| 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 molecular-scale 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 generate a population of single Zinnia elegans plant cells that were synchronized throughout cell wall deposition during xylogenesis was developed in the 1980s (Fukuda and Komamine, 1980). Mesophyll cells isolated from the leaves of Zinnia and cultured in the presence of phytohormones will transdifferentiate into tracheary elements (TEs), which are individual components of the xylem vascular tissue (Fukuda and Komamine, 1980). During this transdifferentiation process, TEs gradually develop patterned secondary wall thickenings, commonly achieving annular, spiral, reticulate, scalariform, and pitted patterns (Bierhorst, 1960; Falconer and Seagull, 1988; Roberts and Haigler, 1994). These secondary wall thickenings serve as structural reinforcements that add strength and rigidity to prevent the collapse of the xylem under the high pressure created by fluid transport. During the final stages of transdifferentiation, TEs accumulate lignin in their secondary walls and undergo programmed cell death, which results in the removal of all cell contents, leaving behind a “functional corpse” (Roberts and McCann, 2000; Fukuda, 2004).In broad terms, the primary cell wall of higher plants is mainly composed of three types of polysaccharides: cellulose, hemicelluloses, and pectins (Cosgrove, 2005). Cellulose is composed of unbranched β-1,4-Glc chains that are packed together into fibrils by intermolecular and intramolecular hydrogen bonding. Hemicelluloses and pectins are groups of complex polysaccharides that are primarily composed of xyloglucans/xylans and galacturonans, respectively. Hemicelluloses are involved in cross-linking and associating with cellulose microfibrils, while pectins control wall porosity and help bind neighboring cells together. The patterned deposits of secondary wall in Zinnia TEs primarily consist of cellulose microfibrils, along with hemicelluloses, and also lignin, a complex aromatic polymer that is characteristic of secondary walls and provides reinforcement (Turner et al., 2007). All the molecular components in the cell wall correspond to a multitude of different polysaccharides, phenolic compounds, and proteins that become arranged and modified in muro, yielding a structure of great strength and resistance to degradation.Currently, electron microscopy is the primary tool for structural studies of cell walls and has provided remarkable information regarding wall organization. Fast-freeze deep-etch electron microscopy in combination with chemical and enzymatic approaches have generated recent models of the architecture of the primary wall (McCann et al., 1990; Carpita and Gibeaut, 1993; Nakashima et al., 1997; Fujino et al., 2000; Somerville et al., 2004). Direct visualization of secondary wall organization has been focused toward the examination of multiple wall layers in wood cells (Fahlen and Salmen, 2005; Zimmermann et al., 2006). However, few studies have examined the secondary wall, so our knowledge regarding the higher order architecture of this type of wall is limited. Over the past few decades, atomic force microscopy (AFM) has provided new opportunities to probe biological systems with spatial resolution similar to electron microscopy techniques (Kuznetsov et al., 1997; Muller et al., 1999), with additional ease of sample preparation and the capability to probe living native structures. AFM has been successfully applied to studies of the high-resolution architecture, assembly, and structural dynamics of a wide range of biological systems (Hoh et al., 1991; Crawford et al., 2001; Malkin et al., 2003; Plomp et al., 2007), thus enabling the observation of the ultrastructure of the plant cell wall, which is of particular interest to us (Kirby et al., 1996; Morris et al., 1997; Davies and Harris, 2003; Yan et al., 2004; Ding and Himmel, 2006).To generate more detailed structural models, knowledge about the structural organization of the cell wall can be combined with spatial information about chemical composition. Instead of utilizing chromatography techniques to analyze cell wall composition by extracting material from bulk plant samples (Mellerowicz et al., 2001; Pauly and Keegstra, 2008), Fourier transform infrared (FTIR) spectromicroscopy can be used to directly probe for polysaccharide and aromatic molecules in native as well as treated plant material (Carpita et al., 2001; McCann et al., 2001). FTIR spectromicroscopy is not only able to identify chemical components in a specific system but also can determine their distribution and relative abundance. This technique also improves the sensitivity and spatial resolution of cellular components without the derivatization needed by chemical analysis using chromatography. Polysaccharide-specific probes, such as carbohydrate-binding modules (CBMs), can also be used to understand the chemical composition of the plant cell wall. CBMs are noncatalytic protein domains existing in many glycoside hydrolases. Based on their binding specificities, CBMs are generally categorized into three groups: surface-binding CBMs specific for insoluble cellulose surfaces, chain-binding CBMs specific for single chains of polysaccharides, and end-binding CBMs specific for the ends of polysaccharides or oligosaccharides. A surface-binding CBM with high affinity for the planar faces of crystalline cellulose (Tormo et al., 1996; Lehtio et al., 2003) has been fluorescently labeled and used to label crystals as well as plant tissue (Ding et al., 2006; Porter et al., 2007; Liu et al., 2009; Xu et al., 2009). The binding capacity of the CBM family has been further exploited for the detection of different polysaccharides, such as xylans and glucans, and can thus be used for the characterization of plant cell wall composition (McCartney et al., 2004, 2006).In this study, we used a combination of AFM, synchrotron radiation-based (SR)-FTIR spectromicroscopy, and fluorescence microscopy using a cellulose-specific CBM to probe the cell wall of Zinnia TEs. The Zinnia TE culture system proved ideal for observing the structure and chemical composition of the cell wall because it comprises a single homogeneous cell type, representing a simpler system compared with plant tissues, which may contain multiple cell types. Zinnia TEs were also advantageous because they were analyzed individually, and population statistics were generated based on specific conditions. Furthermore, cultured Zinnia TEs were used for the consistent production of cell wall fragments for analysis of the organization of internal secondary wall structures. In summary, we have physically and chemically dissected Zinnia TEs using a combination of imaging techniques that revealed primary and secondary wall structures and enabled the reconstruction of TE cell wall architecture. |
