We are indebted to the Research Federation FRABio and the TisBio imaging platform (Univ. Lille, CNRS, FR 3688, FRABio, BiochimieStructurale et Fonctionnelle des Assemblages Biomol&#233;culaires) for providing the technical environment conducive to achieving this work.

NOTE: Liquid and solid &#189; MS media must be prepared beforehand as described in Supplemental Table 1.
1. Plant Culture
<ol>
	<li>Culture of 2 month-old flax plants
	<ol>
		<li>Sow flax (Linum usitatissimum L.) seeds in plastic pots using potting compost.</li>
		<li>Grow flax in growth chambers at 22 &#176;C with a photoperiod of 16 h/8 h day/night.</li>
		<li>Equip plants with vertical support after 1 month and grow until they are 2 month-old.</li>
	</ol>
	</li>
	<li>Culture of 2 week-old flax seedlings
	<ol>
		<li>Wrap 12 flax seeds in a piece of cheese cloth and fix with a rubber band to make a bundle. 
		NOTE: Carry out the next steps under sterile conditions under a laminar flow fume hood.</li>
		<li>Place the seed bundle in a previously autoclaved 250 mL glass bottle.</li>
		<li>Add 70 mL of 70% EtOH in the bottle and gently stir for 1 min.</li>
		<li>Carefully remove the EtOH by decantation while leaving the bundle in the bottle.</li>
		<li>Add 100 mL of 2% sodium hypochlorite and stir gently for 10 min.</li>
		<li>Remove the sodium hypochlorite solution by decantation while leaving the bundle in the bottle.</li>
		<li>Repeat steps 1.2.5-1.2.6.</li>
		<li>Add 100 mL of sterile ultrapure water and stir for 1 min.</li>
		<li>Repeat step 1.2.8 3 times, on increasing the rinsing time for each rinse (5, 10 and 15 min).</li>
		<li>Warm solid &#189; MS medium, pour it in a sterile plant culture box to a height of 2 cm and let it cool down until solidification.</li>
		<li>Delicately place each seed on solid medium using sterile forceps.</li>
		<li>Close the sterile box.</li>
		<li>Transfer the box to a growth chamber at 22 &#176;C with a photoperiod of 16 h/8 h day/night and grow flax seedlings for 2 weeks.</li>
	</ol>
	</li>
</ol>
2. Metabolic Incorporation of Chemical Reporters
NOTE: Three experimental models are presented below: i) metabolic labeling of stem cross-sections, ii) whole stems and iii) plant seedlings. Each protocol is presented for one biological replicate and quantities can be adapted to the number of required biological replicates. The monolignol solutions are prepared from stock solutions that are made as described in Table 2 prior to the experiment. The stock solutions can be stored several months at -20 &#176;C. The HAZ:GALK or H:G proportions can be adjusted to fit all custom-made experimental designs.
<ol>
	<li>Chemical reporter incorporation into 2 month-old flax stem cross-sections
	<ol>
		<li>Prepare a 300 &#181;L chemical reporter solution containing 10 &#181;M of GALK and 10 &#181;M of HAZ in liquid sterile &#189; MS medium.</li>
		<li>Vortex the HAZ/GALK solution and transfer it to one well of a 48-well plate with a micropipette.</li>
		<li>Prepare a 300 &#181;L negative control solution containing 10 &#181;M of G and 10 &#181;M of H in liquid sterile &#189; MS medium.</li>
		<li>Vortex the H/G solution and transfer it to a second well of the same 48-well plate.</li>
		<li>Cut the stem of a 2 month-old flax plant at 10 cm above soil level using a new razor blade.</li>
		<li>Delicately prepare 50 freehand transversal cross-sections of the stem using the razor blade (approximately 150-250 &#181;m thick).</li>
		<li>Immediately place the cross-sections in liquid sterile &#189; MS medium in a watch glass.</li>
		<li>Visually inspect and select intact whole cross-sections and randomly distribute 10 of them in the well filled with HAZ/GALK solution.</li>
		<li>Repeat step 2.1.8 for the well filled with H/G solution (as negative control).</li>
		<li>Repeat step 2.1.8 for a third well filled with 300 &#181;L of &#189; MS medium (as background control to adjust the fluorescence baseline during the subsequent data processing of confocal microscopy images).</li>
		<li>Incubate the plate in continuous light in a growth chamber at 20 &#176;C for 20 h.</li>
		<li>Remove the monolignol solution in each well with a micropipette.</li>
		<li>Add 500 &#181;L of &#189; MS medium to each well, stir gently for 10 min and remove this rinsing solution with a micropipette. Repeat 4 times and proceed to BLISS labeling immediately (section 3).</li>
	</ol>
	</li>
	<li>Chemical reporter incorporation into 2 month-old flax whole stems
	<ol>
		<li>Prepare a 5 mL chemical reporter solution containing 10 &#181;M of GALK and 10 &#181;M of HAZ in liquid sterile &#189; MS medium.</li>
		<li>Vortex the HAZ/GALK solution and transfer it to a 20-cm-high glass test tube with a pipette.</li>
		<li>Prepare a 5 mL negative control solution containing 10 &#181;M of G and 10 &#181;M of H in liquid sterile &#189; MS medium.</li>
		<li>Vortex the H/G solution and transfer it to a 20-cm-high glass test tube.</li>
		<li>Prepare a third glass test tube containing 5 mL of &#189; MS medium (as background control to adjust the fluorescence baseline during the subsequent data processing of confocal microscopy images).</li>
		<li>Cut the stem of a 2 month-old flax plant at 10 cm above soil level using a new razor blade. Discard the root system and lower part of the stem and proceed to the next step with the upper part of the plant stem.</li>
		<li>Immerse the base of the whole cut stem in the tube containing the HAZ/GALK solution. Place the stem in its incubation solution immediately after removal of the root system in order to prevent it from drying. 
		NOTE: If the method is applied to younger/older plants and/or other species, the volume of monolignol solutions must be adjusted according to the size / age of the plant and the diameter of its stem.</li>
		<li>Attach the stem to a vertical support to keep it straight.</li>
		<li>Seal the glass tube with parafilm to avoid evaporation of the solution.</li>
		<li>Repeat steps 2.2.6-2.2.9 for the negative control sample with the H/G solution.</li>
		<li>Repeat steps 2.2.6-2.2.9 for the fluorescence background control sample containing the &#189; MS medium.</li>
		<li>Incubate each stem for 20 h in continuous light using a neon light.</li>
		<li>After 20 h of metabolic incorporation, remove the stem incubated in the HAZ/GALK solution and rinse it with &#189; MS medium.</li>
		<li>Cut and discard the bottom 1 cm of the stem using a razor blade and then prepare a 1 cm high cylinder from the base of the remaining stem.</li>
		<li>Carefully prepare 20 freehand transverse cross-sections (approximately 150-250 &#181;m thick) from this cylinder and place them immediately in a watch glass filled with &#189; MS medium.</li>
		<li>Put 500 &#181;L of &#189; MS medium in one well of a 48-well plate.</li>
		<li>Visually inspect and select intact whole cross-sections and randomly distribute 10 of them in the well filled with &#189; MS solution.</li>
		<li>Repeat steps 2.2.13-2.2.17 for the negative control stem incubated with the H/G solution.</li>
		<li>Repeat steps 2.2.13-2.2.17 for the fluorescence background control stem incubated with &#189; MS medium.</li>
		<li>Carefully remove the solution in each well with a micropipette.</li>
		<li>Add 500 &#181;L of &#189; MS medium to each well, stir gently for 10 min and remove this rinsing solution with a micropipette. Repeat 4 times and proceed to BLISS labeling immediately (section 3).</li>
	</ol>
	</li>
	<li>Chemical reporter incorporation into 2 week-old flax seedlings 
	NOTE: The following steps are all carried out under sterile conditions.
	<ol>
		<li>Prepare a 2 mL chemical reporter solution containing 10 &#181;M of GALK and 10 &#181;M of HAZ in liquid sterile &#189; MS medium.</li>
		<li>Vortex the HAZ/GALK solution and transfer it to a 20 cm-high glass test tube with a pipette.</li>
		<li>Prepare a 2 mL negative control solution containing 10 &#181;M of G and 10 &#181;M of H in liquid sterile &#189; MS medium.</li>
		<li>Vortex the H/G solution and transfer it to a 20 cm-high glass test tube.</li>
		<li>Prepare a third 20-cm-high glass test tube containing 2 mL of &#189; MS medium (as background control to adjust the fluorescence baseline during the subsequent data processing of confocal microscopy images).</li>
		<li>Delicately remove a 2-week-old flax seedling from the solid &#189; MS medium using a long pair of tweezers (section 1.2. above).</li>
		<li>Gently transfer the seedling to the tube containing the HAZ/GALK solution, ensuring that its roots are immersed in the solution.</li>
		<li>Close the glass tube with a plastic cap to avoid evaporation.</li>
		<li>Repeat steps 2.3.6-2.3.8 for the negative control tube with the H/ G solution.</li>
		<li>Repeat steps 2.3.6-2.3.8 for the fluorescence background control tube containing the &#189; MS medium.</li>
		<li>Incubate each seedling in continuous light in a growth chamber for 20 h at 20 &#176;C.</li>
		<li>Delicately remove the seedling incubated in the HAZ/GALK solution and rinse it with &#189; MS medium.</li>
		<li>Delicately prepare 20 freehand cross-sections (approximatively 150-250 &#181;m thick) of the hypocotyl.</li>
		<li>Put 500 &#181;L of &#189; MS medium in one well of a 48-well plate.</li>
		<li>Visually inspect and select intact whole cross-sections and randomly distribute 10 of them in the well filled with &#189; MS solution.</li>
		<li>Repeat steps 2.3.12-2.3.15 for the negative control seedling incubated with the H/G solution.</li>
		<li>Repeat steps 2.3.12-2.3.15 for the fluorescence background control seedling incubated with &#189; MS medium.</li>
		<li>Carefully remove the solution in each well with a micropipette.</li>
		<li>Add 500 &#181;L of &#189; MS medium to each well, stir gently for 10 min and remove this rinsing solution with a micropipette. Repeat 4 times and proceed to BLISS labeling immediately (section 3).</li>
	</ol>
	</li>
</ol>
3. Dual Fluorescence Labeling of Plant Cross-section Samples by SPAAC and CuAAC
NOTE: The BLISS labeling protocol is identical for all 3 experimental models (section 2).
<ol>
	<li>Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) labeling
	<ol>
		<li>Prepare 1 mL of a SPAAC solution containing 5 &#181;M of DBCO-PEG4-5/6-carboxyrhodamine 110 in liquid sterile &#189; MS medium. Vortex the solution and keep it in the dark.</li>
		<li>After the final washing steps of the metabolic incorporation protocol [2.1.13, 2.2.21 or 2.3.19 depending on the chosen experimental design], remove the &#189; MS medium and add 300 &#181;L of the SPAAC solution per well with a micropipette.</li>
		<li>Shield the 48-well plate from light by covering with aluminum foil or by placing it in a box.</li>
		<li>Gently stir the plate on a mechanical shaker for 1 h in the dark at ambient temperature.</li>
	</ol>
	</li>
	<li>Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) labeling
	<ol>
		<li>Wash each well 4 times as described in step 2.1.13 while keeping the plate in the dark.</li>
		<li>Prepare 1 mL of a CuAAC solution containing 2.5 mM sodium ascorbate, 0.5 mM CuSO4 and 5 &#181;M of 5-TAMRA-PEG3-azide in liquid sterile &#189; MS medium. Vortex the solution and keep it in the dark. 
		NOTE: This CuAAC solution must be freshly prepared prior to use and cannot be stored more than a few days at 4 &#176;C.</li>
		<li>Remove the &#189; MS medium in each well and directly add 300 &#181;L of the CuAAC solution per well using a micropipette.</li>
		<li>Gently stir the plate on a mechanical shaker for 1 h in the dark at ambient temperature.</li>
		<li>Remove the CuAAC solution using a micropipette.</li>
		<li>Using a micropipette, add 500 &#181;L of &#189; MS, stir in the dark for 10 min then remove this washing solution. Repeat twice.</li>
		<li>Add 500 &#181;L of a 7:3 MeOH/water solution, stir in the dark for 1 h then remove the solution 
		CAUTION: Methanol is toxic by skin contact or inhalation, and is designated as a human carcinogen. Its utilization requires chemical fume hoods and gloves.</li>
		<li>Add 500 &#181;L of &#189; MS, stir in the dark for 10 min then remove the solution. Repeat 4 times.</li>
	</ol>
	</li>
</ol>
4. Mounting of Samples on Microscope Slides
<ol>
	<li>Place 5 drops of mounting medium on a glass microscope slide.</li>
	<li>Carefully deposit each HAZ/GALK tagged cross-section on the slide.</li>
	<li>Apply nail polish on two opposite sides of the slide.</li>
	<li>Cover the slide with a coverslip. Be careful to avoid air bubbles.</li>
	<li>Remove excess mounting medium using a paper towel.</li>
	<li>Seal the sample with nail polish on all 4 sides. Let the nail varnish dry at room temperature (approximately 30 min).</li>
	<li>Repeat steps 4.1-4.6 for the negative control H/G cross-sections.</li>
	<li>Repeat steps 4.1-4.6 for the fluorescence background control cross-sections.</li>
	<li>Store the slides at 4 &#176;C in the dark until observation under a confocal microscope. 
	NOTE: Prepared slides can be stored for several weeks to several months depending on the quality of the preparation. If a new observation is to be made after storage, ensure that the samples have not dried up.</li>
</ol>
5. Image Acquisition by Fluorescence Confocal Microscopy
<ol>
	<li>Turn on all necessary units of the confocal microscopy system in the right order according to the manufacturer&#39;s instructions.</li>
	<li>Select the desired objective with maximal numerical aperture and adapted excitation and emission wavelengths (e.g., obj. 60x, N.A. 1.2. Autofluorescence channel: &#955;ex 405 nm, &#955;em 450 nm /25; SPAAC HAZ channel: &#955;ex 488 nm, &#955;em 525 nm /25; CuAAC GALK channel: &#955;ex 561 nm, &#955;em 595 nm /25 ; sequential mode).</li>
	<li>Select the desired parameters for generation of images in the software (12- or 16-bit required, pixel size adapted to the Nyquist criterion).</li>
	<li>Place the HAZ/GALK slide on the microscope stage and position the sample under the objective lens.</li>
	<li>Observe and find the desired region of the plant tissue.</li>
	<li>Acquire high quality confocal images of doubly labeled HAZ/GALK sample.
	<ol>
		<li>Select the most fluorescent plane in the Z-axis.</li>
		<li>Adjust gain, offset and laser power to achieve optimal signal distribution along the whole grey level range for each channel (autofluorescence, HAZ and GALK). The same parameters must be applied for all following acquisitions.</li>
		<li>Launch the acquisition of the selected image and save the file. Repeat for each relevant area of the sample. 
		NOTE: 3D Z-stack reconstructions may also be acquired if necessary.</li>
	</ol>
	</li>
	<li>Using the same settings, acquire images of untagged sample only incubated in &#189; MS to determine the autofluorescence level (fluorescence background control as described in section 2).</li>
	<li>Using the same settings, acquire images for the negative control samples incubated with native monolignols H/G to determine non-specific fluorophore adhesion to the sample.</li>
	<li>Apply data treatment to the acquired images to substract background autofluorescence signals in both H/G negative control images and HAZ/GALK images.</li>
</ol>

<ol>
	<li>Grammel, M., Hang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+reporters+for+biological+discovery.">Chemical reporters for biological discovery.</a> Nat Chem Biol. 9 (8), 475-484 (2013).</li><li>Prescher, J. A., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+in+living+systems.">Chemistry in living systems.</a> Nat Chem Biol. 1 (1), 13-21 (2005).</li><li>Sletten, E. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+chemistry:+fishing+for+selectivity+in+a+sea+of+functionality.">Bioorthogonal chemistry: fishing for selectivity in a sea of functionality.</a> Ange Chemie (Int Ed. Engl). 48 (38), 6974-6998 (2009).</li><li>Chang, P. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+labeling+of+sialic+acids+in+living+animals+with+alkynyl+sugars.">Metabolic labeling of sialic acids in living animals with alkynyl sugars.</a> Ange Chemie (Int Ed. Engl). 48 (22), 4030-4033 (2009).</li><li>Gilormini, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sequential+bioorthogonal+dual+strategy:+ManNAl+and+SiaNAl+as+distinct+tools+to+unravel+sialic+acid+metabolic+pathways.">A sequential bioorthogonal dual strategy: ManNAl and SiaNAl as distinct tools to unravel sialic acid metabolic pathways.</a> Chem. Commun. 52 (11), 2318-2321 (2016).</li><li>Mbua, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+accumulation+and+recycling+of+glycoproteins+visualized+in+Niemann-Pick+type+C+cells+using+the+chemical+reporter+strategy.">Abnormal accumulation and recycling of glycoproteins visualized in Niemann-Pick type C cells using the chemical reporter strategy.</a> Proc Natl Acad Sci U S A. 110 (25), 10207-10212 (2013).</li><li>Anderson, C. T., Wallace, I. S., Somerville, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+click-labeling+with+a+fucose+analog+reveals+pectin+delivery,+architecture,+and+dynamics+in+Arabidopsis+cell+walls.">Metabolic click-labeling with a fucose analog reveals pectin delivery, architecture, and dynamics in Arabidopsis cell walls.</a> Proc Natl Acad Sci U S A. 109 (4), 1329-1334 (2012).</li><li>Tobimatsu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+click+chemistry+strategy+for+visualization+of+plant+cell+wall+lignification.">A click chemistry strategy for visualization of plant cell wall lignification.</a> Chem. Commun. 50 (82), 12262-12265 (2014).</li><li>Bukowski, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+clickable+designer+monolignol+for+interrogation+of+lignification+in+plant+cell+walls.">Development of a clickable designer monolignol for interrogation of lignification in plant cell walls.</a> Bioconjugate Chem. 25 (12), 2189-2196 (2014).</li><li>Pandey, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+click-compatible+monolignol+probe+to+study+lignin+deposition+in+plant+cell+walls.">A versatile click-compatible monolignol probe to study lignin deposition in plant cell walls.</a> PLOS ONE. 10 (4), e0121334 (2015).</li><li>Pandey, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+biochemical+and+developmental+dependencies+of+lignification+with+a+click-compatible+monolignol+analog+in+Arabidopsis+thaliana+stems.">Investigating biochemical and developmental dependencies of lignification with a click-compatible monolignol analog in Arabidopsis thaliana stems.</a> Front Plant Sci. 7, (2016).</li><li>Zhu, Y., Wu, J., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+labeling+and+imaging+of+N-linked+glycans+in+Arabidopsis+thaliana.">Metabolic labeling and imaging of N-linked glycans in Arabidopsis thaliana.</a> Ange Chemie (Int Ed. Engl). 55 (32), 9301-9305 (2016).</li><li>Boerjan, W., Ralph, J., Baucher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignin+biosynthesis.">Lignin biosynthesis.</a> Annu Rev Plant Biol. 54, 519-546 (2003).</li><li>Weng, J. -. K., Chapple, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+evolution+of+lignin+biosynthesis.">The origin and evolution of lignin biosynthesis.</a> New Phytol. 187 (2), 273-285 (2010).</li><li>Mottiar, Y., Vanholme, R., Boerjan, W., Ralph, J., Mansfield, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designer+lignins:+harnessing+the+plasticity+of+lignification.">Designer lignins: harnessing the plasticity of lignification.</a> Curr Opin Biotechnol. 37, 190-200 (2016).</li><li>Rinaldi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paving+the+way+for+lignin+valorisation:+recent+advances+in+bioengineering,+biorefining+and+catalysis.">Paving the way for lignin valorisation: recent advances in bioengineering, biorefining and catalysis.</a> Ange Chemie (Int Ed). 55 (29), 8164-8215 (2016).</li><li>Wratil, P. R., Horstkorte, R., Reutter, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+glycoengineering+with+N-acyl+side+chain+modified+mannosamines.">Metabolic glycoengineering with N-acyl side chain modified mannosamines.</a> Ange Chemie (Int Ed. Engl). 55 (33), 9482-9512 (2016).</li><li>Feng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bifunctional+unnatural+sialic+acids+for+dual+metabolic+labeling+of+cell-surface+sialylated+glycans.">Bifunctional unnatural sialic acids for dual metabolic labeling of cell-surface sialylated glycans.</a> J Am Chem Soc. 135 (25), 9244-9247 (2013).</li><li>Dumont, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+cell+wall+imaging+by+metabolic+click-mediated+labelling+of+rhamnogalacturonan+II+using+azido+3-deoxy-+d+-+manno+-oct-2-ulosonic+acid.">Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy- d - manno -oct-2-ulosonic acid.</a> Plant J. 85 (3), 437-447 (2016).</li><li>Niederwieser, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-color+glycan+labeling+of+live+cells+by+a+combination+of+Diels-Alder+and+click+chemistry.">Two-color glycan labeling of live cells by a combination of Diels-Alder and click chemistry.</a> Ange Chemie Int Ed. 52 (15), 4265-4268 (2013).</li><li>Sp&#228;te, A. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+potential+of+norbornene-modified+mannosamine+derivatives+for+metabolic+glycoengineering.">Exploring the potential of norbornene-modified mannosamine derivatives for metabolic glycoengineering.</a> Chem Bio Chem. 17 (14), 1374-1383 (2016).</li><li>Sp&#228;te, A. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Labeling+of+metabolically+engineered+cell-surface+glycoconjugates+with+a+carbamate-linked+cyclopropene+reporter.">Rapid Labeling of metabolically engineered cell-surface glycoconjugates with a carbamate-linked cyclopropene reporter.</a> Bioconjugate Chem. 25 (1), 147-154 (2014).</li><li>Lion, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLISS:+a+bioorthogonal+dual-labeling+strategy+to+unravel+lignification+dynamics+in+plants.">BLISS: a bioorthogonal dual-labeling strategy to unravel lignification dynamics in plants.</a> Cell Chem Biol. 24 (3), 326-338 (2017).</li><li>del R&#237;o, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+characterization+of+guaiacyl-rich+lignins+in+flax+(Linum+usitatissimum)+fibers+and+shives.">Structural characterization of guaiacyl-rich lignins in flax (Linum usitatissimum) fibers and shives.</a> J Agric Food Chemist. 59 (20), 11088-11099 (2011).</li><li>Huis, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+hypolignification+is+associated+with+extensive+oligolignol+accumulation+in+flax+stems.">Natural hypolignification is associated with extensive oligolignol accumulation in flax stems.</a> Plant Physiol. 158 (4), 1893-1915 (2012).</li><li>Chantreau, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+lignification+in+the+flax+lignified+bast+fiber1+mutant+stem+is+associated+with+tissue-specific+modifications+in+gene+expression+and+cell+wall+composition.">Ectopic lignification in the flax lignified bast fiber1 mutant stem is associated with tissue-specific modifications in gene expression and cell wall composition.</a> The Plant Cell. 26 (11), 4462-4482 (2014).</li><li>Terashima, N., Ko, C., Matsushita, Y., Westermark, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolignol+glucosides+as+intermediate+compounds+in+lignin+biosynthesis.+Revisiting+the+cell+wall+lignification+and+new+13C-tracer+experiments+with+Ginkgo+biloba+and+Magnolia+liliiflora.">Monolignol glucosides as intermediate compounds in lignin biosynthesis. Revisiting the cell wall lignification and new 13C-tracer experiments with Ginkgo biloba and Magnolia liliiflora.</a> Holzforschung. 70 (9), 801-810 (2016).</li><li>Noel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+CMP-sialic+acid+donor+specificity+of+two+human+&#946;-d-galactoside+sialyltransferases+(ST3Gal+I+and+ST6Gal+I)+selectively+acting+on+O-+and+N-glycosylproteins.">Probing the CMP-sialic acid donor specificity of two human &#946;-d-galactoside sialyltransferases (ST3Gal I and ST6Gal I) selectively acting on O- and N-glycosylproteins.</a> Chembiochem: Eur J Chem Biol. , (2017).</li></ol>

The authors declare that they have no competing financial interests.

As previously mentioned, the dual labeling BLISS protocol presented in this paper is one of the first examples of a SPAAC/CuAAC combination in vivo12,23. Each step was thoroughly optimized and validated, and it is very important that the order in which the two click chemistry labeling reactions are sequentially performed is respected (i.e., SPAAC first, followed by CuAAC). All cross-controls showed that each labeling step is specific when the BLISS protocol is applied23 : carrying out the SPAAC step first leads to the highly chemoselective labeling of HAZ azide functions by the cyclooctyne-functionalized fluorophore through a [3+2] cycloaddition reaction with fast kinetics. Once HAZ units are tagged, the CuAAC step necessitating copper(I) catalyzed activation of GALK terminal alkynes to generate triazole links by reaction with the azide-fluor 545 probe can be carried out. In contrast, the reverse order (i.e., CuAAC first, followed by SPAAC) should not be used as it leads to GALK and HAZ unit cross-coupling, which competes with fluorophore ligation and induces a dramatic loss of signal. It is also important to stress the necessity of the intermediate washing steps to avoid non-specific staining.
We have shown that our method can be applied to various biological experiment designs. The BLISS labeling protocol was first applied to freehand cross-sections of flax stems (approximately 150-250 &#181;m thick) that were previously cut and incubated with the click-ready monolignols. Although this design has the advantage of minimizing the needed quantities of chemical reporter (as incubation volumes are reduced) and of facilitating the production of statistical replicates, it is not, strictly speaking, an in vivo system and in some cases, may not reflect all aspects of real spatio-temporal lignification dynamics. In a second experimental design, we therefore adapted the BLISS protocol to a method that was previously used to study the incorporation of radiolabeled monolignols in pine and gingko27. In this approach, the roots and stem of the plant are physically separated and the base of the whole stem is incubated in the monolignol solution in what may be dubbed the &#39;flower vase&#39; approach. After leaving the stems the desired (incubation) time, cross-sections are cut and the BLISS protocol performed. This allowed us to show (i) that the modified monolignols are transported through the living stem and are incorporated in growing lignin polymers within the cell walls and (ii) that the localization pattern was essentially identical to that of the cross-section approach. This type of experiment has the merit of being performed in a real living plant / live cell approach allowing longer experiments and more in-depth studies, but requires greater quantities of chemical reporter. Finally, the BLISS protocol was also used with flax plant seedlings, representing a real living plant model in which the chemical reporters must be absorbed through the roots before being transported up the stem. While this model has the clear advantage of being performed in living plants, in practice, it is limited to young seedlings and is not really suitable for investigating lignification dynamics in older plants for practical reasons (long incubation time, elevated quantity of chemical reporters). Nevertheless, these three experiment designs are complementary and all have their pros and cons with regards to practical aspects and biological significance depending on the type of biological question to be answered.
Developed to study the lignification dynamics in flax, our protocol is highly adaptable, not only in terms of biological experiment design but also in terms of its application to other plant species and organs/tissues. For instance, BLISS can easily be transferred to the Arabidopsis or the Populus genera that are more amenable to studies with knock-out or knock-down mutants for various genes. In principle, dual labeling studies with our approach can also be extended to other biomolecules by using two distinct modified precursors of plant cell wall polymers - including all three main monolignols or their metabolic precursors as well as various monosaccharides that constitute the polysaccharide matrix. Since its inception, bioorthogonal chemistry has indeed been mainly developed to investigate glycans/polysaccharides through metabolic oligosaccharide engineering (MOE)4,5,17,28, but surprisingly there have been only very few applications to plant biology so far7,8,9,10,11,12. In terms of compatibility of the reactions, the study of lignin was indeed a complex case to solve as both chemical reporters are incorporated into the same reticulated polymer. The possibility of unlabeled HAZ-GALK cross-link formation was the major issue to overcome due to the spatial proximity of GALK and HAZ units within the 3D structure of lignin23, a limitation that may not be present if the two chemical reporters are not incorporated in the same type of biopolymer or in the same spatial region of any given cell.
In a wider scope the BLISS methodology could essentially be applied to any two-color fluorescence imaging study in bacterial or animal models using two distinct chemical reporters bearing an azide and terminal alkyne tag, respectively.

Over the past two decades, the chemical reporter strategy has emerged as a powerful two-step methodology to investigate the dynamics and functions of non-genetically encoded biomolecules.1,2,3 In this strategy a synthetic analogue of the biomolecule of interest with a small modulation &#8211; the chemical reporter &#8211; is first metabolized by the living organism, and then a chemical probe (e.g., a fluorophore for fluorescence confocal microscopy imaging) is covalently linked to the incorporated reporter via bioorthogonal click chemistry. The probe must react rapidly and specifically with the introduced chemical modification while being inert to any biomolecules present in the living system. In many ways, this method overcomes the limitations of common bioconjugation techniques through the use of highly specific click chemistry ligations thereby providing the opportunity to track metabolites or biomacromolecules that were previously inaccessible in living systems4,5,6.
Despite the fast-growing popularity of this powerful method in bacterial and animal cells, reports describing its use in plant biology are surprisingly few and far between7,8,9,10,11,12. We were particularly interested in applying this strategy in plants to study the formation of lignin, one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass.13,14 Lignin is a phenolic polymer that plays a vital structural and protective role in the development and life of higher plants.
It is generally composed of three 4-hydroxyphenylpropanoid moieties: H (p-hydroxyphenyl), G (guaiacyl) and S (syringyl) units respectively derived from three &#39;monolignols&#39; (p-coumaryl, coniferyl and sinapyl alcohols) that are synthesized via the phenylpropanoid pathway in the cytoplasm of the cell (Figure 1). After being exported to the cell wall, monolignols are oxidized to radicals by peroxidases or laccases after which they undergo purely chemical radical coupling reactions to polymerize to lignin polymers, a process termed lignification.15,16 Although lignins strongly impact the industrial valorization of many plant-derived products, the scientific community still has a long way to go to decipher the intricate mechanisms regulating lignification.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56947/56947fig1.jpg" /> 
Figure 1: The lignification process in plant cells. Monolignols are biosynthesized from phenylalanine in the cytosol. After being exported to the cell wall, monolignols are oxidized to radicals by peroxidases or laccases after which they undergo purely chemical radical coupling reactions to polymerize to lignin polymers, a process termed lignification. <a href="//cloudflare.jove.com/files/ftp_upload/56947/56947fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although reports on the use of bioorthogonal reactions for glycan analysis are numerous,2,3,17 their application examples to other types of biomolecules are fewer. The use of bioorthogonal chemistry for lignin bioimaging purposes was only recently pioneered by Tobimatsu et al.8 in Arabidopsis thaliana to provide information about the incorporation of coniferyl alcohol surrogates into the lignin polymer where it forms the G units,8,9 thus demonstrating the proof of concept that chemical reporter strategies are applicable in this context. The use of CuAAC was also illustrated using a different coniferyl alcohol derivative a few month later by Bukowski et al.9 However, lignin also contains H and S units and a deeper understanding of the lignification process requires more knowledge about how all of the monolignols are incorporated into the polymer and what factors may control its composition. New advances in this field currently depend on the development of efficient methodologies to track multiple chemical reporters simultaneously in living systems. Even though a few articles on glycans have laid the groundwork in recent years18,19,20,21,22, dual labeling approaches remain a major challenge in bioorthogonal chemistry. If a reproducible single-labeling click protocol is hard to develop, then dual labeling approaches that require the optimization in tandem of two mutually compatible bioorthogonal reactions on two separate chemical reporters are even harder. The few examples that pioneered this aspect used a combination of strain-promoted azide-alkyne cycloaddition (SPAAC) and alkene-tetrazine inverse electronic demand Diels-Alder (DAinv) reactions to study glycans in animal cells. However, we thought that the bioorthogonality of the DAinv reaction might not be guaranteed in this application owing to the structural features of lignin (which consists of electron-rich substituted cinnamyl-type monomers that can react with electron-poor dienes such as the tetrazine probes used in DAinv reactions) and that this may generate non-specific labeling. In addition, the DAinv reaction requires chemical handles that are synthetically difficult to access, as well as being bulky and lipophilic thereby raising the possibility that the rate of incorporation, transport and/or localization of the chemical reporter in vivo may be affected. As we considered that the latter aspect was particularly relevant in the case of a click chemistry approach for studying lignification, we chose a different direction and developed a Bioorthogonal Ligation Imaging Sequential Strategy (BLISS) using a combination of Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) and Copper Catalysed Azide-Alkyne Cycloaddition (CuAAC) in vivo.23
These two reactions are indeed the two main bioorthogonal click reactions that have been used to date, and more particularly in the few examples of lignin imaging that were recently published.8,9 Our dual labeling strategy enables the use of an azide moiety on one monolignol reporter and a terminal alkyne on the other, two chemical handles that are i) unreactive towards biologically relevant structures and ii) very small in size (Figure 2). As a result, the impact of these synthetic modifications on the physicochemical properties of the biomolecule under study is minimized thus reducing possible discrepancies between the unnatural and natural monolignol substrates in terms of transport and metabolization rates during the metabolic incorporation step. Although the combination of SPAAC and CuAAC seems very intuitive at first glance, it is to our knowledge only the second example of dual marking using this strategy and the first application on structures other than glycans.12,23
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56947/56947fig2.jpg" /> 
Figure 2: BLISS dual labeling strategy. Chemical reporters HAZ and GALK are tagged analogs of the native H and G monolignols. They are first incorporated into the growing lignin polymers of the cell walls by exogenous feeding (step 1). Cyclooctyne- and azide-functionalized fluorescent probes are then sequentially ligated to the incorporated reporters by bioorthogonal click chemistry: the SPAAC reaction (step 2) is highly specific of HAZ units and is followed by a CuAAC reaction (step 3) that is specific of GALK units (step 3), thus allowing the specific localization of both reporters independently in the same sample. <a href="//cloudflare.jove.com/files/ftp_upload/56947/56947fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
We firstly designed and validated the azide-tagged monolignol reporter HAZ (surrogate of p-coumaryl alcohol and precursor of lignin H units), and then devised the BLISS dual labeling strategy in which it is used in tandem with the previously reported alkyne-tagged GALK,9 (surrogate of coniferyl alcohol and precursor of lignin G units). In this reproducible protocol developed and tested in flax, an economically important plant species, the dual metabolic incorporation of HAZ and GALK into lignin is first achieved prior to sequential SPAAC/CuAAC labeling. Here, tagged HAZ units are first specifically labeled via the SPAAC ligation of a cyclooctyne-functionalized fluorophore, followed by CuAAC-mediated ligation of a second fluorescent probe on tagged GALK units. This method was used to investigate the dynamics of lignification processes within plant cell walls and can be applied in vivo to stem cross-sections, living stems as well as seedlings of different plant species.

<table><tbody><tr><td>(E)-4-(3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)prop-1-en-1-yl)phenol (&lt;strong&gt;H&lt;sub&gt;AZ&lt;/sub&gt;&lt;/strong&gt;)</td><td></td><td></td><td>Synthesized as in Lion &lt;em&gt;et al. Cell Chem. Biol. 2017, 24, 3, 326-338&lt;/em&gt;</td></tr><tr><td>(&lt;em&gt;E&lt;/em&gt;)-4-hydroxy-3-propargyloxycinnamyl alcohol (&lt;strong&gt;G&lt;sub&gt;ALK&lt;/sub&gt;&lt;/strong&gt;)</td><td></td><td></td><td>Synthesized as in Lion &lt;em&gt;et al. Cell Chem. Biol. 2017, 24, 3, 326-338&lt;/em&gt;</td></tr><tr><td>2% sodium hypochlorite</td><td></td><td></td><td></td></tr><tr><td>20 cm high glass tube</td><td></td><td></td><td></td></tr><tr><td>250 mL Schott glass bottle</td><td></td><td></td><td></td></tr><tr><td>48-well Plate</td><td></td><td></td><td></td></tr><tr><td>5/6-TAMRA-PEG&lt;sub&gt;3&lt;/sub&gt;-Azide</td><td>Jena Bioscience</td><td>CLK-AZ109-1</td><td></td></tr><tr><td>Aluminium foil</td><td></td><td></td><td></td></tr><tr><td>Cheese cloth</td><td></td><td></td><td></td></tr><tr><td>Compost containing clay</td><td></td><td></td><td></td></tr><tr><td>Coniferyl alcohol (&lt;strong&gt;G&lt;/strong&gt;)</td><td>Sigma Aldrich</td><td>MFCD00002922</td><td></td></tr><tr><td>Copper (II) sulfate pentahydrate</td><td></td><td></td><td></td></tr><tr><td>DBCO-PEG&lt;sub&gt;4&lt;/sub&gt;-5/6-Carboxyrhodamine 110</td><td>Jena Bioscience</td><td>CLK-A127-1</td><td></td></tr><tr><td>Milli-Q Ultrapure water</td><td></td><td></td><td></td></tr><tr><td>Eppendorf 1,5 mL</td><td></td><td></td><td></td></tr><tr><td>EtOH</td><td></td><td></td><td></td></tr><tr><td>Flax seeds (&lt;em&gt;L. usitatissimum&lt;/em&gt; L.)</td><td></td><td></td><td></td></tr><tr><td>Fluoromount-G&amp;trade; Slide Mounting Medium</td><td>Electron Microscopy Sciences</td><td>17984-25</td><td></td></tr><tr><td>Glass coverslip</td><td></td><td></td><td></td></tr><tr><td>Glass microscope slide</td><td></td><td></td><td></td></tr><tr><td>Growth chamber</td><td>CLF-Plant Climatics</td><td></td><td>For 2-week-old plants culture</td></tr><tr><td>Growth chamber</td><td>Angelantoni Life Sciences</td><td></td><td>For 2-month-old plants culture</td></tr><tr><td>Magenta plant culture box</td><td></td><td></td><td>For 2-week-old seedling culture</td></tr><tr><td>Methanol</td><td></td><td></td><td>Toxic (SGH02, SGH06, SGH08), work with gloves under a hood</td></tr><tr><td>Micropipette</td><td></td><td></td><td></td></tr><tr><td>Nail polish</td><td></td><td></td><td></td></tr><tr><td>Nikon A1R confocal microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Orbital shaker</td><td></td><td></td><td></td></tr><tr><td>Parafilm</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-Coumaryl alcohol (&lt;strong&gt;H&lt;/strong&gt;)</td><td>Carbosynth</td><td>FC145653</td><td></td></tr><tr><td>Plastic cap</td><td></td><td></td><td></td></tr><tr><td>Plastic pipette</td><td></td><td></td><td></td></tr><tr><td>Plastic pot</td><td></td><td></td><td>For 2-month-old plants culture</td></tr><tr><td>Razor blade</td><td></td><td></td><td></td></tr><tr><td>Rubber band</td><td></td><td></td><td></td></tr><tr><td>Sodium Ascorbate</td><td></td><td></td><td></td></tr><tr><td>Sterile clamp</td><td></td><td></td><td></td></tr><tr><td>Vertical support</td><td></td><td></td><td></td></tr><tr><td>Vortex</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents for liquid and solid &amp;frac12; MS medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>KNO&lt;sub&gt;3&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>MnSO&lt;sub&gt;4&lt;/sub&gt;.H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>H&lt;sub&gt;3&lt;/sub&gt;BO&lt;sub&gt;3&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>KI</td><td></td><td></td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;.5H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>CoCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;EDTA.2H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>FeSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>Thiamine.HCl</td><td></td><td></td><td></td></tr><tr><td>Pyridoxine.HCl</td><td></td><td></td><td></td></tr><tr><td>Glycine</td><td></td><td></td><td></td></tr><tr><td>Nicotinic acid</td><td></td><td></td><td></td></tr><tr><td>Myo-inositol</td><td></td><td></td><td></td></tr><tr><td>Saccharose</td><td></td><td></td><td></td></tr><tr><td>MES hydrate</td><td></td><td></td><td></td></tr><tr><td>Agar</td><td></td><td></td><td></td></tr></tbody></table>

visualizing lignification dynamics in plants with click chemistry: dual labeling is bliss!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital structural and protective role in the development and life of higher plants. Although the intricate mechanisms regulating lignification processes in vivo strongly impact the industrial valorization of many plant-derived products, the scientific community still has a long way to go to decipher them. In a simple three-step workflow, the dual labeling protocol presented herein enables bioimaging studies of actively lignifying zones of plant tissues. The first step consists in the metabolic incorporation of two independent chemical reporters, surrogates of the two native monolignols that give rise to lignin H- and G-units. After incorporation into growing lignin polymers, each reporter is then specifically labeled with its own fluorescent probe via a sequential combination of bioorthogonal SPAAC/CuAAC click reactions. Combined with lignin autofluorescence, this approach leads to the generation of three-color localization maps of lignin within plant cell walls by confocal fluorescence microscopy and provides precise spatial information on the presence or absence of active lignification machinery at the scale of plant tissues, cells and different cell wall layers.

By using the presented BLISS protocol involving synthetic monolignol analogs and bioorthogonal click chemistry, it is possible to visualize the dynamics of the lignification process in living plants. Unlike established techniques for lignin visualization (such as histochemical staining, immunolocalization or autofluorescence), this &#39;double click&#39; protocol exclusively targets the lignin produced de novo during the metabolic incorporation step and differentiates it from the preexisting lignin of the sample with triple-channel cellular imaging by confocal fluorescence microscopy (Figure 3). HAZ-units are detected using the excitation and emission wavelengths of the DBCO-PEG4-5/6-carboxyrhodamine 110 that is specifically clicked onto azide functions of incorporated HAZ molecules during the SPAAC step (&#955;ex 501 nm /&#955;em 526 nm, green channel), whereas GALK-units are similarly detected at the characteristic wavelengths of the azidefluor 545 probe that is specifically clicked onto the incorporated terminal alkyne tags of GALK during the CuAAC step (&#955;ex 546 nm /&#955;em 565 nm, red channel); the third channel corresponds to pre-existing lignin, which is detected using its intrinsic autofluorescence at 405 nm (blue channel). This approach leads to the generation of three-color localization maps of lignin within plant cell walls that provide precise spatial information on the presence or absence of active lignification machinery between different tissues of an organ (Figure 3A), between different cell types within the same tissue (Figure 3B-C) and within different wall layers of the same cell (Figure 3D). In other words, this methodology allows us to highlight and specifically localize the &#39;active&#39; lignification sites (HAZ and GALK channels) by differentiating them from zones where lignin was formed at an earlier stage of plant development (autofluorescence channel). In addition, the sensitivity of the technique is dramatically enhanced when compared to autofluorescence, and much smaller amounts of freshly synthesized lignin can be detected (Figure 3B).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56947/56947fig3.jpg" /> 
Figure 3: Imaging of incorporated monolignol chemical reporters in Flax Stems. (A) Half of a hand-made transversal section of a flax stem, reconstructed picture. (B) Close-up of the first layers of differentiating xylem. Left panel: lignin autofluorescence (blue, 405 nm), middle panel: merged lignin autofluorescence (blue) and HAZ fluorescence (green, 526 nm) channels, right panel: merged lignin autofluorescence (blue) and GALK fluorescence (red, 565 nm) channels. The arrow indicates the first labeled cell wall from the cambium illustrating the increased sensitivity of BLISS compared with autofluorescence. (C) Labeling in different cell types of secondary xylem and (D) close up on secondary xylem cells illustrating the labeling variations in different layers of the same cell wall. Left panel: merged lignin autofluorescence (blue) and HAZ fluorescence (green, 526 nm) channels, middle panel: merged lignin autofluorescence (blue) and GALK fluorescence (red, 565 nm) channels, right panel: merged lignin autofluorescence (blue), HAZ (green) and GALK (red) fluorescence channels. HAZ and GALK co-localization is depicted in yellow. <a href="https://cloudflare.jove.com/files/ftp_upload/56947/56947fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Chemical reporter strategies such as the BLISS method presented here or the single-labeling procedures previously published by Tobimatsu et al.8 can therefore allow a much finer study than previously accessible methods such as immuno- or histochemical staining while being very simple to implement. For instance, Figure 4 illustrates that the signal intensity for HAZ/GALK incorporation in fiber tracheids/vessels (FT) of the flax secondary xylem is very high in the first few cell walls from the cambium but progressively decreases in older cells. This profile is opposite to that of the autofluorescence and indicates that maximum lignification is very rapidly reached in the first two to three FT cell layers from the cambium. In contrast, ray (R) cells appear to continue lignification to much later stages of their development as HAZ/GALK incorporation is much more constant in the walls of all cells from the cambium to the pith. It is not surprising that FT and R cell types display contrasted lignification dynamics, since these cell types have different biological roles with associated differences in their developmental program. FTs are indeed elongated tubes that mature and die rapidly, leaving dead empty cells with thick lignified walls that play essential roles in both mechanical support and vertical transport of water and minerals, whereas the narrow rows of R cells that compose the xylem rays are alive in their mature and functional state without having thick lignified walls.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56947/56947fig4.jpg" /> 
Figure 4: Cell-specific monolignol reporter incorporation in flax xylem. Bright-field confocal microscopy view (A) of part of a freehand cross-section from a flax stem. Pink (ray parenchyma cells, R) and yellow (fiber tracheid cells, FT) arrows indicate vectors spanning from the cambial zone toward the pith and scanned for lignin autofluorescence at 405 nm (B), HAZ fluorescence at 526 nm (C) and GALK fluorescence at 565 nm (D). (E) View of the secondary xylem. Left panel: merged lignin autofluorescence (blue), HAZ (green), and GALK (red) fluorescence channels. HAZ and GALK co-localization is depicted in yellow. Right panel: schematic illustration of the secondary xylem structure in the flax stem. V, vessel; FT, fiber tracheid; R, ray parenchyma cell. This figure has been modified from Lion et al.23 <a href="https://cloudflare.jove.com/files/ftp_upload/56947/56947fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In addition, the use of two distinct chemical reporters corresponding to H- and G-units with two bioorthogonal reactions in the BLISS protocol allows more quantitative results to be obtained. Multiplexed labeling strategies such as BLISS could provide additional insights on the control of monolignol incorporation ratios in various conditions and could contribute to deciphering the elusive lignification process. If the relative percentages of H, G and S monolignols in lignins are indeed very variable according to the species, tissue, age or environmental conditions of the plant, the intricate mechanisms that regulate this composition are still not completely understood. The dual labeling technology represents a powerful way of investigating some of the parameters that regulate lignin composition. For example, varying the HAZ:GALK ratio with BLISS allowed us to demonstrate that lignin composition in secondary xylem tissues is directly dependent upon monolignol availability within the cell walls, rather than on peroxidase/laccase specificity (Figure 5).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56947/56947fig5.jpg" /> 
Figure 5: Effect of Incubating Flax Stem Sections with Different Percentage Ratios of HAZ and GALK. HAZ:GALK% ratios are given above each column of figures. Top: merged HAZ and GALK channels. Bottom: histograms indicating average fluorescence intensity of HAZ and GALK for the different% ratios. Values are expressed as mean of the mean fluorescence intensity in gray levels &#177; SD. Scale bar = 100 &#181;m. This figure has been modified from Lion et al.23 <a href="https://cloudflare.jove.com/files/ftp_upload/56947/56947fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Flax is grown for its bast fibers (BF) that are used to manufacture textiles, luxury papers or environmentally-friendly composite materials. An important aspect of their industrial valorization is that they contain very low levels of lignin in their cell walls, which are characterized by an extremely thick S2/G secondary layer19,24. BLISS can highlight lignification dynamics differences between the different layers of the same cell wall. Figure 6A shows that HAZ and GALK reporter incorporation are limited to the cell corners and middle lamella/primary cell wall of some but not all bast fibers. The total absence of lignification in the thick bast fiber secondary cell wall layer even when monolignol chemical reporters are exogenously supplied reveals that their hypolignified state arises from the absence of a molecular environment suitable for enzymatically-mediated oxidation and incorporation of monolignols into a growing polymer chain, and that it is not just due to transcriptional regulation of monolignol biosynthesis genes as previously reported25. This observation also correlates well with the fact that flax peroxidase genes are up-regulated in the outer stem tissues of the flax chemical lbf1 mutant that possesses lignified bast fibers26.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56947/56947fig6.jpg" /> 
Figure 6: BLISS highlights cell wall substructure- or layer-specific differences. (A) Left panel: bright-field image of a flax stem section. The circle indicates a fiber bundle. Right panel: close up on bast fibers. Merged HAZ and GALK channels (with and without bright-field) showing that lignification is limited to cell corners (<img alt="Equation" src="/files/ftp_upload/56947/56947eq1.jpg" />) and middle lamella/primary cell wall of some bast fibers. BF, bast fiber; Par, parenchyma cell; M, middle lamella; P, primary cell wall; S1, first layer of secondary cell wall; S2/G, secondary layer/gelatinous layer of secondary cell wall. (B) 2D slice and 3D reconstruction of confocal z-stack zoom of flax root endodermis region. The Casparian strip (<img alt="Equation" src="/files/ftp_upload/56947/56947eq1.jpg" />) only displays autofluorescence and does not incorporate HAZ or GALK. The associated fluorogram shows a GALK/HAZ anti-correlation: high green fluorescence is associated to low red signal (cortex) and vice-versa (endodermis). Pericycle (P), Endodermis (E), Cortex (C). <a href="https://cloudflare.jove.com/files/ftp_upload/56947/56947fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Finally, this two-color methodology can also provide valuable information on the &#39;lignification state&#39; of cell wall substructures at various developmental stages and in plant organs other than the stem. For example, the endodermis of flax roots is characterized by the existence of a Casparian band in its radial and transverse cell walls during the early stages of development. This band of hydrophobic biopolymer, made of suberin and/or lignin, prevents water and solutes taken up by the root from entering passively through the apoplast and forces them to pass through the plasma membrane via a symplastic route thereby contributing to the selective uptake capacities of plant roots. When applied to flax roots, our strategy showed that HAZ and GALK are incorporated in the tangential walls of endodermal cells as well as in parts of the radial walls where the Casparian band is absent (Figure 6B). However, total absence of monolignol reporter incorporation in the Casparian band itself indicates that it is mature at this developmental stage while the other walls are still the site of further biopolymer deposition. The reconstructed 3D view of a z-stack clearly brings the Casparian band to light. Interestingly, the walls of some cortical cells adjacent to the endodermis also proved capable of incorporating HAZ preferentially (thereby indicating the presence of lignin/suberin in the flax root cortex) but not GALK. Co-localization analysis showed an anti-correlation between HAZ and GALK, which suggest the existence of cell-specific wall structure/enzymatic machinery in these two adjacent cell types.
Supplemental Table 1: Preparation of solid &#189; MS Medium <a href="http://cloudflare.jove.com/files/ftp_upload/56947/renamed_f17ea.xls">Please click here to download this file.</a>
Supplemental Table 2: Preparation of chemical reporter stock solutions <a href="http://cloudflare.jove.com/files/ftp_upload/56947/Table_2_-_Preparation_of_chemical_reporter_stock_solutions.xls">Please click here to download this file.</a>

Watch this Scientific Journal Video about Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS! at JoVE.com

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

BLISS, a dual labeling protocol for studying lignification dynamics, was developed. Using synthetic monolignol reporters and a sequential combination of SPAAC and CuAAC bioorthogonal click reactions, this methodology paves the way to in-depth analysis of the factors that regulate the biogenesis of lignins in planta.

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital ...

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11.9K Views.  Université de Lille. BLISS, a dual labeling protocol for studying lignification dynamics, was developed. Using synthetic monolignol reporters and a sequential combination of SPAAC and CuAAC bioorthogonal click reactions, this methodology paves the way to in-depth analysis of the factors that regulate the biogenesis of lignins in planta.

Video: Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The analysis does not usually require extensive sample preparation; structural and chemical information can be obtained without labeling. However, each Raman image contains thousands of spectra; this raises difficulties when extracting hidden information, especially for components with similar chemical structures. This work introduces a multivariate analysis to address this issue. The protocol establishes a general method to visualize the main components, including lignin, cellulose, and hemicellulose within the plant cell wall. In this protocol, procedures for sample preparation, spectral acquisition, and data processing are described. It is highly dependent upon operator skill at sample preparation and data analysis. By using this approach, a Raman investigation can be performed by a non-specialist user to acquire high-quality data and meaningful results for plant cell wall analysis.

This protocol aims to present a general method to visualize lignin, cellulose, and hemicellulose in plant cell walls using Raman imaging and multivariate analysis.

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The ...

Biochemistry

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

Biology

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In general, for observations over a long period, the technique requires transformation of a fluorescent protein; however, for most systems, genetic transformation is either time-consuming or technically unavailable. This manuscript presents a protocol for 3-D time-lapse imaging of cell wall dynamics over a 3 day period using calcofluor dye (which stains cellulose in the plant cell wall), developed in the moss Physcomitrium patens. The calcofluor dye signal from the cell wall is stable and can last for 1 week without obvious decay. Using this method, it has been shown that the detachment of cells in ggb mutants (in which the protein geranylgeranyltransferase-I beta subunit is knocked out) is caused by unregulated cell expansion and cell wall integrity defects. Moreover, the patterns of calcofluor staining change over time; less intensely stained regions correlate with the future cell expansion/branching sites in the wild type. This method can be applied to many other systems that contain cell walls and that can be stained by calcofluor.

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

This manuscript presents a detailed protocol to image the 3-D cell wall dynamics of living moss tissue, allowing the visualization of the detachment of cell walls in ggb mutants and thickening cell wall patterns in the wild type during development over a long period.

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In ...

Developmental Biology

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Expansion Microscopy for Visualizing Intracellular Sialylated Glycans via MOE

Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes within cells through click and bioorthogonal chemistry. Metabolic oligosaccharide engineering (MOE) has attracted considerable interest due to the essential role of glycosylation in numerous biological processes that involve molecular recognition and its impact on pathologies ranging from cancer to genetic disorders to viral and bacterial infections.
Although MOE is better known for the detection of cell surface glycoconjugates, it is also a very important methodology for the study of intracellular glycans in physiological and pathological contexts. Such studies greatly benefit from high spatial resolution. However, super-resolution microscopy is not readily available in most laboratories and poses challenges for daily implementation. Expansion microscopy is a recent alternative that enhances the resolution of microscopy by physically enlarging biological specimens labeled with fluorescent markers. By embedding the sample in a swellable gel and causing it to expand isotropically through chemical treatment, subcellular structures can be visualized with enhanced precision and resolution without the need for super-resolution techniques.
In this work, we illustrate the capacity of expansion microscopy to visualize intracellular sialylated glycans through the combined use of MOE and click chemistry. Specifically, we propose a procedure for bioorthogonal labeling and expansion microscopy that employs a reporter targeting sialylation, which may be associated with immunofluorescence for co-localization studies. This protocol enables localization studies of sialoconjugate biosynthesis, intracellular trafficking, and recycling.

Here, we propose a simple protocol combining metabolic oligosaccharide engineering, click chemistry, and expansion microscopy that allows bioimaging of intracellular sialylated N-glycoproteins with improved resolution using routine microscopy equipment.

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes ...

Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs that mimic the structure of natural biomolecules. Cells utilize analogs in their endogenous biochemical processes, producing compounds that are labeled. The label allows for the incorporation of detection and affinity tags, which can then be used to elucidate metabolic pathways using other biochemical analytical techniques, such as SDS-PAGE and NMR.
This video introduces the concepts of metabolic labeling and show two general procedures.&#160; The first uses isotopic-labeling, to characterize the phosphorylation of a protein. The second covers a photoreactive labeling to characterize protein-protein interaction within a Also three applications of metabolic labeling are presented: labeling plant material, labeling RNA to measure kinetics and labeling glycans in developing embryos.

<div class="chapter_text" id="script_0">
Metabolic labeling is used to investigate the machinery of a cell. This is accomplished using chemical analogs to probe the biochemical transformations and modifications that occur. This video will show the principles of metabolic labeling, typical isotopic and photoreactive labeling procedures, and some applications.
</div>
<div class="chapter_text" id="script_1">
Metabolic labeling can be conducted using a number of strategies. Here we will describe isotopic, photoreactive, and bio-orthogonal labeling.
Isotopic labeling is performed using structural analogs that are chemically identical to their natural counterparts, but have uncommon isotopes incorporated into their structure. In this L-lysine analog the carbon and nitrogen atoms are replaced with carbon-13 and nitrogen-15. Cells cultured in the presence of isotopic analogs will incorporate them into their biochemical structures. Metabolites are collected from the cells and purified for analysis. Samples with stable isotopes are analyzed using techniques such as mass spectrometry or NMR spectroscopy. Samples with radioactive labels are analyzed using liquid scintillation counting and x-ray films, which will be demonstrated in the isotopic labeling protocol.
Photoreactive labels are functional groups incorporated into proteins, which are stable until exposed to ultraviolet light. The functional group forms a reactive radical, which binds to the nearest protein. A common example, L-photo-leucine, contains a diazirine ring, which is a photoreactive crosslinker. In contrast to isotopic labeling, there is some chemical dissimilarity between photo-reactive chemical analogs and their natural counterparts. Cells may preferentially incorporate natural compounds over analogs. Therefore, it is important to perform photo-reactive labeling in medium free of the compound being mimicked. Once exposed to ultraviolet radiation, the photo-reactive groups in a labeled protein become unstable and highly reactive, causing it to cross-link with interacting proteins, creating a protein complex. Cross-linked complexes, act as snapshots that can then be analyzed using SDS-PAGE and mass spectrometry methods. This provides insights into what reactions are occurring in the metabolic pathway by identifying reaction species and how they interact by determining binding sites.
Bioorthogonal labeling strategies utilize analogs with small functional groups that have little to no reactivity with natural biomolecules. For example, azides are small functional groups, whose reactivity is said to be orthogonal to biochemical reactions. In the Staudinger ligation, a phosphine group attacks the azido group. This yields a transition state that intramolecularly reacts with a nearby ester, resulting in an amine-bonded ligand. Bioorthogonal functional groups incorporated into biomolecules can be ligated with detection tags such as fluorescent functional groups, and affinity tags such as antigens.
Now that some concepts and strategies for metabolic labeling have been discussed, let&#39;s look at the process in the laboratory.
</div>
<div class="chapter_text" id="script_2">
The first step in a metabolic labeling experiment is to collect the protein of interest. To do this, cells are grown on a plate, and an expression method is used to promote synthesis of the desired protein. In this example, leucine-rich repeat kinases, or LRRK, are expressed. Disodium phosphate, containing radioactive phosphorus-32, is used as the analog. Proper measures must be taken to protect against ionizing radiation. This includes setting up a work area, wearing proper protective equipment, and checking for radioactive contamination. Once safety measures have been taken, the medium containing the isotopic analogs is prepared. The medium from the culture is removed and, replaced with one containing the isotopic chemical analogs and then incubated. Following incubation, the cells are lysed. The lysate is collected and purified.
After purification, proteins are resolved using SDS-PAGE and then transferred to a PVDF membrane. Autoradiography is performed by exposing the membrane to x-ray film and measured using a phosphor imager. Western blotting is used to measure relative protein levels in the PVDF membrane. In this example the phosphorylation levels of leucine-rich repeat kinases synthesized in 293T cells were measured. The autoradiogram shows how much phosphorous was incorporated into the protein. Western blotting elucidates the levels of the LRRK proteins. Image analysis software is used to obtain quantitative data of phosphorylation levels of the proteins.
</div>
<div class="chapter_text" id="script_3">
In this next procedure, photoreactive labeling is demonstrated. First, the cells are prepared and cultured. The photoreactive analog is added to the cells at the mid-log phase and incubated. In this procedure p-benzoylphenylalanine is used. Samples are collected over intervals and put on ice. The samples are then exposed to obtain snapshots of the biochemical pathways over time. The proteins of interest are then purified and resolved using SDS-PAGE.
A photoreactive labeling strategy was used to identify compounds that interact with the protein of interest. Immunodetection with Western blotting shows protein bands that indicate higher molecular weight proteins are present in the irradiated samples. These are from cross-linking due to protein-protein interaction occurring during the UV irradiation.
</div>
<div class="chapter_text" id="script_4">
Now that we&#39;ve reviewed metabolic labeling procedures, let&#39;s look at some of the ways the process is used.
Metabolic labeling concepts can be extended to multicellular organisms. Plants are grown in a sealed environment, rich in stable isotopes to produced labeled plant material. Carbon dioxide containing carbon-13 is added to the enclosure, while nitrogen-15 rich fertilizer is used. The resulting harvested plant material can help answer questions about carbon and nitrogen cycling from the ecosystem.
Labeling enables the separation of newly synthesized RNA from older RNA. By changing the initial concentration of analog, the kinetics of new RNA synthesis can be determined. The results show that the concentration of 4-thiouridine affects how much new RNA is transcribed. Additionally, incorporation rates of the label into RNA can be directly quantified with a spectrophotometer.
Using biorthogonal click chemistry, glycans in a zebra fish embryo can be labeled. The eggs are injected with a labeling compound that results in alkyne labels on the glycans. The glycans in the larvae are then ligated to a dye compound at the desired development stage. The glycans in the embryos are then imaged. Glycans produced at different time points can be identified by labeling using different colors at different stages of embryo development.
</div>
<div class="chapter_text" id="script_5">
You&#39;ve just watched JoVE&#39;s video on metabolic labeling. This video described the concepts behind metabolic labeling and their strategies, went over two general procedures, and covered some of the uses of the techniques.
Thanks for watching!
</div>

Isotopic, Photoreactive and Bioorthogonal Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs ...

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Wiesner Staining for Visualizing Lignin: A Technique to Stain Lignin Deposits Present in Stems of Arabidopsis Thaliana

Source: Pradhan Mitra, P. et al., <a href="https://www.jove.com/v/51381">Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements.</a> J. Vis. Exp. (2014).
In this video, we describe the Wiesner staining technique for staining the lignin present in specialized plant cells. This staining technique helps to visualize the lignified cell walls in Arabidopsis thaliana stems and helps to quantify the lignification in the cell walls of different types of cells.

Source: Pradhan Mitra, P. et al., Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements. J. Vis. Exp. (2014).
In this video, we ...

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Biomolecule staining

Maule Staining for Lignin: A Technique to Characterize Lignin Distribution in Arabidopsis Thaliana Stem Sections

Source: Pradhan Mitra, P. et al., <a href="https://www.jove.com/v/51381">Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements.</a> J. Vis. Exp. (2014).
In this video, we describe the Maule staining technique for staining syringyl lignin present in the specialized plant cells. This staining technique helps to characterize the lignin distribution in the xylem and interfascicular fibers.

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

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Chapters in this video

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Label-free in situ Imaging of Lignification in Plant Cell Walls

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy

Metabolic Labeling

Wiesner Staining for Visualizing Lignin: A Technique to Stain Lignin Deposits Present in Stems of Arabidopsis Thaliana

Maule Staining for Lignin: A Technique to Characterize Lignin Distribution in Arabidopsis Thaliana Stem Sections