Study of Lignification by Noninvasive Techniques in Growing Maize Internodes
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Plant Physiol. (1997) 114: 1123-1 133
Study of Lignification by Noninvasive Techniques in
Growing Maize Internodes
An lnvestigation by Fourier Transform lnfrared Cross-Polarization-Magic Angle Spinning
3C-Nuclear Magnetic Resonance Spectroscopy and lmmunocytochemical Transmission
Electron Microscopy
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Jean-Paul Joseleau* and Katia Rue1
Centre de Recherches sur les Macromolécules Végétales, Centre National de Ia Recherche Scientifique,
Associé 2 I'Université Joseph Fourier, BP 153, 38041 Grenoble cedex 9, France
state concentrations, the local pH, and the environment at
Noninvasive techniques were used for the study in situ of ligni- the site of polymerization.
fication in the maturing cell walls of the maize (Zea mays L.) stem. The molecular diversity of lignins is usually expressed in
Within the longitudinal axis of a developing internode all of the terms of the relative content of the three phenylpropane
stages of lignification can be found. The synthesis of the three types building units, resulting from the dehydrogenative poly-
of lignins, p-hydroxyphenylpropane (H), guaiacyl (C), and syringyl merization of the three hydroxycinnamyl alcohols (Higu-
(S), was investigated in situ by cross-polarization-magic angle spin-
chi, 1985, 1990), p-coumaryl, coniferyl, and sinapyl. The
ning '3C-solid-state nuclear magnetic resonance, Fourier transform
infrared spectroscopy, and immunocytochemical electron micros-
co-polymerization of the three monolignol precursors gives
copy. The first lignin appearing in the parenchyma i s of the G-type, rise to lignins with H, G, and S unit compositions
preceeding the incorporation of S nuclei in the later stages. How- (Yamamoto et al., 1989; Higuchi, 1990; Terashima et al.,
ever, in vascular bundles, typical absorption bands of 5 nuclei are 1993). In spite of the tissue and cell wall heterogeneity,
visible in the Fourier transform infrared spectra at the earliest stage lignins from gymnosperms are predominantly composed
of lignification. lmmunocytochemical determination of the three of G units, whereas lignins from angiosperms consist es-
types of lignin i n transmission electron microscopy was possible sentially of the mixed GS-type. Large variations in the S to
thanks to the use of antisera prepared against synthetic H, C, and G ratio are observed between species. In grasses and cere-
the mixed GS dehydrogenative polymers (K. Ruel, O. Faix, J.P.
als, however, the three types of units are always found
Joseleau [19941 J Trace Microprobe Tech 12: 247-265). The spec-
ificity of the immunological probes demonstrated that there are
with H < G < S (Lewis and Yamamoto, 1990). The dehy-
differences in the relative temporal synthesis of the H, C, and CS drogenative polymerization of monolignols via random
lignins in the different tissues undergoing lignification. Considering coupling of transient free radicals leads to a variety of
the intermonomeric linkages predominating in the antigens used for interunit substructures (Nose et al., 1995). The diversity of
the preparation of the immunological probes, the relative intensities assembly of these substructures is the cause of an addi-
of the labeling obtained provided, for the first time to our knowl- tional leve1 of complexity in lignins, which lies in their
edge, information about the macromolecular nature of lignins (con- macromolecular heterogeneity.
densed versus noncondensed) in relation t o their ultrastructural Because of the high n-electron density at the phenolic
localization and developmental stage.
oxygen atom, the interunit linkage involving the phenoxy
position has the highest frequency in lignins (Glasser and
Glasser, 1980).This mode of coupling is the most represen-
Lignin is a complex phenyl propanoid polymer, which is tative of the noncondensed linkages. This is opposed to the
integrated into the wall framework of vascular plant cells. couplings involving the C-3 or C-5 position of the phenolic
This polymer is complex and heterogeneous with respect to ring and leading to the condensed linkages. A11 three of the
the relative proportions of its three constituting monolignol monolignol precursors do not show an identical reactivity
units (Higuchi, 1990). Variations in lignin composition oc- of their radical forms. Consequently, the H units tend to
cur within plant species, tissues, cell types, and also ac- form more condensed linkages than the G lignins, and the
cording to developmental stage (Campbell and Sederoff, S units tend to form more noncondensed substructures.
1996).The ultrastructural heterogeneity of lignin in second- These features are also influenced by the conditions of
ary walls may be more complex than has so far been
demonstrated, because it may be influenced by various
Abbrevia tions: CP-MAS I3C-NMR, cross-polarization-magic an-
factors such as the nature of phenoxy radicals, their steady-
gle spinning I3C-NMR; DHP, dehydrogenative polymer; FTIR,
Fourier transform IR; G, guaiacyl; H, p-hydroxyphenylpropane;
* Corresponding author; e-mail joseleau@cermav.cnrs.fr;fax 33- HRP, horseradish peroxidase; S, syringyl; TEM, transmission elec-
476-54-72-03. tron microscopy; zl, Zulauf method; zt, Zutropf method.
11231 124 Joseleau and Rue1 Plant Physiol. Vol. 114, 1997
polymerization (Sarkanen, 1971; Terashima, 1989). Severa1 the technique it was deduced that lignin in the middle
important factors may influence the polymerization pro- lamella was of the condensed type (Fukushima and Ter-
cess: nature and activity of peroxidases (Van Huystee, ashima, 1990; Terashima et al., 1993). Such a conclusion
1987), rate of generation of hydrogen peroxide at the site of about the macromolecular features of lignin may be inter-
polymerization, pH, and template effect of the pre-existing preted as the consequence of the polymerization of mono-
polysaccharide framework already synthesized in the cell lignols within the pectin gel of the middle lamella. To our
wall (Siegel, 1957; Higuchi, 1985). Most important, how- knowledge, this is the only reference to the condensed
ever, is the steady-state concentration of the phenoxy rad- versus noncondensed type of lignin obtained by an in situ
icals (Sarkanen, 1971). observation. In spite of this improvement in its specificity,
Another level of complexity in the biogenesis of the microautoradiographic observation does not provide a res-
lignified plant cell walls concerns the ultrastructural dis- olution that could allow observation of lignin at the ultra-
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tribution of lignins. A large uncertainty remains concern- structural scale of the microfibrillar organization of the
ing the temporal aspects of lignification in a developing lignified cell wall (Ruel et al., 1979). Another drawback of
tissue, and the relation between the ultrastructural local- this technique is that it can only be performed on living
ization and the chemical nature of the deposited lignin. plant material in the active phase of lignification.
There is evidence that different compositions in monomer In an attempt to obtain probes that could be specific for
units may characterize different cell types and different cell each type of lignin and that could be used at the ultrastruc-
wall localizations. Lignins found in the primary wall, sec- tural level in TEM, we prepared novel immunogold probes
ondary wall, middle lamella, and cell corners are a11 dif- by raising antibodies against pure H-, G-, and mixed GS-
ferent (Hardell et al., 1980; Saka and Goring, 1985, 1988; types of lignins that correspond to the most common types
Eom et al., 1987). The chemical nature of lignin also de- of lignins found in nature. Since there are no known pure
pends on the age of the cell (Terashima et al., 1993). Thus, extracted lignins, the antigens used in this study were
when radioactive-labeled 3H-monolignol glucosides were synthetic lignins (DHPs) prepared by in vitro dehydroge-
administered to various angiosperm trees, the incorpora- native polymerization of the corresponding monolignols
tion of radioactivity was found in the order of the H, G, and (Ruel et al., 1994). In the present study the three probes
S units in different stages of the cell wall formation. The were applied to a developing internode of maize used as a
incorporation of the H unit was the earliest and was limited model to gain insight into the lignification of the different
to the cell corners and middle lamellae. The G units were tissues during cell differentiation. The relative distribution
deposited throughout the vessel and fiber walls. The S of the three types of lignin is discussed with reference to
lignin predominated in the secondary wall of fibers (Ter- the biochemical analysis data obtained by CP-MAS I3C-
ashima et al., 1986; Fukushima and Terashima, 1990).Sim- NMR and FTIR spectroscopy. Because of the macromolec-
ilarly, in monocotyledons structural heterogeneity with re- ular structure of the DHPs used as antigens (i.e. the relative
spect to morphological region was shown to differ among proportion of noncondensed structures), indications on the
fibers, vessel, and parenchyma cell walls (He and Ter- nature of the interunit linkages of the Iignins labeled by
ashima, 1990). the probes could be obtained. This approach represents a
To better describe the relationship between lignin heter- novel in situ analytical and ultrastructural determination
ogeneity and cytological and ultrastructural localization, of native lignin deposition during the course of cell
noninvasive methods are of particular interest. Contrasting differentiation.
reagents for the lignin used in TEM are not numerous and
specific probes corresponding to the different lignin units
do not exist. The nondestructive techniques that have been
applied to study lignins in situ, such as CP-MAS 13C-NMR MATERIALS AND METHODS
(Himmmelsbach et al., 1983) or FTIR and Raman spectros- Biological Material
copies (Agarwal and Atalla, 1986) have insufficient resolu-
tion to prove the ultrastructural scale of the microfibrillar The maize (Zea mays L.) inbred line W 401 was grown in
organization of the lignified plant cell wall (Ruel et al., a field trial at Tienen (Belgium) by the Zeneca Seeds Soci-
1979). Lignin distribution at the cytological level has been ety. The plants were harvested 5 d after anthesis. The
studied in the different morphological regions and cell fourth internode from the top of the culm was used for
types by various techniques such as UV microscopy pho- electron microscopy studies.
tometry (Fergus and Goring, 1970) and a combination of Stalk samples were dissected into nodes and internodes
selective bromination and TEM energy-dispersive x-ray and the nodes were discarded. The upper internodes were
analysis (Saka and Goring, 1988). used for the study for CP-MAS 13C-NMR spectra; 5-mm-
The administration of radioactive-labeled monolignol thick sections were dissected at various distances from the
glucosides and the detection of their incorporation into the base of the internode. For FTIR spectra of parenchyma,
cell walls was utilized (Terashima et al., 1986; He and handmade thin sections (0.5 cm in thickness) were cut at 1,
Terashima, 1990) for the localization of the H, G, and S 2, 4, 7, and 9 cm from the base of the internode. For FTIR
lignins. The technique allowed visualization of the progres- spectra of vascular bundles, handmade dissections were
sive deposition of lignin in specific morphological regions performed under a magnifying lens for separation of the
of the differentiating tissues. From further improvement of tissues.Study of Lignification by Noninvasive Technique 1125
DHPs copy. The affinity was revealed by complexation with a
secondary marker conjugated to gold colloid (protein
The three DHPs used in this study were gifts from Prof.
A-gold), as described above.
O. Faix (Hamburg, Germany). They were synthesized from
coniferyl alcohol, an equimolecular mixture of coniferyl
alcohol and sinapyl alcohol, and p-coumaryl alcohol. Solu- Comparison o f Labeling lntensities
tions of the hydroxycinnamic alcohols in phosphate buffer For each antiserum the dilution corresponding to the
were pumped into a solution of HRP in the same buffer, optimal labeling was determined by applying a series of
and hydrogen peroxide was simultaneously added over a dilutions of the antisera to thin sections of the antigens.
period of 120 h, according to the zt, as described in Ruel et This was also done with a series of ultrathin sections of the
al. (1994). The DHPs were characterized as described in same plant material. The intensity of the labeling was
Faix (1986).
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estimated for each dilution by manual counting of the
number of gold grains per surface unit.
Antibodies
Each polyclonal antiserum was raised in two New Zea- lmmunochemical Controk
land rabbits injected intradermally. The first injection was Severa1 control experiments were performed for each
diluted with Freund's complete adjuvant. Booster injec- polyclonal antibody used in this study. The controls in-
tions were done with the antigen diluted in incomplete cluded the omission of the primary antibody from the
Freunds adjuvant every 2 weeks. Blood samples were immunolabeling experiments or the substitution of the pri-
collected altemately, 1 week after each injection, and rab- mary antibody with the nonimmune serum, and incubation
bits were bled after 6 months. with the primary antibody that had first been saturated
with the corresponding antigen. AI1 of the tests resulted in
Electron Microscopy no detectable gold deposits.
Fixation and Embedding
Lignin Analysis
Samples were cut just after harvesting and immediately
fixed in a freshly prepared mixture of 0.3% glutaraldehyde Lignin analysis was performed by the acetyl bromide
and 4% paraformaldehyde in a 0.05 M phosphate buffer. method (Iiyama and Wallis, 1988). Finely ground tissues
Samples dehydrated up to ethanol 70% were then embed- (inferior 180 F ) were oven-dried at 6OOC. The sawdust (10
ded in London Resin White (hard mixture) and polymer- mg) was placed in screw-cap tubes, and a solution of 25%
ized for 24 h at 50°C. (w/w) acetyl bromide in acetic acid (5 mL) containing
perchloric acid (70%,0.2 mL) was added. The tightly sealed
tubes were placed in an oven at 70°C for 30 min. The
lmmunocytochemical Labeling
resulting solution was transferred to a flask containing 2 M
Thin sections floating on plastic rings were labeled ac- sodium hydroxide (10 mL) and acetic acid (25 mL). The
cording to the protocol described in Ruel and Joseleau volume was adjusted to 100 mL with acetic acid. The lignin
(1993) with a few modifications: BSA was replaced with 5% content was measured at 280 nm using a standard curve
nonfat dried milk in both TBSSOO (0.01 M trisphosphate established with bamboo lignin (a gift from Professor T.
buffer, pH 7.5, containing 0.5 M NaC1) and Tris buffer (0.01 Higuchi, Wood Research Institute, Kyoto, Japan).
M, pH 7.5). The antisera were diluted 1/30 for the anti-H
and anti-G antisera and 1/50 for the anti-GS. The second- CP-MAS 13C-NMR Spectroscopy
ary marker was protein A (PA,, or PA,) (Janssen Pharma-
ceuticals, Geel, Belgium) diluted 1/20 in the Tris buffer Samples of the maize internode were cut at various
containing 0.5% fish gelatin plus 0.02% PEG or Tris buffer distances from the intercalary meristem. The samples were
(milk 0.5%).After rinsing in Tris buffer, sections were fixed washed with hot water and extracted with ethano1:toluene
in 2.5% glutaraldehyde, rinsed in water and transferred to (2:1, v/v) in a Soxhlet apparatus (Prolabo, Grenoble,
carbon-coated copper grids. Poststaining was in 2.5% aque- France).
ous uranyl acetate. Observations were performed with an Natural-abundance CP-MAS I3C-NMR spectra were re-
electron microscope (400T or CM200-cryo, Philips) operat- corded at ambient temperature on a spectrometer (MSL
ing at 80 kV. 200, Bruker Instruments, Inc., Bilerica, MA) operating in a
magnetic field of 4.7 teslas. Transfer of polarization be-
The Specificity of Antisera
tween protons and carbon nuclei, at the frequency of 200
MHz, involves the dipolar interaction between the two
Preincubation of GS antiserum with G homopolymer spin systems. Chemical shifts were referenced to tetra-
was carried out to suppress any possible competitive la- methylsilane via hexamethylbenzene as an externa1 stan-
beling of homoguaiacyl lignin. The specificity of polyclonal dard (aliphatic peak at 17.4 ppm). Each spectrum was
antibodies was assayed by an affinity test in electron mi- acquired with 3000 to 4000 scans. For dipolar dephasing,
croscopy in which the antigens were embedded in London the same technique was applied, allowing a decoupling
Resin White (no aldehyde fixation), and thin sections sub- delay of 40 FS for carbon-proton contact. This gave spectra
jected to the antisera were examined by electron micros- in which nonquaternary 13C resonances were eliminated.1126 Joseleau and Rue1 Plant Physiol. Vol. 114, 1997
FTlR Spectroscopy Table 1. CP-MAS 13C-NMRassignments for the principal signals of
Spectra were recorded on an FTIR spectrometer (model the H-, G-, and S-types of lignins
1700 X, Perkin-Elmer). For collecting parenchyma spectra, 6 Linnin Assianment
hand-cut thin sections were mounted in the sample card, PPm
and the selected parenchyma area was centered in the laser 153-1 52 S C3, C5 in p - 0 - 4
beam. Transmission spectra were recorded between 4000 148-1 47 G C3-etherified
and 600 cm-’. For the spectra of young vascular bundles, 136-1 35 S C4 in p-0-4
the tissues were suspended in ethanol and ground in liquid 128 H C2, C6
nitrogen. The ground material was then mounted in the 1 16-1 15.5 G c5
sample card as above. For the more mature vascular bun- H c3, c 5
dles, the standard KBr pellet technique was used. 57-56 G, S aromatic OCH,
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31-32 Alkyl CH,
RESULTS A N D DISCUSSION
Lignin Analysis (CP-MAS 13C-NMR and FTlR Spectroscopy) found between the base and the top of the internode, as
indicated by in situ detection by CP-MAS I3C-NMR spec-
During the temporal development of a maize internode troscopy recorded from crude samples. This technique pro-
a11 of the stages of cellular differentiation and lignification vided fingerprint indication of the predominating lignin
are spanned from the base to the top of the internode types that are present in the different parts of the internode
(Joseleau et al., 1976; Scobbie et al., 1993). Chemical analy- (Fig. 1).
sis of the basal, middle, and top region of a 10-cm-long Assignment of the principal signals of lignin in the re-
internode of maize showed that the lignin content gradu- gion of the spectrum between 110 and 160 ppm (Bardet et
ally increased upward, as the distance from the intercalary al., 1985; Manders, 1987; Almendros et al., 1992) (Table 1)
meristem increased. Thus, lignin determination by the suggested that the nature of the lignin deposited in the
acetyl bromide method (Iiyama and Wallis, 1988) gave a early lignifying tissues near the intercalary meristem dif-
lignin content of 5% at the base of the internode, 6.5%in the fered from that of the fully lignified tissues of the top of the
middle part, and 9.5% in the top part. The progressive internode. This suggests that the activities of monolignol-
lignification within one internode is accompanied by synthesizing enzymes are not uniform as the internode
changes in the relative proportions of the H, G, and S units extends. Thus, in the basal region signals corresponding to
units H and G are visible, whereas S is also present but
does not predominate. Conversely, in the top region lignin
of the H-type is only weakly represented compared with G,
and the S-type is predominant as shown by signals at 153,
136, and 57 ppm. Between the base and the top, the relative
intensities of the signals at 153 and 148 ppm (Fig. 1),
respectively, assigned to oxygen-linked carbons in etheri-
fied S and G units (Table I), reflect the more active synthe-
sis of S lignin as maturation of the internode increases. The
part of the spectra between around 105 and 60 ppm
changes dramatically from the basal to the top part of the
region. This corresponds to the enhanced deposition of
polysaccharides, particularly of the cellulose in the top
part, the signals of which tend to dominate over the signals
of the other wall polysaccharides.
For FTIR spectra, parenchyma and vascular bundles
were analyzed separately. Only a few IR spectral features
allow a distinction between G and S nuclei, the most in-
tense bands in young tissues being those of polysacchar-
ides (Séné et al., 1995).G and S nuclei were assigned on the
bands due to the aromatic C-H out-of-plane deformations
of the G rings at 810 cm-’, with a shoulder at 870 cm-’, as
opposed to the band at 840 cm-’ given by the S rings
(Evans, 1991; Faix, 1991,1992).Confirmation was provided
l f ~ l l l l l l ~ l
by
l
the intensities of the bands at 1270 and 1325 cm-’,
200 100 O
respectively, assigned to deformation of the G and S nuclei.
PPm The parenchyma was examined at five stages of devel-
Figure 1. CP-MAS 13C-NMR spectra of maize sections from the base opment of the maize internode, corresponding to hand-
(B) and from the top (T) of the internode. In the more mature tissues made sections cut at 0.5,1,2,5, and 9 cm, respectively, from
situated at the top of the internode, characteristic signals of the S the intercalary meristem at the base of the internode (Fig.
nuclei become conspicuous. 2). The first section (0.5 cm) did not show the typical bandsStudy of Lignification by Noninvasive Technique 1127
lmmunogold Labeling of Lignins of Types H, C, and CS in
Transmission Electron Microscopy
Three DHPs were synthesized in vitro by polymerization
of the corresponding monolignols catalyzed by HRP in the
presence of hydrogen peroxide. Thus, a G homopolymer
(G-DHP), an H homopolymer (H-DHP), and a mixed GS
polymer (GS-DHP) were obtained, respectively, from the
polymerization of coniferyl alcohol, of p-coumaryl alcohol,
and of a mixture of the coniferyl and sinapyl alcohols in
which sinapyl alcohol predominated (Faix, 1991; Ruel et al.,
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1994). The mode of polymerization of the DHPs according
t to the zt (Sarkanen, 1971) by dropwise addition of the
monolignols to the medium containing the peroxidase gen-
erally favors the production of the uncondensed type of
linkages, i.e. lignins enriched in 0-0-4 structures. This af-
fects the conformation of the resulting macromolecules
(Sarkanen, 1971). This is as opposed to the zl of polymer-
ization, in which batch addition of the monolignols that
generally leads to macromolecules enriched in condensed
linkages. Polymerization of p-coumaryl alcohol has the
highest tendency of the three monolignol precursors to
V form condensed linkages, whereas the polymerization of
coniferyl alcohol may lead to either condensed or noncon-
, ,
1600 800
densed interunit linkages, with the predominating type of
cm-1 macromolecule being influenced by severa1 factors control-
Figure 2. FTlR spectra of parenchyma tissues at increasing distances ling the polymerization (Terashima, 1989). In particular,
from the intercalary meristem. T h e age of the tissues increases from the Zutropf polymerization of the G-DHP used for the
a to e. Spectra were recorded in transmittance percent. preparation of our immunoprobe is considered to give a
polymer enriched in noncondensed subunits (Sarkanen,
1971; Terashima, 1989). This is even more the case for
of lignin. In the spectra of the following sections (1 and 2 sinapyl alcohol, which polymerizes in a pattern favoring
cm), peaks at 810, 870, and 1270 cm-' revealed that lignin P-0-4 linkage formation.
early synthesized in parenchyma cell walls is of the G-type. The mixed GS-DHP fraction used in this study, which
At 5 cm from the base the appearance of a band of low contained 75% S units, was characterized by a significant
intensity at 840 cm-' was indicative of S nuclei beside the proportion of noncondensed units, as shown by its low
bands at 810 and 870 cm-' of the G nuclei. Lignin of the proportion of free phenolic hydroxyls (O. Faix, personal
S-type was confirmed by the absorption band at 1325 cm-'. communication).
The same peaks revealing the increasing proportion of S For immunization of rabbits each of the DHPs was used
units were present in the spectrum of the most mature without conjugation to a carrier. The three resulting poly-
section of the internode (at 9 cm from the base). The pro- clonal antisera were assayed by use of an electron micros-
gressive synthesis of S nuclei in the maturing parenchyma copy affinity test. This test is used for insoluble substrates
cells of the 5- and 9-cm sections was again confirmed by the that cannot be conveniently assayed by ELISA (Ruel and
presence of more clearly defined bands at 1510 and 1595 Joseleau, 1993; Ruel et al., 1994). Each DHP preparation
cm-' in which the band at 1595 cm-' was of higher inten- was embedded in London Resin White and thin sections
sity (Evans, 1991; Faix, 1991). The intensity of the band at were exposed to the antisera. The affinity was revealed by
1235 cm-', assigned to phenolic hydroxyls, increased with the application of a secondary marker conjugated to gold
maturation of the tissue. It must be noted that a11 of the colloid (Fig. 3 ) .
above signals of lignin were relatively weak. No cross-reactivity was observed between anti-H and the
In comparison, the spectra given by the vascular bundles G and GS antisera. Only a slight cross-reactivity could be
gave stronger lignin-absorption bands. In this case the detected between the anti-GS(zt) and anti-G(zt) antisera.
spectra of the youngest tissues, isolated from sections at 0.5 To suppress this slight competitive labeling of homoguai-
to 1.0 cm from the base of the internode, showed bands of acyl lignin, the GS(zt) antiserum was first incubated with
S nuclei at 840 and 1325 cm-l, together with a slightly the G(zt) homopolymer before being used for thin-section
positive ratio of the intensities of the bands at 1600 and labeling. It is important to note that the anti-G(z1) did not
1510 cm-'. The presence of predominating S lignin was label the mixed, noncondensed GS(zt) antigen. Only a very
obvious in a11 of the lignifying vascular bundles examined weak labeling was observed when the GS(z1) (i.e. con-
in the upper sections of the internode, the intensity of the densed) antiserum was applied on the GS noncondensed
bands becoming stronger beyond 2 cm from the intercalary antigen. It can therefore be concluded that the specificity of
meristem. the GS(zt) antiserum is'directed against the noncondensed,1128 Joseleau and Ruel Plant Physiol. Vol. 114, 1997
Figure 3. Specificity of antisera as assayed by
an affinity test in TEM. All antisera were applied
to each DHP antigen enrobed in London Resin
White. Secondary label was protein A coupled
to gold 10 nm. Bar = 0.2 /xm; ND, not deter-
mined.
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mixed GS lignin. It is worth noting that the absence ence of the mixed GS-type lignin in the more lignified
of cross-reactivity between the G and GS antisera and parenchyma cell walls of the upper part of the internode
the H-antiserum excludes the possibility of interference was well demonstrated by the stronger labeling given by
due to entrapped residual HRP in the antigen preparation, the GS antiserum (Table II). This was particularly clear in
since all three of the antigens were polymerized with the the pluristratified parenchyma cells (Fig. 4). It is interesting
same HRP. to note that the anti-H antiserum gave a poor labeling in
the first stages of lignification of the internode, about 2.0 to
Lignification of Parenchyma Cell Walls 2.5 cm from the base, but that the labeling became more
Of the three antisera used, the anti-GS antiserum gave significant at the top of the internode. This result is some-
the most significant, although discrete, labeling of the pa- what surprising, since it is generally admitted (He and
renchyma at the base of the internode, whereas the pres- Tarashima, 1990; Terashima, 1993) that p-coumaryl alcohol
Table II. Relative intensities of immunolabeling in the walls of the different tissues of the maize maturing internode
Parenchyma Fiber Tracheid Vessel Phloem Cell Corner'1
Antisera
Young Mature Young Mature Young Mature Young Mature Young Mature Young Mature
Anti-H tb +c + + + + + + + + + + - - + +
Anti-G -d t t + + + + + + + - - - +
Anti-GS t + ++ +++ ++ +++ +++ +++ -
a c
The results concern cell corners (tricellular junctions) between fibers, ' t, Trace. The number of plus signs refers to the intensity of
d
the labeling. -, No labeling.Study of Lignification by Noninvasive Technique 1129
fc „
AS
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0.2 pr 0.2pm
B I
Figure 4. Labeling with anti-GS antiserum. A, In parenchyma cells from the young tissues at the base of the internode, the
anti-GS probe is only faintly reactive. On the other hand, in the more mature parenchyma cells from the top of the internode
(B) the labeling (performed at the same dilution of the antiserum) becomes strongly positive. Magnification, X17,500. AS,
Air space.
is incorporated during cell wall development, and that it is also observed in the tricellular junction between fibers,
especially localized in the middle lamella. On the other suggesting that lignin of the H-type is present in this
hand, considering that p-coumaric acid esterifies directly to region. With the anti-G probe, no significant labeling in the
maize lignin (Ralph et al., 1994), and the fact that these younger fibers from the base of the internode was ob-
p-coumarate groups are incorporated in abundance during served. However, G units could be detected in the mature
the later stages of lignification, it could be that our H probe fibers from the upper parts of the internode. In these cells
had some affinity for these groups. This needs to be veri- the labeling appeared heterogenous in the polylamellar cell
fied in future work. A surprisingly low labeling was ob- walls (Fig. 6B), in agreement with earlier observations of
served with G-antiserum at any stage of lignification of the the uneven distribution of lignin in polylamellate struc-
parenchyma. This observation is in apparent contradiction tures (Parameswaran and Liese, 1982). It must be noted
with the preceding FTIR spectra of parenchyma, which
demonstrated that the G lignin appeared in the lignifying
cells before the S units. The lack of significant labeling by
the anti-G probe used in the present study may be attrib-
uted to the fact that the anti-G probe is preferentially
directed toward noncondensed G lignin. This observation
correlates with the results reported by Terashima et al.
(1993) showing that the early formed G lignin is a con-
densed polymer. Application of the GS antiserum preincu-
bated with the G antigen resulted only in a slight weaken- TC
ing of the labeling. This suggests that the lignin of ground
parenchyma is essentially a mixed GS lignin in which the
proportion of G nuclei is low.
Lignification in Fibers
Using the immunological probe for H units, a faint la-
beling of H lignin was visible at the earliest stage of fiber
lignification. The intensity of the labeling increased with 0.2pm
maturation, and in the most lignified fibers a uniform
distribution of gold particles was observed in the S2 layers
(Fig. 5). This supports the proposal that H lignin forms Figure 5 Labeling with anti-H antiserum. H lignin is clearly visible in
early in fibers and is then actively deposited during sec- maturing fiber walls from the top of the internode. Note that the
ondary thickening, as demonstrated by microautoradiog- tricellular junction responds positively to the anti-H antiserum. Mag-
raphy studies (He and Terashima, 1990). The labeling was nification, XI 6,100. TC, Tricellular junction.1130 Joseleau and Ruel Plant Physiol. Vol. 114, 1997
LG 1
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OL
0.2pm
Figure 6. Reactivity of fibers toward anti-G antiserum. A, Heterogenous distribution of the labeling in fibers from the top of
the internode. In the polylamellar fiber bordering the lacuna (Lc, upper part of the micrograph) the more recently deposited
layer is almost not labeled (arrowheads). In the fiber of the lower part of the micrograph, only the first deposited outer layer
(OL) shows a significant presence of the homoguaiacyl polymer. The tricellular junction (TC) is slightly labeled. B, Detail of
a vessel of metaxylem. The presence of the G units is strongly underlined by the anti-G serum. G lignin is deposited
throughout the secondary wall, although with less intensity over the last deposited inner layers (IL). Magnifications: A,
XI 7,500; and B, X 12,950.
that the labeling provided by our G probe was consistently tion that polymerization of secondary wall lignin within
weak in most cell types, except vessels of the metaxylem the template of cellulose microfibrils likely favors the for-
(Table II). mation of the extended macromolecule of noncondensed
Since G lignin has been shown to be present in develop- lignin (Siegel, 1957), rather than that of the bulky macro-
ing maize internode (Gaudillere and Monties, 1989), and molecule of condensed lignin (Higuchi, 1990; Terashima et
this was confirmed by the preceding FOR and CP-MAS al., 1993). It is noteworthy that the intensity of the labeling
13
C-NMR analyses, the weak labeling was ascribed to the provided by the GS antiserum was only slightly affected by
higher affinity of the G probe for the noncondensed lignin. preincubation with the G antigen (Fig. 7D). This supports
Consequently, we suggest that G lignin in most cell types the idea that the lignin depicted by the GS probe corre-
comprises a significant proportion of the condensed link- sponds to a true mixed-type polymer. This result also
ages. This contention may be supported by the fact that in agrees with the only slight affinity of anti-GS for the pure
an acidic medium having a pH similar to that in secondary G homopolymer (Fig. 3).
walls, coniferyl alcohol gives j3-5 dimers, i.e. condensed For immunological control of the specificity of the label-
dimer (Yoshida et al., 1994). Similarly, the acidic pectic ing of lignin in maize tissue sections with the various lignin
environment in the middle lamella may also facilitate the antisera, several experiments were performed. Omission of
formation of condensed G lignin in this area (Terashima, the primary antibody resulted in the complete absence of
1989). In the vessels of metaxylem the intensity of the labeling (Fig. 8B). Substitution of any of the lignin antibod-
labeling remained about the same all along the maturing ies with nonimmune serum gave negative labeling (Fig.
internode, suggesting that in vessels of metaxylem noncon- 8C). Preincubation of the different antibodies with the dif-
densed G lignin is regularly synthesized, in addition to the ferent corresponding antigens abolished the labeling of
condensed form described by other authors (Saka and Gor- maize cell walls (Fig. 8D).
ing, 1985).
Labeling with the anti-GS antiserum demonstrated that
Lignin in the Cell Corners Is a Primarily Condensed
GS lignin was already synthesized in fibers at the earliest
Polymer and Differs from Lignin of the Cell Walls
stage of lignification (Fig. 7, A-C). The intensity of this
labeling was the strongest given by any of the three anti- There have been several indications that lignification
sera all along the fibers within the internode, indicating starts in the cell corners (Fergus and Goring, 1970; Saka and
that the more methoxylated monolignol precursors are in- Goring, 1985) and then proceeds in the middle lamellae. The
corporated in the fibers as soon as lignification proceeds. only consistent labeling obtained in the cell corners with the
Moreover, the strongly positive labeling provided by our three antisera was provided by the anti-H probe. That was
GS(zt) probe suggests that noncondensed GS lignin is the case in both the youngest and the oldest tissues of the
abundant in fibers. This is in agreement with the proposi- internode. Virtually no labeling in this region could be ob-Study of Lignification by Noninvasive Technique 1131
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Lc
0.2 p<
Figure 7. Early synthesis of CS lignin in fibers and vessels. The labeling is done with anti-GS antiserum with Protein A-gold
(10 nm). A, Very young fibers give a positive labeling with anti-GS. The younger fibers are less densely labeled (arrows), the
vessel wall (V) is strongly reactive, and the tricellular junction (TC) gives a negative response. B, At an early stage of
maturation, sclerenchyma walls show a positive reaction to the anti-GS antiserum. The tricellular junction is totally negative.
C, Three fibers near the lacuna at the base of the internode. The secondary thickenings (arrows) show a heavier labeling for
the mixed GS lignin than the compound middle lamella-primary wall. The fiber bordering the lacuna is less labeled than the
two other fibers. Again, the lignin in the tricellular junction is unreactive to the anti-GS antiserum. D, Three fibers of the top
showing a polylamellar organization. The GS lignin shows a concentric distribution. The preincubation of the anti-GS
antiserum with the G antigen has only faintly affected the intensity of the labeling. This is an indication that the mixed GS
lignin type is actively synthesized in these tissues. Magnifications: A, X18,550; B, X16,800; C, XI9,600; and D, X 18,200.
served with the two other probes at any stage of lignifica- tion, it may therefore be inferred that lignin in a cell corner
tion. This confirmed that a specific lignification occurs in the is a condensed lignin. Recent results obtained with immu-
cell corners, and that the first to be synthesized is an H lignin noprobes directed against condensed DHPs confirmed the
(Terashima, 1989). Moreover, the absence of methoxylation presence of condensed G lignin in cell corners (J.P. Joseleau,
at C-3 and C-5 of the aromatic ring of the p-coumaryl mono- V. Burlat, and K. Ruel, data not shown).
lignol makes this unit more susceptible to forming con-
densed interunit linkages than the substituted ring of the G CONCLUSION
and S precursors. From that it can be deduced that our
anti-H probe has a good affinity for condensed H lignin. The developing maize internode is a convenient model
This being valid for in vitro as well as in vivo polymeriza- for studying the biogenesis of lignified plant cell walls,1132 Joseleau and Ruel Plant Physiol. Vol. 114, 1997
Figure 8. Immunological controls. A, Labeling
with the GSZT antiserum; B, omission of the
primary antibody; C, substitution of the primary
antibody with the nonimmune; and D, pread-
sorption control: labeling with the GSzt anti-
serum that had been preadsorbed with the GSzt
antigen. Bar = 0.1 /urn.
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because it offers several degrees of complexity relative to of the parenchyma. In tracheids of protoxylem and in
the diversity of cell types and tissues and to the heteroge- vessels of metaxylem, the earliest synthesized lignin of the
neous structure of lignins (Scobbie et al., 1993). In this secondary walls was found to be already highly heteroge-
actively growing material maturation of lignin globally neous with significant proportions of H units mixed with
corresponds to an increase in synthesis of methoxylated GS units, but lower proportions of homopolymeric G lig-
monolignols (Joseleau et al., 1976; Higuchi, 1990). This nin. This ultrastructural heterogeneity of lignin in the sec-
trend of progressive methoxylation accompanying matura- ondary walls could not be demonstrated without the
tion was verified by CP-MAS13C-NMR spectroscopy in the proper probes for TEM.
samples in which all of the tissues were mixed. However, The conjunction of analytical techniques, such as FTIR
this only corresponds to an averaged estimation of the spectroscopy and CP-MAS 13C-NMR, with an ultrastruc-
lignin composition at different stages of development in tural analysis by immunocytochemical methods in TEM
the maize internode. Higher resolving techniques such as has the potential to provide in vivo new insights into the
FTIR spectroscopy and immunogold TEM, because they biosynthetic pathways involved in the complex lignifica-
can be applied to specific tissues and specific ultrastruc- tion of plants. The new markers of lignins that were used in
tural areas of the cell walls, respectively, showed that the this study allowed refined correlations between the chem-
general concept of increased methoxylation with matura- ical nature of lignins and their ultrastructural distribution.
tion is not representative of the temporal evolution of An interesting aspect of these immunological probes is
lignin in all of the tissues. When parenchyma cell walls their capacity to differentiate between macromolecular fea-
begin to lignify, G monomers are synthesized first. The tures of condensed and noncondensed lignins. Although
appearance of S nuclei in the spectra of the maturing the epitopes that are recognized by the polyclonal antisera
tissues was visible in the upper half of the internode. This are not precisely known, it remains that the fact that two
may correspond to the biphasic thickening of the wall different antisera label different zones of the cell wall dem-
associated with the laying down of structural carbohy- onstrates that the nature of lignin in these zones is defi-
drate, followed by a slow increase of lignification (Scobbie nitely not identical. The immunolabeling brings evidence
et al., 1993). Lignification appears to be controlled differ- that the cell wall microenvironment influences lignin po-
ently in vascular bundles, where S units are synthesized lymerization mechanisms differently according to the cell
from the beginning of lignification. type, the tissue, and the wall layer in which the synthesis
The novel immunological probes that were used in this occurs.
study allowed visualization of the progression of lignin
deposition during maturation in each cell type, and char-
acterization of structural variations in the lignin macromol-
ecule as lignification proceeded. Our results show that all ACKNOWLEDGMENTS
tissues do not undergo lignification simultaneously and at We wish to thank Prof. O. Faix (Institut fur Holzchemie und
the same rate (Table II), indicating that lignification is a chemische Technologie des Holzes, Hamburg, Germany) for the
spatial and temporal phenomenon, which varies according gift of DHPs, and Dr. P. Balls (Zeneca Seeds Society, Tienen,
to tissues and cell walls. Conducting and supporting tis- Belgium) for the gift of the maize stalks. We acknowledge the
sues such as fibers, vessels, and tracheids showed a preco- assistance of Mr. A. Rousseau (Centre d'Etudes Nucleaires de
cious lignification of their secondary walls relative to those Grenoble) for recording the solid-state NMR spectra.Study of Lignification by Noninvasive Technique 1133
Received December 23, 1996; accepted March 24, 1997. Arundo donax lignin in relation of growth. Holzforschung 31:
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