Conservation of IRE1-Regulated bZIP74 mRNA Unconventional Splicing in Rice (Oryza sativa L.) Involved in ER Stress Responses

Molecular Plant

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Volume 5

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Number 2

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Pages 504–514

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March 2012

RESEARCH ARTICLE

Conservation of IRE1-Regulated bZIP74 mRNA Unconventional Splicing in Rice (Oryza sativa L.) Involved in ER Stress Responses Sun-Jie Lu, Zheng-Ting Yang, Ling Sun, Le Sun, Ze-Ting Song and Jian-Xiang Liu1 State Key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, 200433, China

Key words: mRNA unconventional splicing; unfolded protein response; bZIP transcription factor; IRE1; heat stress; salicylic acid (SA); Oryza sativa.

INTRODUCTION Plants are sessile and their development and productivity are severely affected by many environmental stress factors, such as drought, salinity, temperature extremes, and pathogen attack. Hundreds of genes and products are regulated in responding to adverse environments at both transcriptional and translational levels, some of which are quite important for stress tolerance in plants (Zhu, 2002; Fujita et al., 2006; Nakashima et al., 2009). Protein homeostasis, a delicate balance between protein folding capability and folding demands, is sophisticatedly maintained by regulatory networks that control the protein synthesis and degradation. This process is easily interrupted by many environmental stress factors, leading to the unfolded protein response (UPR) in the endoplasmic reticulum (ER). UPR mitigates the ER stress by up-regulating the expression of genes encoding protein components in protein folding machinery and the ER-associated degradation (ERAD) system (Liu and Howell, 2010b). UPR is well conserved from yeast to mammalian cells. In yeast (Saccharomyces cerevisiae), the IRE1-HAC1 pathway mediates

the signal transduction from the ER to the nucleus (Sidrauski and Walter, 1997). In mammals, besides the IRE1-XBP1 mRNA splicing pathway, ATF6 (activating transcription factor 6) proteolytic activation is also important for the transcriptional control of the ER stress response (Ye et al., 2000; Yoshida et al., 2001; Calfon et al., 2002). The counterparts of the IRE1-XBP1 and ATF6 pathways, IRE1-bZIP60 and bZIP28 pathways, have recently been discovered to be also conserved in the model plant Arabidopsis (Arabidopsis thaliana) (Liu et al., 2007a; Iwata et al., 2008; Liu and Howell, 2010a; Deng et al., 2011; Nagashima et al., 2011). Interestingly, although the proteins AtbZIP60 and AtbZIP28 have little sequence similarities to their yeast and

1 To whom correspondence should be addressed. E-mail jianxiangliu@fudan. edu.cn, tel. 86-21-65642163, fax 86-21-65642163.

ª The Author 2011. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi: 10.1093/mp/ssr115, Advance Access publication 22 December 2011 Received 24 November 2011; accepted 6 December 2011

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ABSTRACT Protein folding in the endoplasmic reticulum (ER) is a fundamental process in plant cells that is vulnerable to many environmental stresses. When unfolded or misfolded proteins accumulate in the ER, the well-conserved unfolded protein response (UPR) is initiated to mitigate the ER stress by enhancing the protein folding capability and/or accelerating the ER-associated protein degradation. Here, we report the conservation of the activation mechanism of OsbZIP74 (also known as OsbZIP50), an important ER stress regulator in monocot plant rice (Oryza sativa L.). Under normal conditions, OsbZIP74 mRNA encodes a basic leucine–zipper transcription factor with a putative transmembrane domain. When treating with ER stress-inducing agents such as tunicamycin and DTT, the conserved double stem-loop structures of OsbZIP74 mRNA are spliced out. Thereafter, the resulting new OsbZIP74 mRNA produces the nucleus-localized form of OsbZIP74 protein, eliminating the hydrophobic region. The activated form of OsbZIP74 has transcriptional activation activity in both yeast cells and Arabidopsis leaf protoplasts. The induction of OsbZIP74 splicing is much suppressed in the OsIRE1 knockdown rice plants, indicating the involvement of OsIRE1 in OsbZIP74 splicing. We also demonstrate that the unconventional splicing of OsbZIP74 mRNA is associated with heat stress and salicylic acid, which is an important plant hormone in systemic acquired resistance against pathogen or parasite.

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transduction and gene regulation in UPR in monocots is very limited compared to the dicots Arabidopsis, although chronic UPR has been observed in maize since the early 1990s (Boston et al., 1991). The monocot rice (Oryza sativa L.) only has one IRE1 homolog in the genome. The N-terminal region of this ER-localized OsIRE1 protein has the ability to sense the ER stress in yeast while the C-terminal region has in vitro autophosphorylation activity. The physiological function and target of OsIRE1 are largely unknown (Okushima et al., 2002). In the present study, we report that the rice bZIP transcription factor OsbZIP74 is the equivalent of Arabidopsis bZIP60, thus experimentally demonstrating conservation of IRE1-bZIP mRNA splicing in monocot rice plants. The OsbZIP74 splicing is developmentally regulated and also induced by ER stress agents such as tunicamycin (TM) and DTT, environmental stress, such as heat stress, and salicylic acid (SA), an important phytohormone involved in plant defense responses in plants.

RESULTS Rice bZIP74 mRNA Is Subjected to Splicing in Response to ER Stress Recently, we have reported the Arabidopsis bZIP60 as the functional homolog of yeast HAC1 and mammalian XBP1, which are very important for the signal transduction and gene regulation in the UPR (Deng et al., 2011). In this work, we are interested in finding the equivalent pathway in rice, the important cereal crop that feeds more than half of the world’s population. The Arabidopsis bZIP60 mRNA was used for BLAST search in the Michigan State University (MSU) rice genome annotation database (http://rice.plantbiology.msu.edu/) and the top hit with the highest score (E value = 1.3e-07) was LOC_Os06g41770, which encodes a rice bZIP transcription factor bZIP74 (Guedes Correa et al., 2008), also known as bZIP50 (Os06g0622700 in the Rice Annotation Project Database) (Wakasa et al., 2011) with 28.52% identity and 47.77% similarity to the Arabidopsis inactivated bZIP60 protein. There is one full-length cDNA (AK107021) for OsbZIP74 deposited in the GenBank. Using the RNA secondary structure prediction program M-fold (Zuker, 2003), we found that, in the predicted lowest free energy (dG = –416.30) form of OsbZIP74 mRNA (AK107021), the twin kissing stem loops that are essential for yeast HAC1, mammalian XBP1, and Arabidopsis bZIP60 mRNA splicing (Yoshida et al., 2001; Oikawa et al., 2010; Deng et al., 2011) are also conserved (Figure 1A and Supplemental Figure 1). Like the one in Arabidopsis bZIP60, the left stem (S1) is shorter while the right loop (L2) is larger in rice bZIP74 than the counterparts in humans (Homo sapiens) XBP1. The spacer between two stem-loops in rice is the shortest, with only two nucleotides, while there are five and six nucleotides in Arabidopsis and human, respectively. The spacer in yeast is even larger. Experiments have demonstrated that, in response to ER stress, 26 nucleotides and 23 nucleotides of the target mRNAs are spliced out in human and Arabidopsis, respectively.

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mammalian functional homologs, the components that are required for their activation such as IRE1 (inositol-requiring enzyme 1), S1P (site-1 protease), and S2P (site-2 protease) are much conserved (Koizumi et al., 2001; Liu et al., 2007b; Che et al., 2010). The PERK (protein kinase RNA (PKR)-like ER kinase) pathway, which is responsible for the translational attenuation through phosphorylation of the translation initiation factor 2 alpha subunit (eIF2a), has only been found in mammalian cells so far (Harding et al., 1999). Like the ATF6 in mammals, the Arabidopsis bZIP28 is an ER membrane protein under normal growth conditions with its N-terminal DNA binding domain facing the cytosol and C-terminal stress-sensing domain facing the ER lumen. When the unfolded or misfolded proteins accumulate in the ER, the AtbZIP28 is translocated from the ER to the Golgi, where it is subjected to the regulated proteolysis executed by the two Golgi-resident proteases AtS1P and AtS2P (Liu et al., 2007a; Tajima et al., 2008; Che et al., 2010). The activated form of AtbZIP28, containing the DNA binding domain and transcriptional activation domain, could form the homo-dimer and interacts with the AtNF-Y (nuclear factor Y) trimer AtNF-YA4/AtNF-YB3/ AtNF-YC2 in the nucleus to activate downstream genes through binding to the ERSE (CCAAT-N10-CACG) cis-element (Liu and Howell, 2010a). The yeast IRE1 and mammalian IRE1a are also ER membrane proteins but the ER lumen-facing N-termini serve as the sensor domains and the cytosol-facing C-termini have the protein kinase and endoribonuclease activity (Credle et al., 2005; Zhou et al., 2006; Poothong et al., 2010). Dimerization/oligomerization and autophosphorylation of yeast IRE1 or mammalian IRE1a activate the endoribonuclease activity, enabling the splicing of HAC1 and XBP1 mRNA targets, respectively, independently of the splicesome (Sidrauski and Walter, 1997; Lee et al., 2008; Korennykh et al., 2009). Mammalian cells have another IRE1b. In response to ER stress, IRE1b induces transla_ tional repression through 28S ribosomal RNA cleavage (Iwawaki et al., 2001). The Arabidopsis plant also has two IRE1 homologs, IRE1A and IRE1B. Both of them localize in the ER and their N-terminal putative sensor domains function as actual sensors in yeast (Koizumi et al., 2001; Noh et al., 2002). However, Arabidopsis IRE1A and IRE1B are functionally redundant in terms of splicing the AtbZIP60 mRNA. The AtbZIP60 mRNA encodes 295 amino acids with a putative transmembrane domain near its C-terminus. Under ER stress conditions, 23 ribonucleotides of the AtbZIP60 mRNA are spliced out by AtIRE1A and AtIRE1B, which causes an open reading frame shift immediately before the hydrophobic region, thus producing a new protein with 258 amino acids without the transmembrane domain (Deng et al., 2011; Nagashima et al., 2011). The activated form of AtbZIP60 encoded by the spliced mRNA enters the nucleus and actives the downstream UPR genes, probably through both ERSE andUPREcis-elements(IwataandKoizumi,2005;Iwataetal.,2008). Angiosperms or flowering plants are generally grouped into two major groups or classes: monocotyledon (monocots) and dicotyledon (dicots) plants. Our understanding on signal

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However, only 20 nucleotides are predicted to be spliced out if there is IRE1-like activity in rice (Figure 1A). One pair of primers flanking the predicted OsbZIP74 splice sites were designed and used for RT–PCR analysis. When 2-week-old rice roots were treated with either 5 lg ml 1 TM or 2 mM DTT for 4 h, an excess band with faster migration was detected. DNA sequencing

results revealed that the predicted 20 nucleotides were indeed spliced out in response to ER stress agents TM and DTT (Figure 1B). Additional specific primers were generated to discriminate the unspliced and spliced forms of OsbZIP74 (Figure 2A). The forward primer of bZIP74-SPU crosses the spliced ‘intron’ and bZIP74-SPS bridges the ‘intron’. The amplification specificity was tested using plasmid DNA containing unspliced or spliced form of OsbZIP74 as the template, respectively (Figure 2B). The above specific primer assays were performed to recheck the OsbZIP74 splicing under ER stress

(A) The predicted twin stem-loop structures of human XBP1 mRNA (HsXBP1) and rice bZIP74 mRNA (OsbZIP74). Each of the structure contains two stems (S1 and S2) and two loops (L1 and L2). The conserved nucleotides in each loop are boxed. The spliced and predicted cleavage sites are highlighted with arrows and arrow heads, respectively. (B) OsbZIP74 mRNA splicing in response to ER stress agents. Primers immediately flanking the splicing sites (arrows) were used for the splicing assay; the spliced region is highlighted with bold lines (upper). Electrophoresis pattern of RT–PCR results from untreated (CK) or treated (DTT or TM) rice roots. Bands 1 and 2 are equal to the size of unspliced and spliced products, respectively (lower). (C) Partial nucleotides and encoding amino acid sequences of the unspliced or spliced form of rice bZIP74. Arrows indicate the splicing sites inferred from the DNA sequencing results in (B). Arrow head indicates the joint site. The putative transmembrane domain of the OsbZIP74 unspliced form is boxed (upper) and the new C-terminus derived from splicing is underlined and in bold (lower). An asterisk denotes stop codon.

Figure 2. OsIRE1-Depedent Splicing of OsbZIP74. (A) Primers used for the specific primer assays. The forward primer that crosses the spliced ‘intron’ (bold lines) was used to specifically amplify the unspliced form (SPU) while the forward primer that bridges the spliced ‘intron’ (dash lines) was used to specifically amplify the spliced form (SPS) with the common reverse primer. (B) Testing the specificity of the primers in (A) with PCR using the plasmid DNA harboring the unspliced and spliced sequences of OsbZIP74, respectively. (C) Time course experiment of the OsbZIP74 splicing in response to ER stress. (D) OsIRE1-regulated splicing of OsbZIP74. The expression level of OsIRE1 was checked with RT–PCR in OsIRE1 knock-down (KD) and overexpression (OE) transgenic plants. The specific primers assay was employed to evaluate the role of OsIRE1 in OsbZIP74 splicing. Rice roots were treated with DTT and TM with different time and the SPU and SPS primers were used for the assays. Actin primers were used as the control.

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Figure 1. Splicing of Rice bZIP74 mRNA.

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with higher accuracy. Both TM and DTT induced the splicing in rice roots, and a slight increase in the unspliced form was also detected after stress treatments (Figure 2C).

Rice bZIP74 mRNA Splicing Is Dependent on Rice IRE1

Rice bZIP74 mRNA Splicing Produces the Active Form of Transcription Factor bZIP74 The unspliced form of OsbZIP74 encodes 304 AA protein residues with a putative transmembrane domain near the C-terminus, which is well conserved among plant homologs (Figure 1C and Supplemental Figure 3). Splicing out 20 nucleotides generates a frame shift that results in the loss of 87 AA C-termini including the transmembrane domain but a gain of 44 AA protein residues with no predictable hydrophobic region. The new C-terminus in the spliced form of OsbZIP74 also contains additional putative bipartite nuclear localization signal (NLS) RRKR (Figure 1C). In order to know the subcellular localization of the unspliced and spliced form of OsbZIP74, yellow fluorescence protein (YFP) fusion proteins were produced in tobacco epidermis cells and observed under laser confocal microscopy. The full-length unspliced form YFP–bZIP74(P) is localized outside the nucleus while the spliced form YFP– bZIP74(A) and the truncated form that lacks the transmembrane domain and C-terminus, YFP–bZIP74(D), are largely localized in the nuclei (Figure 3A). The transcriptional activation activity was tested in yeast when the unspliced OsbZIP74(P), truncated OsbZIP74(D), and the spliced OsbZIP74(A) were fused to GAL4 DNA binding domain, respectively. Compared to the empty vector control (only containing GAL4 DNA binding domain), the OsbZIP74(P), OsbZIP74(D), and OsbZIP74(A) fusion proteins all had the transcriptional activation

Figure 3. Subcellular Localization and Transcriptional Activation Activity of OsbZIP74. (A) Protein localization of different YFP fusion forms of full-length unspliced OsbZIP74(P), truncated OsbZIP74(D), and the spliced OsbZIP74(A). Arrow heads point to the nuclei. Bar = 50 lm. (B) Transcriptional activation activity assay in yeast cell for the OsbZIP74(P), OsbZIP74(D), and OsbZIP74(A). Series of yeast cell dilution were used in the study. (C) Regulation of the rice BiP (LOC_Os08g09770) promoter by ER stress and different forms of OsbZIP74. RT–PCR was used for detection of OsBiP expression in rice roots in responding to DTT treatment (left) and transient expression assays were carried out in Arabidopsis leaf protoplasts (right). Instead of DTT treatment, effector plasmids including empty vector (control) and OsbZIP74(D) and OsbZIP74(A) were co-transformed with the reporter plasmid, respectively. Reporter activity was ratio of the BiP-promoter-linked firefly luciferase activity divided by the constitutive renilla luciferase activity. Relative reporter activity represents the reporter activities relative to the basal expression that was obtained from co-transformation with the effector vector control and reporter vector control. Bars indicate the standard errors (n = 3).

activity in terms of turning on HIS3 and LacZ reporter genes (Figure 3B). The transmembrane domain in OsbZIP74(P) did not affect the activation activity in yeast, most probably because of the strong NLS in the yeast GAL4 DNA binding domain. The OsbZIP74 (P) yeast cells grew even faster on -TRP-HIS plates than

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Rice has only one IRE1 homolog, IRE1 (LOC_Os07g28820, or Os07g0471000 in Rice Annotation Project Database), which is ER localized, and the N-terminal domain could substitute for Ire1p in yeast cells and the C-terminal fragment of OsIRE1 has auto-phosphorylation activity in vitro (Okushima et al., 2002). Rice IRE1 has an N-terminal predicted signal peptide and a transmembrane domain in the middle. Although the N-terminal region sequences are quite divergent, the C-terminal protein kinase domains and the globular kinaseextension nuclease (KEN) domains are well conserved in yeast, humans, Arabidopsis, and rice (Supplemental Figure 2). In order to know whether the rice IRE1 was responsible for the observed OsbZIP74 splicing, transgenic rice plants with downregulation and up-regulation of OsIRE1 were generated. The OsIRE1 expression was reduced in the knock-down transgenic plants (IRE1KD-6) and enhanced in the overexpression transgenicplants (IRE1OE-2)(Figure2D, upper). Specificprimerassays were employed to compare the OsbZIP74 splicing in OsIRE1 knock-down and overexpression plants along with wild-type control plants under ER stress conditions. Knock-down of OsIRE1 expression suppressed the induction of OsbZIP74 splicing, while overexpression of OsIRE1 constitutively activated the OsbZIP74 splicing under non-stressed conditions (Figure 2D, lower).

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Rice bZIP74 mRNA Splicing Is Induced by Environmental Stresses Previous results have demonstrated the association of UPR with many abiotic stress and biotic stress responses in Arabidopsis (Liu and Howell, 2010b). In order to know which environmental stress could promote the rice bZIP74 mRNA splicing, a series of abiotic stresses including low (4
C) and high (45
C) temperature, high salt (NaCl), osmotic stress (PEG), oxidative stress (H2O2), and plant hormone (ABA, SA, and JA) treatments were applied to rice roots. Like the ER stress-inducing agent DTT, heat stress and SA treatment induced the splicing of OsbZIP74, while salinity, osmotic stress, oxidative stress, and stress hormone ABA and JA did not in our experiments (Figure 4A). Time-course experiments were carried out for DTT, heat, and SA treatments. Interestingly, the splicing reaction was very quick and the spliced form was readily detected within 5 min of treatment (Figure 4B).

Figure 4. Rice bZIP74 Splicing under Stress Conditions. (A) Rice roots were subjected to different abiotic stresses or phytohormone treatments and the specific primers assay was used to detect the OsbZIP74 splicing. (B) Time course experiment of OsbZIP74 splicing. RNA from rice roots treated with DTT, heat, or SA at different time points was analyzed with a specific primers assay. Actin was used as the control.

Rice bZIP74 mRNA Splicing Is Developmentally Regulated The transcript of OsIRE1 ubiquitously accumulates in various organs and is not induced by TM (Okushima et al., 2002). We were interested in knowing whether OsbZIP74 splicing is regulated under physiological growth conditions. When grown in ½ MS solid medium in well-controlled growth conditions, OsbZIP74 splicing was not detected. However, when plants were grown in the field condition, OsbZIP74 splicing was readily observed in mature roots and leaf sheathes, panicles before heading and grains at filling stages (Figure 5).

DISCUSSION The UPR is one of the protein quality control steps for ensuring the correct protein folding in the ER. The comparable human IRE1a-XBP1_ pathway for ER stress signaling was recently reported to be preserved in dicot plant Arabidopsis (Deng et al., 2011; Nagashima et al., 2011). In the current study,

Figure 5. Developmental Regulation of Rice bZIP74 Splicing. Different rice tissues were sampled from different growth stages and the specific primers assay was used to detect the OsbZIP74 splicing. Actin was used as the control.

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OsbZIP74(D) and OsbZIP74(A) yeast cells, which could be explained by the fact that OsbZIP74(D) and OsbZIP74(A) yeast cells grew more slowly than OsbZIP74(P) yeast cells and the control on -TRP plates. It is possible that the OsbZIP74(D) and OsbZIP74(A) had toxicity in yeast while OsbZIP74(P) did not. In fact, when the growth rate difference was potentially eliminated in the X-gal assay, OsbZIP74(P), OsbZIP74(D), and OsbZIP74(A) had similar activation activity. The transcriptional activation activity was also tested in the dual-luciferase assay in the Arabidopsis leaf protoplasts. The promoter sequences of the OsBiP (LOC_Os08g09770 or Os08g0197700) gene whose expression was up-regulated by DTT in the rice roots (Figure 3C) were placed before the firefly luciferase gene to make the reporter. Various effectors were co-transformed with the reporter in the protoplasts and it was found that both OsbZIP74(D) and OsbZIP74(A) could turn on the OsBiP promoter and that OsbZIP74(D) had higher transcriptional activity than OsbZIP74(A) in protoplasts (Figure 3C).

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3-D structure of the plant IRE1s with their mRNA substrates bound in the KEN domain is awaited for the further understanding of the interaction between the IRE1 enzyme and the mRNA target, and for its comparison to yeast and mammalian counterparts. In the IRE1 splicing, different sizes of nucleotides are excised from yeast to plants according to the distances between the two stem-loop structures (Figure 6A and 6B). Even in plants, three more nucleotides are spliced out or are predicted to be spliced out in dicot plants than that in monocot plants (Figure 6B). Nevertheless, the spliced nucleotides encode hydrophobic amino acids in all the plant species that have the sequences available (Figure 6C and Supplemental Figure 3). The hydrophobic region (HR) of unspliced XBP1 protein in the C-terminus was reported to have the trend to be targeted to the ER membrane, and the HR anchoring was thought to improve the XBP1 mRNA splicing efficiency by bringing the mRNA target to IRE1 enzyme during the protein translational process on the ribosome in the ER vicinity (Yanagitani et al., 2011). The HR of plant XBP1 functional homologs (Figure 6C) might have a similar role in IRE1 mRNA splicing. The Leucine rich repeat LxxxLxxLxL is the prototypic nuclear export signal (NES) that interacts with the exportin to prevent nuclear localization (Macara, 2001). The conserved HR in plant species has the consensus motif LxxxxLxLxxLL in dicots and LxxxLxLxxLL in monocots (Figure 6C). Whether this Leucine-rich consensus sequence is a new NES that prevents the unspliced form of OsbZIP74 orthologs from entering the nucleus needs further experimental testing. Nonetheless, the spliced form of rice bZIP74 has an additional nuclear localization signal (NLS) in the new C-terminus, although all of the plant bZIP74 orthologs have the NLS in their bZIP binding domain in the N-termini (Supplemental Figure 3); indeed, the truncated form of OsbZIP74 has the ability to enter the nucleus (Figure 3A). The IRE1 splicing in mammals produces the active form of the transcription factor by adding the transcriptional activation domain (TAD) in the newly formed longer C-terminus (Figure 6A). However, the rice bZIP74 and Arabidopsis bZIP60 already have TAD in their N-termini and the spliced forms have shorter C-termini. The newly added C-termini do not contribute to either nucleus-localization or transcriptional activation activity. In protoplasts without ER stress, the transcriptional activation activity of the spliced form is lower than that of the truncated form, which suggests that the new C-terminus of OsbZIP(A) might have the regulatory effect on the transcriptional activity in vivo under ER stress conditions. Splicing of HAC1 in yeast also expands the C-terminus but does not cause the open reading frame shift (Figure 6A). The unspliced 252 nucleotides ‘intron’ in the 3’ end of yeast HAC1 mRNA anchors to the 5’-UTR region of HAC1 mRNA, which inhibits the HAC1 protein translation. The abiotic and biotic stresses are great threats to agriculture worldwide. Protein folding in the ER is such a fastidious process that any environmental stress has the potential to break the balance between protein folding demands and

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our results demonstrated the conservation of a similar pathway in the monocot plant rice, one of the most important crops with a fully sequenced genome. First, we provided evidence for the existence of rice bZIP74 mRNA splicing during ER stress response triggered by TM or DTT treatment. Subsequently, we showed the regulation of rice bZIP74 mRNA splicing by IRE1. In addition, we demonstrated that the rice bZIP74 mRNA splicing produced a nucleus-localized active form of bZIP74 transcription factor. We also showed evidence for the association of bZIP74 mRNA splicing with heat stress response and SA treatment; this plant hormone is very important for systemic acquired resistance (SAR) against pathogens or parasites. IRE1 plays essential important roles in the UPR in yeast and mammals by splicing the HAC1 and XBP1 mRNA, respectively (Sidrauski and Walter, 1997; Yoshida et al., 2001). Although there are no existing close orthologs of HAC1 or XBP1 in the sequenced Arabidopsis and rice genomes, the Arabidopsis bZIP60 and rice bZIP74 serve as the functional homologs in the ER stress response and their activations require the function of IRE1, which is similar to the counterparts in yeast or mammals. Yeast has only one IRE1 but mammals have two: IRE1a and IRE1b. IRE1b has been shown to be involved in UPR through _ _ regulation of mRNA decay (Iwawaki et al., 2001). Although Arabidopsis encodes two IRE1s, IRE1A and IRE1B, both are reported to be functional in AtbZIP60 mRNA splicing (Deng et al., 2011; Nagashima et al., 2011). The rice has only one IRE1 and here we demonstrate its involvement in OsbZIP74 mRNA splicing. It seems that the IRE1b pathway has not been _ evolved in plants such as monocot rice and dicot Arabidopsis. The protein kinase and endoribonuclease domains are well conserved at protein sequence level among yeast, mammals, and plants; however, the N-terminal region where the ER stress-sensing domain should be localized is quite divergent (Supplemental Figure 2). It is possible that the 3-D structure formed by the N-terminus amino acids, rather than the secondary structure, senses the ER stress or interacts with the unfolded proteins. Previously, the ER-resident chaperone BiP was thought to be responsible for detecting the unfolded proteins, and releasing BiP from IRE1 might activate IRE1 (Bertolotti et al., 2000). Recently, the direct binding of the unfolded proteins by the core ER-lumenal domain (cLD) of IRE1 in yeast cells was reported (Gardner and Walter, 2011). The crystal structure of Arabidopsis or rice IRE1 luminal domain is needed to clarify the N-termini divergence. The two stem-loop structures provide the physical basis and cleavage specificity for IRE1 function (Oikawa et al., 2010), and such a structure is also predicted to be conserved in 16 plant species (Supplemental Figure 6 in Nagashima et al., 2011). Interestingly, the first stem is shorter and the second loop is larger in plants than in yeast or mammals; the spacer between two stem-loop structures in rice is the shortest (Figures 1A and 6B). It is reasonable to predict that the catalytic surface that binds the mRNA target in the RNase domain of plant IRE1s should be different from that in yeast or mammals (Lee et al., 2008; Korennykh et al., 2009). The

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(A) Comparison of IRE1 splicing in yeast, worm, mammals, and plants. The encoded open reading frame (ORF) of the unspliced and spliced form is beneath the cDNA, respectively. The spliced ‘intron’ sequences and their encoding amino acids are highlighted in red. Some important nucleotides and the respective positions are also depicted. The number of spliced nucleotides is presented beside the arrow. (B) Comparison of the mRNA splicing between monocots and dicots plants. Human (H. sapiens) partial XBP1 mRNA was also listed below. The nucleotides in the stems and the conserved nucleotides in the loops of the stem-loop structures are highlighted in red and blue, respectively, and the (predicted) cleaved sequences are boxed. (C) Comparison of the putative transmembrane domains of the unspliced bZIP proteins between monocots and dicots plants: castor

(R. communis) RcbZIP60 (XP_002510740), poplar tree (P. trichocarpa) PtbZIP60 (XP_002307824), Arabidopsis (A. thaliana) AtbZIP60 (AT1G42990), tomato (S. lycopersicum) SlbZIP60 (AK323903), potato (S. Chacoense) ScbZIP60 (ACB32232), pepper (C. annuum) CabZIP60 (AAX20030), tobacco (N. tabacum) NtbZIP60 (AB281271), soybean (G. max) GmbZIP68 (ABI34650), sorghum (S. bicolor) SbbZIP60 (XP_002437297), maize (Z. mays) ZmbZIP60 (ACR36817 ), Switch grass (P. virgatum) PvbZIP60 (60301256), wheat (T. aestivum) TabZIP60 (AK330972), barley (H. vulgare) HvbZIP60 (BAJ96708), and rice (O. sativa) OsbZIP74 (Os06g41770). The conserved leucine amino acids are highlighted in red.

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Figure 6. Conservation and Divergence of IRE1-Regulated bZIP mRNA Splicing.

capability. The UPR helps to establish the new balance by either improving the protein folding efficiency or accelerating the degradation of the misfolded/unfolded proteins. The UPR has been reported to be activated not only by ER stress agents like TM and DTT, but also by environmental stresses such as heat stress and salt stress (Liu et al., 2008; Liu and Howell, 2010b). The Arabidopsis bZIP60 splicing is initiated by heat stress (Deng et al., 2011) and rice bZIP74 splicing is also induced by heat stress and with even faster response. It is well known that heat stress prevents protein folding and promotes protein denaturing in the cytosol, and the hallmark of heat stress response is the activation of heat shock transcription factors (HSFs) and up-regulation of heat shock proteins (HSPs)/ molecular chaperones. It is possible that heat stress has partial effects on protein folding/denaturing in the ER, therefore triggering the UPR by activation of the IRE1 splicing of bZIP60/ bZIP74 mRNA, and/or proteolytic activation of Arabidopsis bZIP28 (Gao et al., 2008) and its orthologs (OsbZIP16 and OsbZIP17) in rice. SAR is a much conserved induced systemic resistance across diverse plant families. It is a ‘state of enhanced defense capability’ elicited by environmental stimuli such as virulent, avirulent, and non-pathogenic microbes or artificially with chemicals such as SA whereby the plant’s innate defenses are potentiated against subsequent biotic challenges (van Loon et al., 1998; Vallad and Goodman, 2004). The coordinated accumulation of SA is essential for the SAR and any disruption in the plant’s ability to accumulate SA results in the loss of pathogenesis-related gene expression and attenuation of the SAR response, when pathogens are used for induction (Gaffney et al., 1993; Lawton et al., 1995). Interestingly, the rice bZIP74 mRNA splicing is induced in roots by SA treatment but not JA treatment (Figure 4), indicating that the bZIP74 is specifically activated during SAR. Using microarray analysis, it is reported that, besides the NPR1-regulated pathogenesisrelated (PR) genes, several genes encoding the secretory pathway proteins were induced in the SAR, and some of the UPR components were required for the full induction of those genes and resistance (Wang et al., 2005). Tateda and colleagues found that silencing the rice bZIP74 ortholog in tobacco plant (Nicotiana benthamiana) made it more susceptible to infection with soil bacterium (Pseudomonas cichorii) (Tateda et al., 2008). Recently, the expression of rice bZIP74 ortholog was reported to be elevated in tobacco (Nicotiana

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METHODS Plant Growth and Stress Treatments The rice (subsp. Nipponbare or Kitaake) seeds were sown on 1.5% Agar solidified half-strength MS salt supplemented with 30 g l 1 sucrose. The seedlings grew in a controlled growth room at 30 6 2
C with a photoperiod of 16-h light and 8-h dark. Two-week-old seedlings were used for stress treatments and RNA extraction. For various stress treatments, the seedlings were transplanted to ½ MS liquid medium with specific concentration of reagents as follows: DTT 2 mM, TM 5 lg ml 1, PEG4000 15%, NaCl 250 mM, ABA 0.1 mM, SA 1 mM, JA 0.1 mM, H2O2 10 mM. For heat and cold stress treatments, the seedling roots were immerged in ½ MS liquid medium pre-warmed or pre-chilled to 45 or 4
C, respectively. The seedlings transplanted to ½ MS liquid medium were used as controls. For splicing analysis at different developmental stages, rice plants were grown in a lowland paddy field in Shanghai, China, and sampled around heading or after heading.

mRNA Splicing Assay and RT–PCR Total RNAs were extracted from various tissues. One microgram total RNA was applied for first-strand cDNA synthesis using Moloney murine leukemia virus (M-MLV, Invitrogen) reverse transcriptase according to the manufacturer’s instructions. For RT–PCR analysis, the rice actin1 was used for normalization of cDNA quantity. For flanking primer assay, primer pairs used were: 5’-GTTGAAGGATAGGCCTGTCGG-3’ and 5’-ACCGGGAG-

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TAGGCACACGAT-3’. For specific primer assays, primer pairs used were: 5’-GAAATGGAGACTGCCAAGACC-3’ and 5’-TTGGCAATCCACATCTGCTG-3’ for rice actin1; 5’-GCCGTACTCACGGAAACCCT-3’ and 5’-CTAGCAAGCAGCTGCTGCTAAA-3’ for unspliced form of OsbZIP74; 5’-CCATGCAGGAGTCTGCCGCTG-3’ and 5’-CTAGCAAGCAGCTGCTGCTAAA-3’ for spliced form of OsbZIP74. For rice BiP gene (LOC_Os08g09770) expression assay, primer pairs used were: 5’-GAAGACGCAGGTGTTCACCAC-3’ and 5’GCGTGTTCTTGATGTTGTAG-3’.

Transgenic Rice Generation OsIRE1 knockdown rice plants were generated by RNA interference. For the expression of intron-containing hairpin RNA, OsIRE1 cDNA region was amplified by PCR with primers: 5’GAGCTCTAGATCCTTCGGCACAACTTCTAAAGCTG-3’ and 5’AGATCTAGTCAGGTAAAAGATTTGATTTACTATAAG-3’. The cDNA region was linked to the intron sequence for the rice aspartic protease gene. This construct was linked to the ubiquitin promoter and inserted into a p35SHPTAg7GW binary vector (Wakasa et al., 2006) by using Gateway LR clonase reaction (Invitrogen). For OsIRE1 overexpression lines, OsIRE1 cDNA was amplified by PCR with primers 5’-AGATCTGCAAAGCAAAGCAGCGAATCCGGCTTCAC-3’ and 5’-TCTAGAAGTCAGGTAAAAGATTTGATTTACTATAAG-3’. The cDNA was linked to the ubiquitin promoter, and inserted into a p35SHPTAg7GW binary vector that same as OsIRE1 knockdown construct. Transgenic rice plants (Oryza sative L. cv. Kitaake) were generated by Agrobacterium-mediated transformation.

Agrobacterium Infiltration and Protein Subcellular Localization CaMV 35S promoter and NOS terminator were cut from pBI121 by HindIII, XbaI and SacI, EcoRI, respectively, and inserted into pCAMBIA1300 successively to generate the pCAMBIA1300– CaMV35S–NOS construct. YFP coding sequence without stop codon was amplified and inserted into the pCAMBIA1300– CaMV35S–NOS between XbaI and BamHI to produce the pCAMBIA1300–CaMV35S–YFP–NOS vector. To construct the YFP fusion vectors for subcellular localization, bZIP74(D), bZIP74(A), and bZIP74(P) cDNA were cut from pGreenII 62-SK recombinant plasmids and inserted into the pCAMBIA1300– CaMV35S–YFP–NOS vector between BamHI and KpnI. The primer pair used for YFP were: 5’-ATATCTAGAATGGTGAGCAAGGGCGAGGA-3’ and 5’- TATGGATCCAGATCTCTTGTACAGCTCGT-3’. These vectors were transformed to Agrobacterium (Agrobacterium tumefaciens) strain EHA105 through a freeze–thaw method. The Agrobacterium containing different vectors were incubated in LB overnight shaking at 250 rpm, harvested, and re-suspended in 10 mM MgSO4 to an ultimate concentration of OD600 = 0.5. Agrobacterium infiltration method was applied to transfer the constructs to tobacco (Nicotiana benthamiana) leaf cells; the infiltrated plants were placed in a greenhouse for an additional 3 d before detection of YFP fluorescence with a confocal microscope (Zeiss LSM A710).

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benthamiana) infected with potato virus X (PVX). Silencing that ortholog led to the suppression of BiP and SKP1 transcript levels (Ye et al., 2011). Previously, Lee et al. (2006) had shown that the rice bZIP74 ortholog in pepper (Capsicum annuum) called CabZIP1 was up-regulated during an attack of pathogens (Xanthomonas campestris pv. Vesicatoria or Pseudomonas fluorescens). It is evident that the UPR is activated in SAR and that up-regulation and activation of bZIP74 orthologs are common as part of that process in plants. The UPR plays important roles in plant development, especially in tissues with high secretory activity (Vitale and Boston, 2008), and the activation of bZIP60 was observed in Arabidopsis anthers (Iwata et al., 2008). Interestingly, we have detected bZIP74 splicing in rice reproductive tissues such as young panicles and developing seeds. Our observation adds more information on the association of UPR with high secretory activity. Indeed, the transcript levels of OsbZIP74 and other UPRrelated genes were up-regulated in transgenic rice plants with high-level accumulation of seed storage proteins in previous experiments (Wakasa et al., 2011). The OsbZIP74 splicing was not detected in the panicle at anthesis, indicating the appearance of splicing in panicle before heading was most likely related to flower development. OsbZIP74 splicing was high in field-grown mature rice roots, emphasizing the important roles of the IRE1–bZIP pathway for crops in reality.

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Transcriptional Activation Activity Assay in Yeast For transcriptional activation activity assay in yeast, bZIP74(D), bZIP74(A), and bZIP74(P) cDNA were inserted into pGBKT7 (Clontech) between NdeI and BamHI. The forward primers were: 5’-ATACATATGATGGATGTAGAGTTCTTCGCC-3’ for three amplicon, reverse primers were: 5’-ATAGGATCCCTAAGACTCCTGCATGGCTGTGG-3’ for bZIP74(D), 5’-ATTGGATCCCTAGCAAGCAGCTGCTGCTAAA-3’ for bZIP74(A) and bZIP74(P) cDNA. Plasmids were transferred to yeast cells by the standard LiAc–PEG-mediated transformation procedure. Transcriptional activation activity was evaluated based on the activation of the HIS3 and LacZ reporters.

Protoplast Transformation and Luciferase Activity Assay

SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online.

FUNDING This project is funded by the National Natural Science Foundation of China (31070233, 31171157), Shanghai Pujiang Talent Program (11PJ1400700), and partly supported by the National Basic Research Program of China (973 Program, 2012CB910500), all granted to J.X.L.

ACKNOWLEDGMENTS We would also like thank Drs Yuhya Wakasa and Fumio Takaiwa for providing the OsIRE1 transgenic rice seeds. No conflict of interest declared.

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