Atypical endo-β-1,4-mannanases are necessary for normal glucomannan synthesis in Arabidopsis

The molecular mechanisms underlying the synthesis of large cell wall polysaccharides in plant cells are not fully understood. Here, we report that 2 atypical endo-β-1,4-mannanases (MANs), which are not secreted and do not degrade glucomannan in the cell wall, play a role in glucomannan synthesis. Among the 6 MANs in Arabidopsis (Arabidopsis thaliana), AtMAN2 and AtMAN5 contain a transmembrane domain at their N-terminal region instead of a signal peptide. Subcellular localization using MAN protein fused to a fluorescent protein demonstrated that AtMAN2 localizes to the endomembrane system, including the Golgi apparatus, in xylem and interfascicular fiber cells. An Arabidopsis man2 man5 double mutant lost 65% of glucomannan in the cell walls of the inflorescence stem. Immunostaining and immunoelectron microscopic observation also revealed that the man2 man5 double mutant loses glucomannan in the cell walls to about the same extent as the csla2 csla9 double mutant, which lacks major glucomannan synthases. Gene complementation experiments showed that the enzymatic activities of AtMAN2 and AtMAN5 are important for the synthesis of cell wall glucomannan. Arabidopsis possesses another atypical MAN, AtMAN6, with an HDEL retention signal at its C-terminus. However, mutation of AtMAN6 did not affect glucomannan content in the cell walls, suggesting distinct functions for these MANs. This study has identified AtMAN2 and AtMAN5 as factors necessary for normal glucomannan synthesis in Arabidopsis, along with GDP-mannose-generating enzymes and CslAs, and suggests that glucomannan hydrolysis by these MANs contributes to maintaining glucomannan synthesis.