutant, it is obviously insufficient, but the strength of the
model lies in the questions raised. Is NIN a DNA-binding central
regulator or a modest transcriptional co-activator, and if so which
genes are activated? What are the immediate upstream partners, and is
Nin itself activated? To approach such questions, a careful
investigation of the post-translational regulation of NIN implicated by
the predicted transmembrane domains and identification of the first
cells expressing active protein will be necessary. Clues may also come
from the temporal-spatial expression patterns. The observed expression
of Nin in uninfected roots makes it possible to test if NIN
has a role either upstream or downstream of the LCO-induced auxin
pulse, using an auxin sensitive reporter (Mathesius et al., 1998).
Considering the overlap in expression and suggested functions, NIN may
interplay with the Enod40 gene product. Absence of
observable cell divisions in nin mutants would place
Nin upstream of Enod40 assuming that otherwise endogenous Enod40 expression would induce cell
proliferation. However, the effect of expressing Enod40 in
roots of nin mutants should be tested. Eventually the
ordering of Nin and symbiotic loci represented in mutant
collections into pathway(s) of upstream and downstream actors will
provide invaluable information.
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Figure 4.
Structure of the NIN protein and a working model
for the role in early phases of root nodule initiation. A, The
LCO-dependent root hair deformation observed on the nin
mutants, together with the apparent lack of cell division in the outer
cortex, place the Nin gene in signal transduction or gene
activation downstream of LCO perception. Domains in NIN have similarity
to transcription factor domains, suggesting a function in activation of
genes required for nodule initiation. To explain the lack of infection
thread formation in nin mutants, a secondary positive signal
is envisaged to be necessary for this process. This signal may at the
same time repress further root hair deformation explaining the
excessive root hair response of the nin mutants (Schauser et
al., 1999). B, The present resolution of events during nodule inception
predicts that hormonal changes, Enod40, and Nin
are involved at about the same developmental stage. Vertical hatching,
Putative transmembrane domains; black, acidic activation domains; gray,
putative DNA-binding/dimerization domain.
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NODULIN PROMOTERS: CONSERVATION OF REGULATORY ELEMENTS |
In an attempt to unravel the regulatory cascade controlling
genes activated during nodule development and at the same time describe
putative targets for signal transduction pathways, promoter regions of
nodulin genes expressed early and late in the developmental process
were characterized. Promoters from the soybean N23 and leghemoglobin
lbc3 genes (Stougaard et al., 1990) together with the
Sesbania Srglb3 (Szczyglowski et al., 1994) were most
extensively analyzed. Several cis-acting regulatory sequences were
delineated by deletion, hybrid promoter, and point mutational studies
of promoters fused to reporter genes. The type and localization of cis-acting elements in the leghemoglobin promoters were remarkably similar as exemplified by the lbc3 promoter in Figure
5. The lbc3 promoter has a
strong positive element (enhancer), a weak-positive element (WPE), and
an organ-specific element containing the highly conserved
AAAGAT-taTTGT-CTCTT box within a 2-kb promoter region (Ramlov et al.,
1993; Szczyglowski et al., 1994).
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Figure 5.
Schematic representation of the cis-acting
promoter elements and DNA-binding proteins of the soybean
lbc3 and the pea Enod12B promoters. The
lbc3 strong positive element (SPE) contains three half-sites
of an inverted repeat (invXY) suggested to interact with
trans-factors, whereas the WPE has two binding sites for the general
trans-activator NAT2. Highly conserved regions important for promoter
function are located in the organ-specific elements (OSE) and negative
elements (NE). The three proteins, Cys-rich polycomb-like protein
(CPP1), nodule homeo-domain-like (NDX), and leghemoglobin-binding
factor (LBF) bind at as yet undefined sites of the 280 minimal
promoter. In the Enod12B promoter, a short positive element
binding the ENBP1 protein is sufficient for LCO tissue specific
expression.
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Trans-acting proteins binding at or in the vicinity of these DNA
elements were identified either by direct-binding studies using gel
retardation or in South-western hybridization. Two high-mobility group
I-like proteins (NAT1, LAT1) were found in nodules and leaves. A
similar nodule NAT2 protein binds at AT-rich sequences bordering the
soybean lbc3 WPE and activates a minimal lbc3
promoter in nodules as well as the tobacco rbcs-8B promoter
in leaves. Hence, NAT2 is a general transactivator. Another soybean
protein, CPP1, binding to a minimal 280 lbc3 promoter
contains two Cys-rich domains present in polycomb proteins like
Drosophila Ez (Cvitanich et al., 2000). Polycomb proteins
usually restrict expression of developmental regulators, and there is
some support for negative regulation of lbc3 expression by
CPP1 (Cvitanich et al., 2000). Additional lbc3 trans-factors
currently analyzed at the functional levels are homeobox-like proteins
and pathogenesis-related activator proteins (Fig. 5).
The pea early nodulin promoter from Enod12B was analyzed by
a similar approach. A short 139 promoter region was found to suffice
for both nodule expression, activation in LCO-induced primordia, and
interaction with an early nodulin-binding protein (ENBP1) protein. When
mutated binding site motifs were analyzed for ENBP1 binding in vitro
and in vivo a correlation between binding and promoter activity was
seen (Hansen et al., 1999), but ENBP1 was not limiting for
Enod12 expression in transgenic plants. The ENBP1 protein
contains typical AT hooks shown to be required for promoter binding and
a zinc finger domain. Both the MsEnod40-1 and MsEnod12A gene
promoters respond to LCO and cytokinins, but well-defined regulatory
DNA elements operating in phytohormone controlled gene expression were
not reported (Bauer et al., 1996; Fang and Hirsch, 1998). Additional
promoter analysis may eventually draw a direct link between
phytohormone physiology and gene expression during nodule organogenesis.
| |
PERSPECTIVES AND CONCLUSIONS |
The description of the LCO Nod factor molecules and their
morphogenic effects on legume roots raised the possibility of a more
general role as a hitherto undiscovered class of endogenous plant
growth regulators. Now, 10 years later, it is still an open question
whether nodule induction is a specialized effect or a unique event.
LCOs similar to the compounds synthesized by Rhizobium remain undescribed in plants and the evidence of their existence circumstantial. As a recent example, a study of pea Enod12
promoter activity in rice concluded that the LCO perception mechanism
is functional in a monocot plant. (Reddy et al., 1998; for
additional observations, see Spaink, 1996). Arabidopsis, which has
contributed new firm evidence for the role of brassinosteroids in
plant development, has remained unusually silent in relation to LCOs.
Genes with similarity to bacterial nod genes so far were not
reported from the Arabidopsis genome sequencing program even though a
gene with similarity to nodC was found in Xenopus
(Spaink, 1996; Schultze and Kondorosi, 1998). Arabidopsis may still
contribute but we may also have to await the characterization of the
legume LCO receptor(s) and signal transduction pathway(s) before the
question can be fruitfully approached in non-legumes. The promiscuous
mycorrhiza-plant interaction is another opening for approaching
symbiotic functions and genes with a more general role. Several
non-nodulating plant mutants are also impaired in the mycorrhizal
colonization. This, together with expression of Enod12,
Enod40, and Enod2 in roots colonized by
mycorrhiza, points at overlaps in the programs governing endosymbiosis.
In this broader context comparative genomic analysis of symbiotic genes
is bound to add significantly to the understanding of plant
development. Taking Nin as an example, several
uncharacterized Arabidopsis genes similar to the Nin gene
can be identified from the genome sequence and the functional analysis
of these genes in Arabidopsis is now an obvious task. This analysis can
subsequently be taken back to help in determining the function of
Nin and its paralogs in legume development and nodulation.
With the emerging new tools for functional genomics in model legumes,
analysis of the plant contribution to symbiosis will gain speed.
Transposon and T-DNA tagging together with map-based cloning of
symbiotic loci are approaches bound to identify novel genes in the root
nodule regulatory system. The combination of sequence information
generated by EST sequencing and expression analysis using microarrays
or DNA chips will provide information of global gene activities under
various conditions, improve mutant characterization, and allow pathway
members to be identified from co-expression data. Proteomics will
complement this analysis and add to the biochemistry. In the coming
years, mechanisms will be added to many of the detailed and careful
descriptive studies of the past. Functional and comparative genomics
will be major contributors in elucidation of signaling, gene
regulation, and gene function in symbiosis. With a more comprehensive
understanding of legume symbiosis, the puzzling question why only
Parasponia andersonii (Ulmacea) among the
non-legumes, forms nodules with Rhizobium, and the equally
intriguing question of the genetic differences between legumes and
other plants could be approached.
| |
ACKNOWLEDGMENTS |
I am grateful to the colleagues who generously provided
reprints or preprints for this paper. I apologize to the many
colleagues whose publications I was unable to include or cite. I thank
Dr. Leif Schauser for the pictures used in Figures 1 and 2 and
collaborators at the Laboratory of Gene Expression for comments on the manuscript.
| |
FOOTNOTES |
Received May 3, 2000; accepted June 22, 2000.
*
E-mail stougaard{at}mbio.aau.dk; fax 45-8620-1222.
| |
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© 2000 American Society of Plant Physiologists
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INTRODUCTION
HISTOLOGY AND NODULE...
