Udvardi 2013 Revisao Rizobio Leguminosa

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Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401;
email: mudvardi@noble.org
2
John Innes Center, Norwich NR4 7UH, United Kingdom
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Annu. Rev. Plant Biol. 2013. 64:781–805 Keywords

First published online as a Review in Advance on nodule, nitrogen fixation, symbiosome, bacteroid, genetics, genomics
March 1, 2013
The Annual Review of Plant Biology is online at Abstract
plant.annualreviews.org
Symbiotic nitrogen fixation by rhizobia in legume root nodules injects
This article’s doi:
10.1146/annurev-arplant-050312-120235
approximately 40 million tonnes of nitrogen into agricultural systems
each year. In exchange for reduced nitrogen from the bacteria, the plant
Copyright c 2013 by Annual Reviews.
All rights reserved
provides rhizobia with reduced carbon and all the essential nutrients
required for bacterial metabolism. Symbiotic nitrogen fixation requires
exquisite integration of plant and bacterial metabolism. Central to this
integration are transporters of both the plant and the rhizobia, which
transfer elements and compounds across various plant membranes and
the two bacterial membranes. Here we review current knowledge of
legume and rhizobial transport and metabolism as they relate to sym-
biotic nitrogen fixation. Although all legume-rhizobia symbioses have
many metabolic features in common, there are also interesting differ-
ences between them, which show that evolution has solved metabolic
problems in different ways to achieve effective symbiosis in different
systems.
781
and potentially extending them to nonlegumes,

Contents such as cereals (8). Physiological and biochem-
ical approaches dominated early SNF research
INTRODUCTION . . . . . . . . . . . . . . . . . . 782
and led to fundamental concepts that have stood
NODULE DEVELOPMENT
the test of time. The molecular biology revo-
AND TYPES . . . . . . . . . . . . . . . . . . . . . . 783
lution of the 1980s and 1990s, including ad-
NODULE PHYSIOLOGY AND
vances in bacterial genetics, added greater detail
BIOCHEMISTRY . . . . . . . . . . . . . . . . 784
to models of nodule metabolism, including the
CLASSICAL GENETICS OF
identification of specific genes (mainly bacte-
NODULE METABOLISM
rial) that are crucial for SNF. The genomic rev-
AND TRANSPORT . . . . . . . . . . . . . . 787
olution that began around the turn of the cen-
FUNCTIONAL GENOMICS OF
tury has produced complete genome sequences
NODULE DEVELOPMENT
for many rhizobial species and near-complete
AND DIFFERENTIATION . . . . . . 789
genomes for at least five legume species. Com-

BACTEROID DEVELOPMENT . . . . 790
plementary technologies to quantify gene tran-
Oxygen and Nitrogen Fixation . . . . . 790
scripts, proteins, and metabolites on a global
Symbiotic Auxotrophy . . . . . . . . . . . . . 791
scale have helped flesh out important aspects of

Homocitrate Synthesis . . . . . . . . . . . . . 792
legume nodule transport and metabolism. Fi-
Global Changes in Bacteroid
nally, the development of facile tools for classi-
Metabolism . . . . . . . . . . . . . . . . . . . . . 792
cal and reverse genetics of model legumes in the
BACTERIAL TRANSPORT . . . . . . . . . 792
past decade has begun to bear fruit by identi-
Metal and Ion Transport . . . . . . . . . . . 792
fying plant genes encoding enzymes and trans-
Ammonium and Amino Acid
porters that are crucial for SNF. Importantly,
Transport and Assimilation . . . . . . 793
analysis and understanding of plant and bacte-
Dicarboxylate Transport . . . . . . . . . . . 794
rial mutant symbiotic phenotypes require the
BACTERIAL METABOLISM . . . . . . . . 794
integration of molecular, biochemical, cellular,
Bacterial Carbon Metabolism . . . . . . . 794
and physiological methods, which is good and
Gluconeogenesis . . . . . . . . . . . . . . . . . . . 795
necessary if we hope to develop holistic, quanti-
Redox Balance and Storage
tative, and predictive models of the SNF system
Polymers . . . . . . . . . . . . . . . . . . . . . . . 795
in legumes.
FUTURE DIRECTIONS . . . . . . . . . . . . 796
This review focuses on recent advances in
our understanding of legume nodule trans-
port and metabolism that have been facilitated
INTRODUCTION largely by the genomics revolution and the de-
Symbiotic nitrogen fixation (SNF) by rhizobia velopment or deployment of genetic resources
in legume root nodules injects approximately and tools. We also provide context for this re-
40 million tonnes of nitrogen into agricultural cent work by describing nodule development
systems each year (56). In contrast to industrial and types, nodule physiology, and older, sem-
nitrogen fixation for fertilizer production, SNF inal work on plant and bacterial metabolism.
by legumes is essentially free and environmen- At the most basic level, symbioses between
tally benign. SNF provides residual fixed nitro- legumes and rhizobia involve the exchange of
gen to subsequent nonlegume crops, which is reduced carbon from the plant for reduced ni-
why legumes have been used in crop rotations trogen from the bacteria. SNF is powered by
for thousands of years. the sun via photosynthesis in the plant. Approx-
SNF in legumes has been the focus of in- imately 6 g of photosynthetic carbon is used per
tense research for decades as we have strived to gram of nitrogen fixed (154). Rhizobia rely on
SNF: symbiotic
nitrogen fixation understand its molecular and genetic basis with the plant not only for carbon but also for all
a view toward improving existing symbioses other elements required for metabolism. The
782 Udvardi · Poole

trade of reduced nitrogen for everything else development (14, 97). Nod-factor signaling
is not unfair, however, as nitrogen is a limiting also leads to dedifferentiation and division of SM: symbiosome
nutrient in many soils, and legumes have a com- root cortical cells, which in some species give membrane
petitive advantage over other plant families in rise to spherical, determinate nodules and in IRLC: inverted
such soils by virtue of the fixed nitrogen they others give rise to elongated or branched, in- repeat–lacking clade
receive from rhizobia. determinate nodules that contain one or more
Legumes receive the bulk of nitrogen fixed persistent meristems (Figure 1). Although pu-
by rhizobia in the form of ammonia, which rified Nod factors alone can trigger nodulation,
is incorporated into organic form before be- they also represent a key that unlocks a plant
ing exported from nodules. Ammonia assimila- door to rhizobial entry into root epidermal and
tion within nodules also requires carbon com- underlying cortical cell layers (79, 97). Colo-
pounds in the form of organic acids. Much of nization of root cells by rhizobia is facilitated by
the research on nodule metabolism has focused infection threads—tubular structures formed
on primary carbon and nitrogen metabolism— via invagination of the cell wall and plasma
especially sugar, organic acid, and amino acid membrane near the tip of epidermal root hair
metabolism—because of their roles in fueling cells following the binding of rhizobia there
SNF and nitrogen assimilation and export to (Figure 2). Infection threads grow toward the
the rest of the plant. Consequently, this review base of root hair cells and eventually into under-
focuses mainly on primary carbon and nitrogen lying cells, and bacteria grow and divide within
metabolism in nodules and the transporters that the thread. Infection threads ramify within the
facilitate SNF. root cortical tissue and ultimately deposit their
SNF exhibits a few universal features in all bacterial cargo within multiple cortical cells via
legumes studied to date, including the estab- endocytosis. This places the rhizobia within the
lishment of a microaerobic environment within cytoplasm of cortical cells, albeit surrounded
nodules as a prerequisite for bacterial nitro- by a plant membrane called the symbiosome
gen fixation; transport of reduced carbon from membrane (SM). The SM together with the
the plant to rhizobia, principally as dicarboxylic enclosed bacteria is called a symbiosome.
acids; and transport of fixed nitrogen from the Rhizobia continue to grow and divide along
rhizobia to the plant, principally as ammonia. with the SM until infected plant cells are
However, there are interesting differences be- packed with thousands of symbiosomes.
tween symbioses that show that metabolic and Rhizobia within symbiosomes eventually
other problems related to symbiotic harmony differentiate into a nitrogen-fixing form known
were solved in different ways during the evo- as the bacteroid, which involves the induction
lution of SNF. Some of these differences are of genes for nitrogen fixation (nif genes) and
highlighted below. associated processes ( fix genes) (see below). In
many legumes, bacteroids are of similar size
and shape to their free-living counterparts in
NODULE DEVELOPMENT the soil. However, in a subset of closely related
AND TYPES legume species, known as the inverted repeat–
Legume nodules are the culmination of a lacking clade (IRLC), rhizobia undergo multi-
developmental program that is activated in ple rounds of endoreduplication (chromosome
roots by signals received from rhizobia at the replication without cell division), which leaves
root surface. Key among these signals are them with multiple copies of their chromo-
lipochitooligosaccharide Nod (nodulation) somes and enlarged relative to their free-living
factors (33) that bind to specific plant receptor progenitors. Details of bacteroid development
kinases on the plasma membrane and trigger and its regulation are presented below.
a Ca2+ -signaling pathway, which activates Mature, nitrogen-fixing nodules consist
the transcription of genes involved in nodule of central infected tissue made up of infected
www.annualreviews.org • Legume-Rhizobia Transport and Metabolism 783

nitrogen fixation ceases (156). In indeterminate

a b M nodules, this spatial organization facilitates
physical dissection and functional analysis of
INV
VBs discrete symbiosis stages. Analogous studies
IZ can be performed on determinate nodules by
FZ analyzing nodules at different time points (days)
following inoculation of roots with rhizobia.
NP Rhizobial colonization of developing
nodules takes just a few days. Before SNF,
however, a profound change in nodule phys-
VBs iology occurs, associated with the appearance
FZ of nodule-specific proteins called legume
c hemoglobins (leghemoglobins). This change
and some of its consequences for bacterial and

plant metabolism are described below.
UCs
ICs NODULE PHYSIOLOGY AND

BIOCHEMISTRY
SZ
The nitrogenase enzyme complex that reduces
50 μm 100 μm N2 to NH3 is oxygen labile:
Figure 1 N2 + 8H+ + 8e− + 16ATP → 2NH3 + H2

Legume nodule ultrastructure. (a) Longitudinal cross section of a determinate + 16ADP + 16Pi .
soybean nodule. (b) Longitudinal cross section of an indeterminate Medicago
nodule. (c) A higher magnification of the highlighted area in panel b. The Bacteria avoid oxygen inactivation of nitroge-
determinate soybean nodule consists of a central nitrogen fixation zone (FZ) nase in a variety of ways, ranging from the
surrounded by nodule parenchyma (NP) and vascular bundles (VBs). In the “choice” of anaerobic or microaerobic habitats
fixation zone, infected cells are dark and uninfected cells are light. The to spatial separation of nitrogenase in hetero-
indeterminate Medicago nodule maintains an active meristem and continues to
cyst cells from oxygenic, photosynthetic cells
grow throughout its development, displaying a succession of developmental
zones: the meristem (M), invasion zone (INV), interzone (IZ), nitrogen fixation of cyanobacteria (122). As rhizobia are obli-
zone (FZ), and senescence zone (SZ). Infected and uninfected cells (ICs and gate aerobes that require oxygen for respiration
UCs, respectively) are highlighted in panel c. Dark starch granules occur and energy metabolism, they face an obvious
predominantly in uninfected cells. Images in panels b and c courtesy of Dr. conundrum under nitrogen-fixing conditions.
Catalina Pislariu.
Resolution of this conundrum involves the con-
certed efforts of both plant and bacteria during
plant cells interspersed with uninfected cells, SNF in nodules. Three processes intersect to
surrounded by uninfected tissues (including produce a microaerobic environment around
phloem and xylem tissues) that connect with nitrogen-fixing rhizobia in nodules: A barrier
the root vascular system and transport ele- to gaseous diffusion in the nodules’ outer cell
ments and compounds into and out of the layers limits the rate of oxygen influx to the
nodules (Figure 1). Indeterminate nodules are central infected tissue; bacteroids and plant mi-
organized into zones: the meristematic zone tochondria, with their high respiration rates,
at the growing tip; the invasion zone, which consume oxygen as fast as it can enter the nod-
contains infection threads that deliver rhizobia ules; and plant hemoglobins bind oxygen in
to plant cells; a transition or interzone, in which the cytoplasm with high affinity and deliver
rhizobia undergo differentiation; the nitrogen it rapidly to mitochondria and bacteroids of
fixation zone; and a senescence zone proximal infected cells. Steady-state concentrations of
to the root, in which rhizobia are degraded and free oxygen in the infected zones of legume

nodules are typically in the tens of nanomolar

a b
(69), approximately four orders of magnitude
lower than equilibrium levels in water.
Leghemoglobins are the most abundant
plant proteins in nodules. Their crucial role
in SNF was demonstrated relatively recently
via reverse genetics (101). Suppression of
leghemoglobin-encoding gene expression in OC
Lotus japonicus via RNA interference (RNAi) re-
sulted in the loss of leghemoglobin in nodules,
higher steady-state levels of free oxygen but
lower ATP/ADP ratios, and a complete absence NP
of nitrogenase activity. The increase in free-
oxygen concentration in the “mutant” nodules

profoundly affected transcription of both plant
and bacterial genes, including the bacterial nif
genes (100). In fact, low oxygen and possibly as-

sociated changes in cellular redox are key signals 100 μm 100 μm
that induce nif and fix gene expression during
Figure 2
bacteroid development, as described below.
Root infection structures containing rhizobia. (a) Curled epidermal root hair
Sucrose derived from shoot photosynthesis
with infection thread containing LacZ-expressing rhizobia. Histochemical
is the primary source of reduced carbon for X-gal staining highlights rhizobia in blue. (b) Infection threads traversing the
nodule metabolism (67). It is cleaved by su- outer cortical cell layers (OC) and ramifying through dividing cortical cells of a
crose synthases and metabolized further via the developing Medicago nodule. Abbreviation: NP, nodule primordium. Images
glycolytic pathway, mainly in the cytoplasm of courtesy of Dr. Catalina Pislariu.
plant cells in the infected zone (135). The gly-
colytic pathway is upregulated at the transcrip- Overwhelming evidence points to dicar-
tional level during nodule development (22). A boxylic acids, especially malate, as the primary
Pisum sativum (pea) mutant with severely resource of carbon provided by the plant for
duced nodule sucrose synthase activity is defec- bacteroid nitrogen fixation. The strongest
tive in SNF (48). Differences between infected evidence comes from bacterial genetics, which
and uninfected cells in the activities of enzymes has shown that genes for the transport and
involved in glycolysis highlight the compart- metabolism of dicarboxylic acids are indispens-
mentalization of sugar metabolism within nod- able for SNF, whereas those for sugar transport
ules (29). Differences between these cell types and metabolism, for example, are not (see be-
in sugar uptake (106) also indicate specialization low). In addition, the SMs of all studied legumes
of carbon metabolism in different cell types. contain a dicarboxylate transporter of sufficient
In nodules, phosphoenolpyruvate carboxylase activity to account for known SNF rates (148,
(PEPC) and malate dehydrogenase activities di- 150). Genes encoding the SM dicarboxylate
vert carbon flux away from glycolysis to form transporter have not been identified in legumes.
malate, which is considered to be the primary However, a dicarboxylate transporter of the
source of carbon transported to bacteroids (see nonlegume Alnus glutinosa’s SM (AgDCAT1)
below). Numerous studies have shown tran- has been cloned and characterized, and be-
scriptional upregulation of genes for PEPC longs to the peptide transporter (PTR) family
and malate dehydrogenase as well as those for (64). Interestingly, PTR-encoding genes
carbonic anhydrase, which converts CO2 into are induced strongly during legume nodule
HCO3 − for use by PEPC during nodule devel- development (10, 22), so they are reason-
opment (22, 153, 154). able suspects in the hunt for legume SM

dicarboxylate transporters. The SM is weakly via protein channels that are not specific to
permeable to sugars and amino acids, which ammonia. In contrast, two potential modes of
GS: glutamine
synthetase means that these compounds are unlikely to be ammonia transport across the SM have been
major sources of carbon for SNF (148, 151). identified: an NH3 channel (94) and a cation
GOGAT: glutamate
synthase However, there is solid evidence that amino channel that transports K+ , Na+ , and NH4 +
acids are provided to bacteroids by the plant, (146). Aquaporin-like channels are among the
presumably as a source of nitrogen before most abundant proteins in the SM and have
SNF and possibly for other reasons afterward been shown to transport ammonia and other
(see below). Nodule transport and metabolism solutes (31, 63). Therefore, they may be an im-
prior to the onset of SNF remain poorly portant conduit for ammonia export from sym-
understood and warrant greater attention. biosomes. Strengthening this idea is the recent
Bacteroid differentiation during nodule de- finding that one such aquaporin, nodulin 26
velopment programs bacteroids to fix nitrogen from soybean, interacts physically with GS, the
but avoid assimilating ammonia into an organic cytosolic enzyme that assimilates ammonia into
form (see below). Ammonia assimilation is left glutamine (82). The molecular identity of the
to the plant, and a suite of plant genes are SM NH4 + channel remains to be determined.
induced during nodule development, includ- Ammonium transporters of the AMT family
ing those encoding glutamine synthetase (GS), are expressed in legume nodules (131), as are
glutamate synthase (GOGAT), and aspartate K+ transporters of the KUP family (34), which
amino transferase, which incorporate ammo- may also transport NH4 + . However, indi-
nia into amino acids for export from nodules rect evidence indicates that both LjAMT2 and
(153). Nodules of some legumes—especially LjKUP are plasma membrane located, pointing
those of tropical origin, such as Glycine max to possible roles in ammonium recovery from
(soybean) and Vigna unguiculata (cowpea)— the apoplast and potassium homeostasis in
export fixed nitrogen primarily as ureides (103). plant cells, respectively, rather than roles in am-
Genes and enzymes involved in ureide biosyn- monium transport across the SM. Nonetheless,
thesis are known to be induced during the de- the locations of these and other transporters in
velopment of such nodules (133). Interestingly, nodules should be reassessed using more direct
ureide biosynthesis in legume nodules exhibits methods, such as immunolocalization with spe-
compartmentalization within and between in- cific antibodies or transporter–green fluores-
fected and uninfected cells (133). Variations in cent protein (GFP) fusions expressed in nodules
the type and relative amount of organic nitro- under the control of the native promoter of each
gen compounds exported from nodules via the transporter-encoding gene. It is not inconceiv-
xylem in different species reflect subtle or ma- able that the targeting of proteins to different
jor differences in nodule nitrogen metabolism. membrane systems is altered during nodule
This illustrates that a variety of solutions to development and symbiosome proliferation,
the problem of transferring fixed nitrogen to as is the case during arbuscule development in
the whole plant were found during evolution arbuscular mycorrhizal symbiosis (116).
of SNF. Recognition that such differences exist The plant SM controls fluxes not only of
may be important for future efforts to improve carbon and nitrogen to and from the bac-
SNF. teroids, but also of all elements required for
How is ammonia transported across the SM bacterial metabolism, including phosphorus,
and bacteroid membranes? Rhizobia possess sulfur, potassium, sodium, calcium, vanadium,
genes encoding ammonia transporters of the iron, molybdenum, nickel, and cobalt (124).
AMT family, but their expression is downreg- Iron, sulfur, and molybdenum, for instance,
ulated in bacteroids. Instead, it appears that are required in nitrogenase cofactors and are
ammonia is lost from bacteroids via simple therefore essential for SNF in bacteroids. The
diffusion, either across the lipid bilayer or SM is energized by a P-type ATPase (and

possibly other types of energy-dependent pro- surround all nodule organelles and cells, and
ton pumps) that generates both electrical po- transporters in these membranes integrate
tential (ψ) and chemical potential differences metabolism within and between cells, facilitat-
(pH) across the SM, which in turn drive the ing nutrient flow to and from nodules. Few
transport of various solutes across the mem- transport systems of other plant membranes
brane (39, 147, 148). An anion carrier of the have been characterized biochemically in nod-
soybean SM has been characterized that trans- ules. However, one exception is sugar (glucose
ports NO3 − , NO2 − , Cl− , and malate− into sym- and sucrose) transport across the plasma mem-
biosomes at the expense of ψ (149). More brane of nodule cells. Whereas isolated unin-
recently, the nodulins GmN70 and LjN70, fected cells of broad-bean nodules were able
which belong to the major facilitator super- to import both glucose and sucrose, infected
family (MFS), were shown to be inorganic an- cells were unable to do so (106). Thus, infected
ion transporters with substrate specificities sim- cells may be dependent on uninfected cells for
ilar to those of the soybean SM anion carrier(s) carbon supply in the form of sugars (via sym-
(158). Localization of GmN70 to the SM (158) plastic transport) or sugar-metabolism prod-
suggests that it accounts for at least part of the ucts. Similar results were obtained for amino
anion carrier activity measured on this mem- acid transport, with isolated infected cells in-
brane earlier (149). competent to import (and presumably export)
Iron transport across the SM and bacteroid amino acids from (to) the apoplast (107). The
membranes of soybean nodules has been char- specific transporters responsible for such sugar
acterized biochemically, revealing systems for and amino acid transport have not been identi-
Fe2+ and Fe3+ transport on both membranes fied, although many potential genes have been
(71, 86, 87). A nodule-induced Fe2+ transporter identified in recent transcriptome studies (see
of the SM, GmDMT1, has been cloned (65). below). Nitrogen is exported from nodules of
GmDMT1 also transports Zn2+ and probably the tropical legume Phaseolus vulgaris (common
Mn2+ and Cu2+ as well, indicating that it plays bean) mainly as ureides, and a ureide (allan-
a more general role in supplying metal ions to toin) transporter, PvUPS1, has been isolated
bacteroids. Very recently, a nodule-specific pu- and characterized from this species (108). Ex-
tative Fe3+ transporter of the SM, GmYSL7A pression of the PvUPS1 gene in nodule phloem
of the oligopeptide transporter (OPT) family, tissue indicated that this transporter is proba-
was characterized in soybean (P. Smith, per- bly involved in phloem loading and ureide ex-
sonal communication). It will be interesting to port to other organs. Subsequent cellular and
use the new tools of reverse genetics (see below) genetic work on two related UPS transporters
to test the relative contributions that DMT1 in soybean showed that they are located in the
and YSL1 transporters make to bacteroid iron plasma membranes of cortical and vascular en-
nutrition. A second zinc transporter of soy- dodermal cells and indeed play a critical role in
bean, GmZIP1, is also located on the SM and ureide export from nodules (23).
is expressed in a nodule-specific manner (88).
K+ channel activities have been characterized
in the SMs of soybean, L. japonicus, and Vicia
faba (broad bean) (3, 121, 146), but the corre- CLASSICAL GENETICS OF
sponding genes remain to be identified. Cal- NODULE METABOLISM
cium channels and transporters have also been AND TRANSPORT
characterized in the SMs of L. japonicus and Given that legume-rhizobia symbioses are built
Lupinus luteus (yellow lupine) (2, 121), but again on metabolic cooperation, it should come as
the corresponding genes remain unknown. no surprise that mutations in key metabolic or
Although the SM plays a central role in transport genes disrupt or completely abolish
nodule transport and metabolism, membranes SNF. This was first demonstrated for bacterial

genes, including the nif and fix genes (see zymes. One of these is LjSST1 of the SulP trans-
EMS: ethyl
methanesulfonate below). porter family, which encodes an SM sulfate
Until recently (see Functional Genomics of transporter that supplies bacteroids with sulfate
Nodule Development and Differentiation, be- and possibly molybdate, although the latter re-
low), classical genetics work in legumes relied mains to be proven (68). Map-based cloning of
heavily on chemical or physical mutagens, such LjFEN1 revealed that it encodes a homocitrate
as ethyl methanesulfonate (EMS) and fast- synthase (54). Homocitrate, an essential com-
neutron bombardment, to generate point muta- ponent of the iron-molybdenum cofactor of
tions or deletions in legume genomes (109, 134, nitrogenase, cannot be produced by many rhi-
136). Mutant populations created by EMS or zobia (see below). The inability of the L. japon-
fast-neutron bombardment have been instru- icus fen1 mutant to fix nitrogen demonstrates
mental in identifying genes involved in signal that the plant provides bacteroids with homo-
reception and transduction leading to nodule citrate and implies the existence of homocitrate
development (97), although map-based cloning transporters on both the SM and bacteroid
of such genes typically takes years. Map-based inner membrane, although these have not been
cloning of EMS mutants defective in SNF but identified (Figure 3). Cloning of the LjSEN1
not nodule development per se has identified gene required for SNF showed that it encodes a
several genes encoding transporters or en- protein of the vacuolar iron transporter (VIT)
CO2
Sucr
Oxaloacetate PEP Hexoses
ose
Malate +
ATP H
Dct? ADP+Pi Acetyl-CoA
DctA Fen1
Aap α-Ketoglutarate
H+ Homocitrate
Bra ?
A H+
Ald Ala H+
Pyr
?
Acetyl-CoA PHB
Amino acid
OAA e– and Nitrogenase pool Asn
ATP complex Glu
TCA GS/ H+ AS
cycle GOGAT GS
Ile Aap N2 NH3 NH4+ Gln
Leu
Val d
Bra plicate
Endore osome
ne
? chrom
ra
Sst1 b
Mol m em
SO42– ? me
MgtE ioso
? Symb
?
MoO42– Dmt1 Mg2+
Fe2+
Phloem
Xylem
Symbiotic plant cell
Figure 3
Transport and metabolism in an infected nodule cell. Sucrose from the shoot is converted to malate in the plant and imported across
the symbiosome membrane and into bacteroids, where it fuels nitrogen fixation. The product of the nitrogen fixation is then exported
back to the plant, where it is assimilated into Asn for export to the shoot (blue arrows). In many legumes, such as soybean, the export
products are ureides instead of Asn. The plant must provide metals and ions to the bacteroid, although only some of the transport
systems on the symbiosome and bacteroid membranes are defined. Many rhizobia lack the ability to make homocitrate or become
symbiotic auxotrophs for supply of branched-chain amino acids and become dependent on the plant. Adapted from Reference 97.

family related to an iron transporter of genes that are expressed and regulated dur-
Arabidopsis and to an iron and manganese ing nodule development (11, 58, 73, 89, 128,
transporter of yeast (55). Although the sub- 138). Many of these genes are involved in trans-
strate(s) and intracellular location of LjSEN1 port and metabolism, and the broad overview of
have not been determined, localized expres- metabolic pathways provided by genome-wide
sion in infected cells points to a role of the transcriptome data has revealed that several
transporter in metal ion supply to bacteroids. of these pathways are transcriptionally upreg-
Analysis of L. japonicus photorespiratory mu- ulated during nodule development, including
tants defective in plastid glutamine synthetase pathways for glycolysis, carbon dioxide fixation,
(GS2) showed that GS2 is required for effective amino acid biosynthesis, and purine, heme, and
SNF (45). Likewise, analysis of the pea rug4 redox metabolism (22). Global changes in gene
mutant defective in a nodule-enhanced form expression also provide insight into the physio-
of sucrose synthase showed that it is essential logical conditions that may prevail within nod-
for SNF in this species (48). In contrast, loss ules. For instance, in addition to genes required
of nodule-enhanced LjSUS3 sucrose synthase for metabolism under hypoxia, other genes as-
activity in the sus3 mutant of L. japonicus had sociated with plant cell responses to phospho-
no effect on SNF, indicating that other sucrose rus limitation and osmotic stress are induced
synthases contribute to sucrose cleavage in this during the development of L. japonicus nod-
species (61). Differences in the constituents ules (22). It has also been shown that bacteroids
of nodule metabolism in different species may are a major phosphorus sink in infected plant
prove to be the norm rather than the excep- cells and that the SM contains galactolipids,
tion. Diversity in the evolutionary trajectories which plants produce instead of phospholipids
of nodule metabolism in different species may under phosphorus-limited conditions to con-
help to identify bottlenecks that limit SNF in serve phosphorus for other essential processes
particular species. (46).
Proteomic and metabolomic studies using
mass spectrometry to identify and quantify pro-
FUNCTIONAL GENOMICS OF teins and metabolites have so far had a more
NODULE DEVELOPMENT modest impact on the field of nodule trans-
AND DIFFERENTIATION port and metabolism, in part because they have
The scope of research on nodule transport and been confined to a few hundred of the most
metabolism has changed in the past decade abundant proteins and metabolites. By and
as scientists have completed the genome se- large, the results of proteomic and metabolomic
quences of both rhizobia and plant species studies support existing schemes of nodule
and have employed high-throughput methods metabolism (e.g., 22, 35, 95). Steady-state lev-
to analyze thousands of transcripts, proteins, els of metabolites, especially substrate/product
and metabolites in parallel. The genome se- ratios, have provided some insight, albeit in-
quences of many rhizobial species and five plant direct, into changes in metabolic fluxes (e.g.,
species have been published, including those of through glycolysis) that accompany nodule
the two model legumes L. japonicus and Med- development (22). However, whole-nodule
icago truncatula and the crop species G. max metabolome studies lack the necessary resolu-
(soybean) (125, 126, 167). Genome annotation tion to ascribe particular metabolic pathways
has produced inventories of genes involved in to specific cell types and subcellular compart-
plant transport and metabolism (e.g., 10), which ments. This remains a challenge for the future.
then serve as a starting point to decipher gene More progress has been made on compartmen-
function in nodules. Genome-wide expression tation of transport and metabolism in the area
(transcriptome) databases for several legumes of proteomics, with reports on the proteomes
have been used to identify thousands of plant of symbiosomes and the SM (e.g., 18, 102, 165)

and nodule mitochondria (57). Among the pro- essential for SNF (163). Another remarkable
teins to be identified on the SMs of different development for reverse genetics in legumes
species are H+ -ATPases, aquaporins, and the was the establishment of a Tnt1-insertion
TILLING:
targeting-induced sulfate transporter LjSST1 (18, 165), which is mutant population of M. truncatula (137),
local lesions in crucial for SNF (68). Thus, proteomics will which can now deliver knockout mutations in
genomes likely be instrumental in placing transport and almost any selected gene. The roles of several
metabolism into a cellular context. symbiosis genes have been tested using this
Information from genomics, transcrip- resource (70, 92), and similar resources for
tomics, proteomics, and metabolomics enables L. japonicus and other legumes are on the hori-
us to contemplate genes and proteins in a zon (152). We have begun a systematic study of
broader molecular-biological context and to many transporter-encoding genes induced dur-
formulate hypotheses about their roles in SNF. ing nodule development in Medicago, including
Until recently, the main approaches to deter- those encoding putative sugar, dicarboxylate,
mining the functions of specific genes in planta amino acid, and metal transporters. Tnt1 inser-
involved RNAi or antisense RNA, which trig- tions in some of these genes lead to defects in
ger the degradation of target gene transcripts SNF (I. Kryvoruchko & M. Udvardi, unpub-
and reduce or eliminate the production of the lished results). Facile tools for reverse genetics
cognate protein. These approaches have shown in legumes will enable us to decipher the roles
that the following enzymes (among others) are of many enzymes and transporters in nodules in
necessary for effective SNF: PEPC (127) and the coming years, which will lead to a much bet-
GOGAT (25) in Medicago sativa (alfalfa), and ter understanding of the nitrogen-fixing system
sucrose synthase (5) and γ-glutamylcysteine embodied by legume-rhizobia symbioses.
synthetase (involved in glutathione biosyn-
thesis) (38) in M. truncatula. Interestingly,
overexpression of γ-glutamylcysteine syn- BACTEROID DEVELOPMENT
thetase led to enhanced SNF in Medicago,
Oxygen and Nitrogen Fixation
at least under laboratory conditions (38),
indicating that there may be scope to improve In rhizobia, the induction of nif and fix genes
SNF in this and other legume species. required for nitrogen fixation is controlled by
The results of RNAi and antisense RNA oxygen rather than nitrogen status (36), which
are not always unambiguous, because target explains how bacteroids act like ammoniaplasts
mRNA levels are rarely reduced to zero to supply the plant with ammonia without as-
(leaving the possibility of residual protein similating nitrogen. The free-oxygen concen-
activity) and because transcripts of multiple tration in soybean nodules is only 57 nM (69).
sequence-related genes may be reduced. New The signaling systems in rhizobia that regu-
technologies for reverse genetics have solved late nif and fix gene expression in response to
both of these problems. low oxygen are extraordinarily complex, differ-
Targeting-induced local lesions in genomes ing considerably even between strains of the
(TILLING) uses EMS-mutant populations same species. Because these systems were re-
to identify loss-of-function mutations in cently reviewed (141), we do not cover the many
specific genes. TILLING resources have been complex variations in rhizobia here. The ba-
established for soybean (24), L. japonicus (110), sic features were first discovered in Sinorhizo-
and other legumes and have been used to assess bium meliloti, where FixL is the oxygen sen-
the contributions of various plant genes to sor. It is anchored in the membrane, and
SNF. For instance, nodule-enhanced sucrose in the absence of oxygen it autophosphory-
synthase is not required for SNF in L. japonicus, lates and then transfers this phosphate to the
as it is in pea (61). Likewise, the invertase transcriptional regulator FixJ (47, 78). Phos-
LjINV1, which also cleaves sucrose, is not phorylation of FixJ induces dimerization and

disrupts the inhibitory interface between the N-

terminal receiver domain and C-terminal tran- CONTROL OF BACTEROID DEVELOPMENT
scriptional activator domain of FixJ (27). DNA BY PLANT PEPTIDES
sites that bind FixJ are found mostly on pSymA
(41), and this was confirmed by transcriptional Legumes from the IRLC clade, including Medicago, Pisum sativum
analysis (12). Genes directly regulated by FixJ (pea), Vicia faba (broad bean), and Trifolium repens (white clover),
include nifA (which regulates nifHDK) and fixK produce several hundred nodule cysteine-rich antimicrobial pep-
(which encodes a Crp/Fnr homolog regulating tides (NCRs), which induce chromosome endoreduplication in
fixNOQP) (43). FixNOQP is a high-affinity ter- bacteroids of up to 24C compared with 1–2C for laboratory-
minal oxidase critical for microaerobic respira- cultured bacteria (84). NCR peptides cause the bacteria to swell,
tion in rhizobia. develop leaky membranes, and become terminally differentiated.
Major variations on this system include The DNF1 signal peptidase of Medicago truncatula targets NCR
the soluble FixL proteins of Rhizobium etli peptides to the symbiosome membrane (155, 161). bacA mutation
and Rhizobium leguminosarum, which lack increases the sensitivity of Sinorhizobium meliloti to NCR peptides
transmembrane domains. These organisms (53), which explains why bacteroids of the IRLC clade require
also have two Crp/Fnr proteins, FnrN and BacA for development but those of, for example, the phaseoloid
FixK, and mutations in both are needed to clade do not. Given that M. truncatula produces more than 300
prevent nitrogen fixation (105). FixJ is missing NCR peptides, the effects on bacterial growth and metabolism
in R. etli and R. leguminosarum, but FxkR was will be a complex amalgam of many different peptides. Different
recently identified as the activator transducing peptides appear to be produced in slightly different regions of the
the signal from FixL to FixKf (172). Bradyrhi- nodule. Furthermore, the sites of action of individual peptides are
zobium japonicum has significant variations, likely to be very different.
including regulation of fixR-nifA by a two-
component sensor-regulator pair, RegS/RegR,
as well as a FixL cascade regulating FixJ (74).
Key components such as NifA and FixK2 shutdown of bacterial synthesis of amino acids
are also regulated at the posttranslational in bacteroids (115). Preventing branched-chain
level (85). In terms of bacterial metabolism, a amino acid uptake by nodule bacteria leads to
critical aspect of this complex fine-tuning of amino acid starvation, failure to fully develop,
oxygen-dependent regulation is that rhizobia reduced size, and endoreduplication of chro-
must respire at a very low oxygen tension to mosomes. Thus, bacteroids surrender control
prevent oxygen inactivation of nitrogenase. of the biosynthesis of essential compounds to
This explains the need for leghemoglobin plants, thereby acting like organelles. Sym-
(see above) but also the importance of precise biotic auxotrophy also occurs in determinate
redox balance in bacteroids, whether through common bean infected with R. leguminosarum
metabolic turnover or through the synthesis of bv. phaseoli and so is independent of nodule
carbon and redox storage products (see below). cysteine-rich antimicrobial peptides (NCRs)
and nodule type (113) (see sidebar, Control
Symbiotic Auxotrophy of Bacteroid Development by Plant Peptides).
In R. leguminosarum, two broad-specificity However, alfalfa bacteroids did not show
amino acid transporters, AapJQMP and symbiotic auxotrophy when aap and bra (liv)
BraDEFGC (both belonging to the ABC were mutated. This may be because only a
family), are required for effective SNF (62, very low rate of branched-chain amino acid
75). Functional Aap and Bra are needed for transport is required to alleviate amino acid
the transport of branched-chain amino acids. starvation, and transport systems other than
This phenomenon was named symbiotic aux- Aap/Bra may enable sufficient branched-chain
otrophy because bacteria become auxotrophic amino acid transport in S. meliloti to prevent
only in symbiosis with plants, owing to the symbiotic auxotrophy.

Homocitrate Synthesis A fundamental step in bacteroid develop-

ment is the switching off of ammonium as-
Homocitrate is a component of the iron-
similation accompanying nitrogen fixation (re-
molybdenum cofactor of nitrogenase; free-
viewed in 104). Thus, peas infected by bacte-
living diazotrophs synthesize it by condensing
ria that cannot assimilate ammonium because
2-oxoglutarate and acetyl-coenzyme A (CoA),
they are mutated in gltB (encoding GOGAT)
catalyzed by homocitrate synthase (NifV). Cu-
and aldA (encoding alanine dehydrogenase)
riously, most rhizobia do not contain nifV. In-
are unaltered in nitrogen fixation and plant
stead, homocitrate is synthesized by the plant,
growth (91). Mature bacteroids are effectively
as shown in L. japonicus, where fen1 mutants
ammoniaplasts that release ammonia to the
did not fix nitrogen when infected by Mesorhi-
plant without assimilation. However, this pro-
zobium loti (54). FEN1 encodes a homocitrate
cess may become misregulated, as shown by
synthase expressed in nodules (54). Thus, like
a mutant deleted in the N-terminal uridylyl-
symbiotic auxotrophy, homocitrate synthesis is
transferase domain of the nitrogen-sensing pro-

another example of the plant assuming control
tein GlnD in S. meliloti. This mutation causes
of a biochemical function essential for bacteroid
severe nitrogen starvation in the plant even
nitrogen fixation.
though 15 N2 is fixed at wild-type rates (171).

The fate of incorporated 15 N is unknown; it
Global Changes in Bacteroid is initially present in shoots but is lost within
Metabolism 24 h.
The control of the bacterial cell cycle by
plant-derived NCR peptides in IRLC legumes,
development of symbiotic auxotrophy, and
plant production of homocitrate all suggest BACTERIAL TRANSPORT
that bacteroids behave like organelles. Further
weight is given to this hypothesis by a substan-
Metal and Ion Transport
tial reduction in most aspects of cell growth and In B. japonicum, molybdate is transported by
division, including the synthesis of ribosomal the high-affinity ABC-type ModABC system
and outer membrane proteins, peptidoglycan, (32). mod mutants show reduced symbiotic
and nucleic acid (including repair) during bac- performance under limiting molybdate in the
teroid development (7, 9, 16, 20, 66). However, plant growth medium. The severity of the
specific nif and fix genes are induced, enabling growth defect in limiting molybdate is depen-
nitrogen fixation. Although transcription is dent on the sulfate concentration in the growth
largely reduced in mature bacteroids, there is medium, suggesting that these two anions are
an earlier transcriptomic burst in developing transported by a common sulfate transporter.
bacteroids [5–7 days postinoculation (dpi)], Phosphate is transported by three systems in
including induction of regulatory proteins, S. meliloti: the low-affinity OrfA-Pit and the
inner membrane, and transport proteins (16, high-affinity PstSCAB and PhoCDET (168).
66). These changes probably coincide with PhoCDET is essential for symbiosis between S.
bacteroid development, although a clear meliloti 1021 and alfalfa (6); however, S. meliloti
temporal and spatial map of gene expression is 1021 has a pstC point mutation that causes inac-
lacking. Most of the data come from microar- tivation and complex regulatory changes in the
rays on whole nodules where cells in infection pho regulon (168). These changes lead to the
threads and early and mature bacteroids are Fix− phenotype and are corrected by restora-
mixed. We clearly need much finer temporal tion of the wild-type pstC sequence. Thus, root
resolution of changes in bacterial transcription nodule bacteria are not phosphate limited, and
and translation as well as a map of ligand the low-affinity OrfA-Pit is the main system in
availability. planta.

Very little is known about magnesium trans- they also secrete amino acids such as alanine
port in rhizobia, although a miniTn5 mutant in and aspartate (1, 72). It was initially reported
a gene with high identity to an MgtE-like mag- that soybeans secrete alanine as the main export
nesium transporter was Fix− on peas (66). MgtE product of nitrogen fixation (162), but this was
has been confirmed to be required for magne- incompatible with a reassessment of the prod-
sium accumulation (G.A. Hood, personal com- ucts secreted by soybean bacteroids (72). Fur-
munication). Interestingly, this system is con- thermore, in pea, mutation of alanine dehydro-
stitutively expressed and not further induced in genase, which prevents alanine synthesis, had
nodules. no effect on symbiosis (1). Given the pivotal role
B. japonicum has a single Nramp-family of ammonia secretion from bacteroids to plants,
MntH transporter for manganese, which we know very little about ammonia transfer out
is not required for symbiosis with soybean of bacteroids. It is assumed to passively diffuse
(60). B. japonicum also has a specific porin for across the bacteroid membrane as ammonia and
manganese uptake (but not for cobalt or copper be trapped by protonation to ammonium in the
uptake) across the outer membrane (59). This is acidic peribacteroid space (30). Active ammo-
the first characterized porin to be highly solute nia uptake by rhizobia requires AmtB, which is
specific. S. meliloti has a single high-affinity expressed only under nitrogen limitation and is
manganese transporter (SitABCD) of the absent from bacteroids (104).
ABC-type family, mutation of which causes a It is widely accepted that ammonia assimi-
variable symbiotic phenotype depending on the lation is reduced in bacteroids as they become
bacterial strain and/or plant cultivar (21, 28, ammonium-secreting ammoniaplasts. Ammo-
111). Although these results reflect differences nium assimilation in laboratory-cultured rhizo-
between bacterial strains and plant cultivars, bia occurs through the GS/GOGAT systems
they do suggest that manganese accumulation (17, 40, 91). R. etli gltB (encoding GOGAT)
can be important, probably for resistance to mutants either increase nitrogen supply to the
oxidative stress in symbiosis. R. leguminosarum plant (17) or lower acetylene reduction to 25%
has both MntH and SitABCD, with mutation of the wild-type level (40). R. leguminosarum
of both generating a severe Fix− phenotype gltB mutants have a complex phenotype, fail-
(G.A. Hood, personal communication). ing to grow on most organic nitrogen sources
Iron is a key metal for bacteria and for bac- because amino acid transporters are posttran-
teroid function, and although we know a great scriptionally inhibited (91). However, suppres-
deal about the genetic regulation of its acqui- sor mutants were obtained in hfq, ntrC, and
sition in cultured rhizobia, we do not know glnB, which increased transport and restored
which systems are used in bacteroids (117, 132, nitrogen fixation. Thus, although a gltB mutant
144, 157). This is because Fe3+ , the normal perturbs regulation of nitrogen metabolism and
form found in air, is extremely insoluble, often hence nitrogen fixation, there is no requirement
leading to limitation and the requirement for per se for ammonium assimilation. Intriguingly,
siderophore production. None of the systems gsh (encoding glutathione synthase) and gor (en-
induced by iron limitation in the laboratory are coding glutathione reductase) mutants of R. etli
induced in root nodule bacteria (7, 9, 16, 20, also have a severe inhibition of amino acid up-
66), so the transport systems needed for iron take and reduced symbiotic performance (139).
accumulation remain unidentified. Although it is apparent that gsh, gor, and gltB
mutants lead to transport inhibition, probably
by perturbation of a key intracellular metabo-
Ammonium and Amino Acid lite such as glutamine or glutathione, they do
Transport and Assimilation not reveal the signaling mechanism. However,
Ammonium is the principal export product mutations in the PtsNtr system result in post-
of nitrogen fixation from bacteroids, although translational inhibition of ABC transporters but

not of proton-coupled transport systems (114). understandable as they drive nitrogen fixation.
This may be the mechanism by which changes Although we also focus on the metabolism of
in amino acid or glutathione levels regulate the mature bacteroids, it is important to consider
activity of amino acid transporters (91, 139). that rhizobial metabolism must adapt to several
different environments, from the rhizosphere
of host legumes to infection threads, symbio-
Dicarboxylate Transport somes, and finally the mature nitrogen-fixing
The dicarboxylate transport (Dct) system con- bacteroid. A recent study used comparative ge-
sists of an MFS membrane transporter (DctA) nomics to examine the rhizosphere metabolism
and a two-component sensor-regulator system of R. leguminosarum inoculated onto pea (host
(DctBD) that induces dctA in response to C4 legume), alfalfa (nonhost legume), and Beta
dicarboxylates (169). S. meliloti dctA mutants vulgaris (sugar beet) (nonlegume control) (118).
with a partial ability to transport dicarboxy- Carbon metabolism was dominated by organic
lates form nodules with more starch in infected acids, with a strong bias toward aromatic amino
cells compared with dctA null mutants (170). acids, C1 compounds, and C2 compounds. A
dctA mutants form swollen bacteroids that fill common core of rhizosphere-induced genes
plant cells, although they tend to senesce early was identified, including a large number of
(123, 164). Thus, an active Dct system or dicar- specific transport systems. However, many
boxylate accumulation may be a developmental genes were legume specific and others were
checkpoint in the final stages of bacteroid de- species specific. The plasmid pRL8 was des-
velopment. It would be informative to know the ignated as rhizosphere specific, enabling the
endoreduplication state of dct bacteroids and adaptation of R. leguminosarum to pea, with
their transcriptional profile in young nodules mutation of many of the upregulated genes
to see whether they are fully developed. reducing competitiveness for pea rhizosphere
DctB is the membrane-bound sensor colonization. Comparing this gene expression
protein that detects dicarboxylates outside pattern with that of root nodule bacteria
the cell. It has a periplasmic binding domain obtained from early nodules (7 dpi) reveals a
(DctBp) that has been crystallized in the dramatic reduction in the number of genes
apo- and succinate-bound states (174). It has coding for catabolic pathways and transport
two PAS domains: a membrane-distal and a systems expressed in early root nodule bacteria
membrane-proximal site, with ligand binding (66, 118).
occurring at the proximal site. When measured In addition, there are large changes in the
in solution, the binding of a dicarboxylate to gene expression of bacteroids of different ages.
DctBp caused the monomer to dimerize (93). Notably, dicarboxylate, phenylalanine, and
This is consistent with a rearrangement at the malonate catabolism are expressed in early root
dimerization interface of DctBp driving signal nodule bacteria (7 dpi), whereas only dicarboxy-
transduction to the cytosolic histidine kinase late catabolism remains strongly expressed in
domain, resulting in autophosphorylation. mature bacteroids (>15 dpi), which is in agree-
DctB then transfers this phosphate to the ment with a large body of evidence showing
cognate receiver domain in DctD, activating that Rhizobium bacteroids transport and catab-
σ 54 -dependent transcription of dctA. olize dicarboxylic acids (reviewed in 141). In
keeping with this, both the dicarboxylate trans-
port system and malic enzyme [either alone or
BACTERIAL METABOLISM in conjunction with PckA (PEP carboxykinase)]
are essential for nitrogen fixation (90, 173).
Bacterial Carbon Metabolism
Malic enzyme or the combined activities of
Most studies have concentrated on the PckA and pyruvate kinase convert malate to
metabolism of mature bacteroids, which is pyruvate needed for acetyl-CoA synthesis. This

is condensed with oxaloacetate derived from Gluconeogenesis

malate dehydrogenase to feed the TCA cycle.
Although rhizobia receive dicarboxylates as the
In S. meliloti, NAD+ (Dme)-malic enzyme
carbon source for nitrogen fixation, they pre-
rather than NADP+ (Tme)-malic enzyme is
sumably need sugar for general biosynthesis or
essential for direct oxidative decarboxylation to
for polymerization into storage compounds like
pyruvate (173). However, although Dme is the
glycogen. Because the peribacteroid membrane
favored pathway in pea bacteroids, the com-
has limited permeability to sugars, this suggests
bined activities of PckA and pyruvate kinase can
that gluconeogenesis should be important. In-
partially substitute (60% acetylene reduction),
deed, S. meliloti 1021 mutants in the glycolytic
and only a double mutant of dme and pckA is
enzymes enolase, glyceraldehyde-3-phosphate
Fix− (90). Sinorhizobium sp. NGR234 also uses
dehydrogenase, and 3-phosphoglycerate kinase
both of these pathways on a variety of hosts, and
are Fix− on alfalfa (42), whereas Bradyrhizobium
M. loti and B. japonicum USDA110 may also use
sp. ORS278 mutants in enolase and phospho-
both pathways, on the basis that dme mutants

glycerate kinase have only 5% of the wild-type
retain some nitrogen fixation (26, 142). Dme
acetylene reduction rate (13). However, these
is a complex protein with N-terminal, malic
enzymes are needed for sugar interconversion
enzyme, and C-terminal phosphotransacety-

and catabolism as well as gluconeogenesis. The
lase (Pta) domains. Deleting the Pta domain
committed step of gluconeogenesis is catalyzed
increased the peak acetylene reduction rate
by PckA (see above), and an R. etli pckA mu-
in four-week-old pea plants to 140–150% of
tant was Fix− in symbiosis on beans (140). In
the wild-type rate, which was accompanied by
Rhizobium sp. NGR234, pckA mutants are re-
increased nodule mass. Plants infected with
duced for nitrogen fixation on Leucaena leu-
Pta-deletion mutants did not have increased
cocephala and Macroptilium atropurpureum but
dry weight, so there is no sustained change
Fix− on V. unguiculata (98). However, S. meliloti
in nitrogen fixation throughout growth.
and R. leguminosarum pckA mutants show re-
This indicates a complex relationship be-
duced and unaffected acetylene reduction, re-
tween pyruvate, nitrogen fixation, and plant
spectively (42, 90). Some of the phenotypic vari-
growth, with the Pta domain of malic
ability of pckA strains may be due to a second
enzyme having an important regulatory
gluconeogenic pathway capable of producing
function.
PEP from pyruvate via pyruvate orthophos-
The TCA cycle appears to be essential for
phate dikinase (99), and mutants in Bradyrhi-
nitrogen fixation in many rhizobia, including R.
zobium sp. ORS278 reduced acetylene at only
leguminosarum, S. meliloti, and Bradyrhizobium
34% of the wild-type rate (13). Overall, root
sp. ORS278, based on mutation of TCA-cycle
nodule bacteria appear to have a clear require-
enzymes leading to a Fix− phenotype (13, 37,
ment for sugar, which is commonly provided
159). However, several mutations of isocitrate
by gluconeogenesis, although sufficient sugar
dehydrogenase, aconitase, or a component of
for bacteroid metabolism may be provided by
the 2-ketoglutarate dehydrogenase complex
some plants.
are Fix+ in B. japonicum USDA110 (50, 129,
143; reviewed in 141). This suggests that
mature soybean bacteroids may use a very
different pathway for pyruvate metabolism. Redox Balance and Storage Polymers
A bypass pathway has been proposed for the Carbon supplied by the plant may be stored in
2-ketoglutarate dehydrogenase step in B. bacteroids as poly-β-hydroxybutyrate (PHB),
japonicum, although this would not explain the glycogen (reviewed in 77), or even lipid droplets
lack of effect on nitrogen fixation in isocit- in peanuts (130). PHB is synthesized from two
rate dehydrogenase and aconitase mutations molecules of acetyl-CoA to form acetoacetyl-
(51). CoA, which is reduced and polymerized by

PHB synthase (77). Thus it is a store of both and nodule number (81). However, in R. legumi-
carbon and reductant. Bacteroids from deter- nosarum A34, both a glycogen synthase mutant
minate nodules such as those of common bean and a double glycogen synthase and PHB syn-
or soybean store large amounts of PHB, unlike thase mutant were unaltered in acetylene reduc-
those from indeterminate nodules (e.g., pea or tion or pea plant dry weight (76). These PHB
alfalfa). However, PHB granules are detectable mutants lack visible starch granules in nodules,
when carbon metabolism is perturbed or PHB whereas a glycogen synthase mutant showed a
degradation is prevented in bacteroids of inde- large starch accumulation (76). PHB may ac-
terminate nodules of pea or alfalfa, indicating cumulate in R. leguminosarum during infection,
that under normal conditions PHB is made and and this stored carbon could be utilized dur-
turned over rapidly (77, 145). In both pea (in- ing bacteroid differentiation. The reduction in
determinate) nodulating strain A34 and bean plant starch granules when PHB synthesis is
(determinate) nodulating strain 4292, phaC (en- blocked in root nodule bacteria may reflect their
coding PHB synthase) mutants are Fix+ (76).
mobilization to fuel bacteroid differentiation.

Likewise, S. meliloti mutants unable to make In contrast, glycogen synthase ( glgA1) mutants
PHB or degrade it are Fix+ (4, 15, 112, 145, of S. meliloti, like PHB synthase mutants, have
166). However, S. meliloti mutants unable to reduced symbiotic performance on both alfalfa
make PHB do have significantly reduced sym- and M. truncatula (28, 160). However, glgA mu-
biotic performance on alfalfa and M. truncat- tants are more sensitive to hydrogen perox-
ula, whereas mutants unable to degrade PHB ide, suggesting their reduced symbiotic perfor-
are unaltered relative to the wild type (4, 145, mance may occur because glycogen increases
160). Thus, PHB synthesis is important for the stress resistance (28). Glycogen synthase mu-
nitrogen-fixing efficiency of symbioses involv- tants of S. meliloti do not accumulate starch in
ing S. meliloti, whereas degradation is less im- the plant, but PHB synthase mutants do. This
portant, suggesting it acts as a store for excess inversion of the starch storage response to bac-
reductant rather than as a carbon buffer. terial mutants in alfalfa nodules relative to pea
A PHB synthase mutant of Azorhizobium nodules suggests that whereas R. leguminosarum
caulinodans was Fix− both ex planta and in sym- uses glycogen as its main carbon store, S. meliloti
biosis with Sesbania rostrata (80). R. etli phaC uses PHB.
mutants, in contrast, had increased acetylene Overall, carbon storage compounds—
reduction and plant nitrogen content (19). whether PHB, glycogen, lipid, or Calvin-
The difference between symbionts is neatly Benson-cycle products—are important to
illustrated by photosynthetic Bradyrhizobium rhizobia to regulate carbon flux and act as
sp. ORS278, which requires the Calvin-Benson reductant stores. However, which compound
cycle for efficient SNF (13, 49). Mutants of or compounds are used clearly varies between
cbbK (encoding phosphoglycerate kinase) different bacterial-plant symbioses. Interest-
and cbbL [encoding the large chain of ribu- ingly, the role of storage polymers in nocturnal
lose 1,5-bisphosphate carboxylase oxygenase nitrogen fixation (where stored carbon might
(RuBisCO)] reduce nitrogen fixation on support nitrogen fixation when photosynthesis
Aeschynomene indica, and a mutant in cbbE has stopped) has not been reevaluated with pha
(encoding ribulose-5-phosphate-3-epimerase) and glgA mutants.
was Fix− . The Calvin-Benson cycle consumes
3 mol of ATP and 2 mol of NAD(P)H per mole
of carbon dioxide, suggesting that Bradyrhizo- FUTURE DIRECTIONS
bium sp. ORS278 uses the Calvin-Benson cycle It is clear that a more systems-based approach
as a reductant store. is needed to understand the complex changes
In Rhizobium tropici, glycogen synthase in metabolism that occur in bacteroids and in
( glgA) mutants have increased plant dry weight plant cells during nodule development, both

before and after SNF onset. A desirable end will also be greatly assisted by high-quality
point would be to develop a model nodule, al- analysis of metabolite levels in the major nodule
though there clearly needs to be more than one compartments. This will identify metabolites
model as there are major variations between being made specifically in bacteroids as op-
symbioses of different species. Not the least of posed to in the various plant compartments.
these are differences between determinate and It will also link transcriptional and proteomic
indeterminate nodules of the common crop and data to the active production of metabolites.
pasture legumes (e.g., Medicago and pea versus Regarding the temporal and spatial orga-
soybean and L. japonicus). Although the suc- nization of transport and metabolism in nod-
cess of the model legumes M. truncatula and L. ules, it is clear that greater resolution of gene
japonicus for genetic analysis might suggest con- expression and protein location in the various
centrating effort on these plants in the future, plant cell types and bacteroids during nodule
much of the biochemical heavy lifting needed development is required for a complete un-
for quantitative measurements is easier to per- derstanding of how the metabolisms of the
form in pea, common bean, and soybean. plant and bacteria integrate to achieve effective
So far, there have been efforts to model the SNF. Laser capture microdissection of specific
metabolic flux of R. etli and S. meliloti using flux plant cell types is one way to achieve this for
balance analysis (120). These purely theoretical transcriptome analysis (138). Isolation and pu-
approaches have also been modified by the rification of bacteroids and specific plant or-
inclusion of transcriptomic and proteomic ganelles will also facilitate this for proteome
data for R. etli (119). However, it would be analysis (and for transcriptome analysis, in the
desirable to conduct metabolic flux reaction case of bacteroids). Proteomics also promises
analysis using 13 C-labeled substrates, as has to identify posttranslational modifications of
been done for S. meliloti growing on glucose many proteins—e.g., phosphorylation, nitra-
(44). This approach has also worked well for tion, and sulfonation (52, 83, 96)—which may
analyzing the growth of cultured R. legumi- reveal important new control points in trans-
nosarum on 13 C succinate but is more difficult port and metabolism.
for bacteroids, as it relies on steady-state label- Finally, knowledge of how transport and
ing that requires long-term incubation in 13 C metabolism are integrated to achieve effective
substrate (G. Radcliffe & N. Kruger, personal SNF and how they vary in different symbioses
communication). Success will require improve- may help us to identify bottlenecks in SNF in
ments in the stabilization of isolated bacteroids specific legume-rhizobia systems that could be
or the labeling of whole plants or nodules. overcome by breeding to enhance SNF in the
Transcriptomic, proteomic, and flux analysis future.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We thank Catalina Pislariu for help in preparing Figures 1 and 2, and the National Science
Foundation, Samuel Roberts Noble Foundation (M.U.), and Biotechnology and Biological Sci-
ences Research Council (P.S.P.) for funding. We are also grateful to Drs. Mechthild Tegeder,

Vagner Benedito, Igor Kryvoruchko, Daniel Roberts, and Alison East for useful comments on the
manuscript and to Dr. Penny Smith for sharing unpublished data.
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PP64-frontmatter ARI 25 March 2013 10:21
Annual Review of
Plant Biology
Contents Volume 64, 2013
Benefits of an Inclusive US Education System

Elisabeth Gantt p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Plants, Diet, and Health

Cathie Martin, Yang Zhang, Chiara Tonelli, and Katia Petroni p p p p p p p p p p p p p p p p p p p p p p p p p19
A Bountiful Harvest: Genomic Insights into Crop Domestication

Phenotypes
Kenneth M. Olsen and Jonathan F. Wendel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p47
Progress Toward Understanding Heterosis in Crop Plants
Patrick S. Schnable and Nathan M. Springer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p71
Tapping the Promise of Genomics in Species with Complex,
Nonmodel Genomes
Candice N. Hirsch and C. Robin Buell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p89
Understanding Reproductive Isolation Based on the Rice Model
Yidan Ouyang and Qifa Zhang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 111
Classification and Comparison of Small RNAs from Plants
Michael J. Axtell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 137
Plant Protein Interactomes
Pascal Braun, Sébastien Aubourg, Jelle Van Leene, Geert De Jaeger,
and Claire Lurin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 161
Seed-Development Programs: A Systems Biology–Based Comparison
Between Dicots and Monocots
Nese Sreenivasulu and Ulrich Wobus p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 189
Fruit Development and Ripening
Graham B. Seymour, Lars Østergaard, Natalie H. Chapman, Sandra Knapp,
and Cathie Martin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 219
Growth Mechanisms in Tip-Growing Plant Cells
Caleb M. Rounds and Magdalena Bezanilla p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 243
Future Scenarios for Plant Phenotyping
Fabio Fiorani and Ulrich Schurr p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 267
v
Microgenomics: Genome-Scale, Cell-Specific Monitoring of Multiple

Gene Regulation Tiers
J. Bailey-Serres p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 293
Plant Genome Engineering with Sequence-Specific Nucleases
Daniel F. Voytas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 327
Smaller, Faster, Brighter: Advances in Optical Imaging
of Living Plant Cells
Sidney L. Shaw and David W. Ehrhardt p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 351
Phytochrome Cytoplasmic Signaling
Jon Hughes p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 377
Photoreceptor Signaling Networks in Plant Responses to Shade
Jorge J. Casal p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 403

ROS-Mediated Lipid Peroxidation and RES-Activated Signaling
Edward E. Farmer and Martin J. Mueller p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 429

Potassium Transport and Signaling in Higher Plants
Yi Wang and Wei-Hua Wu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 451
Endoplasmic Reticulum Stress Responses in Plants
Stephen H. Howell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 477
Membrane Microdomains, Rafts, and Detergent-Resistant Membranes
in Plants and Fungi
Jan Malinsky, Miroslava Opekarová, Guido Grossmann, and Widmar Tanner p p p p p p p 501
The Endodermis
Niko Geldner p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 531
Intracellular Signaling from Plastid to Nucleus
Wei Chi, Xuwu Sun, and Lixin Zhang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 559
The Number, Speed, and Impact of Plastid Endosymbioses in
Eukaryotic Evolution
Patrick J. Keeling p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 583
Photosystem II Assembly: From Cyanobacteria to Plants
Jörg Nickelsen and Birgit Rengstl p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 609
Unraveling the Heater: New Insights into the Structure of the
Alternative Oxidase
Anthony L. Moore, Tomoo Shiba, Luke Young, Shigeharu Harada, Kiyoshi Kita,
and Kikukatsu Ito p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 637
Network Analysis of the MVA and MEP Pathways for Isoprenoid
Synthesis
Eva Vranová, Diana Coman, and Wilhelm Gruissem p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 665
vi Contents
Toward Cool C4 Crops

Stephen P. Long and Ashley K. Spence p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 701
The Spatial Organization of Metabolism Within the Plant Cell
Lee J. Sweetlove and Alisdair R. Fernie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 723
Evolving Views of Pectin Biosynthesis
Melani A. Atmodjo, Zhangying Hao, and Debra Mohnen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 747
Transport and Metabolism in Legume-Rhizobia Symbioses
Michael Udvardi and Philip S. Poole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 781
Structure and Functions of the Bacterial Microbiota of Plants
Davide Bulgarelli, Klaus Schlaeppi, Stijn Spaepen, Emiel Ver Loren van Themaat,
and Paul Schulze-Lefert p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 807
Systemic Acquired Resistance: Turning Local Infection

into Global Defense
Zheng Qing Fu and Xinnian Dong p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 839
Indexes
Cumulative Index of Contributing Authors, Volumes 55–64 p p p p p p p p p p p p p p p p p p p p p p p p p p p 865

Cumulative Index of Article Titles, Volumes 55–64 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 871
Errata
An online log of corrections to Annual Review of Plant Biology articles may be found at
http://www.annualreviews.org/errata/arplant
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PP64CH31-Udvardi ARI 25 March 2013 16:50

• Our comprehensive search 1

Annu. Rev. Plant Biol. 2013. 64:781–805 Keywords

and potentially extending them to nonlegumes,

genomes for at least ﬁve legume species. Com-

scale have helped ﬂesh out important aspects of

782 Udvardi · Poole

www.annualreviews.org • Legume-Rhizobia Transport and Metabolism 783

nitrogen ﬁxation ceases (156). In indeterminate

and some of its consequences for bacterial and

ICs NODULE PHYSIOLOGY AND

Figure 1 N2 + 8H+ + 8e− + 16ATP → 2NH3 + H2

784 Udvardi · Poole

nodules are typically in the tens of nanomolar

oxygen concentration in the “mutant” nodules

genes (100). In fact, low oxygen and possibly as-

www.annualreviews.org • Legume-Rhizobia Transport and Metabolism 785

786 Udvardi · Poole

www.annualreviews.org • Legume-Rhizobia Transport and Metabolism 787

Symbiotic plant cell

788 Udvardi · Poole

www.annualreviews.org • Legume-Rhizobia Transport and Metabolism 789

790 Udvardi · Poole

disrupts the inhibitory interface between the N-

www.annualreviews.org • Legume-Rhizobia Transport and Metabolism 791

Homocitrate Synthesis A fundamental step in bacteroid develop-

transferase domain of the nitrogen-sensing pro-

though 15 N2 is ﬁxed at wild-type rates (171).

792 Udvardi · Poole

www.annualreviews.org • Legume-Rhizobia Transport and Metabolism 793

794 Udvardi · Poole

is condensed with oxaloacetate derived from Gluconeogenesis

both pathways, on the basis that dme mutants

enzyme, and C-terminal phosphotransacety-

www.annualreviews.org • Legume-Rhizobia Transport and Metabolism 795

mobilization to fuel bacteroid differentiation.

796 Udvardi · Poole

www.annualreviews.org • Legume-Rhizobia Transport and Metabolism 797

ations in the symbiotic transcriptome and metabolome. Plant Physiol. 145:1600–18

798 Udvardi · Poole

www.annualreviews.org • Legume-Rhizobia Transport and Metabolism 799

800 Udvardi · Poole

teroids from soybean root nodules. Microbiology 148:1959–66

www.annualreviews.org • Legume-Rhizobia Transport and Metabolism 801

802 Udvardi · Poole

www.annualreviews.org • Legume-Rhizobia Transport and Metabolism 803

804 Udvardi · Poole

zobium) meliloti and characterization of phbC mutants. Can. J. Microbiol. 44:554–64

www.annualreviews.org • Legume-Rhizobia Transport and Metabolism 805

Contents Volume 64, 2013

Beneﬁts of an Inclusive US Education System

Plants, Diet, and Health

A Bountiful Harvest: Genomic Insights into Crop Domestication

Microgenomics: Genome-Scale, Cell-Speciﬁc Monitoring of Multiple

Jorge J. Casal p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 403

Edward E. Farmer and Martin J. Mueller p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 429

Toward Cool C4 Crops

Systemic Acquired Resistance: Turning Local Infection

Zheng Qing Fu and Xinnian Dong p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 839

Cumulative Index of Contributing Authors, Volumes 55–64 p p p p p p p p p p p p p p p p p p p p p p p p p p p 865

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