Engineered glycoside hydrolases as fluorescent probes reveal the spatial distribution of the pectic polysaccharide rhamnogalacturonan II in plant cell walls

Abstract

Abstract Plant cell walls are dynamic composites whose architecture determines growth, mechanics, and environmental resilience. Efforts to link pectin structure to function have been limited by the lack of molecular probes with sufficient specificity, a gap that becomes even more pronounced for the intricately branched rhamnogalacturonon-II (RG-II) subclass. Here we report the first fluorescent probes with defined specificity to RG-II, engineered from catalytic site mutants of Bacteroides thetaiotaomicron glycoside hydrolases BT1010 and BT0996. These enzyme-derived probes bind RG-II monomer with high affinity, discriminate against dimeric forms, and localize to cell corners and junctions in Arabidopsis thaliana stems, consistent with RG-II’s unique ability among wall polysaccharides to form borate-mediated, covalent crosslinkages between molecules. Application of these probes revealed spatial partitioning distinct from the homogalacturonan (HG)- and rhamnogalacturonan I (RG-I)-enriched middle lamella, highlighting functional specialization among pectic domains, with RG-II reinforcing cell junctions while HG and RG-I mediate wall flexibility. Our work establishes a generalizable framework for transforming CAZymes into high-precision imaging reagents, enabling molecular-level visualization of structurally complex polysaccharides in the cell wall.

Plant phenotyping relevance

RG-IIを特異的に可視化する蛍光プローブを開発し、植物細胞壁内の空間分布という植物状態を画像で測定する手法を示しているため、フェノタイピング手法が中心的です。

Code and data availability

The paper deposits its raw microscopy z-stacks and maximum projections on OSF and its custom MATLAB image-analysis code on GitHub, both with explicit availability statements and public URLs.