Download Regulators and Regulation of Legume Root

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
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 de* E-mail [email protected]; fax 45– 8620 –1222.
velopmental 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.
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 nitrogenfixing 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 plantderived 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
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Stougaard
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.
MUTUAL SIGNAL EXCHANGE STARTS
THE PROCESS
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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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
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Plant Physiol. Vol. 124, 2000
Regulators and Regulation of Legume Root Nodule Development
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).
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 MNNN1008 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.
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 LCOdependent fashion in root epidermal cells (Webb et
al., 2000).
THE ELUSIVE NOD FACTOR RECEPTOR
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.
Plant Physiol. Vol. 124, 2000
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, sug-
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Stougaard
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.
DEVELOPMENTAL CROSS TALKING: CHECKS
AND BALANCES
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.
gesting 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 noncatalytic 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; Dı́az 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, nonspecific 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
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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
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Plant Physiol. Vol. 124, 2000
Regulators and Regulation of Legume Root Nodule Development
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.
SECONDARY SIGNALING THROUGH
PHYTOHORMONES
Most of the plant architecture is formed by postembryonic 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 upregulated 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-␣-(2aminoethoxyvinyl)-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.,
Plant Physiol. Vol. 124, 2000
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.
NITROGEN CONTROL AND AUTOREGULATION
OF NODULATION
Successful nodulation occurs under nitrogenlimiting 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).
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
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Stougaard
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).
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.
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,
Table I. List of genes and proteins analyzed in planta to determine a role in root nodule development or function
Gene or Protein
Function
Pea lectin, psl
Mitogen enhancer?
Soybean lectin, Le 1
Cell attachment
Alfalfa Enod12
Vicia ENBP1
Alfalfa Enod40
LjNin
Mszpt2-1
Cell wall synthesis?
trans-Factor
Cell activation
Regulator of nodule initiation
Cell differentiation
Soybean NAT2
trans-Factor
Soybean CPP1
Alfalfa ccs52
trans-Factor
Mitotic inhibitor
Pea rug4
GmRab1p, Rab7p
Suc synthase
GTP-binding proteins
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In Planta Role, Effect, or Phenotype
Increases response to unspecific LCOs. Extends host range of clover
to include R. leguminosarum by viciae, M. loti, S. meliloti.
Extends host range of L. corniculatus to allow B. japonicum to form
empty nodule structures.
Dispensable in alfalfa plants homozygous for null allele.
Binding sites required for Enod12 promoter activity.
Cell division in axenic roots and accelerated nodulation.
nin mutants have root hair response but are non-nodulating.
Antisense suppression arrests differentiation of nodule central zone
cells and inhibits bacterial release.
Binds a positive regulatory element and activates promoters in leaf
and nodule.
Binds the lbc3 minimal promoter.
Antisense suppression decreases the number of endoreplication
cycles during differentiation.
Ineffective nitrogen fixation.
Involved in biogenesis of peribacteroid membrane.
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Regulators and Regulation of Legume Root Nodule Development
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.
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).
Trans-acting proteins binding at or in the vicinity
of these DNA elements were identified either by directbinding studies using gel retardation or in Southwestern 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
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.
Plant Physiol. Vol. 124, 2000
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Copyright © 2000 American Society of Plant Biologists. All rights reserved.
537
Stougaard
PERSPECTIVES AND CONCLUSIONS
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.
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.
538
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
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Copyright © 2000 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 124, 2000
Regulators and Regulation of Legume Root Nodule Development
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.
Received May 3, 2000; accepted June 22, 2000.
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