india:1 Journal of Experimental Biology
Vol. 42, February 2004, pp. 138-142
Review Article
Nitrogen fixation and carbon metabolism in legume nodules
Neera Garg>~<, Ranju Sing Ia & Geetanjali
Department of Botany, Punjab University, Chandigarh 160014, Indi a
A large amount of energy is utilized by legume nodules for the fixation of nitrogen and assimilation of fixed nitrogen
(ammoni a) into organic compounds . The source of energy is provided in the form of photosynthates by the host plant.
Phosphoenol pyruvate carboxylase (PEPC) enzyme, which is responsible for carbon dioxide fixation in C4 and crassulacean
acid metabolism plants, has also been found to play an important role in carbon metabolism in legume root nodule. PEPCmcdiated co~ fixation in nodules results in the synthesis of c4 dicarboxylic acids, viz. aspartate, malate, fumarate etc. which
can be transported into bacteroids with the intervention of dicarboxylate transporter (OCT) protein. PEPC has been purified
from the root nodules of few legu me species. Information on the relationship between nitrogen fixation and carbon
metabolism through PEPC in leguminous plants is scanty and incoherent. This review summarizes the various aspects of
carbon and nitrogen metabolism in legume root nodules .
Keywords: Bacteroids, Carbon metaboli sm, Legume, Nodule, Nitrogen fixation, Phosphocnol pyruvate carboxylase
Nitrogen fixed symbiotically in the nodules of legumes
accounts for over half of nitrogen fixed in nature
annually. The bacteria are released into newly formed
nodule initials and become bacteroids sutTounded by
plant host tissue. The legume nodules use large amounts
of carbohydrates for the fixation of nitrogen and
assimilation of fixed ammonia into organic compounds
as well as for nodule growth and maintenance.
The photosynthates translocated from the leaves
provide carbon skeleton, reducing power and energy
req uired for sy mbiotic nitrogen fixation 1• Sucrose from
the shoot is convet1ed to organic acids, principally
dicarboxylates, which are supplied to bacteroids . to
provide reductant for the support of key enzyme
nitrogenase 2. It has been established during the last two
decades that an anapleurotic carbon dioxide fixation
takes place in nodules, via phosphoenol pyruvate
carboxyl ase (PEPC), a key enzyme for carbon dioxide
fixation in plants, algae, cyanobacteria and bacteria
located in the cytoplasm of host cellsH .
Nitrogen fixation and metabolism
The process of sy mbiotic nitrogen fixation involves
the reduction of dinitrogen to ammonia by nitrogenase
enzy me complex which is composed of Fe and MoFe
proteins.
8H++ 8e· + N2 + 16ATP---t2NH 3 + 1-h + l6ADP+ l6Pi
Ammonia produced by nitrogen fixation is
converted to organic nitrogen before it is exported
from the nodule for utilization by the host plant9 .
*Correspondent author:
E-mail : [email protected]
The initial organic product is the amino acid
glutamine; the assimilation of ammonia into
glutamine is catalysed by two enzymes-glutamine
synthetase (GS) and glutamate synthase (GOGAT) 10 .
In higher plants, GOGAT occurs in two distinct
isoforms, NADH-GOGAT and ferredoxin-dependent
GOGA T (Fd-GOGA T). These differ in molecular
mass, subunit composition, enzyme kinetics, antigenic
and reductant specificity, and metabolic function. Fdand NADH- GOGAT are encoded by distinct genes 11 ' 18 .
One of the most critical problems faced by a
nitrogen-fixing organism is the senstttvtty of
nitrogenase to molecul ar oxygen. Both the Fe protein
and the MoFe protein are rapidly and irreversibly
inactivated by molecular oxygen 19 . This extreme
sensitivity of nitrogenase to oxygen raises a problem
for nitrogen fixi~g organisms since the large amounts
of energy required (in form of ATP and reductant) are
produced through cellular respiratory pathway that
can only operate efficiently when molecular oxygen is
present.
In legume nodules, the oxygen suppl y is regul ated
to a large extent by an oxygen-binding protein called
leghemoglobin which is produced by host plant and
located within the bacteroid-infected host ce11 20 . lt
constitutes as much as 30% of the host cell protein. Its
function is to bind oxygen and control release of
oxygen in the region of the bacteroid. The equilibrium
concentration of oxygen in the bacteroid zone is thus
kept at a level (about lOnM) sufficient to support
bacteroid respiration 21 and the production of ATP and
reducing potential, while preventing excess oxygen
from inactivating dinitrogenase. Oxygen levels must
GARGet. al.: NITROGEN FIXATION AND CARBON METABOLISM IN LEGUME NODULES
be carefully balanced because a too low oxygen
concentration can also limit dinitrogenase activity in
nodules. Thi s is made possible by the ex istence of a
variable nodul e oxygen permeability (Po), which acts
as an oxygen diffusion barrier22 . The large carbon
req uire me nt imposed by symbiosis may be a
conseq uence of limited ATP production by the plant
res ultin g from low oxygen i.1duced fermentative
pathways lead in g to C 4 -dicarboxylic ac id synthesis 23 ·
Carbon supply to bacteroids
The energy for N2 fixation is derived primarily
from plant-produced dicarboxylic acids which are
taken up by the bacteroids. The dicarboxylic acids are
most likely derived from host cytosol, as low oxygen
te11 sions in infected zone of the nodule may limit
mitochondrial respiration.
Although it has been shown that nitrogen fixation
is fuelled by recently synthesized sucrose translocated
24
to the root nodul e , neither sucrose nor hexoses are
readily metabo li sed by isolated bacteroids at rates
capable of su pporting nitrogenase 25-29 . Also the
movement of neutral sugars across the peribacteroid
membrane (PB M) of isolated soybean sy mbiosomes
is only by slow passive diffusion with rates being
03
inadequate to support nitrogenase activit/ · '. In
contrast, there is evidence to prove that dicarboxylates
play a major and essential role in supporting nitrogen
fixation 7 .
14
Tracer studies on the metabolism of C0 2 in
32 33
. The incorporated
alfalfa nodules have been done
radioactivity was found to be higher when the nodules
were attached to roots than when they were isolated
and on fresh weight basis the incorporated
radioactivity was 8-fold higher in the nodules than the
14
C0 2 metaboli sm stud ies do not indicate
roots.
radioactivity associated with glucose and fructose,
most of the radioactivity being in malate and some in
. 34-36
g I utamate, aspartate an d asparagtne
.
Malate, malonate and succinate are abundant in
37
legume nodules 26 · . Of these, malate and succinate
are transpotted rapidl y across the PBM of soybean 3x-4o
41
and French bean • In vitro and in vivo radioactive
studi es of 14C02 metabolism showed high PEPC
activity in the nodul es of genetically diverse c ulti vars
of chickpea ; this activity increased further in the
tolerant cultivars when the plants were subj ected to
42 43
salt stress ' Simil ar genetic variability associated
with host plant in their tolerance to sa linity and
drought have been reported among different legume
44 45 Tl
. and cu ItlVars
.
spectes
· . . 1ese resu Its .m d.tcate t I1e
139
prese nce of a distinct PBM dicarboxyl ate carrier.
Isolated bacteroids also possess a dicarbox ylate
transporter (Oct) capable of rapid rates of malate and
46
.
succmate
transport-? 7· an d dct- mutants o f rI11-zo b.ta
47 49
form ineffective nodules - . Recently the dct operon
has been implicated in the nifA regulatory system of
rhizobia 50 further emphasizing the importance of
dicarboxylates in sy mbiosis. Thus, malate and
succinate are the most effective substrates for
supporting the nitrogenase activity of isolated
bacteroids.
Nodule phosphoenol pyruvate carboxylase
Nodule PEPC constitutes upto 1-2% of total
soluble proteins of efficient nodules 5 1. 52 . Thi s e nzyme
is a tetramer of identical monomers 53 -56 . PEPC is
ubiquitous and especially known as a carbon dioxide
harvesting enzyme. It is located in the mesophyll cells
of c4leaves and also occurs in an inactive state in c3
plants; PEPC works exclusively during ni ght in CAM
plants. The PEPC activity of nodule expressed on
fresh weight basis is as high as the activity generally
found in leaves of C 4 plants'. Unlike the bacterial
enzyme which is allosteric in nature, PEPC
responsible for carbon dioxide fixation in root nodul es
of legumes is non-allosteric plant enzyme.
Oicarboxylic acids resulting from PEPC activity
have been suggested to support bacteroid metaboli sm
7
in support of nitrogenase activiti . Based on
ox idati on of dark carbon dioxide fixation products in
soybean root nodules, !t has been estimated that the
dicarboxylic acids produced by PEPC activity would
be capable of providing 48 % of e nergy for
nitrogenase acti vi ti 8 .
The first product of carbon dioxide fixation via PEPC
is oxaloacetic acid which is hi ghly unstable and is
rapidly reduced into malate by malate dehydrogenase
(MOH) or transaminated into asprutate by aspartate
aminotransferase'· 36 . The malate formed can be utili zed
in three different ways in nodule metabolism. A patt of
malate formed can be used as a carbon and energy
source for the nitrogen fixing bacteroids. Malate can
enter tricarboxylic acid (TCA) cycle to p:·oduce energy
(ATP), reducing power and carbon skeletons used in the
functioning of nitrogenase and biosynthesis of amino
acids from ammonium. Malate could also play a role in
adj ustment of charge balance in vacuoles and in xylem
fluid 38 .
PEPC-gene regulation
Many plants possess multiple isoform s of PEPC,
perhaps each being associated with a diffe,·en:
140
INDIAN J EXP BIOL, FEBRUARY 2004
metabolic pathway. Although PEPC clearly plays a
central ro le in symbiotic nitrogen fixation, little is
known about the mechanisms underlying PEPC gene
expression during establishment and maintenance of
symbiosis.
PEPC from photosynthetic and non-photosynthetic
tissues of a number of C 3 , C 4 and CAM species were
analysed and based on kinetics and ion-exchange
chromatography at least four distinct isoforms of
PEPC were proposed 59 ·60 . These isoforms are the C 4
photosynthetic PEPC present in leaves of C 4 plants,
C 3 PEPC in leaves of C 3 , C 4 and CAM plants, CAM
PEPC in leaves of CAM plants and non-autotrophic
PEPC present in the roots of all plants. The
functionally active form of all these PEPCs consists
of a heterotetramer with a monomeric molecular mass
of 100 kDa.
Studies on PEPC gene expression in nitrogen
fixing (effective) and non-nitrogen fixing (ineffective)
alfalfa nodules suggest that full gene expression is
associated first
with
nodule initiation
and
development and then with initiation and maintenance
of effective symbiosis 61 • Several workers 62 ' 64 have
isolated PEPC cDNAs and genes from a wide variety
of plants. They isolated and characterized an alfalfa
PEPC gene (PEPC-7) whose transcripts were found at
elevated leve ls in nodules, relative to either roots or
leaves. The intron/exon structure of this gene was
identical to that of most other plant PEPC genes
except for the presence of an additional intron in 5'
untranslated region. The analysis of promoter
deletions suggests that the region between - 634 and536 is of particular importance in directing
transcriptional activity to the infected zone of
nodules. Thus, PEPC-7 has a central role in nitrogen
fixing nodules and the regulation of transcription is an
important determinant of its activity.
Conclusion
The limited need of oxygen for proper functioning
of nitrogenase results in energy burden on symbiotic
nitrogen fixation. This need for energy is
compensated by carbon dioxide fixation via PEPC in
plant part of nodules. The dicarboxylic acids supplied
to bacteroids provide reductants for support of
nitrogenase. However, detailed information on the
structure, function and genetic control of PEPC
activity in a wide spectrum of legume species is
lacking. Such information is expected to be helpful in
the breeding programmes to improve symbiotic
nitrogen fixing efficiency of legumes.
Acknowledgement
We thankfully acknowledge Dr. Carro ll P. Vance,
US Department of Agriculture, Univ . of Minnesota,
USA and Dr. R. Serraj, ICRISAT, Patancheru for
their suggestions on the manuscript. We also thank
Prof. G. S. Randhawa, IIT, Roorkee for critical
reading and necessary corrections of the manuscript.
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