Journal of Experimental Botany, Vol. 52, No. 355, pp.
285±293, February 2001
Abscisic acid induces a decline in nitrogen fixation that
involves leghaemoglobin, but is independent of sucrose
synthase activity
Esther M. GonzaÂlez, Loli GaÂlvez and Cesar Arrese-Igor1
Departamento de Ciencias del Medio Natural, Universidad PuÂblica de Navarra, Campus de ArrosadõÂa,
E-31006 Pamplona, Spain
Received 11 April 2000; Accepted 18 September 2000
Abstract
Sucrose synthase (SS) activity has been suggested to
be a key point of regulation in nodule metabolism
since this enzyme is down-regulated in response to
different stresses which lead to decreased nitrogen
fixation. In soybean, a dramatic decline of SS tran-
scripts has been observed within 1 d from the onset
of drought. Such a quick response suggests media-
tion by a signal transduction molecule. Abscisic acid
(ABA) is a likely candidate to act as such a molecule
as it mediates in a significant number of plant
responses to environmental constraints. The hypo-
thesis of ABA controlling nodule metabolism was
approached in this work by assessing nodule
responses to exogenous ABA supply in pea. Under
the experimental conditions, ABA did not affect plant
biomass, nodule numbers or dry weight. However,
nitrogen fixation rate was reduced by 70% within 5 d
and by 80% after 9 d leading to a reduced plant
organic nitrogen content. Leghaemoglobin (Lb)
content declined in parallel with that of nitrogen
fixation. SS activity, however, was not affected by
ABA treatment, and neither were the activities of
the enzymes aspartate amino transferase, alkaline
invertase, malate dehydrogenase, glutamate syn-
thase, uridine diphosphoglucose pyrophosphorylase,
isocitrate dehydrogenase, and glutamine synthetase.
Nodule bacteroid-soluble protein content was
reduced in nodules only after 9 d of ABA treatment.
These results do not support the hypothesis that
ABA directly regulates SS activity. However, they do
suggest the occurrence of at least two different
control pathways in nodules under environmental
constraints, which include ABA being involved in a Lb/
oxygen-related control of nitrogen fixation.
1
To whom correspondence should be addressed. Fax: q34 948 168930. E-mail: cesarai@unavarra.es
ß Society for Experimental Biology 2001
Key words: Abscisic acid, leghaemoglobin, nodule meta-
bolism, Pisum sativum L., sucrose synthase.
Introduction
Nitrogen ®xation in legume nodules is severely affected
by different environmental constraints, such as drought,
salinity, defoliation, darkness, chilling, and nitrate
supply. This decline in nitrogen ®xation in response to
stress has been explained by means of a closure of the
oxygen diffusion barrier to reduce oxygen ¯ux into the
nodule and avoid nitrogenase damage (Witty et al., 1986;
Hunt and Layzell, 1993), although the mechanism of such
a response is not yet determined. Recently, it has been
proposed that nodule metabolism may respond to the
different stresses regardless of the changes in nodule
permeability (Diaz del Castillo et al., 1994; Diaz del
Castillo and Layzell, 1995). GonzaÂlez et al. found a rapid
decline of both sucrose synthase (SS) activity and protein
content in soybean plants subjected to a moderate water
stress (GonzaÂlez et al., 1995). Durand et al. and Djekoun
and Planchon showed that nitrogen ®xation was more
sensitive than photosynthesis to moderate water depriva-
tion (Durand et al., 1987; Djekoun and Planchon, 1991),
thus SS decline does not seem to be triggered by
photosynthate shortage. Moreover, sucrose is accumu-
lated in nodules subjected to water stress (GonzaÂlez et al.,
1995). Thus, it has been suggested that the impairment
of sucrose metabolism within the nodule could be
responsible for the nitrogen ®xation decline by limiting
the carbon ¯ux for bacteroid respiration (GonzaÂlez et al.,
1995; Gordon et al., 1997; Arrese-Igor et al., 1999).
Gordon et al. found that the decline in SS activity in
response to moderate water stress, was related to the
286 GonzaÂlez et al.
down-regulation of the SS gene within 1 d (Gordon et al.,
1997). Also, salt and nitrate supply affect SS at the
expression level. Altogether these data point to the key
role of SS in the regulation of nodule metabolism.
However, the signal transduction pathway that links the
perception of these environmental stresses and the decline
of SS activity remains unknown.
Levels of the plant hormone abscisic acid (ABA)
increase as a result of water stress, playing an import-
ant role in the plant response to drought, salinity and
cold; stresses that involve cellular water stress. Since
levels of endogenous ABA increase in tissues subjected to
osmotic stress (Henson, 1984, Mohapatra et al., 1988), it
appears to mediate physiological processes in response to
osmotic stress. Under these conditions, speci®c genes are
expressed that can also be induced in unstressed tissues
by the application of exogenous ABA (GoÂmez et al.,
1988; Mundy and Chua, 1988).
It is thought that some of these ABA-responsive genes
may encode proteins with osmoregulatory or other pro-
tective functions. Guan and Scandalios found that ABA
leads to an increase of superoxide dismutase expression
(Guan and Scandalios, 1998) which suggests that ABA-
mediated metabolic changes lead to increases in the anti-
oxidant defence system. Furthermore, it has been shown
that ABA disrupts cortical actin ®laments (Eun and Lee,
1997), which may lead to structural regulation of ion-
channel activity or cytoskeletal regulation of signalling
molecules, such as protein kinases and phosphatases. SS
activity was shown to be modulated by a reversible phos-
phorylation mechanism in maize leaves (Huber et al.,
1996) and the same post-transcriptional regulation in soy-
bean nodule SS has also been found (Zhang and Chollet,
1997). Recently, SS regulation has been examined fur-
ther and it was found that phosphorylation modi®es the
hydrophobicity of the enzyme, suggesting that regulation
could be mediated by partitioning between soluble and
membrane-bound enzyme (Zhang et al., 1999).
Although ABA is the major signal transduction
operating during drought stress, not all drought-induced-
genes are regulated by ABA (see review by Shinozaki
and Yamaguchi-Shinozaki, 1997, and references therein).
Studies conducted on ABA-de®cient or ABA-insensitive
Table 1. Effect of ABA on plant growth
Plant dry weight, shooturoot ratio, nodule dry weight and numbers, and shoot and root organic nitrogen content of pea plants watered with nutrient
solution (initial control at day 0 and ®nal control after 9 d or with nutrient solution containing ABA for 9 d (ABA). Values represent mean"standard
error (n ˆ 9). For each parameter, numbers followed by a different letter are signi®cantly different at PF0.05.
Plant dry weight (g)
Shooturoot ratio (g g 1)
Nodule dry weight (mg)
Nodule numbers
Organic nitrogen shoot (mg g 1 DW)
Organic nitrogen root (mg g 1 DW)
mutants have indicated that several drought- anduor
cold-induced genes are expressed independently of ABA
(Nordin et al., 1991; Gosti et al., 1995). A desiccation
responsive gene in Arabidopsis is rapidly induced by
water and salt stresses in an ABA-independent manner
(Yamaguchi-Shinozaki and Shinozaki, 1993). A tran-
script of a MAP kinase gene in alfalfa accumulates after
drought and cold treatment, but not in response to ABA,
indicating that this kinase pathway mediates drought
and cold signalling independently of ABA (Jonak et al.,
1996). Recently, it has been found that two SS genes of
Arabidopsis were modulated by sugaruosmoticum levels in
an ABA-independent manner (DeÂjardin et al., 1999).
The primary nodule response observed following
the occurrence of abiotic stresses has been the down-
regulation of SS, but as yet there is no direct evidence as
to which signal triggers this mechanism. The aim of this
study has been to ascertain whether ABA is involved
in the signal transduction pathway of down-regulation of
SS activity in nodules. Time-course activities of key
enzymes of carbohydrate and nitrogen metabolism were
examined in relation to nitrogen ®xation rates and levels
of carbohydrates and amino acids in plants supplied with
exogenous ABA.
Materials and methods
Growth conditions and experimental procedures
Pea plants (Pisum sativum L. cv. Sugar snap) were inoculated
with Rhizobium leguminosarum biovar viciae strain NLV 8. This
strain is Hup±, according to Southern-blot analysis of EcoRI-
digested total DNA using a hup-speci®c DNA probe prepared
by dioxigenin labelling of cosmid pAL618 containing the entire
hup gene cluster from Rhizobium leguminosarum bv. viciae strain
UPM791 (Matamoros et al., 1999). Plants were grown in 1 dm3
pots, with a 2 : 1 (v : v) mixture of vermiculite:perlite as the sub-
strate, with a nutrient solution lacking nitrogen (Rigaud and
Puppo, 1975) in a controlled environment chamber (22u18 8C
dayunight temperature, 70% relative humidity, 500 mmol m±2 s±1
(PPF), and 15 h photoperiod).
When plants were 4-weeks-old, a set was watered daily with
50 ml of a nutrient solution containing 100 mmol m 3 ABA and
a control set was watered daily with the same volume of nutrient
solution. This represents twice the daily transpiration rate obser-
ved in these plants. ABA was supplied in the form of "ABA
Initial control Final control ABA
0.67"0.06 b 0.95"0.11 a 1.13"0.09 a
2.60"0.17 a 3.09"0.11 a 2.99"0.18 a
64"3 b 73"5 a 69"3 ab
246"20 a 233"14 a 242"17 a
39.7"2.1 a 41.4"1.9 a 34.7"0.8 b
26.4"0.8 a 24.9"1.0 ab 23.5"0.6 b

(Sigma Chemical Co). This concentration has been shown to be
adequate to simulate the regulation of the plant water status in
legumes (Pardossi et al., 1992). Data were collected in three
independent experiments. Data in Table 1 re¯ects the mean of
the three experiments, whilst the remaining data were collected
in two out of the three experiments performed. Physiological
determinations were carried out at days 0, 1, 5, and 9. Leaf and
nodule samples for protein extraction and analytical determina-
tion were frozen in liquid nitrogen and stored at 80 8C.
Nodules, roots and shoots were separated and dried for 48 h at
70 8C for dry weight determinations.
Water relations and gas exchange measurements
Leaf water potential was measured 2 h after the beginning of the
photoperiod using a pressure chamber (Soil Moisture Equip-
ment, Santa Barbara, CA) as described previously (Scholander
et al., 1965), assuming that total leaf c is in equilibrium with
petiole c. Osmotic potential was determined on the same leaf
with a vapour pressure osmometer (Wescor Inc. 5500, Logan,
UT), after samples were frozen and thawed (Frechilla et al.,
1999). The turgor potential was calculated by the difference
between water and osmotic potential.
Net CO2 assimilation rate, leaf conductance and intercellular
CO2 were measured with a portable IRGA (LI-6200, Li-Cor,
Lincoln, NE). Transpiration rate per plant was calculated gra-
vimetrically each day of the experiment; a substrate-®lled pot
containing no plants was used to estimate evaporation losses.
For nitrogen ®xation determinations, H2 evolution of the
intact plants, whose root systems were sealed into the growth
pots and housed in the growth chamber, was measured in an
open ¯ow-through system (according to Witty and Minchin,
1998) using an electrochemical H2 sensor (Qubit System Inc.,
Canada). The detector was calibrated with high purity gases
(Praxair, Madrid, Spain) using a gas mixer (Air Liquid, Madrid,
Spain) ¯owing at the same rate as the sampling system
(100 ml min 1). Apparent nitrogenase activity (ANA) was deter-
mined under N2 : O2 (79% : 21%). After reaching steady-state
conditions total nitrogenase activity (TNA) was determined
under Ar : O2 (79% : 21%). Nitrogen ®xation rate (NFR) was
calculated as (TNA ANA)u3. Electron allocation coef®cient to
N2 (EAC ) was calculated as a percentage of (1 (ANAuTNA)).
Extraction and assay of enzymes
Nodules were homogenized in a mortar and pestle with
50 mol m 3 MOPS, 10% PVPP, 10 mol m 3 DTT, 1 mol m 3
EDTA, 20 mol m 3 KCl, 5 mol m 3 MgCl2, pH 7 at 0±2 8C
(5 cm3 g 1 fresh weight). The homogenate was centrifuged for
30 min at 20 000 g, 2 8C.
Samples (50 mm3) of the supernatant were retained for
the phosphoenol pyruvate carboxylase (EC 4.1.1.31) assay
(GonzaÂlez et al., 1995) and for protein (Bradford, 1976) and
leghaemoglobin (Lb) determinations (Appleby and Bergersen,
1980). One cm3 aliquots were desalted by low speed centrifuga-
tion (180 g, 1 min) through 5 cm3 columns of Bio Gel P6DG
(BioRad) equilibrated with the extraction buffer (see above)
without PVPP. The desalted extract was used to determine the
following enzyme activities: alkaline invertase (EC 3.2.1.26),
aspartate amino transferase (EC 2.6.1.1), glutamate synthase
(EC 1.4.1.14), glutamine synthetase (EC 6.3.1.2), malate
dehydrogenase (EC 1.1.1.37), sucrose synthase (EC 2.4.1.13)
and uridine diphosphoglucose pyrophosphorylase (EC 2.7.7.9)
(according to GonzaÂlez et al., 1998) and NADP-dependent
isocitrate dehydrogenase (EC 1.1.1.42) (according to Chen et al.,
1988).
Abscisic acid and nitrogen fixation 287
The pellet remaining after removing the host plant soluble
proteins by centrifugation (see above) was washed (1 cm3
extraction buffer) and centrifuged three times for 10 min at
14 000 g, on each occasion discarding the washings. The pellet
was then resuspended in extraction buffer without PVPP
(5 cm3 g 1 original nodule fresh weight) and the bacteroids
broken by sonication (10 3 30 s, 2 8C). The supernatant after
centrifugation (20 000 g, 2 8C 30 min) was used for bacteroid
protein determination (according to Bradford, 1976).
Analytical determinations
Fresh material was exhaustively extracted in boiling 80% (vuv)
ethanol. Ethanol soluble extracts were dried in a Turbovap LV
evaporator (Zymark Corp, Hopkinton, MA) and soluble com-
pounds were redissolved with 4 cm3 of distilled water, mixed
and centrifuged at 20 000 g for 10 min. Total soluble sugars
were measured in the supernatant by the anthrone method
(Spiro, 1966) and sucrose (according to GonzaÂlez et al., 1995).
Free amino acids were assayed using the acid ninhydrin method
of Yemm and Cocking (Yemm and Cocking, 1955). Malate was
also analysed in this supernatant by ion chromatography in
a DX-500 system (Dionex, Salt Lake City, UT) by gradient
separation with a Dionex IonPac AS11 column (2.5 mol m 3
NaOHu18% methanol to 45 mol m 3 NaOHu18% methanol in
13 min).
The ethanol-insoluble residue, remaining after the extraction
of soluble compounds, was extracted for starch (as in MacRae,
1971), and the glucose produced was analysed enzymatically
(as described by GonzaÂlez et al., 1995).
Organic nitrogen content was determined by Kjeldahl analysis
(AOAC, 1990).
Statistical analysis
Results were examined by one-way analysis of variance. All
effects discussed in this study were signi®cant at PF0.05 in
Fisher's (protected) least signi®cant difference (LSD) tests
between means.
Results
The effect of ABA on plant growth was monitored
throughout the 9 d of the experimental period. The effect
of the daily supply of 50 ml of nutrient solution contain-
ing ABA was monitored in relation to controls, where
the same volume of nutrient solution without ABA was
given. Table 1 summarizes growth parameters at the end
of the experimental period. ABA supply did not affect
plant dry weight, shoot to root ratio, nodule numbers or
nodule dry weight (Table 1). Conversely, organic nitro-
gen content declined signi®cantly in shoots as a con-
sequence of ABA treatment, although organic nitrogen
content of roots was less affected (Table 1).
Transpiration rate, calculated gravimetrically, of ABA-
treated plants declined gradually from the onset of ABA
supply to day 5 and afterwards it remained at values that
represented c. 20% of the initial controls (Fig. 1B). This
was in agreement with the decline of stomatal conduc-
tance in ABA-treated plants within 1 d of ABA supply
to 65% of controls, with a further decline thereafter
288 GonzaÂlez et al.
(Fig. 1A). Stomatal closure led to a slight decrease in
intercellular CO2 concentration in ABA-treated plants
that also progressed with time (Fig. 1D). Despite the
rapid stomatal closure and the decreased intercellular
CO2 concentration, photosynthesis was maintained at
rates c. 90% of control values (Fig. 1C). The photosyn-
thetic rate is likely to be only slightly affected under
these conditions because the decreased intercellular CO2
concentration following stomatal closure allowed the
maintenance of CO2 ¯ux into the leaves. Under these
conditions, the relatively low rates of carbon assimilation
(Fig. 1C) are likely to be still CO2-saturated, possibly
associated with a moderate photosynthetic photon ¯ux in
the growth chamber, and it remains to be determined
Fig. 1. Effect of ABA on plant gas exchange. Stomatal conductance,
gravimetric transpiration rate, net photosynthesis and intercellular CO2
concentration in pea plants watered with nutrient solution containing
ABA and their corresponding controls throughout the study period.
Values represent mean"standard error (n ˆ 6), except for transpiration,
where n reduces from 24 (0 d) to 6 (9 d).
Fig. 2. Effect of ABA on plant water status. Leaf water potential, osmotic potential and turgor potential in pea plants watered with nutrient solution
containing ABA and their corresponding controls throughout the study period. Values represent mean"standard error (n ˆ 6).
whether this moderate effect on photosynthesis also
occurs under saturating light intensity.
The water potential of ABA-treated plants was slightly
higher than that of control plants throughout the study
(Fig. 2A). Plants experienced a rapid stomatal closure
in the presence of ABA (Fig. 1A) whilst plants did not
experience water shortage. Thus, both osmotic (Fig. 2B)
and turgor potential (Fig. 2C) showed a transient varia-
tion after 1 d of ABA treatment, possibly re¯ecting the
balance between full availability of water and stomatal
closure, but came back to control values during the study
period.
Apparent and total nitrogenase activities were not
affected within 1 d after ABA supply, but they declined
Fig. 3. Effect of ABA on nitrogen ®xation. Apparent nitrogenase
activity (ANA), total nitrogenase activity (TNA), nitrogen ®xation rate
(NFR) and electron allocation coef®cient (EAC) of pea plants watered
with nutrient solution containing ABA and their corresponding controls
throughout the study period. NDW denotes nodule dry weight. Values
represent mean"standard error (n ˆ 6).

signi®cantly after 5 d (Fig. 3A, B). Nitrogen ®xation rate,
calculated as the difference between TNA and ANA and
the theoretical relationship between H2 and N2 reduction,
showed a similar pattern to the above parameters
(Fig. 3C). However, EAC was stable within the ®rst 5 d
and showed a signi®cant decline at day 9 (Fig. 3D).
The Lb content of ABA-treated plants showed a signi-
®cant decline after 5 d (Fig. 4B). At that time, nodule
plant fraction and bacteroid soluble protein contents did
not show any signi®cant effect by ABA, whilst the latter
only declined by day 9 (Fig. 4C, D). Conversely to Lb, SS
activity was virtually unchanged throughout the experi-
ment (Fig. 4A). Measured SS activity was completely
abolished by the glycoside arbutin (data not shown),
showing that the lack of changes in SS activity was not
due to artefactual, side reactions. Other enzyme activities
involved in nitrogen and carbon metabolism in nodules
of ABA-treated plants (alkaline invertase (0.17), UDP-
glucose pyrophosphorylase (1.22), phosphoenol pyruvate
carboxylase (0.21), malate dehydrogenase (15.7), isocit-
rate dehydrogenase (0.32), glutamine synthetase (0.14),
glutamate synthase (0.018), and aspartate amino trans-
ferase (0.61, all in mmol product mg 1 protein min 1)) did
not show any signi®cant variation during the studied
period.
ABA supply led to a transient total soluble sugar
accumulation in the leaf (Fig. 5A). However, as control
plants also showed a trend to accumulate total soluble
sugars with age, control and treated plants did not show
any difference at the end of the experiment. Likewise,
Fig. 4. Effect of ABA on nodule sucrose synthase activity, leghaemo-
globin and protein content. Sucrose synthase activity, leghaemoglobin
content, protein content of the plant and bacteroid fractions determined
in nodules of pea plants watered with nutrient solution containing ABA
and their corresponding controls throughout the study period. Values
represent mean"standard error (n ˆ 6).
Abscisic acid and nitrogen fixation 289
starch content increased in leaves in response to ABA
(Fig. 5C). Conversely, leaf total free amino acids of ABA-
treated plants was signi®cantly lower than controls at the
end of the study period, in agreement with values of shoot
organic nitrogen content (Table 1), as a consequence of
the sharp decline in nitrogen ®xation experienced by the
ABA-treated plants after 5 d of treatment (Fig. 3).
No signi®cant changes were found in nodule carbo-
hydrates (Fig. 5B) and starch (Fig. 5D). Despite the
reduced nitrogen ®xation rate found in ABA-treated
plants, amino acid content was not affected in nodules,
although a transient increase could be detected after 1 d
of ABA supply (Fig. 5F), possibly re¯ecting a decreased
xylem ¯ux, as a consequence of stomatal closure
(Fig. 1A).
Nodule sucrose content was not affected by ABA
treatment, with values almost constant c. 3 mg g 1 nodule
fresh weight, suggesting that nodules did not experience
photosynthate shortage. Moreover, the concentration of
malate, the organic acid that fuels bacteroid metabolism,
was also unaffected by the ABA treatment, with values
c. 1.4 mg g 1 nodule fresh weight.
Fig. 5. Effect of ABA on leaf and nodule carbohydrates and amino
acids. Total soluble sugars, starch and amino acids content in leaves and
nodules of pea plants watered with nutrient solution containing ABA
and their corresponding controls throughout the study period. Values
represent mean"standard error (n ˆ 6).
290 GonzaÂlez et al.
Discussion
Roots in drying soil produce ABA (Cornish and
Zeevaart, 1985), which is transported to leaves (Zhang
and Davies, 1989). The increased concentration of ABA
stimulates stomatal closure and reduces transpirational
water loss from leaves, therefore preventing plant's
drying. Thus, plants integrate the opposite demands of
maximizing CO2 uptake and minimizing transpiration
water loss (Cowan, 1982; Socias et al., 1997). It has been
shown that nodulated legumes showed a better gas
exchange response to drought than non-nodulated plants,
a fact that is related to the hormone balance (Goicoechea
et al., 1997) and to complex interactions with photo-
respiratory ¯ux and stomatal conductance (Frechilla et al.,
2000). In this study, transpiration rate and conductance
were signi®cantly reduced by ABA supply, showing that
ABA was ef®ciently translocated through the xylem,
although photosynthesis rate was only slightly affected
throughout the study (Fig. 1). Under these experimental
conditions, total soluble sugar accumulated in the leaf
within 1 d from the onset of ABA supply (Fig. 5A) and
leaf starch content was also higher than control values
throughout the study period (Fig. 5C), which con®rms
that CO2 ®xation was not markedly affected by this
treatment. In addition, plant growth was not affected by
ABA (Table 1), which is consistent with an uninhibited
photosynthetic process. Since the aim of this study was to
examine ABA effects on nodule metabolism, this experi-
mental situation where photosynthesis was unaffected
was an important requisite in order to avoid interference
between the ABA effect and a possible limitation of pho-
tosynthate supply to nodules. As expected, the stomatal
closure triggered by ABA (Fig. 1) in an environment
where plants do not actually experience water shortage,
leads to an increase in leaf water potential (Fig. 2A). This
is the opposite of the situation found in water-stressed
plants and, therefore, the conclusions drawn from this
study should be understood in the context of the likely
involvement or not of ABA in the signal transduction
pathway that links drought perception and a decreased
SS activity which, in turn, leads to a decline in nodule
nitrogen ®xation. However, at the whole-plant level, it
cannot be expected that exogenous ABA supply would
mimic every physiological plant response to drought.
A signi®cant reduction both in ANA and TNA was
described in a conventional closed standard system of
ABA-treated plants of Faba vulgaris, without any marked
effect on growth (Bano and Hillman, 1986). However, the
interpretation of such results is complicated in view of the
problems associated with the standard assay (Minchin
et al., 1983). Indeed, they suggested a lack of photo-
synthate supply to nodules as the main cause of nitrogen
®xation decline, since lower leaves of ABA-treated plants
showed an early senescence. However, the ABA treatment
performed in this study did not affect nodule mass (in
agreement with the results of Bano and Hillman,1986),
but, in addition, no photosynthate shortage to nodule
metabolism is evident in view of the concentrations of
total soluble sugars (Fig. 5B), sucrose and malate.
It was found that SS may be regulated in soybean
nodules by reversible phosphorylation (Zhang and
Chollet, 1997) and, moreover, at least two different sites
were found to be susceptible to phosphorylation by
a calcium-dependent protein kinase (Zhang et al., 1999).
ABA activation of protein kinases has been described in
different systems (Esser et al., 1997; Burnett et al., 2000;
Hong et al., 1997). However, SS activity was not affected
by ABA when nitrogen ®xation was inhibited by 80%,
ruling out the hypothesis of an ABA-mediated SS
response to environmental stresses. DeÂjardin et al. found
expression of two SS genes in Arabidopsis differentially
expressed in relation to wateruosmoticum stresses and
both were independent of ABA signals (DeÂjardin et al.,
1999), Sus1 expression was related to the perception of
osmotic potential decreases and Sus2 was independent of
sugar and osmoticum signal. In other plant systems, SS is
known to be induced by changes in carbohydrate availab-
ility (Koch et al., 1992; Heim et al., 1993; Koch, 1996) and
by low O2 environments (Taliercio and Chourey, 1989;
Ricard et al., 1991; Xue et al., 1991). However, both
regulation mechanisms seems to have a limited role in
nodules (Arrese-Igor et al., 1999).
Lb changes have been suggested as the main effect on
nodule metabolism caused by severe drought in indeter-
minate nodules (Guerin et al., 1991; Irigoyen et al., 1992),
suggesting that the decline in nitrogen ®xation was due
to a reduction in Lb content which would limit oxygen
supply to bacteroids and affect respiration and energy
production. Indeed, under those conditions an increase
in nodule malate content was reported (Irigoyen et al.,
1992). This would be consistent with a decreased oxygen
supply and the induction of fermentative pathways (De
Vries et al., 1980). Lb changes have been also described
following other environmental stresses, such as salinity
(Abd-Alla, 1992), darkness (Gogorcena et al., 1997) or
nitrate (Escuredo et al., 1996) and related, at least in
part, with the decline in nitrogen ®xation. However,
such effects are not evident when these stresses occur
in a moderate and gradual situation (GonzaÂlez et al.,
1995, 1998; Gordon et al., 1997). Moreover, preliminary
data suggest that the down-regulation of SS during
drought is followed by a depletion of organic acids in
nodules under mild drought (L GaÂlvez, EM GonzaÂlez, C
Arrese-Igor, unpublished results). Lb declined in nodules
of ABA-treated plants (Fig. 4B) in parallel to nitrogen
®xation (Fig. 3) after 5 d of ABA treatment prior to any
detectable change in the protein content of the plant
and bacteroid fractions of nodules (Fig. 4C, D). The
pathway of Lb degradation in vivo is largely unknown.

Most studies suggest that Lb degradation is due to
a particularly rapid activation or decompartmentation
of proteases located in the infected cells that have a high
af®nity for Lb at the acidic intracellular pH that may
occur under different stresses (Pladys et al., 1991).
Substantial decline of the bacteroid fraction protein
was observed after 9 d treatment, suggesting irreversible
damage caused by ABA (Fig. 4D). This is further sup-
ported by a marked decline in the EAC (Fig. 3D). A
marked reduction in the functional bacteroid tissue was
also described after preliminary examination of nodule
anatomy of plants treated with ABA for 14 d (Bano and
Hillman, 1986). An unexpected feature was the low EAC
shown by this symbiosis (Fig. 3D). Although the theor-
etical value for EAC is 0.75, in vivo losses of H2 from
legumes are often much greater than 25% of the nitro-
genase electron ¯ux. Moreover, EAC seems to respond to
a variety of factors, such as temperature and ATP and
reducing power availability. It has also been shown that
the same bacterial strain may display different relative
ef®ciencies when nodulating different hosts (LoÂpez et al.,
1982). Indeed, when this strain nodulates another pea
variety (Frilene) EAC was shown to be 0.67. Moreover,
changes in carbon availability lead to a decrease in EAC
to 0.52 (PM Cabrerizo, EM GonzaÂlez, PM Aparicio-Tejo,
C, Arrese-Igor, unpublished results). The basis for this
effect is not understood yet.
It has been shown that a moderate and progressive
water de®cit stress, triggers the SS response in nodules
(GonzaÂlez et al., 1995, 1998; Gordon et al., 1997). In these
studies, SS activity and expression were down-regulated,
whilst Lb was not affected. Thus, exogenous ABA supply
seems to simulate a situation of abrupt stress. A severe
drought might also lead to Lb reduction (Guerin et al.,
1991; Irigoyen et al., 1992) mediated by a rapid rise in
ABA levels. Therefore, the stress intensity may determine
nodule responses. It remains to be determined whether
the decline in nitrogen ®xation is solely caused by the
decreased Lb levels. Indeed, at day 5, when nitrogen
®xation declined by 70% (Fig. 3C), Lb concentration
only declined by 50% (Fig. 4B). Thus, it cannot be dis-
carded that a second factor, such as direct effects of ABA
on nodule O2 diffusion may also be involved in the
decline of nitrogen ®xation. In conclusion, the occurrence
of at least two different control pathways in nodules
under environmental constraints is put forward, with
ABA being involved in a Lbuoxygen-related control of
nitrogen ®xation under abrupt or severe stresses, whilst
mild or gradually developed stresses would be mediated
by an ABA-independent SS pathway.
Acknowledgements
Authors thank Anthony J Gordon, Pedro M Aparicio-Tejo and
Mercedes Royuela for their critical reading of the ms, Monica
Abscisic acid and nitrogen fixation 291
Ayerra for technical assistance, Leszek Kleczkowski for provid-
ing results prior publication, TomaÂs Ruiz-ArguÈeso for valuable
advice on relative ef®ciency and the unknown referees for
their constructive comments. Seeds were kindly supplied by
Bonduelle (Milagro, Spain). Loli GaÂlvez was the holder of a
grant from the Spanish Ministry of Education (Plan FPU). This
work was supported by DGESIC (PB98-0545) and CICYT
(AGF97-0458).
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