A Functional Unfolded Protein Response Is Required for Normal Vegetative Development

Abstract

The unfolded protein response (UPR) is activated in plants in response to endoplasmic reticulum stress and plays an important role in mitigating stress damage. Multiple factors act in the UPR, including the membrane-associated transcription factor, BASIC LEUCINE ZIPPER 17 (bZIP17), and the membrane-associated RNA splicing factor, INOSITOL REQUIRING ENZYME1 (IRE1). We have analyzed an Arabidopsis (Arabidopsis thaliana) ire1a ire1b bzip17 triple mutant, with defects in stress signaling, and found that the mutant is also impaired in vegetative plant growth under conditions without externally applied stress. This raised the possibility that the UPR functions in plant development in the same manner as it does in responding to stress. bZIP17 is mobilized to the nucleus in response to stress, and through the analysis of a mobilization-defective bZIP17 mutant, we found that to support normal plant development bZIP17 must be capable of mobilization. Likewise, through the analysis of ire1 mutants defective in either protein kinase or RNase activities, we found that both must be operative to promote normal development. These findings demonstrate that the UPR, which is associated with stress responses in plants, also functions under unstressed conditions to support normal development.

Figure 1.

Root elongation assay for UPR mutants. A to C, Various single and higher order mutants of UPR signaling pathway components (ire1a, ire1b, bzip17, and bzip28) were grown vertically on one-half-strength Murashige and Skoog (MS) medium with a 16-h-light/8-h-dark cycle for 7 d, and representative images are shown. D, Root lengths at 7 d of the different genotypes shown in A to C. The bars are means ± sd of three independent experiments. One-way ANOVA with Tukey’s method was used to examine significant differences at P < 0.01 in pairwise comparison and classified by a, b, or c. WT, Wild type.

Figure 2.

Phenotypes of the 17ab mutant at different growth stages. A, 17ab and wild-type (WT) seedlings were grown vertically on one-half-strength MS medium with a 16-h-light/8-h-dark cycle for 7 d. B and C, 17ab and wild-type plants were grown in soil under short-day (8 h of light and 16 h of dark; B) and long-day (16 h of light and 8 h of dark; C) conditions for 45 d. Rosette images in B were digitally abstracted for comparison. D, 17ab and wild-type plants were grown in soil under long-day conditions for 2, 3, and 4 weeks, and representative images are shown.

Figure 3.

Root elongation assay for bZIP60 mutants. A and B, Wild-type (WT), ire1a ire1b, bzip17 bzip60, and bzip28 bzip60 seedlings were grown vertically on one-half-strength MS medium with a 16-h-light/8-h-dark cycle for 7 d. C, Root lengths at 7 d for different genotypes shown in A and B. The data are means ± sd of three independent experiments. One-way ANOVA with Tukey’s method was used to examine significant differences at P < 0.01 in pairwise comparison and classified by a or b.

Figure 4.

Complementation of 17ab with various UPR constructs. The YFP-tagged constructs include full-length bZIP17(fl); the processed form, bZIP17(p); the bZIP17 point mutant bZIP17^G372A, which prevents processing; and the processed and activated forms, bZIP28(p) and bZIP60(s), respectively. A, Wild-type (WT), 17ab, and different transgenic plants were grown under short-day conditions (8 h of light and 16 h of dark). Rosette images were digitally abstracted for comparison. B, Seedlings representing different genotypes were grown vertically on one-half-strength MS medium with a 16-h-light/8-h-dark cycle for 7 d, and a representative image is shown. C, Root lengths for the seedlings shown in A and B. The data are means ± sd of three independent experiments. One-way ANOVA with Tukey’s method was used to examine significant differences at P < 0.01 in pairwise comparison and classified by a, b, or c.

Figure 5.

Subcellular localization of mobilization-defective bZIP17. Arabidopsis leaf protoplasts were cotransformed with YFP-bZIP17(fl) or YFP-bZIPG372A and with either RFP-tagged ER markers (A and C) or Golgi markers (B and D; Nelson et al., 2007). Protoplasts were treated overnight with 2 mM DTT to induce ER stress, and protoplasts were observed by confocal microscopy. Bars = 10 μm.

Figure 6.

Complementation of 17ab with various mutant forms of IRE1b. IRE1b, IRE1b^{D608N, K610N}, IRE1b^D628A (the latter two are protein kinase dead), and IRE1b^N820A (RNase dead) were introduced into the 17ab mutant background by agrobacteria-mediated transformation. A, Reverse transcription (RT)-PCR analysis of bZIP60(s) (spliced form of bZIP60 mRNA) and IRE1b in the wild type (WT), 17ab, and different transgenic plants. ACTIN2 was used as a loading control. B, The wild type, 17ab, and different transgenic plants were grown under short-day conditions (8 h of light and 16 h of dark) for 45 d. Rosette images were digitally abstracted for comparison. C, Seedlings of different genotypes listed above were grown vertically on one-half-strength MS medium with a 16-h-light/8-h-dark cycle for 7 d. D, Root lengths of the different genotypes shown in B and C. Data represent means ± sd of three independent experiments. One-way ANOVA with Tukey’s method was used to examine significant differences at P < 0.01 in pairwise comparison and classified as a or b.

Figure 7.

Proposed model for the function of IRE1 and bZIP17 under stressed and unstressed conditions. Under stressed conditions, IRE1 up-regulates the expression of the canonical UPR genes in a bZIP60-dependent manner. Under these conditions, the RIDD activity of IRE1 is also activated, leading to the degradation of the RNAs of many secreted protein genes. In addition, bZIP17 is mobilized and functions along with bZIP60 to up-regulate canonical and noncanonical UPR genes. Under unstressed conditions, the RIDD activity of IRE1 presumably leads to the degradation of unidentified mRNAs, but likely those encoding secreted protein. bZIP17 is also modestly active, leading to the production of some noncanonical UPR proteins.