Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina

ARTICLE IN PRESS Journal of Plant Physiology 164 (2007) 214—220

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SHORT COMMUNICATION

Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina Qinghua Hea, Dairong Qiaoa, Linhan Baia, Qinglian Zhangb, Wanggui Yanga, Qian Lia, Yi Caoa, a

Key Laboratory of Bio-resources and Eco-environment (Sichuan University), Ministry of Education, Chengdu, Sichuan Province 610064, PR China b Department of lab medicine, Chengdu Medical College, Chengdu, Sichuan Province 610083, PR China Received 27 November 2005; accepted 10 April 2006

KEYWORDS Dunaliella salina; Glycerol; Glycerol 3-phosphatase; Glycerol 3-phosphate dehydrogenase; Osmotic stress

Summary A cDNA encoding a nicotinamide adenine dinucleotide (NAD+)-dependent glycerol 3-phosphate dehydrogenase (GPDH) has been cloned by rapid amplification of cDNA ends from Dunaliella salina. The cDNA is 3032 base pairs long with an open reading frame encoding a polypeptide of 701 amino acids. The polypeptide shows high homology with published NAD+-dependent GPDHs and has at its N-terminal a chloroplast targeting sequence. RNA gel blot analysis was performed to study GPDH gene expression under different conditions, and changes of the glycerol content were monitored. The results indicate that the cDNA may encode an osmoregulated isoform primarily involved in glycerol synthesis. The 701-amino-acid polypeptide is about 300 amino acids longer than previously reported plant NAD+-dependent GPDHs. This 300-amino-acid fragment has a phosphoserine phosphatase domain. We suggest that the phosphoserine phosphatase domain functions as glycerol 3-phosphatase and that, consequently, NAD+-dependent GPDH from D. salina can catalyze the step from dihydroxyacetone phosphate to glycerol directly. This is unique and a possible explanation for the fast glycerol synthesis found in D. salina. & 2006 Elsevier GmbH. All rights reserved.

Abbreviations: bp, base pair; cDNA, DNA complementary to RNA; DHAP, dihydroxyacetone phosphate; EST, expression sequence tag; GPDH, glycerol 3-phosphate dehydrogenase; NAD, nicotinamide-adenine dinucleotide; ORF, open reading frame; RACE, rapid amplification of cDNA ends Corresponding author. Tel.: +86 028 85412842; fax: +86 028 85405541. E-mail address: [email protected] (Y. Cao).

Introduction The enzyme that catalyzes the reversible conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate, DHAP reductase, also known as glycerol 3-phosphate dehydrogenase (GPDH)

0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.04.004

ARTICLE IN PRESS Cloning and characterization of Dunaliella salina GPDH (EC 1.1.1.8), is found in many eubacteria and eukaryotes. Nicotinamide-adenine dinucleotide (NAD+)-dependent GPDH is an important enzyme in glycerol metabolism. In algae Dunaliella, the reversible conversions between glycerol and DHAP are metabolized by two distinct reactions. For glycerol synthesis, DHAP is converted to glycerol 3-phosphate catalyzed by NAD+-dependent GPDH, and then glycerol 3-phosphate is converted to glycerol catalyzed by glycerol 3-phosphatase. For glycerol dissimilation, glycerol is converted to dihydroxyacetone catalyzed by glycerol dehydrogenase, and then dihydroxyacetone is converted to DHAP catalyzed by dihydroxyacetone kinase (Ben-Amotz and Avron, 1981; Haus and Wegmann, 1984; Sussman and Avron, 1981). The roles of NAD+-dependent GPDH isoenzymes in yeast, Saccharomyces cerevisiae, have been detailed (Albertyn et al., 1994; Ansell et al., 1997). It is known that heterologous expression of GPDH genes in yeast can increase glycerol production (Watanabe et al., 2004). Therefore, the NAD+-dependent GPDH genes were considered as the key genes of glycerol synthesis. In higher plants and algae, GPDH is referred to as DHAP reductase, because at physiological pH and substrate, the enzyme is essentially inactive as a dehydrogenase (Gee et al., 1988a, b). Dunaliella salina, a photosynthetic organism, devoid of a rigid cell wall, has the remarkable characteristic of surviving large osmotic stresses, by adjusting intracellular levels of glycerol to concentrations balancing the external osmotic pressure. Therefore, the cells maintain a constant volume independent of the external salinity (Avron, 1986; Sadka et al., 1991). Based on the previous studies, we hypothesize that the mechanism of glycerol synthesis in D. salina is similar to S. cerevisiae, in that different GPDH isoforms plays different roles (Ansell et al., 1997). Although three isoforms of DHAP reductases in Dunaliella tertiolecta have been separated by a diethylaminoethyl cellulose column and characterized individually (Gee et al., 1989, 1993; Ghoshal et al., 2002), there are no correspondent genes or expression information about these isoforms. In order to understand the glycerol synthesis mechanism in Dunaliella, it is essential to clone and characterize these GPDH genes.

Materials and methods Algae and growth condition D. salina strain 435 was obtained from Institute of Hydrobiology, Chinese Academy of Sciences. The

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algae grew in a controlled-environment chamber with 16 h lighting at 25 1C and 8 h darkness at 15 1C. The composition of the growth medium was 1.5 M NaCl, 5.0 mM NaNO3, 5.0 mM MgSO4  7H2O, 0.1 mM NaH2PO4  2H2O, 1.0 mM KCl, 10.0 mM NaHCO3, 0.3 mM CaCl2  2H2O and a mixture of micronutrients (Pick et al., 1986). Cells in exponential phase were used for treatment. Oxidative stress treatment was performed by adding 5 mL H2O2 to 50 mL cell culture (0.2 mM H2O2 in medium). Osmotic stress treatment was performed by adding 5.85 g NaCl to 50 mL cell culture (3.5 M NaCl in medium).

Isolation of GPDH expression sequence tag (EST) from D. salina The GPDH EST was isolated by random sequencing of clones from a cDNA library prepared from D. salina cells grown in 1.5 M NaCl. The cDNA library was constructed as part of D. salina EST sequencing program that was devoted to comprehensive characterization of gene expression when external NaCl concentration changed. The PCR primers used for EST amplification were: primer E1 (5-CGTATCCTGGGCATCGACTG-3) and primer E2 (5-GAAGAAGGTGTCATCGCGCA-3).

Rapid amplification of cDNA ends (RACE) 30 RACE was performed using the 30 RACE kit (3 -Full RACE Core Set) (TaKaRa). 50 RACE was performed using the 50 RACE kit (SMART RACE cDNA amplification kit) (Clontech). Total RNA was extracted using trizol reagent (Invitrogen) and was used for cDNA synthesis. For 30 RACE, first-strand synthesis was carried out in the presence of oligo-dT primers (TaKaRa). PCR amplification of the 30 end of D. salina GPDH cDNA was carried out with the following primers: genespecific forward primer F1 (5-AGTTCATCTCCCCCTCAGTGCGCGA-3), gene-specific forward nested primer F2 (5-AGGCCTGGGCCCAGAAGAGGATCG-3) and 3sites Adaptor primer (TaKaRa). The PCR product with F1 primer and 3sites Adaptor primer was diluted at the ratio of 1:100 and re-amplified with F2 primer and 3sites Adaptor primer. For 50 RACE, first-strand synthesis was carried out in the presence of 50 CDS primer (Clontech) and the SMART II primer (Clontech). PCR amplification of the 50 end of D. salina GPDH cDNA was carried out with the following primers: gene-specific reverse primer R1 (5-CGCCCGGCACATCCGCAAGCAG-3), gene-specific reverse nested primer R2 (5-TGCCCAGGATACGGGACACCAT-3) and universal primer UPM (Clontech). The PCR product with R1 primer 0

ARTICLE IN PRESS 216 and UPM primer was diluted at the ratio of 1:100, and re-amplified with R2 primer and UPM primer. Because only a fragment of about 1 kb was amplified in the first round of 50 RACE, two additional gene-specific reverse primers (R3 and R4) were designed based on the 1-kb fragment to amplify the rest 50 end of D. salina GPDH cDNA. The PCR product with R3 (5-TCGTTGCATGACGCCACCGAAG-3) primer and UPM primer was diluted at the ratio of 1:100, and re-amplified with R4 (5-TCTTGAGGTGGGAAGCAATGGG-3) primer and UPM primer. The PCR products were purified by gel extraction, cloned into Escherichia coli (JM109) using TA-clone kit (pMD 18-T Vector) (TaKaRa) and sequenced. Primers W1 (5-GGATGCTTCTCCAGAAAGGAAAC-3) and W2 (5-CGCATTTTATCGCACGTTAGTCTC-3) were designed based on the sequences of the 50 RACE fragment and 30 RACE fragment to get the fulllength cDNA. The full-length cDNA was named DsGPDH2. Sequencing was performed at Sangon (Shanghai, China). All the clones were sequenced on both strands using M13 primers. Sequence data from this article have been deposited at NCBI under accession number AY845323.

Preparation of RNA, gel electrophoresis, RNA gel blotting and hybridization Total RNA was extracted using trizol reagent (Invitrogen) and was separated on a MOPS-formaldehyde agarose gel according to Sambrook et al. (1989). Total RNA was transferred onto nylon membranes (Roche) via vacuum blotting and fixed by UV-crossing at 0.5 J for 3 min. Hybridization was performed with a 500 base pairs (bp) cDNA fragment obtained by RT-PCR from D. salina as a probe. The probe was randomly primed and labeled with 32p-dCTP according to TaKaRa Random Primer DNA Labeling Kit. Pre-hybridization lasted for 10 h at 42 1C, then the single-stranded radioactive probe was added and incubation lasted for 16 h at 42 1C. Membranes were washed with 1  SSC and 0.1  SDS at 55 1C. The blots were exposed to X-ray film in a cassette equipped with an intensifying screen at 80 1C for an appropriate time (1–24 h).

Measurement of glycerol content Sample cell suspensions of 1 mL were analyzed for total (intra- plus extracellular) glycerol content. The cell suspensions were heated in boiling water for 5 min, placed in ice water for 2 min and centrifuged at 10,000g for 5 min, and the supernatants were collected and used for detection. The

Q. He et al. glycerol contents of the supernatants were measured using a commercial glycerol analysis kit (Roche). Reproducibility was confirmed by independent duplicate experiments. At the same time, sample cell suspensions of 40 mL were used to calculate the gram yield of wet cells. The cell suspensions were centrifuged at 1000g for 10 min, most of the supernatant was removed carefully, leaving approximate 1 mL liquids for suspension. The remaining cell suspensions were transferred to a 1.5 mL Eppendorf tube, centrifuged at 6000g, all liquids were removed as much as possible and the cell pellet was used to calculate the gram yield of wet cells (total gram yield). Then 1 mL cell culture contains 1/40 fold total gram yield of wet cells.

Results Cloning of DsGPDH2 and sequence analysis The DsGPDH2 EST is 682 bp long fragment. This EST is highly homological to those of previously reported NAD+-dependent GPDHs (not shown) by BLASTX. Based on the EST sequence, 30 and 50 RACE were performed to clone the 30 and 50 regions of DsGPDH2 cDNA. A fragment of 1.1 kb was amplified by 30 RACE, and two fragments of 1 kp (first round of 50 RACE) and 737 bp (second round of 50 RACE) were amplified after two rounds of 50 RACE. These fragments were assembled and confirmed by PCR using primers W1 and W2. The full-length cDNA with 30 and 50 untranslated regions is 3032 bp long. The cDNA has an open reading frame (ORF) encoding a polypeptide of 701 amino acids. The amino acid sequence alignment (Fig. 1) of DsGPDH2 with some published GPDHs displays high homology. According to the plant organellar targeting sequence prediction program (chlorop v1.1) (see http://aramemnon.botanik.uni-koeln.de), the DsGPDH2 protein has at its N-terminal an apparent chloroplast targeting sequence (Fig. 2). The 701-amino-acid polypeptide is about 300 amino acids (characterized in bold in Fig. 2) longer than previously reported NAD+-dependent GPDHs. This 300-aminoacid fragment shows sequence homology with published phosphoserine phosphatases (Fig. 3). Conserved domain search at NCBI shows that this 300-amino-acid fragment includes a complete serB (phosphoserine phosphatase) domain (Fig. 4).

Expression studies of DsGPDH2 Total RNA preparations from D. salina cells grown at different conditions were used for RNA gel blot

ARTICLE IN PRESS Cloning and characterization of Dunaliella salina GPDH

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290 300 310 320 330 340 350 360 376 (278) 278 Escherichia coli NAD (1) -------------------------------------------------MNQRNASMTVIGAGSYGTALAITLARNGHEVVLWGHDP-----------Arabidopsis (1) MRFRSFFFSSSIFSLSHSRSPSLSSSRFSSLSAAMSPALEKSRQGNGGCNDDSKSKVTVVGSGNWGSVAAKLIASNALKLPSFHDEVR----MWVFEEV Drosophila virilis (1) --------------------------------------------------MAEKVNVCIVGSGNWGSAIAKIVGANAAALPEFEKRVT----MFVYEEM Gpd1p [Saccharomyces cerevisiae]. (1) --------------------MSAAADRLNLTSGHLNAGRKRSSSSVSLKAAEKPFKVTVIGSGNWGTTIAKVVAENCKGYPEVFAPIV----QMWVFEE Homo sapiens (1) -------------------------------------------------MAAAPLKVCIVGSGNWGSAVAKIIGNNVKKLQKFASTVK----MWVFEET DsGPDH2 (278) MVGDGFSDLEAMQGSPDGADAFICFGGVMQRPAVASQADWFVRSYDELMAKLKRYKVTMVGSGAWACTAVRMVAQSTAEAAQLPGSVFEKEVTMWVHEE Consensus (278) A A KK KVTVVGSGNWGSAIAKIVA NA LP F V MWVFEE 390 400 410 420 430 440 450 460 475 (377) 377 Escherichia coli NAD (39) -----EHIATLERDRCNAAFLPDVPFPDTLHLESDLATALAASRNILVVVPSHVFGEVLRQIKPL--MRPDARLVWATKGLEAETGR-LLQDVAREALG Arabidopsis (96) LPNGEKLNDVINKTNENVKYLPGIKLGRNVVADPDLENAVKDANMLVFVTPHQFMDGICKKLDGK--ITGDVEAISLVKGMEVKKEG-PCMISSLISKQ Drosophila virilis (46) I-DGKKLTEIINETHENVKYLKGHKLPTNVVAVPDLVEAAKNADILIFVVPHQFIPNFCKQLLGK--IKPNAIAISLIKGFDKAEGGGIDLISHIITRH Gpd1p [Saccharomyces cerevisiae]. (76) EINGEKLTEIINTRHQNVKYLPGITLPDNLVANPDLIDSVKDVDIIVFNIPHQFLPRICSQLKGH--VDSHVRAISCLKGFEVGAKG-VQLLSSYITEE Homo sapiens (47) V-NGRKLTDIINNDHENVKYLPGHKLPENVVAMSNLSEAVQDADLLVFVIPHQFIHRICDEITGR--VPKKALGITLIKGIDEGPEG-LKLISDIIREK DsGPDH2 (377) KHSGRNLIEYINENHENPIYLPGIDLGENVKATSDLIEAVRGADALIFCAPHQFMHGICKQLAAARVVGRGVKAISLTKGMRVRAEG-PQLISQMVSRI VRAISLIKGMEVG EG L LISSIISR Consensus (377) I NGRKLTEIIN HENVKYLPGIKLPDNVVA SDLIEAVKDADILVFVIPHQFI ICKQL GK V 490 500 510 520 530 540 550 560 574 (476) 476 Escherichia coli NAD (130) DQIPLAVISGPTFAKELAAGLPTAISLASTDQTF--------ADDLQQLLHCGKSFRVYSNPDFIGVQLGGAVKNVIAIGAGMSDGIGFGANARTALIT Arabidopsis (192) LGINCCVLMGANIANEIAVEKFSEATVGYRGSRE--------IADTWVQLFSTPYFMVTPVHDVEGVELCGTLKNVVAIAAGFVDGLEMGNNTKAAIMR Drosophila virilis (142) LKIPCAVLMGANLANEVAEGNFCETTIGCTDKK---------YGKVLRDLFQANHFRVVVVEDADAVEVCGALKNIVACGAGFVDGLKLGDNTKAAVIR Gpd1p [Saccharomyces cerevisiae]. (172) LGIQCGALSGANIATEVAQEHWSETTVAYHIPKDFRGEGKDVDHKVLKALFHRPYFHVSVIEDVAGISICGALKNVVALGCGFVEGLGWGNNASAAIQR Homo sapiens (142) MGIDISVLMGANIANEVAAEKFCETTIGSKVMEN---------GLLFKELLQTPNFRITVVDDADTVELCGALKNIVAVGAGFCDGLRCGDNTKAAVIR DsGPDH2 (475) LGIDCSVLMGANIAGDIAKEELSEAVIAYANRES---------GSLWQQLFQRPYFAINLLADVPGAEMCGTLKNIVAVGAGIGDGLGVGPNSKASILR K G LWK LFQ PYFRVTVVEDVDGVELCGALKNIVAIGAGFVDGLGLG NTKAAIIR Consensus (476) LGI CAVLMGANIANEVA E FSETTIAY 590 600 610 620 630 640 650 660 673 (575) 575 580 Escherichia coli NAD (221) RGLAEMSRLGAALG--ADPATFM-GMAGLGDLVLTCTDNQSR--NRRFGMMLG-----QGMDVQSAQEKIGQVVEGYRNTKEVRELAHRFGVEMPITEE Arabidopsis (283) IGLREMKALSKLLFPSVKDSTFF-ESCGVADVITTCLGGRNRRVAEAFAKSRGK----RSFDELEAEMLQGQKLQGVSTAREVYEVLKHCGWLEMFPLF Drosophila virilis (232) LGLMEMIRFVDVFYPGSKLSTFF-ESCGVADLITTCYGGRNRRVSEAFVTSGK------TIEDLEKEMLNGQKLQGPPTAEEVNYMLKNKGLEDKFPLF Gpd1p [Saccharomyces cerevisiae]. (271) VGLGEIIRFGQMFFPESREETYYQESAGVADLITTCAGGRNVKVARLMATSGK------DAWECEKELLNGQSAQGLITCKEVHEWLETCGSVEDFPLF Homo sapiens (232) LGLMEMIAFARIFCKGQVSTATFLESCGVADLITTCYGGRNRRVAEAFARTGK------TIEELEKEMLNGQKLQGPQTSAEVYRILKQKGLLDKFPLF DsGPDH2 (565) QGLSEMRKFCKFISPSVRDDTFF-ESCGVADLIASSYGGRNRRVAEAWAQKRIAGDDQVTFEKLEKEMLNGQKLQGVLTSDEVQEILHARGWELEFPLF TIEELEKEMLNGQKLQGV TAKEVYEILK KGLED FPLF Consensus (575) LGL EMIRFGKIFFP SKDSTFF ESCGVADLITTCYGGRNRRVAEAFA SGK 620 630 640 650 660 670 680 690 700 712 (614) 614 Escherichia coli NAD (257) QSR--NRRFGMMLG-----QGMDVQSAQEKIGQVVEGYRNTKEVRELAHRFGVEMPITEEIYQVLYCGKNAREAALTLLGRARKDERSSH--------Arabidopsis (321) RNRRVAEAFAKSRGK----RSFDELEAEMLQGQKLQGVSTAREVYEVLKHCGWLEMFPLFSTVHQICTGRLQPEAIVQYRENKL--------------Drosophila virilis (270) RNRRVSEAFVTSGK------TIEDLEKEMLNGQKLQGPPTAEEVNYMLKNKGLEDKFPLFTAIHKICTNQLKPKDLIDCIRNHPEHMQTL--------Gpd1p [Saccharomyces cerevisiae]. (310) RNVKVARLMATSGK------DAWECEKELLNGQSAQGLITCKEVHEWLETCGSVEDFPLFEAVYQIVYNNYPMKNLPDMIEELDLHED----------Homo sapiens (271) RNRRVAEAFARTGK------TIEELEKEMLNGQKLQGPQTSAEVYRILKQKGLLDKFPLFTAVYQICYESRPVQEMLSCLQSHPEHT-----------DsGPDH2 (603) RNRRVAEAWAQKRIAGDDQVTFEKLEKEMLNGQKLQGVLTSDEVQEILHARGWELEFPLFTTINRIIHGEVPPTMILRYRVACSMPSMPPARRVVNDYY TIEELEKEMLNGQKLQGV TAKEVYEILK KGLED FPLFTAVHQICY NLPP LL I A EH Consensus (614) RNRRVAEAFA SGK

Figure 1. Comparison of deduced amino acid sequence of DsGPDH2 with GPDHs from Arabidopsis (NP_198877), Drosophila virilis (CAA41800), Escherichia coli (AAN82866), Saccharomyces cerevisiae (GPD1) (NP_010262) and Homo sapiens (AAH28726). Gaps introduced to maximize sequence homology are indicated by dashes. Amino acid residues identical in all six sequences are highlighted. The putative NADH-binding domain is indicated by star.

1 51 101 151 201 251 301 351 401 451 501 551 601 651 701

MLLQKGNIGK ERGSPALLKR EQVLDLWQQA EINLTKAFED VEVFLISGGF MTRAAESHFK CFGGVMQRPA AQSTAEAAQL DLGENVKATS ISLTKGMRVR IAYANRESGS GLGVGPNSKA GGRNRRVAEA HARGWELEFP Y

GIAQPVQRRG QRALDVVLRA DAVCFDVDRT RLAKLNFTPT REMALPIASH SRAIERIRRK VASQADWFVR PGSVFEKEVT DLIEAVRGAD AEGPQLISQM LWQQLFQRPY SILRQGLSEM WAQKRIAGDD LFTTINRIIH

VPSALRHAPL AETEQEAENA VTTDASVGLL DIDRFLEEHP LKIPAKNVFC YPYNNIIMVG SYDELMAKLK MWVHEEKHSG ALIFCAPHQF VSRILGIDCS FAINLLADVP RKFCKFISPS QVTFEKLEKE GEVPPTMILR

ANKVATPAVA GTVVPGDGWE AKFMGIEDEA AHTRLVPGVE NTMSWQLDDH DGFSDLEAMQ RYKVTMVGSG RNLIEYINEN MHGICKQLAA VLMGANIAGD GAEMCGTLKN VRDDTFFESC MLNGQKLQGV YRVACSMPSM

PQGLLRPILS SFPPPPYEPS QSLTEQANRG NLIAALKARG GEPVRLQGLD GSPDGADAFI AWACTAVRMV HENPIYLPGI ARVVGRGVKA IAKEELSEAV IVAVGAGIGD GVADLIASSY LTSDEVQEIL PPARRVVNDY

Figure 2. Deduced amino acid sequence of DsGPDH2. Amino acid residues predicted to be a chloroplast targeting signal are underlined. The extra segments compared to previously reported plant GPDHs are in bold.

analysis. It was found that the expression of DsGPDH2 could be significantly induced when treated with high concentration NaCl (3.5 M NaCl) (Fig. 5).

Glycerol production analysis Osmotic stress significantly induced glycerol synthesis in D. salina cells (Fig. 6). When D. salina cells were treated with 3.5 M NaCl, the glycerol production increased constantly and reached 2.5 folds compared with the control (0 h) after 4 h.

Discussion The DsGPDH2 has an ORF encoding a polypeptide of 701 amino acids. The length of this ORF is significantly long compared to previously reported NAD+-binding GPDHs. It is about 300 amino acids longer than Arabidopsis thaliana NAD+-binding GPDH (Fig. 2). Conserved domain analysis shows that this 300-amino-acid fragment has a phosphoserine phosphatase domain (Fig. 4). Gee et al.

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DsGPDH2 Caenorhabditis elegans Drosophila melanogaster Homo sapiens Arabidopsis thaliana Consensus

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MLLQKGNIGKGIAQPVQRRGVPSALRHAPLANKVATPAVAPQGLLRPILSERGSPALLKRQRALDVVLRAAETEQEAENAGTVVPGDGWESFPPPPYEPSEQVLDL ------------------------------------------------------MQQHQQQYYLFLATLIMIRVALPTTASAIPRSISTSPGETISKNHEEEVKRV -------------------------------------------------MSGSVLSLARPAAATNGHNLLAKQLNCNGNGTTGGAAKTTVASAITPPKQPQLAAKV ------------------------------------------------------------------------------------------------MVSHSELRKL --------------------------MEALTTSRVVPVQVPCRKLSSLFANFSCLELRRYPCRGLVSIMNHPKLLRPVTASVQPHELSTLGHEGNIVPS-KEILDL L L R V L L AS ST EV KL

(107) DsGPDH2 (107) Caenorhabditis elegans (53) Drosophila melanogaster (58) Homo sapiens (11) Arabidopsis thaliana (80) Consensus (107)

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WQQADAVCFDVDRTVTTDASVGLLAKFMGIEDEAQSLTEQANRGEINLTKAFEDRLAKLNFTPTDIDRFLEEHPAHTRLVPGVENLIAALKARGVEVFLISGGFRE WRKADAVCFDVDSTVCQDEGIDELAAYLGVGEAVANVTRTAMNGNARFRDALAARLQVMKPNHEQLEQFVNISKPK--LTVGIRELVSRLHARGTHVYLVSGGFRR IQQSQIVCFDVDSTVICEEGIDELAEYCGKGSEVARVTKEAMGGAMTFQDALKIRLNIIRPTQQQVRDFIQERPST--LSKNVKRFVSHLKAEGKQVYLISGGFDC FYSADAVCFDVDSTVIREEGIDELAKICGVEDAVSEMTRRAMGGAVPFKAALTERLALIQPSREQVQRLIAEQPPH--LTPGIRELVSRLQERNVQVFLISGGFRS WRSVEAVCFDVDSTVCVDEGIDELAEFCGAGKAVAEWTARAMGGSVPFEEALAARLSLFKPSLSKVEEYLDKRPPR--LSPGIEELVKKLRANNIDVYLISGGFRQ W ADAVCFDVDSTV DEGIDELA FCGVGDAVA VTR AMGGAV F DAL RLALIKPS QVE FI E PP LSPGIRELVSRLKARGV VYLISGGFR

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MALPIASHLKIPAKNVFCNTMSWQLDDHGEPVRLQGLDMTRAAES-----------HFKSRAIERIRRKYPYNNIIMVGDGFSDLEAMQGSPDGADAFICFGGVMQ LILPVAELLGIEKSRIYANEILFDKFGKYHGFDTSELTSDSGSKELKSSITNLFGPTGKPAVIALLKKMYNYKTVVMVGDGATDVEASPP----ADAFIGFGGNVI LIAPVANELGIPLKNVYANKMLFDYLGEYDSFDINQPTSRSGGK--------------AEAIALIRKENNDDSLITMIGDGATDLEAVPP----ANYFIGFGGNVV IVEHVASKLNIPATNVFANRLKFYFNGEYAGFDETQPTAESGGK---------------GKVIKLLKEKFHFKKIIMIGDGATDMEACPP----ADAFIGFGGNVI MINPVASILGIPRENIFANNLLFGNSGEFLGFDENEPTSRSGGK--------------AKAVQQIRKGR-LYKTMAMIGDGATDLEARKPG--GADLFICYAGVQL LI PVAS LGIP NVFAN LLF GEY GFD N PTS SGGK AVI ILK KY YK IIMIGDGATDLEA PP ADAFIGFGGNVI

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RPAVASQADWFVRSYDELMAKLKRYKVTMVGSGAWACTAVRMVAQSTAEAAQLPGSVFEKEVTMWVHEEKHSGRNLIEYINENHENPIYLPGIDLGENVKATSDLI REGVKARAKWYVTDFDVLRKDLDHDESDIDDE-------------------------------------------------------------------------RPEVYRRAQYYVTDFEQLMGQ------------------------------------------------------------------------------------RQQVKDNAKWYITDFVELLGELEE---------------------------------------------------------------------------------REAVAANADWLIFKFESLINSLD----------------------------------------------------------------------------------R AV ANA WYVTDFD LMG LD

Figure 3. Comparison of deduced amino acid sequence of DsGPDH2 with phosphoserine phosphatases from Arabidopsis thaliana (AAF98410), Drosophila melanogaster (AAF14696), Caenorhabditis elegans (NP_502581) and Homo sapiens (AAH63614). Gaps introduced to maximize sequence homology are indicated by dashes. Amino acid residues identical in all five sequences are highlighted. 1

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Figure 4. Conserved domain analysis. The figure comes directly form NCBI conserved domain search. The deduced 701-amino-acid sequence is used to search. Hydrolase (haloacid dehalogenase-like hydrolase), SerB (phosphoserine phosphatase), NAD_Gly3P_dh (NAD-dependent GPDH) and GpsA (GPDH) are complete domains. COG4359, Gph and COG4030 are incomplete domains. It is interesting that the Hydrolase family (pfam00702) includes phosphatases.

(1993) suggested that Dunaliella DHAP reductase (NAD+-dependent GPDH) may be in a complex with or tightly associated with the glycerol 3-phosphatase. Their reasons are: (1) they found that the Km of glycerol 3-phosphatase is too high compared with the free glycerol 3-phosphate pool in this algae, it is difficult to understand how substantially large rates of glycerol synthesis occur if DHAP reductase and glycerol 3-phosphatase exist independently of each other; (2) the chloroplastic osmoregulator DHAP reductase and glycerol 3-phosphatase are both stimulated by NaCl and have a pH optimum around 7.0. Based on the

Figure 5. RNA gel blot analysis of osmotic stress inducibility of DsGPDH2. D. salina cells grew in growth medium (1.5 M NaCl) to exponential phase and then was treated with high NaCl medium (3.5 M NaCl). Total RNA was extracted from cells 0, 1, 2 and 4 h (lanes from the left) after transferred to high NaCl medium. Ethidium bromide-stained RNA is shown as a loading control.

sequence information, we suggest that DsGPDH2 encoding one protein that has two function domains that can catalyze the step from DHAP to glycerol directly. In other words, the DsGPDH2 phosphoserine phosphatase domain functions actually as glycerol 3-phosphatase, which provides a possible explanation for the fast glycerol synthesis in D. salina. The chimeric structure of DsGPDH2

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Glycerol (g/g wet cell)

Cloning and characterization of Dunaliella salina GPDH Salt Stress Treatment

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Figure 6. Kinetics of glycerol production in D. salina cells after high NaCl treatment. These treatment conditions were the same as RNA gel blot experiments. The amount of algae (1 mL cell suspension) used for glycerol analysis is 0.75 mg (wet cell).

seems to be an adaptation of D. salina to high osmotic environment. The DsGPDH2 structure enables fast glycerol synthesis under high osmotic stress. DsGPDH2 may be a new (young) gene (Long et al., 2003). It seems that a part of the phosphoserine phosphatase gene was transferred to the 50 position of NAD+-dependent GPDH gene. Whether DsGPDH2 can catalyze the step from DHAP to glycerol directly and the origin of DsGPDH2 is currently under study. Osmotic stress significantly induced the DsGPDH2 expression. When confronting osmotic stress, D. salina cells need rapid synthesis of glycerol to balance osmotic difference across plasma membrane (Ben-Amotz and Grunwald, 1973). However, we found that oxidative stress decreased significantly the mRNA level of DsGPDH2 and induced glycerol synthesis (not shown). Glycerol is actually required to protect cells from oxidative damage (Eastmond, 2004). It is possible that another GPDH isoform functions as glycerol synthesis under oxidative stress. Studies in yeast support this hypothesis. GPD1 plays a role in osmoadaptation; GPD2 plays a role in Redox regulation and is controlled by another signaling pathway (Ansell et al., 1997). Gee et al. (1988c, 1989, 1993) and Ghoshal et al. (2002) reported that there were three DHAP isoforms based on the elution profile, localization and characteristics of different isoforms from the leaves of higher plants and from D. tertiolecta. They concluded that (1) the chloroplast osmoregulatory form is the isoform from Dunaliella chloroplast that plays a role in osmoregulation, is stimulated by salts; (2) the chloroplast glyceride form is the isoform from Dunaliella chloroplast during active growth that plays a role in glyceride synthesis, is inhibited by salts; (3) the cytosolic glyceride form is the third minor isoform from the

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cytosol of Dunaliella that also plays a role in glyceride synthesis. Based on the expression profile of DsGPDH2, characteristics of glycerol synthesis and analysis of cDNA sequence, we suggest that DsGPDH2 encodes an osmoregulatory isoform that is primarily involved in glycerol synthesis. The reasons are: (1) osmotic stress can significantly induce the expression of DsGPDH2 and synthesis of glycerol; (2) DsGPDH2 has a phosphatase domain, which is useless to GPDH glyceride isoform. We suggest that there should be at least two isoforms (encoded by different genes) functioning in glycerol synthesis, because under oxidative condition, the DsGPDH2 expression decreased and glycerol production still increased (not shown). We have already cloned another GPDH EST from D. salina, named DsGPDH1, which has the same gene structure of DsGPDH2. We suggest that DsGPDH1 also functions in glycerol synthesis.

Acknowledgments We thank R. Hong for the technical support on RNA gel blots, Y. Gu and S. Liu for critical reading of the manuscripts. Funds were provided by 863 National High Technology Project (2001AA212161) and National Science Funds Committee (NSFC) (30270711).

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