The overall goal of this dual labeling methodology is to observe actively lignifying zones of plant tissues. In this workflow, the chemical reporter strategy is applied using two distinct chemical reporters in a sequential combination of bio-orthogonal click chemistry reactions. This method can help decipher key questions in the plant biology field such as determining what factors regulate the spatial or temporal formation of lignin within plants and walls.
The main advantage of this technique is that it exclusively targets the lignin that is produced during metabolic Incorporation. It differentiates it from any preexisting lignin in the sample. Although we originally developed this method to study lignification flags, it could be used with other species such as arabidopsis, poplar, or indeed any other plant that produces lignin.
To present this technique we have Clemence Simon who's a PhD student working at the interface between chemistry and biology and who is using this technique to study lignification dynamics. First, prepare a 300 microliter chemical reporter solution containing ten micromuller of alkine-tagged G reporter. And ten micromuller of azide-tagged H reporter and liquid sterile 1/2 MS medium.
Vortex the solution and transfer it to one well of a 48-well plate with a transfer pipette. Prepare a 300 microliter negative control solution containing ten micromuller of G and ten micromuller of H and liquid sterile 1/2 MS medium. Vortex the solution and transfer it to a second well of the same 48-well plate.
Using a clean razor blade, cut the stem of a two month old flax plant at ten centimeters above soil level. Diligently prepare 50 free hand transversal cross sections of the stem using the razor blade and place them immediately in a 1/2 MS medium. Then, select intact, whole cross sections and randomly distribute ten of them in the well filled with the chemical reporter solution, taking care not to damage the tissue.
The cross sections must be cut firmly and cleanly and the cross section has to be straight. You need a steady hand and a sharp razor blade in order to keep the tissues intact. It's advised to practice beforehand.
After distributing ten intact cross sections in the negative control solution, incubate the plate in continuous light in a growth chamber at 20 degrees Celsius for 20 hours. On the following day, remove the monolignal solution in each well with a micropipette. Add 500 microliters of 1/2 MS medium to each well and stir gently for ten minutes.
Then, remove the rinsing solution with a micropipette. Prepare one milliliter of a strain-promoted azide-alkine cycloaddition solution. Containing five micromuller of cyclooctene-tagged fluorescent dye and liquid sterile 1/2 MS medium.
Then, vortex the solution. After the final washing steps of the metabolic incorporation protocol, remove the 1/2 MS medium and add 300 microliters of the SPAAC solution to each well with a micropipette. Shield the 48-well plate from light by placing it in a box.
Then, gently stir the plate on a mechanical shaker for one hour in the dark at ambient temperature. Following this, wash each well four times with 1/2 MS medium as previously described, while keeping the plate in the dark. Prepare one milliliter of copper catalyzed azide alkine cycloaddition solution containing sodium ascorbate, copper sulfate, and azide-tagged fluorescent dye, and liquid sterile 1/2 MS medium.
Then vortex the solution. After removing the 1/2 MS medium, place the plate in a box and add 300 microliters of the CuAAC solution to each well with a micropipette. Gently stir the plate on a mechanical shaker for one hour in the dark at ambient temperature.
Remove the plate from the mechanical shaker and remove the CuAAC solution from each well with a micropipette. Then, add 500 microliters of 1/2 MS medium and stir the plate on the mechanical shaker for ten minutes in the dark. After washing two more times with 1/2 MS medium, add 500 microliters of a 7:3 methanol and water solution to each well.
Stir the plate on the mechanical shaker for one hour in the dark. Then, remove the solution from the wells. Next, add 500 microliters of 1/2 MS medium to each well and stir in the dark for ten minutes.
After repeating this wash four times, keep the cross sections stored in 1/2 MS medium, protected from light. Place five drops of mounting medium on a glass microscope slide. Then, carefully deposit each chemical reporter-tagged cross section on the slide.
Apply nail polish on two opposite sides of the slide. Cover the slide with a cover slip, taking care to avoid air bubbles. Then, remove excess mounting medium using a paper towel.
Following this, seal the sample with nail polish on all four sides. After the nail varnish is dry, store the slides at four degrees Celsius in the dark until observation under a confocal microscope. Plant sections have a tendency to get crisp when they dry.
Make sure to always keep them in solution and to seal the cover slips with nail polish on all sides. It increases the shelf life of the mounted slides. Place the sample slide on the microscope stage under the objective lens.
Observe and spin the sample to identify the desired region of the plant tissue. Next, select the most fluorescent plane in the Z-axis. Adjust the gain, offset, and laser power to achieve the optimal signal distribution along the whole gray level range for each channel.
Finally, acquire high-quality confocal images. BLISS provides three color localization maps of lignin within plant cell walls, exclusively targets the lignin produced in novo during metabolic incorporation and differentiates it from the preexisting lignin in the sample. It highlights the active lignification sites within different tissues of an organ within different cell types of a tissue, or within different wall layers or substructures of a cell.
The alkine-tagged G and azide-tagged H reporter incorporation profiles from the cambium to the pith within the flax secondary xylem indicate that maximum lignification is rapidly reached in fiber tracheids during development, whereas race cells continue to incorporate lignin at much later stages of their life. Varying the chemical reporter ratio demonstrates that lignin composition in secondary xylem tissues is directly dependent upon monolignal availability within the cell walls. Rather than on peroxidase or laccase specificity during lignin polymerization.
Chemical reporter incorporation is limited to the cell corners and middle lamella primary cell wall of some, but not all bast fibers. In flax roots, the chemical reporters are incorporated in the walls of endodermal cells where the casparian band is absent. For better visualization of tissue architecture, 3D Z stack reconstructions can also be generated.
Following this visual, comprehensive data processing can also be performed. All precautions taken, it is possible to obtain constant results by relative comparison, reading a sample between different samples. It can be done in addition to the localization of active leading of varying areas.
This method is a wonderful tool for plant biologists like myself who study the cell wall. Once it's mastered, it can be done in a few hours. It's very sensitive and it can be applied to a whole range of different in vitro in vivo experimental model systems.
Applying the chemical reporter strategy to lignin paves the way for chemical biologist to explore lignification dynamics and find out what controls this polymerization process, which in many respects, still constitutes uncharted territory.
