Abstract
Plant cellulose is synthesized by rosette-structured cellulose synthase (CESA) complexes (CSCs). Each CSC is composed of multiple subunits of CESAs representing three different isoforms. Individual CESA proteins contain conserved catalytic domains for catalyzing cellulose synthesis, other domains such as plant-conserved sequences, and class-specific regions that are thought to facilitate complex assembly and CSC trafficking. Because of the current lack of atomic-resolution structures for plant CSCs or CESAs, the molecular mechanism through which CESA catalyzes cellulose synthesis and whether its catalytic activity influences efficient CSC transport at the subcellular level remain unknown. Here, by performing chemical genetic analyses, biochemical assays, structural modeling, and molecular docking, we demonstrate that Endosidin20 (ES20) targets the catalytic site of CESA6 in Arabidopsis (Arabidopsis thaliana). Chemical genetic analysis revealed important amino acids that potentially participate in the catalytic activity of plant CESA6, in addition to previously identified conserved motifs across kingdoms. Using high spatiotemporal resolution live cell imaging, we found that inhibiting the catalytic activity of CESA6 by ES20 treatment reduced the efficiency of CSC transport to the plasma membrane. Our results demonstrate that ES20 is a chemical inhibitor of CESA activity and trafficking that represents a powerful tool for studying cellulose synthesis in plants.
Highlights
Cellulose is a polymer of b-1,4-D-Glc that serves as an essential cell wall component for controlling the directional growth of plant cells
Our results demonstrate that ES20 is a chemical inhibitor of cellulose synthase (CESA) activity and trafficking that represents a powerful tool for studying cellulose synthesis in plants
When we extended the treatment to 2 h with 6 mM ES20, PMlocalized cellulose synthase complex (CSC) were completely depleted from the plasma membrane (PM), and the abundance of CESA compartments associated with microtubules in the cortical cytoplasm increased (Figures 5H to 5J; Supplemental Movie 3)
Summary
Cellulose is a polymer of b-1,4-D-Glc that serves as an essential cell wall component for controlling the directional growth of plant cells. Cellulose is synthesized at the plasma membrane (PM) by a cellulose synthase complex (CSC) comprising a 25-nmdiameter rosette of subunits in a hexagonal array that can be observed in numerous plant cell types (Mueller et al, 1976; Giddings et al, 1980; Mueller and Brown, 1980). Each CSC is predicted to contain at least 18 monomeric cellulose synthases (CESAs). GT2 proteins are thought to share a common GT-A catalytic fold that has been observed in multiple GT2 family proteins (Charnock and Davies, 1999; Cantarel et al, 2009). CESAs across kingdoms contain multiple transmembrane domains and a cytoplasmic catalytic domain (McNamara et al, 2015). High-resolution structural analysis of Rhodobacter sphaeroidesin CESA (RsBcsA) revealed the detailed
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