Production and function of jasmonates in nodulated roots of soybean plants inoculated with Bradyrhizobium japonicum.

Abstract

Little is known regarding production and function of endogenous jasmonates (JAs) in root nodules of soybean plants inoculated with Bradyrhizobium japonicum. We investigated (1) production of jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) in roots of control and inoculated plants and in isolated nodules; (2) correlations between JAs levels, nodule number, and plant growth during the symbiotic process; and (3) effects of exogenous JA and OPDA on nodule cell number and size. In roots of control plants, JA and OPDA levels reached a maximum at day 18 after inoculation; OPDA level was 1.24 times that of JA. In roots of inoculated plants, OPDA peaked at day 15, whereas JA level did not change appreciably. Shoot dry matter of inoculated plants was higher than that of control at day 21. Chlorophyll a decreased more abruptly in control plants than in inoculated plants, whereas b decreased gradually in both cases. Exogenous JA or OPDA changed number and size of nodule central cells and peripheral cells. Findings from this and previous studies suggest that increased levels of JA and OPDA in control plants are related to senescence induced by nutritional stress. OPDA accumulation in nodulated roots suggests its involvement in "autoregulation of nodulation."

Full text

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Arch Microbiol (2012) 194:837–845
DOI 10.1007/s00203-012-0817-y
123
ORIGINAL PAPER
Production and function of jasmonates in nodulated roots
of soybean plants inoculated with Bradyrhizobium japonicum
María Emilia Costanzo · Andrea Andrade ·
María del Carmen Tordable · Fabricio Cassán ·
Guillermina Abdala
Received: 13 June 2011 / Revised: 28 February 2012 / Accepted: 11 April 2012 / Published online: 1 May 2012
© Springer-Verlag 2012
Abstract
tion of endogenous jasmonates (JAs) in root nodules of
soybean plants inoculated with Bradyrhizobium japonicum.
We investigated (1) production of jasmonic acid (JA) and
12-oxophytodienoic acid (OPDA) in roots of control and
inoculated plants and in isolated nodules; (2) correlations
between JAs levels, nodule number, and plant growth dur-
ing the symbiotic process; and (3) eVects of exogenous JA
and OPDA on nodule cell number and size. In roots of con-
trol plants, JA and OPDA levels reached a maximum at day
18 after inoculation; OPDA level was 1.24 times that of JA.
In roots of inoculated plants, OPDA peaked at day 15,
whereas JA level did not change appreciably. Shoot dry
matter of inoculated plants was higher than that of control
at day 21. Chlorophyll a decreased more abruptly in control
plants than in inoculated plants, whereas b decreased grad-
ually in both cases. Exogenous JA or OPDA changed num-
ber and size of nodule central cells and peripheral cells.
Findings from this and previous studies suggest that
increased levels of JA and OPDA in control plants are
related to senescence induced by nutritional stress. OPDA
accumulation in nodulated roots suggests its involvement in
“autoregulation of nodulation.”
Little is known regarding production and func-
Keywords
Bradyrhizobium · Jasmonates · Soybean
Abbreviations
AOC
AON
AOS
DM
JA
JAs
LOX
MeJA
OPDA 12-Oxophytodienoic acid
Allene oxide cyclase
Autoregulation of nodulation
Allene oxide synthase
Dry matter
Jasmonic acid
Jasmonates
Lipoxygenase
Methyl-jasmonate
Introduction
The Gram-negative soil bacterium Bradyrhizobium japoni-
cum induces root nodulation and nitrogen (N2) Wxation in
soybean [Glycine max (L.) Merrill], its principal legumi-
nous host. Formation of an N2-Wxing root nodule is a
complex developmental event that depends on a speciWc
chemical “dialogue” between a prokaryote (microsymbi-
ont) and a eukaryote (macrosymbiont). Various phytohor-
mones (auxins, cytokinins, gibberellins) have long been
known as “dialogue molecules” or “key signals” for nodule
development. The roles of molecules other than these “tra-
ditional phytohormones” in legume nodule development
have received increasing attention during the last two
decades. In particular, the group of lipid hormones known
as jasmonates (JAs) has been found to be involved in sev-
eral aspects of soybean–bacteria interaction and proposed
as a new class of naturally occurring inducers of Nod factor
production, one of the Wrst steps in the interaction between
root cells and rhizobial bacteria (Rosas et al. 1998; Hause
and Schaarschmidt 2009; Ferguson et al. 2010).
JAs belong to a family of oxygenated fatty acid deriva-
tives, collectively termed oxylipins, that are produced via
Communicated by Ursula Priefer.
M. E. Costanzo · A. Andrade · M. del Carmen Tordable ·
F. Cassán (&) · G. Abdala
Departamento de Ciencias Naturales, Facultad de Ciencias
Exactas, Físico-Químicas y Naturales, Universidad Nacional
de Río Cuarto, 5800 Río Cuarto, Córdoba, Argentina
e-mail: fcassan@exa.unrc.edu.ar

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838Arch Microbiol (2012) 194:837–845
123
oxidative metabolism of polyunsaturated fatty acids. Syn-
thesis and signal transduction pathways of JAs were
recently reviewed (Wasternack and Kombrink 2010). The
initial substrates are ?-linolenic acid (?-LeA; C18:3) or
hexadecatrienoic acid (C16:3) released from plastidial
galactolipids by phospholipases. Following oxidation of
?-LeA by lipoxygenase (LOX) to 13(S)-hydroperoxyocta-
decatrienoic acid (13(S)-HPOT), the Wrst committed step of
jasmonic acid (JA) biosynthesis is conversion of the LOX
product to the allene oxide 12,13(S)-epoxyoctadecatrienoic
acid (12,13(S)-EOT) by allene oxide synthase (AOS). This
unstable compound is enzymatically cyclized by allene
oxide cyclase (AOC) to cis-(+)-12-oxophytodienoic acid
((9S,13S)-OPDA), which is the end-product of the plastid-
localized part of the JA biosynthesis pathway, and has the
same stereochemical conWguration as naturally occurring
(+)-7-iso-JA. Translocation of 12-oxophytodienoic acid
(OPDA) into peroxisomes, where the subsequent part of the
JA biosynthesis pathway occurs, is mediated by the ABC
transporter COMATOSE and/or an ion-trapping mecha-
nism (Theodoulou et al. 2005). The Wnal step is reduction
of the cyclopentenone ring, catalyzed by a peroxisomal
OPDA reductase (OPR), to yield JA.
After JA is formed, methylation by a JA-speciWc methyl
transferase produces methyl-jasmonate (MeJA) (Seo et al.
2001).
JAs aVect nodulation in legume–bacteria interactions in
several ways. Rosas et al. (1998) found that exogenous
application of JA caused induction of nod gene in Rhizo-
bium leguminosarum. Mabood and Smith (2005) showed
that JA and methyl-jasmonate (MeJA) induced expression
of nodulation genes in B. japonicum and that pre-incuba-
tion with JAs enhanced root nodulation, N2 Wxation, and
plant growth in soybean under controlled environmental
conditions. Mabood et al. (2006a) found subsequently that
inoculation of soybean plants with B. japonicum cells incu-
bated with MeJA alone, or in combination with genistein
(GE), caused increases in nodule number, nodule dry mat-
ter (DM) per plant, and seasonal N2 Wxation, in comparison
with control cells. JAs also induced production and secre-
tion of lipo-chitooligosaccharides (LCOs), a Nod factor. JA
and MeJA were more eVective inducers of LCO production
than GE, and JA or MeJA plus GE was more eVective than
JA or MeJA alone (Mabood et al. 2006b). Pre-incubation of
B. japonicum with MeJA increased plant growth, DM accu-
mulation, and grain yield of soybean under short-season
Weld conditions (Mabood et al. 2006c). Findings by other
groups suggest that JA regulates Nod factor-induced signal-
ing and nodulation in both synergistic and antagonistic
manners with others regulators, for example, ethylene (Sun
et al. 2006; Zhao and Qi 2008). Aside from these studies of
early Nod factor responses, however, little is known about
the role of JAs in nodule development and function in
legumes.
The present study was designed to: (1) measure and
compare production of JA, and its precursor OPDA, in con-
trol versus nodulated roots from soybean plants inoculated
with B. japonicum and in isolated nodules; (2) establish
correlations between JAs production, nodule number, and
plant growth during legume–bacteria symbiosis; and (3)
quantify the eVects of exogenously applied JA and OPDA
on nodule cell number and size.
Materials and methods
Plant material
Soybean seeds [G. max (L.) Merr.] cv. Don Mario, with
high purity and 95 % germination capacity according to the
International Seed Test Association (ISTA), were inocu-
lated with agronomic dose (3 ml kg¡1) of B. japonicum
E109 culture, in early stationary growth phase containing a
titer of 5 £ 109cfu ml¡1. B. japonicum E109 (formerly
USDA138 of the NRS collection, USA) provided by Ing.
Agr. Alejandro Perticari, Instituto de Microbiología y Zoo-
logía Agrícola, Instituto Nacional de Tecnología Agropec-
uaria, Castelar, Argentina (Perticari et al. 1996), is one of
the strains most commonly used for soybean inoculation in
Argentina. Bacterial cultures were grown at 28 § 1 °C in
250-ml Xasks containing 100 ml yeast extract mannitol
(YEM) medium (mannitol
MgSO4·7H2O 0.2 g, NaCl 0.1 g, yeast extract 0.4 g, dis-
tilled water 1,000 ml, pH 6.8), on a Orbital Shaker OS-10
(CK-Tech-UE) (150 rpm), in the dark.
10 g, K2HPO4 0.5 g,
Plant growth conditions
Inoculated and non-inoculated soybean seeds were grown
in plastic pots (10 liter volume) containing washed and ster-
ile sand as inert support. Substrate Weld capacity was pre-
adjusted to 60 % with nitrogen-deWcient sterile 25 % (v/v)
Hoagland’s solution and maintained until the end of the
experiment. Seedlings were grown for 21 days in a growth
chamber with photoperiod 16-h light (30 °C)/8-h dark
(20 °C), at 80 % RH, to obtain the maximal and constant
number of nodules per plant according to standard Burton’s
test (Burton et al. 1972) for inoculated legume seeds. Root
samples from control and inoculated plants were harvested
at 6, 9, 12, 15, 18, and 21 days after B. japonicum inocula-
tion. Nodules were excised from plants at day 9 due to mac-
roscopic occurrence of nodule and day 15 due to maximum
nodule number on roots. The experiments were performed
in triplicate.

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Arch Microbiol (2012) 194:837–845839
123
Extraction, puriWcation, and estimation of JA and OPDA
JA and OPDA were extracted and pre-puriWed as described
by Andrade et al. (2005). Control roots, nodulated roots,
and isolated nodules (200 mg DM) were homogenized with
10 ml methanol, 50 ng [2H6]jasmonic acid [(2H6] JA], and
100 ng [2H5]12-oxo-phytodienoic acid [(2H5)OPDA] as
internal standards. The homogenate was Wltered under vac-
uum on a column with cellulose Wlter. The extract was
dried, dissolved with 10 ml methanol, and loaded on a col-
umn Wlled with 3 ml DEAE-Sephadex A25 (Amersham
Pharmacia Biotech AB, Sweden) (Ac¡-form, methanol).
The column was washed with 3 ml methanol and then with
3 ml 0.1 N acetic acid in methanol. Eluents with 3 ml of
1 N acetic acid in methanol, and 3 ml of 1.5 N acetic acid in
methanol, were collected, evaporated, and analyzed by liq-
uid chromatography-electrospray ionization tandem mass
spectrometry (LC–ESI–MS/MS) as described below. Mea-
surements of JAs in control and nodulated roots were taken
in triplicate. For the measurement of JAs in isolated nod-
ules, a combined sample of 30 nodules was obtained ran-
domly from 3 plants.
Liquid chromatography-electrospray ionization tandem
mass spectrometry (LC–ESI–MS/MS)
Mass spectrometric analysis was performed on a quadruple
tandem mass spectrometer (MS–MS, Quattro Ultima,
Micromass, Manchester, UK) Wtted with an electrospray
ion source (ESI). A mixture of all unlabeled compounds
and internal standards was separated by reversed-phase
high-performance liquid chromatography (HPLC) and ana-
lyzed by tandem mass spectrometry with multiple reaction
monitoring (MRM) to determine retention times for all
compounds. The spectrometer software used was Mass-
Lynx™ v. 4.1 (Micromass, Manchester, UK). Response
was calculated as product ion peak area x (IS concentration/
IS product ion peak area), where IS concentration is the
amount of internal standard added. JA and OPDA were
separated from tissues by HPLC. An Alliance 2695 separa-
tion module (Waters, Milford, MA, USA) equipped with a
100 £ 2.1 mm, 3-?m RESTEK C18 column was used to
maintain performance of the analytical column. Fractions
were separated using a gradient of increasing methanol
concentration, constant glacial acetic acid concentration
0.2 % in water, and initial Xow rate 0.2 ml min¡1. The gra-
dient was increased linearly from 40 % methanol/60 %
water–acetic acid at 25 min to 80 % methanol/20 % water–
acetic acid. After 1 min, initial conditions were restored,
and the system was allowed to equilibrate for 7 min. MRM
mode was used for the determination of JA and OPDA.
These compounds were monitored at m/z transitions of 210/
59 and 292/165, with retention times of 14.20 and
18.70 min, respectively. Collision energies used were 20
electron volts (eV) for JA and 30 eV for OPDA, and cone
voltage was 35 V.
Growth parameters and pigment analysis
Four control and 4 inoculated plants were separated into
roots and shoots, and fresh matter (FM) mass and length
were recorded. Samples were dried in an oven at 60 °C
until constant dry matter (DM) mass was obtained. FM and
DM were expressed as g plant¡1. Chlorophyll a and b were
extracted from 100 mg FM of unifoliate leaves and esti-
mated as described by Porra (2002). For this, leaves were
homogenized in a mortar with 10 ml acetone 80 %, centri-
fuged 5 min at 5,000 rpm, and Wltered. Pigment levels were
measured using a spectrophotometer, with wavelengths of
646.6 and 663.6 nm corresponding to chlorophyll a and b,
respectively. Acetone–water (80:20) was used as a blank
control. Results were quantiWed as described by MacKin-
ney (1941) and Vernon (1960). Experiments were per-
formed in triplicate.
Exogenous application of JA and OPDA
Nodules were chosen at the similar developmental stage.
Nodules size was measured with a caliber (2 mm), excised
from nodulated roots at day 21 after inoculation, washed in
distilled water, and placed in 25-ml Erlenmeyer Xasks
containing JA [(§)-1?-2?-3-oxo-2-[cis-2-pentenyl] cyclo-
pentaneacetic acid], 99 % purity, or OPDA [12-oxo-
phytodienoic acid], 95 % purity, kindly provided by
Dr. O. Miersch, Institute of Plant Biochemistry, Halle,
Germany. Based on previous reports on exogenous applica-
tion of MeJA (Yoon et al. 2009) or OPDA (Fliegmann et al.
2010) to soybean seedlings or cell suspension cultures, we
used concentrations of 10¡4, 10¡6, and 10¡8M for JA, and
10¡6 and 10¡8M for OPDA, applied in water solutions.
Water alone was used as a control. Flasks containing two
nodules were incubated for 5 days at 25 °C, in the dark, and
the material was then processed for histological studies.
Experiments were performed in duplicate; results presented
are means of four subsamples of four nodules.
Nodule histological studies
Nodules were Wxed in FAA (ethanol–water–formaldehyde–
acetic acid, 50:35:10:5), dehydrate in a graded series of
ethanol and xylol, starting with alcohol 70 %, and embed-
ded in Histowax™. Longitudinal and transverse serial sec-
tions of the whole nodule, ranging from 8 to 10 ?m in
thickness, were cut with a rotary microtome. Sections were
stained with hematoxylin-safranin-Fast Green and mounted
in Depex™ (Johansen 1940; O’Brien and McCully 1981).

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840Arch Microbiol (2012) 194:837–845
123
Photomicrographs were taken with an Axiophot Carl Zeiss
microscope equipped with AxioCam HRC camera, com-
puter image capture, and digitization by AxioVision 4.3
program. Size and number of cells in the infected area were
measured. Longitudinal and transverse sections with diam-
eter »2 mm were obtained from each nodule. For the deter-
mination of cell number, 10 sections from each nodule
were analyzed. In the central region of the nodule, two
zones were deWned: Zone I, central area; Zone II, peripheral
area adjacent to cortex. Areas studied were »62,000 ?m2.
Results were expressed as means of cell number recorded
for Zone I versus Zone II and for various treatments. For
the determination of cell size, four sections of the central
region were observed for each nodule. In each section,
twenty infected cells were selected at random and the long
axis was measured.
Statistical analysis
Shoot DM was analyzed by nonparametric Kruskal–Wallis
test, with a posteriori Dunn test; p < 0.05 considered
signiWcant. Results from chlorophyll quantiWcation, and
exogenous application of OPDA and JA to nodules, were
subjected to ANOVA analysis, with a posteriori Tukey’s
test.
Results
Endogenous jasmonates
OPDA and JA were detected in roots of control plants, in
roots nodulated by B. japonicum, and in isolated nodules.
Levels of both compounds diVered between control versus
nodulated roots.
In control roots, an abrupt peak of OPDA was observed
at day 18. In nodulated roots, OPDA level reached a maxi-
mum at day 15. OPDA peaked earlier in nodulated roots
(day 15) than in control roots (day 18, Fig. 1a).
JA in control roots also peaked at day 18. Level of
endogenous JA was low and did not show signiWcant varia-
tion in nodulated roots during the experiment (Fig. 1b).
In nodulated roots, the magnitude of OPDA increase
(Fig. 1a) was greater than that of JA, for example, 5.5 times
at day 15 (Fig. 1b), whereas in control roots, OPDA level
was 1.24 times that of JA at day 18.
Nodule formation was evident in taproot at day 9 and in
lateral root at day 12. Total nodule number showed a steady
increase until day 15, when it reached its maximal value of
13 nodules plant¡1 (Fig. 2). The maximal nodule number,
occurring when the nodulation system was well established,
coincided with the OPDA peak recorded in nodulated roots
at day 15 (Fig. 1a).
In isolated nodules, OPDA level was very high
(31.484 ng g¡1) at day 9, when nodule formation was evi-
dent in taproot, and much lower (2.998 ng g¡1) at day 15,
when total nodule number was maximal. In contrast, JA
level was high at day 9 (14.443 ng g¡1) and relatively con-
stant until day 15 (12.123 ng g¡1).
Fig. 1 Levels of 12-oxophytodienoic acid (OPDA) and jasmonic
acid (JA) in roots of control soybean plants and plants inoculated with
B. japonicum strain E109, at various times (days) after inoculation.
a OPDA; b JA. Values shown are mean § SD (n = 3)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
6912
Time (days)
151821
OPDA (ng.g-1)
( ) Nodulated plants
( ) Control plants
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
6912
Time (days)
151821
JA (ng.g-1)
( ) Nodulated plants
( ) Control plants
B
A
Fig. 2 Mean numbers of nodules in taproot, lateral root, and total nod-
ules in inoculated plants at various times (days) after inoculation.
Mean § SD (n = 3)
0
5
10
15
20
6912
Time (days)
151821
Nodule number.plant-1
( ) Total
( ) Taproot
(
) Lateralroot

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Arch Microbiol (2012) 194:837–845841
123
Growth of shoots, as DM, increased throughout the
experiment for both control and nodulated plants. Shoot
DM value at day 21 was signiWcantly higher for nodulated
than for control plants (Fig. 3). Shoot length, root length,
and root DM were not aVected by B. japonicum inoculation
(data not shown).
Chlorophyll a level in control plants increased from day
9 to 12 and then decreased steadily through day 21. In
nodulated plants, chlorophyll a level was higher (1.4 times)
than in control plants at day 21 (Fig. 4a). Chlorophyll
b level decreased steadily throughout the experiment in
both control and nodulated plants (Fig. 4b).
Nitrogen (N) status was assessed based on biomass pro-
duction (shoot DM) and chlorophyll concentration in
shoots and leaves, two plant life cycle parameters that are
highly sensitive to presence/absence of N. Chlorophylls in
particular are considered direct “N status markers,” whose
levels reXect early N deWciency. For example, Evans
(1989) reported that total chlorophyll content is closely
related to leaf N in C3 species and that photosynthetic
machinery accounts for >50 % of leaf N content.
EVect of exogenous jasmonates (JAs)
In view of the eVect of JAs on cell expansion in various
plant tissues, and the high levels of JAs observed inside
nodules, we studied the eVect of exogenously applied
OPDA and JA on isolated nodules. We used 10¡4, 10¡6,
and 10¡8M for JA and 10¡6 and 10¡8M for OPDA, based
on the reports of Yoon et al. (2009) and Fliegmann et al.
(2010) about JAs exogenous application to soybean seed-
lings or cell suspension cultures. Both number and size of
central and peripheral cells were altered by these treat-
ments. JA at 10¡4 M concentration increased central and
peripheral cell number dramatically (Fig. 5a-b) and cell
size to a lesser extent (Fig. 5c). JA at lower concentrations
(10¡6 and 10¡8M) aVected number of peripheral cells and
cell size (Fig. 5b-c), but not number of central cells
(Fig. 5a). OPDA at 10¡6 M caused signiWcant increase in
number of peripheral cells (Fig. 5b), but did not aVect cell
size, or number of central cells (Fig. 5a-c). OPDA at lower
concentration (10¡8 M) enhanced all three parameters
(Fig. 5a-c); central cell number increased 128 %, and cell
size increased 110 % relative to control.
EVects of 10¡6 M JA and 10¡6 M OPDA on the above
parameters were further studied by photomicrography.
General sections from cortical and central regions of nod-
ules are shown in Fig. 6a, c, e. Central region Zones I and II
were used for analysis. Detailed views of central regions
are shown in Fig. 6b, d, f. Infected cells, containing symbi-
osomes, display dense cytoplasm and disappearance of the
central vacuole. Uninfected cells retain a typical vacuole
throughout nodule development.
Discussion
Many plant species have developed mutualistic interactions
with nitrogen-Wxing bacteria and/or arbuscular mycorrhizal
Fig. 3 Shoot dry matter (g) of control plants (black bars) and nodulat-
ed plants (white bars) at various times (days) after inoculation.
Mean § SD (n = 3). Values indicated by diVerent letters are signiW-
cantly diVerent at (p < 0.05)
6912
Time (days)
15
18
21
0.2
0.3
0.4
B
Control plants
Nodulated plants
a
b
b
b
c
c
d
d
d
d
d
d
Shoot dry matter (g)
Fig. 4 Levels of chlorophylls in control plants and nodulated plants at
various times (days) after inoculation. a chlorophyll a; b chlorophyll
b. Mean § SD (n = 3). Values indicated by diVerent letters are signiW-
cantly diVerent at p < 0.001
9
121518 21
40
60
80
100
Control plants
Nodulated plants
A
a
b
b
bc
c
cd
d
de
e
f
Time (days)
μg chlorophyll a . g DM-1
9 121518 21
40
60
80
100
Control plants
Nodulated plants
B
a
a
b bc
c
d
d
dd
d
Time (days)
μg chlorophyll b . g DM-1

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842Arch Microbiol (2012) 194:837–845
123
fungi. These relationships are based on mutual recognition
and a high degree of coordination that depends on activity
of a number of signaling molecules, including JAs. In the
Bradyrhizobium–soybean symbiosis, JAs induce transcrip-
tion of nodulation genes (Mabood and Smith 2005).
Studies so far are limited to eVects of exogenous applica-
tion of JAs, particularly JA and MeJA, on early events of
nodulation, N2 Wxation, and N partitioning (Rosas et al.
1998; Rossato et al. 2002; Mabood and Smith 2005;
Mabood et al. 2006a, b). In cultured soybean cells (Flieg-
man et al. 2010), JA, OPDA, and coronalon (a synthetic
jasmonate analogue) all induced accumulation of 7,4?-
dihydroxyXavone, a compound involved in plant–microbe
interactions (Martens and Mithöfer 2005). There are limited
data regarding endogenous levels of JAs in nodules and
nodulated roots, and the role of JAs in nodule formation
and function during Bradyrhizobium–soybean interaction.
The senescence visually observed in control plants by
day 18 may be associated with accumulation of OPDA and
JA, and with N starvation caused by absence of Bradyrhiz-
obium and loss of chlorophyll, particularly chlorophyll
a. Low N availability may trigger production of JAs in
roots and consequent senescence process in leaves. JA bio-
synthesis is up-regulated at the transcriptional level follow-
ing nutrient or mineral deprivation (Pawuels et al. 2009).
Transcription of genes for JA biosynthetic enzymes (e.g.,
LOX2, AOS, AOC) is enhanced by K+ starvation (Armen-
gaud et al. 2004) and by sulfur starvation (Hirai et al. 2003;
Nikiforova et al. 2003), indicating a role of JA in plant min-
eral nutrition.
A role of JAs in initiation and progression of leaf senes-
cence, although still controversial, has been suggested for
many plant species (Sembdner and Parthier 1993; He et al.
2001; Seltmann et al. 2010). In monocarps, such as soy-
bean, senescence is a highly regulated process, involving
decreased photosynthesis, loss of chlorophyll, and break-
down of CO2-Wxing enzymes such as ribulose bisphosphate
carboxylase oxygenase (Rubisco).
In the present study, high levels of JAs in control plants
were correlated with reduced chlorophyll levels and N star-
vation, indicating a connection between JAs and senes-
cence. Seltmann et al. (2010) demonstrated a similar
decline in total chlorophyll in Arabidopsis thaliana leaves
during senescence, coinciding with increased levels of JA
and OPDA. In Brassica napus L., addition of MeJA to
nutrient solution reduced photosynthetic activity, chloro-
phyll content, and nitrate uptake (Rossato et al. 2002);
the latter eVect was partially reversed when MeJA was
removed.
In the present study, in contrast to control plants, inocu-
lated plants showed increase of shoot DM at day 21 and did
not show symptoms of senescence until the end of the
experiment.
Nodule development is an energetically expensive pro-
cess, and the number of nodules is typically controlled by
the host plant, through an “autoregulation of nodulation
(AON)” process involving
(Ferguson et al. 2010). In studies of M. truncatula, reduc-
tion of JA levels seems to aVect arbuscular mycorrhization,
but not nodulation (Zdyb et al. 2011). The diVerent reaction
long-distance signaling
Fig. 5 EVect of exogenously applied JA and OPDA on number and
size of cells of nodules. a number of central cells (mean § SD, n = 20).
b number of peripheral cells (mean § SD, n = 20). c cell size
(mean § SD, n = 80). * p < 0.05, ** p < 0.001
Control
10-4 M 10-6M10-8 M10-6M10-8 M
60
80
100
120
140
JA
OPDA
**
**
A
Exogenous concentration
Central cell number
Control
10-4 M 10-6M 10-8 M10-6M10-8 M
60
80
100
120
140
JA
OPDA
**
B
*
**
Exogenous concentration
Peripheral cell number
Control
10-4 M 10-6M 10-8 M10-6M10-8 M
30
32
34
36
38
40
JA
OPDA
**
C
**
*
**
Exogenous concentration
Cell size (μm)

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Arch Microbiol (2012) 194:837–845843
123
of soybean may be due to the diVerence between the induc-
tion of indeterminated (M. truncatula) versus determinated
(soybean) nodules.
Nevertheless, JA appeared to act as a negative regulator
of nodulation, by inhibiting expression of early nodulation
genes (Sun et al. 2006). There is some recent evidence that
JA level is positively correlated with nodulation in soybean
(Seo et al. 2007; Kinkema and Gresshof 2008). In contrast,
Nakagawa and Kawaguchi (2006) reported that shoot-
applied MeJA exhibited a strong inhibitory eVect on
nodulation in the wild type and even in the hypernodulating
phenotype of the L. japonicus har1-4 mutant. However,
several studies gave conXicting results about JAs participa-
tion in nodulation.
We propose that JAs function as part of a hormonal net-
work to control nodule development through AON, consis-
tent with the Wndings of Kinkema and GresshoV (2008).
The accumulation of OPDA we observed at day 15, coin-
ciding with maximal nodule number in nodulated roots,
suggests that OPDA is involved in AON as a signal mole-
cule, independent of JA.
The high levels of OPDA and JA found in isolated nod-
ules suggest that these compounds play a greater role in
Bradyrhizobium–soybean symbiosis than previously sus-
pected. Consistent with our Wndings, Mohammadi et al.
(2003) observed activity of LOX enzymes in eVective nod-
ules induced by B. japonicum strains 2122 and 2143 in soy-
bean. LOX1-speciWc activity was several-fold higher in
nodules than in adjacent root tissue and appeared to be a
result of plant–symbiont interaction. Using microarray
analysis, Hayashi et al. (2008) identiWed genes associated
with nodule development in soybean. LOX expression dur-
ing nodulation was relatively complex; at least eight diVer-
ent LOX genes were expressed in nodules, some of which
were probably involved in nodule development. The high
LOX activity and high level of JAs in nodules conWrm par-
ticipation of these partner components in steps of Brady-
rhizobium–soybean interaction.
Fig. 6 Photomicrographs of
longitudinal and transversal sec-
tions of nodules. a General
view: control, b detailed view:
control, c general view: JA 10¡6
M, d detailed view: JA 10¡6 M,
e general view: OPDA 10¡6 M,
and f detailed view: OPDA 10¡6
M. Central zone (Zone I) and
peripheral zone (Zone II) are
marked with black. Area shown
»62,000 ?m2. cer central re-
gion, cor cortical region, ic in-
fected cell, uc uninfected cell

Page 8

844Arch Microbiol (2012) 194:837–845
123
Levels of JAs inside nodules are high, and previous stud-
ies have shown that JA and MeJA induce cell expansion in
potato tubers (Takahashi et al. 1994), corpus cells of tuber
buds (Castro et al. 1999), and meristematic regions of sto-
lons (Cenzano et al. 2003). We therefore examined eVects
of exogenous OPDA and JA in soybean nodules and found
that these compounds increased number and size of cells to
various degrees, depending on concentration. Levels of JAs
during early stages of nodule development appear to aVect
cell growth processes. Along this line, Hause and
Schaarschmidt (2009) proposed that JAs aVect nodule cell
growth, and the aging process, by inXuencing antioxidant
metabolism. Our knowledge of the function of JAs in mutu-
alistic symbioses in general remains highly fragmentary,
but the present Wndings support their involvement in Brady-
rhizobium–soybean interaction.
Acknowledgments
CYT-UNRC and ANPCYT to G.A. The authors thank Dr. S. Anderson
for English editing.
This work was supported by grants from SE-
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