Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1 A903V and CESA3 T942I of cellulose synthase

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 13C solidstate 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 A903Vand CESA3 T942I have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.Fil: Harris, Darby M.. University of Kentucky; Estados UnidosFil: Corbin, Kendall. University of Kentucky; Estados UnidosFil: Wang, Tuo. University of Iowa; Estados UnidosFil: Gutierrez, Ryan. Carnegie Institution for Science; Estados UnidosFil: Bertolo, Ana L.. Cornell University; Estados UnidosFil: Petti, Carloalberto. University of Kentucky; Estados UnidosFil: Smilgies, Detlef M.. Cornell University; Estados UnidosFil: Estevez, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Bonetta, Dario. University Of Ontario Institute Of Technology; CanadáFil: Urbanowicz, Breeanna R.. Cornell University; Estados Unidos. University of Georgia; Estados UnidosFil: Ehrhardt, David W.. Carnegie Institution for Science; Estados UnidosFil: Somerville, Chris R.. University of California at Berkeley; Estados UnidosFil: Rose, Jocelyn K. C.. Cornell University; Estados UnidosFil: Hong, Mei. University of Iowa; Estados UnidosFil: DeBolt, Seth. University of Kentucky; Estados Unido