title: Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase.
abstract: The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1(A903V) and CESA3(T942I) in Arabidopsis thaliana. Using (13)C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1(A903V) and CESA3(T942I) displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1(A903V) and CESA3(T942I) have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.
authors.affiliation: Department of Horticulture, University of Kentucky, Lexington, KY 40546, USA.
authors.forename: Darby M
authors.initials: DM
authors.lastname: Harris
authors.forename: Kendall
authors.initials: K
authors.lastname: Corbin
authors.forename: Tuo
authors.initials: T
authors.lastname: Wang
authors.forename: Ryan
authors.initials: R
authors.lastname: Gutierrez
authors.forename: Ana L
authors.initials: AL
authors.lastname: Bertolo
authors.forename: Carloalberto
authors.initials: C
authors.lastname: Petti
authors.forename: Detlef-M
authors.initials: DM
authors.lastname: Smilgies
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Estevez
authors.forename: Dario
authors.initials: D
authors.lastname: Bonetta
authors.forename: Breeanna R
authors.initials: BR
authors.lastname: Urbanowicz
authors.forename: David W
authors.initials: DW
authors.lastname: Ehrhardt
authors.forename: Chris R
authors.initials: CR
authors.lastname: Somerville
authors.forename: Jocelyn K C
authors.initials: JK
authors.lastname: Rose
authors.forename: Mei
authors.initials: M
authors.lastname: Hong
authors.forename: Seth
authors.initials: S
authors.lastname: Debolt
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002482:Cellulose / Q000096:biosynthesis / Q000737:chemistry*; D003460:Crystallization; D004351:Drug Resistance / Q000187:drug effects; D005799:Genes, Dominant / Q000235:genetics; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D020894:Microfibrils / Q000737:chemistry* / Q000187:drug effects; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D021381:Protein Transport / Q000187:drug effects; D011804:Quinolines / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D029681:Arabidopsis Proteins; D050505:Mutant Proteins; D011804:Quinolines; D002482:Cellulose; C516179:At5g05170 protein, Arabidopsis; C110227:CESA1 protein, Arabidopsis; D005964:Glucosyltransferases; C470613:quinoxyfen