Biology subjects

Kotake, T.

Publications and source records attributed to Kotake, T..

5 recordsLinked to original sources

bioRxiv2025

Atypical endo-β-1,4-mannannases 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 two atypical endo-{beta}-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 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 endomembrane system including the Golgi apparatus in xylem 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 and immunoelectron microscopic observation also revealed that the man2 man5 double mutant lost 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 function in synthesis of glucomannan for the cell wall. 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 novel factors necessary for the normal glucomannan synthesis in Arabidopsis, along with GDP-mannose generating enzymes and CslAs, and suggests that the glucomannan hydrolysis by these MANs contributes to the maintenance of glucomannan synthesis.

plant biology↗

bioRxiv2025

Defective pollen meiosis in Arabidopsis due to combined arabinan and galactan insufficiency

The development of pollen is critical or seed plants, and depends on precise cellular and molecular mechanisms. It is known that the cell wall plays a key role in the progression of pollen maturation. An earlier phase in pollen development is the meiosis of pollen mother cells (PMCs), the fundamental process for producing viable pollen grains. However, the significance of the cell wall during pre-meiosis processes has remained unclear. Pectin, a major component of the cell wall, is known to accumulate abundantly in pollen. To investigate the significance of the cell wall during pollen meiosis, we generated an arad1 gals2 gals3 triple mutant of Arabidopsis thaliana, lacking the genes responsible for normal synthesis of arabinan and galactan, which constitute the side chains of pectin. Although vegetative growth and cell wall properties were comparable in wild-type (WT) and arad1 gals2 gals3, the pollen development in the mutant failed during meiosis. Immunohistochemical analysis showed that pectic arabinan and galactan accumulated in WT PMCs before meiosis, but this was not observed in mutant PMCs. We believe that this study is the first to demonstrate the critical role of cell wall components, specifically pectic arabinan and galactan, in the pre-meiosis processes of pollen development. HighlightPectic arabinan and galactan accumulate on the surface of pollen mother cells and this accumulation is essential for pollen meiosis.

plant biology↗

bioRxiv2024

Glucomannan engineering highlights the role of galactosyl modification in fine-tuning cellulose-glucomannan interaction in Arabidopsis cell walls

Widely found in most plant lineages, {beta}-mannans are structurally diverse polysaccharides that can bind to cellulose fibrils to form the complex polysaccharide architecture of the cell wall. Glucomannan backbones acquire variable patterns of galactosyl substitutions, depending on plant developmental stage and species. How the changes in polysaccharide structure influence its cell wall solubility or promote appropriate interaction with cellulose fibrils is poorly understood. Here, we show that fine-tuning of galactosyl modification on glucomannans is achieved by the differing acceptor recognition of mannan -galactosyltransferases (MAGTs). Biochemical analysis and 13C solid-state nuclear magnetic resonance spectroscopy of Arabidopsis with cell wall glucomannan engineered by MAGTs revealed that the degree of galactosylation strongly affects the interaction with cellulose. The findings indicate that plants tailor galactosyl modification on glucomannans for constructing an appropriate cell wall architecture, paving the way to convert properties of lignocellulosic biomass for better use.

bioengineering↗

bioRxiv2023

Cell wall pectic β-1,4-galactan contributes to increased plant freezing tolerance induced by cold acclimation

Subzero temperatures are often lethal to plants. Many temperate plants have a mechanism called cold acclimation that allows them to sense a drop in temperature and prepare for freezing stress through accumulation of soluble sugars and cryoprotective proteins. Plant cells are surrounded by a cell wall and as ice formation primarily occurs in the extracellular matrix (the apoplast), cell wall properties are important for plant freezing tolerance. Although previous studies have shown that the amounts of constituent sugars of the cell wall, in particular those present in pectic polysaccharides, are altered by cold acclimation, the significance of this change during cold acclimation has not been clarified. We found that {beta}-1,4-galactan, which forms neutral side chains of pectin, accumulates in the cell walls of Arabidopsis and various freezing tolerant vegetables during cold acclimation. The gals1 gals2 gals3 triple mutant, which has reduced {beta}-1,4-galactan in the cell wall, exhibited reduced freezing tolerance compared with wild-type Arabidopsis during initial stages of cold acclimation. Expression of genes involved in the galactan biosynthesis pathway such as GALACTAN SYNTHASES and UDP-glucose 4-epimerases were induced during cold acclimation in Arabidopsis explaining galactan accumulation. Cold acclimation resulted in a decrease in extensibility and an increase in rigidity of the cell wall in the wild type, whereas these changes were not observed in the gals1 gals2 gals3 triple mutant. These results indicate that the accumulation of pectic {beta}-1,4-galactan contributes to acquired freezing tolerance by cold acclimation via changes in cell wall mechanical properties.

plant biology↗

bioRxiv2020

Crystal structure of GH43 exo-β-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium provides insights into the mechanism of bypassing side chains

Arabinogalactan proteins (AGPs) are functional plant proteoglycans, but their functions are largely unexplored, mainly because of the complexity of the sugar moieties, which are generally analyzed with the aid of glycoside hydrolases. In this study, we solved the apo and liganded structures of exo-{beta}-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. It is composed of a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family (CBM) 35 binding domain. GH43_sub24 lacks the catalytic base Asp that is conserved among other GH43 subfamilies. Crystal structure and kinetic analyses indicated that the tautomerized imidic acid function of Gln263 serves instead as the catalytic base residue. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite -1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the {beta}-1,6-linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s. Some of the residues involved in ligand recognition differ from those of galactomannan-binding CBM35, including substitution of Trp for Gly, which affects pyranose stacking, and substitution of Asn for Asp in the lower part of the binding pocket. Pc1,3Gal43A WT and its mutants at residues involved in substrate recognition are expected to be useful tools for structural analysis of AGPs. Our findings should also be helpful in engineering designer enzymes for efficient utilization of various types of biomass.

molecular biology↗