Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides

Abstract

BackgroundCarbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques.ResultsArabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, l-fucose, and l-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions.ConclusionsOur results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands the molecular toolbox for direct glycan imaging in plants, from three to eight glycan analogs, which enables more extensive future studies of spatiotemporal glycan dynamics in a wide variety of plant tissues and species. We also show, for the first time in metabolic labeling and imaging of plant glycans, the potential of two copper-free click chemistry methods that are bio-orthogonal and lead to more uniform labeling. These improved labeling methods can be generalized and extended to already existing and future click chemistry-enabled monosaccharide analogs in Arabidopsis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0907-0) contains supplementary material, which is available to authorized users.

One, Two, Three: A Bioorthogonal Triple Labelling Strategy for Studying the Dynamics of Plant Cell Wall Formation In Vivo

“…Cell wall formation in plants is ahighly regulated process and lignin deposition is coordinated with the formation of other cell wall polymers.W et herefore investigated whether this triple labelling approach could be used to simultaneously investigate the dynamics of both lignin and non-cellulosic polysaccharide (NCP) polymer deposition in flax stem cell walls.F or this we used the H AZ 4 and S CP 6 monolignol reporters together with peracetylated alkyne-tagged fucose reporter Fuc ALK 8.Fucose (7)ispresent in both the side chains of xyloglucan hemicelluloses and in pectin rhamnogalacturonan Ia nd II (RGI, RGII) motifs, [25,26] and previous reports have shown that Fuc ALK 8 can be successfully used in ab ioorthogonal reaction to label plant NCP cell wall polymers. [4,5] In contrast to the incorporation of monolignol reporters directly into the cell wall, NCP labelling requires that the reporter be metabolized into the cytoplasm and transported to the cell wall by the Golgi apparatus,a nd we therefore reasoned that the entire stem in vivo system might be more appropriate than the cross-section model.…”

“…[11] Since lignin properties are influenced by monomer ratio and the nature of inter-unit bonds,improved knowledge of lignification would contribute to better understanding of plant physiology and factors affecting biomass quality. Although there are various reports of dual labelling methods that combine two biorthogonal reactions both in vitro and in vivo, [6,7,[13][14][15][16][17][18][19] the only instance where three such reactions were used concurrently within the same sample was based on asequence of CuAAC, DAR inv ,a nd Staudinger-Bertozzi ligation in an activity-based protein profiling assay in solution. [13] Theuse of two different cycloaddition reactions is necessary for dual lignin labelling as the monolignol mimics 4 and 5 are incorporated into the same polymer.B ecause of their spatial proximity,u sing consecutive CuAACl igations indeed leads to cross-linking reactions that compete with the desired probe ligation.…”

“…[1][2][3] Many groups have focused on the use of reaction-based fluorescent probes for chemoselective bioimaging in mammalian living systems,but plants have generally received less attention despite the fact that they are complex multicellular eukaryotes.Plant cells are characterized by the presence of an extra-cellular matrix called the cell wall that is made up of different polymers (e.g., cellulose,h emicelluloses,p ectins, lignin). To date,bioorthogonal approaches have been used in plants to investigate polysaccharide and lignin biosynthesis using tagged sugars or monolignols as reporters in in vivo labelling experiments, [4][5][6][7][8][9] as well as auxin. [10] Lignin plays vital roles in plants and is also of considerable industrial importance.Itforms in the cell wall when hydroxycinnamyl alcohols (monolignols) are enzymatically oxidised by peroxidases and/ or laccases and undergo polymerisation ( Figure 1).…”

“…To develop at riple labelling strategy for the three main monolignols,wetested whether the Diels-Alder reaction with inverse electronic demand (DAR inv ) could be used to label the lignin S-unit and successfully combined with SPAACa nd CuAAC. Although there are various reports of dual labelling methods that combine two biorthogonal reactions both in vitro and in vivo, [6,7,[13][14][15][16][17][18][19] the only instance where three such reactions were used concurrently within the same sample was based on asequence of CuAAC, DAR inv ,a nd Staudinger-Bertozzi ligation in an activity-based protein profiling assay in solution. [20] Although three-color imaging using compatible reactions seems intuitively possible,tothe best of our knowledge it has never been applied to track three distinct molecules within asingle living sample.H erein, we demonstrate the feasibility of triple biorthogonal labelling in vivo by sequentially exploiting the major triad of biorthogonal reactions (DAR inv ,S PA AC,a nd CuAAC) to track the three main lignin monomers ( Figure 1).…”

Monitoring Polysaccharide Dynamics in the Plant Cell Wall

“…The RG I and RG II probes have been detected via copper-catalyzed click reactions, which are toxic to Arabidopsis seedlings and may damage the wall. However, two alternative methods to detect click-compatible analogs not requiring copper were developed recently and could be used to study the long-term dynamics of extracellular glycans (Hoogenboom et al, 2016).…”