Abstract
The current dogma for cell wall polysaccharide biosynthesis is that cellulose (and callose) is synthesized at the plasma membrane (PM), whereas matrix phase polysaccharides are assembled in the Golgi apparatus. We provide evidence that (1,3;1,4)-β-D-glucan (mixed-linkage glucan [MLG]) does not conform to this paradigm. We show in various grass (Poaceae) species that MLG-specific antibody labeling is present in the wall but absent over Golgi, suggesting it is assembled at the PM. Antibodies to the MLG synthases, cellulose synthase-like F6 (CSLF6) and CSLH1, located CSLF6 to the endoplasmic reticulum, Golgi, secretory vesicles, and the PM and CSLH1 to the same locations apart from the PM. This pattern was recreated upon expression of VENUS-tagged barley (Hordeum vulgare) CSLF6 and CSLH1 in Nicotiana benthamiana leaves and, consistent with our biochemical analyses of native grass tissues, shown to be catalytically active with CSLF6 and CSLH1 in PM-enriched and PM-depleted membrane fractions, respectively. These data support a PM location for the synthesis of MLG by CSLF6, the predominant enzymatically active isoform. A model is proposed to guide future experimental approaches to dissect the molecular mechanism(s) of MLG assembly.
Highlights
The primary plant cell wall is a mechanical network of rigid cellulose microfibrils embedded within a reinforced gel-like phase of matrix polysaccharides
The discrepancy in the cellular distribution of mixed-linkage glucan (MLG) in barley and maize outlined above was attributed by Carpita and McCann (2010) to either fixation artifacts or a possible timing issue, where it was proposed that sampled tissues had ceased synthesizing MLG and would not contain MLG in the endomembrane system
The quality of tissue preservation is evidenced by the plasma membrane (PM) being appressed to the wall, delineation of membranes with a smooth rather than wavy appearance, Golgi stacks with clearly resolved cisternae, and a uniformly electron-dense cytosol rather than condensed and/or electron-lucent regions (Figure 1; Supplemental Figure 1)
Summary
The primary plant cell wall is a mechanical network of rigid cellulose microfibrils embedded within a reinforced gel-like phase of matrix (noncellulosic and pectic) polysaccharides. It is vital to plant growth and development, as it determines the functional specialization of cells through regulation of their shape, permeability, and mechanical properties. Walls and their constituent polysaccharides, including mixed-linkage glucan (MLG), have important roles in the agrifood industry and in human health (Collins et al, 2010). Polysaccharide biosynthesis is largely attributed to two major classes of enzymes: several large families of polysaccharide synthases (the cellulose synthase [CesA] superfamily, GT2, and the glucan synthase-like family [GSL], GT48), which are integral transmembrane proteins found in both the plasma membrane (PM) and the Golgi apparatus, and multiple families of type II glycosyltransferases (CAZy, www.cazy.org; Lombard et al, 2014) largely found in the Golgi. The CesA superfamily comprises the cellulose synthases (CesAs) and a large family of CELLULOSE SYNTHASE-LIKE (CSL) genes that encode the backbones of a range
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