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ChemComm
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COMMUNICATION
Journal Name
combined in a plant model, and more widely in a eukaryotic was done at RT as a proof of concept and it should be noted that
DOI: 10.1039/D0CC05218C
model. In vivo bioorthogonal spin labeling has however, also imaging samples at cryogenic temperature would allow an
been very recently applied to proteins in bacteria using CuAAC improvement of the signal to noise ratio of the image in future
ligation 21
.
studies.
In conclusion, we have demonstrated that the
Although the presence of residual unbound probe in the sample
is usually considered a hindrance in data processing, we were
able to use it to our advantage in order to quantify the amount
of labelled SCP within the sample. For this, a calibration curve
was produced by plotting signal intensity vs. probe
concentration in the 1 to 200 µM range (Fig. S5, ESI), which then
allowed us to determine the concentration of IIIA in the
measured samples. Given the relative weights calculated for
each species, this technique allows us to quantify the amount
of immobilized probe IIIB specifically linked to SCP monomers
polymerized into lignin. Although 4 also gave a specific signal by
EPR and led to similar results (Fig. S7A, ESI), a significant
difference arose in the acquired spectra (Table 1). The
simulations calculated from the acquired spectra clearly show
that there are two major immobilized contributors to the
spectrum IVB and IVC, in addition to the residual signal for the
free probe IVA (Table 1 and Fig. S7B, ESI). Whereas IVA exhibits
a mobility very similar to the previously observed IIIA (Azz = 17
G, τc = 2 and 3 ns, respectively), the two other contributors IVB
and IVC to the EPR signal are even more immobilized (τc = 46 and
87 ns, respectively). It could be hypothesized that the length of
the PEG4 spacer arm and its flexibility allows probe 4 to explore
a larger environmental pocket around the tagged lignin site
thereby enabling it to enter into denser and/or smaller cell wall
spaces that are less accessible to 3. We then went on to use the
nitroxide radical footprint of 3 specifically labeled to lignin in the
plant cell wall to perform EPR imaging (Fig. 3C). The EPR images
were obtained on two sets of 9 flax stem cross-sections with
(left), or without (right) prior metabolic incorporation of 2.
Careful examination indicated that the high signals observed in
the left-hand image are associated with plant samples and most
likely result from the detection of labelled lignin in flax xylem
tissue. Differences in signal intensity observed between
samples can be related to the fact that the flax cross-sections
are orientated differently in the tube (Fig. S8, ESI). It is unlikely
that these differences reflect variation in SCP incorporation as
DARinv fluorophore ligation (Fig. S3, ESI) gave highly
reproducible results in different sections in agreement with our
previous studies in this species.7,8,13 In contrast, a much lower
overall signal was observed in samples that had not
metabolically incorporated 2. Interestingly, a closer look at the
observed signal indicated that the marking spots were between
5 and 10µm in size suggesting that there are privileged zones in
which the probe is located. Since these dimensions are
comparable to those of the cell walls in the lignifying region of
the young xylem (Fig. S3, ESI) it is tempting to speculate that
these zones demonstrate the imaging of de novo lignification at
the cellular level. However, further refinement of this approach
and continued experimentation are clearly necessary to confirm
this hypothesis. Although EPR imaging has previously been used
in plants, to our knowledge, this result is the first example of its
application to plant cell wall polymers ,22-24 and the only one
treating with the labelling of exogenous reporters. Our work
bioorthogonal chemical reporter strategy can be successfully
combined with EPR spectroscopy in plant tissues to detect the
incorporation of a cyclopropenyl-tagged monolignol into the
lignin polymer. We therefore show that the production of de
novo lignin can be detected by another method in addition to
fluorescence. Simulation of the recorded EPR spectrum
provides mobility parameters about the paramagnetic probe
specifically tagged to lignin and should allow us to obtain spatial
information about its polymerization dynamics in future
studies. The spin labeling strategy presented here could also be
transposed to other major plant cell wall polymers like pectin or
hemicelluloses of importance in the context of lignocellulosic
biomass valorization.
Conflicts of interest
There are no conflicts to declare.
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