In the grass species Brachypodium distachyon, the production of mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) occurs in the Golgi apparatus

Abstract

Mixed-linkage (1,3;1,4)-β-glucan (MLG) is a glucose polymer with beneficial effects on human health and high potential for the agricultural industry. MLG is present predominantly in the cell wall of grasses and is synthesized by cellulose synthase-like F or H families of proteins, with CSLF6 being the best-characterized MLG synthase. Although the function of this enzyme in MLG production has been established, the site of MLG synthesis in the cell is debated. It has been proposed that MLG is synthesized at the plasma membrane, as occurs for cellulose and callose; in contrast, it has also been proposed that MLG is synthesized in the Golgi apparatus, as occurs for other matrix polysaccharides of the cell wall. Testing these conflicting possibilities is fundamentally important in the general understanding of the biosynthesis of the plant cell wall. Using immuno-localization analyses with MLG-specific antibody in Brachypodium and in barley, we found MLG present in the Golgi, in post-Golgi structures and in the cell wall. Accordingly, analyses of a functional fluorescent protein fusion of CSLF6 stably expressed in Brachypodium demonstrated that the enzyme is localized in the Golgi. We also established that overproduction of MLG causes developmental and growth defects in Brachypodium as also occur in barley. Our results indicated that MLG production occurs in the Golgi similarly to other cell wall matrix polysaccharides, and supports the broadly applicable model in grasses that tight mechanisms control optimal MLG accumulation in the cell wall during development and growth. This work addresses the fundamental question of where mixed linkage (1,3;1,4)-β-glucan (MLG) is synthesized in plant cells. By analyzing the subcellular localization of MLG and MLG synthase in an endogenous system, we demonstrated that MLG synthesis occurs at the Golgi in Brachypodium and barley. A growth inhibition due to overproduced MLG in Brachypodium supports the general applicability of the model that a tight control of the cell wall polysaccharides accumulation is needed to maintain growth homeostasis during development.

A Trihelix Family Transcription Factor Is Associated with Key Genes in Mixed-Linkage Glucan Accumulation

“…In the cellulose synthase-like gene family, CSLF genes are only found in grasses and CSLF6 is the most highly expressed member of this gene family (Burton et al, 2008;Christensen et al, 2010;Kaur et al, 2017). The location of CSLF6 and MLG synthesis is controversial, as one study in barley (Hordeum vulgare) found that MLG is synthesized at the plasma membrane (Wilson et al, 2015), while another study using tobacco (Nicotiana tabacum) epidermal cells expressing B. distachyon CSLF6 and B. distachyon plants overexpressing CSLF6 demonstrated that MLG is synthesized in the Golgi apparatus (Kim et al, 2015(Kim et al, , 2018, supporting the conclusion of Carpita and McCann (2010), who had shown by in vivo radiolabeling that MLG is synthesized in the Golgi apparatus in maize (Zea mays).…”

Grass secondary cell walls, Brachypodium distachyon as a model for discovery

“…By far the best characterized enzyme in MLG synthesis is CslF6, which has been studied in barley, wheat (Triticum aestivum), rice, maize and B. distachyon (Nemeth et al, 2010;Vega-Sanchez et al, 2012;Kim et al, 2015Kim et al, , 2018. BdCslF6 protein is localized to the Golgi membrane, with an external catalytic domain (Kim et al, 2015(Kim et al, , 2018. Antibody detection of (1,3;1,4)-b-glucan in maize also supports a Golgi-localized synthesis of MLG (Carpita & McCann, 2010).…”

“…Members of CslF/H/J clades have been shown to be capable of synthesizing (1,3;1,4)-b-glucan when heterologously expressed, but it is not clear whether all of these groups are responsible for native MLG synthesis. By far the best characterized enzyme in MLG synthesis is CslF6, which has been studied in barley, wheat (Triticum aestivum), rice, maize and B. distachyon (Nemeth et al, 2010;Vega-Sanchez et al, 2012;Kim et al, 2015Kim et al, , 2018. BdCslF6 protein is localized to the Golgi membrane, with an external catalytic domain (Kim et al, 2015(Kim et al, , 2018.…”

“…Among the genes co-expressed with B. distachyon CSLF6, a predominant MLG synthase, was a trihelix family transcription factor (BdTHX1) (Fan et al, 2018;Kim et al, 2018). This is the first THX protein associated with cellwall biosynthesis and the first shown to bind directly to a CSLF gene.…”

The trans-Golgi sorting and the exocytosis of xylogalacturonan from the root border/border-like cell are conserved among monocot and dicot plant species

“…19 Furthermore, amounts of pectic polysaccharides are lower in the type II cell wall than in the type I cell wall 20 and mixed-linkage β-glucan abundant in the grass cell wall is produced in the Golgi. 21 These indicate that the Golgi organization in maize cells involved in cell wall polysaccharide synthesis differs significantly from that in Arabidopsis and alfalfa cells. However, remodeling of the Golgi stack associated with the synthesis and secretion of XGA in the border/border-like cells of the three plant species seems to be conserved.…”