Atypical endo-β-1,4-mannannases are necessary for normal glucomannan synthesis in Arabidopsis
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The molecular mechanisms underlying the synthesis and secretion of large cell wall polysaccharides in plant cells are not fully understood. In this study, we report that two atypical endo-β-1,4-mannanases (MANs), which are not secreted and do not degrade glucomannan in the cell wall, play a novel role in glucomannan synthesis. Among the six MANs in Arabidopsis, AtMAN2 and AtMAN5 contain a transmembrane (TM) domain at their N-terminal region instead of a signal peptide. Subcellular localization using MAN protein fused with fluorescent protein demonstrated that AtMAN2 localizes to the Golgi apparatus in vascular and interfascicular fiber cells. We found that an Arabidopsis man2 man5 double mutant lost 65% of glucomannan in the cell walls of the inflorescence stem. Immunostaining revealed that the double mutant retained a small amount of glucomannan in the vacuole instead of secreting it to the cell walls. 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 revealed that the function of AtMAN2 and AtMAN5 is necessary for normal glucomannan synthesis. We suggest that these man mutants point to an uncharacterized transport pathway in plant cells, through which glucomannan is trafficked to the vacuole instead of the cell walls.
SIGNIFICANCE STATEMENT
Cell wall polysaccharides are synthesized from building block nucleotide sugars by the action of glucosyltransferases, but other factors facilitating the synthesis and secretion of large polysaccharides without aggregation remain to be identified. Here, we focused on the importance and functions of two atypical Arabidopsis endo-β-1,4-mannanases that have a transmembrane domain instead of a secretion signal, and which are widely distributed in angiosperms but have not been well characterized. Arabidopsis mutants impaired in these endo-β-1,4-mannanases lost 65% cell wall glucomannan, indicating that the hydrolysis by these enzymes is necessary for normal synthesis. Enhanced retention of glucomannan inside cells in the mutant hints at a transport route to deliver glucomannan to the vacuole.
