Hironori Takasaki

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-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 six 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.

<jats:title>ABSTRACT</jats:title><jats:p>Pesticide contamination in freshwater ecosystems poses a significant threat to water quality and aquatic life, potentially disrupting entire food webs. Fluxametamide (FMT), a novel insecticide regarded as safe for nontarget species, has not been extensively studied in aquatic environments, raising concerns about its potential toxicity to organisms such as <jats:italic>Daphnia magna</jats:italic> (<jats:italic>D. magna</jats:italic>). This study investigates the acute toxicity of FMT, the potential of ultrafine bubbles (UFB), and the associated morphological alterations on <jats:italic>D. magna</jats:italic>. This result revealed that acute toxicity tests showed FMT is very toxic to <jats:italic>D. magna</jats:italic>, with LC<jats:sub>50</jats:sub> 24–48 h values of 0.051 and 0.013 µg/L, respectively. However, UFB has the potential to reduce the toxicity of FMT by twofold compared to control water in <jats:italic>D. magna</jats:italic>. Morphological analysis in the presence of UFB revealed carapace and body damage at high concentrations and prolonged exposure. In contrast, control water resulted in pesticide accumulation throughout the body, leading to more rapid tissue destruction even at lower concentrations and shorter exposure durations. In conclusion, UFB has the potential to alleviate the toxic effects of FMT by modifying its chemical structure, enhancing detoxification mechanisms, and minimizing its overall detrimental effect on <jats:italic>D. magna</jats:italic>. These findings represent an initial report on the effect of UFB on FMT toxicity in <jats:italic>D. magna</jats:italic>, providing essential theoretical foundations for the application of FMT in ecotoxicology.</jats:p>