Observing Biosynthesis of Lignocellulosic Biomass in situ
The pollen grains of Arabidopsis thaliana produce a fast-growing pollen tube. The cell wall of this pollen tube can model lignocellulosic biomass. This project utilized a fucose analog to label the distribution of fucose in pollen tube cell wall during biosynthesis. Pollen grains were germinated on medium containing this alkyne-modified fucose analog, called FucAl. This allowed pollen grains to incorporate FucAl, rather than fucose, into cell walls. Following tube growth, cell walls containing FucAl were exposed to Alexa 594 and Alexa 647 Azides in a copper-catalyzed “click” reaction. This reaction between the azide and alkyne yields fluorescent product at the FucAl molecule’s location. This allowed us to localize where fucose belongs in the cell wall. These results confirm the success of the click reaction and suggest that fucose may be taken up by the pollen grain during tube elongation. Further studies are needed to shed light on the critical steps of this process. Once the methodology is optimized, it will be expanded to include biomass degradation. This simple biomass model was suitable for effective mapping of polysaccharide distribution during growth, and this project utilized correlative optical and fluorescence microscopy to study fucose delivery and utilization during cell wall biosynthesis.