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

BMC Plant Biology

Methodology Article

Dario Cramer¹  Rene Geurts²  Han Zuilhof³  Jorin Hoogenboom³  Nathalja Berghuis³  Tom Wennekes⁴ 

[1] Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands;Department of Plant Science, Laboratory of Molecular Biology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands;Laboratory of Organic Chemistry, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The Netherlands;Laboratory of Organic Chemistry, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The Netherlands;Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands;

关键词: Click chemistry;    Arabidopsis thaliana;    Cell wall;    Glycans;    -Arabinofuranose;    -Glucosamine;    -Galactosamine;    -Fucose;    Metabolic oligosaccharide engineering;   

DOI  :  10.1186/s12870-016-0907-0

 received in 2016-07-15, accepted in 2016-09-26,  发布年份 2016

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© The Author(s). 2016