Post-translational Regulation of CSLF6, the Major Mixed Linkage (1,3;1,4)-Β-glucan Synthase, in Barley (hordeum Vulgare) Seedlings

Mixed linkage (1,3;1,4)-β-glucan (MLG) is a major non-cellulosic polysaccharide of commelinid monocot cell walls and an important soluble dietary fibre component found in cereal grains, including those of barley. Despite its importance, relatively little is known about the post-translational regulation of cellulose synthase-like F6 (CSLF6), the major MLG synthase (MLGS) isoform involved in MLG biosynthesis in developing grain and young vegetative tissues. Following the detection of higher-order CSLF6-containing protein complexes under non-denaturing conditions, CSLF6 co-immunoprecipitation (co-IP) using detergent-solubilised barley microsomal membranes (MM) identified 16 high confidence (class I) potential candidate HvCSLF6-interacting proteins and 37 medium-confidence (class II) candidate interactors. Biochemical analyses of in vitro MLGS activity in 4-day-old barley seedling MMs, as a proxy for CSLF6 activity, indicated that alkaline pH in the presence of Mg2+ and Ca2+ enhanced MLGS activity. The effect of a buffer-soluble cytoplasmic protein fraction (>100,000 g supernatant) and a mild detergent-treated soluble fraction previously shown to impact MLGS activity in maize coleoptiles was investigated. A higher stimulatory effect on MLGS activity was observed when the cytoplasmic-soluble protein fraction, lacking CSLF6, was incubated with MM compared to MM assayed alone. Based on our co-IP results and MLGS activity reconstitution assays in barley seedlings (and other grass species), we conclude that ancillary protein factor(s) are likely involved in CSLF6 regulation.