Plant Physiol. (1999) 121: 113-122
Flavan-Containing Cells Delimit Frankia-Infected
Compartments in Casuarina glauca Nodules1
Laurent Laplaze,
Hassen Gherbi,
Thierry Frutz,
Katharina Pawlowski,
Claudine Franche,
Jean-Jacques Macheix,
Florence Auguy,
Didier Bogusz, and
Emile Duhoux*
Physiologie Cellulaire et Moléculaire des Arbres, GeneTrop
Institut de Recherche pour le Développement, 911 Avenue
Agropolis, 34032 Montpellier cedex 1, France (L.L., H.G., T.F., C.F.,
F.A., D.B., E.D.); Department of Molecular Biology, Agricultural
University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands (K.P.); Biochemie der Pflanze, Albrecht-von-Haller-Institut für
Pflanzenwissenschaften, Untere Karspüle 2, 37073 Göttingen,
Germany (K.P.); and Laboratoire de Biotechnologie et Physiologie
Végétale Appliquée, Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier cedex 5, France
(J.-J.M.)
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ABSTRACT |
We investigated the involvement of
polyphenols in the Casuarina
glauca-Frankia symbiosis. Histological analysis
revealed a cell-specific accumulation of phenolics in C. glauca nodule lobes, creating a compartmentation in the cortex.
Histochemical and biochemical analyses indicated that these phenolic
compounds belong to the flavan class of flavonoids. We show that the
same compounds were synthesized in nodules and uninfected roots.
However, the amount of each flavan was dramatically increased in
nodules compared with uninfected roots. The use of in situ
hybridization established that chalcone synthase transcripts accumulate
in flavan-containing cells at the apex of the nodule lobe. Our findings
are discussed in view of the possible role of flavans in plant-microbe
interactions.
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INTRODUCTION |
Flavonoids are secondary metabolites derived from the
phenylpropanoid pathway. They are involved in various biological
processes, including flower pigmentation (anthocyanins) and protection
against UV irradiation (Shirley, 1996
). Flavonoids play key roles at
different levels of plant-microbe interactions (Dixon and Paiva, 1995;
Shirley, 1996). In legumes the accumulation of flavonoid compounds,
identified as phytoalexins, occurs in response to pathogen attack.
These compounds have been shown to prevent the spread of the pathogen (Dixon and Paiva, 1995). Particular flavonoids have also been implicated in the establishment of pathogenic and symbiotic
plant-microbe interactions, in particular, in the
Rhizobium-legume symbiosis (Peters and Verma, 1990).
Specific flavonoids released from the roots of legumes interact with
the NodD protein of Rhizobium to activate transcription of
other nod genes responsible for the synthesis of Nod factors
(Dénarié et al., 1996; Long, 1996). Aside from their role
as signal molecules in root exudates, flavonoids might also be involved
in the morphogenesis of legume nodules. Since specific flavonoids can
inhibit polar auxin transport (Jacobs and Rubery, 1988), they were
proposed to change the phytohormone balance during nodule initiation
(Hirsch et al., 1989; Yang et al., 1992; Mathesius et al., 1998).
Moreover, Charrier et al. (1998) suggested a role of flavonoids in
modulating nutrient exchanges and enzymatic activities in legume
nodules. Furthermore, the accumulation of flavonoids in some
ineffective symbioses have been related to a host defense response
elicited by rhizobia, which could be part of the regulation of
nodulation (Grosskopf et al., 1993).
The flavonoid biosynthetic pathway has been extensively studied in
legumes (see e.g. Paiva et al., 1991; Maxwell et al., 1993; McKahn and
Hirsch, 1994; Charrier et al., 1995; Djordjevic et al., 1997). Chalcone
synthase (CHS), which catalyzes the first step of flavonoid
biosynthesis, is a key enzyme in this pathway. It condenses three
molecules of malonyl CoA with 4-coumaroyl CoA to produce chalcones
(Martin, 1993). CHS genes have been cloned from many plants and are
often encoded by small gene families (Martin, 1993). Several authors
have reported that CHS is a key regulatory enzyme of flavonoid
biosynthesis (Hahlbrock and Scheel, 1989) and that the study of CHS
gene expression is a good molecular marker of flavonoid production. CHS
gene expression is subject to complex developmental and environmental
regulation (Hahlbrook and Scheel, 1989; Dixon and Paiva, 1995). Its
expression is induced by nitrogen deprivation in alfalfa (Coronado et
al., 1995) and upon inoculation by rhizobia in soybean and vetch
(Estabrook and Sengupta-Gopalan, 1991; Recourt et al., 1992). In pea
the highest level of chs expression was found in uninfected
apical parts of the nodule, suggesting a developmental function of
flavonoids (Yang et al., 1992).
Whereas rhizobia interact with legumes, Frankia strains
establish root nodule symbioses with eight different angiosperm
families collectively called actinorhizal plants (Benson and Silvester, 1993). The mode of infection depends on the host plant. Two modes of
infection of actinorhizal plants have been described: intercellular and
intracellular (Berry and Sunell, 1990; Franche et al., 1998). Intracellular infection (e.g. of Casuarina glauca) starts
with root hair curling induced by an unknown Frankia signal.
Bacteria penetrate a curled root hair and grow intracellularly. Root
hair infection induces limited cell divisions in the cortex, giving rise to the so-called prenodule. Frankia infects some of the
prenodule cells. Concomitantly with prenodule formation, cell divisions occur in the pericycle opposite to a protoxylem pole, leading to the
formation of a nodule primordium. Its cells become infected by
bacterial hyphae coming from the prenodule. The involvement of
flavonoids in actinorhizal symbioses is poorly understood. Because of
the similarities of the infection process between some actinorhizal
plants and legumes, flavonoids were proposed to act as plant signals
activating the production of a Frankia root hair-deforming factor (Prin and Rougier, 1987; Van Ghelue et al., 1997).
As part of our investigation into plant responses to Frankia
infection, we characterized the major polyphenol compounds and monitored chs gene expression in C. glauca
nodules. We report a cell-specific accumulation of phenolic compounds
in cell layers delimiting Frankia-infected areas of the
nodule cortex. Histochemical and biochemical analyses indicated that
compounds found in these cell strands belong to the flavan group of
flavonoids. We also report that high levels of CHS transcripts were
found in the uninfected tannin-containing cells at the apex of the
nodule lobes. Finally, we discuss the possible functions of flavans in
the C. glauca-Frankia interaction.
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MATERIALS AND METHODS |
Plant Material
Casuarina glauca plants were grown in a greenhouse and
inoculated with Frankia as previously described (Gherbi et
al., 1997). Mature nodules (three to six lobes) from 5- to 6-month-old
plants were harvested 3 to 4 weeks after inoculation and used for the experiments.
Histochemistry
Plant material was fixed and embedded in paraffin as described
previously (Gherbi et al., 1997). Sections (7 µm) were cut with a
microtome (Jun GRM 2055, Leica Microsystems, Wetzlar, Germany). After
staining, sections were mounted in the staining reagent or in glycerine
plus water (15%, v/v) and examined with a light microscope (model
DMR13, Leica). Two filter sets were used: a UV filter set with a 340- to 380-nm excitation and a 425-nm barrier filter, and a blue filter set
with a 450- to 490-nm excitation filter and a 515-nm barrier filter.
For the structural study, sections were stained with 0.025% (w/v)
toluidine blue.
Flavonoid compounds were detected with Neu's reagent (Neu, 1956).
Sections were immersed in 1% (w/v) 2-aminoethyl-diphenyl borinate
(Fluka, Milwaukee, WI) in absolute methanol for 2 to 5 min, mounted in
glycerine water, and observed with epifluorescence. The results were
confirmed with Wilson's reagent (Hariri et al., 1991).
A reagent of vanillin-HCl (Sarkar and Howarth, 1976) was employed for
analysis of catechins and condensed tannins. Sections were immersed in
10% (w/v) vanillin in 1 volume of absolute ethanol mixed with 1 volume
of concentrated HCl, mounted in this reagent, and observed with a light
microscope. Results were confirmed using 4-dimethyl-aminocinnamaldehyde
staining (Feucht and Treutter, 1990) on handmade thick sections.
Hydrolyzable tannins were detected according to the method of Schneider
(1977).
Extraction and Analysis of Soluble Phenolics
Plant material was ground in cold ethanol:water (4:1) containing
2% (w/v) K2SO3. The
extract was agitated for 15 min at 4°C and vacuum filtered. This step
was repeated three times. After ethanol evaporation, the aqueous phase
was extracted four times with EtOAc containing 20% (w/v)
(NH4)2SO4
and 2% (w/v) metaphosphoric acid. The organic phase was then reduced
to dryness in vacuo and the residue was dissolved in MeOH. Root
and nodule extractions were repeated two times using different tissue
samples.
Methanolic extracts were analyzed by HPLC. The liquid chromatograph
(model 600E, Waters, Milford, MA) was equipped with a 5-mm
C18 column (250 × 5 mm; Spherisorb, Machery
and Nagel, Düren, Germany). The following linear gradient
elution system was applied at a flow rate of 1 mL
min
1: within 13 min from 7% to 15% solvent B
(acetonitrile) in solvent A (water), within 17 min at 15% B,
within 5 min from 15% to 19% B, within 15 min at 19% B,
within 5 min from 19% to 27% B, within 5 min at 27% B, within 5 min at 80% B, within 5 min from 80% to 100% B. Injections of 5 µL were carried out using an automatic sampler (model U6K, Waters).
Detection of compounds was done photometrically by a photodiode array
detector (model 990, Waters). (+)-Catechin and (
)-epicatechin were
identified and quantified by chromatographic and spectrophotometric
comparison with standards from Extrasynthese (Genay, France) and Sigma
(St. Louis), respectively. Other flavans were expressed as (+)-catechin
equivalents from internal standardization.
Isolation of a C. glauca CHS cDNA Clone
A 
gt10 cDNA library was prepared from C. glauca root
nodule RNA as described elsewhere (Gherbi et al., 1997). The library was screened by differential hybridization of randomly picked clones
with biotin-labeled root or nodule cDNA (Gherbi et al., 1997). The
insert of cgCHS1 was subcloned into pGEM-T vector (Promega, Madison, WI). Both strands of the insert were sequenced using an
automatic sequencing system (model 373A, Applied Biosystems, Foster
City, CA). Sequence data were analyzed by the BLAST program (Altschul
et al., 1990).
Southern-Blot Analysis
Genomic DNA of C. glauca was isolated using a plant
mini kit (Dneasy, Qiagen, Courtaboeuf, France) according to the
manufacturer. For Southern-blot analysis, 10 µg of DNA was cut by
BamHI and HindIII, separated on an 0.8%
(w/v) agarose gel, and blotted as described previously (Gherbi
et al., 1997). Prehybridization and hybridization were performed at
65°C using 6× SSC, 5× Denhardt's solution, 1% (w/v) SDS,
and 0.1 mg/mL of salmon-sperm DNA. The insert of cgCHS1 was
labeled with [32P]dCTP using an oligolabeling
kit (Pharmacia, Piscataway, NJ). After hybridization the filters were
washed at 65°C successively using 2× SSC, 0.1% SDS (twice for 20 min), 1× SSC, 0.1% SDS (twice for 20 min), and 0.5× SSC, 0.1% SDS
(twice for 20 min).
Isolation of Total RNA and Reverse Transcription (RT) PCR
Analysis
Total RNA was isolated from nodules, uninfected roots, and
stems/leaves using the procedure of Bugos et al. (1995). Total RNA (2.5 µg) was reverse transcribed using an mRNA capture kit (Boehringer
Mannheim, Basel) to avoid DNA contamination in a total volume of 50 µL as described by the manufacturer. The reaction mixture was
incubated at 42°C for 1 h and then for 5 min at 94°C. The PCR
reactions were performed with 5 µL of the cDNA solution in 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2, 0.1% Triton X-100, 0.2 mM each dNTPs, 0.2 mM of each primer, and 1 unit of Taq polymerase (Promega) in a total volume of 50 µL. A 745-bp fragment was amplified using two primers with sequence
similarity to the cgCHS1 coding region (forward:
5
-CTGTCTCGACCAAAGCAC-3, reverse: 5-TGAGATGAGCCCAGGAAC-3) within 35 cycles (94°C, 1 min; 55°C, 1 min; and 72°C, 1 min). As an
internal control, PCR was performed simultaneously using ubiquitin
primers (Horvath et al., 1993). PCR products (5 µL) were separated by
electrophoresis on a 1.6% (w/v) agarose gel, followed by blotting onto
a positive membrane (Appligene Oncor, Heidelberg). The
cgCHS1 or ubiquitin products were detected by hybridization
with 32P-labeled inserts of the corresponding
cDNA clones. Control reactions in which RNA was treated as above but
without reverse transcriptase gave no signal after hybridization.
RT-PCR experiments were repeated using at least two independent RNA
preparations.
In Situ Localization of cgCHS1 mRNA
In situ hybridization was performed as previously described
(Gherbi et al., 1997). For generation of an RNA probe,
cgCHS1 was amplified with two primers homologous to the
coding region (5-TAGTGGTGTGGACATGCC-3 and 5-TGAGATGAGCCCAGGAAC-3).
The 435-bp product was cloned in pGEM-T (Promega). Two
clones were selected, resulting in plasmids pGEM/CHS26 (antisense) and
pGEM/CHS2 (sense). For antisense and sense RNA production, pGEM/CHS26
and pGEM/CHS2 were cut with PstI and in vitro transcribed by
T7 polymerase. Radioactive labeling was performed as previously
described (Gherbi et al., 1997).
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RESULTS |
Histological Study of C. glauca Nodule
Compartmentalization
Actinorhizal nodules consist of multiple lobes, each representing
a modified lateral root without a root cap, with a superficial periderm
and infected cells in the expanded cortex. Longitudinal sections of
mature C. glauca nodules stained with toluidine blue showed
the presence of large amounts of phenolic compounds (green color in
Fig. 1, A-C). These compounds were
abundant in endodermal cells and in a few layers of cortical cells
below the periderm. In the cortex, cells containing phenolics formed
continuous files from the apex to the base of the nodule lobe (Fig. 1,
A-C). These uninfected cells containing phenolics bound layers of
infected and uninfected cells (Fig. 1, A and B). In each layer,
Frankia grew acropetally and never crossed files of
phenolic-filled cells (Fig. 1, A and B) except during early layer
infection (Fig. 1C). In this last case, hyphae went through a
phenolic-free cell at the base of the file. Transverse sections of
C. glauca nodule lobes show that cortical cells accumulating
phenolics are organized in concentric layers (data not shown).
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| Figure 1.
Localization and histochemical characterization of
C. glauca nodule phenolics. A, Longitudinal section of a
nodule lobe stained with toluidine blue showing a central vascular
bundle (V), a ramification of the nodule lobe (R), and the basis of the
nodular root (NR). Purple cells are Frankia-infected
cells (I). Phenolics (green) accumulate in the endoderm (white arrows),
below the periderm (double black arrowheads), and between the nodule
lobe and the nodule root (black arrowheads). Bar = 100 µm. B,
Detail of a longitudinal section of a nodule lobe stained with
toluidine blue showing Frankia infecting acropetally a
cortical compartment surrounded by phenolics containing cells arranged
in files. Bar = 100 µm. C, Longitudinal section of a young
nodule lobe stained with toluidine blue showing cortical files of cells
accumulating phenolic compounds. Frankia infection
progresses acropetally in each layer. Bar = 100 µm. D,
Longitudinal section of a nodule lobe stained with Wilson's reagent
showing that flavonoids accumulate in the endoderm, below the periderm,
and in the cortical cell files. Bar = 200 µm. E, Transversal
section of a nodule lobe stained with vanillin-HCl reagent. Positive
reaction (red color) is found within phenolic globules. Bar = 100 µm. F, Detail of a longitudinal section of a nodule lobe stained with
Schneider's reagent. A positive reaction (black deposit) is found just
below the periderm (arrows). Bar = 100 µm.
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An endophyte-free nodule root is formed at the apex of a nodule lobe.
This root has specific features (i.e. agravitropism, aerenchyma), and
is thought to facilitate oxygen access to the nodule lobe. Figure 1A
shows the presence of phenolics in cells located at the boundary
between the nodule lobe apex and the nodule root.
Taken together, these results show accumulation of phenolic compounds
in the endodermis, below the periderm, and in cortical cells
compartmentalizing the infected cortical tissue. In the cortical
tissue, continuous layers of phenolic-containing cells separate layers
containing both infected and uninfected cells. The boundary between the
apex of the infected nodule lobe and the uninfected nodule root is also
marked by a few layers of phenolic-containing cells. Thus, accumulation
of phenolics creates a compartmentation of C. glauca root
nodules. Nearly cylindrical layers of cells accumulating phenolics
limit different cortical areas where Frankia infection takes
place.
Histochemistry of C. glauca Nodule Phenolics
To characterize phenolic compounds involved in this
compartmentation process, histochemical analyses were conducted on
sections of nodule lobes. Autofluorescence and histochemical data are
listed in Table I.
White-blue autofluorescence was seen in the cell walls of periderm and
endodermal cells, while a white-yellow fluorescence was detected in
infected cell walls after excitation at 365 nm, indicating the presence
of lignin and/or suberin. The color of the infected cell walls
suggested that other phenolics might also be present. These findings
are in agreement with data published by Berg and McDowell (1987) on the
cell wall cytochemistry of Frankia-infected cells in
C. glauca nodules.
Weak orange autofluorescence after excitation at 365 nm and bright
yellow fluorescence after excitation at 420 nm were observed in
phenolic-containing cells. Treatment with either Neu's or Wilson's reagent (Fig. 1D) gave the same results. These tests suggested the
presence of flavonoids, presumably flavan derivatives that are
known to be poorly fluorescent under UV light. This hypothesis was
confirmed by using vanillin-HCl and
4-dimethyl-aminocinnamaldehyde reagents. Positive reactions were
found for both reagents in all cells containing flavonoids (Fig. 1E),
confirming that flavans are present in these cells. These flavans may
be both monomeric flavan-3-ols (catechins) and oligomeric and polymeric
forms (condensed tannins). Dark staining with Schneider's reagent
(Fig. 1F) was detected in cell layers just below the periderm and
endodermis, suggesting that these cells also contain hydrolyzable
tannins.
Histochemical analyses suggest that condensed tannins (flavans) are the
major components of the deposits of phenolic compounds found
in uninfected nodule cells. Gallic tannins accumulated specifically at
the boundaries of the infected cortex, i.e. in the endodermis and below
the periderm.
HPLC Analysis of Root and Nodule Phenolic Compounds
We further characterized soluble phenolics from C. glauca nodules using HPLC. Alcoholic extracts of nodules were
analyzed on a reverse-phase C18 HPLC column. As
shown in Figure 2, HPLC analysis of
nodule extracts showed 10 major peaks. Spectral data indicated that the
two first peaks were not phenolic compounds (data not shown). Based on
retention time, co-injection experiments, and absorption spectra, peaks
5 and 8 were identified as soluble (+)-catechin and ()-epicatechin,
respectively. All other compounds presented the same UV spectrum as
catechins, with a maximum at 280 nm, suggesting that they belong to the
flavan class of flavonoids. Because of their long retention time, peaks
9 and 10 might be polymerized forms of flavan (i.e. condensed tannins).
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| Figure 2.
HPLC analysis of C. glauca nodule
phenolics analyzed at 280 nm. Products 5 and 6 were identified as
(+)-catechin and ()-epicatechin, respectively. The amounts of
flavonoids were calculated from the area of the peaks detected by
analytical HPLC.
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To compare flavonoid metabolism in nodules and uninfected roots, the
same biochemical studies were conducted with root material. The same
profile was observed in roots as in nodules (data not shown). Products
3 to 10 were quantified by integration and are expressed as catechin
equivalents. Table II shows that there
was a strong increase in flavan content on nodules compared with
uninfected roots. These results indicate that nodules accumulated the
same compounds as roots but in a greater amount. Moreover, there were changes in the relative proportion of products: while in
uninfected roots compound 10 was the most abundant, compound 9 was the
major component in nodules. Also, (+)-catechin and ()-epicatechin
amounts were similar in roots, whereas (+)-catechin was about two times more abundant in nodules. This indicated some subtle reorientations of
metabolic fluxes in the different pathways.
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Table II.
Flavonoid content of roots and nodules
Peaks 5 and 8 were identified as soluble (+)-catechin and
()-epicatechin, respectively. All other peaks are catechins belonging
to the flavan class of flavonoids.
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cgCHS1: Isolation and Analysis of Expression in
C. glauca Nodules
Since the synthesis and accumulation of secondary metabolites can
take place at different sites (Reinold and Hahlbrock, 1997), and since
flavans are derived from the normal flavonoid pathway, we chose a CHS
cDNA clone as a marker of flavonoid production to determine in which
cell type flavan synthesis occurs.
The cDNA clone corresponding to a CHS gene was isolated from a C. glauca nodule cDNA library as described previously (Gherbi et al.,
1997). This cDNA, designated cgCHS1, was 1,407 bp long with
an ORF of 1,170 bp corresponding to 389 amino acids (accession no.
AJ132323). A search of the nucleic acid and protein databases showed
high sequence similarity to CHS genes of other plants. For
example, the ORFs in Juglans sp. (J. nigra × J. negia) JSPCHS1 and Vigna unguiculata VUCHSCH showed a
high degree of similarity to the sequence of cgCHS1: 84%
and 72% at the nucleotide level and 91% and 83% identity at the
amino acid level (Fig. 3), respectively.
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| Figure 3.
Sequence alignment of the chalcone synthase cDNAs
from C. glauca (cgCHS1), Juglans
sp. (J. nigra × J. negia) (JSPCHS1; accession no.
X94995), and V. unguiculata (VUCHSCH; accession no.
X74821). Shaded boxes indicate conserved nucleotides. Putative
initiation and termination codons are marked by boxes.
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To determine the number of CHS genes in the C. glauca
genome, DNA was digested with BamHI and HindIII,
and a Southern transfer experiment was performed with the entire
cgCHS1 cDNA clone as a probe. Several bands were detected
with both enzymes (Fig. 4) suggesting
that there are several CHS genes in C. glauca, as in other
plants (Martin, 1993).
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| Figure 4.
Southern-blot hybridization of genomic DNA
isolated from C. glauca. Ten micrograms of DNA was
digested with BamHI (Ba) and BgIII (Bg)
and probed with 32P-labeled cgCHS1 cDNA.
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We were unable to detect cgCHS1 transcripts using the
northern blot technique. Therefore, RT-PCR, a more sensitive technique, was used to determine the expression of cgCHS1. As shown in
Figure 5A, a 745-bp fragment was
amplified from aerial parts, uninfected roots, and nodule RNA. Thus,
cgCHS1 is expressed in all tested organs. No PCR product was
observed when the reverse transcription step was omitted (Fig. 5A),
indicating the absence of any DNA contamination. PCR on genomic DNA
using the same primers amplified a product of about 1,100 bp,
suggesting that at least one intron was present in the amplified
fragment (data not shown). cgCHS1 transcripts were localized
in C. glauca nodules by in situ hybridization with sense and
antisense RNA probes. As shown in Figure
6, cgCHS1 mRNA is
present in phenolic-containing cell layers between the nodule lobe and
the nodule root, below the periderm, in the endoderm, and in the
apical part of the cortical cell files. cgCHS1 mRNA was
also detected at the apex of young nodule lobes before nodule root
formation (data not shown). Thus, in situ hybridization studies showed
that the expression of cgCHS1 occurred in cells where
flavans were detected. However, cgCHS1 expression was
restricted to the apical part of the cortical flavonoid-containing cell
layers, suggesting that in the cortex, flavonoid biosynthesis is
restricted to these cells. Low levels of CHS transcripts were also
present in infected cells. No signal was found in hybridizations with sense CHS RNA (data not shown).
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| Figure 5.
RT-PCR analysis of cgCHS1
expression in C. glauca aerial parts (AP), uninfected
roots (R), and nodules (N). A, cgCHS1 expression was
found in all tissue tested. No amplification was found when the reverse
transcription step was omitted. B, Amplification of ubiquitin RNA used
as an internal control.
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| Figure 6.
Localization of CgCHS1 transcripts
in C. glauca nodules. Shown are bright- (A) and
dark-field (B) micrographs of 7-µm sections hybridized with
35S-labeled antisense transcripts. Arrowheads on the bright-field
micrograph indicate the locations of tannin accumulation. White grains
on the dark-field micrograph (highlighted by white arrows) indicate the
locations of hybridizing 35S-labeled transcripts. E, Endodermis; P,
periderm; C, cortical cell strands; B, boundary between the nodule lobe
and the nodule root; I, infected cells. Bar = 100 µm.
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DISCUSSION |
We have shown that in C. glauca nodules,
Frankia-infected cells occur in layers surrounded by
tannin-containing cell layers located below the periderm, in the
endodermis, and in the cortex. This characteristic distribution of
phenolics was also observed in the early steps of nodule development,
i.e. in the prenodule and in the nodule primordia (data not shown).
Interestingly, GUS reporter gene expression driven by the CaMV 35S
promoter in transgenic C. glauca nodules within the nodule
cortex was limited to tannin-containing cells, confirming the specific
behavior of these cells (C. Franche, unpublished data). Thus, in
addition to infected and uninfected cells, cells containing phenolics
represent a third specialized cell type in the cortex of C. glauca nodules.
No such organized tannin accumulation is found in C. glauca
pseudonodules induced by auxin transport inhibitors (E. Duhoux, unpublished data). Regular layers of phenolic-containing cells in the
nodule cortex are also found in other actinorhizal nodules, e.g. those
of Alnus glutinosa. There, the regular pattern of layers of
cells containing phenolics is absent in ineffective nodules induced by
Frankia strains not capable of symbiotic nitrogen fixation (Guan et al., 1996). Therefore, the existence of these cell layers seems to be the result of signal exchange with the endophyte. However,
this compartmentation is initiated prior to infection of the nodule
lobe and therefore is not solely a response to Frankia.
A similar accumulation of phenolics has previously been described for
the early steps of the intracellular infection process in
Casuarina cunninghamiana (Torrey, 1976),
Comptonia peregrina (Callaham and Torrey, 1977), and
Alnus glutinosa (Angulo Carmona, 1974). Furthermore,
accumulation of phenolics was also observed during intercellular
infection of Eleagnus angustifolia (Miller and Baker, 1985)
and Parasponia rigida (Lancelle and Torrey,
1984), the only non-legume nodulated by Rhizobium,
suggesting that this phenomenon is a common feature in non-legume root
nodules.
In some legume systems, it has been shown that proanthocyanidins
(condensed tannins) are present in the root cortex (Stafford, 1997). In
Medicago spp., flavonoids are detected in the cortex and in
the nodule meristem (Charrier et al., 1998). Furthermore, in
Acacia mangium, a legume tree, polyphenols accumulate at the site of root hair infection, and the developing nodule primordium is
surrounded by cortical cells containing large amounts of polyphenols (Y. Prin and E. Duhoux, unpublished data). Therefore, the accumulation of phenolics is not restricted to the root nodules of non-legumes, but
seems to play a larger role in woody than in herbaceous plants.
HPLC elution profiles of root and nodule extracts gave the same 10 components. Histochemical and biochemical analyses revealed that all
soluble phenolic compounds in nodules belong to the flavan class of
flavonoids. Our data indicate that (+)-catechin and ()-epicatechin are the major monomeric flavans, and that polymerized flavans (i.e.
condensed tannins) are also present (Fig. 2, peaks 9 and 10). Although
in the alcoholic extract, flavans were the only soluble flavonoids
detected, cell wall-bound flavonoid compounds might also be located
within the cell walls of Frankia-infected cells, as
suggested by autofluorescence and histochemical data. The observed
increase in flavan content in nodules and the different proportions of
the different compounds in root and nodule suggest that
Frankia infection stimulates the expression of genes
encoding flavan-biosynthetic enzymes. However, recent results from Guo et al. (1998) showed that in the soybean cell-suspension-culture system, posttranscriptional mechanisms are responsible for isoflavonoid phytoalexin accumulation. Identification of the flavan monomers and
oligomers found in nodule extracts and the isolation of C. glauca genes involved in their biosynthesis will help us to gain an understanding of the regulatory mechanisms responsible for the
symbiotic induction of flavan production.
High amounts of phenolics in actinorhizal nodules might restrict the
endophyte to certain regions in the cortex, preventing its spread in
the vascular tissue, periderm, and meristem. Moreover, it has been
shown that phenolics can influence Frankia growth in vitro
(Perradin et al., 1982). Therefore, it will be interesting to determine
whether flavans isolated from C. glauca nodules can influence the growth of C. glauca-infective
Frankia strains. Weiss et al. (1997) reported an
accumulation of phenylpropanoids in larch mycorrhizae. They observed a
significant accumulation of (+)-catechin and ()-epicatechin in the
inner cortex, endodermis, and root apex that remained free of fungi.
Since (+)-catechin and ()-epicatechin have been shown to inhibit
fungal growth, they suggested that this accumulation might control
fungal invasion. In the legume Lotus corniculatus, flavans
accumulate in cells arranged in files near the root vascular bundle
(Stafford, 1997). Altogether, these data suggest that flavan synthesis
is a constitutive plant defense response in roots of C. glauca, and that Frankia infection increases this
reaction. This increase might be mediated by a symbiosis-specific
regulation of genes of the flavan biosynthetic pathway. Alternatively,
these polyphenol layers might contribute to the protection against
secondary infection and/or limit oxygen penetration in the nodule.
Several authors have suggested that flavonoid and isoflavonoid
biosynthesis in response to biotic or abiotic stresses is regulated via
CHS expression (Martin, 1993). The localization of CHS mRNA by in situ
hybridization indicated that cgCHS1 is expressed in the
flavan-containing cells of the apex of the nodule lobe. This expression
pattern was correlated with the detection of flavans in the same cell
type. This observation implies that flavonoid synthesis depends on the
developmental stage of the cortical cells. The inability to detect
cgCHS1 transcripts on northern blot might be due to the fact
that cgCHS1 is expressed transiently in a few specialized
cells of the apex. Interestingly, in parsley, several enzymes of the
flavonoid pathway appeared much more stable than their corresponding
mRNAs (Reinold and Hahlbrock, 1997).
It is therefore possible that in C. glauca nodules
chs expression appears transiently at the apex and is still
active in cell derivatives within the nodule lobe to produce the
tannin-containing cell layers. This hypothesis can be tested using
antibodies directed against CgCHS1. A low level of cgCHS1
expression was also found in the infected cortical cells. Similarly,
flavonoid biosynthetic mRNAs were detected in cells of Medicago
truncatula colonized by the mycorrhizal fungus Glomus
versiforme (Harrison and Dixon, 1994). In this case, it was
clearly demonstrated that expression was not linked to a defense
response. It was proposed that flavonoids might influence carbon
metabolism in these cells. As suggested by our histochemical data,
chs expression in infected cells of C. glauca
nodules might be associated with the presence of flavonoids in their
cell walls. These wall-bound flavonoids have been suggested to function
in limiting oxygen access to the places of nitrogen fixation (Berg and
McDowell, 1987; Silvester et al., 1990).
In conclusion, during C. glauca nodule formation,
cell-specific flavan biosynthesis and accumulation delimit cortical
compartments containing Frankia-infected cells and might
restrict endophyte invasion. Recent reports on mycorrhiza-induced
biosynthesis and tissue-specific accumulation of phenolics in larch
(Weiss et al., 1997) suggest that the same type of strategy is used to
control ectomycorrhizal interaction. However, it should be pointed out that catechin and epicatechin accumulate mainly in the endodermis of
the mycorrhizal roots or in the calyptra surrounding the root tips, and
not in the vicinity of the symbiotic cells (Weiss et al., 1997). The
fact that high amounts of phenolics are also found in root nodules of
P. rigida and A. mangium and not in nodules of
herbaceous legumes suggests that the woody nature of the host plant is
an important factor.
Layers of polyphenol-containing cells lead to a compartmentation of the
cortex of the nodule lobe that is composed of several infected areas
delimited by flavan-containing cell layers. The most
recently infected compartment seems to be the innermost
one. The meaning of this second compartmentation is not understood, but
obviously some signal exchange with the endophyte is needed for its
development.
| |
FOOTNOTES |
1
This research was supported by the Institut de
Recherche pour le Développement.
*
Corresponding author; e-mail duhoux{at}mpl.ird.fr; fax
33-4-67-63-82-65.
Received February 3, 1999; accepted June 7, 1999.
| |
ACKNOWLEDGMENTS |
We acknowledge Dr. M. Nicole (Institut de Recherche pour le
Développement-GeneTrop, Montpellier, France) and Dr. C. Andary (Faculté de Pharmacie, Montpellier, France) for helpful
discussions. We thank Magali Derroja for help with biochemical
analysis.
| |
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Abstract
Introduction