Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase

• Published in: Proceedings of the National Academy of Sciences - PNAS 2012-03, Vol.109 (11), p.4098-4103
• Main Authors: Harris, Darby M, Corbin, Kendall, Wang, Tuo, Gutierrez, Ryan, Bertolo, Ana L, Petti, Carloalberto, Smilgies, Detlef-M, Estevez, José Manuel, Bonetta, Dario, Urbanowicz, Breeanna R, Ehrhardt, David W, Somerville, Chris R, Rose, Jocelyn K. C, Hong, Mei, DeBolt, Seth
• Format: Article
• Language: English
• Subjects: Alleles; Amino Acid Sequence; Amino Acid Substitution - genetics; Arabidopsis - drug effects; Arabidopsis - enzymology; Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; bacteria; Biological Sciences; biosynthesis; Cell Membrane - drug effects; Cell Membrane - enzymology; cellulose; Cellulose - biosynthesis; Cellulose - chemistry; cellulose synthase; crystal structure; Crystallization; Drug Resistance - drug effects; Genes, Dominant - genetics; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; image analysis; Magnetic Resonance Spectroscopy; Microfibrils - chemistry; Microfibrils - drug effects; Molecular Sequence Data; Mutant Proteins - chemistry; Mutant Proteins - metabolism; mutants; Mutation - genetics; nuclear magnetic resonance spectroscopy; plasma membrane; point mutation; polymerization; Protein Transport - drug effects; Quinolines - chemistry; Quinolines - pharmacology; saccharification; Structure-Activity Relationship; X-ray diffraction
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1200352109

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 CESA1A903V and CESA3T942I in Arabidopsis thaliana. Using 13C 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 CESA1A903V and CESA3T942I 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 CESA1A903V and CESA3T942I have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.