Article Figures & Data
Figures
- Figure 1. Arabidopsis IRE1C does not contribute to the UPR.
(A) Structures of Arabidopsis IRE1A, IRE1B, and IRE1C proteins. (B) Phylogenetic tree of IRE1 proteins from mammals (Homo sapiens), fungi (Saccharomyces cerevisiae and Saccharomyces pombe) and plants was constructed by UPGMA using MEGA 6 (Tamura et al, 2013). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. (C) Wild-type (WT) and ire1 mutant plants 40 d after germination (DAG). Bar = 10 mm. (D) DTT sensitivity of the ire1 mutants. Seedlings at 15 DAG of the indicated lines were treated with or without 1 mM DTT. (E) The relative mRNA levels of BiP3 (upper), bZIP60s (middle), and PR-4 (lower) in WT and ire1 mutants. RNA from seedlings at 10 DAG were treated with 5 mg/l Tm, 2 mM DTT, or mock for 5 h and subjected to qPCR. Data are means ± SEM of three independent experiments. Different letters within each treatment indicate significant differences (P < 0.05) by the Tukey–Kramer Honestly Significant Difference (HSD) test. SS, signal sequence; TM, transmembrane domain.
- Figure S1. Characterization of ire1 mutants.
(A) Schematic representation of T-DNA insertion sites in ire1a, ire1b, and ire1c. Grey and white boxes indicate coding sequences and untranslated regions, respectively. Arrows indicate the positions of primers (Table S1) used for genotyping. (B) Tm sensitivity of the ire1 mutants. Seedlings at 15 DAG of the indicated lines were treated with or without Tm. (C) Relative shoot fresh weight of seedlings at 15 DAG treated with 0.1 mg/l Tm (left) or 1 mM DTT (right) compared with untreated seedlings. Data are means ± SEM of three independent experiments. Different letters within each treatment indicate significant differences (P < 0.05) by the Tukey–Kramer HSD test. (D) Genotyping of wild-type, ire1a, ire1b, ire1c, ire1a ire1b, ire1a ire1c, and selfed siblings (ire1c/+ or +/+) of ire1a ire1b ire1c/+. Lanes 1–3, three independent plants for each mutant. Lane M, 100-bp DNA ladder. M, mutant; W, wild-type.
- Figure 2. ire1a ire1b homozygous and ire1c heterozygous plants show developmental defects.
(A) Self-pollinated progenies of ire1a ire1b ire1c/+ mutant plants at 40 DAG. Genotypes are shown on the left. Bar = 10 mm. (B) Siliques of ire1a ire1b ire1c/+ (left) and ire1a ire1b +/+ (right) plants. Bar = 10 mm. (C) Reproductive development of ire1 mutants. Flowers at stage 14 (Smyth et al, 1990; upper; bar = 1 mm), siliques (middle; bar = 3 mm), and anthers at stage 12 stained with Alexander’s stain (lower; bar = 100 μm) from wild-type (WT) and ire1 mutants are shown.
- Figure S2. T-DNA constructs of the binary vectors used in this study.
(A) IRE1C promoter-driven GUS reporter gene construct. IRE1C promoter region within pENTR vector was transferred into pSMAB-GW-GUS binary vector through Gateway LR reaction. (B) FLAG-tagged IRE1A and IRE1B constructs. Genomic regions of the IRE1A and IRE1B genes were cloned into pENTR vector and transferred into pSMAB-GW binary vector. Modifications are indicated in each panel. (C) A CRISPR/Cas9 binary vector containing gRNA1 and gRNA2 targeting the sensor domain of the IRE1B gene (see Fig 7A).
- Figure S3. Tissue-specific expression of IRE1C gene.
(A, B, C) GUS histochemical staining of transgenic Arabidopsis containing IRE1C promoter–GUS fusion construct in floral tissues (A), ovules, and embryo (B). Bar = 100 μm. (C) 8-d-old seedlings treated with or without Tm for 5 h, and 11-d-old seedlings treated with glycerol for 3 d. Bar = 5 mm. (D) WT (upper), ire1a ire1b (middle), and ire1a ire1b ire1c/+ (lower) plants at 30 DAG cultured on 1/2 MS plate containing 1% sucrose. Plants were cultured on medium containing 2% sucrose for 10 d and then transferred to the 1% sucrose medium. (E, F) The relative mRNA level of IRE1C in WT, ire1a ire1b, ire1a ire1b ire1c/+, and ire1c plants (E) as indicated in (D) or flower bud tissues (F). Data are means ± SEM of four independent experiments. Different letters within each treatment indicate significant differences (P < 0.05) by the Tukey–Kramer HSD test.
- Figure 3. Transgenic ire1a ire1b plants expressing FLAG-tagged wild-type or mutant IRE1.
(A) Schema of FLAG-tagged IRE1 proteins. Mutations of kinase and RNase domains are shown as arrowheads. (B) Detection of FLAG-IRE1 proteins in the transgenic ire1a ire1b plants with anti-FLAG antibody. Ponceau S staining was used as loading control. (C) RNA blot analysis of BiP3 and PR-4 in wild-type (WT), ire1a ire1b mutant, and transgenic ire1a ire1b plants. Seedlings at 10 DAG were treated with (+) or without (−) 5 mg/l Tm for 5 h. (D) Detection of FLAG-IRE1B(WT) and FLAG-IRE1B(K487A) with anti-FLAG antibody in the transgenic ire1a ire1b plants treated with Tm or DTT. Samples were resolved on Phos-tag SDS–PAGE to detect the phosphorylated FLAG-IRE1B (p-IRE1B). (E) DTT and Tm sensitivities of the transgenic ire1a ire1b plants. Seedlings at 15 DAG of the indicated lines were treated with or without 1 mM DTT or 0.1 mg/l Tm. SS, signal sequence; TM, transmembrane domain.
- Figure 4. Phenotypic complementation of ire1a ire1b ire1c/+ mutants by FLAG-IRE1B(WT) or ΔLD.
Phenotypes of the transgenic ire1a ire1b ire1c/+ plant having FLAG-IRE1B(WT) (left) or ΔLD (center), and ire1a ire1b ire1c/+ plant (right). (A, B, C) Plants at 40 DAG. Bar = 10 mm. (D, E, F) Flowers at stage 14. Bar = 500 μm. (G, H, I) Siliques. Bar = 3 mm. (J, K, L) Anthers at stage 12 stained with Alexander’s stain. Bar = 100 μm. (M, N, O) Tetrads stained with toluidine blue. Bar = 20 μm.
- Figure 5. Abnormal pollen development in ire1a ire1b ire1c/+ is partially complemented by ΔLD.
Transverse sections of developing anthers at stages 8, 9, and 11 in WT (left), ire1a ire1b ire1c/+ mutant (middle), and transgenic ire1a ire1b ire1c/+ plants having ΔLD (right). Arrowheads indicate collapsed pollen grains. Bar = 50 μm.
- Figure 6. Glycerol treatment stimulates IRE1 kinase and RNase activities.
(A) Percentages of saturated fatty acids (16:0 and 18:0) in WT and ire1a ire1b plants at 10 DAG treated with (G) or without (C) glycerol for 3 d. Error bars represent SD (n = 6). Different letters within each treatment indicate significant differences (P < 0.01) by the Tukey–Kramer HSD test. (B, C) Detection of bZIP60 mRNA splicing in WT, ire1a ire1b plants (B), and FLAG-IRE1 transgenic ire1a ire1b plants (C) at 10 DAG. RT-PCR was performed using bZIP60s-specific primers. Actin2 (Act2) was used as an internal control. (B) Glycerol treatment was performed for 0–4 d. (C) Plants were treated with (+) or without (−) glycerol for 3 d. (D) Detection of FLAG-IRE1B(WT) with anti-FLAG antibody in the transgenic ire1a ire1b plants treated with glycerol for 0–3 d. Samples were resolved on SDS–PAGE (upper) and Phos-tag SDS–PAGE (lower) followed by immunodetection with anti-FLAG antibody. An equal loading was shown by CBB staining after SDS–PAGE (middle). (E) RNA blot analysis of PR-4, PRX34, and MBL1 in WT and ire1a ire1b plants at 10 DAG. Plants were treated with or without glycerol for 3 d and plants at 10 DAG were treated with cordycepin (Cord) for 0–5 h. (F) Relative mRNA levels of PR-4, PRX34, and MBL1 in FLAG-IRE1B(WT, K821A, ΔLD) transgenic ire1a ire1b plants. 3-d glycerol-treated plants at 10 DAG were treated with Cord for 1 and 3 h and subjected to qPCR. log2 fold change was calculated by dividing average mRNA level of Cord 3 h by that of Cord 1 h. Data are means ± SEM of 3–6 independent experiments. (G, H) Detection of ΔLD with anti-FLAG antibody in the transgenic ire1a ire1b plants treated with glycerol, Tm, or DTT. (G) Samples were resolved on SDS–PAGE followed by immunodetection with anti-FLAG antibody. CBB staining was used as loading control. (H) Samples were resolved on Phos-tag SDS–PAGE to detect the phosphorylated ΔLD (p-ΔLD). Asterisks indicate possible degradation products of ΔLD. (I) The relative mRNA expression levels of FLAG-IRE1B(WT, ΔLD) transgenes. RNA from seedlings at 10 DAG treated with (G) or without (C) glycerol for 3 d was subjected to qPCR. Data are means ± SEM of four independent experiments. Different letters within each treatment indicate significant differences (P < 0.05) by the Tukey–Kramer HSD test. AP, alkaline phosphatase-treated sample.
- Figure S4. Effect of RNase and sensor domain of IRE1B on bZIP60 splicing and RIDD under glycerol treatment.
(A) Detection of bZIP60s mRNA splicing in WT, ire1a ire1b, and FLAG-IRE1B(WT, ΔLD) transgenic ire1a ire1b plants at 10 DAG. RT-PCR was performed using bZIP60s-specific primers. Actin2 (Act2) was used as an internal control. Glycerol treatment was performed for 0, 1, and 3 d. (B, C, D) The relative mRNA levels of bZIP60s (B), cytosolic marker protein genes (C), and RIDD target genes (D) in WT, ire1a ire1b, and FLAG-IRE1B(WT, K821A, ΔLD) transgenic ire1a ire1b plants. RNA from seedlings at 10 DAG treated with (+) or without (−) glycerol for 3 d was subjected to qPCR. Data are means ± SEM of four independent experiments. Different letters within each treatment indicate significant differences (P < 0.05) by the Tukey–Kramer HSD test.
- Figure 7. CRISPR/Cas9-mediated IREB gene editing in ire1a ire1c mutant.
(A) Schema of hypothetical IRE1B products in the IRE1B-mutated ire1a ire1c T3 plant lines #2–5 and #1–10. Positions of gRNA target sites are shown as arrowheads. (B) Tm sensitivity of the ire1a ire1b, ire1a ire1c, and IRE1B-mutated ire1a ire1c plants. Seedlings at 15 DAG of the indicated lines were treated with or without 0.1 mg/l Tm. (C) RNA blot analysis of BiP3 and PR-4 in WT, ire1a ire1b, ire1a ire1c mutant, and IRE1B-mutated ire1a ire1c plants. Seedlings at 10 DAG were treated with (+) or without (−) 5 mg/l Tm for 5 h. (D) Detection of bZIP60 mRNA splicing in WT, ire1a ire1b, ire1a ire1c, and IRE1B-mutated ire1a ire1c plants at 10 DAG treated with (+) or without (−) glycerol for 3 d. (E) RNA blot analysis of PR-4 and PRX34 in WT, ire1a ire1b, ire1a ire1c, and IRE1B-mutated ire1a ire1c plants at 10 DAG treated with glycerol for 3 d. The samples were treated with (+) or without (−) Cord for 2 h immediately before sampling. SS, signal sequence; TM, transmembrane domain.
- Figure S5. Characteristics of the CRISPR/Cas9-mediated IRE1B mutant lines #2–5 and #9–6.
(A) Genotyping of IRE1A-C genes in wild-type, ire1a ire1b, ire1a ire1c mutants, and T3 plants of lines #2–5 and #9–6. Lane M, 100 bp DNA ladder. Lanes 1–6, six independent plants for each line. Note that PCR amplification of Cas9 gene was performed to detect T-DNA and that #9–6 lane 7 (asterisk) is a T2 sibling plant having T-DNA used as a control. (B) RT-PCR of IRE1B mRNA in ire1a ire1c, ire1a ire1b mutants, and T3 plants of #2–5 and #9–6. Actin2 (Act2) was used as an internal control. (C) DTT sensitivity of the ire1a ire1b, ire1a ire1c mutants, and T3 plants of #2–5 and #9–6. Seedlings at 15 DAG were treated with or without 1 mM DTT. M, mutant; W, wild-type.
- Figure S6. #2–5 and #9–6 lines retain RIDD activity and show no phenotypic abnormality.
(A) The relative mRNA levels of RIDD target genes in WT, ire1a ire1b, ire1a ire1c, and ire1a ire1c with IRE1B mutation T3 lines #2–5 and #9–6. RNA from seedlings at 10 DAG treated with glycerol for 3 d was subjected to qPCR. Data are means ± SEM of four independent experiments. Different letters indicate significant differences (P < 0.05) by the Tukey–Kramer HSD test. (B) T3 plants of lines #2–5 and #9–6 and ire1a ire1b ire1c/+ mutant at 40 DAG. Bar = 10 mm. (C) Siliques of the lines #2–5 (left) and #9–6 (right) plants. Bar = 3 mm.
Tables
- Table 1.
Transmission of the ire1c allele through the male and female gametophyte in the progenies of the ire1a ire1b ire1c/+ mutants crossed with the ire1a ire1b mutant or self-pollination.
Parental Genotype Genotypes of Progeny Observed Ratio Expected Ratio Female Male +/+ c/+ c/c Total +/+:c/+:c/c +/+:c/+:c/c a/a b/b c/+ a/a b/b c/+ 83 25 0 108 1.0:0.30:0a 1:2:1 a/a b/b c/+ a/a b/b +/+ 186 39 0 225 1.0:0.21:0a 1:1:0 a/a b/b +/+ a/a b/b c/+ 119 0 0 119 119:0:0a 1:1:0 ↵a Significantly different from the Mendelian segregation ratio (χ2, P < 0.01).
+, wild-type allele; a, ire1a allele; b, ire1b allele; c, ire1c allele.
- Table 2.
Transmission of the ire1c allele through the male and female gametophyte in the progenies of the ire1a ire1b ire1c/+ mutants having IRE1B or ΔLD transgenes crossed with wild-type or self-pollination.
Parental Genotype Genotypes of Progeny Observed Ratio Expected Ratio Female Male +/+ c/+ c/c Total +/+:c/+:c/c +/+:c/+:c/c +/+ +/+ +/+ a/a b/b c/+ 202 0 0 202 202:0:0a 1:1:0 +/+ +/+ +/+ a/a b/b c/+ IRE1B 43 14 0 57 1.0:0.33:0a 1:1:0 +/+ +/+ +/+ a/a b/b c/+ ΔLD 60 34 0 94 1.0:0.57:0a 1:1:0 a/a b/b c/+ +/+ +/+ +/+ 266 81 0 347 1.0:0.30:0a 1:1:0 a/a b/b c/+ IRE1B +/+ +/+ +/+ 130 32 0 162 1.0:0.25:0a 1:1:0 a/a b/b c/+ ΔLD +/+ +/+ +/+ 135 25 0 160 1.0:0.19:0a 1:1:0 a/a b/b c/+ IRE1B a/a b/b c/+ IRE1B 53 101 0 154 1.0:1.9:0a 1:2:1 a/a b/b c/+ ΔLD a/a b/b c/+ ΔLD 97 140 0 237 1.0:1.4:0a 1:2:1 ↵a Significantly different from the Mendelian segregation ratio (χ2, P < 0.01).
+, wild-type allele; a, ire1a allele; b, ire1b allele; c, ire1c allele; IRE1B, FLAG-IRE1B(WT) transgene (homozygote); ΔLD, ΔLD transgene (homozygote).
