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Journal of Plant Physiology 170 (2013) 1434–1441 Contents lists available at ScienceDirect Journal of Plant Physiology Molecular Biology The ER luminal binding protein (BiP) alleviates Cd2+-induced programmed cell death through endoplasmic reticulum stress–cell death signaling pathway in tobacco cells Hua Xu a,b,1, Wenzhong Xu a,1, Hongmei Xi a,b, Wenwen Ma a, Zhenyan He a, Mi Ma a,∗ a Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China Cadmium (Cd) is very toxic to plant cells and Cd2+ stress induces programmed cell death (PCD) in Nicotiana Received 8 March 2013 tabacum L. cv. bright yellow-2 (BY-2) cells. In plants, PCD can be regulated through the endoplasmic reti- Received in revised form 22 May 2013 culum (ER) stress–cell death signaling pathway. However, the mechanism of Cd2+-induced PCD remains Accepted 24 May 2013 unclear. In this study, we found that Cd2+ treatment induced ER stress in tobacco BY-2 cells. The expres- Available online 16 July 2013 sion of two ER stress markers NtBLP4 and NtPDI and an unfolded protein response related transcription factor NtbZIP60 were upregulated with Cd2+ stress. Meanwhile, the PCD triggered by prolonged Cd2+ stress could be relieved by two ER chemical chaperones, 4-phenylbutyric acid and tauroursodeoxycholic acid. These results demonstrate that the ER stress–cell death signaling pathway participates in the medi- Bright yellow-2 cells ation of Cd2+-induced PCD. Furthermore, the ER chaperone AtBiP2 protein alleviated Cd2+-induced ER stress and PCD in BY-2 cells based on the fact that heterologous expression of AtBiP2 in tobacco BY-2 Programmed cell death cells reduced the expression of NtBLP4 and a PCD-related gene NtHsr203J under Cd2+ stress conditions. In summary, these results suggest that the ER stress–cell death signaling pathway regulates Cd2+-induced PCD in tobacco BY-2 cells, and that the AtBiP2 protein act as a negative regulator in this process.
2013 Elsevier GmbH. All rights reserved.
The ER is a very important organelle for protein synthesis, signal transduction, and Ca homeostasis. ER stress is generally caused by Cadmium (Cd), a toxic heavy metal, has been classified as a an overload of unfolded proteins in the ER, which activates signal human carcinogen. Thus Cd is a tremendous danger to plants and transduction of unfolded protein response (UPR) (Ron and Walter, animals (Bernard, 2008; DalCorso et al., 2010). The damage caused 2007; Zhang and Kaufman, 2004). Inositol-requiring enzyme-1 by Cd2+ in plant cells mainly includes reactive oxygen species (IRE1), activating transcription factor 6 (ATF6), and PKR-like ER (ROS) production, disturbances in photosynthesis, disorder of cal- kinase (PERK) act as transducers in the UPR signaling pathway in cium (Ca) signaling, and induction of programmed cell death (PCD) mammals (Calfon et al., 2002; Harding et al., 1999, 2000; Sidrauski (Chaffei et al., 2004; Ma et al., 2010; Rodriguez-Serrano et al., 2009; and Walter, 1997; Yamamoto et al., 2007; Ye et al., 2000). Then, Zhang et al., 2005). Cd2+-induced endoplasmic reticulum stress (ER the ER luminal binding protein (BiP) and glucose-regulated pro- stress) has been demonstrated in yeasts and mammals (Gardarin tein 94 (GRP94) are induced by the UPR (Yoshida et al., 1998), et al., 2010; Liu et al., 2006), but it has not been reported in plants and they enhance ER protein-folding capacity and maintain stor- age of ER Ca2+ (Lievremont et al., 1997). BiP is a central regulator of the UPR and a classical marker of UPR activation. When misfolded proteins accumulate in the ER, UPR is activated by BiP released from those three ER trans-membrane sensors IRE1, ATF6, and PERK Abbreviations: ATF6, activating transcription factor 6; BiP, luminal binding (Bertolotti et al., 2000). Several UPR-related genes have been iden- protein; BY-2, bright yellow-2; bZIP, basic-leucine zipper; Ca, calcium; Cd, cad- mium; ER, endoplasmic reticulum; GRP94, glucose-regulated protein 94; Hac1, tified in plants in recent years. Two orthologs of IRE1 in Arabidopsis ATF/CREB homolog 1; IRE1, inositol-requiring enzyme-1; NRP, N-rich protein; PCD, (AtIRE1a and AtIRE1b) are expressed in various organs and local- programmed cell death; PDI, protein disulfide isomerase; PERK, PKR-like ER kinase; ized to the perinuclear ER membrane (Koizumi et al., 2001). Moreno UPR, unfolded protein response; Xbp1, X-box binding protein 1.
and Hayashi found that AtIRE1b modulates activation of some Corresponding author at: Institute of Botany, Chinese Academy of Sciences, basic-leucine zipper (bZIP) transcription factors (Hayashi et al., Xiangshan, Beijing 100093, PR China. Tel.: +86 10 62836255; fax: +86 10 62836690.
E-mail address: [email protected] (M. Ma).
2012; Moreno et al., 2012). Moreover, two groups of membrane- 1 These authors contributed equally to this work.
associated bZIP transcription factors play very important roles in 0176-1617/$ – see front matter 2013 Elsevier GmbH. All rights reserved.
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H. Xu et al. / Journal of Plant Physiology 170 (2013) 1434–1441 transducing ER stress signals in plant cells. One group, including promoter and the 3 un-translated end of the nopaline synthase AtbZIP60 and OsbZIP50, is activated by IRE1-mediated mRNA splic- gene present on pSN1301, resulting in pSN1301-BiP2. Using this ing, which is similar to activation of ATF/CREB homolog 1 (Hac1) same process, the AtBiP2 coding region was amplified by PCR with in yeast and X-box binding protein 1 (Xbp1) in animals (Cox and another two primer pairs (BiP2F2 and BiP2R2, BiP2F2 and BiP2R3), Walter, 1996; Shen et al., 2001). The new proteins encoded by the particularly the BiP2R3 primer containing the 6-His-tag codes spliced mRNA are trans-located to the nucleus where they activate (5-GTGGTGGTGGTGGTGGTG-3) before the termination codon transcription of some ER function-related genes (Deng et al., 2011; (Table S1). Those fragments were separately inserted into pER8 Hayashi et al., 2012; Iwata and Koizumi, 2005a; Nagashima et al., (Zuo et al., 2000), which is an estrogen-dependent expression 2011). Activation of the other group of proteins such as AtbZIP28 vector in plants, and were respectively named pER8-BiP2, and and OsbZIP39 depends on regulated intra-membrane proteolysis pER8-BiP2-6H. All plasmids obtained were identified by DNA and resembles the ATF6 process in animals in which site-1 and sequencing before they were transformed into Agrobacterium C58.
site-2 proteases are cleaved in response to ER stress (Liu et al., 2007; Tajima et al., 2008; Takahashi et al., 2012). As one of the most Cell culture conditions important ER chaperone proteins, BiP alleviates ER stress in tobacco (Alvim et al., 2001; Leborgne-Castel et al., 1999). However, PCD Tobacco cell (Nicotiana tabacum L. cv bright yellow BY-2) is the result of prolonged ER stress triggered by multiple stimuli, (Nagata et al., 1992) lines were grown in Murashige and Skoog although the UPR alleviates ER stress damage.
liquid medium supplemented with 256 mg/L KH2PO4, 1 mg/L thi- The main apoptotic cell death signaling pathways demonstrated amine, 100 mg/L myo-inositol, 0.2 mg/L 2,4-dichlorophenylacetic in mammalian cells are dependent on plasma membrane recep- acid, and 30 g/L sucrose. The cells were maintained on a gyratory tors, mitochondria, and the ER (Earnshaw et al., 1999; Nakagawa shaker (120 rpm) in a dark, temperature-controlled room at 25 ◦C et al., 2000). ER-dependent apoptotic pathways have two differ- and sub-cultured weekly with 2% inoculum.
ent modes. The cleavage of procaspase 12 depends on calpain or caspase 7, which is released from ER membranes into the cyto- BY-2 cell transformations sol during prolonged ER stress and forms active caspase 12 to activate downstream apoptotic factors (Rao et al., 2002; Yoneda Each expression vector was transformed into the C58 Agrobac- et al., 2001). BiP acts as an anti-apoptotic moderator and prevents terium rifampicin-resistant strain by electroporation. Two- or activation of procaspase-7 and procaspase-12 by binding them at three-week-old tobacco BY-2 cell clusters were infused into a sus- the ER membrane (Reddy et al., 2003). The mode of the other pension of respective Agrobacterium stains with a BY-2 cell culture ER-dependent pathway is based on the lack of Ca2+ homeostasis.
medium for 20 min. After the clusters grew on the normal medium Whenever prolonged ER stress is triggered by depletion of Ca2+ in for 2 days, they were washed three times, and then dropped into the ER, mitochondria overloaded with Ca2+ release cytochrome c water containing 400 ␮g/mL cefotaxime for 45 min. Finally, the into the cytosol, which causes activation of caspase-9 (Hacki et al., transformants were selected on tobacco BY-2 cell culture medium 2000). Similarly, PCD triggered by ER stress in plants has also been containing 20 ␮g/mL hygromycin B and 400 ␮g/mL cefotaxime.
reported. In soybean cells, cyclopiazonic acid treatment induces ER stress, increases cytoplasmic Ca2+, generates hydrogen peroxide, Semi-quantitative reverse transcription (RT)-PCR induces release of cytochrome c from mitochondria, and activates caspase-like proteases causing PCD (Zuppini et al., 2004). In addi- RNA isolation and the generation of cDNA were performed as tion, tobacco BY-2 cells, treated with a UPR inducer tunicamycin, described above. The primers for each target gene are shown in may also lead to PCD along with up-regulation of NtHsr203J, a Table S2, which also includes the annealing temperature and PCR marker of PCD (Iwata and Koizumi, 2005b).
cycles. The amplified fragments of the target genes, such as NtBLP4 As studies on UPR and PCD become more in-depth, these (Accession No: X60057), NtHsr203J (Accession No: AF212184), and signaling pathways have been revealed in plants as well as ani- NtEF1-˛ (Accession No: D63396), were segregated by agarose gel mals. BiP plays a very important role in stress-induced plant cell electrophoresis under 6 V/cm pressure for 10 min. Gel Doc-ItTM 300 death, such as ER stress, osmotic stress, and water stress (Alvim (Ultra-Violet Products Ltd., Cambridge, UK) was used to scan the et al., 2001; Leborgne-Castel et al., 1999; Reis et al., 2011). More- over, BiP over-expression enhances tolerance to drought stress and delays leaf senescence induced by drought in soybean (Valente Real-time quantitative RT-PCR et al., 2009). The primary focus of the present study was on the pathway through which Cd2+ triggers PCD in plant cells and the RNA isolation and the generation of cDNA were performed as mode of BiP action during this process.
described above. For real-time quantitative RT-PCR, the final primer concentration was 0.2 ␮M in a total 20 ␮L reaction volume, and all primers were listed in Table S3. Real-time quantitative RT-PCR Materials and methods
was performed with the StepOneTM Real-Time PCR System (Applied Biosystems, CA, USA) and prepared with the SYBR Premix Ex TaqTM Plant expression vector construction GC Kit (DRR071A; TaKaRa Biotechnology Ltd, Dalian, China). The efficiencies of all cDNAs amplification were between 90 and 100%.
The reagent of TRIzol (15596026; Life Technologies Corp., Each RNA sample was assayed in triplicate. Expression levels of Carlsbad, CA, USA) was used to extract Arabidopsis total RNA, NtBLP4, NtPDI (Accession No: Y11209), and NtbZIP60 (Accession and full length cDNA was produced using the SuperScript® II No: AB281271) were calculated relative to the standard sample for Reverse Transcriptase kit (18064-014; Life Technologies Corp.).
calibration and then normalized to the NtEF1-˛ gene.
The BiP coding the isoform AtBiP2 (at5g42020) region was ampli- fied by polymerase chain reaction (PCR) with the BiP2F1 and Protein gel blot hybridization BiP2R1 primers (Table S1) to create two KpnI (E.C.3.1.23.26) sites located in front of the translation initiation codon and after Each treated cell line was collected and quickly frozen in liquid the stop codon, respectively. After KpnI digestion, this fragment nitrogen. Frozen samples were ground and transferred to tubes.
was inserted between the cauliflower mosaic virus (CaMV) 35S Protein extraction buffer (100 mM HEPES, pH 7.5, 5 mM EDTA, pH

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H. Xu et al. / Journal of Plant Physiology 170 (2013) 1434–1441 8.0, 5 mM EGTA, pH 7.0, 50 mM glycerol-p, 10 mM DTT, 5% glyc- erol, 1 mM PMSF) was added to each tube, shaken well, kept in ice-water for 40 min, and then centrifuged at 13,000 rpm for 40 min.
Cell lysates containing proteins were transferred to new tubes. Pro- tein concentrations were determined using the CWBIO Bradford assay reagent.
Proteins in 12% sodium dodecyl sulfate–polyacrylamide gels were transferred to polyvinylidene fluoride membranes and blocked with Tris-buffered saline (TBST: 50 mM Tris, pH 7.5, 150 mM NaCl, and 0.05% Tween 20) containing 5% nonfat dry milk at 4 ◦C overnight. The membranes were then incubated in TBST con- taining 0.5% nonfat dry milk BIP goat polyclonal antibody (1:3000; aC-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a dilu- tion of 1:4000 for mouse tubulin and His-tag monoclonal antibody (AT819 and AH367; Beyotime Institute of Biotechnology, Shanghai, China). After 1 h, three 10-min washes were performed with TBST, and then the membranes were incubated at 1:4000 in horseradish peroxidase-conjugated anti-goat or anti-mouse secondary anti- body (A0181 or A0216; Beyotime Institute of Biotechnology) in TBST containing 0.5% nonfat dry milk for 45 min. Three 10-min washes were performed in TBST, the antigen–antibody complexes were detected using an Enhanced Chemiluminescence-Plus Detec- tion kit (P0018; Beyotime Institute of Biotechnology), and the images were recorded on film.
Fig. 1. (A) The amounts of NtBLP4 and NtPDI transcripts were detected by real-time
quantitative RT-PCR at each time point with the treatment of 100 ␮M CdCl2. NtBLP4 and NtPDI expression were separately normalized to NtEF1-˛. The values at 0 h were further normalized to 1. Error bars represent SE for three independent replicates.
The significant differences between 0 h and the other time points were marked by PCD using Hoechst 33342 and propidium iodide (PI) co-staining asterisks (*: p < 0.05; **: p < 0.01). (B) BLP protein was inducible expressed in BY-2 was detected based on a method described previously (Ormerod cells treated with CdCl2. Cells were harvested at 3, 6 and 12 h after treatment with et al., 1993; Yuan et al., 2002). The Apoptosis and Necrosis Assay 100 ␮M CdCl2 (Cd2+) or not (control). Total protein was separated by SDS-PAGE and analyzed by protein gel blot hybridization using anti-BiP and anti-␤-tubulin kit (C1056; Beyotime Institute of Biotechnology) was used as a antibodies. The amount of ␤-tubulin protein was used as a loading control. (C) The method to detect cell death. Samples were collected from each amount of NtbZIP60 transcripts was detected by real-time quantitative RT-PCR at treatment, washed twice with BY-2 cell culture medium, incu- each time point with the treatment of 100 ␮M CdCl2. The data normalization and bated in buffer-containing Hoechst 33342 and PI at 4 ◦C for 30 min, significant analysis were the same as above.
and then washed twice with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4). All Cd2+ induces PCD in BY-2 tobacco cells stained cells were observed under a fluorescence microscope (Zeiss Axioskop 40; Carl Zeiss, Oberkochen, Germany) with a 353–377- BY-2 cells treated with 150 ␮M CdCl2 (Fig. 2C) or 0.2 mg/L tuni- nm excitation filter. The percentages of cells undergoing PCD were camycin (Fig. 2D) were observed to undergo PCD, whereas bright calculated by randomly observing >750 cells in each treatment.
blue fluorescence did not appear in BY-2 cells under normal cul- ture (Fig. 2A and B). Furthermore, the number of cells undergoing Statistical analysis PCD increased continuously from 24 h to 48 h under treatment with 150 ␮M CdCl2 or 0.2 mg/L tunicamycin (Fig. 2E). The percentages of SPSS 16.0 (SPSS Inc., Chicago, IL, USA) was used for statistical BY-2 cells undergoing PCD exceeded 80% when they were treated analyses, and p values were calculated using one-way analysis of with 150 ␮M CdCl 0.2 mg/L tunicamycin for 48 h.
variance. A p < 0.05 was considered significant.
ER chemical chaperones relieve the PCD induced by Cd2+ Two ER chemical chaperones, 4-phenylbutyric acid and tau- Cd2+ leads to ER stress in BY-2 tobacco cells roursodeoxycholic acid, which have the ability to relieve ER stress (Oezcan et al., 2006), were used to investigate whether The UPR pathway is activated during ER stress, and then induces Cd2+ induces PCD by triggering ER stress. In this experiment, the expression of ER chaperone proteins, such as BiP and pro- both 4-phenylbutyric acid and tauroursodeoxycholic acid mod- tein disulfide isomerase (PDI). Consequently, the up-regulations estly reduced the number of tobacco cells treated with 0.2 mg/L of BiP and PDI are considered as the occurrence of ER stress in tunicamycin undergoing PCD (Fig. 3). The PCD percentages of cells cells. NtBLP4 and NtPDI were homologous genes of BiP and PDI pre-treated with 500 ␮M 4-phenylbutyric acid or 100 ␮M tau- in N. tabacum, and the amounts of NtBLP4 and NtPDI transcripts roursodeoxycholic acid for 12 h respectively decreased to 4.56% were examined by real-time quantitative RT-PCR (Fig. 1A). Both (13.41 ± 2.03%) and 12.45% (5.52 ± 1.10%) at 24 h, compared with NtBLP4 and NtPDI transcripts gradually increased with 100 ␮M 17.97 ± 1.77% of the cells with 0.2 mg/L tunicamycin treatment.
CdCl2 treatment. Moreover, BLP proteins also accumulated grad- A similar situation was observed after 36 h of treatment, and the ually following the 100 ␮M CdCl2 treatment (Fig. 1B). Additionally, percentages of cells undergoing PCD were 45.16 ± 2.48% (tuni- the NtbZIP60 was considered as the UPR-related transcription fac- camycin alone), 36.88 ± 2.76% (pre-treated with 4-phenylbutyric tor in N. tabacum, and its transcript was induced to increase with acid) and 26.87 ± 2.05% (pre-treated with tauroursodeoxycholic 100 ␮M CdCl2 treatment (Fig. 1C). These results indicate that Cd2+- acid), respectively, at 36 h. Almost all tobacco BY-2 cell under- induced ER stress in BY-2 tobacco cells.
went PCD after being treated for 48 h. These results indicate

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H. Xu et al. / Journal of Plant Physiology 170 (2013) 1434–1441 Fig. 2. The cells with bright blue fluorescence in their nucleus were considered as undergoing programmed cell death (PCD), whereas bright red fluorescence in cells indicated
that cells had been already dead. However, no bright blue and red fluorescence could be detected in the cells alive. The images exhibited the fluorescence of BY-2 cells treated for 36 h under the conditions of normal culture (control) (A), dimethyl sulfoxide (DMSO) (B), 150 ␮M CdCl2 (Cd2+) (C) or 0.2 mg/L tunicamycin (Tm) (D). Bar 200 ␮m. (E) The statistical analysis of cells undergoing PCD was based on the examinations of more than 750 cells in each experiment. Each value and its standard error was calculated from three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 3. The images exhibited the fluorescence of BY-2 cells treated for 36 h under the conditions of dimethyl sulfoxide (DMSO) (A), 0.2 mg/L tunicamycin (Tm) (B), 0.2 mg/L
tunicamycin (Tm) besides having been pretreated for 12 h with 500 ␮M 4-phenylbutyric acid (4-PBA) (C), or 100 ␮M tauroursodeoxycholic acid (TUDCA) (D). Bar 200 ␮m.
(E) At three different time points, the ratios of each treatment cell lines undergoing programmed cell death (PCD) were shown and the calculation of data was operated just as above. The significant differences between pretreated cells and non-pretreated cells were marked by asterisks (*: p < 0.05; **: p < 0.01).
that 4-phenylbutyric acid and tauroursodeoxycholic acid partially 48 h. However, the number of pretreated cells undergoing PCD was relieved ER stress-induced PCD in tobacco BY-2 cells. BY-2 cells less than half of the non-pretreated cells at 36 h, and it was slightly treated with 150 ␮M CdCl2 instead of 0.2 mg/L tunicamycin were more than a half of the non-pretreated cells at 48 h. As a result, ER used to observe PCD (Fig. 4). The percentages of cells undergoing chemical chaperones had a great effect on relieving PCD in BY-2 PCD decreased by 12.81% (11.47 ± 3.48%) and 19.26% (5.02 ± 1.10%) cells induced by Cd2+.
in the 4-phenylbutyric acid and tauroursodeoxycholic acid pre- treated groups compared with that in the non-pretreated group BiP is capable of downregulating Cd2+-induced ER stress (24.28 ± 1.74%) when 150 ␮M CdCl2 was added to the culture medium for 24 h. Then, the number of cells undergoing PCD Constitutively expressing cell lines BiP2-Ox were used in which increased in both non-pretreated and pretreated groups at 36 and AtBiP2 was driven by the CaMV 35S promoter to identify the role Fig. 4. The images exhibited the fluorescence of BY-2 cells treated for 36 h under the conditions of normal culture (control) (A), 150 ␮M CdCl2 (Cd2+) (B), 150 ␮M CdCl2 (Cd2+)
besides having been pretreated for 12 h with 500 ␮M 4-phenylbutyric acid (4-PBA) (C), or 100 ␮M tauroursodeoxycholic acid (TUDCA) (D). Bar 200 ␮m. (E) At three different time points, the conditions of each treatment cell lines undergoing programmed cell death (PCD) were shown, and the method of statistical analysis was the same as before.
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H. Xu et al. / Journal of Plant Physiology 170 (2013) 1434–1441 Fig. 6. The transformed cells named BiP2-In-6H were able to express 6-His tagged
AtBiP2 protein under the induction of 17-␤-estradiol. The 6-His tag was located just in front of C-terminal four peptides (HDEL) in AtBiP2 protein. (A) The BiP2-In- 6H cells were divided into an untreated group named control and a 150 ␮M CdCl2 treated group named Cd2+. Both groups of cells were collected at each time point, and then their proteins were prepared for protein gel blot hybridization. Anti-BiP antibody could detect both NtBLP and AtBiP2 protein, and Anti-His antibody was used to immunize 6-His tagged AtBiP2 alone. The amount of ␤-tubulin protein was Fig. 5. Two transgenic cell lines were used in the experiment. The cell line trans-
used as a loading control. (B) All procedures were the same as the description in (A) formed by an empty plant expression vector SN1301 was named EV, and the other except that transgenic cells had been pretreated with 5 ␮M 17-␤-estradiol for 2 h.
cell line named BiP2-Ox was able to express AtBiP2 driven by a CaMV 35S promoter.
(A) The expression of NtBLP4 was detected by semi-quantitative RT-PCR at each time point with treatment of 150 ␮M CdCl2 in both cell lines. The detection of AtBiP2 tran- script was used to distinguish two cell lines. The amount of NtEF1-˛ transcript was Cd2+-induced ER stress seemed somewhat impaired in the BiP2- considered as an internal control. (B) NtBLP4 transcripts were detected by real-time Ox cell lines. However, constitutively expressing AtBiP2 partially quantitative RT-PCR at each time point with the treatment of 150 ␮M CdCl2. NtBLP4 repressed the transcription of NtBLP4 even under normal culture expression was normalized to NtEF1-˛. The values in EV cell lines at 0 h were fur- condition (Fig. 5A and B). To eliminate such impact, a chemically ther normalized to 1. Error bars represent SE for three independent replicates. The inducible expression vector pER8 was introduced. The expression significant differences between two cell lines were marked by asterisks (*: p < 0.05; **: p < 0.01). (C) The quantity of BiP protein in both transgenic cell lines treated with of AtBiP2 in tobacco cells was induced by the chemical inducer (17- 150 ␮M CdCl2 was measured by protein gel blot hybridization. Anti-BiP antibody ␤-estradiol). The AtBiP2 protein was 6-His tagged at its C terminus was used to detect NtBLP alone in EV cell line and totality of AtBiP2 and NtBLP pro- to detect the amount of AtBiP2 protein. After being treated with tein in BiP2-Ox cell line. The same procedure was used just as the description in 150 ␮M CdCl AtBiP2 and NtBLP were detected with anti-BiP and AtBiP2 was also detected with anti-His antibodies. As shown in Fig. 6A, NtBLP expression increased gradually under the 150 ␮M CdCl2 treatment, and 6-His tagged AtBiP2 was not expressed with- out 17-␤-estradiol. This result suggests that ER stress occurred of AtBiP2 in Cd2+-induced ER stress, while the control cell lines acutely without 6-His tagged AtBiP2 expression in cells cultured transformed empty vector were named EV cell lines. Furthermore, with 150 ␮M CdCl 6-His tagged AtBiP2 protein accumulated along NtBLP4 is considered as an ER stress marker in N. tabacum, and with 17-␤-estradiol induction in the control group (Fig. 6B) and the detection of its expressing is used to report the occurrence NtBLP expression did not increase. Then, 6-His tagged AtBiP2 in the of ER stress. The amounts of AtBiP2 and NtBLP4 transcripts in the Cd2+ group (Fig. 6B) was almost the same as that the former group.
transgenic cell lines were detected by RT-PCR (Fig. 5A). The amount However, levels of the AtBiP2 and NtBLP proteins were no obvi- of AtBiP2 transcript driven by the CaMV 35S promoter was stable ous differences between the control and Cd2+ groups, although the with or without treatment with 150 ␮M CdCl 2 (Fig. 5A). However, NtBLP protein should be induced to increase under CdCl the amount of NtBLP4 transcript in the BiP2-Ox cell lines was dis- (Fig. 6B). Based on these results, the 6-His tagged AtBiP2 protein tinctly lower than that in the EV cell lines at each time point (except was demonstrated to be functional for suppressing Cd2+-induced 36 h). Moreover, the data of real-time quantitative RT-PCR further confirmed the results of RT-PCR (Fig. 5B). At each time point, the differences of NtBLP4 transcript between two cell lines were signif- icant (p < 0.01). Additionally, anti-BiP antibody was used to detect BiP is efficient at alleviating PCD triggered by Cd2+ the target protein in two cell lines. The blots detected by anti-BiP antibody indicated the amount of NtBLP protein in the EV cell line; The constitutively expressing cell lines BiP2-Ox were used to while the blots detected by anti-BiP antibody contained both AtBiP2 examine the function of BiP for alleviating PCD induced by Cd2+ and NtBLP protein in the BiP2-Ox line, since anti-BiP antibody can- stress. The percentage (16.63 ± 1.95%) of cells undergoing PCD not distinguish the NtBLP from AtBiP2. As shown in Fig. 5C, the in EV lines was more than twice higher than the BiP2-Ox lines intensities of the blots detected by anti-BiP antibody had no obvi- (7.49 ± 1.65%) when BY-2 cells were treated with 150 ␮M CdCl2 ous difference between two cell lines through the entire treatment for 24 h. Furthermore, the number of cells undergoing PCD contin- (Fig. 5C). We therefore inferred that the amount of NtBLP protein in ued to increase and exceeded one-third of all cells in the EV group, the BiP2-Ox line was less than that in the EV line at each time point.
whereas the percentage in BiP2-Ox lines was only 21.21 ± 1.48% Based on the transcriptional and translational detections, NtBLP at 36 h. About 90% of cells underwent PCD in EV cell lines after a expression was less induced in transformed AtBiP2 cells under the 48 h treatment, and the percentage of BiP2-Ox lines was approxi- 150 ␮M CdCl2 treatment. In other words, the overexpressing cells mately 10% lower (Fig. 7A). As shown in Fig. 7B, Cd2+ was capable of appeared to have less capacity to induce ER stress in response to inducing NtHsr203J transcription, which was considered as a PCD- related gene in BY-2 cells. In addition, the amount of NtHsr203J Author's personal copy
H. Xu et al. / Journal of Plant Physiology 170 (2013) 1434–1441 Fig. 7. (A) Cells were treated with 150 ␮M CdCl2 for 24, 36, or 48 h, and then
observed through a fluorescence microscope. The percentages of cells undergoing programmed cell death (PCD) in EV and BiP2-Ox cell lines at each time point were shown. The statistical analysis of cells undergoing PCD was based on the examina- tions of more than 750 cells in each experiment. Each value and its standard error were calculated from three independent experiments. The significant differences between two cell lines were marked by asterisks (*: p < 0.05; **: p < 0.01). (B) Both kinds of transgenic cells were treated with 150 ␮M CdCl2. The changes of NtHsr203J expression were detected by semi-quantitative RT-PCR at five different time points.
The amount of NtEF1-˛ transcript was considered as an internal control.
transcript increased gradually in the two kinds of transformed cell lines under the 150 ␮M CdCl2 treatment. However, clear dif- ferences were observed between the EV and BiP2-Ox lines. The amount of NtHsr203J transcript increased distinctly at 12 h in EV cell lines, whereas the increase appeared at 24 h in the transformed AtBiP2 cells. Thus, the BiP2-Ox cell lines had more capacity to resist Fig. 8. (A) BY-2 cells named BiP2-In were able to express AtBiP2 protein under the
Cd2+-induced PCD than that of the EV cell lines.
induction of 17-␤-estradiol. The cells without pre-treatment were signed as BiP2-In.
The cells pre-treated with 5 ␮M 17-␤-estradiol for 2 h were signed as BiP2-In (5) and Next, the inducible expressing AtBiP2 cell line BiP2-In was used.
20 ␮M 17-␤-estradiol as BiP2-In (20). The chart exhibited the percentages of BiP2- After being treated with 150 ␮M CdCl2 for 36 h, the percentage of In cells undergoing programmed cell death (PCD) with treatment of 150 M CdCl2 cells undergoing PCD was 47.14 ± 2.64% in the non-17-␤-estradiol at 36 h. The statistical analysis of cells undergoing PCD was based on the exam- treated group, whereas it decreased about 10% (37.24 ± 3.75%) inations of more than 750 cells in each experiment. Each value and its standard in the 5 ␮M 17-␤-estradiol-treated group and by more than error were calculated from three independent experiments. The significant differ- ences between pretreated cells and non-pretreated cells were marked by asterisks half (15.78 ± 4.71%) in the 20 ␮M 17-␤-estradiol-treated group (*: p < 0.05; **: p < 0.01). (B) In the same way, the percentages of cells undergoing (Fig. 8A). These results suggest that inducible expression of AtBiP2 PCD in BiP2-In-6H were shown in (B). The same statistical analysis was used just as enabled BY-2 cells have higher resistance to PCD triggered by CdCl2.
Furthermore, the more chemical inducer added, the stronger resis- tance tobacco cells got. The same experiment was carried out et al., 2012; Shen et al., 2001). Tateda et al. cloned a UPR-related to examine whether the 6-His tagged AtBiP2 protein remained transcription factor NtbZIP60, which was the AtbZIP60 homolog in functional in BiP2-In-6H cell lines. The results showed that the N. tabacum. Although the NtbZIP60 mRNA splicing just as AtbZIP60 non-17-␤-estradiol-treated group was 41.17 ± 3.03%, the 5 ␮M has not been verified, NtbZIP60 transcript was induced by ER stress 17-␤-estradiol-treated group was 23.63 ± 2.49%, and the 20 ␮M inducer such as tunicamycin and dithiothreitol (Deng et al., 2011; 17-␤-estradiol-treated group was 13.20 ± 2.69% (Fig. 8B), indicat- Tateda et al., 2008). In this paper, CdCl also triggered the ing that the tagged protein was effective in reducing the number accumulation of NtbZIP60 mRNA, and two ER stress marker genes of cells undergoing PCD and that the 6-His tag had no deleterious NtBLP4 and NtPDI were upregulated by Cd2+ stress as well (Fig. 1).
effect on AtBiP2 function. Thus, BiP had a good effect on alleviating Together with those results, it indicated that Cd2+ stress led to ER PCD triggered by Cd2+ stress.
stress in BY-2 tobacco cells. Based on current works, however, it was still uncertain that Cd2+ stress directly activated ER stress or not.
Prolonged ER stress determines the occurrence of PCD in both animals and plants. In this study, the existence of the ER stress–PCD Several studies reported that Cd2+ leads to ER stress in yeast and pathway was supported by new evidence. Tunicamycin, an inducer mammals (Gardarin et al., 2010; Liu et al., 2006; Yokouchi et al., of ER stress, led to PCD in BY-2 tobacco cells (Fig. 2). Moreover, PCD 2007, 2008). BiP, a major marker of ER stress in mammalian cells, triggered by tunicamycin was markedly attenuated when BY-2 cells is up-regulated following Cd2+ treatment (Liu et al., 2006). Three were pretreated with 4-phenylbutyric acid and tauroursodeoxy- main sensors of the UPR such as PERK, ATF6, and IRE1 are acti- cholic acid, which are two types of ER chemical chaperones (Fig. 3).
vated by CdCl2 (Yokouchi et al., 2007). AtbZIP60 is the first reported Consequently, BY-2 cells responded with ER stress–cell death UPR-related transcription factor in Arabidopsis, and the amount of signaling. Our examination of Cd2+-induced PCD in BY-2 cells was its transcript was in response to ER stress (Iwata and Koizumi, the same as that of some other investigation, such as those using 2005a). Furthermore, AtbZIP60 was activated by IRE1-mediated DNA laddering and terminal deoxynucleotidyl transferase dUTP mRNA splicing, which was similar to activation of Hac1 in yeast and nick end labeling assays (Kuthanova et al., 2008; Ma et al., 2010). As Xbp1 in animals (Cox and Walter, 1996; Deng et al., 2011; Moreno expected, if either 4-phenylbutyric acid or tauroursodeoxycholic Author's personal copy
H. Xu et al. / Journal of Plant Physiology 170 (2013) 1434–1441 acid were added to the culture medium, the number of cells under- After analyzing the BY-2 cell phenotype following Cd2+ treat- going PCD clearly decreased (Fig. 4). However, the 4-phenylbutyric ment, we proposed that the over-expressing cell lines had greater acid and tauroursodeoxycholic acid mechanisms were different to resistance to PCD than that of the control (Fig. 7A). Furthermore, the maintain ER function. Tauroursodeoxycholic acid treatment led to AtBiP2 protein inhibited PCD, and this ability was positively corre- increased stability of the mutant protein, whereas 4-phenylbutyric lated with AtBiP2 protein quantity (Fig. 8). The results of NtBLP acid facilitated degradation of misfolded proteins (de Almeida expression indicated that exogenous BiP proteins were able to et al., 2007). Based on these results, we predicted that Cd2+- inhibit the UPR activation in constitutively expressing and instantly triggered ER stress was probably the result of an accumulation expressing cell lines. So, it is reasonable to assume that the Cd2+- of misfolded proteins in the ER, and 4-phenylbutyric acid and induced ER stress–cell death pathway depended on UPR signaling tauroursodeoxycholic acid treatment delayed PCD. In other words, together with the 4-phenylbutyric acid and tauroursodeoxycholic the UPR-dependent signaling of ER stress-induced cell death acid results. In addition, the amount of NtHsr203J transcript as a clearly participated in the regulation of Cd2+-induced PCD.
PCD-related marker gene in tobacco increased more quickly in The BiP is one of the most important chaperone proteins the control cell lines compared with that in the over-expressing that assist in maintaining physiological function of the ER. The cell lines. However, the PCD-related NtHsr203J may be induced BiP protein not only protects peptide folding and preserves Ca2+ by ER stress through a pathway independent of the UPR (Iwata homeostasis but also acts as a modulator of the UPR and ER and Koizumi, 2005b). Therefore, determining whether the Cd2+- stress–cell death signaling pathways. The amount of NtBLP4 tran- induced ER stress–cell death occurs through another signaling script was less than that in control cells under CdCl2 treatment pathway requires further research. Some researchers have found when AtBiP2 was constitutively expressed in BY-2 cells (Fig. 5A and that signaling of plant-specific N-rich protein (NRP) mediates ER B). Therefore, the exogenous BiP protein reduced ER damage caused stress- and osmotic stress-induced cell death pathways, which by Cd2+ stress. The reason might be that the exogenous BiP pro- diverged from a branch of the UPR (Costa et al., 2008). Furthermore, tein partly took the place of the endogenous BLP protein, which BiP acts as a negative regulator of the NRP-mediated cell death would maintain the UPR in a less activated state. Above all, this response (Reis et al., 2011). The senescence-associated marker result indicated that exogenous BiP protein expression regulated gene GmCystP, DNA fragmentation, and stress-triggered cell death activation of the UPR to alleviate Cd2+-triggered ER stress. Addi- were all less induced in over-expressing BiP lines. These discov- tionally, the amount of NtBLP4 transcript in the AtBiP2 expressed eries have provided a new direction to research the action of BiP cell lines was clearly less than that in the control even under in Cd2+-induced PCD. The BiP protein relieved Cd2+-induced ER normal growth conditions (Fig. 5A and B). This interesting phe- stress, which was reasonable considering that the anti-PCD action nomenon was quite similar with a previous study showing that on Cd2+ stress originated from alleviating ER stress by the BiP pro- BiP over-expression leads to down-regulation of endogenous BiP tein. Nevertheless, the BiP protein also acted as an ER apoptotic mRNA and a reduction in the UPR in tobacco (Leborgne-Castel modulator by inhibiting activation of the BIK pro-apoptotic fac- et al., 1999). Thus, a transcriptional regulatory mechanism might tor, which is a member of the Bcl-2 family in mammals. Moreover, maintain the quantity of BiP in the ER. However, feedback regu- some researchers have reported that several homologs in the Bcl- lation at the translational level in HeLa cells was more efficient 2-associated athanogene (BAG) family affect ER stress and PCD in at controlling BiP expression (Gulow et al., 2002). The efficiency Arabidopsis, particularly AtBAG7 interacting with AtBiP2 in the UPR of translational regulation represented rapid control of the total (Williams et al., 2010). Consequently, it is quite probable that the BiP balance when it was disturbed by transient expression of BiP protein might use this mechanism as well, although a homolo- exogenous BiP protein. The mechanism of transcriptional regula- gous pro-apoptotic factor has not been discovered in plants.
tion seemed to be a long-term adjustment compared with that In summary, Cd2+ treatment induced ER stress and activated of translational control. However, both transcriptional and trans- the UPR. Cd2+-induced PCD was dependent on the ER stress–cell lational regulation lost control when ER stress was triggered by death signaling pathway in BY-2 tobacco cells. Furthermore, the BiP a stimulator. As shown in Fig. 5C, total BiP expression accumu- protein as a Ca2+ binding chaperone and regulator of both the UPR lated gradually in tobacco BY-2 cells treated with CdCl2. Although and ER stress–cell death signaling played an extremely important constitutive expression of AtBiP2 led to transcriptional regulation role in maintenance of the ER. Thus, BiP alleviated ER stress and of NtBLP4 expression in tobacco BY-2 cells, the expressed AtBiP2 delayed the appearance of Cd2+-induced PCD.
cell lines actually had stronger resistance to ER stress than that of control cells before CdCl2 treatment. An estrogen-dependent inducible expression vector pER8 (Zuo et al., 2000) was introduced to eliminate the difference between the two cell lines, which also The authors thank Prof. Xuejun Hua (Key Laboratory of Plant enabled us to control the timing and quantity of AtBiP2 expres- Resources, Institute of Botany Chinese Academy of Sciences, Beijing sion. It was useful to help examine whether BiP directly alleviated 100093, PR China) for providing the tobacco BY-2 cells. Prof. Nam- Cd2+-induced ER stress. AtBiP2 reduced the quantity of endoge- Hai Chua (Laboratory of Plant Molecular Biology, The Rockefeller nous BLP induced by Cd2+ stress when comparing AtBiP2 and University, New York, New York 10065) gave us the expression vec- NtBLP expression at two levels of estrogen-dependent induction tor of pER8 as a gift, which was very helpful in our research. We also (Fig. 6). Based on this result, BiP probably functioned by eliminat- appreciate the suggestions on paper writing that Prof. Jinxing Lin ing damage to the ER environment before the Cd2+-induced UPR.
(Key Laboratory of Plant Molecular Physiology, Institute of Botany Thus, rapid expression of the AtBiP2 protein relieved Cd2+-induced Chinese Academy of Sciences, Beijing 100093, PR China) advised us.
ER stress through another mechanism. In addition, translational This work was supported by the National Natural Science Founda- regulation mentioned above lost control of total BiP expression tion of China (31170164 and 90713030).
under the induction of 17-␤-estradiol in the control cell lines (Fig. 6B). It was probably because the induction was so strong that the translational regulation was overridden. Although excess and Appendix A. Supplementary data
unnecessary BiP proteins are induced to degrade under normal growth conditions (Gulow et al., 2002), BiP proteins make a con- Supplementary data associated with this article can be found, tribution to maintaining the ER environment at the initiation of in the online version, at http://dx.doi.org/10.1016/j.jplph.2013.
Author's personal copy
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