Gene 482 (2011) 51–58

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Methods paper

Identification and characterization of genes encoding two novel LEA proteins inAntarctic and temperate strains of Chlorella vulgaris

Xiaoxiang Liu, Yali Wang, Hong Gao, Xudong Xu ⁎State Key laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei 430072, China

Abbreviations: aa, amino acid residue(s); BCIP,phosphate; bp, base pair(s); cDNA, complementary DNCTAB, hexadecyl trimethyl ammonium bromide; EDTacid; EST, expressed sequence tag; 5′ RACE, rapid ampliglutathione-S-transferase; h, hour; kb, kilobase(s); kdehydrogenase; LEA, late embryogenesis abundant; mitetrazolium chloride; nt, nucleotide; ORF, open readinggel electrophoresis; POPP, Protein or Oligonucleotide Pbinding protein 1; RT-PCR, reverse-transcription polsodium dodecyl sulfate; SSH, suppression subtractivepoint; poly (A), polyadenylation; rRNA, ribosomal RNA;⁎ Corresponding author at: Institute of Hydrobiology,

Wuhan, Hubei 430072, China. Tel.: +86 27 68780659.E-mail address: xux@ihb.ac.cn (X. Xu).

0378-1119/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.gene.2011.05.006

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 16 May 2011Available online 24 May 2011

Received by A.J. van Wijnen

Keywords:Chlorella vulgarisAntarctic strainFreeze toleranceLate embryogenesis abundant proteinsCryoprotective activity

An Antarctic strain (NJ-7) of Chlorella vulgaris possesses the same 18S rRNA sequence as that of a temperatestrain (UTEX259), but shows significantly higher freezing tolerance than the latter. Suppression subtractivehybridization (SSH) was performed to identify genes of intensified expression in NJ-7 relative to UTEX259.Among the genes identified, Ccor1 and Ccor2, co-organized in the same gene cluster Ccor1–Ccor2–Ccor1–Ccor2, showed much higher expression levels in NJ-7 than in UTEX259 at both 20 °C and 4 °C. As detected byNorthern blot andWestern blot analyses, the two genes were cold-inducible in NJ-7 but almost not expressedin UTEX259. Their encoded products are predicted to share 55.7% identity to each other and possessphysicochemical characteristics similar to that of late embryogenesis abundant (LEA) proteins in plants. Thepurified recombinant Ccor1 and Ccor2 showed high heat-stability and could act as cryoprotectants to lactatedehydrogenase in vitro. Based on their expression patterns and protein characteristics, we propose that Ccor1and Ccor2 are two novel LEA proteins and are related to the greatly enhanced freezing tolerance in theAntarctic strain.

5-bromo-4-chrolo-3-indolylA; COR, cold-regulated gene;A, ethylenediaminetetraaceticfication of 5′-cDNA ends; GST,Da, kilodalton; LDH, L-lactaten, minute; NBT, tetranitroblueframe; PAGE, polyacrylamiderobability Profile; Rbp1, RNA-ymerase chain reaction; SDS,hybridization; pI, isoelectricUTR, untranslated region.Chinese Academy of Sciences,

l rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Extremely low temperature is the major environmental stress towhich organisms inhabiting in Antarctica are exposed. Cold-adaptedorganisms, psychrophilic or psychrotrophic, have developed a variety ofstrategies at the molecular level for successful colonization in theAntarctic environments. The adaptive strategies include increasingmembrane fluidity (Henderson et al., 1998; Mock and Kroon, 2002;Thomson et al., 2004), producing cold-adapted enzymes (Loppes et al.,1996; di Rigano et al., 2006), accumulating antifreeze proteins andcryoprotectants (Gilbert et al., 2004; D'Amico et al., 2006). Some studieshave shown that accumulated LEA (late embryogenesis abundant)proteins can act as cryoprotectants and enhance the freezing tolerancein plants (Honjoh et al. 1999; Kim et al., 2005; Peng et al., 2008).

However, there is a paucity of reports on the roles of LEA proteins inadaptation to Antarctic environments.

LEA proteins were first found in cotton (Galau et al. 1986), and laterin other higher plants, algae, fungi, cyanobacteria and nematodes (Closeand Lammers, 1993;Honjoh et al., 1995;Ukaji et al., 2001; Browne et al.,2002; Abba' et al., 2006). On the basis of similarities in amino acidsequences, they can be divided into different groups (Colmenero-Floreset al., 1997; Cuming, 2005; Battaglia et al., 2008). Members of eachgroup are characterized by the presence of particular sequence motifs.Alternatively, the group nomenclature can be performed by usingbioinformatics tools, such as POPP (Protein or Oligonucleotide Proba-bility Profile) analysis (Wise, 2003). Although often no significanthomology can bedetected betweendifferent groups of LEA proteins, thebiased amino acid composition, high hydrophilicity and heat stability insolutions are considered as their common features (Hundertmark andHincha, 2008). Most of them are predicted to possess random coilstructures and are thought to be intrinsically unstructured (Tunnacliffeand Wise, 2007).

LEAproteins can be induced in response towater deficit (IngramandBartels, 1996; Abba' et al., 2006) or cold (Hara et al., 2001; Kosová et al.,2007). Reyes et al. (2005; 2008) suggested that LEA proteins can protectenzymes against the effects of water limitation and freeze-inactivation.For examples, a cold-responsive group-2 LEA protein in the higher plantCitrus unshiuwas shown to possess the cryoprotective activity (Hara etal., 2001), and a desiccation-responsive group-3 LEA protein from thenematode Aphelenchus avenae was shown to prevent a wide range ofproteins from aggregation (Chakrabortee et al., 2007). It has beenhypothesized that LEAproteins canmaintain the functions or stability of

52 X. Liu et al. / Gene 482 (2011) 51–58

cellular or molecular structures by replacement of hydrogen bondingfunction of water, sequestration of ions, or refolding of denaturedproteins (Bray et al., 2000; Cuming, 2005).

Chlorella vulgaris is often used as a eukaryotic model to investigatethe acquisition of freezing tolerance by cold hardening. HiC6 andHiC12,showing similarity to group-3 LEA proteins, are two hardening-inducedproteins identified in this species (Honjoh et al., 1995). The twoproteinsexhibit cryoprotective activities on lactate dehydrogenase, and over-expression of hiC6 in yeast or tobacco plant can enhance their freezingtolerance (Honjoh et al., 1999, 2000, 2001). An Antarctic strain of C.vulgaris, namely NJ-7, has been isolated (Hu et al., 2008), showing thesame 18S rRNA sequence as that of C. vulgaris SAG211-11b/UTEX259, atemperate strain isolated from the Netherlands. NJ-7 showed muchhigher freezing tolerance thanUTEX259with orwithout cold hardening(Hu et al., 2008; Li et al., 2009), and the greatly enhanced freezingtolerance was proposed to be based on intensified expression of someanti-freeze protein genes rather than mutations in their encodingsequences (Li et al., 2009).

Cold-induced proteins or genes have been identified in C. vulgarisusing 2D gel protein analysis or suppression subtractive hybridization(SSH) (Honjoh et al., 1995; Machida et al., 2008). Because the twostrains NJ-7 and UTEX259 show identical 18S rRNA sequences andhighly conserved encoding regions of some genes, and because NJ-7 ismuch more tolerant to freeze than UTEX259 even without coldhardening, we performed SSH with mRNA samples prepared from thetwo strains cultured at 20 °C to identify novel genes involved in freezetolerance. Among the genes identified, Ccor1 and Ccor2 (Chlorella cold-regulated) are co-organized in the same gene cluster in both strains butalmost exclusively expressed in NJ-7. Even though no significanthomology was found to known genes, their products showed typicalfeatures of LEA proteins, including high hydrophilicity, heat stability,cryoprotective activity on lactate dehydrogenase.

2. Materials and methods

2.1. Algal strains, culture conditions and cold treatment

C. vulgaris NJ-7 was isolated from the surface of wet rocks near theZhongshan station of the Antarctica (Hu et al., 2008); strain UTEX259was purchased from the Algal Culture Collection of University of Texas,and confirmed by sequencing of 18S rRNA. Algal cells were grown inBG11 (Stanier et al., 1971) under the light of 50 μE m−2 s−1 at 20 °Cwith aeration. NJ-7 and UTEX259 grown at 20 °Cwere rapidly cooled to4 °C in a water bath and then transferred to a refrigerator of 4 °C withillumination of 50 μE m−2 s−1 for different periods of time. Twohundred milliliters of algal cells were harvested at each time point bycentrifugation at4991 g for 3 min and frozen in liquid nitrogenuntil use.

2.2. Construction of a suppression subtractive hybridization (SSH)library

In order to identifying genes with higher expression level in theAntarctic strainNJ-7 than in the temperate strainUTEX259, a PCR-basedcDNA subtractionwas performed using PCR-SelectTM cDNA SubtractionKit (Clontech) according to themanufacturer's instructions. Briefly, totalRNA of NJ-7 and UTEX259 grown at 20 °C was extracted using Trizolreagent (Invitrogen), mRNA was isolated from total RNA using PolyATract mRNA Isolation System (Promega), tester cDNA was preparedfrom 2 μg of NJ-7 mRNA, driver cDNA was synthesized from UTEX259mRNA, and subtracted cDNA fragments were amplified and cloned intoa pMD18-T vector (TaKaRa).

2.3. Differential screening by dot-blot hybridization

Dot blot hybridization was performed to screen the library toidentify positive clones. PCR amplified cDNA inserts of the clones were

denatured and blotted onto two identical positively charged nylonmembranes (Millipore). The membranes were hybridized with DIG-labeled probes prepared from forward subtracted cDNA (NJ-7 tester,UTEX259 driver) and reverse subtracted cDNA (UTEX259 tester, NJ-7driver) respectively. Labeling, hybridization and detection wereperformed with DIG High Prime DNA Labeling and Detection StarterKit I (Roche) according to the manufacturer's recommendation. Thehybridization temperaturewas 65 °C and thewashing temperaturewas68 °C. The clones showing a significantly increased hybridization signaldetected with forward subtracted cDNA probe over the reversesubtracted cDNA probe were selected for sequencing.

2.4. Rapid amplification of 5′-cDNA ends (5′-RACE)

5′-RACE was performed using SMART RACE cDNA Amplification kit(Clontech) according to the manufacturer's instructions. The gene-specific primer A524-4 (5′-CACACGCTCCTTCACCACT-3′) was designedon the basis of a conserved region of Ccor1 and Ccor2 cDNA fragments,and was paired with the end primer UPM (5′-CTAATACGACTCACTA-TAGGGC-3′) provided with the kit to amplify the 5′ portions of cDNA.Both cDNA fragments possessed 3′-ends already. Full length cDNAsequences were obtained by juxtaposing sequences of 5′ and 3′portions.

2.5. Bioinformatics analyses

Similarity searches were performed using SIB BLAST NetworkService (http://expasy.org/tools/blast/). The physico-chemical parame-ters of a protein were predicted with ProtParam tool (http://www.expasy.ch/tools/protparam.html). The hydropathy profiles of theputative proteins were constructed by the ProtScale method (http://expasy.org/tools/protscale.html). Multiple sequence alignment wascarried out using the CLUSTALW 1.81 program (http://clustalw.genome.jp/). Secondary structures were predicted using SOPMA(http://npsa-pbil.ibcp.fr/cgi-bin/npsa _automat.pl?page=npsa_sopma.html). The disordered characteristics were predicted using IUPred(http://iupred.enzim.hu/) and PONDR (http://www.pondr.com). Thesearch for putative cis elements in the promoter region was carried outusing PLACE (http://www.dna.affrc.go.jp/PLACE/signalscan.html). DNAsequences were deposited in the NCBI GenBank under accessionnumbers: HQ832412 (NJ-7 Ccor1/Ccor2 gene cluster), HQ832413(UTEX259 Ccor1/Ccor2 gene cluster), HQ833824 (NJ-7 Ccor1 (NB)cDNA), HQ833825 (NJ-7 Ccor2 (NE) cDNA), HQ850451 (NJ-7 NA cDNA).

2.6. Southern blot analysis

High-molecular-weight DNA of C. vulgaris NJ-7 and UTEX259 wasextracted using the CTAB method (Murray and Thompson, 1980). Tenmicrograms of genomic DNA of NJ-7 or UTEX259 was digestedcompletely with one or two restriction enzymes. The digested DNAsamples were separated by electrophoresis on 0.7% agarose gels andwere blotted onto Immobilon-Ny+ membrane (Millipore) by capillarytransfer (Sambrook et al., 1989). The DIG-labeled Ccor2 probe forhybridization was prepared by PCR using A524-1 (5′-CTCATCGCCTA-CACGCAGAC-3′) and A41 (5′-GTTCACAGCCAGGTGGATACA-3′) asprimers and cDNA of UTEX259 as the template. Labeling, hybridizationand detection were performed with DIG High Prime DNA Labeling andDetection Starter Kit I (Roche) according to the manufacturer'srecommendation.

2.7. Northern blot analysis

After exposure to 4 °C for different periods of time, algal cells wereharvested by centrifugation at 4991 g for 3 min and frozen in liquidnitrogen until use. Total RNA was extracted using Trizol reagent(Invitrogen) from 200 ml of C. vulgaris NJ-7 and UTEX259 according to

53X. Liu et al. / Gene 482 (2011) 51–58

the manufacturer's instructions, separated by electrophoresis onagarose/formaldehyde gel and blotted onto Immobilon-Ny+ mem-branes (Millipore) by capillary transfer. DIG-labeling of DNA probe,hybridization and detection were performed similarly as described inSouthern blot analysis. The transcription of Ccor1 and Ccor2was probedby a 706-bp fragment internal to the NJ-7 Ccor1 cDNA (nt. 13-717),generated by PCR using cDNA clone A524 as the template and NestedPCR primer 1 (5′-TCGAGCGGCCGCCCGGGCAGGT-3′)/Nested PCR prim-er 2R (5′-AGCGTGGTCGCGGCCGAGGT-3′) as the primers, or a 258-bpfragment internal to the UTEX259 Ccor1 cDNA (nt. 30-287), generatedby PCR using total cDNA as the template and A524-1 and A524-4 as theprimers. 18S rRNA used as the internal control was probed by a PCRfragment generated using genomic DNA as the template and 18S-1 (5′-TGGTTGATCCTGCCAGTAGTC-3′)/18S-2 (5′-TGATCCTTCTGCAGGTT-CACC-3′) as the primers.

2.8. Western blot analysis

The frozen algal cells were broken by grinding in liquid nitrogen andsuspended in protein extraction buffer (0.1 M Tris–HCl, pH 7.5, 0.1 mMEDTA), and the cell debriswas removed by centrifugation at 17,226 g for30 min at 4 °C. Proteins in the supernatant were separated by 15% SDS-PAGE and electro-blotted onto a nitrocellulose membrane (Millipore).Ccor1 and Ccor2 proteins were detected with the rabbit antiserumraised against the GST-Ccor2 (UTEX259) fusion protein, and visualizedusing alkaline phosphatase-conjugated secondary antibody specific forrabbit IgG (Pierce) with NBT and BCIP (Amresco) as substrates. Inaddition to quantification with Bradford's method, protein sampleswere examined by SDS-PAGE and Coomassie brilliant blue-staining tomake sure equal amounts of total proteins were loaded.

GST-Ccor2 was produced in E. coli BL21 (DE3) harboring pET-GST-Ccor2, which was constructed by inserting a NcoI- and HindIII-digested PCR fragment containing UTEX259 Ccor2 into pET41a(Novagen). The PCR fragment was generated using A524-16 (5′-AGACCATGGCACACAAGATGGGCATTGGC-3′) and A524-17 (5′-TGCAAGCTTCGTGCTGTGGTGTCCTGTG-3′) as the primers and a UTEX259Ccor2 fragment cloned in pMD18-T (Takara) as the template.Expression of GST-Ccor2 in E. coli was induced with 1 mM IPTG at37 °C for 3 h. Cells broken by sonication were centrifuged at 13,000 gfor 5 min to remove cell debris. The fusion protein was purified fromtotal soluble proteins using His·Bind resin (Novagen) according to themanufacturer's recommendation followed by separation with SDS-PAGE. The gel slice containing the recombinant fusion protein wasused to generate the rabbit antiserum.

2.9. Expression of Ccor1 and Ccor2 in E. coli and boiling-stability analysis

Ccor1 and Ccor2were expressed in E. coli BL21 (DE3) harboring pET-Ccor1 andpET-Ccor2 respectively. pET-Ccor1was constructedby cloninga PCR fragment, which was generated using A524-9 (5′-GGACATATG-CAGAAGGTCAAGGAA GTCG-3′) and A524-10 (5′-GAGCTCGAGTCAG-TACGGAGTCGTGCCAT-3′) as the primers and total cDNA as thetemplate, into the NdeI and XhoI sites of pET21b (Novagen). The PCRfragment for construction of pET-Ccor2 was generated using primersA524-9 and A524-12 (5′-GACAAGCTTTCAGAGAGTACGCCCCGTG-3′).

Total soluble proteinswere extracted from E. coli expressingCcor1orCcor2, boiled for 5 to 20 min and centrifuged at 13,000 g for 5 min. Thesupernatant and precipitates were subjected to analysis with 15% SDS-PAGE.

2.10. Purification of recombinant proteins Ccor1-His and Ccor2-His andcryoprotection assay

Ccor1-His and Ccor2-His were expressed in E. coli BL21 (DE3)harboring pET-Ccor1-His and pET-Ccor2-His respectively. pET-Ccor1-His was constructed by cloning a PCR fragment, which was generated

using A524-9 and A524-11 (5′-CTGCTCGAGGTACGGAGTCGTGC-CATCC-3′) as the primers and total cDNA as the template, into theNdeI and XhoI sites of pET21b (Novagen). The PCR fragment forconstruction of pET-Ccor2-His was generated using primers A524-9and A524-13 (5′-CTGAAGCTTGAGAGTACGCCCCGTGCT-3′). The solu-ble fractions of IPTG-induced E. coli cells were boiled for 10 min andthen centrifuged at 13,400 g for 5 min to eliminate insoluble proteins.The boiling-stable proteins Ccor1 and Ccor2 were purified using theHis·Bind resin and dialyzed overnight at 4 °C against 3 l of potassiumphosphate buffer (25 mM, pH 7.5).

The cryoprotective activities of Ccor1 and Ccor2 were assayed asdescribed (Honjoh et al., 2000) with a few modifications. Briefly, thefreeze-labile enzyme lactate dehydrogenase (LDH, Fluka) was dilutedto 16.6 μg/ml (500 nM) in 10 mM sodium phosphate buffer (pH 7.5).Five hundred microliters of this solution was mixed with an equalvolume of cryoprotectant solution and frozen at −20 °C for 24 h. Thefrozen protein samples were thawed at 20 °C, and the LDH activitieswere measured as described (Honjoh et al., 2000) using a spectro-photometer (Shimadzu).

3. Results

3.1. Identification of two genes, Ccor1 and Ccor2, exclusively expressed inC. vulgaris NJ-7

In C. vulgaris NJ-7, genes possibly related to freezing tolerance,such as hiC6 and hiC12, show increased mRNA levels relative to thosein UTEX259 even at 20 °C (Li et al., 2009). Because the two strainspossess identical 18S rRNA and highly similar sequences of encodinggenes, it would be possible to employ Suppression SubtractiveHybridization (SSH) to identify genes whose expression levels aredifferent between them. The SSH was performed with pools of cDNAsof the two strains grown at 20 °C to search for genes showing higherexpression levels in NJ-7 than in UTEX259.

One of the identified cDNA fragments (706 bp, clone A524 in theSSH library) of NJ-7 showed similarity to Dehydrin 5, a group-2 LEAprotein from Hordeum spontaneum (Morrell et al., 2003). Northernblot analysis using the labeled cDNA fragment revealed a transcriptof ~700 nt that was exclusively expressed in NJ-7 and up-regulatedupon exposure to low temperature (Fig. 1A). The transcript wasaccumulated to its maximal level after 6 h of cold treatment at 4 °C,decreased at 24 h and returned to the background at 72 h. On thecontrary, no signal was detected in UTEX259 with or without coldinduction. Similar results were found in Northern blot hybridizationusing the homologous cDNA fragment of UTEX259 as the probe(Fig. 1A).

In the SSH library, another cDNA clone (no. A41) of 627 bp showedsimilarity to the cDNA fragment of no. A524. Both clones contained 3′ends of cDNA. 5′ RACE was performed to obtain the 5′-end sequences.Full length cDNA sequences were obtained by juxtaposing sequencesof the two portions of cDNA (Fig. S1). Genes corresponding to A524and A41 clones were named as Ccor1 and Ccor2 respectively. Due tosimilar sizes, their transcripts could not be resolved in the Northernblot analysis shown in Fig. 1A.

We also examined the expression of Ccor1 and Ccor2 in the twostrains at protein level by Western blot analysis using antiserumraised against Ccor2 of UTEX259 (Fig. 1B). Ccor1 and Ccor2, withapparent molecular weights of ~21 kDa and ~19 kDa respectively,both could be detected using the antiserum against the purifiedrecombinant Ccor2. The abundance of the two proteins was rapidlyincreased in NJ-7 within 6 h after exposure to 4 °C and reached themaximal level at 24 h. Consistent with Northern blot analysis, noCcor1 or Ccor2 was detectable in UTEX259. Ccor1 and Ccor2 wereexclusively expressed in NJ-7 and up-regulated in response to coldtreatment.

18S

A

B

UTEX259 20 4NJ-7 20 4

Ccor1

Ccor1 Ccor2

21 kDa19 kDa

0 2 6 24 72 0 2 6 24 72 h

UTEX259 20 4

0 2 6 24 72 h

NJ-7 20 4

0 2 6 24 72

I

II

Fig. 1. Expression of Ccor1 and Ccor2 in C. vulgaris strains NJ-7 and UTEX259 during cold induction at 4 °C. (A) Northern blot analysis. Twenty five micrograms of total RNAs wereloaded to each lane and a 706-bp fragment of NJ-7 Ccor1 cDNA (I) or a 258-bp fragment of UTEX259 Ccor1 cDNA (II) was used as the probe. 18S rRNAwas used as the internal control.(B) Western blot analysis. Fifty micrograms of total soluble proteins were loaded to each lane and the anti-Ccor2 (UTEX259) rabbit antiserum was used in the detection. Proteinconcentrations were determined by the Bradford's method and verified by SDS-PAGE. Soluble proteins extracted from E. coli producing NJ-7 Ccor1 (lane 1) or Ccor2 (lane 2) wereused as the positive control.

0 20 40 60 80 100 120 140 160 180

Dis

ord

er t

end

ency

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Ccor1

Ccor2

B

Residue position

-3

1

0.5

0

-0.5

-1

-1.5

-2

-2.5

A

Hyd

rop

ath

ic in

dex

Fig. 2. Hydropathy and disorder predictions of Ccor1 (dark line) and Ccor2 (gray line)from C. vulgaris NJ-7. (A) Hydropathy predictions with ProtParam. Regions withhydropathic indices below zero are considered to be hydrophilic. (B) Disorderpredictions with IUPred. Regions with scores above 0.5 are considered to be disordered.

54 X. Liu et al. / Gene 482 (2011) 51–58

3.2. Predicted similarities of Ccor1 and Ccor2 to LEA proteins

The full-length cDNA of Ccor1 was 717-bp long, with a 477-bpORF, a 75-bp 5′-UTR, a 165-bp 3′-UTR carrying poly (A) tail. Ccor1encodes a protein of 158 aa (Fig. S1A), with a predicted molecularmass of 16.9 kDa and pI of 5.31. A similarity search using SIB BLASTNetwork Service showed amino acid sequence homology betweenCcor1 and Dehydrin 5, a group-2 LEA protein from Hordeumspontaneum (Morrell et al., 2003). The highest similarity wasfound between residues 10 to 155 of Ccor1 and residues 184 to317 of Dehydrin 5 with 29% identities and 37% positives, but notypical domains of dehydrins or motifs of LEA groups wereidentified in Ccor1.

The full-length of cDNAof Ccor2was 686-bp long,with a 375-bpORF,a 74-bp 5′-UTR, a 237-bp 3′-UTR carrying poly (A) tail. Ccor2 encodes aprotein of 124 aa (Fig. S1B), with a predictedmolecularmass of 13.8 kDaandpI of 5.9. TheCcor2amino acid sequenceshared55.7% identitieswiththat of Ccor1.

The two proteins are predicted to show some common physico-chemical characteristics of LEA proteins. Ccor1 is rich in Gly (15.2%) andGlu (10.8%) residues and is devoid of Cys and Trp. Similarly, Ccor2 is alsorich in Gly (11.3%) and Glu (13.7%) residues and is devoid of Cys andTrp. Both proteins contained 77.4% hydrophilic amino acid residues (A,R, N, D, Q, E, G, H, K, P, S and T). As shown with their hydropathy plots(Kyte and Doolittle, 1982), the two proteins are highly hydrophilic(Fig. 2A). Secondary structure analysis using SOPMA indicates that65.82% of Ccor1 and 41.94% of Ccor2 are random coil structure.Consistently, Ccor1 and Ccor2 are predicted to be highly disorderedproteins by using IUPred (Dosztanyi et al., 2005) (Fig. 2B). Similardisorder prediction results were obtained by using PONDR (Romeroet al., 2004).

3.3. The genomic organization of Ccor1 and Ccor2 in NJ-7 and UTEX259

In order to obtain the chromosomal DNA sequences of Ccor1 andCcor2 from NJ-7 and UTEX259, cosmid libraries of the two C. vulgarisstrains were constructed (Wang and Xu, unpublished) and screened by

PCR using primers A524-1 (5′-CTCATCGCCTACACGCAGAC-3′) andA524-4 (5′-CACACGCTCCTTCACCACT-3′), which were designed basedon the identical regions of Ccor1 and Ccor2 cDNA sequences.

6.6

23.1

9.4

4.4

2.32.0

NJ-7

BamH I

UTEX259

Ahd IEcoR I+

EcoR V

Ahd I+

Spe I

kb

Fig. 4. Southern blot analyses of Ccor1/Ccor2 and homologous sequences in two strains ofC. vulgaris. High-molecular-weight DNA from NJ-7 was digested with BamHI or EcoRI+EcoRV, DNA from UTEX259 was digested with AhdI or AhdI+SpeI. A PCR-generated Ccor2cDNA fragment of UTEX259 was used as the probe. The restriction enzymes were chosenaccording to the gene cluster sequences of the two strains.

55X. Liu et al. / Gene 482 (2011) 51–58

One clone containing Ccor1 or Ccor2 as shownwith PCRwas isolatedfrom the NJ-7 cosmid library and mapped with restriction enzymes(data not shown). A 13723-bp region containing the target DNA wassequenced. Fig. 3 shows that five genes (denoted as NA, NB, NC, ND andNE), identical or similar to Ccor1 or Ccor2, are arrayed in tandem in thesame direction (Fig. 3). The structure of NA to NE region was confirmedby a series of PCR examinations using NJ-7 chromosomal DNA as thetemplate (data not shown). NB to NE forms the gene cluster Ccor1–Ccor2–Ccor1–Ccor2, and each Ccor1 or Ccor2 has five exons and fourintrons. NA is predicted to encode a 174-aa protein with 39.7% identityto Ccor1 and 43.7% identity to Ccor2. Unlike Ccor1 and Ccor2, however, ithas six exons and five introns. Typical splicing motifs are present at the5′ (GT) and 3′ (AG) ends of almost all the introns. The predictedproducts of NA to NE share a conserved region of about 50 aa (Fig. S2).However, only Ccor1 and Ccor2 (NB to NE) were detectable in Westernblot analysis (Fig. 1B).

Two overlapping clones were isolated from the UTEX259 cosmidlibrary and a 14209-bp region was sequenced. In addition to five genessimilar to NA to NE, which are denoted as UA to UE respectively, anoppositely oriented ORF named UF is located upstream of the genecluster (Fig. 3). The structure of this region was also confirmed by PCRexaminations using UTEX259 chromosomal DNA as the template (datanot shown). UA toUE shows very high similarity (92.0%–96.0% identity)to their counterparts in NJ-7 at the amino acid level. UF shows a lowersimilarity (35.9%–44.3% identity) to them at the amino acid level. Evenso, like UA to UE, UF also contains the 50-aa conserved region (Fig. S2).

To find out whether there were any more copies of Ccor1/Ccor2 orhomologue(s) in the two strains, Southern blot analyses wereperformed using Ccor2 cDNA of UTEX259 as the probe (Fig. 4). Aspredicted from the sequence ofCcor1/Ccor2 region, an11.5-kbbandwasfound inBamHI-cut genomicDNAofNJ-7. In addition, twoweakerbandsof ~9 kb and ~1.8 kb were found, indicating the presence of additionalCcor1 or Ccor2 or homologues in NJ-7. In AhdI-cut genomic DNA ofUTEX259, however, a singlebandof12.4 kbwas foundaspredicted fromthe sequence of the Ccor1/Ccor2 region, suggesting that no other copy ofCcor1 or Ccor2 was present in the genome.

Each copy of Ccor1 and Ccor2 in NJ-7 possesses a 133-bp highlyconserved sequence immediately upstream of the transcriptional startpoint (Fig. S3). Possibly, this stretch of sequence is a key portion of thepromoter. A search of the PLACE database (Higo et al., 1999; Prestridge,1991) revealed potential regulatory elements. There was no typicalTATAboxat−30site, but twoMYCrecognition sites (CANNTG,N=A/T/G/C) were found at −71 and −101 sites, which had been shown tomediate cold and dehydration transcriptional responses in Arabidopsisthaliana (Chinnusamy et al., 2003; Iwasaki et al., 1995). In addition, aGCC-box (GCCGCC) at −79 site was identified. Such a box had beenshown to function as ethylene-responsive element mediating theregulation of defense-related gene expression (Chakravarthy et al.,2003). UTEX259 possesses similar conserved regions and putativeregulatory sites in upstream regions of Ccor1 and Ccor2 genes.

0 1 2 3 4 5 6 7 8

BamHI Not I Not

EcoRI EcoRI Not I Not

NJ-7

UTEX259Ahd I

NA NB

UF UA UB

Fig. 3. The genomic regions containing the Ccor1–Ccor2–Ccor1–Ccor2 cluster in the two straUTEX259 are named as UA to UF. NB/UB and ND/UD are the two copies of Ccor1 (dark), NC/positions and directions of genes. Solid lines indicate the region already sequenced, dashed

3.4. Heat stability and cryoprotectant activities of Ccor1 and Ccor2

Because Ccor1 and Ccor2 were predicted to have physicochemicalcharacteristics of LEA proteins, and because most LEA proteins showheat stability (Hundertmark and Hincha, 2008), we tested the boiling-stability of Ccor1 and Ccor2. Total soluble proteins of E. coli cellsproducing Ccor1 or Ccor2were heated in boilingwater. Ccor1 and Ccor2remained soluble after boiling for 20 min, while the majority of E. coliproteins became aggregated and precipitated (Fig. 5).

It was noticeable that the apparent molecular masses of the twoproteins were higher than the predicted (Ccor1, 21 versus 17 kDa;Ccor2, 19 versus 14 kDa) (Fig. 5). The discrepancy could be due totheir high affinities to SDS, which is often found in LEA proteins withskewed amino acid compositions (Kosová et al., 2007, Battaglia et al.,2008).

9 10 11 12 13 14 15 16

I Not I Not I BamHI BamHIEcoRV

I EcoRI EcoRI

17 18kb

EcoRV

Ahd ISpe I

NC ND NE

UC UD UE

ins of C. vulgaris. Genes of this genomic region in NJ-7 are named as NA to NE, those inUC and NE/UE are two copies of Ccor2 (gray). Boxes with arrowheads stand for relativelines indicate the region not sequenced.

2A B

Ccor1 Ccor2

66

31

43

2014

97

kDa

1 3 4 5 6 7 21 3 4 5 6 7

Fig. 5. Heat-stability analyses of Ccor1 and Ccor2 expressed in E. coli. lane 1, total soluble proteins of E. coli cells induced by IPTG; lanes 2–4, the supernatant resulted fromcentrifugation of total soluble proteins after boiling for 5 min, 10 min or 20 min; lanes 5–7, the sediments resulted from the same experiment as lanes 2–4. (A) E. coli BL21 (DE3)(pET-Ccor1). (B) E. coli BL21 (DE3) (pET-Ccor2).

56 X. Liu et al. / Gene 482 (2011) 51–58

Wewondered if Ccor1 andCcor2were involved in cryoptrotection ofprotein activities in Chlorella. The cryoprotective activities wereevaluated with purified recombinant Ccor1 and Ccor2 proteins basedon their abilities to protect LDH from freeze-inactivation. Therecombinant proteins were both fused with a stretch of 6 histidineresidues at the 3′ ends, therefore, denoted as Ccor1-His and Ccor2-Hisrespectively. With no cryoprotectant, a freeze–thaw cycle resulted inloss of over 80% of LDH activity, while recombinant Ccor1 or Ccor2,similar to BSA,were able to fully protect LDH from freeze-inactivation atthe concentration of 40 μg/ml (Fig. 6A). BSA is often used as the positivecontrol in such assays (Ukaji et al., 2004; Nakayama et al., 2007). Withthe decrease of concentration, Ccor1, Ccor2 and BSA showed reducedcryoprotective activities in parallel (Fig. 6A). Synechocystis Rbp1 andsucrose, however, showed almost no cryoprotective activities. Thepurities and apparent molecular weights of proteins are shown inFig. 6B. Deduced from the experimental data, to maintain 50% LDHactivity in a freeze–thaw cycle, 11 μg/ml of purified Ccor1-His or 17 μg/ml of purified Ccor2-His or 18 μg/ml of BSA is needed.

4. Discussion

LEA proteins are often involved in adaptive responses to freezing,dehydration or other abiotic stresses (Tunnacliffe and Wise, 2007). Inthis study, two novel LEA proteins Ccor1 and Ccor2 were identifiedand characterized in NJ-7, the Antarctic strain of Chlorella vulgaris.Without typical motifs or conserved sequences, the two proteins werepredicted to have the common features of LEA proteins, such as highhydrophilicity and high degree of disordered structure. The physico-

A

Cryoprotectant concentration (

LD

H r

esid

ual

act

ivit

y (%

)

0

20

40

60

80

100

120

140

40 20

Fig. 6. Cryoprotection of LDH by recombinant Ccor1 and Ccor2. (A) LDH solutions were froz20 °C. The relative residual activity after a freeze–thaw treatment is expressed as the perceexperiments with standard deviations. (B) The SDS-PAGE analysis showing that the protein

chemical characteristics of Ccor1 and Ccor2 were experimentallyinvestigated. Both were stable in boiling water and showed significantcryoprotective activities over LDH, and the activities were comparableto that of other LEA proteins, such as PCA60 (Wisniewski et al., 1999),WAP18 (Ukaji et al., 2004) and Cor15am (Nakayama et al., 2007), butappeared to be lower than the reported activities of HiC6 and HiC12from C. vulgaris C-27 (Honjoh et al., 2000).

NJ-7 shows higher freezing tolerance than UTEX259, the temper-ate strain of the same green alga, and the freezing tolerance can befurther increased by cold hardening (Hu et al., 2008; Li et al., 2009).We hypothesized that the difference in freezing tolerance is due todifferences in contents of antifreeze or cryoprotective proteins in cells.Before our studies, LEA proteins had been found to be correlated to theanti-freeze capacities of some higher plants (Minami et al., 2005;Rorat et al., 2006; Nakayama et al., 2007; Peng et al., 2008). In thisstudy, we showed that the levels of Ccor1 and Ccor2were correlated tothe freeze tolerance in the two Chlorella strains. In NJ-7, Ccor1 andCcor2 were up-regulated at both mRNA and protein levels upon coldinduction. In UTEX259, however, their expression with or withoutcold induction was detectable by neither Northern blot nor Westernblot analysis, even though transcripts of Ccor1 and Ccor2 could bedetected using RT-PCR (data not shown). Ccor1 and Ccor2 of NJ-7, asnovel LEA proteins, probably play an important role in the enhancedfreezing tolerance. Previously, we reported that other two LEA proteingenes, hiC6 and hiC12, were expressed in NJ-7 at higher levels than inUTEX259 (Li et al., 2009). Compared to hiC6 and hiC12, Ccor1 andCcor2 showed much greater differences in expression between thetwo strains.

116.066.245.035.025.0

18.4

14.4

B

kDa

µg/ml)

10

Ccor1-HisCcor2-HisBSAsucroseRbp1No additive

en at −20 °C with additives Ccor1-His, Ccor2-His, BSA, sucrose or Rbp1 and thawed atntage of the activity of untreated LDH solution. Values are means of three independents of different molecular weights were used in equal amounts (μg/ml).

57X. Liu et al. / Gene 482 (2011) 51–58

In Antarctic notothenioid fish, the enhanced gene expressioninvolved in cold adaptation is correlated to gene duplications (Chen etal., 2008). We wondered if NJ-7 possessed more copies of Ccor1 andCcor2 than UTEX259. Sequence analyses of cosmid clones revealed thatin both strains, the two genes were located in the gene cluster Ccor1–Ccor2–Ccor1–Ccor2. The Southernblot analysis indicated the presence ofextra sequences homologous to Ccor1 or Ccor2 in NJ-7. However,according to the intensities of the extra hybridization bands, theincrease of their copy numbers was slight, if any, in NJ-7 compared tothat in UTEX259, thus, not sufficient to explain the greatly intensifiedexpression of Ccor1 and Ccor2. Instead, the difference in expressionbetween the two strains could be due to transcriptional regulation.

An analysis of the 5′ non-coding sequences showed that all copies ofthe two genes in the cluster have a conserved region with certainpotential regulatory sequences, suchas theMYC recognition site and theGCC box. In higher plants, these regulatory sites are involved in stress-responsive or defense-related gene expression. Most Ccor1 and Ccor2copies in the two strains showed no difference in these sites. The loss ofcold-induced expression of Ccor1 and Ccor2 in UTEX259may not be duetomutations in the promoters. An alternative possibility is, an activatorof the promoters might have been lost or silenced in UTEX259. It hasbeen reported that in Arabidopsis thaliana, a MYC-like transcriptionalactivator, ICE1, binds specifically to the MYC recognition sequences inthe promoter of CBF gene to trigger its expression in response to coldstress, which in turn activates many downstream genes that conferchilling and freezing tolerance to plants (Chinnusamy et al., 2003). Tomake clear why Ccor1 and Ccor2 are so differently expressed in the twostrains, it may be necessary to identify the regulator(s) that bind(s) thepromoters, and compare the expression of the regulator(s) in the twostrains.

So far, four LEA proteins have been identified in different strains of C.vulgaris, namely HIC6, HIC12, Ccor1 and Ccor2. HIC6 and HIC12 werefirst identified to be hardening (cold treatment)-induced proteins in C.vulgaris C-27 by 2D-HPLC and SDS-PAGE (Honjoh et al., 1995), andHIC6was later localized to mitochondria in transgenic plant (Honjoh et al.,2001). At mRNA level, our previous studies showed that hiC6 and hiC12were cold-inducible in both strains and expressed at significantly higherlevels in strain NJ-7 than in UTEX259 (Li et al., 2009). At bothmRNA andprotein levels,wenowshowthatCcor1 andCcor2 are cold-inducible andalmost exclusively expressed in NJ-7. Different LEA proteins could beinvolved in protecting different cellular compartments from freezingdamages. Cellular localization information of different LEA proteins in C.vulgaris would be very useful to understanding the formation of itsfreezing tolerance.

Acknowledgments

This research was supported by the Key Projects KSCX2-YW-G-060and KSCX2-SW-332 of Knowledge Innovation Program of ChineseAcademy of Sciences.

Appendix A. Supplementary data

Supplementary data to this article can be found online at doi:10.1016/j.gene.2011.05.006.

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