The click-compatible sugar 6-deoxy-alkynyl glucose metabolically incorporates into Arabidopsis root hair tips and arrests their growth
Graphical abstract
Arabidopsis root epidermal cells incorporate the click-compatible sugar analog, 6-deoxy-alkynyl glucose, specifically into root hair tip cell walls where it arrests root hair growth.
Introduction
The primary walls of plant cells are dynamic extracellular structures that enable tissue growth and determine cellular and organismal morphology (Cosgrove, 2005). They are composed of carbohydrate polymers that interact with one another to form strong, flexible networks that are reorganized during cell growth (Anderson et al., 2010, Fry, 2004, Somerville et al., 2004). Many of these carbohydrate polymers contain glucose monomers: for example, cellulose, the main load-bearing component of the plant cell wall, is composed of β-1,4-linked glucan chains that coalesce to form partially crystalline microfibrils with high tensile strength (Chanliaud et al., 2003). Xyloglucan, the most abundant hemicellulose in the primary walls of many eudicot plant species, contains a backbone of β-1,4-linked glucose monomers that is decorated with neutral sugar sidechains, whereas the linear polymer, mixed-linkage glucan, contains β-1,4- and β-1,3-linked glucose subunits. Callose, a specialized cell wall polymer that is produced during new cell wall formation and in response to external stimuli such as wounding and pathogen attack, is composed of linear β-1,3-linked glucan chains (Chen and Kim, 2009, Currier, 1957). Although candidate glycosyltransferases for all of these polymers have been identified at the genetic level, many details of where and when these polymers are synthesized, delivered to the cell wall, and modified during cellular development and expansion are unknown (Lerouxel et al., 2006, Worden et al., 2012). Since cell wall carbohydrates embody much of the biomass produced by plants via photosynthesis, improved knowledge of their dynamics will be useful for the sustainable production of food, materials, and renewable bioenergy.
The ability to specifically label newly synthesized carbohydrate polymers with fluorescent tags is an effective method for following their life histories in living organisms. However, because carbohydrate polymers cannot be tagged genetically in the same way that proteins can be tagged with fluorescent markers such as GFP, they must be labeled by other means. One such approach is metabolic labeling, in which sugar analogs act as chemical reporters, becoming incorporated into natural polymers before being covalently linked to fluorescent probes using, for example, a copper-catalyzed cyclization reaction (a subset of “click reactions”) (Kolb et al., 2001, Laughlin and Bertozzi, 2009). This method has been used to investigate the details of glycan synthesis and metabolism across diverse biological taxa (Beatty et al., 2006, Hsu et al., 2007). For example, a click chemistry-based approach to label nascent pectins in root epidermal cell walls of the model plant Arabidopsis thaliana (L.) Heynh. was previously adopted using the click-compatible fucose analog fucose-alkyne. This study found that fucose-alkyne-containing pectins are initially delivered to discrete locations across the cell surface that likely represent sites of exocytic vesicle fusion and that fucose-alkyne-containing pectins become reorganized into linear fibrillar arrangements as cell elongation progresses and they age in the cell wall (Anderson et al., 2012).
In addition to their reorganization during diffuse growth, cell wall components are also reorganized during the transition to tip growth in the same cell. This transition in wall properties occurs in some Arabidopsis root epidermal cells. At a certain point along the root developmental gradient, two distinct cell files become visible, trichoblasts and atrichoblasts, which are distinguishable by the presence of root hair bulges in the former. These files typically alternate along the circumference of the root; their identity is determined by the arrangement of cortex cells below (Dolan et al., 1993). Root hair bulges of trichoblasts are regions of distinct wall biochemistry and mechanics where a subregion of the outer wall changes growth schemes. Unlike the diffuse expansion that had occurred during primary cell expansion, these bulges expand by tip growth, the polarized addition of new wall material to elongate the growing tip (Vissenberg et al., 2001).
Diversifying the click chemistry-based approach to enable the metabolic labeling of cellulose, hemicelluloses, and/or callose would allow for the study of how these polymers change in abundance and distribution over developmental time in any plant species of interest. Although azido-linked analogs for several monosaccharides that are commonly found in cell walls are commercially available, and azido 3-deoxy-d-manno-oct-2-ulosonic acid (KDO-azide) was recently reported to incorporate into pectic rhamnogalacturonan-II (Dumont et al., 2015), it was previously found that fucose-azide is not efficiently incorporated into plant cell walls as compared to fucose-alkyne (Anderson et al., 2012). Thus, there is a need to expand the toolbox of click-compatible analogs of monosaccharides, such as glucose, galactose, xylose, rhamnose, and arabinose, that are present in cell wall carbohydrates. In this work, the synthesis of one such analog, 6-deoxy-alkynyl glucose (6) (6dAG), and its use in metabolic labeling experiments in Arabidopsis roots, is reported. This compound is incorporated into Arabidopsis root epidermal cells in a metabolism-, time-, and concentration-dependent manner, but unexpectedly, 6dAG (6) incorporation results in specific labeling of a subcellular structure, the root hair bulge, and inhibits root hair growth, suggesting that it might be incorporated into a novel cell wall component that affects cell wall expansion in root hairs.
Section snippets
Synthesis and design of 6-deoxy-alkynyl glucose (6)
Synthesis of 6dAG started with commercially available d-glucose (1) (Fig. 1). To differentiate the C6-OH group from the other hydroxy groups, the C6-OH group was first protected with a tert-butyldimethylsilyl (TBS) group following a procedure similar to that reported (Dasgupta and Nitz, 2011), giving intermediate (2). The remaining hydroxy groups were protected by methoxymethyl (MOM) groups, which can be easily deprotected under mild acidic conditions and survive the Seyferth–Gilbert
Conclusion
6dAG (6) has the potential to probe glycan metabolism in many different biological systems, including plants. Click-chemistry is one of few methods compatible with in vivo imaging of polysaccharides, compounds that are otherwise difficult to label (Sletten and Bertozzi, 2009). The toolbox of click-compatible sugar analogs relevant to plant cell wall chemistry is currently restricted to fucose-alkyne and KDO-azide (Anderson et al., 2012, Dumont et al., 2015). Synthesis of 6dAG (6) and the
Seedling culture
All click labeled specimens were 4 d-old A. thaliana Col-0 seedlings. 30% (v/v) NaOCl surface-sterilized seeds were imbibed for at least 3 days, then grown vertically for 4 d on MS plates (0.8% agar-agar, MS salts and vitamins used at half the strength as in (Murashige and Skoog, 1962)) at constant light, 22 °C. 6dAG (6) root elongation assays were performed on vertical MS plates to which filter-sterilized 6dAG in DMSO and/or filter-sterilized glucose (1) in H2O was added during agar cooling.
6-deoxy-alkynyl glucose (6) click-labeling
Acknowledgements
This work was supported as part of The Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences under Award # DE-SC0001090.
We thank the Office of Collaborative Science (OCS), Microscopy Core, NYU Langone Medical Center for sharing an ImageJ macro that facilitated repeated root hair measurements.
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