Article Figures & Data
Figures
- Fig. 1.
Cooperative induction of mRNA of the ER chaperones in Arabidopsis by tunicamycin. Total RNA was extracted from Arabidopsis seedlings after addition of tunicamycin (5 μg mL−1) to the culture medium. Incubation periods after treatment were 0 h (Control), 1 h (Tm 1 h), and 2.5 h (Tm 2.5 h). The RNA blot was probed with the cDNAs of BiP, PDI, calnexin, calreticulin-1, and calreticulin-2.
- Fig. 2.
Alignment of Ire1 homologs from various organisms. Two Arabidopsis homologs (AtIre1-1 and AtIre1-2; this study), yeast Ire1p (Ire1p, accession no. Z11701), human Ire1α (hIre1α, accession no. AF059198), and mouse Ire1β (mIre1β, accession no. AF071777) were aligned using ClustalW software. Amino acid residues conserved in three out of five sequences are boxed. The ATP binding motif (VAVKR) and the Ser/Thr protein kinase motif (DLKPQN) are underlined.
- Fig. 3.
A, Genomic Southern analysis of AtIre1-1 and AtIre1-2. Genomic DNA of Arabidopsis digested with two restriction enzymes was fractionated by agarose gel, blotted, and probed with a fragment of either AtIre1-1 or AtIre1-2 cDNA. B, mRNA abundance analysis of AtIre1-1, AtIre1-2, and BiP. Poly(A+) RNA was isolated from Arabidopsis seedlings with no treatment or treated with tunicamycin (5 μg mL−1 for 4 h). Two micrograms of RNA sample from each treatment was used for northern blotting. Probes used were same as in A for AtIre1-1 and AtIre1-2. BiP probe was the same as in Figure 1.
- Fig. 4.
A and B, Autophosphorylation activity of the C-terminal domain of AtIre1-2. A, Domain structure of Ire1 (top) and of the GST fusions showing the location of the K to A mutation. B, The fusion protein of GST with the C-terminal domain of AtIre1-2 was expressed in E. coli and affinity purified on glutathione-Sepharose. Purified protein incubated with γ-32P ATP was subjected to SDS-PAGE. The gel was Coomassie stained (CBB) and exposed to x-ray film (autoradiogram). The same fraction was used for an immunoblot analysis with anti-GST antibody (anti-GST). In A and B, lane 1 has the wild-type GST-AtIre1-2 fusion and lane 2 has the mutant GST-AtIre1-2 fusion. There is no32P labeling of the fusion polypeptide of the mutant (lane 2 in the autoradiogram).
- Fig. 5.
Localization of AtIre1-1-GFP and AtIre1-2-GFP fusion proteins by fluorescence microscopy. Epifluorescence images of tobacco BY-2 cells (A–H), and purified nuclei from tobacco BY-2 cells (i–l). A, B, and i, BY-2 cells express normal GFP. Note green fluorescence in the nucleoplasm. C, D, and j, BY-2 cells expressing GFP-KDEL. Note absence of fluorescence in the nucleoplasm. E, F, and k, BY-2 cells expressing AtIre1-1-GFP. G and l, Cells expressing AtIre1-2-GFP. H, BY-2 cell expressing the AtIre1-2(K442A)-GFP. Bars = 0.01 mm.
- Fig. 6.
A through C, Complementation of yeastire1 deletion mutant with chimeric constructs of yeast Ire1p and Arabidopsis Ire1 homologs. A, Schematic view of the chimeric constructs used for yeast complementation. Chimeric constructs consisted of the sensor domains of AtIre1-1 or AtIre1-2, and other parts (signal peptide and C-terminal half) of yeast Ire1p. B, Growth ofΔire1 strains of yeast complemented with chimeric constructs in the absence or presence of tunicamycin.Δire1 containing vector only (vector), chimeric constructs for AtIre1-1 (Atire1-ch) and AtIre1-2 (Atire2-ch), and yeastIRE1 were grown on synthetic dextrose plates without tunicamycin (−Tunicamycin) and with tunicamycin (+Tunicamycin, final concentration at 0.2 μg mL−1). Approximately 1 × 105 cells were spotted on the left column of each plate. Series of one-tenth dilution of cells were spotted on the right side. Yeast cells were grown for 3 d at 30°C. C, Activity of β-galactosidase of yeast cells containing each construct. Cells were incubated at 30°C with 2 μg mL−1 tunicamycin for 4 h, or 1 mm dithiothreitol for 2 h, and their β-galactosidase activity was measured.
- Fig. 7.
A through K, Histochemical staining of GUS of Arabidopsis plants transformed with AtIre1-promoter::GUS fusions. A through F, AtIre1-1 and G-K: AtIre1-2. A and B, Seedlings with fully expanded cotyledons (note staining of the meristems); A inset, 48-h-old seedling with stained cotyledons; C, leaf margin; D, flower with immature anthers; E, flower after pollination, only the gynecium is stained and anthers are not longer stained; F. ovules; G, seedling; G inset, root tip; H and I, rosette leaf; J, seedling; K, flowers.
