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First published online March 23, 2007; 10.1104/pp.106.094581 Plant Physiology 144:513-523 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Arabidopsis Cor15am Is a Chloroplast Stromal Protein That Has Cryoprotective Activity and Forms Oligomers1,[W],[OA]The 21st Century Centers of Excellence Program, Cryobiosystem Research Center, Iwate University, Morioka 020–8550, Japan (K.N., K.O., T.K., T.I.); and Department of Material-Process Engineering and Applied Chemistry for Environment, Faculty of Engineering and Resource Science, Akita University, Akita 010–8502, Japan (T.H., H.I.)
Many plants acquire increased freezing tolerance when they are exposed to nonfreezing temperatures of a certain duration. This process is known as cold acclimation and allows plants to protect themselves from freezing injury. A wide variety of polypeptides are induced during cold acclimation, among which is one encoded by COR15A in Arabidopsis (Arabidopsis thaliana). Previous studies showed that the COR15A gene encodes a small, plastid-targeted polypeptide that is processed to a mature form called Cor15am. In this study, we examined the biochemical properties and activities of Cor15am in more detail. We provide evidence that Cor15am localizes almost exclusively to the chloroplast stroma. In addition, the cold-regulated accumulation of Cor15am is affected by chloroplast functionality. Both gel-filtration chromatography and protein cross-linking reveal that Cor15am forms oligomers in the stroma of chloroplasts. Although Cor15am accumulates in response to low temperature, cold acclimation is not a prerequisite for oligomerization of Cor15am. Structural analysis suggests that Cor15am is composed of both ordered and random structures, and can stay soluble with small structural change after boiling and freeze-thaw treatments. Recombinant Cor15am exhibits in vitro cryoprotection of a freeze-labile enzyme, L-lactate dehydrogenase. Furthermore, Cor15am is capable of associating with L-lactate dehydrogenase in vitro and with potential stromal substrates in vivo. On the basis of these results, we propose that Arabidopsis Cor15am is a cryoprotective protein that forms oligomers in the chloroplast stroma, and that direct association of Cor15am with its substrates is part of its cryoprotective mechanism.
When plants are exposed to nonfreezing temperatures of a certain duration, they increase their level of freezing tolerance. This process, known as cold acclimation, appears to involve an alteration of gene expression (Thomashow, 2001  ). Considerable effort has been directed toward uncovering the mechanisms regulating the expression of cold-regulated genes (Shinozaki et al., 2003). Such studies have revealed that CBF/DREB transcription factors are key regulators of cold-regulated gene expression (Stockinger et al., 1997; Jaglo-Ottosen et al., 1998; Liu et al., 1998), although there may be additional regulatory pathways that remain to be characterized. Overexpression of CBF/DREB transcription factors increased the freezing tolerance of Arabidopsis (Arabidopsis thaliana; Jaglo-Ottosen et al., 1998; Liu et al., 1998). In these plants, the downstream target genes for CBF/DREB transcription factors were up-regulated without cold acclimation, resulting in increased freezing tolerance.
Although a number of target genes for CBF/DREB transcription factors have been identified (Maruyama et al., 2004
In Arabidopsis, a small hydrophilic polypeptide, designated as Cor15a, is induced and targeted to chloroplasts during cold acclimation (Lin and Thomashow, 1992a We have been interested in the biochemical activities of Cor15am and the molecular mechanism by which this protein protects the plant cell from freezing injury. In this article, we describe the biochemical properties of Cor15am. We provide compelling evidence that Cor15am is almost exclusively localized to the chloroplast stroma. Using a mutant defective in functional chloroplasts, we demonstrate that the cold-regulated accumulation of Cor15am is affected by chloroplast functionality. Both gel-filtration chromatography and protein cross-linking reveal that Cor15am forms oligomers in the stroma of chloroplasts. However, cold acclimation is not required for the assembly of Cor15am oligomers. We also demonstrate that Cor15am exhibits in vitro cryoprotective activity with a freeze-labile enzyme, and directly associates with potential substrates both in vitro and in vivo.
Precise Localization of Cor15am within Chloroplasts
COR15A was originally identified as a cold-regulated gene in Arabidopsis (Lin and Thomashow, 1992a First, we generated antibody against the mature form of Cor15a, Cor15am. To obtain antigen, Cor15am carrying a C-terminal His-tag was expressed in Escherichia coli (Figs. 1 and 2A ). As shown in Figure 2B, anti-Cor15am serum reacted with Cor15am-His, whereas preimmune serum did not. Furthermore, the serum detected an approximately 10-kD protein in Arabidopsis protein extract only after cold acclimation (Fig. 2C), indicating that the antibody reacts with native Cor15am.
We separated cold-acclimated Arabidopsis chloroplasts into stroma, envelope, and thylakoid fractions by Suc density gradient centrifugation. Each fraction was resolved by SDS-PAGE, blotted onto nitrocellulose membrane, and probed with antibody against Cor15am. The purity of each fraction was confirmed using marker proteins such as the small subunit of Rubisco (SSU; stroma), Tic110 (envelope), and light-harvesting complex protein (LHCP; thylakoid). As shown in Figure 2D, Cor15am was found exclusively in the stromal fraction. As an additional control, we examined AccA protein, the carboxyl transferase  -subunit of acetyl-CoA carboxylase. AccA is a soluble stromal protein (Kozaki et al., 2000) but is also known to associate with envelope membranes (Shorrosh et al., 1996; Thelen and Ohlrogge, 2002; Ferro et al., 2003). Consistent with previous studies, AccA protein was found in the envelope fraction as well as in the stroma (Fig. 2D), supporting the idea that Cor15am does not stably associate with envelope membranes. The stromal localization of Cor15am was further examined by trypsin treatment of isolated intact chloroplasts. Trypsin permeates the outer membrane but not the inner membrane of intact chloroplasts (Schnell et al., 1994; Jackson et al., 1998). Cor15am was resistant to trypsin treatment, whereas the outer membrane component Toc75 was digested (Fig. 2E). This result indicates that Cor15am is indeed localized to the stroma and is not derived from the intermembrane space. Based on these data, we concluded that Cor15am is a soluble protein of the chloroplast stroma.
Previous studies have shown that chloroplasts and light affect the expression of some cold-regulated genes in cereal plants (Chauvin et al., 1993
To further investigate the mechanism regulating Cor15am accumulation in chloroplasts, we measured the expression of the COR15A gene in the ppi2 mutant. Since ppi2 exhibits defects in functional chloroplasts, it was possible that the expression of COR15A was compromised, leading to the reduced accumulation of Cor15am. Indeed, real-time PCR analysis revealed that the cold-induced expression of COR15A in ppi2 was approximately 50% that of wild-type plants (Fig. 3B). These data indicate that the significant decrease in Cor15am accumulation in ppi2 plastids is in part attributable to a reduction of COR15A expression. Taken together, we conclude that the cold-regulated accumulation of Cor15am is affected by chloroplast functionality, and that this regulation occurs in part at the level of mRNA accumulation.
As a next step in our analysis, we wished to establish the biochemical properties of Cor15am. Cor15am runs at approximately 10 kD in SDS-PAGE, but in native-PAGE analysis both chloroplastic and recombinant Cor15am migrated with apparent molecular masses of about 70 kD (Gilmour et al., 1996
To further dissect the oligomeric state of Cor15am, we resolved recombinant Cor15am by gel-filtration chromatography. As shown in Figure 5B , the elution volumes of the Mr markers nicely fitted a regression line (r2 = 0.999) under our experimental conditions. The major peak of recombinant Cor15am appeared to be at approximately 40 kD, accompanied by a minor peak at around approximately 20 kD (Fig. 5A). Since mature Cor15am-His is 10.4 kD, this result suggests that the majority of the recombinant Cor15am had formed homo-oligomers, most likely tetramers. This conclusion is consistent with the apparent molecular mass of complex 3 (C3 in Fig. 4) observed in cross-linking reactions, although we do not exclude the possible existence of other oligomeric forms, such as trimers and dimers. Taken together, we conclude that recombinant Cor15am forms homo-oligomers.
Cold Acclimation Is Not Required for the Formation of Cor15am Oligomers in the Chloroplast Stroma The homo-oligomerization of recombinant Cor15am in vitro suggests that native Cor15am can form homo-oligomers in the chloroplast stroma. However, it is also possible that additional proteins are present in native Cor15am protein complexes and help protect chloroplasts from freezing injury. To test for the presence of other proteins, we performed gel-filtration chromatography on chloroplast stroma isolated from cold-acclimated Arabidopsis. The major peak of authentic Cor15am was centered around fractions 27 and 28 (Fig. 6 ), similar to that of recombinant Cor15am-His. This result suggests that a majority of Cor15am exists as homo-oligomers and does not stably associate with other proteins in the stroma.
Although we established that both recombinant and authentic Cor15am can form oligomers, it was unclear whether in vivo oligomerization of Cor15am requires cold acclimation. Of particular interest, Arabidopsis constitutively overexpressing COR15A still showed increased levels of Cor15am in response to cold acclimation (Artus et al., 1996 ), suggesting that there may be regulation of Cor15am accumulation at the protein level. For example, cold acclimation might be required for efficient oligomerization of Cor15am in the stroma, and Cor15am may not accumulate in untreated plants because it is labile in its monomeric form. To test this hypothesis, we generated transgenic Arabidopsis plants constitutively expressing the COR15A-PROTEIN A fusion gene. As a negative control, we also generated Arabidopsis plants overexpressing protein A (pA) fused to the transit peptide of Rubisco (preSSU-pA). As shown in Figure 7A
, the transgenic plants accumulated similar levels of Cor15am-pA and pA in chloroplasts. When the stroma of cold-acclimated Cor15a-pA plants was resolved by gel-filtration chromatography, the major peak of Cor15am-pA was at approximately 90 kD (Fig. 7B). This result indicates that Cor15am-pA is as capable as endogenous Cor15am of forming oligomers. The oligomerization is mediated by the Cor15am portion of the fusion protein because pA expressed on its own did not form oligomers (Fig. 7C).
To further test whether cold acclimation is a prerequisite for oligomerization of Cor15am in vivo, we isolated the stroma of chloroplasts from nonacclimated plants overexpressing Cor15a-pA. Gel-filtration chromatography revealed that the apparent molecular mass of the Cor15am-pA complex from nonacclimated plants was similar to that from acclimated plants (Fig. 7D). Therefore, we concluded that Cor15am forms oligomers as soon as it is imported into chloroplasts.
Previous literatures (Lin and Thomashow, 1992a
Previous studies showed that Cor15am stayed soluble after boiling (Lin et al., 1990 ; Lin and Thomashow, 1992a). Therefore, we tested whether Cor15am can still retain the observed structure after boiling as well as a freeze-thaw cycle. As shown in Figure 8A, Cor15am exhibited small structural change. The amount of  -strand seemed to be affected particularly (Fig. 8B), although the functional significance of this structural change remains to be characterized. We conclude that Cor15am contains a certain amount of ordered secondary structures as well as random structure and can stay soluble with small structural change after temperature stress.
The structural stability of Cor15am at extreme temperatures is consistent with a previously proposed role in protecting proteins from temperature stress. This hypothesis was originally proposed many years ago based on the fact that in vitro translated Cor15a, the precursor of Cor15am, had potent cryoprotective activity (Lin and Thomashow, 1992b
Cor15am Is Capable of Associating with Other Proteins Both in Vitro and in Vivo To further unravel the molecular mechanism by which Cor15am protects freeze-labile enzymes, we examined whether Cor15am could directly associate with LDH. A mixture of Cor15am and LDH was treated with DSP to stabilize any interactions between proteins, and then the Cor15am complexes were affinity purified using anti-Cor15am IgG Sepharose. As shown in Figure 10A , LDH was copurified with Cor15am, indicating that Cor15am can associate with the proteins it cryoprotects.
The direct association between Cor15am and LDH suggested that Cor15am may associate with stromal proteins. To test this hypothesis, we purified Cor15am-substrate complexes from the stroma of chloroplasts. We used a cross-linker in these experiments because Cor15am did not seem to associate stably with other proteins in the stroma (Fig. 6). Intact chloroplasts isolated from wild-type plants before and after cold acclimation were treated with DSP to chemically cross-link Cor15am with potential substrates. After lysis of chloroplasts, the stromal fraction was subjected to immunoaffinity chromatography. DSP is a cleavable cross-linker, and the disulfide bonds between Cor15am and associated proteins were cleaved by adding dithiothreitol (DTT) before electrophoresis. As shown in Figure 10, B and C, Cor15am was only detected in the eluate of the acclimated plants. This result is consistent with the cold inducibility of Cor15am. In addition, we observed that certain other proteins were significantly enriched in the eluate of the acclimated plants (Fig. 10B, arrows and arrowheads). Among those proteins, two appeared to consistently copurify with Cor15am: the large and small subunits of Rubisco (Fig. 10B, arrows). The consistent association of Cor15am with Rubisco suggests that the latter is a substrate for Cor15am's cryoprotective activity. The amounts of other proteins enriched in the eluate of acclimated plants differed in each experiment (Fig. 10B, indicated by arrowheads). Despite this variability, we do not exclude the possibility that these proteins are also substrates of Cor15am. The validity of the interaction between Cor15am and Rubisco was further confirmed using transgenic lines expressing fusion proteins. When we performed similar immunoaffinity chromatography using stroma isolated from Cor15a-pA-expressing plants, Rubisco was copurified with Cor15am-pA (Fig. 10D, lane 2). The amount of Rubisco in the Cor15am-pA eluate was much more than that in the negative control (Fig. 10D, lane 1), indicating that the interaction between Rubisco and Cor15am-pA is mediated by the Cor15am portion of the fusion protein. Based on these results, we concluded that Cor15am can associate with its substrates in vivo as well as in vitro, and that Rubisco is one of the potential substrates for Cor15am in the stroma of chloroplasts.
Hundreds of cold-regulated genes have been identified so far from a number of plant species. However, only a small number of polypeptides have been characterized at the molecular level, and, therefore, the biochemical activities of cold-regulated proteins remain largely unclear. In this study, we have examined in detail the biochemical properties of a cold-regulated protein, Arabidopsis Cor15am. We demonstrated that Cor15am is a chloroplast stromal protein (Fig. 2). In addition, we showed that the normal accumulation of Cor15am was prevented in the absence of functional chloroplasts (Fig. 3). We also obtained evidence that Cor15am forms oligomers (Figs. 4–6  ), most likely tetramers, both in vitro and in vivo. In transgenic plants expressing a Cor15am fusion protein constitutively, the formation of Cor15am oligomers did not depend on cold acclimation (Fig. 7). Finally, structural and biochemical analysis revealed that Cor15am is a cryoprotective protein containing ordered structures (Figs. 8 and 9) and is capable of associating directly with potential substrates (Fig. 10). Taken together, these data establish that Cor15am is a cryoprotective protein that contains ordered structures and forms oligomers in the stroma of chloroplasts.
We observed that Cor15am was localized exclusively to the stroma of chloroplasts. This raises the question of how Cor15am protects the chloroplast envelope from freezing injury (Steponkus et al., 1998
In an attempt to further clarify the mechanism by which Cor15am protects enzymes from inactivation, we examined the interaction between Cor15am and its substrates by cross-linking. As shown in Figure 10A, Cor15am is capable of associating with LDH in vitro. Furthermore, we identified Rubisco as a potential substrate in the stroma by immunoaffinity chromatography of stromal samples cross-linked in vivo (Fig. 10, B–D). Intriguingly, previous studies have shown that Rubisco activity increases during cold acclimation (Holaday et al., 1992
Structural analysis revealed that Cor15am contains a certain amount of ordered secondary structures, such as
We speculate that Cor15am may exist as homo-oligomers under normal conditions. However, Cor15am may associate with other proteins under conditions where proteins are unstable. Another example of a stress-responsive, boiling-stable protein that forms homo-oligomers is SP1 isolated from aspen (Populus spp.; Wang et al., 2002
Previous studies have shown that chloroplasts and light affect the expression of some cold-regulated genes in cereal plants (Chauvin et al., 1993
In summary, we demonstrated that Cor15am is almost exclusively located in the stroma of chloroplasts. Cor15am contains a certain amount of ordered secondary structures as well as random structure. Unlike most other hydrophilic and stress-inducible proteins, Cor15am is capable of forming oligomers, although cold acclimation is not a prerequisite for oligomerization. Cor15am also has the ability to protect a freeze-labile enzyme from inactivation and to directly associate with potential substrates. This evidence suggests a novel cryoprotective mechanism: that Cor15am protects enzymes in the chloroplast stroma from low- or freezing-temperature stresses. Although we were not able to obtain direct evidence for it, an intriguing hypothesis is that Cor15am can associate not only with freeze-labile stromal enzymes, but also with envelope membranes under certain conditions (Steponkus et al., 1998
Construction of Plant Transformation Vectors and Arabidopsis Transformation All constructs used in this study are summarized in Figure 1. The COR15A cDNA was amplified from Arabidopsis (Arabidopsis thaliana) cDNA by RT-PCR using primers that introduced a 5' NcoI site and a 3' XhoI site. The cDNA was inserted into the NcoI and XhoI sites of pET21d to generate pET-Cor15a-His. For construction of pCAMBIA-Cor15a-pA, the COR15A cDNA was first subcloned into the NcoI and EcoRI sites of pET21d-TEV-pA to generate pET21d-Cor15a-pA. This plasmid was then digested with NcoI and XbaI and the insert subcloned into the NcoI and XbaI sites of pCAMBIA3300.1 to generate pCAMBIA-Cor15a-pA. For expression in Escherichia coli, the mature portion of Cor15a, designated as Cor15am, was amplified by PCR using pET-Cor15a-His as the template. The amplified fragment was subcloned into pET21d to generate pET-Cor15am-His.
All pCAMBIA constructs were introduced into Arabidopsis (ecotype Columbia) via Agrobacterium tumefaciens-mediated transformation by the floral dip method (Clough and Bent, 1998
Total RNA was isolated using an RNAiso kit (TaKaRa) from the aerial tissues of Arabidopsis grown on 0.5x Murashige and Skoog plates supplemented with 1% Suc, before or after cold acclimation. COR15A mRNA was quantified with a LightCycler PCR machine (Roche), using a One Step SYBR RT-PCR kit (TaKaRa) and gene-specific primers. The transcript levels of COR15A were normalized to those of 18S ribosomal RNA.
For bacterial expression, pET21d-Cor15am-His was transformed into E. coli BL21(DE3). Expression was induced with 1 mM IPTG for 3 h at 37°C and the soluble fraction was recovered. The Cor15am-His protein was purified under nondenaturing conditions using His-bind resin (Novagen).
Rabbit polyclonal antibody against Cor15am was produced using Cor15am-His (Fig. 2A) as the antigen. Antibodies against Tic110 (Inaba et al., 2003
Wild-type and transgenic Arabidopsis were grown on 0.5x Murashige and Skoog plates supplemented with 1% Suc. Chloroplasts were isolated from 14- to 18-d-old seedlings as described previously (Smith et al., 2002
Chloroplasts were fractionated into stroma, envelope, and thylakoid fractions as described previously (Smith et al., 2002
For protein cross-linking using glutaraldehyde, 30 µM (on a monomer basis) recombinant Cor15am-His was incubated with 0.125% glutaraldehyde (Wako Chemical) at room temperature. A small aliquot of the sample was taken out at each of the indicated time points and then quenched by adding an equal volume of 2x SDS-PAGE sample buffer containing DTT and incubating for 15 min. For cross-linking reactions using DST and DSP (PIERCE), 2 µM recombinant Cor15am-His was incubated with the indicated concentrations of cross-linker for 30 min at room temperature. The samples were then quenched by adding an equal volume of 2x SDS-PAGE sample buffer (minus DTT) and incubating for 15 min. To examine the interaction between Cor15am and LDH (obtained from Nacalai Tesque), 0.8 µM LDH was incubated with 0.5 mM DSP in the presence or absence of Cor15am (10 µM) for 30 min at room temperature. The reaction was quenched by incubating with Gly at a final concentration of 50 mM for an additional 15 min. The quenched samples were mixed with an equal volume of HEGS buffer (50 mM HEPES-KOH, pH 7.5, 2 mM EDTA, 10% glycerol, and 250 mM NaCl) containing 2% Triton X-100 (w/v). The samples were then analyzed by immunoaffinity chromatography, as described later.
For cross-linking of stromal proteins, isolated chloroplasts (700 µg of chlorophyll) were first incubated in the dark for 30 min to deplete the reducing potential. The depleted chloroplasts were then incubated with 2.5 mM DSP on ice for 15 min in the dark (Inaba et al., 2005
To obtain the stromal fraction, chloroplasts (1 mg chlorophyll/mL) were lysed in 5 volumes of TES buffer containing 15 mM Tricine-KOH, pH 7.5, 2 mM EDTA, and 250 mM NaCl and centrifuged at 100,000g for 20 min. The supernatant was resolved on a Sephacryl S-300 HR column equilibrated with TES buffer using an AKTA-prime chromatography system (GE Healthcare). The first 30 mL of eluate was discarded as void volume because we did not observe any A280 (data not shown). Fractions of 1.5 mL each were collected, and the protein in each fraction precipitated with 10% trichloroacetic acid and resolved by SDS-PAGE.
CD spectra were determined as described previously (Miyazaki et al., 2004
The cryoprotective activity of Cor15am was assayed as described previously (Lin and Thomashow, 1992b
For immunoaffinity chromatography of wild-type plants, anti-Cor15am IgG was affinity purified using a Cor15am antigen column. For immunoaffinity chromatography of transgenic plants expressing pA fusions, anti-pA IgG (obtained from Sigma) was affinity purified using pA Sepharose (GE Healthcare). The purified IgG was dialyzed against phosphate buffer containing 150 mM NaCl and then coupled to CNBr-activated Sepharose (GE Healthcare) according to the manufacturer's recommendations. The cross-linked samples were applied to the antibody column and the column was washed with 20 volumes of TEGS buffer (15 mM Tricine-KOH, pH 7.5, 2 mM EDTA, 10% glycerol, and 250 mM NaCl) containing Triton X-100. The bound proteins were eluted with 0.2 M Gly-HCl, pH 2.2. Cross-links between Cor15am and its substrates were cleaved by adding SDS-PAGE sample buffer containing DTT. After SDS-PAGE, the samples were analyzed by either western blotting or Coomassie Brilliant Blue staining.
The following materials are available in the online version of this article.
We thank Professors Danny J. Schnell, Matsuo Uemura, Yukiko Sasaki, and Yukio Nagano for their invaluable support of our research project. We also thank Mrs. Yuko Suzuki for her technical assistance. LHCP antibody was a kind gift from Prof. Kenneth Cline. Received December 11, 2006; accepted March 20, 2007; published March 23, 2007.
1 This work was supported in part by the 21st Century Centers of Excellence program, the Naito Memorial Foundation, and funding from the President of Iwate University. T.K. is the recipient of a Japan Society for the Promotion of Science fellowship for young scientists.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Takehito Inaba (tinaba{at}iwate-u.ac.jp).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.094581 * Corresponding author; e-mail tinaba{at}iwate-u.ac.jp; fax 81–19–621–6200.
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