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Induction of BiP by sugar independent of a cis-element for the unfolded protein response in Arabidopsis thaliana
BBRC Biochemical and Biophysical Research Communications 346 (2006) 926–930 www.elsevier.com/locate/ybbrc
Induction of BiP by sugar independent of a cis-element for the unfolded protein response in Arabidopsis thaliana Hiromi Tajima, Nozomu Koizumi
*
Department of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0192, Japan Received 29 May 2006 Available online 9 June 2006
Abstract BiP is a molecular chaperone induced in the unfolded protein response (UPR). In mammalian cells, BiP is induced by glucose starvation when it is called glucose-regulated protein 78 (GRP78). In Arabidopsis thaliana, however, we demonstrated that BiP transcripts decreased with sugar depletion and increased with sugar addition. Transcripts for b-glucuronidase (GUS) driven by BiP promoter respond to tunicamycin and sugar, being similar with endogenous BiP transcripts in transgenic A. thaliana. When GUS was regulated by P-UPRE, a cis-element responsible for the UPR identified in BiP promoter, GUS transcripts were accumulated by sugar starvation. Subsequently, transgenic A. thaliana harboring luciferase (LUC) gene regulated by P-UPRE was analyzed. Sugar depletion also increased LUC activity. It is concluded that BiP is induced by sugar independent of the cis-element responsible for the UPR. 2006 Elsevier Inc. All rights reserved. Keywords: Arabidopsis thaliana; BiP; Endoplasmic reticulum; UPR
The endoplasmic reticulum (ER) is the organelle where secretory proteins are synthesized, folded, and assembled. The ER contains molecular chaperones that help proper folding and assembly of proteins synthesized in the ER. If correct folding or assembly of proteins is disturbed by various stresses, a set of the ER chaperones is transcriptionally induced to obviate such stresses. This phenomenon observed among eukaryotes is referred to as the unfolded protein response (UPR) [1–4]. Stresses that induce the UPR are referred to as ER stresses. In the laboratory, treatment by drugs such as tunicamycin (Tm) and dithiothreitol (DTT) is often used to induce the UPR [5–8]. Tm is a potent inhibitor of Nlinked glycosylation that is important for proper folding of proteins in the ER. DTT disturbs disulfide bond formation in protein, resulting in the generation of malfolded
*
Corresponding author. Fax: +81 743 72 5659. E-mail address: [email protected] (N. Koizumi).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.05.189
proteins. Thus, Tm and DTT induce artificial ER stresses [9]. One of the most representative ER chaperones is the luminal binding protein (BiP), an ER cognate of HSP70. BiP is considered to bind nascent polypeptides and assist in their correct folding and assembly [10]. In addition, BiP is thought to play various roles, such as in the ER-associated degradation of unfolded proteins [11]. In addition to BiP, lectin-like chaperones calnexin/calreticulin have roles, especially for folding of glycoproteins [12]. GRP94, an ER homolog of HSP90, is also induced in the UPR although its specific function has not been elucidated [13– 16]. The signaling mechanism of the UPR has been extensively studied in yeast, in animals, and recently in plants. In each case, transcription factors bind to specific cis-elements in promoters of chaperone genes and activate transcription. Common cis-elements are often found in different chaperone genes [17–20]. In plants, at least two cis-elements responsible for the UPR have been identified [21]. One is P-UPRE (ATTGGTCCACGTCATC) and
H. Tajima, N. Koizumi / Biochemical and Biophysical Research Communications 346 (2006) 926–930
another is ERSE (CCAAT-N9-CCACG). The P-UPRE was found in the promoter of two BiP genes in Arabidopsis thaliana. Interestingly, these two cis-elements are almost identical to those identified in animals. Although ER stress in nature has not been determined, in mammalian cells glucose depletion induces ER chaperones such as BiP and GRP94 [22]. In fact, BiP is also called glucose-regulated protein 78 (GRP78), since it was identified as a protein induced by glucose depletion [23]. In plants, Tm and DTT have been shown to induce the UPR, while the effect of sugar has not yet been elucidated. Thus, in the present study, we examined the effect of sugars on the expression of BiP in A. thaliana. Materials and methods Plant materials and growth conditions. All plants used were A. thaliana ecotype Col-0. Seedlings germinated in half-strength Murashige and Skoog medium containing 1% (w/v) sucrose were further cultured in a light/dark cycle of 16/8 h with gentle shaking. For sugar starvation treatment, two-week-old seedlings were washed twice with distilled water and incubated in distilled water in the dark for 24 h with gentle shaking. These seedlings were transferred to half-strength Murashige and Skoog medium containing an appropriate concentration of sugars. Intact plants were grown in soil for 4 weeks in a light/dark cycle of 16/8 h. Rosette leaves of these plants were used for tunicamycin and sugar treatments. Transgenic plants. Transgenic A. thaliana harboring the b-glucuronidase (GUS) gene under regulation by the BiP2 promoter or the P-UPRE hexamer produced previously [21]. The GUS gene under the regulation of the hexamers of P-UPRE was exchanged with firefly luciferase (LUC) gene and the resulting construct was introduced into A. thaliana by in planta infiltration with Agrobacterium tumefaciens according to Clough et al. [24]. Homozygotic lines were selected and used for the LUC assay. Northern blot. Total RNA was extracted according to Gonzalez et al. [25], fractionated by agarose gel electrophoresis, and transferred to nylon membranes (Hybond N, Amersham). Hybridization probes were BiP cDNA and the coding region of the GUS gene labeled with 32P by using a DNA labeling kit (BcaBEST labeling kit, Takara). The membrane was washed in 0.2· SSC and 0.1% (w/v) SDS at 65 C, and exposed to X-ray film. Quantitative GUS assay and LUC assay. Protein was extracted by GUS extraction buffer (50 mM NaHPO4, pH 7, 10 mM b-mercaptoethanol, 10 mM Na2EDTA, 0.1% sodium lauryl sarcosine, and 0.1% Triton X-100). Quantitative assay of GUS activity was performed as described previously [26]. LUC activity was measured using an assay kit (Luciferase assay system, Promega) according to the manufacturer’s protocol.
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BiP rRNA Fig. 1. Various sugars induce BiP transcripts in A. thaliana. (A) Twoweek-old seedlings (lane 1) were deprived of sugar for 24 h and then transferred to water containing 90 mM sucrose in the dark for 0, 10, and 24 h (lanes 2–4). Total RNA was extracted at each time point. (B) Detached rosette leaves of four-week-old plants (lane 1) were deprived of sugar by transfer to water for 24 h in the dark (lane 2). Rosette leaves were then transferred to water (lane 3) and each 90 mM sugar solution (lane 4, sucrose; lane 5, glucose; lane 6, fructose; lane 7, mannitol) in the dark for 10 h. Five micrograms of RNA was placed in each lane and hybridized with the 32P-labeled BiP probe. rRNA was visualized with ethidium bromide staining as a loading control.
Profile of GUS reporter fused with the BiP promoter In order to examine whether induction of BiP transcription by sugar depends on a cis-element responsible for the UPR, transgenic A. thaliana harboring the GUS reporter gene fused with the BiP promoter was analyzed. At first, response of the GUS reporter in the UPR was determined by treatment with tunicamycin. As shown in Fig. 2, GUS transcripts and activity showed good correlation with accumulation of BiP transcripts, indicating that the promoter region used contains regulatory elements for the UPR. Subsequently, the effect of sucrose was examined. As shown in Fig. 3, both BiP and GUS transcripts increased after addition of sucrose, suggesting that the promoter is responsible for sugar induction of BiP in A. thaliana. However, we could not observe proportional GUS activity, probably due to stability of GUS protein (data not shown). In subsequent experiments, GUS transcripts were monitored instead of the activity.
Results Effect of P-UPRE on expression of the GUS reporter Regulation of BiP transcription by sugars Seedlings of A. thaliana growing in liquid medium were treated with sucrose after being depleted of sugar by keeping them in the dark. Expression of BiP was decreased by sugar depletion and increased by addition of sucrose (Fig. 1A). This observation contrasts with the results reported in mammalian cells. Subsequently, the effects of other sugars including glucose were examined (Fig. 1B). Glucose clearly induced accumulation of BiP transcripts, as did fructose, but mannitol did not.
In our previous study, a hexamer of P-UPRE fused with CaMV35S minimal promoter (6· P-UPRE) responded to Tm or DTT treatment in transient assay [21]. Then, we used transgenic A. thaliana harboring 6· P-UPRE with the GUS gene (6· P-UPRE :: GUS) in addition to transgenic plants with the authentic BiP promoter fused with the GUS gene (BiP-pro :: GUS). As shown in Fig. 4A, GUS transcripts regulated by both BiP-pro and 6· P-UPRE were highly induced by Tm. With sucrose treatment, GUS under BiP-pro showed a
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Fig. 2. Tunicamycin induces GUS expression of BiP-pro :: GUS transgenic A. thaliana. (A) Two-week-old seedlings were treated with tunicamycin, which was added to the culture medium at final concentrations of 0, 0.1, 0.3, 1.0, and 3.0 lg mL 1 for 16 h. Five micrograms of RNA was placed in each lane and hybridized with the 32P-labeled BiP and GUS probes. rRNA was visualized with ethidium bromide staining as a loading control. (B) Two-week-old seedlings were treated the same as (A) before GUS assay. GUS activity was calculated by dividing fluorescence intensity by microgram protein. Relative activity represents activities relative to basal activity obtained from seedlings treated without tunicamycin.
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Fig. 4. Sucrose induces BiP expression independently of the UPR. (A) Four-week-old detached rosette leaves of BiP-pro :: GUS plants (lanes 1 and 2) and 6· P-UPRE :: GUS plants (lanes 3 and 4) were treated with (lanes 2 and 4) and without (lanes 1 and 3) 5 lg mL 1 tunicamycin for 24 h. (B) Two-week-old seedlings (lanes 1 and 5) were deprived of sugar for 24 h in the dark (lanes 2 and 6), and then transferred to water (lanes 3 and 7) and water containing 90 mM sucrose (lanes 4 and 8) in the dark for 10 h. Five micrograms of RNA was placed in each lane and hybridized with the 32P-labeled BiP and GUS probes. Lanes 1–4 have BiP-pro :: GUS and lanes 5–8 have 6· P-UPRE :: GUS.
Response of P-UPRE on sucrose depletion In order to confirm the response of P-UPRE to sucrose depletion, transgenic A. thaliana with 6· P-UPRE with the LUC gene (6· P-UPRE :: LUC) was analyzed. When seedlings were transferred to the medium with or without tunicamycin, tunicamycin clearly increased LUC activity (Fig. 5A). Sucrose depletion increased LUC activity progressively as time of sucrose depletion increased. If sucrose was present, however, LUC activity did not increase (Fig. 5B).
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Discussion rRNA Fig. 3. Sucrose induces BiP and GUS transcripts in BiP-pro :: GUS transgenic A. thaliana. Detached rosette leaves of four-week-old plants were deprived of sugar by transfer to water in the dark for 24 h. Rosette leaves were then transferred to water containing 90 mM sucrose in the dark for 0 (lane 1), 5 (lane 2), 10 (lane 3), and 24 (lane 4) h, and total RNA was extracted at each time point. Five micrograms of RNA was placed in each lane and hybridized with the 32P-labeled BiP and GUS probes.
similar expression profile with BiP transcripts, but the expression profile of GUS under 6· P-UPRE was different (Fig. 4B). That is, the GUS transcripts increased in response to sucrose depletion but not due to the addition thereof. This result was the opposite to that for the accumulation profile of GUS transcripts driven by the BiP promoter.
Glucose depletion induces transcription of BiP/GRP78 mammalian cells. This induction has been considered to be due to the UPR [27]. The explanation underlying the occurrence of UPR in response to glucose depletion is related to the amount of energy required for protein folding, vesicle budding, and the prevention of protein aggregation, all of which disrupt ER homeostasis. Alternatively, reduction of cellular glucose levels may limit glycosylation of proteins, preventing folding of glycoproteins [4]. In the present study, however, we demonstrated that sugar depletion decreases BiP transcripts, but sugar addition increases BiP transcripts in a model plant A. thaliana. Although induction of BiP has been often considered to indicate the UPR, BiP induction does not always imply the UPR. For instance, when B lymphocytes differentiate into plasma cells, protein levels of ER chaperones including BiP
H. Tajima, N. Koizumi / Biochemical and Biophysical Research Communications 346 (2006) 926–930
Relative LUC activity
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Fig. 5. Sucrose depletion induces LUC activities of 6· P-UPRE :: LUC transgenic A. thaliana. (A) Two-week-old seedlings of 6· P-UPRE :: LUC plants were treated with and without 5 lg mL 1 tunicamycin for 24 h. Relative activity represents activities relative to basal activity obtained from seedlings treated without tunicamycin. (B) Seedlings were treated with water ( Suc) and water containing 90 mM sucrose (+Suc) for indicated time periods. Relative activity represents activities relative to basal activity obtained from seedlings at 0 h. LUC activity was calculated by dividing luminescence intensity by milligram protein. The results are means ± SE of three independent experiments.
increase before the mass production of immunoglobulins [28]. Similarly, in A. thaliana, induction of BiP precedes accumulation of PR1 protein, a pathogen-related secretory protein involved in defense response [29]. Such induction has been considered independent of the UPR pathway. In other words, those inductions do not depend on cis-elements responsible for the UPR. In order to clarify whether regulation of BiP expression by sugar observed in A. thaliana depends on the cis-element responsible for the UPR, we used transgenic plants harboring BiP pro :: GUS and 6· P-UPRE :: GUS. The P-UPRE is located in both promoters of two BiP genes, BiP1 and BiP2. These two genes are very similar to each other and show considerable homology even in the promoter sequences. In this study we used the BiP2 promoter, the expression profile of which is very similar to that of BiP1 (data not shown). Addition of sugars induced the GUS gene driven by the BiP2 promoter, being consistent with expression of endogenous BiP. On the other hand, sugar depletion induced the GUS gene driven by the P-UPRE. This result clearly indicates that BiP induction by sugar is independent of the UPR. The molecular mechanism to explain this phenomenon is that the BiP2 promoter may contain cis-elements respon-
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sible for sugar induction besides P-UPRE. In sugar-abundant conditions, P-UPRE is not thought to activate the expression of BiP, while another element may enhance the expression, resulting in induction of BiP. In fact promoters of BiP1 and BiP2 contain putative cis-elements responsible for the sugar induction. For instance, four sequences similar to SURE-1 (AATAGAAAA) identified in a potato patatin k2.1 promoter are found in BiP1 promoter [30]. One and two SURE-2 (TACTAATA) are located in BiP1 and BiP2 promoter, respectively [31]. However, it remains to be elucidated whether these sequences in fact function to induce BiP in sugar-abundant conditions. Why does sugar addition induce BiP in plants? One explanation is that addition of sugar increases rate of protein synthesis, which requires more folding machinery. In fact, addition of sugar induces many genes related to protein translation such as ribosomal proteins and elongation factors [32]. Therefore, it is likely that more BiP is necessary under sugar-abundant conditions to promote folding of nascent proteins. The finding that decreased BiP protein is associated with sugar consumption in a culture medium of A. thaliana suspension cells may corroborate our observations [33]. The different effects of sugars on expression of BiP between animals and plants might be due to difference in availability of sugars. Acknowledgments This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (Nos.1638023 and 17028037) to N. Koizumi. References [1] D.T. Rutkowski, R.J. Kaufman, A trip to the ER: coping with stress, Trends Cell Biol. 14 (2004) 20–28. [2] C. Patil, P. Walter, Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals, Curr. Opin. Cell Biol. 13 (2001) 349–355. [3] K. Mori, Tripartite management of unfolded proteins in the endoplasmic reticulum, Cell 101 (2000) 451–454. [4] R.J. Kaufman, D. Scheuner, M. Schroder, X. Shen, K. Lee, C.Y. Liu, S.M. Arnold, The unfolded protein response in nutrient sensing and differentiation, Nat. Rev. Mol. Cell Biol. 3 (2002) 411–421. [5] Y. Okushima, N. Koizumi, Y. Yamaguchi, Y. Kimata, K. Kohno, H. Sano, Isolation and characterization of a putative transducer of endoplasmic reticulum stress in Oryza sativa, Plant Cell Physiol. 43 (2002) 532–539. [6] N. Koizumi, I.M. Martinez, Y. Kimata, K. Kohno, H. Sano, M.J. Chrispeels, Molecular characterization of two Arabidopsis Ire1 homologs, endoplasmic reticulum-located transmembrane protein kinases, Plant Physiol. 127 (2001) 949–962. [7] N. Koizumi, Isolation and responses to stress of a gene that encodes a luminal binding protein in Arabidopsis thaliana, Plant Cell Physiol. 37 (1996) 862–865. [8] Y. Iwata, N. Koizumi, An Arabidopsis transcription factor, AtbZIP60, regulates the endoplasmic reticulum stress response in a manner unique to plants, Proc. Natl. Acad. Sci. USA 102 (2005) 5280–5285. [9] C.E. Shamu, J.S. Cox, P. Walter, The unfolded-protein-response pathway in yeast, Trends Cell Biol. 4 (1994) 56–60.
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[10] B. Kleizen, I. Braakman, Protein folding and quality control in the endoplasmic reticulum, Curr. Opin. Cell Biol. 16 (2004) 343–349. [11] M. Kabani, S.S. Kelley, M.W. Morrow, D.L. Montgomery, R. Sivendran, M.D. Rose, L.M. Gierasch, J.L. Brodsky, Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP, Mol. Biol. Cell 14 (2003) 3437–3448. [12] L. Ellgaard, A. Helenius, Quality control in the endoplasmic reticulum, Nat. Rev. Mol. Cell Biol. 4 (2003) 181–191. [13] S.J. Noh, C.S. Kwon, W.I. Chung, Characterization of two homologs of Ire1p, a kinase/endoribonuclease in yeast, in Arabidopsis thaliana, Biochim. Biophys. Acta 1575 (2002) 130–134. [14] I.M. Martinez, M.J. Chrispeels, Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes, Plant Cell 15 (2003) 561–576. [15] Y. Kozutsumi, M. Segal, K. Normington, M.J. Gething, J. Sambrook, The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins, Nature 332 (1988) 462–464. [16] S. Kamauchi, H. Nakatani, C. Nakano, R. Urade, Gene expression in response to endoplasmic reticulum stress in Arabidopsis thaliana, FEBS J. 272 (2005) 3461–3476. [17] H. Yoshida, K. Haze, H. Yanagi, T. Yura, K. Mori, Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors, J. Biol. Chem. 273 (1998) 33741–33749. [18] K. Mori, A. Sant, K. Kohno, K. Normington, M.J. Gething, J.F. Sambrook, A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins, EMBO J. 11 (1992) 2583–2593. [19] K. Mori, T. Kawahara, H. Yoshida, H. Yanagi, T. Yura, Signalling from endoplasmic reticulum to nucleus: transcription factor with a basic-leucine zipper motif is required for the unfolded proteinresponse pathway, Genes Cells 1 (1996) 803–817. [20] K. Kokame, H. Kato, T. Miyata, Identification of ERSE-II, a new cis-acting element responsible for the ATF6-dependent mammalian unfolded protein response, J. Biol. Chem. 276 (2001) 9199–9205. [21] D.H. Oh, C.S. Kwon, H. Sano, W.I. Chung, N. Koizumi, Conservation between animals and plants of the cis-acting element involved in the unfolded protein response, Biochem. Biophys. Res. Commun. 301 (2003) 225–230.
[22] J. Pouyssegur, K.M. Yamada, Isolation and immunological characterization of a glucose-regulated fibroblast cell surface glycoprotein and its nonglycosylated precursor, Cell 13 (1978) 139–140. [23] R.P. Shiu, J. Pouyssegur, I. Pastan, Glucose depletion accounts for the induction of two transformation-sensitive membrane proteinsin Rous sarcoma virus-transformed chick embryo fibroblasts, Proc. Natl. Acad. Sci. USA 74 (1977) 3840–3844. [24] S.J. Clough, A.F. Bent, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant J. 16 (1998) 735–743. [25] R.G. Gonzalez, R.S. Haxo, T. Schleich, Mechanism of action of polymeric aurintricarboxylic acid, a potent inhibitor of protein– nucleic acid interactions, Biochemistry 19 (1980) 4299–4303. [26] S.R. Gallagher, Quantitation of GUS activity by fluorometry, in: S.R. Gallagher (Ed.), GUS Protocols, Academic Press, San Diego, CA, 1992, pp. 47–59. [27] S. Munro, H.R. Pelham, An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein, Cell 46 (1986) 291–300. [28] E. van Anken, E.P. Romijn, C. Maggioni, A. Mezghrani, R. Sitia, I. Braakman, A.J. Heck, Sequential waves of functionally related proteins are expressed when B cells prepare for antibody secretion, Immunity 18 (2003) 243–253. [29] D. Wang, N.D. Weaver, M. Kesarwani, X. Dong, Induction of protein secretory pathway is required for systemic acquired resistance, Science 308 (2005) 1036–1040. [30] C. Grierson, J.S. Du, M. de Torres Zabala, K. Beggs, C. Smith, M. Holdsworth, M. Bevan, Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene, Plant J. 5 (1994) 815–826. [31] H. Fu, S.Y. Kim, W.D. Park, High-level tuber expression and sucrose inducibility of a potato Sus4 sucrose synthase gene require 5’ and 3’ flanking sequences and the leader intron, Plant Cell 7 (1995) 1387– 1394. [32] A.L. Contento, S.J. Kim, D.C. Bassham, Transcriptome profiling of the response of Arabidopsis suspension culture cells to Suc starvation, Plant Physiol. 135 (2004) 2330–2347. [33] K. Tamura, K. Yamada, T. Shimada, I. Hara-Nishimura, Endoplasmic reticulum-resident proteins are constitutively transported to vacuoles for degradation, Plant J. 39 (2004) 393–402.
Induction of BiP by sugar independent of a cis-element for the unfolded protein response in Arabidopsis thaliana Hiromi Tajima, Nozomu Koizumi
*
Department of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0192, Japan Received 29 May 2006 Available online 9 June 2006
Abstract BiP is a molecular chaperone induced in the unfolded protein response (UPR). In mammalian cells, BiP is induced by glucose starvation when it is called glucose-regulated protein 78 (GRP78). In Arabidopsis thaliana, however, we demonstrated that BiP transcripts decreased with sugar depletion and increased with sugar addition. Transcripts for b-glucuronidase (GUS) driven by BiP promoter respond to tunicamycin and sugar, being similar with endogenous BiP transcripts in transgenic A. thaliana. When GUS was regulated by P-UPRE, a cis-element responsible for the UPR identified in BiP promoter, GUS transcripts were accumulated by sugar starvation. Subsequently, transgenic A. thaliana harboring luciferase (LUC) gene regulated by P-UPRE was analyzed. Sugar depletion also increased LUC activity. It is concluded that BiP is induced by sugar independent of the cis-element responsible for the UPR. 2006 Elsevier Inc. All rights reserved. Keywords: Arabidopsis thaliana; BiP; Endoplasmic reticulum; UPR
The endoplasmic reticulum (ER) is the organelle where secretory proteins are synthesized, folded, and assembled. The ER contains molecular chaperones that help proper folding and assembly of proteins synthesized in the ER. If correct folding or assembly of proteins is disturbed by various stresses, a set of the ER chaperones is transcriptionally induced to obviate such stresses. This phenomenon observed among eukaryotes is referred to as the unfolded protein response (UPR) [1–4]. Stresses that induce the UPR are referred to as ER stresses. In the laboratory, treatment by drugs such as tunicamycin (Tm) and dithiothreitol (DTT) is often used to induce the UPR [5–8]. Tm is a potent inhibitor of Nlinked glycosylation that is important for proper folding of proteins in the ER. DTT disturbs disulfide bond formation in protein, resulting in the generation of malfolded
*
Corresponding author. Fax: +81 743 72 5659. E-mail address: [email protected] (N. Koizumi).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.05.189
proteins. Thus, Tm and DTT induce artificial ER stresses [9]. One of the most representative ER chaperones is the luminal binding protein (BiP), an ER cognate of HSP70. BiP is considered to bind nascent polypeptides and assist in their correct folding and assembly [10]. In addition, BiP is thought to play various roles, such as in the ER-associated degradation of unfolded proteins [11]. In addition to BiP, lectin-like chaperones calnexin/calreticulin have roles, especially for folding of glycoproteins [12]. GRP94, an ER homolog of HSP90, is also induced in the UPR although its specific function has not been elucidated [13– 16]. The signaling mechanism of the UPR has been extensively studied in yeast, in animals, and recently in plants. In each case, transcription factors bind to specific cis-elements in promoters of chaperone genes and activate transcription. Common cis-elements are often found in different chaperone genes [17–20]. In plants, at least two cis-elements responsible for the UPR have been identified [21]. One is P-UPRE (ATTGGTCCACGTCATC) and
H. Tajima, N. Koizumi / Biochemical and Biophysical Research Communications 346 (2006) 926–930
another is ERSE (CCAAT-N9-CCACG). The P-UPRE was found in the promoter of two BiP genes in Arabidopsis thaliana. Interestingly, these two cis-elements are almost identical to those identified in animals. Although ER stress in nature has not been determined, in mammalian cells glucose depletion induces ER chaperones such as BiP and GRP94 [22]. In fact, BiP is also called glucose-regulated protein 78 (GRP78), since it was identified as a protein induced by glucose depletion [23]. In plants, Tm and DTT have been shown to induce the UPR, while the effect of sugar has not yet been elucidated. Thus, in the present study, we examined the effect of sugars on the expression of BiP in A. thaliana. Materials and methods Plant materials and growth conditions. All plants used were A. thaliana ecotype Col-0. Seedlings germinated in half-strength Murashige and Skoog medium containing 1% (w/v) sucrose were further cultured in a light/dark cycle of 16/8 h with gentle shaking. For sugar starvation treatment, two-week-old seedlings were washed twice with distilled water and incubated in distilled water in the dark for 24 h with gentle shaking. These seedlings were transferred to half-strength Murashige and Skoog medium containing an appropriate concentration of sugars. Intact plants were grown in soil for 4 weeks in a light/dark cycle of 16/8 h. Rosette leaves of these plants were used for tunicamycin and sugar treatments. Transgenic plants. Transgenic A. thaliana harboring the b-glucuronidase (GUS) gene under regulation by the BiP2 promoter or the P-UPRE hexamer produced previously [21]. The GUS gene under the regulation of the hexamers of P-UPRE was exchanged with firefly luciferase (LUC) gene and the resulting construct was introduced into A. thaliana by in planta infiltration with Agrobacterium tumefaciens according to Clough et al. [24]. Homozygotic lines were selected and used for the LUC assay. Northern blot. Total RNA was extracted according to Gonzalez et al. [25], fractionated by agarose gel electrophoresis, and transferred to nylon membranes (Hybond N, Amersham). Hybridization probes were BiP cDNA and the coding region of the GUS gene labeled with 32P by using a DNA labeling kit (BcaBEST labeling kit, Takara). The membrane was washed in 0.2· SSC and 0.1% (w/v) SDS at 65 C, and exposed to X-ray film. Quantitative GUS assay and LUC assay. Protein was extracted by GUS extraction buffer (50 mM NaHPO4, pH 7, 10 mM b-mercaptoethanol, 10 mM Na2EDTA, 0.1% sodium lauryl sarcosine, and 0.1% Triton X-100). Quantitative assay of GUS activity was performed as described previously [26]. LUC activity was measured using an assay kit (Luciferase assay system, Promega) according to the manufacturer’s protocol.
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BiP rRNA Fig. 1. Various sugars induce BiP transcripts in A. thaliana. (A) Twoweek-old seedlings (lane 1) were deprived of sugar for 24 h and then transferred to water containing 90 mM sucrose in the dark for 0, 10, and 24 h (lanes 2–4). Total RNA was extracted at each time point. (B) Detached rosette leaves of four-week-old plants (lane 1) were deprived of sugar by transfer to water for 24 h in the dark (lane 2). Rosette leaves were then transferred to water (lane 3) and each 90 mM sugar solution (lane 4, sucrose; lane 5, glucose; lane 6, fructose; lane 7, mannitol) in the dark for 10 h. Five micrograms of RNA was placed in each lane and hybridized with the 32P-labeled BiP probe. rRNA was visualized with ethidium bromide staining as a loading control.
Profile of GUS reporter fused with the BiP promoter In order to examine whether induction of BiP transcription by sugar depends on a cis-element responsible for the UPR, transgenic A. thaliana harboring the GUS reporter gene fused with the BiP promoter was analyzed. At first, response of the GUS reporter in the UPR was determined by treatment with tunicamycin. As shown in Fig. 2, GUS transcripts and activity showed good correlation with accumulation of BiP transcripts, indicating that the promoter region used contains regulatory elements for the UPR. Subsequently, the effect of sucrose was examined. As shown in Fig. 3, both BiP and GUS transcripts increased after addition of sucrose, suggesting that the promoter is responsible for sugar induction of BiP in A. thaliana. However, we could not observe proportional GUS activity, probably due to stability of GUS protein (data not shown). In subsequent experiments, GUS transcripts were monitored instead of the activity.
Results Effect of P-UPRE on expression of the GUS reporter Regulation of BiP transcription by sugars Seedlings of A. thaliana growing in liquid medium were treated with sucrose after being depleted of sugar by keeping them in the dark. Expression of BiP was decreased by sugar depletion and increased by addition of sucrose (Fig. 1A). This observation contrasts with the results reported in mammalian cells. Subsequently, the effects of other sugars including glucose were examined (Fig. 1B). Glucose clearly induced accumulation of BiP transcripts, as did fructose, but mannitol did not.
In our previous study, a hexamer of P-UPRE fused with CaMV35S minimal promoter (6· P-UPRE) responded to Tm or DTT treatment in transient assay [21]. Then, we used transgenic A. thaliana harboring 6· P-UPRE with the GUS gene (6· P-UPRE :: GUS) in addition to transgenic plants with the authentic BiP promoter fused with the GUS gene (BiP-pro :: GUS). As shown in Fig. 4A, GUS transcripts regulated by both BiP-pro and 6· P-UPRE were highly induced by Tm. With sucrose treatment, GUS under BiP-pro showed a
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Fig. 2. Tunicamycin induces GUS expression of BiP-pro :: GUS transgenic A. thaliana. (A) Two-week-old seedlings were treated with tunicamycin, which was added to the culture medium at final concentrations of 0, 0.1, 0.3, 1.0, and 3.0 lg mL 1 for 16 h. Five micrograms of RNA was placed in each lane and hybridized with the 32P-labeled BiP and GUS probes. rRNA was visualized with ethidium bromide staining as a loading control. (B) Two-week-old seedlings were treated the same as (A) before GUS assay. GUS activity was calculated by dividing fluorescence intensity by microgram protein. Relative activity represents activities relative to basal activity obtained from seedlings treated without tunicamycin.
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Fig. 4. Sucrose induces BiP expression independently of the UPR. (A) Four-week-old detached rosette leaves of BiP-pro :: GUS plants (lanes 1 and 2) and 6· P-UPRE :: GUS plants (lanes 3 and 4) were treated with (lanes 2 and 4) and without (lanes 1 and 3) 5 lg mL 1 tunicamycin for 24 h. (B) Two-week-old seedlings (lanes 1 and 5) were deprived of sugar for 24 h in the dark (lanes 2 and 6), and then transferred to water (lanes 3 and 7) and water containing 90 mM sucrose (lanes 4 and 8) in the dark for 10 h. Five micrograms of RNA was placed in each lane and hybridized with the 32P-labeled BiP and GUS probes. Lanes 1–4 have BiP-pro :: GUS and lanes 5–8 have 6· P-UPRE :: GUS.
Response of P-UPRE on sucrose depletion In order to confirm the response of P-UPRE to sucrose depletion, transgenic A. thaliana with 6· P-UPRE with the LUC gene (6· P-UPRE :: LUC) was analyzed. When seedlings were transferred to the medium with or without tunicamycin, tunicamycin clearly increased LUC activity (Fig. 5A). Sucrose depletion increased LUC activity progressively as time of sucrose depletion increased. If sucrose was present, however, LUC activity did not increase (Fig. 5B).
GUS
Discussion rRNA Fig. 3. Sucrose induces BiP and GUS transcripts in BiP-pro :: GUS transgenic A. thaliana. Detached rosette leaves of four-week-old plants were deprived of sugar by transfer to water in the dark for 24 h. Rosette leaves were then transferred to water containing 90 mM sucrose in the dark for 0 (lane 1), 5 (lane 2), 10 (lane 3), and 24 (lane 4) h, and total RNA was extracted at each time point. Five micrograms of RNA was placed in each lane and hybridized with the 32P-labeled BiP and GUS probes.
similar expression profile with BiP transcripts, but the expression profile of GUS under 6· P-UPRE was different (Fig. 4B). That is, the GUS transcripts increased in response to sucrose depletion but not due to the addition thereof. This result was the opposite to that for the accumulation profile of GUS transcripts driven by the BiP promoter.
Glucose depletion induces transcription of BiP/GRP78 mammalian cells. This induction has been considered to be due to the UPR [27]. The explanation underlying the occurrence of UPR in response to glucose depletion is related to the amount of energy required for protein folding, vesicle budding, and the prevention of protein aggregation, all of which disrupt ER homeostasis. Alternatively, reduction of cellular glucose levels may limit glycosylation of proteins, preventing folding of glycoproteins [4]. In the present study, however, we demonstrated that sugar depletion decreases BiP transcripts, but sugar addition increases BiP transcripts in a model plant A. thaliana. Although induction of BiP has been often considered to indicate the UPR, BiP induction does not always imply the UPR. For instance, when B lymphocytes differentiate into plasma cells, protein levels of ER chaperones including BiP
H. Tajima, N. Koizumi / Biochemical and Biophysical Research Communications 346 (2006) 926–930
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Fig. 5. Sucrose depletion induces LUC activities of 6· P-UPRE :: LUC transgenic A. thaliana. (A) Two-week-old seedlings of 6· P-UPRE :: LUC plants were treated with and without 5 lg mL 1 tunicamycin for 24 h. Relative activity represents activities relative to basal activity obtained from seedlings treated without tunicamycin. (B) Seedlings were treated with water ( Suc) and water containing 90 mM sucrose (+Suc) for indicated time periods. Relative activity represents activities relative to basal activity obtained from seedlings at 0 h. LUC activity was calculated by dividing luminescence intensity by milligram protein. The results are means ± SE of three independent experiments.
increase before the mass production of immunoglobulins [28]. Similarly, in A. thaliana, induction of BiP precedes accumulation of PR1 protein, a pathogen-related secretory protein involved in defense response [29]. Such induction has been considered independent of the UPR pathway. In other words, those inductions do not depend on cis-elements responsible for the UPR. In order to clarify whether regulation of BiP expression by sugar observed in A. thaliana depends on the cis-element responsible for the UPR, we used transgenic plants harboring BiP pro :: GUS and 6· P-UPRE :: GUS. The P-UPRE is located in both promoters of two BiP genes, BiP1 and BiP2. These two genes are very similar to each other and show considerable homology even in the promoter sequences. In this study we used the BiP2 promoter, the expression profile of which is very similar to that of BiP1 (data not shown). Addition of sugars induced the GUS gene driven by the BiP2 promoter, being consistent with expression of endogenous BiP. On the other hand, sugar depletion induced the GUS gene driven by the P-UPRE. This result clearly indicates that BiP induction by sugar is independent of the UPR. The molecular mechanism to explain this phenomenon is that the BiP2 promoter may contain cis-elements respon-
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sible for sugar induction besides P-UPRE. In sugar-abundant conditions, P-UPRE is not thought to activate the expression of BiP, while another element may enhance the expression, resulting in induction of BiP. In fact promoters of BiP1 and BiP2 contain putative cis-elements responsible for the sugar induction. For instance, four sequences similar to SURE-1 (AATAGAAAA) identified in a potato patatin k2.1 promoter are found in BiP1 promoter [30]. One and two SURE-2 (TACTAATA) are located in BiP1 and BiP2 promoter, respectively [31]. However, it remains to be elucidated whether these sequences in fact function to induce BiP in sugar-abundant conditions. Why does sugar addition induce BiP in plants? One explanation is that addition of sugar increases rate of protein synthesis, which requires more folding machinery. In fact, addition of sugar induces many genes related to protein translation such as ribosomal proteins and elongation factors [32]. Therefore, it is likely that more BiP is necessary under sugar-abundant conditions to promote folding of nascent proteins. The finding that decreased BiP protein is associated with sugar consumption in a culture medium of A. thaliana suspension cells may corroborate our observations [33]. The different effects of sugars on expression of BiP between animals and plants might be due to difference in availability of sugars. Acknowledgments This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (Nos.1638023 and 17028037) to N. Koizumi. References [1] D.T. Rutkowski, R.J. Kaufman, A trip to the ER: coping with stress, Trends Cell Biol. 14 (2004) 20–28. [2] C. Patil, P. Walter, Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals, Curr. Opin. Cell Biol. 13 (2001) 349–355. [3] K. Mori, Tripartite management of unfolded proteins in the endoplasmic reticulum, Cell 101 (2000) 451–454. [4] R.J. Kaufman, D. Scheuner, M. Schroder, X. Shen, K. Lee, C.Y. Liu, S.M. Arnold, The unfolded protein response in nutrient sensing and differentiation, Nat. Rev. Mol. Cell Biol. 3 (2002) 411–421. [5] Y. Okushima, N. Koizumi, Y. Yamaguchi, Y. Kimata, K. Kohno, H. Sano, Isolation and characterization of a putative transducer of endoplasmic reticulum stress in Oryza sativa, Plant Cell Physiol. 43 (2002) 532–539. [6] N. Koizumi, I.M. Martinez, Y. Kimata, K. Kohno, H. Sano, M.J. Chrispeels, Molecular characterization of two Arabidopsis Ire1 homologs, endoplasmic reticulum-located transmembrane protein kinases, Plant Physiol. 127 (2001) 949–962. [7] N. Koizumi, Isolation and responses to stress of a gene that encodes a luminal binding protein in Arabidopsis thaliana, Plant Cell Physiol. 37 (1996) 862–865. [8] Y. Iwata, N. Koizumi, An Arabidopsis transcription factor, AtbZIP60, regulates the endoplasmic reticulum stress response in a manner unique to plants, Proc. Natl. Acad. Sci. USA 102 (2005) 5280–5285. [9] C.E. Shamu, J.S. Cox, P. Walter, The unfolded-protein-response pathway in yeast, Trends Cell Biol. 4 (1994) 56–60.
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[10] B. Kleizen, I. Braakman, Protein folding and quality control in the endoplasmic reticulum, Curr. Opin. Cell Biol. 16 (2004) 343–349. [11] M. Kabani, S.S. Kelley, M.W. Morrow, D.L. Montgomery, R. Sivendran, M.D. Rose, L.M. Gierasch, J.L. Brodsky, Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP, Mol. Biol. Cell 14 (2003) 3437–3448. [12] L. Ellgaard, A. Helenius, Quality control in the endoplasmic reticulum, Nat. Rev. Mol. Cell Biol. 4 (2003) 181–191. [13] S.J. Noh, C.S. Kwon, W.I. Chung, Characterization of two homologs of Ire1p, a kinase/endoribonuclease in yeast, in Arabidopsis thaliana, Biochim. Biophys. Acta 1575 (2002) 130–134. [14] I.M. Martinez, M.J. Chrispeels, Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes, Plant Cell 15 (2003) 561–576. [15] Y. Kozutsumi, M. Segal, K. Normington, M.J. Gething, J. Sambrook, The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins, Nature 332 (1988) 462–464. [16] S. Kamauchi, H. Nakatani, C. Nakano, R. Urade, Gene expression in response to endoplasmic reticulum stress in Arabidopsis thaliana, FEBS J. 272 (2005) 3461–3476. [17] H. Yoshida, K. Haze, H. Yanagi, T. Yura, K. Mori, Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors, J. Biol. Chem. 273 (1998) 33741–33749. [18] K. Mori, A. Sant, K. Kohno, K. Normington, M.J. Gething, J.F. Sambrook, A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins, EMBO J. 11 (1992) 2583–2593. [19] K. Mori, T. Kawahara, H. Yoshida, H. Yanagi, T. Yura, Signalling from endoplasmic reticulum to nucleus: transcription factor with a basic-leucine zipper motif is required for the unfolded proteinresponse pathway, Genes Cells 1 (1996) 803–817. [20] K. Kokame, H. Kato, T. Miyata, Identification of ERSE-II, a new cis-acting element responsible for the ATF6-dependent mammalian unfolded protein response, J. Biol. Chem. 276 (2001) 9199–9205. [21] D.H. Oh, C.S. Kwon, H. Sano, W.I. Chung, N. Koizumi, Conservation between animals and plants of the cis-acting element involved in the unfolded protein response, Biochem. Biophys. Res. Commun. 301 (2003) 225–230.
[22] J. Pouyssegur, K.M. Yamada, Isolation and immunological characterization of a glucose-regulated fibroblast cell surface glycoprotein and its nonglycosylated precursor, Cell 13 (1978) 139–140. [23] R.P. Shiu, J. Pouyssegur, I. Pastan, Glucose depletion accounts for the induction of two transformation-sensitive membrane proteinsin Rous sarcoma virus-transformed chick embryo fibroblasts, Proc. Natl. Acad. Sci. USA 74 (1977) 3840–3844. [24] S.J. Clough, A.F. Bent, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant J. 16 (1998) 735–743. [25] R.G. Gonzalez, R.S. Haxo, T. Schleich, Mechanism of action of polymeric aurintricarboxylic acid, a potent inhibitor of protein– nucleic acid interactions, Biochemistry 19 (1980) 4299–4303. [26] S.R. Gallagher, Quantitation of GUS activity by fluorometry, in: S.R. Gallagher (Ed.), GUS Protocols, Academic Press, San Diego, CA, 1992, pp. 47–59. [27] S. Munro, H.R. Pelham, An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein, Cell 46 (1986) 291–300. [28] E. van Anken, E.P. Romijn, C. Maggioni, A. Mezghrani, R. Sitia, I. Braakman, A.J. Heck, Sequential waves of functionally related proteins are expressed when B cells prepare for antibody secretion, Immunity 18 (2003) 243–253. [29] D. Wang, N.D. Weaver, M. Kesarwani, X. Dong, Induction of protein secretory pathway is required for systemic acquired resistance, Science 308 (2005) 1036–1040. [30] C. Grierson, J.S. Du, M. de Torres Zabala, K. Beggs, C. Smith, M. Holdsworth, M. Bevan, Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene, Plant J. 5 (1994) 815–826. [31] H. Fu, S.Y. Kim, W.D. Park, High-level tuber expression and sucrose inducibility of a potato Sus4 sucrose synthase gene require 5’ and 3’ flanking sequences and the leader intron, Plant Cell 7 (1995) 1387– 1394. [32] A.L. Contento, S.J. Kim, D.C. Bassham, Transcriptome profiling of the response of Arabidopsis suspension culture cells to Suc starvation, Plant Physiol. 135 (2004) 2330–2347. [33] K. Tamura, K. Yamada, T. Shimada, I. Hara-Nishimura, Endoplasmic reticulum-resident proteins are constitutively transported to vacuoles for degradation, Plant J. 39 (2004) 393–402.