Plant Physiol. (1995) 108: 533-542
Alfalfa Root Flavonoid Production 1s Nitrogen Regulated'
Carmen C ~ r o n a d o José
~ ~ ~ Angelo
,
Silviera Zuanazzi2, Christophe Sallaud, Jean-Charles Quirion, Robert Esnault,
Henri-Philippe Husson, Adam Kondorosi*, and Pascal Ratet
lnstitut des Sciences Végétales (C.C., J.A.S.Z., C.S., R.E., A.K., P.R.) and lnstitut de Chimie des Substances
Naturelles (J.A.S.Z., j.-C.Q., H.-P.H.), Centre National de Ia Recherche Scientifique, Avenue de Ia Terrasse,
F-91198, C i f sur Yvette Cedex, France; and lnstitute of Cenetics, Biological Research Centre, Hungarian
Academy of Sciences, Szeged P.O. Box 521, H-6701 Hungary (A.K.)
Flavonoids produced by legume roots are signal molecules acting
both as chemoattractants and nod gene inducers for the symbiotic
Rhizobium partner. Combined nitrogen inhibits the establishment
of the symbiosis. To know whether nitrogen nutrition could act at
the leve1 of signal production, we have studied the expression of
flavonoid biosynthetic genes as well as the production of flavonoids
in the roots of plants grown under nitrogen-limiting or nonlimiting
conditions. We show here that growth of the plant under nitrogenlimiting conditions results in the enhancement of expression of the
flavonoid biosynthesis genes chalcone synthase and isoflavone reductase and in an increase of root flavonoid and isoflavonoid production as well as in the Rhizobium melilofi nod gene-inducing
activity of the root extract. These results indicate that in alfalfa
(Medicago sativa L.) roots, the production of flavonoids can be
influenced by the nitrogen nutrition of the plant.
Higher plants synthesize a wide variety of phenolic compounds during normal growth and development. These
compounds are products of the phenylpropanoid biosynthetic pathway in both root and shoot tissues. They generally accumulate at relatively high concentrations in the
cell vacuole and are chemically associated with sugars
(McClure, 1975).
In pathogen-plant interactions, certain end products of
the (iso)flavonoid biosynthesis pathway serve as phytoalexins in plant defense reactions (reviewed by McClure,
1975; Hahlbrock and Scheel, 1989; Peters and Verma, 1990;
Rao, 1990), or as chemoattractants toward pathogenic and
symbiotic bacteria (Peters and Verma, 1990), or as signal
molecules required for the induction of essential bacterial
genes, leading to the appropriate interaction with the host
plants (Peters and Verma, 1990).In the association between
Research support was provided by a Commission of the European Countries (BRIDGE BIOT-900159-C) contract to A.K.
J.A.S.Z. was supported by a grant from the Brazilian Conselho
Nacional de Desenvoluimento Cientifico e Tecnológico-Brazil.
C.C. was supported by a fellowship from the Spanish Ministry of
Education.
'The two first authors contributed equally to the work presented here.
Present address: Departamento de Microbiologia y Parasitologia, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla,
Spain.
* Corresponding author; e-mail [email protected];
fax 33-1-69-82-36 -95.
leguminous plants and their symbiotic bacteria of the family Rhizobiaceae, the flavonoids play the dual role of symbiotic signal molecules and phytoalexins (Pankhurst and
Biggs, 1980).
The phenylpropanoid biosynthetic pathway has been
extensively studied (Heller and Forkmann, 1988) and a
number of genes involved in this pathway have been isolated (MOIet al., 1988; Dixon et al., 1992).The enzyme CHS,
which condenses three molecules of malonyl COA with
4-coumaroyl COAto produce chalcones, is a key enzyme in
the pathway leading to the production of flavonoids and
isoflavonoids. Chalcone synthase genes have been cloned
from a wide range of higher plants (MOI et al., 1988). In
severa1 plants, including alfalfa (Medicago sativa), the CHS
genes are represented by a small gene family of six to eight
members (Ryder et al., 1987; Koes et al., 1989; Wingender et
al., 1989; Harker et al., 1990; Estabrook and Sengupta
Gopalan, 1991; Junghans et al., 1993; McKhann and Hirsch,
1994). In soybean (Glycine max), differential regulation of
the CHS transcripts was observed during its interactions
with Rhizobium and Agrobacterium (Wingender et al., 1989;
Estabrook and Sengupta Gopalan, 1991), and the production of the phytoalexin glyceollin I was also enhanced in
root exudates and root hairs of seedlings in response to
infection by the symbiotic Rhizobium or after pathogenic
infection (Schmidt et al., 1992). In the alfalfa-Rhizobium
meliloti and pea-Rhizobium leguminosarum bv viciae symbiosis, certain Fix- mutant bacteria can induce high levels of
CHS expression in nodules, evoking a pathogenic-like response (Yang et al., 1992; Grosskopf et al., 1993). In alfalfa,
the different CHS cDNA clones isolated respond to wounding and elicitor treatment or to infection (Esnault et al.,
1993; Junghans et al., 1993; McKhann and Hirsch, 1994). In
addition to CHS, a number of other genes from the phenylpropanoid pathway have been isolated from M. sativa
(Dixon et al., 1992). Among these, IFR (Paiva et al., 1991),
which establishes the steric configuration of the pterocarpans, is the penultimate enzyme for the synthesis of medicarpin, the major phytoalexin found in alfalfa cell cultures
elicited by funga1 elicitors (Dixon et al., 1992).
The activation of rhizobial nod genes involved in the
synthesis of the Nod factor, which is necessary for initiaAbbreviations: CHS, chalcone synthase; EIMS, electronic impact
mass spectra; FAB, fast atom bombardment; IFR, isoflavone reductase; MeOH, methanol.
533- Published by www.plantphysiol.org
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Copyright © 1995 American Society of Plant Biologists. All rights reserved.
534
Coronado et al.
tion of nodule development (Dénarié and Roche, 1992;
Kondorosi, 1992), requires specific flavonoids released by
roots of the host plant and the product of the regulatory
gene nodD (Kondorosi,1992).Thus, the production of hostspecific flavonoids (Firmin et al., 1986; Djordjevic et al.,
1987; Rossen et al., 1987; Gyorgypal et al., 1991) and their
release from determined sites of the roots (Peters and Long,
1988) are two important factors in the symbiotic interaction
with Rhizobium (Fisher and Long, 1992). In alfalfa roots,
CHS and IFR were shown to be involved in the synthesis of
various nod gene inducers and phytoalexins (Maxwell
et al., 1989; Dixon et al., 1992; Phillips et al., 1993). The
flavonoids luteolin, 4,4’-dihydroxy-2’-methoxy-chalcone,
7,4‘-dihydroxyflavone, and 7,4’-dihydroxyflavanone,isolated from the seeds and roots of M . sativa, plants are
inducers of the R. meliloti nod genes (Phillips et al., 1993),
and some of the phytoalexin precursors are also potent nod
gene inducers (Dakora et al., 1993a).
It has been shown that the plant flavonoid content can
change in response to fertilization with the macro-nutrients
nitrogen, phosphorus, and potassium (McClure, 1975;
Murali and Teramura, 1985; Nair et al., 1991). In soybean
plants, application of combined nitrogen markedly decreased root isoflavonoid concentration (Cho and Harper,
1991a, 1991b; Morandi and Gianinazzi-Pearson, 1993). In
Lotus corniculatus, the content of root polyphenols (tannins)
was correlated to the nitrogen nutrition status of the plant
(Pankhurst and Jones, 1979), and in Lupinus albus, the presente of nitrate reduced the amount of flavonoids present in
the root exudate or in the root extract (Wojtaszek et al.,
1993).
Growth of the plant under nitrogen-limiting conditions
is a prerequisite for the establishment of the symbiotic
association. Application of utilizable nitrogen inhibits a11
phases of nodulation, including bacterial infection, nodule
development, and nitrogenase function (reviewed by
Streeter, 1988; Carro11 and Mathews, 1990).
Here we show that in M . sativa the production of the
flavonoid signal molecules toward the nitrogen-fixing symbiotic partner is regulated by the nitrogen status of the
plant. In roots of M . sativa plants grown under limited
nitrogen supply, the production of flavonoids, including
the nod gene inducer compounds, as well as the expression
of the genes involved in their biosynthesis, were found to
be induced.
MATERIALS A N D M E T H O D S
Plant Physiol. Vol. 108, 1995
plants were placed on the lid of the container so that the
roots were constantly dipped in the culture medium spray.
Cuttings of an individual M . sativa ssp. varia cv A2 plant
were maintained in water for rooting, and seedlings of M .
sativa ssp. sativa cv Nagyszenasi plants were germinated in
sterile sand. Once the plants had approximately 3-cm-long
roots, they were placed into the planting holes of the
aeroponic system. A nutrient solution (Blondon, 1964) supplemented with either 0.25 mM KNO, (limited nitrogen
supply) or 10 mM KNO, (nonlimited nitrogen supply) was
used as specified below.
Plants were grown in a growth chamber maintained at
day/night temperatures of 25/20°C and a 14-h photoperiod at 650 pmol photons m-* s-’.
Carbon and Nitrogen Content Determinations
The complete root system of one or two plants harvested
at different time intervals of the experiment was frozen and
ground in liquid nitrogen. One to 2 g of the powder obtained was lyophilized and analyzed by catharometry
(Fraisse and Schmitt, 1977; Pella and Colombo, 1978) to
determine the percentage of carbon and nitrogen present in
each sample. The accuracy of each measurement was 95%.
This analysis was performed by the Service Centrale
d’Analyse, CNRS (Vernaison, France).
Source of Flavonoids
Authentic formononetin, ononin, and 7,4’-dihydroxyflavone were purchased from Extrasynthese SA (Genay,
France). Other flavonoids used were isolated as described
below.
Flavonoid lsolation
Approximately 1 g of the fresh root samples ground in
liquid nitrogen were extracted with 95% aqueous ethanol
(1 g fresh weight/5 mL), and the extract was filtered by
passing it through a 0.2-pm Millipore filter. The ethanol
extracts were analyzed in HPLC analytical systems.
For the separation of the nonglycosylated flavonoids
from the glycosylated forms, the ethanolic fraction obtained from 700 g of roots harvested 10 d after nitrogen
deprivation was dried, resuspended in water, and successively extracted with ether and butanol. The extracts were
first chromatographed on a column of polyamide (size, 23
x 3 cm; ratio, 1O:l) and the flavonoids contained in the pool
fractions were isolated using preparative HPLC.
Plant Material
Flavonoid ldentification
Two alfalfa cultivars, Medicago sativa ssp. varia cv A2
(Deak et al., 1986) and M . sativa ssp. sativa cv Nagyszenasi,
were used for the nitrogen-starvation experiments.
Structural features of purified flavonoids in MeOH, with
reagents used to characterize these type of products, were
examined by UV absorption spectroscopy (Markham and
Mabry, 1975) using a Perkin-Elmer Lambda 5 UV/VIS
spectrophotometer.
EIMS data were collected with an AEI MS-50 spectrometer and FAB-MS with a Kratos (Manchester,UK) MS-80 RF
spectrometer. NMR spectra were obtained with a Bruker
(Wissenbourg,France) AM-300 instrument with the Aspect
Plant Culture
Plants were cultured in an aeroponic system consisting
of a PVC container with sterile nutrient solution at the
bottom part and a vaporizer to spread this solution. The
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Copyright © 1995 American Society of Plant Biologists. All rights reserved.
Nitrogen Regulation of Flavonoid Production in Alfalfa
3000 data system. The 16 K free induction decay was acquired at 300 and 75.5 MHz for 'H and I3C spectra, respectively. Solvents were (d6)-DMSO and (d4)-MeOH and
shifts were measured relative to trimethylsilyl as an interna1 reference.
Analytical and Preparative HPLC
Aliquots were loaded onto a Waters HPLC system (Millipore) fitted with a 3.9 X 150 mm Nova-pak CI8 4-pm
column, preceded by a guard column, eluted at 1 mL/min
from O to 5 min with a linear gradient solvent system from
20:80:0.1 (v/v/v) MeOH:H,O:TFA to 60:40:0.1 (v/v/v)
MeOH:H,O:TFA. From 5 to 20 min, a linear gradient to
1OO:O.l (v/v) Me0H:TFA was applied and the analysis
continued isocratically at that concentration for another 6
min. Eluting compounds were monitored with a Waters
991 photodiode array detector, which measured absorbance (250400 nm) every 2 s with 5 nm resolution.
For the preparative HPLC, we used a Waters system
(Millipore) equipped with a PrepPak 25 X 100 mm C,,
6-pm column and radial compression at 700 to 800 psi,
eluted at 5 mL/min from O to 40 min with a linear gradient
solvent system from 40:60:0.1 (v/v/v) MeOH:H,O:TFA to
80:20:0.1 (v/v/v) MeOH:H,O:TFA. Eluting compounds
were monitored with a Waters 600E system controller and
a Waters 484 Tunable Absorbance Detector/UV, which
measured A,,,, and every peak was separated.
Measurement of nod Cene-lnducing Activity
The nod gene induction by flavonoids was monitored
using strains Rhizobium meliloti 1021, containing plasmid
pRm57 (Mulligan and Long, 1985), or R. meliloti JM57 containing plasmid pKSK5 carrying the nodD2 gene or plasmid
pGM108 carrying the nodD2 gene (Gyorgypal et al., 1991),
as specified. Bacteria were grown overnight to mid-log
phase and diluted to an A,,, of 0.2 in 2 mL of YTB medium
(10 g/L Bacto tryptone, 1 g/L Bacto yeast extract, 1 g/L
NaCl, 1 mM CaCl,, 1 mM MgSO,; Orosz et al., 1973) containing 20 pL of the root extracts to be tested and the
appropriate antibiotics if needed. After incubation for 16 h
at 28"C, the cultures were monitored for P-galactosidase
activity of the nodC::lacZ fusion as described (Miller, 1972).
A11 assays were repeated at least three times and statistical
analysis of the data was performed.
Northern Blot Analysis
Total RNA was isolated from plant roots by the guanidine thiocyanate-CsC1 purification method according to
Sambrook et al. (1989). Total RNA (20 pg/lane) was denatured and subjected to electrophoresis through a formaldehyde 1% agarose gel and transferred to Hybond-N (Amersham) nylon membranes by capillary blotting according to
the manufacturer's protocol. RNA was quantitated spectrophotometrically before loading, and equal loading was
confirmed after electrophoresis by staining with ethidium
bromide and probing the blots with the cDNA clone of
Msc27, a constitutively expressing alfalfa gene (Gyorgyey
et al., 1991). Autoradiograms of the northern blot hybrid-
535
ization were quantified using the Whole Band Analysis
program of the BioImage system of Millipore. The CHS
probe used was a cDNA clone obtained from RNA induced
in alfalfa leaves after infection with Pseudomonas syringae
pv pisi (Esnault et al., 1993). The IFR cDNA probe used in
this study was kindly provided by R. Dixon (Paiva et al.,
1991).
RESULTS
C r o w t h of Alfalfa under Limiting and Nonlimiting
Nitrogen Supply
To know whether the production of root flavonoids is
regulated by the nitrogen nutrition of the plant, the timing
of flavonoid production and that of the induction of flavonoid biosynthetic gene expression were determined in
plants grown under nonlimiting and limiting nitrogen
SUPPlY.
To ensure that the observed results would not be cultivar
dependent, two subspecies, M . sativa ssp. varia cv A2 and
M. sativa ssp. sativa cv Nagyszenasi, were used in these
experiments. Cuttings or seedlings of the two Medicago
subspecies were grown in the aeroponic system for 3 weeks
in a nitrogen nonlimiting plant growth solution (10 mM
KNO,). At this point, a new 10 mM KNO, nutrient solution
was given again to half of the plants, and to the other half,
a 0.25 mM KNO, nutrient solution was added. The complete root system of the plants was harvested at O, 3, 5 , 8,
and 10 d or 2, 3, 6, 8, and 10 d after changing the nutrient
solution, depending on the experiment. In the experiment
using M. sativa ssp. varia cv A2, the carbon:nitrogen ratio
was determined for each sample. This ratio was unaffected
in plants grown in 10 mM KNO, nutrient solution (Fig. 1).
In contrast, the carbon:nitrogen ratio increased during the
experiment for plants grown in 0.25 mM KNO,, confirming
that these growth conditions represented nitrogen limitation and suggesting that major metabolic changes occurred.
After 10 d of growth in the nitrogen-limited solution, yellowing of the leaves and growth inhibition were detectable
(data not shown). In addition, the same M . sativa ssp. varia
plants grown for 15 d in the 0.25 mM KNO, nitrogenlimited solution developed a few spontaneous nodules in
the absence of Rhizobium (Narf phenotype; Truchet et al.,
1989; Caetano-Anollés et al., 1992; data not shown). This
nitrogen concentration-dependent phenomenon also confirmed that plants were grown under limiting nitrogen
SUPPlY.
Flavonoid ldentification
To determine the flavonoid content in the various samples, fresh roots ground in liquid nitrogen were extracted
with 95% ethanol and then analyzed by an HPLC analytical
system. The HPLC profiles of the ethanolic root extracts
from M . sativa ssp. varia cv A2 from each time point of the
experiment revealed the presence of two major peaks (Fig.
2A). Fractionation and HPLC analysis of the fractions revealed that these compounds were present in the butanol
extract (see "Materials and Methods"; data not shown). The
butanol extract of plants harvested 10 d after nitrogen
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Copyright © 1995 American Society of Plant Biologists. All rights reserved.
536
Coronado et al.
(C-ti), 114.6 (C-3’ and C-5’), 104.4 ((249, 101.0 (C-l”), 78.2
(C-5”), 77.4 (C-3’9, 74.1 (C-2”),70.6 (C-4’9, 61.6 (C-6”), and
56.1 (OCH,).
The coupling constant for the anomeric proton in ‘H
NMR spectra (J = 7.0 Hz) established the stereochemistry
of the glycosidic linkage as /3 (Sakagami et al., 1974;
14
O
112
c
B
z
Plant Physiol. Vol. 108, 1995
10
,
.
/
.
I
.
,
.
I
.
/
.
I
.
,
.
I
.
,
.
I
I
I
.
I
.
/
A
3 s
9
------
6
------
-~___-----o-----
/.*
4
O
2
4
6
8
Days after Nitrate Treatment
10
Figure 1. Carbon:nitrogen ratio in M. sativa ssp. varia roots. Root
samples of plants grown in nonlimiting (10 mM KNO,, dashed line)
nitrogen solution or in limiting (0.25 mM KNO,, solid line) nitrogen
solution were frozen in liquid nitrogen and ground. The powder was
lyophilized and analyzed by catharometry to determine the percentage of carbon and nitrogen present in each sample. The carbon:
nitrogen ratio was calculated for each sample.
deprivation (see “Materials and Methods“) was then concentrated and chromatographed on a column of polyamide, eluted first with a O to 100% MeOH gradient in H,O
followed by a O to 5% TFA gradient in MeOH. Forty-two
fractions were collected, each of 30 mL, and were pooled
into five fractions after HPLC analysis. These pools were
fractionated by preparative HPLC and three flavonoids
were isolated. Together, these three compounds represent
approximatively 2% (w/w) of the original ethanolic extract. A11 compounds produced UV spectra nearly identical
to the authentic formononetin (205, 248, and 302[sh] nm).
No effects were observed after addition of the shifts (sodium methoxide and aluminum chloride), indicating the absence of free hydroxyls in the aromatic nucleus.
The EIMS data for the three compounds showed a major
ion at m/z = 268 (100%) and another at m/z = 132.
However, the FAB-MS showed different values for the
molecular ion: {I] = 430, {2] = 516, and {31 = 530. In fact,
the EIMS of the flavonoids having a sugar moiety are
characterized by the presence of the aglycone ion where the
sugar moiety is replaced by hydrogen (Mabry and
Markham, 1975). This and the signal at m/z = 132 (Maxwell and Phillips, 1990) also indicate that these molecules
are glycoconjucates of formononetin.
The three flavonoids had the following NMR signal
resonances:
(1):6’H ppm ([d6]-DMSO);8.43 (lH, s, H-2), 8.06 (lH, d,
J = 8.9 Hz, H-5), 7.54 (ZH, d, J 8.7 Hz, H-2‘-6’), 7.22 (lH,
d, J = 2.0 Hz, H-8), 7.15 (lH, dd, J = 8.9, 2.0 Hz, H-6), 7.00
(ZH, d, J = 8.7 Hz, H-3’-5’),5.12 (lH, br d, J = 7.0 Hz, H-1’9,
3.79 (3H, s, OCH3), 3.75-3.70 (ZH, H-6”), 3.5 to 3.2 (4H, m,
H-2”-5”). SI3C ppm ([d6]-DMSO); 175.5 (C-4), 162.4 (C-7),
160.0 (C-4’), 157.8 (C-9), 154.6 (C-Z), 131.0 (C-2’ and C-69,
127.9 (C-5), 124.7 (C-1’), 123.8 (C-3), 119.2 (C-lO), 116.6
I
10
A
15
Retention Time (min)
4.5
2
4
-?
z
-gEE 3.53
I
s
.- 2.5
5
2
E
1.5
1
O
2
4
6
8
Days after Nitrate Treatment
10
Figure 2. Flavonoid production in M . sativa ssp. varia roots grown
under nonlimiting and limiting nitrogen conditions. A, HPLC analysis
of the ethanol extract of roots grown in 0.25 mM KNO, (solid lhe) or
10 mM KNO, (dashed line) analyzed between 250 and 400 nm.
Peaks (11 and (2) were identified as formononetin 7-O-p-~-glycoside
(malonyl
(ononin) and formononetin 7-O-~-~-glycoside-6”-ma~onate
ononin), respectively. B, Ratio of production of the isoflavonoid
ononin (solid line) or malonyl ononin (dashed line) in plants grown
in limiting (0.25 mM KNO,) nitrogen solution versus nonlimiting (10
mM KNO,) nitrogen solution. The amounts of flavonoids in each
sample were calculated from the area of the peaks detected by
analytical HPLC.
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Nitrogen Regulation of Flavonoid Production in Alfalfa
Markham and Mabry, 1975). On the basis of these data, it
was concluded that the flavonoid (1) is the formononetin7-O-P-~-glycoside (ononin). For confirmation, the flavonoid was compared with an authentic sample for UV,
'H-, and ',C-NMR, EIMS, FAB-MS, and HPLC (data not
shown).
(2):S'H ppm ([d4]-MeOH);8.20 (lH, s, H-2), 8.15 (lH, d ,
J = 8.9 Hz, H-5), 7.48 (2H, d , J = 8.7 Hz, H-2'-6'), 7.25 (lH,
d , J = 2.2 Hz, H-8), 7.18 (lH, dd, J = 8.9, 2.2 Hz, H-6), 6.98
(2H, d , J = 8.7 Hz, H-3'-5'), 5.08 (lH, br d , J = 7.4 Hz, H-l"),
4.60 (lH, dd, J = 11.9, 1.9 Hz, H-6"a), 4.25 (ZH, d d , J = 11.9,
7.1 Hz, H-6"b), 3.82 (3H, S, OCH3), 3.6 to 3.4 (4H, m,
H-2"-5"), 2.66 (s, CH,-malonyl). S13C ppm (ld41-MeOH);
178.9 (C-4), 169.1, and 168.8 (ester malonyl), 164.0 (C-7),
162.0 (C-47, 160.0 (C-9), 156.0 (C-2), 132.2 (C-2' and C-67,
129.2 (C-5), 126.9 (C-l'), 126.3 (C-3), 121.1 (C-lO), 117.9
(C-6), 115.7 (C-3' and C-57, 105.9 (C-8), 102.5 (C-l"), 78.6
(C-S"), 76.5 (C-3"), 75.5 (C-Y), 72.3 (C-4'9, 66.3 (C-6"), 56.6
(OCH,), and 41.3 (CH,-malonyl).
Compared with the flavonoid (11 there was an increment
of 86 atomic mass units consistent with a malonyl moiety.
In 'H NMR spectra, the signals at 4.60 and 4.25 ppm could
be attributed to the protons H-6" a and b of the sugar
moiety and at S 2.66 ppm of the mãlonylkoiety. In 13C
NMR spectra, we observed a signal at 6 41.3 ppm corresponding to the CH, of malonyl. The flavonoid (2) should
then be the ononin-6"-malonate.These results are in agreement with the data of Dakora et al. (1993a). The coupling
constant value (J = 7.4 Hz) in 'H NMR spectra for the
anomeric proton established the stereochemistry of the
glycosidic linkage as P (Sakagami et al., 1974; Markham
and Mabry, 1975). We concluded that the flavonoid 121 is
(malonyl
the formononetin-7-O-P-~-g~ycoside-6"-ma~onate
ononin).
(3):S'H ppm ([d6]-DMSO);8.44 (lH, s, H-2), 8.07 (lH, d ,
J = 8.9 Hz, H-5), 7.54 (2H, d , J = 8.6 Hz, H-2'-6'), 7.22 (lH,
d , J = 2.0 Hz, H-8), 7.15 (lH, d d , J = 8.9, 2.0 Hz, H-6), 7.00
(2H, d, J = 8.6 Hz, H-3'-5'), 5.16 (lH, br d , J = 7.0 Hz, H-l"),
4.41 (lH, d, J = 10.9 Hz, H-6"a), 4.13 (lH, dd, J = 11.9, 6.8
Hz, H-6"b), 3.80 (3H, s, OCH,-formononetin), 3.60 (3H, s,
OCH,-malonyl), 3.5 to 3.2 (4H, m, H-2"-5"), 2.55 (s, CH,malonyl). 613C ppm ([d6]-DMSO); 175.6 (C-41, 167.8 and
167.3 (ester malonyl), 162.1 (C-7), 160.0 (C-4'), 158.0 (C-9),
154.6 (C-2), 131.0 (C-2' and C-67, 128.0 (C-5), 125.0 (C-1'1,
124.4 (C-3), 119.5 (C-lO), 116.4 (C-6), 114.6 (C-3' and C-57,
104.5 (C-8), 100.6 (C-l"), 77.1 (C-5"), 74.7(C-3"), 74.0 (C-Y),
70.2 (C-4), 65.2 (C-6"), 56.1 (OCH,-formononetin), 53.0
(OCH,-malonyl), and 41.8 (CH,-malonyl).
The 'H and I3C spectra of (3) are nearly identical to those
of (21, with the presence of an additional methoxy group
(signals at S ppm 3.70 and 53.0 in 'H and 13C spectra,
respectively). The MS spectra are in agreement with the
formononetin-glycoside-6"-malonateplus 15 atomic mass
units. Based on the results obtained for the flavonoid (31,
we propose the structure of formononetin-7-O-P-~-glycoside-6"-malonate, methyl ester. This product was previously isolated in clover (Beck and Knock, 1971).
The presence of products (1) and (2) but not (3) was
confirmed in the ethanol extracts by analytical HPLC. We
537
concluded, then, that this flavonoid (ononin-6"-malonate,
methyl ester) appears during flavonoid preparation by
modification (methylation) of the formononetin-7-0-P-~glycoside-6"-malonate.
The presence of two other flavonoids, eluting between
ononin and malonyl-ononin in the HPLC analytical chromatogram (Fig. 2A), was also detected, but their complete
spectral analysis could not be determined because of the
small quantity isolated. Based on their UV and EIMS spectra, which are identical to those of compounds (11,(21, and
(31, as well as HPLC analysis in the presence of the authentic formononetin, we suggest that these flavonoids are also
glycoconjugate derivatives of formononetin. The HPLC
analysis and the purification experiments indicate that
ononin and malonyl ononin probably represent the major
isoflavonoids in the M . sativa ssp. varia cv A2 roots.
In addition to these compounds, we identified 7,4'-dihydroxyflavone in the ether fraction of the root extract using
the same procedure as that used for the glycosylated compounds. This flavonoid, already described as one of the R.
meliloti nod gene activators present in alfalfa root exudate
(Maxwell et al., 1989), represented only 0.005% (w/w) of
the original ethanolic fraction, and for this reason is not
detectable by HPLC analysis of this ethanolic fraction. The
purified compound produced UV spectra nearly identical
to the authentic 7,4'-dihydroxyflavone (205, 230[shl,
247[sh], 310[sh], and 327). The EIMS data for this compound showed a major ion at m/z = 254. The NMR signal
resonances were: S'H ppm (CD,OD); 8.07 (lH, d, J = 8.6
Hz, H-5); 7.98 (2H, d , J = 8.6, H-2' and H-6'); 7.08 (lH, d , J
= 2.1 Hz, H-8); 7.04 (2H, d , J = 8.6 Hz, H-3' and H-5'); 7.03
(lH, d d , J = 8.7 and 2.2 Hz, H-6); 6.78 (lH, s, H-3). On the
basis of these data, it was concluded that this flavonoid is
7,4'-dihydroxyflavone. Comparison of this compound with
an authentic sample for UV, EIMS, 'H-NMR, and HPLC
(retention time 13 min) confirmed our conclusion (data not
shown).
Flavonoid Production in Roots Is Nitrogen Regulated
To know whether nitrogen limitation would increase
flavonoid production in the roots of M . sativa ssp. varia cv
A2, the flavonoid content from each sample of the ethanolic
extract was analyzed by analytical HPLC. In the HPLC
profiles analyzed at 250 to 400 nm, the amount of the two
major compounds (ononin and malonyl ononin) was significantly higher in the samples of roots cultured for 10 d in
0.25 mM KNO, compared to those of roots cultured for 10
d in 10 mM KNO, (Fig. 2A).
Figure 2B shows the timing of flavonoid production from
d O to d 10 as calculated from the area of these two major
peaks (ononin and malonyl ononin, (1) and (21, respectively) present in the ethanol extract. Their amounts were
constant in plants grown in 10 mM KNO, but increased in
plants grown in 0.25 mM KNO,. The increase was particularly pronounced after 8 d (see "Discussion"). The R. meliloti nod gene inducer 7,4'-dihydroxyflavone was not detectable by HPLC analysis in the ethanol root extract, but
could be identified in the ether extract. The amount of this
compound also increased 2- to 3-fold in the ether extract of
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Copyright © 1995 American Society of Plant Biologists. All rights reserved.
Coronado et al.
538
FI: 90
-.- 80
3.- 70
B 60
38 50
m
40
,o
-Om 30
2 20
O
2
4
6
8
Days after Nitrate Treatment
10
Figure 3. Rhizobium nod gene-inducing activity of M. sativa root
extracts grown under nonlimiting and limiting nitrogen conditions.
M. sativa ssp. varia plants were grown in nonlimiting (10 mM KNO,,
dashed line) nitrogen solution or in limiting (0.25 mM KNO,, solid
line) nitrogen solution. The ethanol extracts were used to induce a
nodC::lacZ gene fusion in strain Rm1021. The value of the noninduced control was subtracted from each sample value. p-Calactosidase values are expressed in Miller units. Vertical bars represent the
SD for each sample.
roots of plants grown for 10 d under limiting nitrogen
supply (data not shown). Thus, the increase in flavonoid
production was not restricted to the major root flavonoids,
but rather might be a general phenomenon.
Rhizobium nod Gene-lnducing Activity of the Root
Extracts
To investigate whether the production of nod gene inducers was enhanced under nitrogen starvation, ethanol
extracts of M. sativa ssp. varia cv A2 roots were tested for
their nod gene induction capacities in an R. meliloti strain
containing plasmid pRm57 carrying the n o d D l gene
(Mulligan and Long, 1985). A representative experiment is
shown in Figure 3. The level of nod gene induction was
significantly higher in a11 root samples of plants grown in
0.25 mM KNO, during the course of the experiment (Fig. 3),
paralleling the increase of the carbodnitrogen ratio (Fig.
1). This analysis indicates that in roots of plants grown
under limiting nitrogen supply, the production of nod
gene-inducing compounds was significantly enhanced.
To determine whether the two major isoflavonoids
present in the root extract could act as nod gene inducers in
R. meliloti and could be responsible for the observed increase in nod gene-inducing activity, the ethanol extracts of
roots of plants grown for 10 d in nonlimiting or limiting
nitrogen solution were fractionated into an ether fraction
allowing the isolation of nonglycosylated flavonoids, and
into a butanol fraction that should contain the glycosylated
flavonoids (see "Materials and Methods"). By HPLC, glycosylated flavonoids including ononin and malonyl ononin
were detected in the butanol fraction, whereas nonglycosylated flavonoids including 7,4'-dihydroxyflavone were
detected in the ether fraction (see "Materials and Methods"; data not shown). The fractions were evaporated and
resuspended in ethanol, then at a final concentration of 20
Plant Physiol. Vol. 108, 1995
Pg,lmL were used to test their nod gene-inducing capacities
in R. meliloti strain JM57, containing either plasmid pKSK5
(nodD2, Gyorgypal et al., 1991) or plasmid pGM108 (nodD2,
Gyorgypal et al., 1991).
The results shown in Table I confirmed that the production of nod gene inducers was enhanced when plants were
grown in nitrogen-limiting conditions. Moreover, the nod
gene-inducing activity, acting in conjunction with either
NodDl or NodD2, was associated only with the ether
fraction containing the nonglycosylated flavonoids such as
7,4'-dihydroxyflavone, indicating that the two glycosylated
compounds, present in the butanol fraction, are not R.
meliloti nod gene inducers. To rule out the presence of
repressor molecules in the butanol fraction, the purified
compounds (ononin and malonyl-ononin) were also tested
for their nod gene-inducing activity (Table I). Like formononetin (their aglycone form), they did not exhibit detectable nod gene-inducing activity when they were tested
in comparison to the strong nod gene inducer 7,4'-dihydroxyflavone (Table I). Thus, the major compounds present
in the root ethanol extract were not responsible for the nod
gene-inducing activity of this extract.
Expression of Flavonoid Biosynthetic Genes in Roots 1s
Nitrogen Regulated
To know whether the increase in flavonoid and isoflavonoid production correlates with an increase in flavonoid
biosynthetic gene expression, we analyzed the accumulation of transcripts of the flavonoid and isoflavonoid biosynthetic genes CHS and IFR in the roots of the plants of
the two M. sativa subspecies. Northern blots of total RNA
isolated from roots of plants grown as described above
were hybridized successively with the M. sativa CHS (Esnault et al., 1993), IFR (Paiva et al., 1991), and the constitutive Msc27 (Gyorgyey et al., 1991) cDNA probes (Fig. 4,
B and D). The level of expression was quantified from the
autoradiograms and used to determine the level of induction in the course of the experiment. The CHS cDNA hybridized to a 1.4-kb transcript whose accumulation inTable 1. NodDl- and NodD2-mediatednod gene induction by
root extracts of plants grown for 70 d in nonlimiting or limiting
nitrogen solution
The ether and butanol fractions were tested at 20 pg/mL final
concentration for their nod gene-inducing capacities in R. meliloti
strain J M 5 7 containing either plasmid pKSK5 (nodD7) or plasmid
pGM108 (nodD2).P-Galactosidaseactivities are expressed in Miller
units. The value of the noninduced control was subtracted from each
value. SD values are indicated for each value. M-ononin, Malonylononin; 7,4'DHF, 7,4'-dihydroxyflavone.
Fraction Tested
~~~
Ether - NO,
Ether + NO,
Butanol - NO,
Butanol + NO,
M-Ononin 1 p M
O n o n i n 1 PM
Formononetin 1 p~
7,4'DHF 1 PM
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Copyright © 1995 American Society of Plant Biologists. All rights reserved.
NodDl
NodD2
~
140 ? 12
45 2 7
1.2 ? 1
<1
<1
<1
<I
90 ? 37
226
&
7
85 2 12
<1
<1
<1
<I
<1
26 ? 5
539
Nitrogen Regulation of Flavonoid Production in Alfalfa
0
3
Day* after Treatment
5 8 10 3 S 8 10
CHS
Days after Treatment
2
3
«
8 10
2
3
«
I
1*
CHS
IFR
Mac 27
M1C27
•**«•»•«»•
Figure 4. CHS and IFR transcript levels in M. saliva roots grown under nonlimiting and limiting nitrogen conditions. Plants
were grown in nonlimiting (10 mM KNO3) nitrogen solution or in limiting (0.25 mM KNO3) nitrogen solution. A and C,
Enhancement of the CHS and IFR transcripts after different growth periods (in days) in the different nutrient solutions. The
ratio of transcript levels between plants grown in limiting versus nonlimiting nitrogen nutrition was calculated from the
quantification of the signals measured from the northern blot autoradiograms, corrected relative to the values of the
constitutive Msc27 probe. Dashed line, CHS transcript; solid line, IFR transcript. B and D, Northern blot analyses of the
expression of CHS, IFR, and Msc27 genes in roots of M. sativa grown in different nutrient solutions ( + , 10 mM KNO3; -,
0.25 mM KNO3). Experiments in A and B were made with M. sativa ssp. varia cv A2, and those in C and D were made with
M. sativa ssp. sativa cv Nagyszenasi. The transcript sizes are CHS, 1.4 kb; IFR, 1.2 kb; Msc27, 0.8 kb.
creased after 3 to 6 d in the 0.25 mM KNO3 treatment
depending on the subspecies used for the experiment, then
gradually decreased until d 8 and increased again at d 10
(see "Discussion"). In the roots of plants grown in 10 mM
KNO3 nutrient solution, only slight fluctuations in transcript levels were detected (Fig. 4, A and C). The northern
blots were washed and hybridized with a 1.2-kb EcoRI
fragment corresponding to the IFR cDNA clone. The pattern of transcript accumulation was similar to that of CHS.
There was no significant modification in the transcript
level in plants grown in 10 mM KNO3 from d 0 to d 10. In
plants grown under limited nitrogen supply the transcript
level reached a maximum level at d 3 or d 6, depending on
the subspecies, and a second peak reached a maximum
level at d 10. The overall induction of IFR expression was
less pronounced than the increase of the CHS expression.
Thus, the nitrogen deprivation correlates with an increase in (iso)flavonoid biosynthetic gene expression and
the genotype of the plant did not significantly modify the
kinetics of expression of the two genes studied.
lel an increase in the exudate. Moreover, the production of
more than 10 peaks absorbing at a wide UV wavelength
range, detected in the ether extract by HPLC, was enhanced (data not shown) and at least some of them might
be flavonoids, suggesting a general increase in flavonoid
production. We cannot exclude that the synthesis of other
types of (nonflavonoid) molecules reported to act as nod
gene activators (Philips et al., 1992) is induced in the roots
of plants grown under nitrogen-limiting conditions. Nevertheless, the increase in (iso)flavonoid content observed
here correlates with an augmentation of the Rhizobium nod
gene-inducing ability of the root extracts, indicating that,
although the production of both nod gene inducers and
noninducers under nitrogen-limiting conditions can be enhanced, the net balance in root flavonoid content is shifted
to the production of nod gene inducers.
CHS transcripts have been shown to be present in roots
of soybean (Estabrook and Sengupta-Gopalan, 1991), pea
(Harker et al., 1990), and alfalfa (Junghans et al., 1993).
Relatively high IFR transcript levels were observed in roots
of healthy, nodulated alfalfa plants (Paiva et al., 1991),
suggesting the possibility of a constant "elicitation" of the
DISCUSSION
roots either by a component of the culture medium, the
presence of bacteria, or the physical stress accompanying
The results presented here indicate that in alfalfa nitroroot growth and pushing through the soil.
gen deprivation enhances the production of root phenolic
The expression of CHS and IFR detected by northern blot
compounds. The analysis of nitrogen-starved or nitrogenanalysis suggests that under nitrogen limitation the expressupplied plants revealed that the production of the most
sion of the two genes is enhanced in two steps. The first
abundant flavonoids ononin and malonyl-ononin, as well
induction occurs 3 to 5 d after nitrogen depletion. Since the
as the minor flavonoid 7,4'-dihydroxyflavone, was encarbon:nitrogen ratio in the plant is affected only slightly at
hanced. Because these compounds are also present in root
this time, we can speculate that the plants are still not
exudates (Dakora et al., 1993a; Phillips et al., 1993), their
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- Published
by www.plantphysiol.org
under
nitrogen-starving
conditions and that the induction
enhanced synthesis in the root observed
here
might
Copyright © 1995 American Society of Plant Biologists. All rights reserved.
540
Coronado et al.
of the synthesis of these compounds may represent a metabolic adaptation to the new growth conditions or be the
consequence of it. The second step of induction may represent a more general stress response of the plant resulting
from nitrogen starvation. The increase in flavonoid content
of the root paralleled the increase of the carbon:nitrogen
ratio, suggesting an accumulation of these compounds in
the course of nitrogen deprivation.
The induction of flavonoid production does not seem to
be specific for one type of flavonoid, but rather seems to be
general. Our studies indicate that the flavonoids acting as
inducers of nod gene expression in the root extract are
nonglycosylated compounds (Table I). Among them, the R.
meliloti nod gene activator 7,4'-dihydroxyflavone (Phillips
et al., 1993) was identified. In addition to the nod gene
inducers present in these extracts, we have identified two
abundant isoflavonoids, ononin and malonyl ononin,
which are derivatives of the isoflavonoid formononetin.
Our results indicate that despite an increase in their production during growth under limiting nitrogen nutrition,
these two compounds are not involved in R. meliloti nod
gene induction, suggesting that the whole pathway leading
to the production of isoflavonoids is induced under the
conditions tested, although only some of the intermediates
are used for the induction of the nod genes. Our results do
not confirm the results of Dakora et al. (1993a) that malonyl-ononin is a nod gene inducer acting in conjunction with
both NodDl and NodD2 regulatory proteins. Our results
were reproducible using purified compounds as well as
butanol extracts from various samples containing this
compound.
Despite the strong expression of t h e IFR gene in t h e root
extracts of nitrogen-starved plants, we did not find the
pterocarpan medicarpin, described as the major phytoalexin produced in response to pathogenic infection and
abiotic stress (Dixon et al., 1992).An authentic medicarpin
sample run at 14 min in the analytical HPLC system was
used in this study (Fig. 2; data not shown) but did not
correspond to any peaks present in this area. If medicarpin
was present in the extract, its amount was below the detection level. This compound was also not detectable in the
ether extract (data not shown). Since an authentic sample
for medicarpin-3-O-glucoside-6"-O-malonate(Tiller et al.,
1994) was not run in this experiment, we cannot exclude
that in our extracts this compound was present at a very
low level. Nor can we exclude that these compounds could
be present only in M . sativa root exudates, or that other
compounds not detected in this experiment and whose
synthesis requires IFR were produced in the M. sativa ssp.
varia roots. The presence of high amounts of glycosylated
forms of formononetin in the roots of M. sativa ssp. varia
may also indicate that instead of medicarpin, these glycosylated forms may represent end products of the isoflavonoid pathway.
The flavonoid content in the roots is a limiting factor for
efficient nodulation of some plant hosts. Nodulation and
nitrogen fixation was shown to be improved in alfalfa after
addition of luteolin to the plant growth medium (Kapulnik
et al., 1987). Severa1 experiments indicate that flavonoids
Plant Physiol. Vol. 108, 1995
can accumulate in plant leaves in response to different
mineral (nitrogen, phosphorus, and potassium) nutrition
deficiencies (McClure, 1975). The increased synthesis of
flavonoids would then represent a general response to
(NO,) nutrition deficiency. In soybean and lupin, combined nitrogen application also decreased the root isoflavonoid concentration (Cho and Harper, 1991a, 1991b; Morandi and Gianinazzi-Pearson, 1993; Wojtaszek et al., 1993).
These data together with our results support the idea that
the host plant partly controls nodulation in relation to
nitrogen nutrition probably by regulating flavonoid accumulation. Part of this control is exerted by modulating the
level of flavonoid biosynthetic gene expression in the roots.
The observed effect can result from changes in the nitrogen
metabolism of the plant or from other physiological
changes resulting from the presence or the absence of the
NO,- ion in the growth medium. In future studies it will
be interesting to test whether similar responses would
occur in roots of alfalfa plants grown under conditions limiting for other elements, such as phosphorus or
potassium.
Root growth and development is affected by the nitrogen
content of the growth medium (Marriott and Dale, 1977).
Flavonoids can function as auxin transport inhibitors
(Jacobs and Rubery, 1988) and in this way can modify the
hormone balance. We cannot exclude the fact that by altering the hormonal content of the root, the new growth
conditions might modify the expression of the flavonoid
biosynthetic genes and consequently flavonoid production
(Ward et al., 1989; Weiss et al., 1990). Rhizobia may have
evolved to use this increase in flavonoid production as a
symbiotic signal for interacting with the nitrogen-starved
plant.
Dakora et al. (1993a, 199313) showed that alfalfa and bean
respond to their symbiotic bacteria by exuding the phytoalexins medicarpin, daidzein, and coumestrol, the production of which is normally elicited by pathogens. Their
results suggest that the first response of the plant to the
presence of the bacteria might be a pathogen-like response,
and rhizobia can use a precursor of the phytoalexin as a
trigger for inducing symbiotic nod genes. Our results are in
agreement with this idea but may also suggest that rhizobia evolved to use, as symbiotic signal molecules, compounds whose synthesis are triggered during nitrogen starvation. This may represent an additional step in the
regulation of the nodulation process.
ACKNOWLEDCMENT
M.-T. Adeline is greatly acknowledged for her help and advice
in the HPLC analysis.
Received October 21, 1994; accepted February 14, 1995.
Copyright Clearance Center: 0032-0889/95 /108 /O533 / 1O
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