||UDP-D-galactose-dependent organization of the trans-Golgi network & Mechanisms for polar localization of boric acid channels in Arabidopsis thaliana [an abstract of entire text]
Boron (B) is essential for plant development but is toxic when presents inexcess. In Arabidopsis thaliana, a boric acid channel NIP5;1 is required for Buptake under B deficiency. NIP5;1 is expressed in outermost cell layers of rootsand shows polar localization toward the soil-side in the plasma membrane (PM).The polar localization is assumed to be important for the efficient uptake of boricacid from soil solution. However, the molecular mechanisms of PM targetingand polar localization of NIP5;1 and their physiological importance were notestablished.In this study, the nature of a mutant developing intracellular aggregatescontaining GFP-tagged NIP5;1 was investigated. In addition, the mechanism ofpolar localization of NIP5;1 and its physiological importance on plant growthwere elucidated.1. UDP-D-galactose synthesis by UDP-glucose 4-epimerase 4 is requiredfor organization of the trans-Golgi network/early endosome inArabidopsis thaliana root epidermal cellsPreviously, a fluorescence-imaging based screening isolated an Arabidopsismutant developing intracellular aggregates containing GFP-NIP5;1 in epidermalcells of the root elongation zone. This mutant was identified as an allele of UDPglucose4-epimerase 4 (uge4) mutant which has defects in UDP-D-galactosesynthesis and channeling into the Golgi.Here, I further investigated the nature of intracellular aggregates in the uge4mutants. Live-cell imaging indicated that the markers of multi-vesicularbodies/late endosomes (MVB/LEs) and PM were strongly accumulated in theintracellular aggregates in the uge4 mutant. Furthermore, electron microscopyanalysis revealed accumulation of abnormal high-electron-density vesicles inelongating epidermal cells. The abnormal vesicles were often associated orinterconnected with trans-Golgi network/early endosomes (TGN/EEs) and contained ADP-ribosylation factor 1, which is usually localized to the Golgi andthe TGN/EEs. Together, our data indicate the importance of UDP-D-galactosesynthesis by UGE4 for the organization and function of endomembranes,especially TGN/EEs, which are a sorting station of the secretory and vacuolarpathways.2. Polar localization of a boric acid channel is mediated byphosphorylation and plays a crucial role in growth of Arabidopsisthaliana under low boron conditionsTo elucidate the mechanism of the polar localization of NIP5;1, I performedstructure-localization analysis of NIP5;1 by expression of GFP-used homologsand chimera proteins in Arabidopsis thaliana. The N-terminal region of NIP5;1was identified to be required for the polar localization and have the ability totarget non-polar aquaporin proteins to the soli-side PM domain. Further analysisrevealed that phosphorylation in the N-terminal region of NIP5;1 is required forthe polar localization.I then investigated the physiological importance of the polar localization usingtransgenic Arabidopsis lines expressing a non-polar NIP5;1 variant. Althoughthe non-polar variant showed comparable boric acid transport activity inXenopus oocytes, the transgenic lines with the variant developed shorter rootsthan those with the wild-type NIP5;1 under low B conditions.These results revealed that the polar localization of NIP5;1 is mediated byphosphorylation, and plays a crucial role in growth of Arabidopsis thaliana underlow boron conditions probably by improving the efficiency of boric acid uptake inthe roots.
Hokkaido University（北海道大学）. 博士(農学)