Plant Physiol. (1999) 120: 83-92
Nodule-Inducing Activity of Synthetic Sinorhizobium
meliloti Nodulation Factors and Related
Lipo-Chitooligosaccharides on
Alfalfa.
Importance of the Acyl Chain Structure1
Nathalie Demont-Caulet2, 3,
Fabienne Maillet2,
Denis Tailler,
Jean-Claude Jacquinet,
Jean-Claude Promé,
Kyriacos C. Nicolaou,
Georges Truchet,
Jean-Marie Beau, and
Jean Dénarié*
Institut de Pharmacologie et de Biologie Structurale, Centre
National de la Recherche Scientifique (CNRS), 205 Route de Narbonne,
31077 Toulouse cedex, France (N.D.-C., J.-C.P.); Laboratoire de
Biologie Moléculaire des Relations Plantes-Microorganismes,
CNRS-Institut National de la Recherche Agronomique, B.P. 27, 31326 Castanet-Tolosan cedex, France (N.D.-C., F.M., G.T., J.D.); Laboratoire
de Biochimie Structurale, Unité de Recherche Associée
(URA)-CNRS 499, Université d'Orléans, B.P. 6759, 45067 Orléans cedex, France (D.T., J.-C.J., J.-M.B.); Department of
Chemistry, The Scripps Research Institute, 10666 North Torrey Pines
Road, La Jolla, California 92037 (K.C.N.); and Laboratoire de
Synthèse de Biomolécules, URA-CNRS 462, Institut de Chimie
Moléculaire, Université Paris-Sud, 91405 Orsay cedex,
France (J.-M.B.)
 |
ABSTRACT |
Sinorhizobium
meliloti nodulation factors (NFs) elicit a number of symbiotic
responses in alfalfa (Medicago sativa) roots. Using a
semiquantitative nodulation assay, we have shown that chemically
synthesized NFs trigger nodule formation in the same range of
concentrations (down to 10
10 M) as natural
NFs. The absence of O-sulfate or
O-acetate substitutions resulted in a decrease in
morphogenic activity of more than 100-fold and approximately 10-fold,
respectively. To address the question of the influence of the structure
of the N-acyl chain, we synthesized a series of sulfated
tetrameric lipo-chitooligosaccharides (LCOs) having fatty acids of
different lengths and with unsaturations either conjugated to the
carbonyl group (2E) or located in the middle of the chain (9Z). A
nonacylated, sulfated chitin tetramer was unable to elicit nodule
formation. Acylation with short (C8) chains rendered the LCO active at
107 M. The optimal chain length was C16, with
the C16-LCO being more than 10-fold more active than the C12- and
C18-LCOs. Unsaturations were important, and the diunsaturated 2E,9Z LCO
was more active than the monounsaturated LCOs. We discuss different
hypotheses for the role of the acyl chain in NF perception.
| |
INTRODUCTION |
Rhizobia are symbiotic bacteria that elicit on the roots of
specific leguminous hosts the formation of new organs (nodules) in
which they multiply and fix nitrogen. Both loss- and gain-of-function genetic experiments have shown that host recognition, the initiation of
infection thread formation, and the induction of nodules require rhizobial nodulation (nod) genes. Most products of
nod genes are involved in the synthesis and transport of
bacterial symbiotic signals, the NFs (Dénarié et al., 1996
;
Long, 1996). NFs synthesized by all rhizobial species are LCOs. They
consist of a chitin oligomer backbone that is made up of four or five
GlcNAc residues, mono-N-acylated at the nonreducing end and
carry diverse substitutions at both ends. Each rhizobial
species (or biovar) has a defined host range and
produces a set of NFs with specific substitutions (Dénarié et al., 1996). NF structure does not correlate with rhizobial taxonomy
and phylogeny as derived from molecular studies of rRNA, but it
correlates instead with the rhizobial host range, indicating that a
given plant has defined NF structural requirements for the triggering
of symbiotic responses (Dénarié et al., 1996).
Rhizobia can be schematically grouped into two classes according to the
type of N-acylation of their NFs. Rhizobia that nodulate tropical and temperate legumes of the Genisteae and Loteae tribes (Azorhizobium caulinodans, Bradyrhizobium
japonicum, Rhizobium sp. NGR234,
Rhizobium fredii, and Rhizobium loti) produce NFs that are N-acylated with fatty acids of general lipid
metabolism such as C18:1
11Z (vaccenic acid) (Cohn et al., 1998).
In contrast, rhizobia that nodulate legumes of the related Galegeae,
Trifolieae, and Vicieae tribes produce NFs that are
N-acylated by polyunsaturated fatty acids, whose major
species are C16:22E,9Z in Sinorhizobium meliloti;
C18:42E,4E,6E,11Z in Rhizobium leguminosarum bv
viciae and Mesorhizobium huakuii; C20:3 and C20:4
in R. leguminosarum bv trifolii; and C18:2 and
C18:3 in Rhizobium galegae (Lerouge et al., 1990; Spaink et
al., 1991, 1995; van der Drift et al., 1996; G.P. Yang, F. Debellé, A. Savagnac, M. Ferro, O. Schiltz, F. Maillet, D. Promé, M. Treilhou, C. Vialas, K. Lindstrom, and others,
unpublished data). The nodFE genes, which determine the synthesis of these polyunsaturated fatty acids, are major host-range determinants, indicating that the N-acyl-specific structure
(carbon-chain length and degree of unsaturation) is involved in host
specificity (Spaink et al., 1989; Ardourel et al., 1994; Bloemberg
et al., 1995). The use of purified NFs has shown that acylation by
polyunsaturated fatty acids is important for biological activity in
vetch, clover, and alfalfa (Spaink et al., 1991, 1995; Ardourel et al.,
1994).
Strains of S. meliloti, an alfalfa symbiont, produce NFs
that have three specific substitutions (Fig.
1): O-sulfation at the C6
position of the reducing GlcNAc residue, O-acetylation at
the C6 position, and N-acylation by an unsaturated C16 fatty
acid (C16:19Z, C16:22E,9Z, and C16:32E,4E, 9Z, respectively)
of the terminal nonreducing glucosamine residue (Lerouge et al., 1990;
Roche et al., 1991b; Schultze et al., 1992; Demont et al., 1993).
Purified NFs of S. meliloti in the nanomolar to picomolar range elicit various responses on alfalfa roots: electrophysiological changes in root hairs (Ehrhardt et al., 1992, 1996; Felle et al., 1995,
1996, 1998; Kurkdjian, 1995), root hair deformations (Lerouge et al.,
1990), activation of cortical cells (Ardourel et al., 1994), activation
of early nodulin genes (Journet et al., 1994; Fang and Hirsch, 1998),
induction of cell divisions in the inner cortex, and formation of empty
nodules (Truchet et al., 1991). Two approaches have been used to study
the NF structural requirements for the elicitation of alfalfa
responses: treatment of plants with S. meliloti nod mutants
that produce modified NFs and treatment with purified NFs prepared from
these mutants. The requirement for the three NF substitutions seems to
vary with the plant response, suggesting the involvement of different
mechanisms of NF perception and transduction in alfalfa (Ardourel et
al., 1994; Felle et al., 1995) and underlining the importance of
structure-activity studies.
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| Figure 1.
A, Structure of the synthetic S. meliloti NFs. B, Related LCOs with different
N-acyl moieties used in this study.
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An important limitation when using natural NFs to study
structure-activity relationships is that rhizobia produce a mixture of
NFs having different substitutions and it is presently not possible to
separate all of the molecular species. The chemical synthesis of NFs of
S. meliloti (Nicolaou et al., 1992; Wang et al., 1993;
Ikeshita et al., 1994; Tailler et al., 1994) and B. japonicum (Ikeshita et al., 1995) has now been achieved, making possible the synthesis of a variety of chemically pure authentic NFs,
the introduction of single, defined, structural modifications, and the
synthesis of analogs that may not occur naturally. Stokkermans et
al. (1995) used synthetic NFs and related LCOs to study NF structural
requirements to elicit symbiotic responses in soybean, a plant
nodulated by rhizobia that produce fucosylated NFs and do not possess
active nodFE genes. These studies have shown the importance
of the length of the chitin oligomer backbone, the requirement for the
fucosyl group at the reducing end, and the relative unimportance of the
structure of the acyl chain on soybean symbiotic responses.
In the present study we used chemically synthesized S. meliloti NFs and a semiquantitative nodulation bioassay to show
that synthetic NFs have nodule-inducing activities similar to those of
their natural analogs. We also address the role of the
O-sulfate and O-acetate substitutions and use a
set of new synthetic LCOs to address the role of the structure of the
N-acyl moiety in this morphogenic activity.
| |
MATERIALS AND METHODS |
Preparation and Purification of NFs and Related LCOs
Natural NFs were isolated from the overproducing
Sinorhizobium meliloti 2011 (pMH682) strain as previously
described (Roche et al., 1991a). The following LCOs, analogs to natural
NFs, were chemically synthesized by Nicolaou et al. (1992):
LCO-IV(Ac,S,C16:22,9), LCO-IV(S,C16:22,9),
LCO-IV(Ac,C16:22,9), and LCO-IV(C16:22, 9; see Fig. 1). Sulfated
LCO-IV with modified aliphatic chains (described in Fig. 1B) were
synthesized according to a modification of the procedure described by
Tailler et al. (1994). The synthetic intermediate consisting of a
chitotetramer with a free amino group at the nonreducing end and an
O-sulfate group located on C6 of the GlcNAc reducing
residue, was acylated by a variety of fatty acid chlorides as described
below.
To a solution of the sulfated tri-N-acetyl chitotetraose
(20-40 µmol), either in a 5:2 (v/v) dimethylformamide:water mixture (1 µmol 20 µL1) for acylation with the C8:12,
C12:12, C16:12, and C18:12 acyl chlorides or in a 1:1:1 (v/v)
ethyl acetate:methanol:water (25 µL µmol1
oligosaccharide) for acylation with the C16:19, C16:22,9, and C18:22,9 acyl chlorides, we added 3 equivalents of sodium
hydrogenocarbonate and 2 equivalents of the acyl chloride, either as a
0.7 M solution in tetrahydrofuran for the
former solvent system or as a 0.7 M solution in
ethyl acetate for the latter system. The reaction mixture was stirred
at room temperature; we then added 2 equivalents of hydrogenocarbonate
and acyl chloride after 4 and 24 h, respectively. The reaction was
monitored on silica gel 60 F254 TLC plates
(Merck, Darmstadt, Germany) using ethyl acetate:methanol:water (2:1:1, v/v) as an eluant, with detection by charring with
H2SO4:ethanol (10:1).
After 48 h the reaction mixture was concentrated in vacuo and
excess acylating agent was removed with ethyl ether. The acylated products were purified by column chromatography on silica gel (Merck,
40-63 µm, 1.5 g 10 µmol1 starting
oligosaccharide) with ethyl acetate: methanol:water (5:2:1, v/v) as an
eluant, followed by chromatography on a Sephadex G-25 column (1.4 × 28 cm) with water as an eluant. The unreacted amine (20%-30%) was
recovered from the silica gel column. LCOs were isolated as white,
amorphous powders after lyophilization, and we
obtained the following yields: LCO-IV (C8:1,2E), 68%; LCO-IV
(C12:1,2E), 55%; LCO-IV (C16:1,2E), 61%; LCO-IV (C18:1,2E), 65%; LCO-IV (C16:1,9Z), 60%; LCO-IV (C16:2,2E,9Z), 60%; and LCO-IV (C18:2,2E,9Z), 56%. The structure and purity of the LCOs was
determined by proton-NMR and MS. NMR spectra were recorded at 25°C
for samples in 2H2O (LCO-IV
[C8:1,2E]) or methanol-d4 (other LCOs) on a
spectrometer (model AM-300WB, Bruker Instruments, Billerica, MA); for
details, see Tailler et al. (1994) and D. Tailler, J.C. Jacquinet, and J.M. Beau (unpublished data). Mass spectra of LCOs were recorded on a
mass spectrometer (Autospec VG-Analytical, Fisons, Manchester, UK),
equipped with liquid secondary ion MS, as previously described (Demont
et al., 1993).
Plant Assays
Seeds of alfalfa (Medicago sativa cv gemini)
were provided by V. Gensollen (Groupe d'Etude et de Contrôle des
Variétés et des Semences, Montpellier, France). Seeds were
sterilized and germinated, and seedlings were transferred to test tubes
as previously described by Truchet et al. (1985). Plants were grown in
a growth chamber (20°C, 80% RH, 16 h under 300 mE
m2 s1 for 24 h). For each treatment three series of dilutions of LCOs were tested,
ranging from 108 to
1010 M for the LCOs
described in Figure 1B, top, and from 107 to
109 M for the LCOs
described in Figure 1B, bottom. LCOs were applied in melted Fahraeus
agar medium immediately after it was poured into test tubes. To
minimize experimental errors in the preparation of these highly diluted
solutions, we prepared three independent LCO solutions for each
dilution; each of the three served to inoculate 10 test tubes. We used
30 tubes, each containing two seedlings, for each treatment. To limit
subjective bias in the estimation of nodulation, two persons counted
the nodules independently and the results were averaged.
Seeds of common vetch (Vicia sativa subsp. nigra)
were kindly provided by G. Genier (Station d'Amelioration des Plantes,
Institut National de la Recherche Agronomique, Lusignan, France). The
vetch root-hair initiation and thick and short root assays were
performed as previously described by Roche et al. (1991a), except that
Jensen medium was replaced with Fahraeus medium. We used the Fisher's test, with Statview SE+ software (Abacus Koncept, Alfa System Diffusion, Meylan, France), to perform the variance analysis. We
estimated the activity of root-hair initiation with the limit-dilution method, as previously described (Roche et al., 1991a).
Microscopic Methods
Light microscopy of root deformations took place as follows. Roots
were fixed in 2.5% glutaraldehyde buffered at pH 7.2 with sodium
cacodylate (30 min under vacuum and 1 h at room temperature), stained with methylene blue (0.005% in distilled water), cleared 3 min
with sodium hypochlorite (Truchet et al., 1989b), and finally, stained
with potassium iodide (Vasse et al., 1990). Root sections (80 µm
thick) were made after the fixation procedure (Micro-Cut H 1200, Bio-Rad). We viewed the entire plants or the root sections by bright-
or dark-field microscopy with a light microscope (Vanux, Olympus).
| |
RESULTS |
Morphogenic Activity of Synthetic NFs on Alfalfa and the Importance
of O-Sulfate and O-Acetate Groups
Our first goal was to determine whether chemically synthesized NFs
showed the activities and limitations that are characteristic of
biological material. The two major natural NFs produced by S. meliloti are 
-1,4-linked GlcNAc tetramers,
O-6-sulfated on the reducing sugar, N-acylated by
2E,9Z hexadecadienoic acid on the nonreducing sugar, and either
O-6-acetylated or not on C6 of this sugar; these NFs were
termed NodRm-IV(Ac,S,C16:2) and NodRm-IV(S,C16:2), respectively (Roche
et al., 1991a). These two compounds were chemically synthesized by
Nicolaou et al. (1992) (Fig. 1). S. meliloti NFs can induce
the formation on alfalfa seedlings of root-derived structures that
share common features with the nodules induced by bacteria (Truchet et
al., 1991). Nodule formation is NF dose dependent and can be used as a
semiquantitative bioassay. Synthetic LCO-IV(Ac,S,C16:2) and
LCO-IV(S,C16:2) and the mixture of natural NFs were tested at
concentrations varying from 108 to
1010 M. Root-derived
structures having the appearance of small nodules appeared rapidly and
could be observed within 2 weeks at concentrations of
108 and 109
M (Fig. 2A).
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| Figure 2.
Nodule induction on alfalfa by natural and
synthetic S. meliloti NFs. A, Nodulation kinetics at
108 M. B, Nodule number 26 d after
addition of NFs. NodRm, Natural S. meliloti NFs; LCO-IV,
synthetic S. meliloti NFs. All LCOs tested had a
C16:22E,9Z acyl chain (see Fig. 1B). Nodule numbers represent the
average of 30 tubes, each containing two seedlings. Statistical
analysis of nodule numbers was performed separately for each
concentration by analysis of variance with Fisher's test: treatments
with letters in common do not differ significantly at the P = 0.05 level.
|
|
Cytological studies revealed that the root-derived structures induced
by LCO-IV(Ac,S,C16:2), LCO-IV(S,C16:2), and by natural NFs were
indistinguishable and shared a number of features with S. meliloti-induced nodules: cortical origin, peripheral vascular bundles, and endodermis. In contrast to rhizobium-induced nodules, those induced by both synthetic and natural NFs exhibited the following
features: they generally had transient meristems and thus remained
small and nonelongated (Fig. 3, A and C),
and root hairs located at the distal ends of emerging nodules (Fig. 3, A and C) and distally to the meristematic areas elicited in the plant
cortex (Fig. 3, B and D) were extremely long and deformed. These
characteristics allowed us to easily distinguish between NF-induced
nodules and the rare, spontaneous nodules that developed in the absence
of rhizobia (Truchet et al., 1989a). In the control seedlings without
NFs, the formation of non-rhizobium-induced nodules was a rare event:
two or three nodule-like structures could be observed among 60 seedlings approximately 3 weeks after germination. Morphologically,
such spontaneous nodules are glabrous, and because they have a
persistent meristem, they are elongated (Pichon et al., 1994). These
differences suggest that NFs induce a nodule-organogenic program that
does not correspond to the activation of non-rhizobium-induced nodule
formation. These results show that synthetic NFs are responsible for
the induction of nodule morphogenesis and that the alfalfa nodulation
assay can be used to study the structure-activity relationship with
synthetic NFs and related LCOs.
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| Figure 3.
Comparison between biological activities of
natural S. meliloti NFs (A and B) and synthetic NFs and
related LCOs (C-F). After fixation, entire plants (A, C, E, and F) or
root sections (B and D) were cleared with sodium hypochlorite (A-F),
stained with methylene blue (A and C), stained with potassium iodide
(C-F), and finally viewed by dark-field microscopy (A and C) or
bright-field microscopy (B, D, E, and F). A to D, Abundant development
of root hairs at the distal part of nodules (A and C) or in the
vicinity of the root area where cortical cell divisions (asterisks) are
observed. A and B, Plants treated with natural NFs. C and D, Plants
treated with synthetic NFs. E and F, Amyloplasts (arrowheads) in
dividing cortical cells of a plant treated with LCO-IV(S,C16:12E)
(E) and in nondividing inner cortical cells of a plant treated by
LCO-IV(S,C8:12E) (F). In A and C, bars = 250 µm; in B and D,
bars = 100 µm; in E and F, bars = 50 µm.
|
|
At the three concentrations used, the natural NFs elicited the
formation of slightly more nodules than the synthetic ones (Fig. 2B),
suggesting that a mixture of various NFs may be more efficient in
triggering nodule formation than a single molecular species. The
nonacetylated LCO-IV(S,C16:2) was less efficient than the acetylated
form in eliciting nodule formation, confirming the importance of the
O-acetyl substitution for morphogenic activity. Root
treatment with chemically synthesized nonsulfated LCO-IV(Ac,C16:2) and
LCO-IV(C16:2) did not result in a detectable induction of nodule
formation, confirming that O-sulfation of NFs is required for nodule induction in alfalfa.
Activity of Synthetic NFs on Vetch
The original definition of NFs as specific signals was
strengthened by observing the positive and negative relationships
between the NF structures and the host range. S. meliloti
nodH mutants have acquired the ability to infect and nodulate
vetch, a nonhost (Faucher et al., 1988). An S. meliloti
nodH::Tn5 mutant produces nonsulfated NFs that elicit
root-hair initiation and thick and short root formation on vetch
(Faucher et al., 1989; Roche et al., 1991a). Using these two vetch
bioassays we compared the biological activity of synthetic sulfated and
nonsulfated NFs using natural nonsulfated NFs purified from a
nodH mutant as a control. Synthetic, nonsulfated
LCO-IV(C16:2,Ac) and LCO-IV(C16:2) factors induced significant
root-hair initiation (P = 0.01) at concentrations as small as
1011 M, as did the
natural, nonsulfated NFs (Table I). In
contrast, the sulfated NFs had no detectable effect, even at
109 M, showing that the
nonsulfated factors are at least 1000-fold more active than the
sulfated factors in eliciting biological responses on this nonhost.
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|
Table I.
Root-hair initiation assays on vetch
All LCOs tested had a C16:2 (2E,9Z) acyl chain. Ten plants were used
for each treatment and each dilution. Forty untreated control plants
were used to estimate the intrinsic plant variability for the root-hair
initiation character. The responses were classified "+" when the
proportion of root hair initiation was significantly higher (at P = 0.05) among the treated plants compared with the untreated control.
Data were analyzed using Fisher's exact
test.
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Because natural NFs do not elicit nodule formation on vetch (Spaink et
al., 1991), we used the thick and short root assay for an assessment of
NF activity on vetch root morphology. At 109
M, nonsulfated synthetic factors induced a significant
(P = 0.01) thick and short root effect, whereas the sulfated
factors had no detectable effect (Fig.
4). Thus, as is the case with natural NFs, nonsulfated synthetic NFs are able to elicit morphological changes
on the roots of vetch. The natural, nonsulfated NFs had a stronger
thick and short root effect than the synthetic NFs (significant at the
P = 0.01 level), suggesting that in this assay a mixture of NFs,
including tetramers and pentamers and N substitutions by
various unsaturated fatty acids, might be more active than single
molecular species.
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| Figure 4.
Induction of root shortening on vetch by sulfated
and nonsulfated synthetic S. meliloti NFs.
Nat.nodH, Natural nonsulfated NFs from a
nodH mutant of S. meliloti; LCO-IV,
synthetic NFs (see Fig. 2 legend). Vetch roots were treated with
109 M NFs and LCOs. All LCOs tested had a
C16:22E,9Z acyl chain (see Fig. 1A). The vertical bars correspond to
confidence intervals at the 5% probability level.
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Synthesis of LCOs Differing in Their Acyl Moiety
The above results confirm the general usefulness of synthetic
molecules for studies of symbiotic signals (Stokkermans et al., 1995).
Therefore, we chose to use this approach to examine the effects of
N-acyl chain structure in isolation from other changes in
the LCO molecule. S. meliloti nodFE mutants produce NFs that are N-acylated by 18:1 (vaccenic acid ) instead of
unsaturated C16 fatty acids (Demont et al., 1993). These mutants
exhibit a strong decrease in the ability to infect alfalfa root hairs,
indicating that the structure of the N-acyl chain is
important for bacterial entry into root hairs (Ardourel et al., 1994).
NFs from nodFE mutants elicit nodule formation with a
reduced efficiency, suggesting that the structure of the
N-acyl chain is also important at the step of nodule
organogenesis induction (Ardourel et al., 1994).
To obtain reagents to study acyl chain effects, we synthesized LCOs
differing in the length and in the presence of double bonds in the acyl
moiety. The synthesis of the two O-acetylated LCOs described
in the previous section required six chemical steps after the
introduction of the acyl chain (Nicolaou et al., 1992). To provide
direct and easy access to a collection of LCOs with different
N-acyl chains in a single operation with a single precursor, we used another synthetic route in which the last step was the introduction of the acyl chain. This approach was not compatible with
the presence of an O-acetyl group on the same GlcNAc unit because in the basic conditions used the acetyl group either migrated to the amino group or was partially removed. Thus, the synthetic LCOs
described below did not contain an acetyl group at the C6 position of
the nonreducing GlcNAc residue. We synthesized the LCOs by
treating sulfated tri-N-acetyl-chitotetraose with the appropriate acid chlorides following a published procedure (Tailler et
al., 1994). The synthesized compounds are described in Figure 1.
The structure of the purified compounds was confirmed by the
1H-NMR spectra in MeOH-d4 showing the
expected signals for the carbohydrate domain and the olefinic protons
of the acyl chain at 6.83 to 6.80 ppm (H-3, dt, J 15.5, and
7 Hz) and 5.99 to 5.95 ppm (H-2, dt, J 15.5, and 1.5 Hz) for
the E-conjugated double bond (when present) and at 5.35 ppm (H-9 and
H-10, m) for the Z internal double bond (when present). The presence of
the sulfate group was readily ascertained by the downfield shift of the
H-6 protons of the sulfated reducing sugar unit at 4.21 to 4.25 ppm (H-6a of the major 
anomer, dd, J 3.0, and 10.5 Hz) and
4.06 to 4.09 ppm (H-6b of the major anomer, dd, J 2, and
10.5 Hz) (Tailler et al., 1994; D. Tailler, J.C. Jacquinet, and
J.M. Beau, unpublished data). The structure and the purity of each
compound have also been tested by MS in the positive ion mode after
alkali cationization with alkali salts. The specific fragmentations of the molecules resulted in the loss of the sulfate group and the cleavage of the interglycosidic residues corresponding to a tetramer of
GlcNAc and revealed an acyl moiety having the expected masses for the
various derivatives (data not shown).
Acyl Chain Structural Requirements for Alfalfa Nodulation
An exogenous supply of unsubstituted chitin oligomers does not
elicit symbiotic responses in vetch (Spaink et al., 1991) or alfalfa
(Journet et al., 1994). Given the importance of the sulfate group for
inducing symbiotic responses in alfalfa, we first checked whether the
o-sulfation on the C6 of the reducing GlcNAc residue of
chitotetraose having no N-acyl substitution (with a free
NH2 group at the nonreducing end) would be
sufficient to generate activity on this plant. To increase the chance
of detecting a response, a high concentration
(107 M) of
CO-IV(S,NH2) was used and a large number
(n = 100) of alfalfa seedlings were treated. No
significant difference could be detected (P = 0.05) with the
untreated control. This result indicates that CO-IV(S,NH2) neither induced nodule organogenesis
nor increased the frequency of appearance of spontaneous
non-rhizobium-induced nodules. Thus, the presence of an acyl group
seems to be an essential requirement for nodule formation in alfalfa.
The most abundant N-acyl chain found in natural S. meliloti NFs is C16:22,9. To determine the role of the double
bond number and position in the acyl chain, synthetic LCOs containing
C16 acyl chains with different types of unsaturation were tested: LCO-IV(S,C16:22E,9Z), LCO-IV(S,C16:12E), and
LCO-IV(S,C16:19Z) at 107 to
109 M. Results
presented in Figure 5 clearly show that
at all concentrations tested LCO-IV(S,C16:22E,9Z) had a better
nodulation efficiency than the monounsaturated LCO-IV(S,C16:19Z),
with the unsaturation located in the middle of the chain at position
C9. It can be concluded that the presence of the two double bonds in
the acyl moiety of LCO results in an enhanced efficiency of nodulation.
LCO-IV(S,C16:12E), with a double bond conjugated to the carbonyl
group, elicited the formation of slightly more nodules than
NodRmIV(S,C16:19Z) at all concentrations, with the unsaturation
located in the middle of the chain at position C9, but the differences
were not significant at P = 0.05.
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| Figure 5.
Influence of the number and location of
unsaturations on the N-acyl chain on the morphogenic
activity of synthetic LCOs. LCOs are tetrameric and sulfated (see Fig.
1B) and only the structure of the N-acyl chain is
represented. Nodule number 23 d after addition of LCOs (see Fig.
2B for comparison with NF activity) is shown. Experimental treatment
and statistical analysis are the same as in the legend of Figure 2.
|
|
The next series of experiments addressed the question of the
influence of the carbon chain length. We first compared LCOs having C16
or C18 acyl chains unsaturated at positions C2 and C9.
LCO-IV(S,C16:22E,9Z) and LCO-IV(S,C18:22E,9Z) were applied to
alfalfa seedlings at 107 to
109 M. NodRm-IV(S,C16:22E,9Z)
was more active than LCO-IV(S,C18:22E,9Z) at all concentrations, but
the difference was only significant at 108
M (Fig. 5).
We further analyzed the influence of the carbon chain length by testing
a series of LCOs with a single 2 unsaturation and varied carbon
chain lengths: C8, C12, C16, and C18. Figure
6 shows that synthetic C16-LCO was
clearly the most active compound. Comparison of data obtained at
108 to 109
M indicated that the C16-LCO was more than 10 times as
active as the other LCOs. C12-LCO and C18-LCO had similar activities at
108 and 109
M. The activity of the C8-LCO was very weak: nodule
formation could be observed only at 107
M. We can conclude that, of LCOs having one conjugated
double bond and different carbon chain lengths, the LCO with a C16
chain was the most efficient in triggering nodule formation in alfalfa.
View larger version (48K):
[in this window]
[in a new window]
| Figure 6.
Influence of the length of the
N-acyl chain on the morphogenic activity of synthetic
LCOs. LCOs are tetrameric and sulfated (see Fig. 1B), and only the
structure of the N-acyl chain is represented. Nodule
number 26 d after addition of LCOs is shown (see Fig. 2B for
comparison with NF activity). Experimental treatment and statistical
analysis are the same as in the legend of Figure 2.
|
|
Histological studies have determined the ability of LCOs with a single
2E substitution and varied carbon lengths to elicit another cortical
response, the accumulation of starch in plastids (Ardourel et al.,
1994). A correlation was found between the ability of LCOs to trigger
nodule primordium formation and their ability to provoke amyloplast
formation. Strong starch accumulation was observed in plastids of
dividing cortical cells in plants treated with the most morphogenic
compound, LCO-IV(S,C16:12E) (Fig. 3E), whereas the lowest (but still
significant) accumulation was seen in cortical cells of plants treated
with the less morphogenic LCO-IV(S,C8:12E) (Fig. 3F).
| |
DISCUSSION |
Chemically synthesized S. meliloti NFs have been
reported to elicit alfalfa root-hair deformation (Bono et al., 1995).
We have shown here that they elicit nodule organogenesis in alfalfa roots in the same range of concentrations (down to 109
to 1010
M) as their natural analogs. The biological
activity of the synthetic products is definitive evidence that the
chemical structures initially proposed for the natural S. meliloti NFs were correct (Lerouge et al., 1990; Roche et al.,
1991b; Schultze et al., 1992) and that NFs alone act as potent
morphogenic compounds and elicit alfalfa nodule organogenesis (Truchet
et al., 1991).
Natural NFs are made up of a mixture of related LCOs. For example,
S. meliloti NFs are all sulfated but comprise tetra- and pentamers (Roche et al., 1991b; Schultze et al., 1992) with or without
an O-acetyl group (Roche et al., 1991b; Truchet et al., 1991) and are N-acylated by a variety of C16 unsaturated
fatty acids (Lerouge et al., 1990; Schultze et al., 1992; Demont et al., 1993) or a series of (
-1)-hydroxylated fatty acids (Demont et
al., 1994). We found that such a mixture of NFs was slightly more
active than the synthetic analog of the major natural product, NodRm-IV(Ac,S,C16:22,9), a single molecular species. Similar results
have already been described with different legume hosts. Mixtures of
NFs from B. japonicum elicited the transcription of early
nodulin genes in soybean more efficiently than did single molecular
species (Minami et al., 1996a), which could explain the diversity
observed in NFs synthesized by a single rhizobial strain: different NFs
could differentially activate plant genes involved in the symbiotic
process.
The NF structural requirements to trigger responses in alfalfa are
varied. Most responses, e.g. root-hair membrane depolarization, root-hair deformation, induction of ENOD12 transcription,
and nodule formation, require the presence of the O-sulfate
substitution. In contrast, with regard to the structure of the acyl
chain, two types of responses have been identified. Nonstringent
responses that seem not to require the presence of the specific
unsaturated C16 acyl moiety include root-hair deformation (Ardourel et
al., 1994), induction of ENOD12 transcription in epidermal
cells (Journet et al., 1994), and starch accumulation in cortical cells
(Ardourel et al., 1994). Stringent responses such as root-hair plasma
membrane potential changes (Felle et al., 1995), bacterial entry into
root hairs, and initiation of infection thread formation and nodule formation (Ardourel et al., 1994) require the presence of the polyunsaturated C16:2 chain. These differences are not tissue specific,
because both stringent and nonstringent requirements have been found in
both epidermal and cortical cells. The use of synthetic LCOs has
confirmed that induction of nodule formation is also a stringent
response and we now can address the question of the respective roles of
carbon chain length and the degree and position of unsaturations.
Requirement for the Presence of an
N-Acyl Group
All NFs identified so far from various rhizobial strains are
N-acylated. This observation is not due simply to a bias in
the NF-extraction procedures that eliminated the nonacylated chitin oligosaccharides. A major nodulation gene, nodA, was present
in all rhizobial strains studied so far and this gene specifies the transfer of an acyl moiety on the amino group of the terminal, nonreducing GlcNAc residue of the oligochitin backbone (Atkinson et
al., 1994; Röhrig et al., 1994; Debellé et al., 1996;
Ritsema et al., 1996; Roche et al., 1996). When supplied exogenously to legume roots, unsubstituted chitin oligomers were unable to elicit plant responses such as root-hair deformations, cortical cell divisions, and the formation of a nodule primordium (Lerouge et al.,
1990; Spaink et al., 1991; van Brussel et al., 1992; Relic et al.,
1993). Similarly, O-acetylated chitin oligomers were unable to trigger cortical cell divisions when added exogenously (Schlaman et
al., 1997). Given the importance of the sulfate moiety in triggering responses in alfalfa, we have tested the morphogenic activity of a
chitin tetramer O-sulfated at C6 of the reducing GlcNAc
residue and no nodule or bump could be detected. The addition of a
short (C8) acyl chain was sufficient to provide some morphogenic
activity at a high concentration (107
M). Thus, the presence of an acyl moiety seems
essential for triggering symbiotic responses when LCOs are added
exogenously. It is worth noting that a simple, nonacylated chitin
pentamer was found to transiently elicit ENOD40
transcription in soybean roots (Minami et al., 1996b). However,
sustained expression of ENOD40 required an acylated Nod
signal (Minami et al., 1996b), and ENOD40 could also be
expressed in nonsymbiotic conditions (Asad et al., 1994; Papadopoulou
et al., 1996).
In rhizobia that produce NFs acylated by fatty acids of the general
lipid metabolism, such as 18:1 (vaccenic acid), 16:0 (palmitic acid),
and 18:0 (stearic acid) , it is unlikely that the acyl moiety plays an
important role in specific recognition by the host plant. The same
fatty acid substitutions are present in NFs of diverse rhizobial
strains irrespective of their host range, and the nature of the fatty
acid chain does not seem to play a significant role in biological
activity (Stokkermans et al., 1995; Cohn et al., 1998). However, the
presence of an acyl group is nonetheless essential for NF activity.
What is the role of the acyl chain? We hypothesize that one of the
functions of the N-acyl chain is to provide a hydrophobic
tail, making possible the insertion of NF into the lipid bilayer of the
plant plasma membrane (lipid trapping of the ligand). Such an insertion
would facilitate two-dimensional lateral diffusion of NFs toward a
(putative) membrane receptor and would also give an orientation to NFs
that might facilitate ligand-receptor interactions.
N-acylation could also influence the stability of the signal
against degradation by chitinases (Staehelin et al., 1994).
Recently, it was shown that, whereas O-acetylated (and
nonacylated) chitin oligomers do not induce cortical cell division in
vetch when applied exogenously, they do act as mitogens when delivered
inside the roots by ballistic microtargeting (together with uridine, a
putative stele factor required for cortical cell division; Schlaman et
al., 1997). This result suggests that the acyl group of NFs is involved
in the transport (internalization) of the signal and that substituted
oligosaccharides could activate an intracellular receptor(s). A
transport role of the NF hydrophobic tail is also supported by the
recent observation, using fluorescent probes, that the acyl chain was
required for uptake of LCOs by root cells (Philip-Hollingsworth et al.,
1997).
Influence of the Structure of the
N-Acyl Group
In rhizobia that nodulate legumes of the tribes Galegeae,
Trifolieae, and Vicieae, NFs are acylated by specific polyunsaturated fatty acids. In these symbiotic associations the structure of the acyl
chain appears to be important for host specificity and biological
activity. We have observed that the presence of two double bonds, one
conjugated to the carbonyl group (2E) and the other located in the
middle of the acyl chain (9Z), makes the LCO more active than an LCO
with a single double bond. The 2E-conjugated unsaturation might
influence the rotation of the acyl chain around the chitin
backbone.
The length of the acyl chain is very important for morphogenic
activity. If a short C8 chain was sufficient to provide some activity,
increasing the chain length to C12 and C16 resulted in a dramatic
increase. C16 was clearly the optimal length, about 10-fold more active
than C18 and C12. It is unlikely that the NF acyl chain length
corresponds to an adaptation to the fatty acid composition of the host
plasma membrane. The genera Medicago and
Trifolium are closely related within the Trifolieae tribe, and fatty acid composition studies of plants do not suggest drastic differences between related plants of similar habits, with a majority of C16 in alfalfa and C20 in clover. This suggests that a specific recognition mechanism of subtle structural differences in the structure
of the NF acyl chain exists in the roots of alfalfa and of other
legumes of the Galegeae, Trifolieae, and Vicieae tribes. We are
currently using a genetic approach to identify Medicago
truncatula genes involved in NF perception and transduction and
have found M. truncatula ecotypes exhibiting different
requirements for the O-acetyl and N-acyl
substitutions. Their genetic analysis should allow the identification
of a gene(s) involved in N-acyl chain recognition and open
the way to positional cloning and molecular characterization of these
genes.
| |
FOOTNOTES |
1
This work was supported by grants from the Human
Frontier Science Program (no. RG-372/92) and the European Communities
BIOTECH program (no. PTP CT93-0400).
2
These two authors contributed equally to the
paper.
3
Present address: Laboratoire de Biochimie
Génétique Institut Jacques Monod-Centre National de
la Recherche Scientifique, Université Paris 7, 2 Place Jussieu,
75251 Paris cedex 05, France.
*
Corresponding author; e-mail denarie{at}toulouse.inra.fr; fax
33-561-28-50-61.
Received December 7, 1998;
accepted February 1, 1999.
| |
ABBREVIATIONS |
Abbreviations:
LCOs, lipo-chitooligosaccharides.
NF, nodulation
(Nod) factor.
X:Y, a fatty acyl group containing X carbon atoms and Y
cis double bonds.
| |
ACKNOWLEDGMENTS |
We are grateful to Sharon Long, Julie Cullimore, and
Frédéric Debellé for critically reviewing the
manuscript. We thank Justin Teissié for stimulating discussions.
| |
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[Abstract/Free Full Text]
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Abstract
Introduction