Imaging Cell Wall Architecture in Single Zinnia elegans Tracheary Elements  

Abstract

The chemical and structural organization of the plant cell wall was examined in Zinnia elegans tracheary elements (TEs), which specialize by developing prominent secondary wall thickenings underlying the primary wall during xylogenesis in vitro. Three imaging platforms were used in conjunction with chemical extraction of wall components to investigate the composition and structure of single Zinnia TEs. Using fluorescence microscopy with a green fluorescent protein-tagged Clostridium thermocellum family 3 carbohydrate-binding module specific for crystalline cellulose, we found that cellulose accessibility and binding in TEs increased significantly following an acidified chlorite treatment. Examination of chemical composition by synchrotron radiation-based Fourier-transform infrared spectromicroscopy indicated a loss of lignin and a modest loss of other polysaccharides in treated TEs. Atomic force microscopy was used to extensively characterize the topography of cell wall surfaces in TEs, revealing an outer granular matrix covering the underlying meshwork of cellulose fibrils. The internal organization of TEs was determined using secondary wall fragments generated by sonication. Atomic force microscopy revealed that the resulting rings, spirals, and reticulate structures were composed of fibrils arranged in parallel. Based on these combined results, we generated an architectural model of Zinnia TEs composed of three layers: an outermost granular layer, a middle primary wall composed of a meshwork of cellulose fibrils, and inner secondary wall thickenings containing parallel cellulose fibrils. In addition to insights in plant biology, studies using Zinnia TEs could prove especially productive in assessing cell wall responses to enzymatic and microbial degradation, thus aiding current efforts in lignocellulosic biofuel production.The organization and molecular architecture of plant cell walls represent some of the most challenging problems in plant biology. Although much is known about general aspects of assembly and biosynthesis of the plant cell wall, the detailed three-dimensional molecular cell wall structure remains poorly understood. The highly complex and dynamic nature of the plant cell wall has perhaps limited the generation of such detailed structural models. This information is pivotal for the successful implementation of novel approaches for conversion of biomass to liquid biofuels, given that one of the critical processing steps in biomass conversion involves systematic deconstruction of cell walls. Therefore, a comprehensive understanding of the architecture and chemical composition of the plant cell wall will not only help develop molecularscale models, but will also help improve the efficiency of biomass deconstruction.The composition and molecular organization of the cell wall is species and cell type dependent (Vorwerk et al., 2004). Thus, the development of a model plant system, which utilizes a single cell type, has enhanced our capacity to understand cell wall architecture. The ability to genera...

Formation of plant tracheary elements in vitro – a review

“…The patterned deposits of secondary wall in TEs primarily consist of polysaccharides (cellulose microfibrils and hemicelluloses) and lignin, a complex aromatic polymer (Turner et al 2007;Wagner et al 2012). These cell wall components result in a structure of great strength and resistance to degradation (Lacayo et al 2010). Deposition of cell wall polymers prevents the collapse of the xylem under the high pressure created by fluid transport Fukuda 2004) and reinforces these cells enabling the TEs to withstand the negative pressure generated during transpiration.…”

“…Cellulose can be localised in cell walls after staining with calcofluor white (Pesquet and Tuominen 2011). Fluorescence microscopy can be used to detect crystalline cellulose by labelling TEs with CtCBM3-GFP (green fluorescent protein), a fluorescently labelled family-3 carbohydrate binding module that binds to cellulose (Lacayo et al 2010). Lignin can be localised under a fluorescence microscope, due to autofluorescence of lignin in the green excitation range.…”

“…Ideally this will involve in vitro cell cultures that produce TEs in a synchronous manner to provide access to high percentages of TEs that are in specific stages of development. Examples of successfully established in vitro TE systems include Zinnia elegans L. or Arabidopsis thaliana L. These model systems have been instrumental in obtaining an improved understanding of cell wall architecture, composition and molecular organisation of cell walls in TEs (Lacayo et al 2010;Höfte 2010;Oda and Fukuda 2012).…”

“…Lignin can be localised under a fluorescence microscope, due to autofluorescence of lignin in the green excitation range. Cell-wall polysaccharides can be analysed using fluorescence microscopy after labelling with a green fluorescent protein-tagged carbohydrate-binding molecule specific for cellulose (Lacayo et al 2010). Cell-wall monosaccharide composition can be determined after hydrolysis and gas chromatography analysis (Möller et al 2003).…”

“…Atomic force microscopy (AFM) has been used to reveal the topography of TE cell walls (Lacayo et al 2010). Mild sonication was used to physically dissect TEs prior to microscopic analysis.…”