An alternatively spliced heat shock transcription factor, OsHSFA2dI, functions in the heat stress-induced unfolded protein response in rice.

Plant Biology ISSN 1435-8603

RESEARCH PAPER

An alternatively spliced heat shock transcription factor, OsHSFA2dI, functions in the heat stress-induced unfolded protein response in rice Q. Cheng, Y. Zhou, Z. Liu, L. Zhang, G. Song, Z. Guo, W. Wang, X. Qu, Y. Zhu & D. Yang State Key Laboratory of Hybrid Rice, Engineering Research Center for Plant Biotechnology and Germplasm Utilization, Ministry of Education, College of Life Sciences, Wuhan University, Wuhan, China

Keywords Heat shock transcription factor; heat stress; Oryza sativa (rice); unfolded protein response. Correspondence D. Yang, State Key Laboratory of Hybrid Rice, Engineering Research Center for Plant Biotechnology and Germplasm Utilization, Ministry of Education, College of Life Sciences, Wuhan University, 430072 Wuhan, China. E-mail: [email protected] Editor E. Flemetakis Received: 24 May 2014; Accepted: 9 September 2014 doi:10.1111/plb.12267

ABSTRACT As sessile organisms, plants have evolved a wide range of defence pathways to cope with environmental stress such as heat shock. However, the molecular mechanism of these defence pathways remains unclear in rice. In this study, we found that OsHSFA2d, a heat shock transcriptional factor, encodes two main splice variant proteins, OsHSFA2dI and OsHSFA2dII in rice. Under normal conditions, OsHSFA2dII is the dominant but transcriptionally inactive spliced form. However, when the plant suffers heat stress, OsHSFA2d is alternatively spliced into a transcriptionally active form, OsHSFA2dI, which participates in the heat stress response (HSR). Further study found that this alternative splicing was induced by heat shock rather than photoperiod. We found that OsHSFA2dI is localised to the nucleus, whereas OsHSFA2dII is localised to the nucleus and cytoplasm. Moreover, expression of the unfolded protein response (UNFOLDED PROTEIN RESPONSE) sensors, OsIRE1, OsbZIP39/OsbZIP60 and the UNFOLDED PROTEIN RESPONSE marker OsBiP1, was up-regulated. Interestingly, OsbZIP50 was also alternatively spliced under heat stress, indicating that UNFOLDED PROTEIN RESPONSE signalling pathways were activated by heat stress to re-establish cellular protein homeostasis. We further demonstrated that OsHSFA2dI participated in the unfolded protein response by regulating expression of OsBiP1.

INTRODUCTION Plants are frequently exposed to various biotic and abiotic stresses, including extreme temperatures, drought, salinity and heavy metal stresses. These stresses perturb cellular protein homeostasis and lead to incorrect protein synthesis and degradation. A range of mechanisms has evolved in plants to cope with these environmental stresses. Heat stress, one of the most common stresses, triggers the heat stress response (HSR), which triggers rapid production of heat shock proteins (HSPs) to facilitate the folding of accumulated unfolded proteins (Morimoto 1998; Hartl & Hayer-Hartl 2002; Baniwal et al. 2004; Wang et al. 2004; Kotak et al. 2007). HSP expression is primarily regulated by heat shock transcription factors (HSFs; Wu 1995; Morimoto 1998; Nover et al. 2001; Baniwal et al. 2004; Miller & Mittler 2006; Kotak et al. 2007), which recognise conserved promoter elements [heat shock elements (HSEs)] comprising at least three continuous inverted repeats of the sequence 50 -nGAAn-30 (Pelham 1982; Nover 1987; Yamamoto et al. 2005, 2009; Yamamoto & Sakurai 2006; Sakurai & Takemori 2007). The basic structure of HSFs is conserved throughout the eukaryotic kingdom, comprising a DNA-binding domain (Damberger et al. 1994), an oligomerisation domain (HR-A/B regions; Peteranderl et al. 1999) and a C-terminal activation domain (AHA motif; Treuter et al. 1993). Based on their DNA-binding domain and flexible linkers in the HR-A/B

regions, plant HSFs can be categorised into three classes: A, B and C. The AHA motif is essential for transcriptional activation activity and is exclusively found in class A HSFs, whereas the absence of AHA motifs in classes B and C results in a lack of transcriptional activation (Nover et al. 2001; Bharti et al. 2004). HSFA2 is the dominant HSF in thermotolerant cells of tomato and Arabidopsis (Nishizawa et al. 2006; Charng et al. 2007; von Koskull-Doring et al. 2007; Yokotani et al. 2008; Chan-Schaminet et al. 2009). Under stress conditions, the protein folding and degradation machinery in the endoplasmic reticulum (ER) lumen become overwhelmed, as the demand for protein folding and degradation exceeds the capacity of the system, which leads to the accumulation of unfolded and/or mis-folded proteins in the ER and triggers the ER stress response (Malhotra & Kaufman 2007). The unfolded protein response is then activated to mitigate this ER stress by inducing a set of genes and pathways to enhance protein folding, accelerate ER-associated degradation and/or reduce cellular protein synthesis (Harding et al. 1999; Rutkowski & Kaufman 2004; Ron & Walter 2007; Liu & Howell 2010b). The unfolded protein response is conserved in eukaryotic organisms. Two classes of ER stress sensors that function in the unfolded protein response signalling pathway have been reported in rice: OsIRE1 (Nijhawan et al. 2008; Wakasa et al. 2011; Hayashi et al. 2012; Lu et al. 2012) and OsbZIP39/OsbZIP60 (Takahashi et al. 2012). These sensors can transduce the

Plant Biology 17 (2015) 419–429 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

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Alternatively spliced OsHSFA2dl in rice

Cheng, Zhou, Liu, Zhang, Song, Guo, Wang, Qu, Zhu & Yang

stress signal from the ER to the nucleus and are involved in transcriptional regulation (Liu et al. 2007a; Iwata et al. 2008; Tajima et al. 2008; Che et al. 2010; Liu & Howell 2010a; Deng et al. 2011). The signalling mechanisms of the unfolded protein response in rice also involve the splicing of OsbZIP50 (also known as OsbZIP74; Hayashi et al. 2012; Lu et al. 2012; Takahashi et al. 2012). Additionally, OsBiP1, the most abundant ER chaperone protein, is a unfolded protein response marker that assists in protein folding in the ER (Brandizzi et al. 2003; Foresti et al. 2003; Noh et al. 2003; Mainieri et al. 2004). However, the signalling pathway crosstalk between different environmental stimuli and the unfolded protein response is not well understood. In this work, we found that OsHSFA2d was alternatively spliced to encode two different proteins with different expression profiles, subcellular localisations, and functions. We further analysed the connection between environmental stress (heat stress and long-day light stress) and the unfolded protein response. The results show that heat stress triggers the unfolded protein response and that OsHSFA2d functions as a transcriptional activator of OsBiP1, indicating that OsHSFA2d connects the HSR and unfolded protein response signalling pathways by regulating expression of the unfolded protein response marker OsBiP1. MATERIAL AND METHODS Material Nongken 58S (Oryza sativa L. ssp. japonica), three other japonica varieties, Nongken58 (NK58), Nipponbare (NIPP) and Taibei 309 (TP309), and four indica varieties, Aizizhan (AZZ), Minghui 86 (MH86), Teqing (TQ) and 93-11, were used in this study. All of the plants were grown in a growth chamber under a 12-h light/12-h dark cycle at 26 °C. The heat-stress (HS) and long-day (LD) treatments were performed when the seedlings had more than seven leaves. For heat-shock expression analysis, the leaves of seedlings were harvested after 0, 15, 30 and 60 min of heat shock at 42 °C, followed by 4 h of recovery at 26 °C. For the LD treatment expression analysis, leaves of seedlings were harvested after 0, 1, 2 and 3 days of LD (16-h light/8-h dark) treatment, followed by 1 day of recovery under a 12-h light/12-h dark cycle. For transient expression, a suspension of tobacco Bright Yellow-2 (TBY-2) cells was cultured at 27 °C, as previously described (De Pinto et al. 2002). Rice protoplast cells derived from 10-day-old etiolated rice seedlings were used for the subcellular localisation study. Briefly, rice seeds were soaked in water for 24 h, and grown in darkness for 10 days at 28 °C after seed germination. Rice protoplast preparation was described previously (Yang et al. 2007; Zhang et al. 2011). RNA isolation and cDNA synthesis Total RNA was isolated from young leaves using the TRIzol Reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The total RNA was then treated with RNase-free DNase I (New England Biolabs, Hitchin, UK) to remove trace DNA contamination. The total RNA (2 lg) was used as template, and an oligo (dT) primer was used as 420

primer for first-strand cDNA synthesis with M-MLV reverse transcriptase (Promega, Madison, WI, USA). Reverse transcription PCR The first-strand cDNA was used for reverse transcription polymerase chain reaction (RT-PCR) assays. The cycling conditions were 94 °C for 5 min, followed by 25 cycles of a 30-s denaturation at 94 °C, a 30-s annealing at 55 °C and a 30-s extension at 72 °C, with a final 5-min extension at 72 °C. The PCR amplicons were analysed using 2% agarose electrophoresis. The amount of first-strand cDNA used for the RT-PCR was determined by the expression of Osactin1. The primer sets used for these experiments are listed in Table S1. Quantitative RT-PCR The quantitative RT-PCR (qRT-PCR) was performed using the StepOne Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) with Platinum SYBR Green qPCR SuperMixUDG (Invitrogen). Each reaction was performed in triplicate, and an average threshold cycle (Ct) was used to determine the fold change in gene expression. The expression level of Osactin1 was used as an internal control in all of experiments, and negative controls with no template DNA were used to ensure that primer dimers did not interfere with the amplification. The qRT-PCR experiments were performed with at least three technical repeats and three biological repeats. The primer sets used for the experiments are listed in Table S1. Transient expression assay For construction of the effector plasmids PCaMV 35S:OsHSFA2dI and PCaMV 35S:OsHSFA2dII, the OsHSFA2dI and OsHSFA2dII cDNAs were amplified by PCR and cloned into the pMF6 vector, a plasmid containing the CaMV 35S promoter and the Nos terminator without a coding region (Goff et al. 1990). To create the reporter constructs POsAPX2:GUS, POsIRE1:GUS, POsBiP1: GUS and POsbZIP60:GUS, promoter regions of approximately 1.5 kb in length that included the 50 upstream regions were amplified from genomic DNA using PCR. The PCR products were digested with HindIII/SmaI, HindIII/NaeI, SphI/NaeI and SphI and these fragments were then cloned into the Gt13a:GUS vector (Ning et al. 2008) to replace the Gt13a promoter. The pAHC18 vector (Christensen & Quail 1996), which contains the luciferase gene driven by a ubiquitin promoter, was used as an internal control. For the negative control, the effector construct was replaced by the pMF6 vector. All of the primer sets used in this study are listed in Table S1. The TBY-2 suspension cells were transformed with the effector, reporter and internal control constructs using particle bombardment performed with the Biolistic PDC-1000/He instrument (Bio-Rad, Hercules, CA, USA), as previously described (Yang et al. 2001; Sambrook & Russell 2006). After the bombardment, the cells were incubated in the dark at 27 °C for 24 h, frozen immediately in liquid nitrogen, and ground into a powder. The powder was then suspended at room temperature in 1 9 lysis buffer (CCLR; Luciferase Assay System; Promega), followed by further homogenisation, and was then briefly centrifuged to remove cellular debris. The resulting supernatant was used for the luciferase and GUS




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Fig. 3. Expression profiles of OsHSFA2d in different rice varieties. Experiments similar to those described in Fig. 2 were performed, but the leaves of four japonica varieties (including NK58S) and four indica varieties were used as samples. A, B: Expression profiles of OsHSFA2dI in the eight varieties during HS (A) or LD (B) treatment. C, D: Expression profiles of OsHSFA2dII and OsHSFA2dIII in the eight varieties during HS (C) or LD (D) treatment.

decreased gradually (Fig. 6A). In contrast, expression levels of the unfolded protein response sensors and OsBiP1 did not change after LD treatment, indicating that these unfolded protein response-associated genes are not involved in the response to photoperiod (LD) treatment (Fig. 6B). To further investigate whether the splicing event of OsbZIP50 mRNA occurs under HS, PCR was performed using a specific set of primers (Zhang et al. 2013). The results showed that OsbZIP50 splicing was induced after 15 min of 42 °C HS but was not affected by LD treatment (Fig. 6C), which also demonstrates that the unfolded protein response is induced by HS.

The OsHSFA2dI protein acts as an activator of OsBiP1 expression In our study, OsIRE1, OsbZIP39/OsbZIP60 and OsBiP1 were induced with HS (Fig. 6A), implying that a connection exists between the HSR and unfolded protein response signalling pathways. Thus, we analysed promoter regions of these four unfolded protein response-associated genes, and found a consensus sequence characteristic of HSEs within the promoter region of OsBiP1, suggesting that OsBiP1 might be a target gene of OsHSFA2d. To confirm this, we first performed qRT-PCR to

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Fig. 4. The subcellular localisation of OsHSFA2dI and OsHSFA2dII. The subcellular localisation of OsHSFA2dI (A) and OsHSFA2dII (B) in rice protoplast cells. The cells are shown at a magnification of 6009. Bars = 5 lm. The subcellular localisation of OsHSFA2dI (C) and OsHSFA2dII (D) in onion epidermis cells is presented. Magnification of images in C and D are 100 9 (left) and 400 9 (right, bar = 500 lm). Plant Biology 17 (2015) 419–429 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands


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Fig. 1. The three transcripts of OsHSFA2d. A: Genomic DNA or cDNA was used as the template for PCR amplification with one set of primers (P-F, P-R) corresponding to part of the OsHSFA2d gene coding region. The amplified fragments were analysed using 2% agarose gel electrophoresis. B–D: Basic structures of the three forms of the mature OsHSFA2d transcript. The 946-bp intron is removed from the OsHSFA2dI mRNA (B). In contrast, 676 bp (left) and 176 bp (right) are spliced out of the 946 bp intron in the OsHSFA2dII mRNA, leaving 94 bp (middle) as a new exon and introducing a new stop codon (C). Only 676 bp (left) are removed from the OsHSFA2dIII mRNA, introducing the same stop codon as that in the OsHSFA2dII mRNA (D). The additional splice sites are denoted by lowercase letters, and the new exons indicated in bold and uppercase letters. The new stop codons are underlined. DBD, DNA binding domain; HR-A/B, oligomerisation domain; NLS, nuclear localisation signal; NES, nuclear export signal; AHA, activation domain. The amplicon sizes using the primer pairs (P-F/P-R) are presented in panel B. E: OsHSFA2dI and OsHSFA2dII protein sequences. The coding regions of OsHSFA2dII and OsHSFA2dIII are the same and generate the OsHSFA2dII protein. The conserved domains are shown in bold.

under LD treatment (Fig. 2A and B). To further verify these results, two primer sets, one for specific amplification of OsHSFA2dI (P8-F/R; Table S1) and the other for OsHSFA2dII and OsHSFA2dIII (P9-F/R; Table S1) were used for qRT-PCR. These results showed that OsHSFA2dI was induced only by HS, whereas OsHSFA2dII and OsHSFA2dIII were mainly induced by LD and only slightly by HS (Fig. 2C and D). These results confirm that OsHSFA2dI is a specific response to HS, while OsHSFA2dII/III exhibits diverse expression in HS and LD conditions. Taken together, our results show that three OsHSFA2d transcripts are generated by alternative splicing in response to different environmental stimuli: OsHSFA2dI responds to heat stress only and OsHSFA2dII and OsHSFA2dIII mainly respond to LD light stress. To explore the profile of the alternative spliced transcripts in different rice varieties that respond differently to temperature 422

and photoperiod, we investigated expression profiles of the three OsHSFA2d transcripts in four japonica varieties and four indica varieties (Fig. 3). The expression profiles of the OsHSFA2dI gene were similar in all eight rice varieties, while no induction of OsHSFA2dI was found under LD condition (Fig. 3A and B). In contrast, expression profiles of OsHSFA2dII and OsHSFA2dIII response to HS varied greatly in the eight varieties. Two varieties, NK58 and AZZ, with strong photoperiodism had a stronger response to HS, while the other varieties were categorised into weaker responses to HS (Fig. 3C). The tendencies to respond to HS among the varieties was strongly correlated with traits such as strong photoperiodism (NK58 and AZZ), weak photoperiodism (93-11, TP309 and TQ) and no response (NIPP and MH86). However, the expression profiles of OsHSFA2dII and OsHSFA2dIII in response to LD can be divided into two groups: in the first group with four japonica



varieties NK58, Nk58s, TP309 and NIPP, the expression of OsHSFA2dII and OsHSFA2dIII transcripts rapidly peaked after 1 day of LD treatment and then gradually decreased to very low levels (Fig. 3D); while in the second group with all the indica varieties, TQ, AZZ, 93-11 and MH86, a very weak response to LD was observed (Fig. 3D). These results reveal that the expression profiles of OsHSFA2dII and OsHSFA2dIII were likely intrinsic to different subspecies. Taken together, we conclude that the alternative spliced product OsHSFA2dI was consistently and specifically induced by HS in all subspecies, while OsHSFA2dII and OsHSFA2dIII expression is more intrinsically correlated to different subspecies. Furthermore, the expression profile of OsHSFA2dII and OsHSFA2dIII were highly correlated to subspecies of rice, rather than to response to HS. Subcellular localisation of OsHSFA2dI and OsHSFA2dII Our results showed that OsHSFA2d produced two different proteins with alternative splicing. The OsHSFA2dI protein contains all of the conserved domains that are characteristic of class A HSFs (Fig. 1B and E). In contrast, the OsHSFA2dII protein has neither a nuclear localisation signal nor a nuclear export signal (Fig. 1C–E). Because the structures of OsHSFA2dI and OsHSFA2dII are presumably different, we hypothesised that the subcellular localisation of these proteins might also differ. To confirm this hypothesis, the expression constructs of OsHSFA2dI and OsHSFA2dII fused to RFP or GFP were constructed and transformed into rice protoplasts or onion epidermis cells. We found that OsHSFA2dI was localised to the nucleus in rice protoplast cells (Fig. 4A), whereas subcellular localisation of OsHSFA2dII was in both the nucleus and cytoplasm (Fig. 4B). The same results were observed in the onion epidermal cells (Fig. 4C and D). Overexpression of OsHSFA2dI activates OsAP2 gene expression The OsHSFA2dI protein possesses an AHA motif that is essential for class A HSF transcription activator function, whereas OsHSFA2dII lacks this motif (Fig. 1E). Because the structures of OsHSFA2dI and OsHSFA2dII are different, we speculated that these proteins might play different roles in cellular events.


Therefore, we investigated the transcriptional activation activity of OsHSFA2dI and OsHSFA2dII using transient expression assays with the cytosolic ascorbate peroxidase 2 (OsAPX2) gene as a reporter gene, which has been reported to be directly regulated by OsHSFA2 (Nishizawa et al. 2006). The results showed no change in relative GUS activity when the effector OsHSFA2dII was co-expressed with the reporter gene (Fig. 5A). Conversely, relative GUS activity exhibited a six-fold increase when OsHSFA2dI was co-expressed with the reporter gene (Fig. 5A). These results demonstrate that OsHSFA2dI functions as a transcriptional activator, and that OsHSFA2dII has no transcriptional activation activity, consistent with the sequence of the alternative splicing product OsHSFA2dI that encodes a full-length protein. To further verify transcriptional activation activity of OsHSFA2dI, we performed yeast one-hybrid assays with the OsAPX2 gene promoter (Fig. 5B). The one-hybrid assay showed that the yeast strain with the bait reporter gene driven by the OsAPX2 promoter grew well on media containing the AbA antibiotic when co-transformed with the OsHSFA2dI gene (Fig. 5B). These results demonstrate that OsHSFA2dI can activate OsAPX2 gene expression. Heat stress induces the unfolded protein response Protein folding is important for cellular function, and a variety of environmental stresses disturb cellular protein homeostasis, leading to accumulation of unfolded proteins in the ER, which induces the unfolded protein response in eukaryotes. To explore the connection between environmental stresses, such as HS and LD light stress, and the unfolded protein response in rice, we analysed expression profiles of the unfolded protein response sensors OsIRE1, OsbZIP39/OsbZIP60 and OsBiP1 and the splicing of OsbZIP50 mRNA during HS or LD treatments. The qRT-PCR results showed that expression levels of these four genes rapidly increased after 15 min of 42 °C HS treatment; levels then decreased to normal after 4 h of recovery (Fig. 6A), indicating that the unfolded protein response signalling pathways were activated by HS treatment. Interestingly, although the expression level of all four of the unfolded protein response-related genes peaked after 15 min of HS treatment, expression levels of the three unfolded protein response sensors (OsIRE1 and OsbZIP39/OsbZIP60) decreased immediately after peaking, whereas the expression level of the chaperone OsBiP1

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Fig. 2. Expression profiles of OsHSFA2d. Nongken 58S leaves were used as samples. A, B: Expression levels of the three OsHSFA2d transcripts were examined using RT-PCR during HS (A) or LD (B) treatment. C, D: qRT-PCR during HS (C) or LD (D) treatment was performed using two primer sets. OsHSFA2dI represents expression of OsHSFA2dI, whereas OsHSFA2dII+OsHSFA2dIII represents expression of OsHSFA2dII and OsHSFA2dIII. The relative amounts were calculated and normalised to that of Osactin1 mRNA (=100%). Bars represent SD. Plant Biology 17 (2015) 419–429 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

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Fig. 3. Expression profiles of OsHSFA2d in different rice varieties. Experiments similar to those described in Fig. 2 were performed, but the leaves of four japonica varieties (including NK58S) and four indica varieties were used as samples. A, B: Expression profiles of OsHSFA2dI in the eight varieties during HS (A) or LD (B) treatment. C, D: Expression profiles of OsHSFA2dII and OsHSFA2dIII in the eight varieties during HS (C) or LD (D) treatment.

decreased gradually (Fig. 6A). In contrast, expression levels of the unfolded protein response sensors and OsBiP1 did not change after LD treatment, indicating that these unfolded protein response-associated genes are not involved in the response to photoperiod (LD) treatment (Fig. 6B). To further investigate whether the splicing event of OsbZIP50 mRNA occurs under HS, PCR was performed using a specific set of primers (Zhang et al. 2013). The results showed that OsbZIP50 splicing was induced after 15 min of 42 °C HS but was not affected by LD treatment (Fig. 6C), which also demonstrates that the unfolded protein response is induced by HS.

The OsHSFA2dI protein acts as an activator of OsBiP1 expression In our study, OsIRE1, OsbZIP39/OsbZIP60 and OsBiP1 were induced with HS (Fig. 6A), implying that a connection exists between the HSR and unfolded protein response signalling pathways. Thus, we analysed promoter regions of these four unfolded protein response-associated genes, and found a consensus sequence characteristic of HSEs within the promoter region of OsBiP1, suggesting that OsBiP1 might be a target gene of OsHSFA2d. To confirm this, we first performed qRT-PCR to

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Fig. 4. The subcellular localisation of OsHSFA2dI and OsHSFA2dII. The subcellular localisation of OsHSFA2dI (A) and OsHSFA2dII (B) in rice protoplast cells. The cells are shown at a magnification of 6009. Bars = 5 lm. The subcellular localisation of OsHSFA2dI (C) and OsHSFA2dII (D) in onion epidermis cells is presented. Magnification of images in C and D are 100 9 (left) and 400 9 (right, bar = 500 lm). Plant Biology 17 (2015) 419–429 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands



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Fig. 5. Transcriptional activity of OsHSFA2dI and OsHSFA2dII. A: Transcriptional activation of OsHSFA2d was analysed using GUS transient expression assays. Upper panels show schematic diagrams of the effector and reporter constructs. Lower panels show relative levels of GUS activity. B: Transcriptional activation of OsHSFA2dI was analysed using yeast one-hybrid assays. Upper panels show schematic diagrams of the bait and prey constructs, respectively. The bait plasmid P53:AUR1-C, p53 control and pGADT7-Rec AD vectors were used as positive controls. The consensus sequence of the heat shock element (HSE) is shown in bold. “*” indicates 0.05 significance.

examine the expression profile of OsAPX2, which is directly regulated by OsHSFA2d, and compared the expression profiles of the four unfolded protein response-associated genes. OsBiP1 and OsAPX2 exhibited similar expression patterns (Fig. 7A); expression levels of OsBiP1 and OsAPX2 rapidly peaked after 15 min of HS treatment and then gradually decreased to baseline. This suggests that OsBiP1 might be a target gene of OsHSFA2d. To investigate the effects of OsHSFA2dI or OsHSFA2dII on expression of OsBiP1, OsIRE1 or OsbZIP60, we then bombarded TBY-2 suspension cells with the PCaMV 35S:OsHSFA2dI or PCaMV 35S:OsHSFA2dII effector construct and the GUS reporter constructs driven by OsBiP1, OsIRE1 or OsbZIP60 promoters and analysed relative GUS activity in the TBY-2 cells. The relative GUS activity markedly increased 11-fold when the cells were co-expressed with effector OsHSFA2dI and POsBiP1:GUS reporter genes. The results indicate that OsHSFA2dI activated expression of OsBiP1 via binding to the

OsBiP1 promoter (Fig. 7B). In contrast, no GUS relative activity was observed when the GUS reporter gene was driven via either the OsIRE1 promoter or OsbZIP60 promoter, compared to the negative control group (Fig. 7C and D). These results demonstrate that OsHSFA2dI regulates expression of the unfolded protein response marker OsBiP1. To further verify the transcription activation activity of OsHSFA2dI via HSE on the promoter, we replaced GAA with GAC or CAA of the HSE core sequence and conducted yeast one-hybrid assays. The yeast strain with the bait reporter gene driven by the OsBiP1 promoter with core HSE grew well on media containing the AbA antibiotic when co-transformed with the OsHSFA2dI gene (Fig. 7E). However, a 2 bp replacement on HSE caused a decrease of transcription activation activity (Fig. 7E middle panel). These results again demonstrate that OsHSFA2dI can activate OsBiP1 gene expression via binding to the HSE of the OsBiP1 promoter sequence.

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Fig. 6. Expression profiles of unfolded protein responseassociated genes and splicing of OsbZIP50 during HS or LD treatment. Leaf samples used were the same as those indicated in Fig. 2. A, B: Expression profiles of unfolded protein response-associated genes (OsIRE1, OsbZIP60/ OsbZIP39 and OsBiP1) were examined using qRT-PCR during HS (A) or LD (B) treatment. The relative amounts of mRNA were measured and normalised to that of Osactin1 (=100%). Bars represent SD. C: Splicing of OsbZIP50 mRNA during HS or LD treatment. The PCR products were analysed using 2% agarose gel electrophoresis.

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Fig. 7. The effect of OsHSFA2d on the expression of unfolded protein response-associated genes. A: Expression patterns of unfolded protein response-associated genes and OsAPX2; B–D: transcriptional activation of OsHSFA2d on promoters of unfolded protein responseassociated genes is presented. The promoter regions (including the 50 upstream region) of OsBiP1, OsIRE1 or OsbZIP60 were used to show activation activity. E: Transcriptional activation of OsHSFA2dI with yeast onehybrid assays. Three tandem copies of an 861-bp promoter region of OsBiP1 with or without point mutations in the HSE are presented in bait constructs. The HSE sequence is shown in bold; the underlined sequence indicates the HSE mutated bases.

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DISCUSSION The HSFA2 is the dominant HSF in thermotolerant cells. In this work, we found that OsHSFA2d RNA could be alternatively spliced within the conserved introns of the DNA binding domain coding region, and that these splicing events can introduce one or two small exons with premature stop codons, resulting in three alternative splicing products, OsHSFA2dI, OsHSFA2dII and OsHSFA2dIII. A similar phenomenon was recently observed for HSF1 in Medicago sativa (He et al. 2007), HSFA1d and HSFA2 in Arabidopsis (Sugio et al. 2009) and HSFA2a2 in Potamogeton (Amano et al. 2012). The new HSF splice products that contain premature stop codons are degraded through the nonsense-mediated mRNA decay pathway (He et al. 2007; Sugio et al. 2009; Amano et al. 2012). These findings indicate that the alternative splicing of the HSF intron might be a conserved mechanism at the post-transcriptional level for HSF expression regulation in plants. We also found that the three alternatively spliced transcripts encode two proteins: OsHSFA2dI encodes a 357-amino acid protein (OsHSFA2dI) that contains all of the conserved domains exhibited a characteristic of class A HSFs, whereas the other 426

two transcripts encode the same small 113-amino acid protein (OsHSFA2dII), which contains a deficient DNA binding domain and no oligomerisation or C-terminal activation domain. OsHSFA2dI localised to the nucleus and function as a transcriptional activator stimulated by heat stress. In contrast, OsHSFA2dII localised to the nucleus and cytoplasm. Further study revealed that the expression level of OsHSFA2dI is specifically induced by heat shock, while OsHSFA2dII is induced by photoperiod. According to our data, we propose that the OsHSFA2d gene might be constitutively transcribed and spliced to generate the inactive forms of OsHSFA2dII and OsHSFA2dIII, encoding the OsHSFA2dII protein that lacks transcriptional activity. This alternative splicing mechanism in response to environmental stimuli could be the consequence of long-term evolution in higher plants, thereby activating the unfolded protein response signalling pathway to adapt to the environment. The subcellular localisation of HSFA2 has been exhaustively studied in tomato and Arabidopsis (Scharf et al. 1998; Heerklotz et al. 2001; Port et al. 2004; Meiri & Breiman 2009). A complicated pattern of subcellular localisation was observed for HsfA2 in tomato; HsfA2 was localised mainly to the cytoplasm at 25 °C yet to the nucleus under HS conditions and also



formed heat stress granules (HSGs) with other heat-inducible proteins during the heat stress and recovery period (Scharf et al. 1998; Heerklotz et al. 2001; Port et al. 2004). Arabidopsis HSFA2 is regulated in a different manner and is localised to both the nucleus and cytoplasm, with an obvious preference for the nucleus (Meiri & Breiman 2009). However, our findings indicated that OsHSFA2dI, with both nucleus localisation signal and nucleus export signal, is localised to the nucleus, whereas OsHSFA2dII contains neither nucleus localisation signal nor nucleus export signal and is localised to the nucleus and cytoplasm. It is possible that there may be an unknown weak nucleus localisation signal in OsHSFA2dII. The other possibility is that OsHSFA2dII could be recruited into the nucleus by other nuclear proteins, as reported in the tomato HSF system (Scharf et al. 1998; Heerklotz et al. 2001). We also found that the subcellular localisation of both proteins was not affected by HS (data not shown). Until now, only one HSFA2 has been identified in tomato and Arabidopsis (Scharf et al. 1990; Nover et al. 2001; Baniwal et al. 2004; Kotak et al. 2004; von Koskull-Doring et al. 2007), whereas six OsHSFA2 members have been identified in rice, with OsHSFA2a, OsHSFA2c and OsHSFA2d responding to HS (Baniwal et al. 2004; Xiong et al. 2005; von Koskull-Doring et al. 2007; Gao et al. 2008; Mittal et al. 2009; Wang et al. 2009). Therefore, we speculate that the redundant functions of OsHSFA2a, OsHSFA2c and OsHSFA2d might promote functional diversity via an alternative splicing pattern that alters the protein subcellular localisation under HS. Plants have developed the capacity to cope with various environmental stimuli during evolution. In our data, OsHSFA2dI is specifically induced through HS in different varieties of rice, while the alternative splicing products OsHSFA2dII and OsHSFA2dIII exhibited diverse expression patterns. The expression profiles of OsHSFA2dII and OsHSFA2dIII in HS are highly correlated with photoperiodism of the varieties. It is noteworthy that expression profiles of OsHSFA2dII and OsHSFA2dIII in LD conditions are highly correlated to rice subspecies, as entirely different expression profiles are found between subspecies indica and japonica varieties. Similarly, expression profiles of OsHSFA2dII and OsHSFA2dIII under LD treatment are subspecies-dependent, as both of the transcripts are highly induced by photoperiod in japonica variety, with a delayed response in indica variety. The results imply that HSF gene(s) could be involved in coordination between photoperiodism and thermo-regulation. As sessile organisms, plants have evolved a wide range of defence pathways to cope with biotic and abiotic stresses. Indeed, protein homeostasis is vital for normal cellular function. The accumulation of unfolded and/or misfolded proteins REFERENCES Amano M., Iida S., Kosuge K. (2012) Comparative studies of thermotolerance: different modes of heat acclimation between tolerant and intolerant aquatic plants of the genus Potamogeton. Annals of Botany, 109, 443–452. Baniwal S.K., Bharti K., Chan K.Y., Fauth M., Ganguli A., Kotak S., Mishra S.K., Nover L., Port M., Scharf K.D., Tripp J., Weber C., Zielinski D., von KoskullDoring P. (2004) Heat stress response in plants: a complex game with chaperones and more than


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Heat shock factor and the heat shock response.

Sir2 links the unfolded protein response and the heat shock response in a stress response network.

Antiproliferative prostaglandins activate heat shock transcription factor.

ATF1 modulates the heat shock response by regulating the stress-inducible heat shock factor 1 transcription complex.

The human heat shock protein hsp70 interacts with HSF, the transcription factor that regulates heat shock gene expression.

Evidence for a heat shock transcription factor-independent mechanism for heat shock induction of transcription in Saccharomyces cerevisiae.

Expression and interaction of small heat shock proteins (sHsps) in rice in response to heat stress.

An essential role for heat shock transcription factor binding protein 1 (HSBP1) during early embryonic development.

Elevated transcription of heat shock protein gene in scleroderma fibroblasts.

Characterization of the heat shock response and identification of heat shock protein antigens of Borrelia burgdorferi.

Heat shock-induced interactions of heat shock transcription factor and the human hsp70 promoter examined by in vivo footprinting.

Novel isoforms of heat shock transcription factor 1, HSF1γα and HSF1γβ, regulate chaperone protein gene transcription.

Transcription regulation of HYPK by Heat Shock Factor 1.

Molecular cloning, phylogenetic analysis and heat shock response of Babesia gibsoni heat shock protein 90.

Heat shock transcription factor HSF1 regulates the expression of the Huntingtin-interacting protein HYPK.

The translation elongation factor eEF1A1 couples transcription to translation during heat shock response.

The heat shock response in human phagocytes.

Heat shock factor 1 prevents the reduction in thrashing due to heat shock in Caenorhabditis elegans.

Heat shock factor 1 promotes TERRA transcription and telomere protection upon heat stress.

Identification of heat shock factor binding protein in Plasmodium falciparum.

Physical interaction between heat shock proteins DnaK, DnaJ, and GrpE and the bacterial heat shock transcription factor sigma 32.

Trans-spliced heat shock protein 90 modulates encystation in Giardia lamblia.

Heat shock protein response in phosphorus-deficient heat-stressed broiler chickens.

Quercetin and heat shock response.