identiﬁcation of a novel lea protein involved in freezing ...identiﬁcation of a novel lea...

Identification of a Novel LEA Protein Involved in FreezingTolerance in WheatKentaro Sasaki1,6, Nikolai Kirilov Christov1,2,6, Sakae Tsuda3,4 and Ryozo Imai1,5,*1Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization, Hitsujigaoka 1, Toyohira-ku, Sapporo,062-8555 Japan2AgroBioInstitute, Dragan Tsankov 8, Sofia 1164, Bulgaria3Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Toyohira-ku, Sapporo, 062-8517 Japan4Graduate School of Life Science, Hokkaido University, Kita-ku, Sapporo, 060-0810 Japan5Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo, 060-8589 Japan6These authors contributed equally to this work.*Corresponding author: E-mail, [email protected]; Fax, +81-11-857-9382.(Received August 27, 2013; Revised November 4, 2013)

Late embryogenesis abundant (LEA) proteins are a family ofhyper-hydrophilic proteins that accumulate in response tocellular dehydration. Originally identified as plant proteinsassociated with seed desiccation tolerance, LEA proteinshave been identified in a wide range of organisms such asinvertebrates and microorganisms. LEA proteins are thoughtto protect proteins and biomembranes under water-deficitconditions. Here, we characterized WCI16, a wheat (Triticumaestivum) protein that belongs to a class of plant proteins ofunknown function, and provide evidence that WCI16 sharescommon features with LEA proteins. WCI16 was inducedduring cold acclimation in winter wheat. Based on itsamino acid sequence, WCI16 is highly hydrophilic, like LEAproteins, despite having no significant sequence similarity toany of the known classes of LEA proteins. RecombinantWCI16 protein was soluble after boiling, and 1H-nuclearmagnetic resonance (NMR) spectroscopy revealed that thestructure of WCI16 is random and has no hydrophobicregions. WCI16 exhibited in vitro cryoprotection of thefreeze-labile enzyme L-lactate dehydrogenase as well asdouble-stranded DNA binding activity, suggesting thatWCI16 may protect both proteins and DNA during environ-mental stresses. The biological relevance of these activitieswas supported by the subcellular localization of a greenfluorescent protein (GFP)-fused WCI16 protein in thenucleus and cytoplasm. Heterologous expression of WCI16in Arabidopsis (Arabidopsis thaliana) plants conferredenhanced freezing tolerance. Taken together, our results in-dicate that WCI16 represents a novel class of LEA proteinsand is involved in freezing tolerance.

Keywords: Cold acclimation � Freezing tolerance � LEAprotein.

Abbreviations: BSA, bovine serum albumin; CA, cold accli-mation; DA, de-acclimation; dsDNA, double-stranded DNA;

DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid; GFP, greenfluorescent protein; GST, glutathione S-transferase; IPTG,isopropyl-b-D-thiogalactopyranoside; LDH, L -lactate dehydro-genase; LEA, late embryogenesis abundant; NLS, nuclearlocalization signal; NMR, nuclear magnetic resonance; ORF,open reading frame; ssDNA, single-stranded DNA; WCI16,Wheat Cold Induced 16.

The nucleotide sequence reported in this paper has been sub-mitted to DDBJ under accession number AB830333.

Introduction

Low temperature is a major factor that limits plant growth andproductivity. In overwintering plants, freezing tolerance isincreased after a period of exposure to low, but non-freezing,temperatures (Levitt 1980, Sakai and Larcher 1987, Guy 1990).This process, known as cold acclimation (CA), allows plants todevelop tolerance essential for winter survival through alter-ation of gene expression (Guy 1990, Pearce 1999). CA-inducedgenes have been identified from many plant species includingArabidopsis (Thomashow 1999) and winter wheat (Pearce1999), and the level of freezing tolerance has been reportedto correlate positively with the expression of cold-inducedgenes (Monroy et al. 1993, Houde et al. 1995). Functions infreezing tolerance have been suggested for several of thesecold-induced genes (Uemura et al. 1996, Keresztessy andHughes 1998).

Late embryogenesis abundant (LEA) proteins are a group ofCA-induced genes that encode extremely hydrophilic polypep-tides (Danyluk et al. 1994, Artus et al. 1996). LEA proteins ac-cumulate to high levels in seeds during the late stage ofembryogenesis and in vegetative tissues under dehydrationstress conditions such as cold, drought and high salinity

Plant Cell Physiol. 55(1): 136–147 (2014) doi:10.1093/pcp/pct164, available online at www.pcp.oxfordjournals.org! The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.All rights reserved. For permissions, please email: [email protected]

136 Plant Cell Physiol. 55(1): 136–147 (2014) doi:10.1093/pcp/pct164 ! The Author 2013.

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(Ingram and Bartels 1996, Bray 1997, Thomashow 1999). LEAproteins are hyper-hydrophilic and intrinsically disordered, andtherefore can remain soluble after boiling and freeze–thawtreatments (Dure 1993b). These characteristics have led tothe suggestion that LEA proteins have a protective roleduring dehydration (Baker et al. 1988, Bray 1993, Dure1993b). LEA proteins have been classified into various groupson the basis of amino acid sequence similarity and the presenceof specific motifs (Bray 1993, Dure 1993a, Wise 2003). For ex-ample, group 1 LEA proteins typically contain a hydrophilic 20-mer motif (TRKEQ[L/M]G[T/E]EGY[Q/K]EMGRKGG[L/E])(Espelund et al. 1992, Galau et al. 1992), and group 2 LEA pro-teins (dehydrins) are characterized by a K-segment (EKKGIME/DKIKEKLPG) and a Y-segment ([V/T]D[E/Q]YGNP) (Close1996, Close 1997). Based on individual amino acid sequencesand predicted protein structures, LEA proteins of each grouphave been suggested to have specific functions during dehydra-tion (Dure et al. 1989, Bray 1993, Dure 1993a).

Functional analyses of LEA proteins have been carried outboth in vitro and in vivo. Ectopic overexpression of genesencoding LEA proteins can enhance stress tolerance underwater-deficit conditions. LEA25, a group 4 LEA protein fromtomato (Solanum lycopersicum), can improve tolerance againsthigh salinity and freezing when expressed in Saccharomycescerevisiae (Imai et al. 1996). Overexpression of barley(Hordeum vulgare) HVA1 in wheat (Triticum aestivum) andrice (Oryza sativa) confers enhanced drought tolerance (Xuet al. 1996, Sivamani et al. 2000). In addition, freezing toleranceof Arabidopsis is increased by overexpression of wheat WCS19or Arabidopsis Cor15A (Artus et al. 1996, NDong et al. 2002).On the other hand, overexpression of individual genesencoding LEA proteins from spinach (Spinacia oleracea) andCraterostigma plantagineum in tobacco (Nicotiana tabacum)does not lead to any significant changes in freezing or droughttolerance (Iturriaga et al. 1992, Kaye et al. 1998). These resultsmay indicate either that not all LEA proteins have significantroles in stress tolerance or that they need other factors foractivation (Houde et al. 2004, Hundertmark and Hincha2008). Biochemical analyses showed that several LEA proteinsbehave as cryoprotectants in vitro (Houde et al. 1995,Wisniewski et al. 1999 Nakayama et al. 2007). The maize (Zeamays) LEA protein DHN1 changes conformation when boundto lipid vesicles, suggesting its involvement in membranestabilization under stress conditions (Koag et al. 2003, Koaget al. 2009).

LEA proteins are not restricted to plants; LEA-like proteins,otherwise termed hydrophilins, exist in a range of organismsincluding Escherichia coli, yeast and invertebrates (Garay-Arroyo et al. 2000, Browne et al. 2002, Gal et al. 2004,Kikawada et al. 2006). Reductions in CeLEA1, encoding anLEA-like protein from Caenorhabditis elegans, results in reducedsurvival after desiccation, osmotic and heat stresses (Gal et al.2004). Recently, it has been demonstrated that hydrophilinsare required for desiccation and freezing tolerance in yeast(Dang and Hincha 2011, Lopez-Martinez et al. 2012,

Rodriguez-Porrata et al. 2012). Therefore, the function of LEAproteins in stress tolerance appears to be conserved in a varietyof organisms.

In the present work, we characterize a cold-induced proteinfrom wheat, WCI16, and show that it functions in freezing tol-erance. Our data thus identify a group of proteins of unknownfunction as a novel class of LEA proteins.

Results

cDNA isolation and sequence analysis

We previously performed a macroarray-based screen for genesthat are induced during CA in wheat (Christova et al. 2006). Thescreening identified 69 clones that were up-regulated duringCA. Here, we characterized a clone that contained a 477 bpopen reading frame (ORF) encoding a putative 16.3 kDa pro-tein, and named the corresponding gene Wheat Cold Induced16 (WCI16) (DDBJ accession No. AB830333). The WCI16 proteinis rich in alanine (16.46%), lysine (14.56%), glycine (12.03%) andglutamic acid (12.03%), but lacks cysteine, tryptophan, arginineand histidine residues (Supplementary Fig. S1). WCI16 pos-sesses two alternating lysine and glutamic acid residue-rich re-gions (EKKSVEGIDKEK and KKSGEEEKE) (Supplementary Fig.S1). Hydropathy plots predicted that WCI16 is predominantlyhydrophilic, with few hydrophobic amino acid residues (Fig.1B). This degree of hydrophilicity is reminiscent of that ofLEA proteins, although WCI16 did not show significant se-quence similarity to any known classes of LEA proteins.Homology searches indicated that the amino acid sequenceof WCI16 was similar to those of a group of plant proteinswith unknown function, including rice Os10g0330000 andArabidopsis At1g13930 and At2g03440 (Fu et al. 2010)(Supplementary Fig. S2). At2g03440 was previously namedAtNRP1 (for Arabidopsis thaliana Nodulin-related protein 1)and shows partial similarity to the alfalfa (Medicago sativa)early nodulin 12B precursor (Fu et al. 2010). WCI16 showed57.1, 42.1 and 42.1% identity to Os10g0330000, At1g13930and AtNRP1, respectively. A phylogenetic tree generated withthe sequences of WCI16 homologs from several plant speciesthat are available in the GenBank database showed that thereare at least three groups within the plant WCI16 homologs(Fig. 1A). WCI16 homologs were identified in monocotsand dicots, but not in ferns, mosses, green algae and othernon-plant species (1e�90< E-value< 1e�2 in BLAST searches).

Expression of WCI16 in response tocold treatment

As the WCI16 cDNA was isolated by a differential screening forcold-induced genes, its expression during the course of CA wasexamined by RNA gel blot analysis. Accumulation of the WCI16mRNA in crown tissue reached its maximum after 1 d of CA anddeclined somewhat thereafter, but maintained relatively highlevels during 14 d of CA (Fig. 2A). In contrast, WCI16 mRNAdecreased immediately after 1 d of de-acclimation (DA)

137Plant Cell Physiol. 55(1): 136–147 (2014) doi:10.1093/pcp/pct164 ! The Author 2013.

Novel LEA-class protein confers freezing tolerance

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(Fig. 2A). The short-term response of WCI16 to cold wasdetermined using young seedlings. RNA gel blot analysis indi-cated that the expression of WCI16 mRNA steadily increasedup to 24 h of cold treatment and slightly declined thereafter(Fig. 2B).

Purification and heat stability of intactWCI16 protein

To explore the possible functions and activity of WCI16, a gluta-thione S-transferase (GST)-fused form of WCI16 was produced

Fig. 1 Phylogenic analysis of WCI16 homologs and the hydrophilic nature of WCI16. (A) A phylogenetic tree was generated by the Neighbor–Joining method using the Genious Pro 5.3.4 software. Individual branch lengths indicate evolutionary distance. Two major groupings weredetected within dicot plants (Dicot I and Dicot II). (B) Hydropathy analysis of the WCI16 protein based on Kyte–Doolittle values (Kyte andDoolittle 1982). The window size was set at 9. The values above the zero line indicate predicted hydrophilicity.


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in E. coli and purified as an untagged form after on-columncleavage with PreScission� protease. Although the calculatedmolecular mass of WCI16 is 16.3 kDa, the purified WCI16migrated as a larger protein on SDS–polyacrylamide gels(Fig. 3, lane 4). Similar phenomena have been observed formany LEA proteins (Kovacs et al. 2008, Hara et al. 2009, Koaget al. 2009); because of their amino acid composition, intrinsic-ally unstructured proteins bind less SDS than other proteins doand their apparent molecular mass is often higher than thatcalculated from sequence data (Tompa 2002). RecombinantGST alone was also purified (Fig. 3, lane 1) and was used as anegative control in the following experiments.

We first examined the boiling solubility of WCI16. WCI16and GST were boiled for 20 min, separated into soluble andinsoluble fractions, and analyzed by SDS–PAGE. After boiling,GST was not detected in the soluble fraction but was present inthe insoluble fraction (Fig. 3, lanes 2 and 3), reflecting the heat-labile nature of the protein. In contrast, the WCI16 proteinband was detected in the soluble fraction after boiling (Fig. 3,lane 5) and was not found in the insoluble fraction (Fig. 3, lane6). These data indicate that WCI16 is a boiling-soluble proteinthat shares characteristics with LEA proteins.

WCI16 protects lactate dehydrogenase fromfreeze inactivation

We next evaluated the cryoprotective activity of the purifiedWCI16 protein by testing its ability to protect L-lactatedehydrogenase (LDH), a freeze-labile enzyme, from freeze

inactivation. Equivalent concentrations of bovine serum albu-min (BSA) and sucrose were used as positive and negative con-trols, respectively. Measurements of residual LDH activityindicated that WCI16 had cryoprotection activity comparablewith that of BSA (Fig. 4). In contrast, sucrose failed to protectLDH in an identical freeze–thaw cycle. These data strongly sup-port the hypothesis that WCI16 protects cellular enzymes fromfreeze-induced inactivation.

Structural analysis of WCI16

One of characteristic features of LEA proteins is unstructuredfolding due to amino acid compositions biased toward hydro-philic residues (Dure 1993b). We tested whether WCI16 sharesthis characteristic, using 1H-nuclear magnetic resonance (NMR)analysis of purified recombinant WCI16 dissolved in 10% D2O.The resultant 1H-NMR spectrum was analyzed with empiricalrules that correlate 1H-NMR spectra with protein structuralcomposition (Wishart et al. 1991, Wishart et al. 1992). Asshown in Fig. 5, no significant secondary shifts were observedin the high-field-shifted methyl region (0–1.0 p.p.m.), suggest-ing that hydrophobic side chains including aromatic rings donot form any clustered regions in WCI16. In addition, therewere no shifts in the down-field-shifted CaH region (5.0–6.0 p.p.m.), indicating that WCI16 does not form definitive sec-ondary structure, such as an a-helix or b-strand. Further, nosecondary shift was found in the extremely low-field-shiftedNH-proton region (9.0–12.0 p.p.m.), revealing that there is nosignificant specific three-dimensional structure in WCI16. Theresonances observed between 8.2 and 9.0 p.p.m. are attribut-able to random-coiled NH groups. For WCI16, the total inten-sity of this spectral region was small compared with that ofsimilar sized globular proteins [e.g. hen egg lysozyme (129 resi-dues)], suggesting that most of the amide group of WCI16 iseasily exchanged with solvent water. It is therefore highly likelythat WCI16 does not form any rigid structure but favors arandom state at 4�C.

Fig. 2 Expression of WCI16 in response to cold. (A) RNA gel blotanalysis of wheat crown tissue during cold acclimation (4�C).(B) RNA gel blot analysis of wheat shoot tissue during short-termcold treatment (4�C). rRNAs were visualized by staining with ethidiumbromide to serve as loading controls. CA, cold acclimation; DA,de-acclimation.

Fig. 3 Purification and heat stability of WCI16 protein. RecombinantGST and WCI16 proteins were purified and treated with (+) orwithout (�) boiling (100�C for 20 min). Proteins were subsequentlyseparated by SDS–PAGE. S, soluble fraction after the boiling treat-ment; IS, insoluble fraction after the boiling treatment. Lane 1, purifiedGST; lane 2, soluble fraction of boiling-treated GST; lane 3, insolublefraction of boiling-treated GST; lane 4, purified WCI16; lane 5, solublefraction of boiling-treated WCI16; lane 6, insoluble fraction of boiling-treated WCI16.



Subcellular localization of WCI16

To elucidate further the function of WCI16 in plant cells, wetransiently expressed a green fluorescent protein (GFP)-fusedWCI16 protein. Wheat leaf sheath epidermal cells were trans-formed with the fusion gene by particle bombardment.Fluorescence of WCI16::GFP was detected in cytosolic andnuclear regions of wheat cells (Fig. 6). The GFP-only positivecontrol also showed a cytoplasmic and nuclear localization(Fig. 6). From these data, we conclude that WCI16 localizesto the cytosol and nucleus in wheat cells.

Nucleic acid binding activity of WCI16

Recent bioinformatics analysis predicted that LEA proteinshave DNA binding activity (Wise and Tunnacliffe 2004).

In order to explore the possible function of WCI16 in thenucleus, the nucleic acid binding activity of WCI16 wastested. Recombinant GST:WCI16 protein was purified andgel-shift assays were performed utilizing single- or double-stranded DNAs (ssDNA or dsDNA) as substrates. As shown inFig. 7, shifts of the only dsDNA band were detected whenGST:WCI16 was added. In contrast, shifts of both ssDNA anddsDNA bands were detected when 700 pmol GST:WCSP1(Karlson et al. 2002) was added to the reaction. When GSTwas added as a negative control, no shift was detected foreither ssDNA or dsDNA substrates. RNA binding assays wereperformed using in vitro transcribed luciferase (luc) mRNA as asubstrate. As shown in Fig. 7, no shift was detected withGST:WCI16 or GST, whereas a clear shift was observed withGST:WCSP1. These data indicate that WCI16 specificallybinds to dsDNA.

Overexpression of WCI16 confers freezingtolerance

To test the effects of WCI16 expression on freezing tolerance,we generated transgenic Arabidopsis lines overexpressingWCI16. We analyzed two homozygous 35S:WCI16 transgeniclines (WCI16-15 and -30) (Fig. 8A). The 35S:WCI16 plantsshowed no difference in phenotype compared with the wildtype under normal growth conditions (Fig. 8B). The 35S:WCI16and wild-type plants were then exposed to freezing tempera-tures under non-cold-acclimated conditions. As shown inFig. 8C and D, 35S:WCI16 transgenic plants displayed highersurvival rates than did wild-type plants at �10�C. The WCI16-15 line had a survival rate of 95.8%, while the survival of wild-type plants was 72.2%. Similar results were also observed withthe WCI16-30 line, with 87.5% survival vs. 65.3% for the wild-type plants in these experiments (Fig. 8D). Freezing tolerancewas further examined by electrolyte leakage assay. Rosetteleaves of 35S:WCI16 lines exhibited lower levels of leakage

Fig. 4 Cryoprotection of LDH by recombinant WCI16. An LDH solu-tion was frozen at �30�C for 24 h with varying concentrations ofrecombinant WCI16, BSA or sucrose. The samples were thawed atroom temperature and the LDH activity was measured. The residualactivity represents the LDH activity remaining after a freeze–thawtreatment as a percentage of the enzyme activity before the treat-ment. Values are means of three independent experiments with barsrepresenting the standard errors.

Fig. 6 Subcellular localization of WCI16::GFP fusion protein.Fluorescence images of wheat leaf sheath epidermal cells transformedwith WCI16::GFP or the GFP vector-only control. Scale bar = 20 mm.

Fig. 5 Structural analysis of WCI16. The one-dimensional 1H-NMRspectrum of His-WCI16 was recorded on a 500 MHz spectrometerat 4�C. Measurements were made in 10% D2O with a protein concen-tration of 10 mg ml�1. Chemical shifts are referenced to the trimethyl-silyl resonance of 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS),which was added as an internal standard (0 p.p.m.).


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than those of the wild-type under non-acclimated condition(Fig. 8E). These data demonstrate that overexpression of WCI16confers freezing tolerance to non-acclimated plants.

Discussion

In the present study, we isolated the WCI16 gene encoding ahyper-hydrophilic protein from cold-acclimated wheat. TheWCI16 protein belongs to a group of plant proteins whosefunction is unknown. Hyperhydrophilicity is one of the charac-teristics of LEA proteins; however, the amino acid sequence ofWCI16 showed no significant similarity to any known classes ofLEA proteins. LEA proteins generally share several features(Baker et al. 1988, Dure 1993b): (i) hyper-hydrophilicity;(ii) boiling solubility; (iii) stress inducibility; and (iv) stress-protecting activity. Several lines of evidence in this study dem-onstrate that WCI16 represents a novel class of LEA proteinsand is involved in freezing tolerance.

As shown in Fig. 3, purified WCI16 remained soluble afterboiling for 20 min. Hydropathy plots indicated that the overallprotein lacks hydrophobic regions (Fig. 1B). Our 1H-NMRstructure analysis revealed that WCI16 is unstructured in

solution (Fig. 5). It is likely that the boiling-soluble nature ofWCI16 is due to a lack of hydrophobic interaction and foldedstructures.

WCI16 mRNA expression was strongly induced in crowntissue during CA (Fig. 2A). Crown tissue is important forwinter survival and develops the highest tolerance against freez-ing (Chen et al. 1983, Perras and Sarhan 1989, Houde et al.1995). In addition, WCI16 transcript was induced by cold treat-ment in shoot tissues (Fig. 2B). In contrast, the expression ofWCI16 was not induced by other dehydration-related stressessuch as drought and salt, or by treatment of seedlings with thedehydration-related hormone ABA (data not shown). Thesedata therefore suggest that WCI16 function is mainly associatedwith freezing tolerance. This hypothesis is further supported bythe fact that WCI16 protects LDH from freeze–thaw inactiva-tion (Fig. 4). Functions in protection of LDH during freezinghave been demonstrated for LEA proteins from several plantspecies (Wisniewski et al. 1999, Honjoh et al. 2000, Hara et al.2001, Bravo et al. 2003, Nakayama et al. 2007). The cryoprotec-tive activity of WCI16 protein was comparable with that re-ported for other LEA proteins, such as PCA60 (Wisniewski et al.1999) and Cor15am (Nakayama et al. 2007).

We found that WCI16 protein has dsDNA binding activity(Fig. 7). Recently, it was demonstrated that the lysine and glu-tamic acid cluster in the dehydrin CuCOR15 plays an importantrole in DNA binding (Hara et al. 2009). WCI16 also has lysineand glutamic acid clusters, namely EKKSVEGIDKEK andKKSGEEEKE (Supplementary Fig. S1), which might contributeto the DNA binding activity of WCI16. An increase in reactiveoxygen species production due to environmental stress cancause oxidative modifications to proteins, lipids and DNA(Apel and Hirt 2004, Bartoli et al. 2004). Dps, a non-specificDNA binding protein in E. coli, protects DNA from genotoxicstress caused by oxidative stress (Martinez and Kolter 1997).WCI16 may play a similar role in DNA protection by interactingwith DNA under stress conditions.

WCI16::GFP showed a nucleo-cytoplasmic localization inwheat cells (Fig. 6). This nuclear and cytoplasmic localizationis consistent with roles for WCI16 in protecting proteins andDNA from freezing stress. Sequence analysis of WCI16 did notreveal any known nuclear localization signal (NLS), and it alsolacks the S-segment that is believed to be involved in nuclearlocalization (Jensen et al. 1998). The wheat LEA protein WCS120localizes to the nucleus and cytoplasm, but is similarly devoid ofan NLS or S-segment (Houde et al. 1995). It is possible that anunknown nuclear localization mechanism, independent of NLSsand S-segments (Rorat 2006), delivers WCI16 to the nucleus.

Transgenic Arabidopsis plants overexpressing WCI16 weregenerated in order to test the contribution of WCI16 to freezingtolerance in planta (Fig. 8A). The WCI16-overexpressing plantsdisplayed no morphological or developmental defects, suggest-ing no negative effect of WCI16 overexpression on growthunder non-stress conditions (Fig. 8B). Overexpression ofWCI16 conferred improved freezing tolerance to non-accli-mated plants in both whole plant survival and leaf electrolyte

Fig. 7 Analysis of nucleic acid binding activity of WCI16 by gel shiftassay. Purified GST:WCI16 fusion proteins were incubated withdsDNA, ssDNA or in vitro transcribed luciferase mRNA, and separatedby agarose gel electrophoresis. A range of WCI16 fusion protein con-centrations from 0 to 700 pmol was used for the analyses. Purified GSTand GST:WCSP1 were loaded as negative and positive controls,respectively.




leakage (Fig. 8C�E). Although there is a discrepancy betweenthe temperature ranges of tolerance in the two experiments,the difference can be explained by the effect of sucrose addedto the agar medium, which benefits clear identification of sur-vivors, on lowering the killing temperatures (K. Sasaki,

unpublished observation). LEA proteins are highly divergent,and the contribution of each protein to overall stress toleranceis considered to be rather small. However, several reports haveshown that overexpression of single LEA proteins can conferstress tolerance. Electrolyte leakage tests revealed that the

Fig. 8 Effect of WCI16 overexpression on freezing tolerance. (A) RNA gel blot analysis of 10-day-old seedlings in two independent transgenicArabidopsis lines (WCI16-15 and -30). (B) Phenotype of 3-week-old wild-type and WCI16-overexpressing plants. (C, D) Survival of WCI16overexpressors (WCI16-15 and -30) and wild-type plants after freezing stress. Ten-day-old plants grown on MS medium containing 2% sucrosewere frozen with ice chips and cooled (�1�C h�1) until the designated temperatures were reached. Plates were then maintained in the dark at4�C for overnight and transferred to a growth chamber (16 h light/8 h dark) at 22�C for survival rate determination. The photographs were takenafter 6 d of recovery from freezing treatments (C). Experiments were performed in quadruplicate, and the percentage of plants surviving wascalculated (n = 18 plants used in each treatment). Data represent means ± SE of quadruplicate experiments. * and ** indicate statisticallysignificant differences from the wild type at P< 0.05 and P< 0.01 (Student’s t-test), respectively (D). (E) Electrolyte leakage of 4-week-oldwild-type and 35S:WCI16 plants after exposure to freezing temperatures (�5�C to �8�C). One detached rosette leaf per plant was placed in atube containing 200 ml of ice-containing distilled water for ice nucleation at�2�C. The temperature was lowered at a rate of�1�C h�1. The tubeswere removed from the freezer at the designated temperatures and shaken at 25�C for 4 h before electrical conductivity measurements.


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freezing tolerance of Arabidopsis is increased by ectopic expres-sion of the wheat LEA gene WCS19 or the Rhododendron cat-awbiense LEA gene RcDhn5 (NDong et al. 2002, Peng et al.2008). Recently, it was demonstrated that overexpression ofOpsDHN1, an LEA gene from Opuntia streptacantha, increasesthe survival rate of Arabidopsis at the whole plant level afterfreezing treatment (Ochoa-Alfaro et al. 2012).

A knock-out line of At1g13930, a WCI16 homolog inArabidopsis, is hypersensitive to salt in terms of germination,although the expression of the At1g13930 gene is not inducedby salt treatment (Du et al. 2008). According to data from thepublic microarray database (BAR; http://www.bar.utoronto.ca/), the At1g13930 transcript is 5.2-fold up-regulated inshoots and 3.6-fold up-regulated in roots by 24 h of cold treat-ment. At1g13930 could be involved in CA and the acquisitionof dehydration stress tolerance in Arabidopsis. The expressionof another WCI16-like gene, AtNRP1, is also up-regulated by lowtemperature (Fu et al. 2010). One possible mechanism for theincreased freezing tolerance of Arabidopsis plants overexpres-sing wheat WCI16 is that the overproduction and nucleo-cytoplasmic localization of the WCI16 protein protectsendogenous proteins and DNA from lethal freezing damage.

Our current data, together with the recent findings inArabidopsis, support a functional role for WCI16 family pro-teins in stress tolerance. Together, this work shows that WCI16exhibits the hyper-hydrophilicity, boiling solubility, stress indu-cibility and stress-protecting activity shared by LEA proteins,suggesting that WCI16 is a novel LEA protein that is inducedduring CA.

Materials and Methods

Plant materials

Surface-sterilized seeds of winter wheat (T. aestivum L. cv.Chihoku) were germinated on wet paper and planted on com-mercial soil mix. Plants were grown at 22�C/18�C (16 h light/8 hdark) in a growth chamber. Seeds of Arabidopsis [Arabidopsisthaliana ecotype Columbia (Col-0)] were surface sterilized andplanted on MS medium (Murashige and Skoog 1962) contain-ing 2% sucrose. Plates were maintained in the dark at 4�C for 2 dand were transferred to a growth chamber (16 h light/8 h dark)at 22�C.

Stress treatments

Plants of winter wheat were grown in pots (pot size:60 mm�155 mm�100 mm; four wheat plants per pot) for14 d. CA was performed at 6�C/2�C (8 h light/ 16 h dark at211 mmol m�2 s�1) for an additional 14 d. Subsequently,plants were subjected to DA treatment at 22�C/18�C (16 hlight/8 h dark at 211 mmol m�2 s�1) for 3 d. Crown tissue washarvested before acclimation (NA; day 0), after 1, 3, 7, 10 and14 d of CA, and after 1 and 3 d of DA. Collected plant materialswere frozen in liquid nitrogen and stored at �80�C until pro-cessed for RNA extraction. For short-term cold treatment,

germinated seeds of winter wheat were cultivated hydroponic-ally in water on plastic mesh grids that were suspended justabove the water line in plastic containers. The container wasmaintained in a growth chamber at 22�C under continuousillumination. After growing for 7 d, seedlings were subjectedto cold treatment by transferring them into plastic containerswith cold water (4�C). At each designated time period, shoottissue was collected, frozen with liquid nitrogen, and stored at�80�C until processed for RNA extraction.

cDNA isolation and sequence analyses

Screening of cold-regulated cDNA clones in a cDNA library wasperformed using a macroarray-based differential screeningmethod (Christova et al. 2006). Isolated cDNA clones weresequenced with a 373A DNA sequencer (Applied Biosystems)using a Big Dye Terminator Cycle Sequencing Kit v 1.1 (AppliedBiosystems). Multiple amino acid alignments were performedusing the online ClustalW alignment program at a websitemaintained by the DDBJ (http://clustalw.ddbj.nig.ac.jp/top-j.html). Protein hydropathy was predicted by Kyte–Doolittlehydropathy plot analysis (http://fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1). A phylogenetic tree wascreated using Geneious Pro 5.3.4 software (Biomatters Ltd.).

Total RNA extraction and RNA gel blot analyses

Total RNA was isolated from wheat shoot and crown tissuesusing TRIzol reagent (Invitrogen). Total RNA was separated ona 1.2% agarose gel containing formaldehyde and transferred toHybond N+ membrane (GE Healthcare). Hybridization was per-formed with a full-length WCI16 cDNA probe that was labeledwith the ECL Kit (GE Healthcare) according to the manufac-turer’s instructions. Detection and quantification of the signalswere carried out using a LAS3000 image analyzer (Fuji Film).

Recombinant protein production and purification

The ORF of WCI16 was amplified with 50-GGATCCATGGAGGGTGAGAAGAAC-30 and 50-GTCGACCTACTTCATGAACCCCTGA-30 (underlining denotes restriction sites) and cloned intothe BamHI–SalI site of pGEX6P-3 (GE Healthcare) to producea GST-fused protein (pGEX-WCI16). Construction of pGEX-WCSP1 was described previously (Karlson et al. 2002).Escherichia coli cells containing pGEX-WCI16, pGEX-WCSP1or pGEX6P-3 were cultured in LB medium until the OD600

reached 0.6. Isopropyl-b-D-thiogalactopyranoside (IPTG) wassubsequently added to a final concentration of 1 mM and re-combinant proteins were induced for 3 h. The bacterial cultureswere centrifuged and the resuspended pellets were disruptedwith sonication. The cell lysate was centrifuged and the totalsoluble fraction (supernatant) was affinity purified with a gluta-thione–Sepharose 4B column (GE Healthcare) according to themanufacturer’s instructions. Subsequent to washing, boundGST-fused proteins were eluted or further purified byon-column cleavage with PreScission� protease (GEHealthcare) to eliminate GST according to the manufacturer’s



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http://www.clustalw.ddbj.nig.ac.jp/top-j.html

http://www.clustalw.ddbj.nig.ac.jp/top-j.html

http://www.fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1

http://www.fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1

instructions. Eluted proteins were washed and concentratedwith buffer (10 mM Tris–HCl, pH 7.5). Protein concentrationwas determined with the BioRad Protein Assay Kit (Bio-RadLaboratories) using IgG as a standard.

Boiling treatment

The protein samples were heated to 100�C for 20 min andsubsequently cooled on ice. The samples were centrifuged at12,000�g for 10 min at 4�C in order to separate the soluble andinsoluble fractions. The soluble and insoluble fractions weresuspended with SDS sample buffer, separated by SDS–PAGEand stained with Coomassie Brilliant Blue according tostandard protocols.

Cryoprotection assay

The cryoprotective activities of purified intact recombinantWCI16 were assayed as described previously (Lin andThomashow 1992). Briefly, LDH from Lactobacillus cassei(Nacalai tesque) was diluted to 12.5mg ml–1 in 10 mM KPO4,pH 7.5. A 50ml aliquot of this solution was mixed with an equalvolume of the tested cryoprotectant and frozen at �30�C for24 h. After thawing at room temperature, 20ml of sample wasadded to 1 ml of substrate solution containing 80 mM Tris–HCl,pH 7.5, 100 mM KCl, 2 mM pyruvic acid and 0.3 mM NADH. Theabsorbance decrease at 340 nm was measured using a spectro-photometer (Implen). The rate of decrease in absorbance duringthe first 3 min of the reaction was used to calculate LDH activity.

1H-NMR structure analysis

The ORF of WCI16 was amplified with 50-GGATCCATGGAGGGTGAGAAGAAC-30 and 50-GTCGACCTACTTCATGAACCCCTGA-30 (underlining denotes restriction sites) and cloned intothe BamHI–SalI site of pQE30 (Qiagen) to produce a His-taggedfusion protein (pQE30-WCI16). Escherichia coli cells containingpQE30-WCI16 were cultured until the OD600 reached 0.6. IPTGwas subsequently added to a final concentration of 1 mM andproduction of recombinant proteins was induced for 3 h. Thebacterial cultures were centrifuged and the pellets were dis-rupted with sonication. The cell lysate was centrifuged andthe total soluble fraction (supernatant) was affinity purifiedwith Ni-NTA agarose (Qiagen) according to the manufacturer’sinstructions. Subsequent to washing, bound His-fused proteinswere eluted. Eluted proteins were washed and concentratedwith Milli-Q water. The protein concentration was determinedwith the BioRad Protein Assay Kit (Bio-Rad Laboratories) usingIgG as a standard. For NMR sample preparation, His-WCI16 wasdissolved at 10 mg ml–1 concentration in H2O containing 10%D2O for spectrometer locking. The 1H-NMR measurement wasperformed using a Varian Unity INOVA 500 MHz spectrometer(Varian) at 4�C. The spectrum was acquired with the 1H-carriercentered at the water frequency (5.01 p.p.m.), and water sup-pression was achieved by a low-power (20 Hz) water pre-satur-ation pulse of 1.0–1.5 s duration. The 1H chemical shifts werereferenced to the trimethylsilyl resonance of 2,2-dimethyl-2-

silapentane-5-sulfonic acid (DSS) at 0 p.p.m., which wasadded as an internal standard.

Subcellular localization of GFP-fused protein

A GFP gene, sGFP (S65T), was used as a reporter for an in-frameC-terminal fusion to WCI16 (Niwa et al. 1999). To generatethe GFP fusion construct, the ORF of WCI16 was amplifiedwith primers 50-GCGCGCGGATCCATGGAGGGTGAGAAGAA-30 and 50-GCGCGCCCATGGACTTCATGAACCCCTGAG-30

(underlining denotes restriction sites) that removed thenative stop codon of the ORF and added BamHI and NcoIsites for subsequent cloning. The resulting PCR fragment wasdigested with BamHI and NcoI and ligated into BglII–NcoI sitesof the sGFP (S65T) vector (Niwa et al. 1999). WCI16::GFP plas-mid was bound to 1.0mm gold particles (Bio-Rad Laboratories)according to the manufacturer’s instructions. Particles werebombarded into wheat leaf sheath cells with a PDS-1000 bio-listic delivery system (Bio-Rad Laboratories) with a rupture set-ting of 1,100 p.s.i. After bombardment, the tissues weremaintained in the dark for 16 h at 25�C and viewed on glassslides with a FW4000 fluorescence imaging workstation (Leica).Deconvolution images were obtained with Leica DEBLURsoftware (Leica).

Nucleic acid binding analysis

Gel shift assay with ds/ssDNA substrates was performed as pre-viously described (Nakaminami et al. 2005). ssDNA (M13mp8)or dsDNA (M13mp8 RFI) (Nippon Gene) was incubated withGST–WCI16, GST–WCSP1 or GST in 15ml of binding buffer(10 mM Tris–HCl, pH 7.5) and was kept on ice for 15 min.The samples were then separated on 1% agarose gels andstained with ethidium bromide for visualization of gel shifts.Gel shift assays with mRNA substrates were performed as pre-viously described (Nakaminami et al. 2005). Luciferase mRNAwas in vitro transcribed with the RiboMAX kit (Promega).

Overexpression of WCI16 in Arabidopsis

To generate the construct for overexpression of WCI16 underthe control of the Cauliflower mosaic virus 35S promoter inArabidopsis, the ORF of WCI16 was amplified by PCR withprimers 50-GGATCCATGGAGGGTGAGAAGAACTC-30 and50-CCCGGGACCAGACTTCTACTTCATGA-30 (underliningdenotes restriction sites) and ligated into pGEM-TEasy vector(Promega). The vector was digested with BamHI and SacI andthe resulting fragment, including a part of the multicloning siteof the pGEM-TEasy vector (between SpeI and SacI), wassubcloned into the corresponding sites of the pBI121 vector(Clontech) to generate the 35S:WCI16 construct.Agrobacterium-mediated transformation of Arabidopsis wasperformed using a modified floral dip method (Clough andBent 1998). Agrobacterium culture resuspended in transform-ation buffer [1/2� MS medium, 5% sucrose, 0.05% silwet L-77,44 nM 6-benzylaminopurine (BAP), Gamborg’s vitamins,pH 5.7] was dropped onto young floral buds (before flowering).Inoculated plants were covered with a plastic bag to maintain


K. Sasaki et al.

high humidity and placed in the dark for 12–20 h. Plants weregrown under normal growth conditions (16 h light/8 h dark at22�C) and seeds were harvested. For selection of transformants,T1 seeds were plated on MS medium containing 2% sucrose and50mg ml�1 kanamycin. Expression of WCI16 in independenttransgenic lines was examined by RNA gel blot analysis usingthe ORF region as a probe. Homozygous T4 plants were used forfurther analysis.

Freezing stress tolerance analysis

Whole-plant freezing tolerance was assayed as described previ-ously (Chinnusamy et al. 2003), with some modifications.Non-acclimated 10-day-old seedlings were transferred to a pro-grammed freezer LU-112 (ESPEC) set at �1�C. After 1–2 h, theplates were sprinkled with ice chips and maintained at�1�C forat least 16 h. The temperature was lowered at a rate of�1�C h�1

and plates were removed from the freezer at specific tempera-tures. Plates were thawed overnight at 4�C in the dark and sub-sequently incubated at 22�C. The survival rate of the plants wasscored after 6 d. Electrolyte leakage assays were performed asdescribed previously (Kim et al. 2009). One excised rosette leaffrom 4-week-old Arabidopsis plants was placed in a tube con-taining 200ml of ice-containing distilled water for ice nucleationand the tube was maintained in the programmed freezer withthe temperature set at�2�C. The temperature was lowered at arate of �1�C h�1. The tubes were removed from the freezer atspecific temperatures and placed at 2�C for 1 h. After adding800ml of distilled water, the tubes were shaken at 25�C for 4 h.The electrolyte leakage was measured using a compact conduct-ivity meter C-172 (HORIBA). The tubes were frozen at�80�C for16 h and then shaken at 25�C for 4 h to obtain a value for 100%electrolyte leakage for each sample.

Supplementary data

Supplementary data are available at PCP online.

Funding

This work was supported by the National Agriculture and FoodResearch Organization [NARO; Development of InnovativeCrops through Molecular Analysis of Useful Genes, (grantNo. 3210) to R.I.]. N.K.C. was supported by a JSPS postdoctralfellowship.

Acknowledgments

We are grateful for the technical support provided by KazueSugawara. We would like to thank Dr. Yasuo Niwa (University ofShizuoka) for kindly providing the sGFP (S65T) vector.

Disclosures

The authors have no conflicts of interest to declare.

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