Unraveling mannan biosynthesis in poplar and spruce: functional characterization of Cellulose Synthase-Like family A (CSLA) genes.

Hemicelluloses are key chemical constituents of plant cell walls, influencing structure, strength, and interactions between cellulose and lignin. While xylan has been widely studied and shown to affect cell wall integrity, the function of mannan remains largely unexplored. To address this gap, we identified and functionally characterized putative mannan biosynthetic genes from spruce and poplar, and tested their impact on wood formation and digestibility. Bioinformatic and phylogenetic tools were employed to identify Cellulose Synthase-Like family A (CSLA) genes from spruce and poplar. These genes were then functionally validated by complementing the mannan deficient Arabidopsis csla2,3,9 triple mutants, and creating CRISPR-Cas9 knockouts and ectopic overexpression lines in poplar. Spruce and poplar CSLA candidates restored mannan biosynthesis in the Arabidopsis mutant. In poplar, mannan content was altered without impacting plant growth. Changes in mannan content marginally affected glucose and xylose release and improved cellulose hydrolysis during saccharification assays. This study demonstrates an effective approach to engineering mannan content in trees and highlights that this polysaccharide is not essential for cell wall formation in poplar. Our work provides a platform for further investigating the role of this polymer in tree cell wall structure and stability.