Using monoclonal antibodies to label living root hairs: a novel tool for studying cell wall microarchitecture and dynamics in Arabidopsis

Abstract

BackgroundThe Arabidopsis root hair represents a valuable cell model for elucidating polar expansion mechanisms in plant cells and the overall biology of roots. The deposition and development of the cell wall is central to the root hair expansion apparatus. During this process, incorporation of specific wall polymers into the growing wall architecture constitutes a critical spatio-temporal event that controls hair size and growth rate and one that is closely coordinated with the cell’s endomembrane, cytoskeletal and signal transduction apparatuses.ResultsIn this study, the protocol for live cell labeling of roots with monoclonal antibodies that bind to specific wall polymers is presented. This method allows for rapid assessment of root hair cell wall composition during development and assists in describing changes to cell wall composition in transgenic mutant lines. Enzymatic “unmasking” of specific polymers prior to labeling allows for refined interpretation of cell wall chemistry. Live cell immunofluorescence data may also be correlated with transmission electron microscopy-based immunogold labeling.ConclusionsLive Arabidopsis root hairs may be labeled with cell wall polymer-specific antibodies. This methodology allows for direct visualization of cell wall dynamics throughout development in stable transgenic plant lines. It also provides an important new tool in the elucidation of the specific interactions occurring between membrane trafficking networks, cytoskeleton and the cell wall deposition/remodeling mechanism.

An oligogalacturonide‐derived molecular probe demonstrates the dynamics of calcium‐mediated pectin complexation in cell walls of tip‐growing structures

“…To confirm these observations we labelled fixed whole mount root hairs with the JIM5 and JIM7 mAbs (Figures i,j and S9i,j). No signal could be observed for JIM5, which is in line with previous reports on live non‐sectioned material (Larson et al ., ). On the other hand labelling with JIM7 produced a specific signal which could be observed along the root hair cell wall but often appeared in a punctate pattern, suggesting distinct periodic secretion events (Figure i,j).…”

“…Dynamics of Arabidopsis pollen tube HG dynamics studied in real-time Some of the best models for studying rapid cell wall formation in land plants are tip-growing cellular structures: pollen tubes and root hairs (Gu and Nielsen, 2013;Hepler et al, 2013;Rounds and Bezanilla, 2013;Grierson et al, 2014;Larson et al, 2014). We utilised Arabidopsis pollen tubes and root hairs to test the versatility of the probes further through real-time analysis of HG dynamics.…”

Root hairs enhance Arabidopsis seedling survival upon soil disruption

“… Parameters Seedlings (3–5 DAG) Older plants (10–14 DAG) References a Root hair length b (mm) 0.49 ± 0.11 0.38 ± 0.19 Supplementary Table S1 Root hair length of lateral root b (mm) N.A. 0.28 ± 0.16 Supplementary Table S1 Root hair diameter c (μm) 9.9 ± 2.2 26 – 29 Primary root length c (cm) 0.72 ± 0.4 70.15 ± 0.88 Supplementary Table S1 , 30 – 33 Primary root diameter c (μm) 126.5 ± 36.8 145.3 ± 41.2 Supplementary Table S1 , 27 , 28 , 34 – 39 Total lateral root length c (cm/seedling) N.A. 5.93 ± 0.20 32 , 33 Lateral root diameter c (μm) N.A.…”

Vacuolar Protein Sorting 26C encodes an evolutionarily conserved large retromer subunit in eukaryotes that is important for root hair growth in Arabidopsis thaliana

“…Wild‐type, vps26c‐1 , vti13 and vti13 vps26c seedlings were grown on 1 × Murashige–Skoog ( MS ) medium, pH 6 for 5 days and labeled with LM 15, a monoclonal antibody that recognizes a xyloglucan epitope in cell walls. Root epidermal cells and root hairs of wild‐type and vps26c seedlings label similarly with LM 15, whereas vti13 root epidermal cells and root hairs do not exhibit significant LM 15 labeling (as previously described in Larson et al ., ,b). LM 15 labeling of xyloglucan in root epidermal cells and root hairs is restored in the vti13 vps26c double‐mutant, indicating that the vps26c mutation can suppress the vti13 cell wall phenotype.…”