Determining the subcellular location of synthesis and assembly of the cell wall polysaccharide (1，3; 1，4)-β-D-glucan in grasses.

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.