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UDP-d-galactose synthesis by UDP-glucose 4-epimerase 4 is required for organization of the trans-Golgi network/early endosome in Arabidopsis thaliana root epidermal cells

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Abstract

Endomembrane organization is essential for cell physiology. We previously identified an Arabidopsis thaliana mutant in which a plasma membrane (PM) marker GFP-NIP5;1 and trans-Golgi network/early endosome (TGN/EE) markers were accumulated in intracellular aggregates in epidermal cells of the root elongation zone. The mutant was identified as an allele of UDP-glucose epimerase 4 (UGE4)/root hair defective 1/root epidermal bulgar 1, which was previously described as a mutant with swollen root epidermal cells and has an altered sugar composition in cell wall polysaccharides. Importantly, these defects including aggregate formation were restored by supplementation of d-galactose in the medium. These results suggested that UDP-d-galactose synthesis by UGE4 is important for endomembrane organization in addition to cell wall structure. Here, we further investigated the nature of the aggregates using various markers of endomembrane compartments and BOR1-GFP, which traffics from PM to vacuole in response to high-B supply. The markers of multi-vesicular bodies/late endosomes (MVB/LEs) and BOR1-GFP were strongly accumulated in the intracellular aggregates, while those of the endoplasmic reticulum (ER), the vacuolar membrane, and the Golgi were only slightly affected in the uge4 mutant. The abnormal localizations of these markers in the uge4 mutant differed from the effects of inhibitors of actin and microtubule polymerization, although they also affected endomembrane organization. Furthermore, electron microscopy analysis revealed accumulation of abnormal high-electron-density vesicles in elongating epidermal cells. The abnormal vesicles were often associated or interconnected with TGN/EEs and contained ADP-ribosylation factor 1, which is usually localized to the Golgi and the TGN/EEs. On the other hand, structures of the ER, Golgi apparatus, and MVB/LEs were apparently normal in uge4 cells. Together, our data indicate the importance of UDP-d-galactose synthesis by UGE4 for the organization and function of endomembranes, especially TGN/EEs, which are a sorting station of the secretory and vacuolar pathways.

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Abbreviations

UGE4:

UDP-d-glucose-4 epimerase 4

rhd1 :

Root hair defective (rhd) 1

reb1 :

Root epidermal bulgar

PM:

Plasma membrane

TGN:

Trans-Golgi network

MVB:

Multi-vesicular body

ER:

Endoplasmic reticulum

GFP/YFP:

Green/yellow fluorescence protein

NIP:

Nodulin 26-like intrinsic protein

EE:

Early endosome

LE:

Late endosome

HDVs:

High-electron-density vesicles

BFA:

Brefeldin A

ARF-GEF:

ADP-ribosylation factor-Guanine nucleotide exchange factors

DIC:

Differential interference contrast

FM4-64:

N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide

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Acknowledgments

We acknowledge Tomoko Shimizu for technical assistance, Akira Yoshinari for valuable comments, Niko Geldner (Lausanne University), Takashi Ueda (University of Tokyo), and ABRC for providing materials. This work was supported by the NEXT program from the Japan Society for the Promotion of Science and the Young Investigators Grant from the Human Frontier Science Program, to J. T.

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Authors and Affiliations

  1. Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan

    Sheliang Wang, Masataka Uehara & Junpei Takano

  2. Research Faculty of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan

    Toshiaki Ito, Satoshi Naito & Junpei Takano

  3. Graduate School of Life Science, Hokkaido University, Sapporo, 060-8589, Japan

    Satoshi Naito

  4. Technology Development Center of Sumika Agrotech Co., LTD, Sobiraki, Nihama, 792-0001, Japan

    Masataka Uehara

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  1. Sheliang Wang
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Correspondence to Junpei Takano.

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10265_2015_737_MOESM1_ESM.tif

Fig. S1 Localization of various markers in root epidermal cells of the uge4 mutant. ER-GFP in the wild-type (a) and uge4 mutant (b–d), YFP-Got1p homolog in the wild-type (e) and uge4 mutant (f–h), GFP-VAMP727 in the wild-type (i) and uge4 mutant (j–l), γ-TIP-GFP in the wild-type (m) and uge4 mutant (n–p), and BOR1-GFP in the wild-type (q) and uge4 mutant (r–t). Plant roots were stained with FM4-64 for 30–40 min prior to image with confocal microscopy (TIFF 2029 kb)

10265_2015_737_MOESM2_ESM.mpg

Supplementary material 1 Electron tomography analysis of high-electron-density vesicles in root epidermal cells of the uge4 mutant. An ultrathin section (~300 nm thickness) was imaged at 10,000× from +60.0° to −60.0° at 1.0° intervals (MPG 7266 kb)

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Wang, S., Ito, T., Uehara, M. et al. UDP-d-galactose synthesis by UDP-glucose 4-epimerase 4 is required for organization of the trans-Golgi network/early endosome in Arabidopsis thaliana root epidermal cells. J Plant Res 128, 863–873 (2015). https://doi.org/10.1007/s10265-015-0737-4

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  • DOI: https://doi.org/10.1007/s10265-015-0737-4

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