- Email: [email protected]
Constitutive expression of an endoplasmic reticulum small heat shock protein alleviates endoplasmic reticulum stress in transgenic tomato
ARTICLE IN PRESS Journal of Plant Physiology 164 (2007) 835—841
www.elsevier.de/jplph
Constitutive expression of an endoplasmic reticulum small heat shock protein alleviates endoplasmic reticulum stress in transgenic tomato Chunmei Zhaoa, Mariko Shonob, Aiqing Suna, Shuying Yia, Minghui Lia, Jian Liua, a
College of Life Science, Shandong Normal University, Jinan, Shandong, PR China Okinawa Sub-tropical Station, Japan International Research Center for Agricultual Sciences (JIRCAS) 1091-1, Maezato Kawarabaru, Ishigaki, Okinawa 907-0002, Japan
b
Received 21 March 2006; accepted 20 June 2006
KEYWORDS Endoplasmic reticulum small heat-shock protein; Endoplasmic reticulum stress; Molecular chaperone; Tunicamycin; Unfold protein response
Summary To explore the function of endoplasmic reticulum-located small heat-shock proteins (ER-sHSPs) in ER stress, a putative ER-sHSP cDNA (the driven protein was named as LeHSP21.5 with GenBank accession No. AB026983) was isolated from tomato (Lycopersicon esculentum). Fractionation of the crude microsomes by isopycnic sucrose-gradient centrifugation revealed that LeHSP21.5 was distributed on a density corresponding to the fractions with a higher activity of ER marker enzyme, suggesting the localization of LeHSP21.5 in the ER. Overexpressing LeHSP21.5 in transgenic tomato plants (L. esculentum var. Zhongshu 4) greatly attenuated the lethal effect of tunicamycin on tomato seedlings. Moreover, under the tunicamycin treatment, transcripts of BiP, PDI and calnexin in transgenic tomato plants accumulated to a less level than those in non-transgenic tomato plants. These results suggest that LeHSP21.5 can function to alleviate the tunicamycin-induced ER stress. & 2006 Elsevier GmbH. All rights reserved.
Abbreviations: BiP, binding protein; BLAST, basic local alignment search tool; DTT, dithiothreitol; ER-sHSP, endoplasmic reticulum small heat-shock protein; ER stress, endoplasmic reticulum stress; EST, expressed sequence tag; GST, glutathione S-transferase; PCR, polymerase chain reaction; PDI, protein disulfide isomerase; UPR, unfold protein response Corresponding author. Tel.: +86 531 86180797; fax: +86 531 86180107. E-mail address: [email protected] (J. Liu).
Introduction The endoplasmic reticulum (ER) is the site of an assembly of polypeptide chains destined for secretion or routing into various subcellular compartments. A variety of stress conditions, including the treatment with tunicamycin, cause aberrant
0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.06.004
ARTICLE IN PRESS 836 folding of newly synthesized polypeptides, and the accumulation of unfolded proteins in the ER is referred to as ER stress (Pelham, 1989). Via a signaling transduction pathway termed as unfolded protein response (UPR), the accumulation of unfolded proteins within the ER lumen ultimately induces the expression of a set of ER-located molecular chaperones, such as protein disulfide isomerase (PDI), calnexin and binding protein (BiP), to assist the refolding and assembly of proteins. The increment of ER-resident molecular chaperones can help to prevent the non-productive intermolecular interaction and misaggregation of proteins in the ER (Hammond and Helenius, 1995). The relationship between ER-resident molecular chaperones and UPR in plants, especially the best characterized BiP (Denecke, 1996), has been revealed by several excellent research projects. The overexpression of BiP in tobacco could alleviate UPR, slow down the induction of other ER molecular chaperones and increase the whole plant’s resistance to tunicamycin, which is a potent inducer of UPR (Leborgne-Castel et al., 1999; Alvim et al., 2001). However, most studies mentioned above are focused only on high molecular chaperones, neglecting the small heat shock proteins (sHSPs). Early reports showed a co-sedimentation of sHSP with endomembrane fractions from heat-stressed maize and barley (Cooper and Ho, 1987; LaFayette and Travis, 1990; Sticher et al., 1990). Later there was a report about endomembrane localization of the sHSPs PsHSP22.7 and GmHSP22.0 in heatstressed pea and heat-stressed soybean, respectively (Helm et al., 1993). In potato tubers, a coldinduced gene, C119, has been shown to have homology to genes of heat-shock induced sHSPs localized in the ER of pea (Helm et al., 1993), suggesting that the C119 gene product exists in the ER (van Berkel et al., 1994). The localization of sHSPs in the endomembrane has also been suggested (Helm et al., 1995) in Arabidopsis and recently in cortical parenchyma cells of mulberry trees (Ukaji et al., 1999). All these findings suggest that ER-localized sHSPs accumulate in all higher plants, and that these sHSPs might have important functions in protecting the ER proteins from the heat-induced damage. As an ER-localized molecular chaperone peculiar to plants, ER-sHSP has not been well studied, except for its basic information as described above. Its possible role on ER stress and mediation to UPR has not been demonstrated. Thus, in the present study, we cloned an ERlocalized sHSP gene (named as LeHSP21.5) from tomato, and examined the effect of tunicamycin on LeHSP21.5 transgenic tomato plants with the
C. Zhao et al. purpose of investigating a possible role of ER-sHSP in ER stress.
Materials and methods Isolation of LeHSP21.5 cDNA Two degenerated primers (50 -CTTAAGCTTAARGARACWSCRGARGG-30 and 50 -ATCAAGCTTAGTAAGMACHCCRTTYTC-30 , Hind III underline) were designed, and employed to amplify the mRNA isolated from tomato (Lycopersicon esculentum) leaves which were treated at 39 1C for 3 h. These primers was designed based on the reported cDNA and consensus amino acid sequences at carboxylterminal (C-terminal) and central regions of endoplasmic reticulum small heat shock proteins (ERsHSP) in potato, pea, soybean and Arabidopsis (their GenBank accession numbers are S70186, M33898, X63198 and U11501, respectively). After the first strand of cDNA was synthesized with M-MLV reverse transcriptase (Gibco BRL), the polymerase chain reaction (PCR) was conducted with ExTaq DNA polymerase (Takara). The amplified DNA was digested by Hind III and cloned into pBluescript II (SK)+vector. After the sequencing of the insert, one clone containing the tomato ER-sHSP cDNA conserved region was selected. According to the result of the BLAST retrieval system, the cloned cDNA fragment was believed to be a conservative region in the ER-sHSP gene and was used for the preparation of the probe. A tomato cDNA library was constructed in the l ZAP II vector (Stratagene, La Jolla, CA, USA) with Poly(A)+ RNA prepared from the heat-treated tomato flowers (39 1C/3 h). The library was probed with the cDNA fragment described above. Positive plaques were isolated and converted into the pBluescript (SK) vector by an in vivo excision method according to the manufacturer’s instructions. Sequencing of the cDNA fragment that had been sub-cloned into the pBluescript (SK) vector was performed.
Antibody production To introduce BamH I and EcoR I sites, the full length cDNA of LeHSP21.5 was amplified by PCR with primers (50 -GAAGGATCCATACATATGAGGGTCATCAG-30 and 50 -AGCGAATTCAGCAGCCAACTCAGCTTC-30 ) and Taq DNA polymerase (Takara). After digested with BamH I and EcoR I, the PCR product was sub-cloned into the glutathione S-transferase (GST) vector (pGEX-6P-1) to generate a GST-LeHSP21.5 fusion
ARTICLE IN PRESS Overexpressing LeHSP21.5 alleviates ER-stress protein. The C-terminus of calnexin cDNA (GenBank accession No. AB218598) was amplified with primers (50 -TCGGATCCATTGGCATTGAGATC-30 and 50 -AGGAATTCGACGAGGAGCAGCAC-30 ), and the PCR product digested by BamH I and EcoR I was sub-cloned into the GST vector (pGEX-6P-3) to generate a GSTcalnexin fusion protein. The accuracy of these constructs was verified by DNA sequencing. Recombinant proteins were expressed in strain BL21 (DE3) of Escherichia coli and purified by glutathioneagarose beads affinity chromatography as described previously (Lindahl et al., 2000). Polyclonal antibodies were raised in rabbits against GST-LeHSP21.5 and GST-calnexin, respectively.
Protein gel blot analysis Total protein was extracted from tomato leaves (500 mg) in 500 mL of cold extraction buffer (10 mM Tris-HCl, pH8.0). The protein concentration was determined by the method of Bradford (1976). An equal amount of proteins (10 mg) was separated on 12.5% polyacrylamide-SDS gel and transferred to PVDF membranes with a semi-dry blot system. The subsequent immunoblot analysis was carried out as described by Alvim et al. (2001). The specific antibody (anti-LeHSP21.5 or anti-calnexin) was used at a dilution of 1:100 with 0.05% (v/v) in TBST (20 mM Tris-HCl, pH7.5, and 500 mM NaCl).
Subcellular localization analysis of LeHSP21.5 Microsomal membranes were prepared from excised tomato roots (20 g) which have been pretreated at 39 1C for 3 h, according to the procedure previously described by De Michelis and Spanswick (1986) with a slight modification. After homogenization and centrifugation, membrane pellets were resuspended in a resuspension buffer (1 mL/10 g starting material), containing 10% (w/v) Suc, 1 mM EGTA, 0.1 mM EDTA, and 2 mM DTT. One milliliter of the crude microsome was overlaid on 12 mL of a continuous Suc gradient (20–40%, w/v) containing the resuspension buffer and centrifuged at 25,000 rpm for 4 h at 4 1C (Beckman SW40 Ti Rotor). After the centrifugation, each 1.2 mL of gradient medium was fractionated and used for the following experiments. Activities of marker enzymes, including vanadate-sensitive H+-ATPase, nitrate-sensitive H+-ATPase, antimycin A-insensitive NADH Cytochrome c reductase and Cytochrome c oxidase, were measured in each fraction, as described by Koike et al. (1997).
837
Tomato transformation The complete LeHSP21.5 cDNA fragment with BamH I and Kpn I cohesive ends produced from a pBluescript (SK) plasmid described above was ligated with pROK II vector with the same cohesive ends, forming the reconstructed pROK II binary plasmid under cauliflower mosaic virus (CaMV) 35S promoter. After verified by DNA sequencing, the reconstructed pROK II binary plasmid was transformed into Agrobacterium strain LBA4404 using the method described by Ho ¨fgen and Willmitzer (1988). Cotyledons of 7–10-d-old tomato seedlings (L. esculentum var. Zhongshu 4) were used for transformation. The genetic transformation was performed as previously described by Fillatti et al. (1987). Transformed callus were selected by kanamycin resistance and then transgenic plants were regenerated. The homozygotic lines of T3 generational transgenic tomato plants were used as plant materials for the following experiments.
RNA gel blot analysis Total RNA was extracted from the leaf tissue as described by Logemann et al. (1987). Twenty-five micrograms total RNA of each sample were separated on 1.5% formaldehyde-agarose gels, and then transferred to the nylon membrane and UV fixed. The full-length LeHSP21.5 cDNA, partial length BiP, PDI and calnexin cDNA were amplified, labeled with [a-32P]dCTP and used as the probe, respectively. Hybridization and washing was carried out at 65 1C as described by Sauter (1997). The primers for LeHSP21.5 cDNA were 50 -GAAGGATCCATACATATGAGGGTCATCAG-30 and 50 -AGC0 GAATTCAGCAGCCAACTCAGCTTC-3 . The primers for calnexin cDNA were 50 -TCGGATCCATTGGCATTGAGATC-30 and 50 -AGGAATTCGACGAGGAGCAGCAC-30 . cDNAs of BiP and PDI were amplified from the cDNA library by PCR according to the reported sequences in tomato expressed sequence tag database with primers BiP-1 (50 -AGAGAAGCTGAGCGTGCCAAGAG-30 ) and BiP-2 (50 -CTGGTCCTGGTAAGTGGTGAAGAC-30 ), and primes PDI-1 (50 -GGTCCTGCATCAGCTGAAATCAAGTC-30 ) and PDI-2 ( 50 -CTTGCAGTGGCCACACCAAGGTG-30 ), respectively. The accuracy of these PCR products was verified by DNA sequencing.
UPR analysis Surface-sterilized seeds of T3 generational LeHSP21.5-transformed plants (TP), pROK II vector-transformed plants (VT) and wild-type (WT)
ARTICLE IN PRESS 838
C. Zhao et al.
plants were grown in liquid 1/2 MS0 medium (28 1C) with a gentle shake. Two-week-old seedlings were treated with 10 mg/mL tunicamycin for 24 h and then transferred to tunicamycin-free liquid 1/2 MS0 medium to allow their recovery. Total RNA was extracted at the indicated time for RNA hybridization with probes BiP, PDI and calnexin as described above.
Results LeHSP21.5 is characteristic of ER-resident protein A DNA fragment, corresponding to the conserved amino acid sequences of the ER-sHSP of other organisms, was reproducibly amplified by RT-PCR from tomatoes. Using this DNA fragment as a probe, we isolated a tomato ER-sHSP cDNA clone, designated LeHSP21.5 (GenBank accession No. AB026983). The similarity between the deduced amino acid sequence of LeHSP21.5 and other reported ER-sHSP was well compared and documented by Scharf et al. (2001). To confirm the subcelluar localization of LeHSP21.5, microsomes were isolated from the heat-treated wild-type tomato root by centrifugation and further separated by continuous sucrose gradient centrifugation (20–40% w/v sucrose). Proteins in each fraction were subjected to an immunoblot analysis with polyclonal antibodies against either LeHSP21.5 or calnexin. The distribution profile of LeHSP21.5 in fractions of sucrose gradient corresponded to that of calnexin (Fig. 1B). Moreover, fractions with higher western signals of LeHSP21.5 also possessed higher activities of the ER marker enzymes (Fig. 1A). Together, these data support the interpretation that LeHSP21.5 is a small heat shock protein located in the ER.
Identification of transgenic tomato plants A construct containing the LeHSP21.5 was introduced into the genome of L. esculentum (var. Zhongshu 4). Seven independent transgenic lines (TP) were generated. A pROK II-transfomed line (VT) was also generated to serve as a vectorcontrol. According to the analysis of kanamycinresistant segregation ratio in T1 seeds and the Mendel’s principle law, four lines that appeared to have only one locus of integrated T-DNA on genome were selected. Transgenic plants were screened by Northern and Western analysis in the T2 generation.
Figure 1. Localization of LeHSP21.5 in the crude microsome fractions. Microsomes were isolated from heattreated (39 1C) wild-type tomato roots and overlaid on an isopycnic Suc-density gradient. 1.2 mL fractions were collected from top to bottom and marker-enzyme activities (V-ATPase, P-ATPase, NADH Cyt c reductase and Cyt c oxidase) in four organelles (tonoplast, plasma membrane, ER and MT, respectively) were measured (A). The bottom and top of the fractions are indicated. SDSPAGE of fractionated proteins was performed using 10 mg protein samples in each fraction. An Immunoblot analysis of proteins was performed with anti-LeHSP21.5 and anticalnexin antibodies (B). Molecular masses are shown in kDa.
High LeHSP21.5 transcript and protein levels were confirmed in the T3 generation of TP plants (Fig. 2).
Tunicamycin resistant ability of TP plants To elucidate the role that LeHSP21.5 played in ER-stress, 14-day-old seedlings were treated with tunicamycin, which acted as a potent activator of UPR pathway. The seedlings were treated with 10 mg/mL tunicamycin for 24 h and then recovered in a tunicamycin-free medium for another 72 h. After the treatment, TP plants remained alive with fresh green cotyledons and leaves, a contrast to the distinct necrosis which happened to cotyledons in WT and VT plants (Fig. 3).
Unfold protein response of TP plants It is a known fact that tunicamycin induced the expression of the ER resident molecular chaperones such as BiP as part of the overall UPR of the cells. In order to detect whether an overexpression of
ARTICLE IN PRESS Overexpressing LeHSP21.5 alleviates ER-stress
Figure 2. RNA and protein gel blot analysis of transgenic plants overexpressing LeHSP21.5. (A) Total RNA (20 mg per lane) extracted from four lines of transgenic tomatoes overexpressing LeHSP21.5 (TP 6, 8, 10, 13) and the pROK II-vector transformed plant (VT) were blotted after separation and hybridized with 32P-labeled LeHSP21.5 cDNA probes. rRNA was visualized with the ethidium bromide staining as a loading control. (B) Leaf protein extracts (10 mg per lane) of TP and VT plants were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies raised against recombinant protein GST-LeHSP21.5. WT/HS was a wild-type plant which was heated at 39 1C for 3 h and used as a positive control.
Figure 3. Tunicamycin resistance ability analysis of TP lines and control lines (WT and VT). Fourteen-day-old tomato seedlings growing in liquid MS medium were treated with tunicamycin (10 mg/mL) for 24 h, and then recovered for another 72 h on MS medium without tunicamycin.
LeHSP21.5 in plants could affect the mRNA levels of BiP or other ER chaperones, control plants (WT and VT) and TP plants were first cultivated in liquid MS medium without tunicamycin, and then treated with tunicamycin (10 mg/mL) for different periods of time before RNA extraction. As shown in Fig. 4A, the basal BiP mRNA level is lower in TP plants (cf. first and sixth lanes), and the tunicamycinmediated induction observed in control plants (second lane) was barely noticeable in the transformed plants overproducing LeHSP21.5 (seventh lane). The BiP mRNA level in control plants accumulated quickly and reached the peak at 10 h, whereas in TP plants its level at 10 h (tenth
839
Figure 4. RNA and protein gel blot analysis of tunicamycin-treated plants. Fourteen-day-old tomato seedlings growing in liquid MS medium were treated with tunicamycin (10 mg/mL) for 24 h and total RNA was extracted at the indicated time periods, 20 mg of total RNA was loaded in each lane and hybridized with cDNA probes of BiP (A), PDI (B) and calnexin (C). rRNA was visualized with ethidium bromide staining as a loading control. (D) Leaf protein extracts (10 mg per lane) of tunicamycin-treated TP and control plants were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies raised against recombinant protein GSTLeHSP21.5. CK means control lines (WT and VT); TP means transgenic lines.
lane) was only equal to that of control plants at 4 h (third lane). Hybridization with PDI or calnexinspecific probes showed a similar reduction in the basal and tunicamycin-mediate induction mRNA levels of PDI and calnexin (Figs. 4B and C). Thus, artificially increasing the LeHSP21.5 protein levels led to a down-regulation of the unfolded protein response. The LeHSP21.5 protein level in TP plants remained high during the treatment. However, we did not detect any trace of LeHSP21.5 in tunicamycin-treated control plants (Fig. 4D).
Discussion Several ER-located sHSPs in plants have been reported, and it has been suggested that they might execute molecular chaperone function in stabilizing proteins during stress conditions (Ukaji et al., 1999). However, the evidence that ER-sHSP confers enhanced ER stress tolerance to plants has not been reported. In the present study, we have isolated a tomato cDNA LeHSP21.5 which encodes a putative homolog of ER-sHSP, and introduced the construction of DNA, containing LeHSP21.5 cDNA
ARTICLE IN PRESS 840 under the control of 35S CaMV promoter, into tomato plants by Agrobacterium-mediated transformation. Interestingly, transgenic tomatoes with an over-expression of LeHSP21.5 exhibited the improved tunicamycin-resistance ability. Tunicamycin is an ER stress inducer by blocking the glycosylation of newly synthesized proteins in the ER. ER stress leads to UPR. UPR provides a protective response to ER stress. An increased expression of ER-located molecular chaperons is regarded as the most important indication of UPR (Koizumi et al., 2001; Martı´nez and Chrispeels, 2003). It has been proved that overexpressing BiP in plants leads to the down-regulation of BiP and other ER-located chaperones under normal or tunicamycin stress conditions (Leborgne-Castel et al., 1999; Alvim et al., 2001). In the present study, similar to these previous cases, the overexpression of LeHSP21.5 in plants down-regulated not only the basal but also the inducible mRNA levels of BiP, calnexin and PDI (Fig. 4), which indicated the alleviated ER stress. In addition, Alvim et al. (2001) have confirmed that the effectiveness of BiP protection against ER stress could be extended to the whole organism level, as judged by the results of germination/survival assay in the presence of tunicamycin. Here, we showed that overexpressing LeHSP21.5 in plants also enhanced the whole plant’s resistance to tunicamycin (Fig. 3), which further proved the protective effect of LeHSP21.5 against ER stress. Up to now, the UPR signal transmission pathway in plants has not been revealed completely. The transgenic plants with an overexpression of ER-sHSP may provide another way to study the ER stress in plant cells. Analysis of the promoter regions of UPR-regulated genes in plants has identified the presence of ER-stress response elements (Martı´nez and Chrispeels, 2003). Although artificially increasing the LeHSP21.5 protein levels led to an alleviation of the UPR, the endogenous LeHSP21.5 gene did not respond to tunicamycin (Fig. 4). It may suggest an absence of the ER-stress response element in the promoter of LeHSP21.5. If this claim is true, it further suggestes that LeHSP21.5 is not a UPRregulated gene even though it is a molecular chaperone located in the ER. To our best knowledge, this is the first time to demonstrate that overexpressing an ER-sHSP gene could enhance the ER stress tolerance of the transgenic plants. Recently, it has been proved that the abiotic stress could also induce the UPR in plants (Koiwa et al., 2003). Overexpressing ER molecular chaperones in plants enhances their resistance to tunicamycin as well as to other abiotic stresses (Alvim et al., 2001). Based on the
C. Zhao et al. results obtained in the present study, we can now expect to utilize transgenic tomatoes for demonstrating the precise role of LeHSP21.5 in ER stress or other abiotic stresses.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 30270132).
References Alvim FC, Carolino SMB, Cascardo JCM, Nunes CC, Martinez CA, Otoni WC, et al. Enhanced accumulation of BiP in transgenic plants confers tolerance to water stress. Plant Physiol. 2001;126:1042–54. Bradford M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248–54. Cooper P, Ho THD. Intracellular localization of heat shock proteins in maize. Plant Physiol. 1987;84:1197–203. De Michelis MI, Spanswick RM. H+ pumping driven by the vanadate-sensitive ATPase in membrane vesicles from corn roots. Plant Physiol. 1986;81:542–7. Denecke J. Soluble endoplasmic reticulum resident proteins and their function in protein synthesis and transport. Plant Physiol. Biochem. 1996;34:197–205. Fillatti JJ, Kiser J, Rose R, Comai L. Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Biotechnology 1987;5:726–30. Hammond C, Helenius A. Quality control in the secretory pathway. Curr. Opin. Cell Biol. 1995;7:523–9. Helm KW, LaFayette PR, Nagao RT, Key JL, Vierling E. Localization of small heat shock proteins to the higher plant endomembrane system. Mol. Cell Biol. 1993;13: 238–47. Helm KW, Schmeits J, Vierling E. An endomembranelocalized small heat-shock protein from Arabidopsis thaliana. Plant Physiol. 1995;107:287–8. Ho ¨fgen R, Willmitzer L. Storage of competent cells for Agrobacterium transformation. Nucl. Acids Res. 1988; 16:9877. Koike M, Takezawa D, Arakawa K, Yoshida S. Accumulation of 19-kDa plasma membrane polypeptide during induction of freezing tolerance in wheat suspensioncultured cells by abscisic acid. Plant Cell Physiol. 1997;38:707–16. Koiwa H, Li F, McCully MG, Mendoza I, Koizumi N, Manabe Y, et al. The STT3a subunit isoform of the Arabidopsis oligosaccharyltransferase controls adaptive responses to salt/osmotic stress. Plant Cell 2003;15:2273–84. Koizumi N, Martı´nez IM, Kimata Y, Kohno K, Sano H, Chrispeels MJ. Molecular characterization of two Arabidopsis Ire1 homologs, endoplasmic reticulum-located
ARTICLE IN PRESS Overexpressing LeHSP21.5 alleviates ER-stress transmembrane protein kinases. Plant Physiol. 2001; 127:949–62. LaFayette PR, Travis RL. Soluble and membrane-associated heat shock proteins in soybean root. Protoplasma 1990;156:174–82. Leborgne-Castel N, Jelitto-Van Dooren EPWM, Crofts AJ, Denecke J. Overexpression of BiP in tobacco alleviates endoplasmic reticulum stress. Plant Cell 1999;11: 459–69. Lindahl M, Spetea C, Hundal T, Oppenheim AB, Adam Z, Andersson B. The thylakoid FtsH protease plays a role in the light-induced turnover of the photosystem II D1 protein. Plant Cell 2000;12:419–31. Logemann J, Schell J, Willmitzer L. Improved method for the isolation of RNA from plant tissues. Anal. Biochem. 1987;163:16–20. Martı´nez IM, Chrispeels MJ. Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes. Plant Cell 2003;15:561–76. Pelham HRB. Control of protein exit from the endoplasmic reticulum. Annu. Rev. Cell Biol. 1989;5:1–23.
841 Sauter M. Differential expression of a CAK (cdc2-activating kinase)-like protein kinase, cyclins and cdc2 genes from rice during the cell cycle and in response to gibberellin. Plant J. 1997;11:181–90. Scharf KD, Siddique M, Vierling E. The expanding family of Arabidopsis thaliana small heat stress proteins and a new family of proteins containing a-crystallin domains (Acd proteins). Cell Stress Chaperones 2001;6:225–37. Sticher L, Biswas AK, Bush DS, Jones RL. Heat shock inhibits a-amylase synthesis in barley aleurone without inhibiting the activity of endoplasmic reticulum marker enzymes. Plant Physiol. 1990;92:506–13. Ukaji N, Kuwabara C, Takezawa D, Arakawa K, Yoshida S, Fujikawa S. Accumulation of small heat-shock protein homologs in the edoplasmic reticulum of cortical parenchyma cells in mulberry in association with seasonal cold acclimation. Plant Physiol. 1999;120: 481–9. van Berkel J, Salamini F, Gebhardt C. Transcripts accumulating during cold storage of potato (Solanum tuberosum L.) tubers are sequence related to stressresponsive genes. Plant Physiol. 1994;104:445–52.
www.elsevier.de/jplph
Constitutive expression of an endoplasmic reticulum small heat shock protein alleviates endoplasmic reticulum stress in transgenic tomato Chunmei Zhaoa, Mariko Shonob, Aiqing Suna, Shuying Yia, Minghui Lia, Jian Liua, a
College of Life Science, Shandong Normal University, Jinan, Shandong, PR China Okinawa Sub-tropical Station, Japan International Research Center for Agricultual Sciences (JIRCAS) 1091-1, Maezato Kawarabaru, Ishigaki, Okinawa 907-0002, Japan
b
Received 21 March 2006; accepted 20 June 2006
KEYWORDS Endoplasmic reticulum small heat-shock protein; Endoplasmic reticulum stress; Molecular chaperone; Tunicamycin; Unfold protein response
Summary To explore the function of endoplasmic reticulum-located small heat-shock proteins (ER-sHSPs) in ER stress, a putative ER-sHSP cDNA (the driven protein was named as LeHSP21.5 with GenBank accession No. AB026983) was isolated from tomato (Lycopersicon esculentum). Fractionation of the crude microsomes by isopycnic sucrose-gradient centrifugation revealed that LeHSP21.5 was distributed on a density corresponding to the fractions with a higher activity of ER marker enzyme, suggesting the localization of LeHSP21.5 in the ER. Overexpressing LeHSP21.5 in transgenic tomato plants (L. esculentum var. Zhongshu 4) greatly attenuated the lethal effect of tunicamycin on tomato seedlings. Moreover, under the tunicamycin treatment, transcripts of BiP, PDI and calnexin in transgenic tomato plants accumulated to a less level than those in non-transgenic tomato plants. These results suggest that LeHSP21.5 can function to alleviate the tunicamycin-induced ER stress. & 2006 Elsevier GmbH. All rights reserved.
Abbreviations: BiP, binding protein; BLAST, basic local alignment search tool; DTT, dithiothreitol; ER-sHSP, endoplasmic reticulum small heat-shock protein; ER stress, endoplasmic reticulum stress; EST, expressed sequence tag; GST, glutathione S-transferase; PCR, polymerase chain reaction; PDI, protein disulfide isomerase; UPR, unfold protein response Corresponding author. Tel.: +86 531 86180797; fax: +86 531 86180107. E-mail address: [email protected] (J. Liu).
Introduction The endoplasmic reticulum (ER) is the site of an assembly of polypeptide chains destined for secretion or routing into various subcellular compartments. A variety of stress conditions, including the treatment with tunicamycin, cause aberrant
0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.06.004
ARTICLE IN PRESS 836 folding of newly synthesized polypeptides, and the accumulation of unfolded proteins in the ER is referred to as ER stress (Pelham, 1989). Via a signaling transduction pathway termed as unfolded protein response (UPR), the accumulation of unfolded proteins within the ER lumen ultimately induces the expression of a set of ER-located molecular chaperones, such as protein disulfide isomerase (PDI), calnexin and binding protein (BiP), to assist the refolding and assembly of proteins. The increment of ER-resident molecular chaperones can help to prevent the non-productive intermolecular interaction and misaggregation of proteins in the ER (Hammond and Helenius, 1995). The relationship between ER-resident molecular chaperones and UPR in plants, especially the best characterized BiP (Denecke, 1996), has been revealed by several excellent research projects. The overexpression of BiP in tobacco could alleviate UPR, slow down the induction of other ER molecular chaperones and increase the whole plant’s resistance to tunicamycin, which is a potent inducer of UPR (Leborgne-Castel et al., 1999; Alvim et al., 2001). However, most studies mentioned above are focused only on high molecular chaperones, neglecting the small heat shock proteins (sHSPs). Early reports showed a co-sedimentation of sHSP with endomembrane fractions from heat-stressed maize and barley (Cooper and Ho, 1987; LaFayette and Travis, 1990; Sticher et al., 1990). Later there was a report about endomembrane localization of the sHSPs PsHSP22.7 and GmHSP22.0 in heatstressed pea and heat-stressed soybean, respectively (Helm et al., 1993). In potato tubers, a coldinduced gene, C119, has been shown to have homology to genes of heat-shock induced sHSPs localized in the ER of pea (Helm et al., 1993), suggesting that the C119 gene product exists in the ER (van Berkel et al., 1994). The localization of sHSPs in the endomembrane has also been suggested (Helm et al., 1995) in Arabidopsis and recently in cortical parenchyma cells of mulberry trees (Ukaji et al., 1999). All these findings suggest that ER-localized sHSPs accumulate in all higher plants, and that these sHSPs might have important functions in protecting the ER proteins from the heat-induced damage. As an ER-localized molecular chaperone peculiar to plants, ER-sHSP has not been well studied, except for its basic information as described above. Its possible role on ER stress and mediation to UPR has not been demonstrated. Thus, in the present study, we cloned an ERlocalized sHSP gene (named as LeHSP21.5) from tomato, and examined the effect of tunicamycin on LeHSP21.5 transgenic tomato plants with the
C. Zhao et al. purpose of investigating a possible role of ER-sHSP in ER stress.
Materials and methods Isolation of LeHSP21.5 cDNA Two degenerated primers (50 -CTTAAGCTTAARGARACWSCRGARGG-30 and 50 -ATCAAGCTTAGTAAGMACHCCRTTYTC-30 , Hind III underline) were designed, and employed to amplify the mRNA isolated from tomato (Lycopersicon esculentum) leaves which were treated at 39 1C for 3 h. These primers was designed based on the reported cDNA and consensus amino acid sequences at carboxylterminal (C-terminal) and central regions of endoplasmic reticulum small heat shock proteins (ERsHSP) in potato, pea, soybean and Arabidopsis (their GenBank accession numbers are S70186, M33898, X63198 and U11501, respectively). After the first strand of cDNA was synthesized with M-MLV reverse transcriptase (Gibco BRL), the polymerase chain reaction (PCR) was conducted with ExTaq DNA polymerase (Takara). The amplified DNA was digested by Hind III and cloned into pBluescript II (SK)+vector. After the sequencing of the insert, one clone containing the tomato ER-sHSP cDNA conserved region was selected. According to the result of the BLAST retrieval system, the cloned cDNA fragment was believed to be a conservative region in the ER-sHSP gene and was used for the preparation of the probe. A tomato cDNA library was constructed in the l ZAP II vector (Stratagene, La Jolla, CA, USA) with Poly(A)+ RNA prepared from the heat-treated tomato flowers (39 1C/3 h). The library was probed with the cDNA fragment described above. Positive plaques were isolated and converted into the pBluescript (SK) vector by an in vivo excision method according to the manufacturer’s instructions. Sequencing of the cDNA fragment that had been sub-cloned into the pBluescript (SK) vector was performed.
Antibody production To introduce BamH I and EcoR I sites, the full length cDNA of LeHSP21.5 was amplified by PCR with primers (50 -GAAGGATCCATACATATGAGGGTCATCAG-30 and 50 -AGCGAATTCAGCAGCCAACTCAGCTTC-30 ) and Taq DNA polymerase (Takara). After digested with BamH I and EcoR I, the PCR product was sub-cloned into the glutathione S-transferase (GST) vector (pGEX-6P-1) to generate a GST-LeHSP21.5 fusion
ARTICLE IN PRESS Overexpressing LeHSP21.5 alleviates ER-stress protein. The C-terminus of calnexin cDNA (GenBank accession No. AB218598) was amplified with primers (50 -TCGGATCCATTGGCATTGAGATC-30 and 50 -AGGAATTCGACGAGGAGCAGCAC-30 ), and the PCR product digested by BamH I and EcoR I was sub-cloned into the GST vector (pGEX-6P-3) to generate a GSTcalnexin fusion protein. The accuracy of these constructs was verified by DNA sequencing. Recombinant proteins were expressed in strain BL21 (DE3) of Escherichia coli and purified by glutathioneagarose beads affinity chromatography as described previously (Lindahl et al., 2000). Polyclonal antibodies were raised in rabbits against GST-LeHSP21.5 and GST-calnexin, respectively.
Protein gel blot analysis Total protein was extracted from tomato leaves (500 mg) in 500 mL of cold extraction buffer (10 mM Tris-HCl, pH8.0). The protein concentration was determined by the method of Bradford (1976). An equal amount of proteins (10 mg) was separated on 12.5% polyacrylamide-SDS gel and transferred to PVDF membranes with a semi-dry blot system. The subsequent immunoblot analysis was carried out as described by Alvim et al. (2001). The specific antibody (anti-LeHSP21.5 or anti-calnexin) was used at a dilution of 1:100 with 0.05% (v/v) in TBST (20 mM Tris-HCl, pH7.5, and 500 mM NaCl).
Subcellular localization analysis of LeHSP21.5 Microsomal membranes were prepared from excised tomato roots (20 g) which have been pretreated at 39 1C for 3 h, according to the procedure previously described by De Michelis and Spanswick (1986) with a slight modification. After homogenization and centrifugation, membrane pellets were resuspended in a resuspension buffer (1 mL/10 g starting material), containing 10% (w/v) Suc, 1 mM EGTA, 0.1 mM EDTA, and 2 mM DTT. One milliliter of the crude microsome was overlaid on 12 mL of a continuous Suc gradient (20–40%, w/v) containing the resuspension buffer and centrifuged at 25,000 rpm for 4 h at 4 1C (Beckman SW40 Ti Rotor). After the centrifugation, each 1.2 mL of gradient medium was fractionated and used for the following experiments. Activities of marker enzymes, including vanadate-sensitive H+-ATPase, nitrate-sensitive H+-ATPase, antimycin A-insensitive NADH Cytochrome c reductase and Cytochrome c oxidase, were measured in each fraction, as described by Koike et al. (1997).
837
Tomato transformation The complete LeHSP21.5 cDNA fragment with BamH I and Kpn I cohesive ends produced from a pBluescript (SK) plasmid described above was ligated with pROK II vector with the same cohesive ends, forming the reconstructed pROK II binary plasmid under cauliflower mosaic virus (CaMV) 35S promoter. After verified by DNA sequencing, the reconstructed pROK II binary plasmid was transformed into Agrobacterium strain LBA4404 using the method described by Ho ¨fgen and Willmitzer (1988). Cotyledons of 7–10-d-old tomato seedlings (L. esculentum var. Zhongshu 4) were used for transformation. The genetic transformation was performed as previously described by Fillatti et al. (1987). Transformed callus were selected by kanamycin resistance and then transgenic plants were regenerated. The homozygotic lines of T3 generational transgenic tomato plants were used as plant materials for the following experiments.
RNA gel blot analysis Total RNA was extracted from the leaf tissue as described by Logemann et al. (1987). Twenty-five micrograms total RNA of each sample were separated on 1.5% formaldehyde-agarose gels, and then transferred to the nylon membrane and UV fixed. The full-length LeHSP21.5 cDNA, partial length BiP, PDI and calnexin cDNA were amplified, labeled with [a-32P]dCTP and used as the probe, respectively. Hybridization and washing was carried out at 65 1C as described by Sauter (1997). The primers for LeHSP21.5 cDNA were 50 -GAAGGATCCATACATATGAGGGTCATCAG-30 and 50 -AGC0 GAATTCAGCAGCCAACTCAGCTTC-3 . The primers for calnexin cDNA were 50 -TCGGATCCATTGGCATTGAGATC-30 and 50 -AGGAATTCGACGAGGAGCAGCAC-30 . cDNAs of BiP and PDI were amplified from the cDNA library by PCR according to the reported sequences in tomato expressed sequence tag database with primers BiP-1 (50 -AGAGAAGCTGAGCGTGCCAAGAG-30 ) and BiP-2 (50 -CTGGTCCTGGTAAGTGGTGAAGAC-30 ), and primes PDI-1 (50 -GGTCCTGCATCAGCTGAAATCAAGTC-30 ) and PDI-2 ( 50 -CTTGCAGTGGCCACACCAAGGTG-30 ), respectively. The accuracy of these PCR products was verified by DNA sequencing.
UPR analysis Surface-sterilized seeds of T3 generational LeHSP21.5-transformed plants (TP), pROK II vector-transformed plants (VT) and wild-type (WT)
ARTICLE IN PRESS 838
C. Zhao et al.
plants were grown in liquid 1/2 MS0 medium (28 1C) with a gentle shake. Two-week-old seedlings were treated with 10 mg/mL tunicamycin for 24 h and then transferred to tunicamycin-free liquid 1/2 MS0 medium to allow their recovery. Total RNA was extracted at the indicated time for RNA hybridization with probes BiP, PDI and calnexin as described above.
Results LeHSP21.5 is characteristic of ER-resident protein A DNA fragment, corresponding to the conserved amino acid sequences of the ER-sHSP of other organisms, was reproducibly amplified by RT-PCR from tomatoes. Using this DNA fragment as a probe, we isolated a tomato ER-sHSP cDNA clone, designated LeHSP21.5 (GenBank accession No. AB026983). The similarity between the deduced amino acid sequence of LeHSP21.5 and other reported ER-sHSP was well compared and documented by Scharf et al. (2001). To confirm the subcelluar localization of LeHSP21.5, microsomes were isolated from the heat-treated wild-type tomato root by centrifugation and further separated by continuous sucrose gradient centrifugation (20–40% w/v sucrose). Proteins in each fraction were subjected to an immunoblot analysis with polyclonal antibodies against either LeHSP21.5 or calnexin. The distribution profile of LeHSP21.5 in fractions of sucrose gradient corresponded to that of calnexin (Fig. 1B). Moreover, fractions with higher western signals of LeHSP21.5 also possessed higher activities of the ER marker enzymes (Fig. 1A). Together, these data support the interpretation that LeHSP21.5 is a small heat shock protein located in the ER.
Identification of transgenic tomato plants A construct containing the LeHSP21.5 was introduced into the genome of L. esculentum (var. Zhongshu 4). Seven independent transgenic lines (TP) were generated. A pROK II-transfomed line (VT) was also generated to serve as a vectorcontrol. According to the analysis of kanamycinresistant segregation ratio in T1 seeds and the Mendel’s principle law, four lines that appeared to have only one locus of integrated T-DNA on genome were selected. Transgenic plants were screened by Northern and Western analysis in the T2 generation.
Figure 1. Localization of LeHSP21.5 in the crude microsome fractions. Microsomes were isolated from heattreated (39 1C) wild-type tomato roots and overlaid on an isopycnic Suc-density gradient. 1.2 mL fractions were collected from top to bottom and marker-enzyme activities (V-ATPase, P-ATPase, NADH Cyt c reductase and Cyt c oxidase) in four organelles (tonoplast, plasma membrane, ER and MT, respectively) were measured (A). The bottom and top of the fractions are indicated. SDSPAGE of fractionated proteins was performed using 10 mg protein samples in each fraction. An Immunoblot analysis of proteins was performed with anti-LeHSP21.5 and anticalnexin antibodies (B). Molecular masses are shown in kDa.
High LeHSP21.5 transcript and protein levels were confirmed in the T3 generation of TP plants (Fig. 2).
Tunicamycin resistant ability of TP plants To elucidate the role that LeHSP21.5 played in ER-stress, 14-day-old seedlings were treated with tunicamycin, which acted as a potent activator of UPR pathway. The seedlings were treated with 10 mg/mL tunicamycin for 24 h and then recovered in a tunicamycin-free medium for another 72 h. After the treatment, TP plants remained alive with fresh green cotyledons and leaves, a contrast to the distinct necrosis which happened to cotyledons in WT and VT plants (Fig. 3).
Unfold protein response of TP plants It is a known fact that tunicamycin induced the expression of the ER resident molecular chaperones such as BiP as part of the overall UPR of the cells. In order to detect whether an overexpression of
ARTICLE IN PRESS Overexpressing LeHSP21.5 alleviates ER-stress
Figure 2. RNA and protein gel blot analysis of transgenic plants overexpressing LeHSP21.5. (A) Total RNA (20 mg per lane) extracted from four lines of transgenic tomatoes overexpressing LeHSP21.5 (TP 6, 8, 10, 13) and the pROK II-vector transformed plant (VT) were blotted after separation and hybridized with 32P-labeled LeHSP21.5 cDNA probes. rRNA was visualized with the ethidium bromide staining as a loading control. (B) Leaf protein extracts (10 mg per lane) of TP and VT plants were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies raised against recombinant protein GST-LeHSP21.5. WT/HS was a wild-type plant which was heated at 39 1C for 3 h and used as a positive control.
Figure 3. Tunicamycin resistance ability analysis of TP lines and control lines (WT and VT). Fourteen-day-old tomato seedlings growing in liquid MS medium were treated with tunicamycin (10 mg/mL) for 24 h, and then recovered for another 72 h on MS medium without tunicamycin.
LeHSP21.5 in plants could affect the mRNA levels of BiP or other ER chaperones, control plants (WT and VT) and TP plants were first cultivated in liquid MS medium without tunicamycin, and then treated with tunicamycin (10 mg/mL) for different periods of time before RNA extraction. As shown in Fig. 4A, the basal BiP mRNA level is lower in TP plants (cf. first and sixth lanes), and the tunicamycinmediated induction observed in control plants (second lane) was barely noticeable in the transformed plants overproducing LeHSP21.5 (seventh lane). The BiP mRNA level in control plants accumulated quickly and reached the peak at 10 h, whereas in TP plants its level at 10 h (tenth
839
Figure 4. RNA and protein gel blot analysis of tunicamycin-treated plants. Fourteen-day-old tomato seedlings growing in liquid MS medium were treated with tunicamycin (10 mg/mL) for 24 h and total RNA was extracted at the indicated time periods, 20 mg of total RNA was loaded in each lane and hybridized with cDNA probes of BiP (A), PDI (B) and calnexin (C). rRNA was visualized with ethidium bromide staining as a loading control. (D) Leaf protein extracts (10 mg per lane) of tunicamycin-treated TP and control plants were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies raised against recombinant protein GSTLeHSP21.5. CK means control lines (WT and VT); TP means transgenic lines.
lane) was only equal to that of control plants at 4 h (third lane). Hybridization with PDI or calnexinspecific probes showed a similar reduction in the basal and tunicamycin-mediate induction mRNA levels of PDI and calnexin (Figs. 4B and C). Thus, artificially increasing the LeHSP21.5 protein levels led to a down-regulation of the unfolded protein response. The LeHSP21.5 protein level in TP plants remained high during the treatment. However, we did not detect any trace of LeHSP21.5 in tunicamycin-treated control plants (Fig. 4D).
Discussion Several ER-located sHSPs in plants have been reported, and it has been suggested that they might execute molecular chaperone function in stabilizing proteins during stress conditions (Ukaji et al., 1999). However, the evidence that ER-sHSP confers enhanced ER stress tolerance to plants has not been reported. In the present study, we have isolated a tomato cDNA LeHSP21.5 which encodes a putative homolog of ER-sHSP, and introduced the construction of DNA, containing LeHSP21.5 cDNA
ARTICLE IN PRESS 840 under the control of 35S CaMV promoter, into tomato plants by Agrobacterium-mediated transformation. Interestingly, transgenic tomatoes with an over-expression of LeHSP21.5 exhibited the improved tunicamycin-resistance ability. Tunicamycin is an ER stress inducer by blocking the glycosylation of newly synthesized proteins in the ER. ER stress leads to UPR. UPR provides a protective response to ER stress. An increased expression of ER-located molecular chaperons is regarded as the most important indication of UPR (Koizumi et al., 2001; Martı´nez and Chrispeels, 2003). It has been proved that overexpressing BiP in plants leads to the down-regulation of BiP and other ER-located chaperones under normal or tunicamycin stress conditions (Leborgne-Castel et al., 1999; Alvim et al., 2001). In the present study, similar to these previous cases, the overexpression of LeHSP21.5 in plants down-regulated not only the basal but also the inducible mRNA levels of BiP, calnexin and PDI (Fig. 4), which indicated the alleviated ER stress. In addition, Alvim et al. (2001) have confirmed that the effectiveness of BiP protection against ER stress could be extended to the whole organism level, as judged by the results of germination/survival assay in the presence of tunicamycin. Here, we showed that overexpressing LeHSP21.5 in plants also enhanced the whole plant’s resistance to tunicamycin (Fig. 3), which further proved the protective effect of LeHSP21.5 against ER stress. Up to now, the UPR signal transmission pathway in plants has not been revealed completely. The transgenic plants with an overexpression of ER-sHSP may provide another way to study the ER stress in plant cells. Analysis of the promoter regions of UPR-regulated genes in plants has identified the presence of ER-stress response elements (Martı´nez and Chrispeels, 2003). Although artificially increasing the LeHSP21.5 protein levels led to an alleviation of the UPR, the endogenous LeHSP21.5 gene did not respond to tunicamycin (Fig. 4). It may suggest an absence of the ER-stress response element in the promoter of LeHSP21.5. If this claim is true, it further suggestes that LeHSP21.5 is not a UPRregulated gene even though it is a molecular chaperone located in the ER. To our best knowledge, this is the first time to demonstrate that overexpressing an ER-sHSP gene could enhance the ER stress tolerance of the transgenic plants. Recently, it has been proved that the abiotic stress could also induce the UPR in plants (Koiwa et al., 2003). Overexpressing ER molecular chaperones in plants enhances their resistance to tunicamycin as well as to other abiotic stresses (Alvim et al., 2001). Based on the
C. Zhao et al. results obtained in the present study, we can now expect to utilize transgenic tomatoes for demonstrating the precise role of LeHSP21.5 in ER stress or other abiotic stresses.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 30270132).
References Alvim FC, Carolino SMB, Cascardo JCM, Nunes CC, Martinez CA, Otoni WC, et al. Enhanced accumulation of BiP in transgenic plants confers tolerance to water stress. Plant Physiol. 2001;126:1042–54. Bradford M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248–54. Cooper P, Ho THD. Intracellular localization of heat shock proteins in maize. Plant Physiol. 1987;84:1197–203. De Michelis MI, Spanswick RM. H+ pumping driven by the vanadate-sensitive ATPase in membrane vesicles from corn roots. Plant Physiol. 1986;81:542–7. Denecke J. Soluble endoplasmic reticulum resident proteins and their function in protein synthesis and transport. Plant Physiol. Biochem. 1996;34:197–205. Fillatti JJ, Kiser J, Rose R, Comai L. Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Biotechnology 1987;5:726–30. Hammond C, Helenius A. Quality control in the secretory pathway. Curr. Opin. Cell Biol. 1995;7:523–9. Helm KW, LaFayette PR, Nagao RT, Key JL, Vierling E. Localization of small heat shock proteins to the higher plant endomembrane system. Mol. Cell Biol. 1993;13: 238–47. Helm KW, Schmeits J, Vierling E. An endomembranelocalized small heat-shock protein from Arabidopsis thaliana. Plant Physiol. 1995;107:287–8. Ho ¨fgen R, Willmitzer L. Storage of competent cells for Agrobacterium transformation. Nucl. Acids Res. 1988; 16:9877. Koike M, Takezawa D, Arakawa K, Yoshida S. Accumulation of 19-kDa plasma membrane polypeptide during induction of freezing tolerance in wheat suspensioncultured cells by abscisic acid. Plant Cell Physiol. 1997;38:707–16. Koiwa H, Li F, McCully MG, Mendoza I, Koizumi N, Manabe Y, et al. The STT3a subunit isoform of the Arabidopsis oligosaccharyltransferase controls adaptive responses to salt/osmotic stress. Plant Cell 2003;15:2273–84. Koizumi N, Martı´nez IM, Kimata Y, Kohno K, Sano H, Chrispeels MJ. Molecular characterization of two Arabidopsis Ire1 homologs, endoplasmic reticulum-located
ARTICLE IN PRESS Overexpressing LeHSP21.5 alleviates ER-stress transmembrane protein kinases. Plant Physiol. 2001; 127:949–62. LaFayette PR, Travis RL. Soluble and membrane-associated heat shock proteins in soybean root. Protoplasma 1990;156:174–82. Leborgne-Castel N, Jelitto-Van Dooren EPWM, Crofts AJ, Denecke J. Overexpression of BiP in tobacco alleviates endoplasmic reticulum stress. Plant Cell 1999;11: 459–69. Lindahl M, Spetea C, Hundal T, Oppenheim AB, Adam Z, Andersson B. The thylakoid FtsH protease plays a role in the light-induced turnover of the photosystem II D1 protein. Plant Cell 2000;12:419–31. Logemann J, Schell J, Willmitzer L. Improved method for the isolation of RNA from plant tissues. Anal. Biochem. 1987;163:16–20. Martı´nez IM, Chrispeels MJ. Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes. Plant Cell 2003;15:561–76. Pelham HRB. Control of protein exit from the endoplasmic reticulum. Annu. Rev. Cell Biol. 1989;5:1–23.
841 Sauter M. Differential expression of a CAK (cdc2-activating kinase)-like protein kinase, cyclins and cdc2 genes from rice during the cell cycle and in response to gibberellin. Plant J. 1997;11:181–90. Scharf KD, Siddique M, Vierling E. The expanding family of Arabidopsis thaliana small heat stress proteins and a new family of proteins containing a-crystallin domains (Acd proteins). Cell Stress Chaperones 2001;6:225–37. Sticher L, Biswas AK, Bush DS, Jones RL. Heat shock inhibits a-amylase synthesis in barley aleurone without inhibiting the activity of endoplasmic reticulum marker enzymes. Plant Physiol. 1990;92:506–13. Ukaji N, Kuwabara C, Takezawa D, Arakawa K, Yoshida S, Fujikawa S. Accumulation of small heat-shock protein homologs in the edoplasmic reticulum of cortical parenchyma cells in mulberry in association with seasonal cold acclimation. Plant Physiol. 1999;120: 481–9. van Berkel J, Salamini F, Gebhardt C. Transcripts accumulating during cold storage of potato (Solanum tuberosum L.) tubers are sequence related to stressresponsive genes. Plant Physiol. 1994;104:445–52.