One, Two, Three: A Bioorthogonal Triple Labelling Strategy for Studying the Dynamics of Plant Cell Wall Formation In Vivo

Article abstract of DOI:10.1002/anie.201808493

Reported herein is an in vivo triple labelling strategy to monitor the formation of plant cell walls. Based on a combination of copper-catalysed alkyne–azide cycloaddition (CuAAC), strain-promoted azide–alkyne cycloaddition (SPAAC), and Diels–Alder reacti

Full text of DOI:10.1002/anie.201808493

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International Edition: DOI: 10.1002/anie.201808493
German Edition: DOI: 10.1002/ange.201808493
Bioimaging
One, Two, Three: A Bioorthogonal Triple Labelling Strategy for
Studying the Dynamics of Plant Cell Wall Formation In Vivo
Clemence Simon+, Cedric Lion+,* Corentin Spriet, Fabien Baldacci-Cresp, Simon Hawkins,* and
Christophe Biot
Abstract: Reported herein is an in vivo triple labelling strategy
to monitor the formation of plant cell walls. Based on
a combination of copper-catalysed alkyne–azide cycloaddition
(CuAAC), strain-promoted azide–alkyne cycloaddition
(SPAAC), and Diels–Alder reaction with inverse electronic
demand (DARinv), this methodology can be applied to various
plant species of interest in research. It allowed detection of the
differential incorporation of alkynyl-, azido-, and methyl-
cyclopropenyl-tagged reporters of the three main monolignols
into de novo biosynthesized lignin in different tissues, cell
types, or cell wall layers. In addition, this triple labelling was
implemented with different classes of chemical reporters, using
two monolignol reporters in conjunction with alkynylfucose to
simultaneously monitor the biosynthesis of lignin and non-
cellulosic polysaccharides. This allowed observation of their
deposition occurring contemporaneously in the same cell wall.
to the hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units,
respectively, forming lignin.[11] Since lignin properties are
influenced by monomer ratio and the nature of inter-unit
bonds, improved knowledge of lignification would contribute
to better understanding of plant physiology and factors
affecting biomass quality.[12] We recently reported a dual
labelling method using strain-promoted alkyne–azide cyclo-
addition (SPAAC) and copper-catalysed alkyne–azide cyclo-
addition (CuAAC) to detect incorporated azide-tagged
H-monolignols (HAZ 4) and alkyne-tagged G-monolignols
(GALK 5).[13] The use of two different cycloaddition reactions is
necessary for dual lignin labelling as the monolignol mimics 4
and 5 are incorporated into the same polymer. Because of
their spatial proximity, using consecutive CuAAC ligations
indeed leads to cross-linking reactions that compete with the
desired probe ligation. To develop a triple labelling strategy
for the three main monolignols, we tested whether the Diels–
O
ver the last two decades bioorthogonal reactions, com-
Alder reaction with inverse electronic demand (DARinv)
bined with fluorescence microscopy, have proven to be
a powerful tool for generating novel biological information
in a wide variety of in vivo molecular imaging applications.[1–3]
Many groups have focused on the use of reaction-based
fluorescent probes for chemoselective bioimaging in mam-
malian living systems, but plants have generally received less
attention despite the fact that they are complex multicellular
eukaryotes. Plant cells are characterized by the presence of an
extra-cellular matrix called the cell wall that is made up of
different polymers (e.g., cellulose, hemicelluloses, pectins,
lignin). To date, bioorthogonal approaches have been used in
plants to investigate polysaccharide and lignin biosynthesis
using tagged sugars or monolignols as reporters in in vivo
labelling experiments,[4–9] as well as auxin.[10] Lignin plays vital
roles in plants and is also of considerable industrial impor-
tance. It forms in the cell wall when hydroxycinnamyl alcohols
(monolignols) are enzymatically oxidised by peroxidases and/
or laccases and undergo polymerisation (Figure 1). In angio-
sperms, the three main monolignols, that is, para-coumaryl
alcohol 1, coniferyl alcohol 2, and sinapyl alcohol 3, give rise
could be used to label the lignin S-unit and successfully
combined with SPAAC and CuAAC. Although there are
various reports of dual labelling methods that combine two
biorthogonal reactions both in vitro and in vivo,[6,7,13–19] the
only instance where three such reactions were used con-
currently within the same sample was based on a sequence of
CuAAC, DARinv, and Staudinger–Bertozzi ligation in an
activity-based protein profiling assay in solution.[20] Although
three-color imaging using compatible reactions seems intui-
tively possible, to the best of our knowledge it has never been
applied to track three distinct molecules within a single living
sample. Herein, we demonstrate the feasibility of triple
biorthogonal labelling in vivo by sequentially exploiting the
major triad of biorthogonal reactions (DARinv, SPAAC, and
CuAAC) to track the three main lignin monomers (Figure 1).
Furthermore, we show that the same strategy can be used to
conjointly monitor lignin and cell wall glycan biosynthesis.
We firstly designed a sinapyl alcohol surrogate bearing
a suitable tag for DARinv. For this we opted for the reactive
methylcyclopropenyl moiety[18,21,22] to allow fast kinetics of
the DARinv without introducing a bulky trans-cyclooctene or
norbornene tag that might hamper cell wall phenoloxidase-
initiated oxidation. The new chemical reporter SCP 6 was
synthesized in seven steps from commercially available
sinapic acid (see Scheme S1 in the Supporting Information)
and its reactivity with the selected tetrazine-functionalized
Cy5 fluorescent probe was evaluated by HPLC. Full con-
version occurred rapidly at ambient temperature under
aqueous conditions, and the cycloadduct was identified by
MALDI-TOF mass spectrometry (see Figure S1).
[*] C. Simon,[+] C. Lion,[+] C. Spriet, F. Baldacci-Cresp, S. Hawkins, C. Biot
Universitꢀ de Lille, CNRS, UMR 8576
UGSF—Unitꢀ de Glycobiologie Structurale et Fonctionnelle
59000 Lille (France)
E-mail: cedric.lion@univ-lille.fr
simon.hawkins@univ-lille.fr
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201808493.
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Figure 1. Overview of the bioorthogonal triple labelling strategy. a) General scheme of plant cell wall structure and localisation. b) Chemical
structure of natural molecules of interest and corresponding tagged reporters. c) Chemical reporter strategy with triple labelling: DARINV, SPAAC
and CuAAC.
After confirming its lack of toxicity on living flax seedlings
(see Figure S2), we investigated whether SCP 6 could be
metabolically incorporated into the cell walls of flax plants.
For this we established and fully optimized a single-labelling
protocol in which the tetrazine-Cy5 probe is specifically
ligated to metabolically incorporated lignin SCP units by the
DARinv reaction at ambient temperature.
Subsequent confocal fluorescence microscopy observa-
tion indicates that SCP 6 is incorporated in a time- and dose-
dependent manner into the lignifying walls of differentiating
secondary xylem cells but is not/poorly incorporated into
already lignified mature cell walls (Figure 2). This labelling
pattern is in agreement with our previous observations of the
alkyne- and azide-tagged G- and H-unit reporters 4 and 5,
respectively.[13,23,24] Additional experiments showed that SCP
incorporation is 1) abolished by competition with the natural
S monolignol 3 and 2) almost completely eliminated in
samples when the enzymatic machinery is heat denatured
prior to incubation, indicating that SCP incorporation is under
enzymatic control (see Figures S3 and S4). These results also
indicate that the introduction of the methylcyclopropenyl tag
and spacer arm does not prevent either the phenoloxidase-
mediated oxidation of the sinapylic moiety, or its radical
polymerization.
Before incorporating the DARinv reaction into an in vivo
triple labelling strategy we first evaluated the compatibility of
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Figure 2. Incorporation of SCP monolignol reporter into flax stem cell walls. a) Negative control incorporating unmarked 3. b) Incorporation of
tagged SCP 6. Lignin autofluorescence in blue, tetrazine-Cy5 fluorescence in magenta. Magnified zooms (left, middle) show intensity-dependant
false colouring of differentiating xylem allowing direct comparison of the 3 images and illustrating the greater sensitivity of SCP labelling compared
to autofluorescence. c) SCP concentration- (left) and time- (right) dependant fluorescence. Scale bars (a, b main figures=50 mm, inserts=20 mm).
the three click reactions in this context. DARinv specificity
between SCP 6 and the tetrazine-Cy5 probe was tested in the
presence of HAZ 4 and GALK 5 reporters in solution (see
Figure S5). Results showed that DARinv is orthogonal to the
other two reactions as SCP is fully converted into the
corresponding dihydropyridazine cycloadduct while HAZ
must be performed last, after removal of the excess cyclo-
octyne- and tetrazine-activated probes from the biological
sample. This protocol avoids possible competitive GALK-HAZ
cross-linking that may result in signal loss,[13,23,24] as well as
side-reactions of the tetrazine-functionalized probe in the
presence of copper sulfate that may result in strong back-
ground.[20] Our results (Figure 3; see Figure S6) show that the
triple-labelling protocol can be successfully used to visualize
H-, G-, and S-monolignol reporter incorporation into actively
lignifying cell walls in differentiating flax secondary xylem
using the two experimental systems we previously reported
(i.e., flax stem sections as an easily implemented model, or
entire flax stems as a relevant in vivo system). Crossed
negative controls showed the chemoselective and specific
nature of each reaction for its target reporter (see Figure S7).
This strategy is also applicable to other plant organs, such as
roots (see Figure S8).
and GALK remain untouched. After full conversion of SCP
,
specific SPAAC cycloaddition between HAZ and DBCO-
PEG4-Rhodamine Green occurs without degradation of
either the SCP cycloadduct or of the remaining GALK reporter
in the mixture, and can in turn be linked to TAMRA-azide by
CuAAC as previously reported.[13,23,24] Based on these results
we developed an in vivo, sequential triple-labelling protocol
to visualize lignification in flax plant cell walls. Samples were
incubated for 20 hours with the reporters HAZ 4, GALK 5, and
SCP 6 prior to: 1) DARinv labelling of incorporated SCP units,
2) SPAAC labelling of HAZ units, and 3) CuAAC labelling of
GALK units. As expected, the CuAAC labelling of GALK units
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Figure 3. Bioorthogonal lignin triple (H, G, S) labelling in flax. Lignin autofluorescence (blue), incorporated HAZ 4 (green, SPAAC/ DBCO-PEG4-
Rhodamine Green), GALK 5 (red, CuAAC/ Azide-fluor 545), SCP 6 (magenta, DARinv/ tetrazine-Cy5), and merge channels. Incorporation of reporters
in a) freshly cut transversal cross-sections of flax stems; b) freshly cut longitudinal sections of flax stems; c) living entire flax stems. Focus on cell
walls of differentiating xylem: inserts show intensity-dependant false colouring image of cell walls illustrating differential incorporation of
monolignol reporters in wall sub-layers. Scale bar=50 mm (a,b), 10 mm (c) and 5 mm (insert).
Our triple labelling method offers a number of advantages
for the dynamic study of lignification processes compared to
more conventional approaches based on either autofluores-
cence, histochemistry, or immunolocalization. A first advant-
age is that this metabolic labelling enables us to distinguish
between cell wall layers that are lignified, but not actively
undergoing lignification, from cell wall layers that are still
actively producing lignin (see higher reporter incorporation in
differentiating xylem). The use of specific chemical reporters
for the three main lignin units also allows us to analyse the
capacity of different cell wall layers and/or domains to
incorporate the different monolignols during development.
Nevertheless, even if any potential reporter crosstalk is
extremely unlikely, as reporters will be rapidly incorporated
into cell wall lignin through the apoplastic route and will not
enter the cytoplasm where enzymatic conversion might take
place, future detailed quantitative studies would benefit from
complementation by in-depth metabolomics. Although abso-
lute quantitative data cannot be obtained by direct compar-
ison between the three image channels due to varying
acquisition parameters (e.g., different quantum yields or
excitation conditions), layers showing preferential incorpo-
ration of different reporters can be identified. For example,
Figure 3c clearly illustrates that, in the selected cell, HAZ
reporters are preferentially incorporated in the middle
lamella and bordered pit compared to other cell wall regions.
In contrast GALK reporters are preferentially incorporated
into the primary cell wall/S1 secondary cell wall layer of this
cell and the younger adjacent cell closer to the vascular
cambium. Last, the SCP reporter appears to be very heavily
incorporated into the S1 and S2 secondary cell wall layers of
the selected cell, but not in the wall of the younger adjacent
cell. Although care should be taken when interpreting the
biological significance of observed differences since, strictly
speaking, labelling reflects the incorporation of monolignol
mimics and not native molecules, our results are in agreement
with previous studies using radioactively labelled mole-
cules.[27] Altogether, this technique represents a powerful
tool for investigating the heterogeneity in lignin cell wall
composition.
To evaluate the triple labelling strategy for lignification in
other species we tested three other commonly used plant
models: Arabidopsis thaliana, Nicotiana Benthamiana
(tobacco), and Populus tremula x alba (poplar). In all cases
stem sections incorporated the three reporters into actively
lignifying xylem cell walls (Figure 4). As observed in flax cell
walls, labelling was generally more intense in the cell walls of
young differentiating xylem cells compared to more mature
cells. Lignified cell walls that were no longer undergoing
active lignification could be distinguished from actively
lignifying cell walls by their UVautofluorescence and absence
of reporter signal. This observation was particularly evident in
poplar, but could also be observed in Arabidopsis and
tobacco. Reporters were also incorporated in lignifying
interfascicular fibers (Arabidopsis) and sclerenchyma fibers
(tobacco, poplar) that are more heavily lignified than flax
fibers. A relatively strong labelling was also observed at the
xylem/pith parenchyma junction in tobacco and Arabidopsis
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investigate the dynamics of both lignin and non-cellulosic
polysaccharide (NCP) polymer deposition in flax stem cell
walls. For this we used the HAZ 4 and SCP 6 monolignol
reporters together with peracetylated alkyne-tagged fucose
reporter FucALK 8. Fucose (7) is present in both the side chains
of xyloglucan hemicelluloses and in pectin rhamnogalactur-
onan I and II (RGI, RGII) motifs,[25,26] and previous reports
have shown that FucALK 8 can be successfully used in
a bioorthogonal reaction to label plant NCP cell wall
polymers.[4,5] In contrast to the incorporation of monolignol
reporters directly into the cell wall, NCP labelling requires
that the reporter be metabolized into the cytoplasm and
transported to the cell wall by the Golgi apparatus, and we
therefore reasoned that the entire stem in vivo system might
be more appropriate than the cross-section model. This
approach was successful and our results (Figure 5) allowed us
to distinguish three main zones according to precursor
incorporation profiles: the marking pattern of cell walls in
the phloem and vascular cambium (zone 1 in the Figure)
indicated that fucose reporters had been incorporated into
cell wall NCP polymers, but that these cells are not actively
producing lignin. In the well-developed secondary xylem
(zone 2) only HAZ and SCP monolignol reporters were
incorporated into cell walls, indicating that NCP wall polymer
biosynthesis had stopped in these cells. A third area,
corresponding to the differentiating xylem (zone 3) incorpo-
rated both fucose and monolignol reporters in the same wall
indicating that lignin and NCP polymer deposition occur
contemporaneously in the same cell wall. Although different
lines of evidence have for a long time suggested that such co-
deposition could occur,[27,28] this is the first time, to our
knowledge, that this has been experimentally demonstrated.
This data is the first report of the use of a SPAAC,
CuAAC, and DARinv triple labelling approach to successfully
monitor three distinct molecules in vivo. We used this method
to visualize the incorporation of the three main lignin
monomers in the cell walls of different model plant species
and showed that it can also be exploited to conjointly study
the deposition of lignin and cell wall glycans in the same
sample. The development of this powerful and versatile triple
labelling strategy based upon the use of independent bio-
orthogonal reactions will greatly advance our understanding
of cell wall formation in plants and should also become
a valuable addition to the human and animal imaging toolbox.
Furthermore, the combination of tags used in this study also
theoretically allows the detection and quantification of up to
three mimics by different non-imaging-based analytical
techniques including fluorescence, mass spectrometry, mag-
netic resonance spectroscopies, and vibrational spectroscop-
ies, thereby paving the way to many different applications.
One exciting perspective would be to use the different
emission fluorescence wavelengths to follow the dynamic
release of the different lignin monomers during chemical/
enzymatic degradation of the labelled lignin polymer in plant
lignocellulose biomass.
Figure 4. Bioorthogonal lignin triple H/G/S labelling in other plant
species. HAZ 4 incorporation in green (SPAAC/ DBCO-PEG4-Rhodamine
Green), GALK 5 in red (CuAAC/ Azide-fluor 545), SCP 6 in magenta
(DARinv/ tetrazine-Cy5), and autofluorescence in blue. Cross-section of
a) Arabidopsis thaliana floral hemp; b) tobacco stem; c) poplar stem.
Left panel: low power merge, scale bar=50 mm; right panel: zoom of
xylem vascular bundle (a) or differentiating xylem (b,c), scale
bar=20 mm. Differentiating xylem (arrow), (interfascicular) fibers
(star), xylem/pith junction (triangle), mature vessel (MV), mature
xylem (MX).
in comparison with a much weaker signal in poplar indicating
a potential difference between lignification in woody and
non-woody species. As observed in flax, certain cell wall
layers/regions showed preferential incorporation of one (two)
reporter(s) compared to the other(s) most likely reflecting
heterogenity in the cell wall machinery and/or structure.
Cell wall formation in plants is a highly regulated process
and lignin deposition is coordinated with the formation of
other cell wall polymers. We therefore investigated whether
this triple labelling approach could be used to simultaneously
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Figure 5. Bioorthogonal lignin and polysaccharide triple fucose/H/S labelling in flax. Incorporation of a) FucALK 8 (red, CuAAC/ Azide-fluor 545),
b) HAZ 4 (green, SPAAC/ DBCO-PEG4-Rhodamine Green), c) SCP 6 (magenta, DARinv/ tetrazine-Cy5) and d) merge in phloem/vascular cambium
and differentiating xylem zones in entire living flax stem; e) intensity profile of reporter incorporation in cell walls in vascular cambium (1),
maturing xylem (2) and young xylem (3). Dotted line=profile trace (a–d), arrows=indicated cell walls (a–d) and corresponding peaks (e). Scale
bar=20 mm.
[4] Y. Tobimatsu, D. V. de Wouwer, E. Allen, R. Kumpf, B.
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Accepted manuscript online: October 29, 2018
Version of record online: && &&, &&&&
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Communications
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C. Simon, C. Lion,* C. Spriet,
F. Baldacci-Cresp, S. Hawkins,*
C. Biot
&&&& — &&&&
One, Two, Three: A Bioorthogonal Triple
Labelling Strategy for Studying the
Dynamics of Plant Cell Wall Formation In
Vivo
One, two, three: A triple bioorthogonal
labelling strategy was developed to study
the incorporation of three distinct
monolignol reporters in lignin within
plant cell walls. Alkynylfucose was also
used in conjunction with monolignol
reporters to show that non-cellulosic
polysaccharide and lignin deposition can
occur contemporaneously in the same
cell wall. CuAAC: copper-catalysed
alkyne–azide cycloaddition, SPAAC:
strain-promoted azide–alkyne cycloaddi-
tion, DARinv: Diels–Alder reaction with
inverse electronic demand.
8
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