Click & Sea: using bioorthogonal click chemistry to visualise seaweed cell walls

Abstract Objectives The study of seaweed cell walls, including their metabolism and composition, is crucial to monitor and understand their adaptation to climate change. Microscopy-based techniques that facilitate studies of seaweed cell walls in situ, including staining and immunolabelling, exist but have significant limitations, including that only a few monoclonal antibodies have been developed towards seaweed cell wall components. Furthermore, not all seaweed cell wall components have been fully described. This makes in situ studies focussed on the metabolism of seaweed cell walls particularly challenging. Here, we present a method for labelling seaweed cell walls by incorporating chemical reporters in muro, followed by their association with fluorophores by click chemistry. Methods Two different species of seaweed, representing different groups, i.e. the red seaweed Phycodrys rubens and the green seaweed Ulva spp. were selected for their abundance on the Irish coast and because they have relatively thin tissues, a feature likely to facilitate uptake and labelling using monosaccharides reporters. We selected three different activated sugars (fucose, galactose and glucose analogues) on the basis that they are major components of seaweed polysaccharides. Small sections of the seaweeds were incubated with the activated sugars, and their uptake and incorporation were visualised by attachment to the fluorescent probe AF488 and imaged using confocal microscopy. Results After incubation with the activated sugars, the seaweed cell walls, i.e. the contours of the cells, and, to a lesser extent, some cell organelles, fluoresced at 517 nm (emission wavelength of AF488), suggesting that the seaweed had incorporated the activated sugars. Fiji software was used to remove nonspecific fluorescence (autofluorescence of the seaweed and non-specific binding of the fluorochrome), with the final images suggesting good and specific incorporation of the fluorochrome by the seaweeds. More interestingly, the fluorescence was primarily associated with the cell walls, implying that the activated sugars were predominantly incorporated into cell wall components (most likely either polysaccharides or glycoproteins). Conclusion To date, bioorthogonal click chemistry has not been applied to seaweed but represents a useful tool for phycologists to better understand seaweed cell wall composition and dynamics, for example, throughout the seaweed life cycle or in the face of stresses (biotic or abiotic), including those resulting from climate change.