title: The valine and lysine residues in the conserved FxVTxK motif are important for the function of phylogenetically distant plant cellulose synthases.
abstract: Cellulose synthases (CESAs) synthesize the β-1,4-glucan chains that coalesce to form cellulose microfibrils in plant cell walls. In addition to a large cytosolic (catalytic) domain, CESAs have eight predicted transmembrane helices (TMHs). However, analogous to the structure of BcsA, a bacterial CESA, predicted TMH5 in CESA may instead be an interfacial helix. This would place the conserved FxVTxK motif in the plant cell cytosol where it could function as a substrate-gating loop as occurs in BcsA. To define the functional importance of the CESA region containing FxVTxK, we tested five parallel mutations in Arabidopsis thaliana CESA1 and Physcomitrella patens CESA5 in complementation assays of the relevant cesa mutants. In both organisms, the substitution of the valine or lysine residues in FxVTxK severely affected CESA function. In Arabidopsis roots, both changes were correlated with lower cellulose anisotropy, as revealed by Pontamine Fast Scarlet. Analysis of hypocotyl inner cell wall layers by atomic force microscopy showed that two altered versions of Atcesa1 could rescue cell wall phenotypes observed in the mutant background line. Overall, the data show that the FxVTxK motif is functionally important in two phylogenetically distant plant CESAs. The results show that Physcomitrella provides an efficient model for assessing the effects of engineered CESA mutations affecting primary cell wall synthesis and that diverse testing systems can lead to nuanced insights into CESA structure-function relationships. Although CESA membrane topology needs to be experimentally determined, the results support the possibility that the FxVTxK region functions similarly in CESA and BcsA.
authors.affiliation: Department of Crop Science and Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Slabaugh
authors.affiliation: Department of Biological Sciences, University of Rhode Island, Kingston, RI 02881, USA.
authors.forename: Tess
authors.initials: T
authors.lastname: Scavuzzo-Duggan
authors.affiliation: Department of Biological Sciences, University of Rhode Island, Kingston, RI 02881, USA.
authors.forename: Arielle
authors.initials: A
authors.lastname: Chaves
authors.affiliation: Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Liza
authors.initials: L
authors.lastname: Wilson
authors.affiliation: Department of Crop Science and Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Carmen
authors.initials: C
authors.lastname: Wilson
authors.affiliation: Department of Crop Science and Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Jonathan K
authors.initials: JK
authors.lastname: Davis
authors.affiliation: Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Cosgrove
authors.affiliation: Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Charles T
authors.initials: CT
authors.lastname: Anderson
authors.affiliation: Department of Biological Sciences, University of Rhode Island, Kingston, RI 02881, USA.
authors.forename: Alison W
authors.initials: AW
authors.lastname: Roberts
authors.affiliation: Department of Crop Science and Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA chhaigle@ncsu.edu.
authors.forename: Candace H
authors.initials: CH
authors.lastname: Haigler
mesh_terms: D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D019068:Bryopsida / Q000201:enzymology* / Q000235:genetics; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D014633:Valine / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; C110227:CESA1 protein, Arabidopsis; D005964:Glucosyltransferases; D014633:Valine; D008239:Lysine
keywords: cellulose synthesis; gametophore formation; phenotypic complementation; root elongation; site-directed mutagenesis