Mol Genet Genomics (2008) 279:291–301

DOI 10.1007/s00438-007-0313-2

ORIGINAL PAPER

Cloning and characterization of three genes encoding Qb-SNARE proteins in rice

Yong-Mei Bao · Jian-Fei Wang · Ji Huang · Hong-Sheng Zhang

Received: 28 April 2007 / Accepted: 11 December 2007 / Published online: 16 January 2008© Springer-Verlag 2008

Abstract Qb-SNARE proteins belong to the superfamilyof SNAREs (soluble N-ethylmaleimide-sensitive factorattachment protein receptors) and function as importantcomponents of the vesicle traYcking machinery in eukary-otic cells. Here, we report three novel plant SNARE(NPSN) genes isolated from rice and named OsNPSN11,OsNPSN12 and OsNPSN13. They have about 70% nucleo-tide identity over their entire coding regions and similargenomic organization with ten exons and nine introns ineach gene. Multiple alignment of deduced amino acidsequences indicate that the OsNPSNs proteins arehomologous to AtNPSNs from Arabidopsis, containinga Qb-SNARE domain and a membrane-spanning domain inthe C-terminal region. Semi-quantitative RT-PCR assaysshowed that the OsNPSNs were ubiquitously and diVeren-tially expressed in roots, culms, leaves, immature spikesand Xowering spikes. The expression of OsNPSNs was sig-niWcantly activated in rice seedlings treated with H2O2, butdown-regulated under NaCl and PEG6000 stresses. Tran-sient expression method in onion epidermal cells revealedthat OsNPSNs were located in the plasma membrane.Transformed yeast cells with OsNPSNs had better growthrates than empty-vector transformants when cultured oneither solid or liquid selective media containing various

concentrations of H2O2, but more sensitive to NaCl andmannitol stresses. The 35S:OsNPSN11 transgenic tobaccoalso showed more tolerance to H2O2 and sensitivity to NaCland mannitol than non-transgenic tobacco. These resultsindicate that OsNPSNs may be involved in diVerent aspectsof the signal transduction in plant and yeast responses toabiotic stresses.

Keywords Oryza sativa L. · OsNPSN genes · Qb-SNARE · Expression patterns · Functional characterization

Introduction

The soluble N-ethylmaleimide-sensitive factor attachmentprotein receptors (SNAREs) are important components ofthe vesicle traYcking machinery in eukaryotic cells (Heeseet al. 2001; Wick et al. 2003). In the process of vesicletraYcking, SNAREs anchored on diVerent membranesinteract through their SNARE domains to form a four-helixSNARE bundle, thereby providing energy to drivemembrane fusion. One helix of the SNARE bundle iscontributed by an R-SNARE domain in a vesicle SNARE(v-SNARE) anchored to a vesicle membrane compartment,and the three other helices are added by the SNAREdomains in target membrane SNAREs (t-SNAREs) resid-ing in other membrane compartments, including Qa-,Qb- and Qc-SNARE domains (Fukuda et al. 2000). Asyntaxin, one of the t-SNAREs, contributes one helix(Qa-SNARE domain), whereas the remaining two helices,Qb- and Qc-SNARE domains, are either from oneSNAP25-type protein (synaptosomal-associated protein) orfrom two additional t-SNAREs on the target membrane(Antonin et al. 2000; Fukuda et al. 2000). Sanderfoot et al.

Communicated by H. Ronne.

Electronic supplementary material The online version of this article (doi:10.1007/s00438-007-0313-2) contains supplementary material, which is available to authorized users.

Y.-M. Bao · J.-F. Wang · J. Huang · H.-S. Zhang (&)State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, Chinae-mail: hszhang@njau.edu.cn

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(2000) reported a family of plant t-SNAREs, which have nohomolog in mammalian or yeast genomes, and these novelplant SNAREs were designated NPSNs. Zheng et al. (2000)identiWed three NPSN gene members in Arabidopsis(AtNPSN11, -12, and -13). Uemura et al. (2004) found thatAtNPSNs were located in plasma membranes of Arabidopsisprotoplasts.

Many t-SNAREs in plants are involved in the formationof the cell plates during cell division, and in resistance topathogens (Pratelli et al. 2004). Zheng et al. (2002) reportedthat AtNPSN11, one member of the NPSN gene family inArabidopsis, interacted with the syntaxin KNOLLE tofunction in diverse membrane fusion processes. HvSNAP34,a t-SNARE gene encoding a SNAP25-type protein in barley,was associated with non-host resistance of barley penetratedby the powdery mildew fungus (Blumeriae graminis f. sp.tritici; Collins et al. 2003). Wick et al. (2003) showed thatthe expression of another SNAP25-type protein, AtSNAP33,in Arabidopsis was induced by various pathogens and alsoby mechanical stimulation. We previously cloned aSNAP25-type protein gene OsSNAP32 from rice andshowed that it was involved in rice responses to biotic andabiotic stresses (Bao et al. 2007).

With the release of the complete genomic sequence, riceis now a model organism for studying the physiology,developmental biology, genetics, and evolution of mono-cotyledonous plants (Yu et al. 2002). Here, we report thecloning and characterization of three SNARE protein genesOsNPSN11, OsNPSN12 and OsNPSN13, from rice by bio-informatics and RT-PCR approaches. These three genesmay encode Qb-SNAREs proteins located in the plasmamembrane. The expression of OsNPSN11, OsNPSN12 orOsNPSN13 in transgenic yeast cells may improve their tol-erance to H2O2 stress, but increase their sensitivity to saltand osmotic stresses. The expression of OsNPSN11 intransgenic tobacco showed the same eVect as in transgenicyeast cells.

Materials and methods

Plant materials

Rice (Oryza sativa L. subs. japonica cv. Heikezijing) seedswere sterilized with 0.1% HgCl2 and washed with tap waterand allowed to germinate in an incubator at 28°C. The ger-minated seeds were cultured in liquid nutrient according tothe method of Yoshida (Yoshida et al. 1976) and held in agreenhouse at 30°C/22°C (day/night) and 70% relativehumidity, with a 12 h photoperiod under Xuorescent whitelight (250 �mol m¡2 s¡1). The growth medium was main-tained at pH 5.6 and renewed every 5 days. Seedlings at thethree to four leaves stage were harvested and immediately

stored at ¡80°C for RNA preparation and cloning ofOsNPSNs. Meanwhile, some seedlings were transplanted tothe experimental Weld in Nanjing in late May. The roots,leaves, culms and immature and Xowering spikes fromthese plants were harvested after 2-1/2 months, and imme-diately stored at ¡80°C for expression analyses of OsNPSNsin various rice organs at the adult stage.

In order to detect the expression of OsNPSNs under abi-otic stress conditions, rice seedlings at the three to fourleaves stage were cultured in fresh liquid nutrient contain-ing 250 mM NaCl to induce salt stress, 20% PEG6000 toinduce osmotic stress (Gu et al. 2005; Park et al. 2003), and20 mM H2O2 (Wnal concentration; Desikan et al. 2001),respectively. At various time intervals after culturing, therice seedlings were sampled for expression analysis of thetarget genes.

Cloning of OsNPSNs

Total RNAs from rice tissues or seedlings was extractedusing Trizol reagent (Invitrogen) according to the manufac-turer’s protocol. The Wrst strand cDNA was synthesizedwith 2 �g of puriWed total RNA using an RT-PCR system(Promega, USA) according to the manufacturer’s protocol.Oligo(dT)15 was used as a primer and the reverse transcrip-tion reaction was incubated at 42°C for 1 h in a total vol-ume of 20 �l.

In order to clone OsNPSNs genes, the amino acidsequence of Arabidopsis AtNPSN11 (Zheng et al. 2002)was used as a query probe to search the Rice Genome Data-base of China through the tBLASTn algorithm program.Three contigs (Ctg011788, Ctg011756 and Ctg006721)having high homology with the probe were analyzed withthe FGENES-M program for predicting and assembling thecomplete open reading frame (ORF). The putative ORFsequences were further conWrmed by the expressedsequence tags (ESTs) existing in the rice EST database ofGenBank. Based on the conWrmed ORF sequences, threepairs of primers were designed for cloning the OsNPSNs.The primers, synthesized by BBST (Shanghai), were asfollows: sense1: 5-TGCTTTTGGTTGTTGTGATCC-3,and antisense1: 5-CTGCTCTTGGTTGATTTGTTC-3 forcloning OsNPSN11; sense2: 5-CACCGCTGGAGTAGAGTTC-3, and antisense2: 5-TCGTAGTATCTTATGACCGC-3 for OsNPSN12; and sense3: 5-TGACGAGACGACCAACCAC-3, and antisense3: 5-TCCCACTCGCAATGCTCAG-3 for OsNPSN13.

The PCR conditions were as follows: 0.5 �l RT productwas ampliWed in a 25 �l volume containing 2.5 �l of10 £ PCR buVer with MgCl2, 0.5 �l of 20 mM dNTPs, 1 �lTaq polymerase (DingGuo, Beijing), 0.5 �l of 25 mM ofeach primer and 19.5 �l double distilled water. PCR wasperformed on a DNA ampliWcation machine (MJ, USA) for

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an initial denaturation at 94°C for 5 min; 30 cycles at 94°Cfor 45 s, 60°C for 45 s, 72°C for 90 s and a Wnal 72°C for10 min. The PCR products were run on 1% agarose gelsand puriWed with a gel extraction kit (HuaShun, Shanghai)according to the manufacturer’s protocol. The puriWedproduct was then cloned into the pGEM-T vector(Promega, USA) and sequenced (BBST, Shanghai).

Sequence analysis

The BLAST program at GenBank (http://www.ncbi.nlm.nih.gov/blast) was used to search the Non-Redundant (NR)or dbEST databases. The genome organization of threeOsNPSNs were investigated using FGENES-M software(http://www.softberry.com/berry.phtml) by comparing thecloned cDNAs with the corresponding genome sequencesfrom the Rice Genome Database of China (http://btn.genomics.org.cn/rice) and Rice Genome Project (RGP,http://rgp.dna.affrc.go.jp), respectively. The sequencealignment was performed using the ClustalW (version 1.8) andGeneDoc (version 2.5) programs. The GenBank accessionnumbers and original species of the representative Qb-SNAREfrom several eukaryotes are as follows: OryzaNPSN11,AAU94635 (Oryza sativa); OryzaNPSN12, AAU94636(Oryza sativa); OryzaNPSN13, AAU94637 (Oryza sativa);ArathNPSN11, AAL27494 (Arabidopsis thaliana); Arat-hNPSN12, AF487545 (Arabidopsis thaliana); ArathNPSN13,NP_566578 (Arabidopsis thaliana); ArathGOS11, AAK48904(Arabidopsis thaliana); ArathGOS12, AAK48905 (Arabid-opsis thaliana); ArathMEMB11, AAD31575 (Arabidopsisthaliana); ArathMEMB12, BAB09463 (Arabidopsis thaliana);ArathVTI11, AAF24061 (Arabidopsis thaliana); ArathVTI12,AAF24062 (Arabidopsis thaliana); ArathVTI13, BAB01986(Arabidopsis thaliana); SacceGOS1P, NP_011832 (Saccha-romyces cerevisia); SacceBOS1P, NP_013179 (Saccharomy-ces cerevisia); SacceVTI1P, NP_013924 (Saccharomycescerevisia); HomsaGS28, NP_004862 (Homo sapiens);HomsaMEMBRIN, NP_004278 (Homo sapiens); Hom-saVTI1a, AAH17052 (Homo sapiens); HomsaVTI1b,NP_006361 (Homo sapiens); MusmuGS28, NP_058090(Mus musculus); MusmuMEMBRIN, NP_062624 (Musmusculus); MusmuVTI1a, AAC23482 (Mus musculus);MusmuVTI1b, AAC23483 (Mus musculus).

Expression of OsNPSNs in rice

In order to compare the expression levels of OsNPSNs invarious rice tissues or seedlings, an 800 bp PCR fragmentof the constitutively expressed rice actin gene Rac1 (Martin1999) was ampliWed synchronously as a control. Theprimers for Rac1 were: sense: 5�-GGAACTGGTATGGTCAAGGC-3�; antisense: 5�-AGTCTCATGGATAACCGCAG-3�. The PCR conditions for amplifying the Rac1

fragment were the same as those for cloning the OsNPSNgenes, but the cycles were changed to 25. The OsNPSNgenes were ampliWed by PCR with the same conditions ascloning. The Wgure capture was undertaken using TouchingTransilluminator (Shanghai, China).

Subcellular localization of the OsNPSN proteins

The coding regions of OsNPSNs were ampliWed by PCRcontaining an Xba I site at the 5� end and a BamHI site atthe 3� end, with the primers: sense1: 5-ATTCTAGATGGATTTGGAGTCGGTCA-3 (XbaI); and antisense1: 5-TAGGATCCAAGCAGACGGCGTGCCC-3 (BamH I) forOsNPSN11; sense2: 5-TTCTAGACACCGCTGGAGTAGAGTT-3 (XbaI), and antisense2: 5-AGGATCCTCTGATAATTTCTACCGA-3 (BamH I) for OsNPSN12; sense3: 5-TGATCTAGAGACCAACCACCTCCTG-3 (Xba I), andantisense3: 5-AAGGATCCGAGGCTTCCGAAAGATT-3(BamH I) for OsNPSN13. The resulting fragments werecloned into the vector pGEM-T (Promega, USA), digestedwith XbaI and BamHI, then cloned into the expression vec-tor pBI121 (Clontech). The coding regions of OsNPSNswere fused in frame with the coding region of �-glucuroni-dase (GUS) already present in plasmid pPBI121, yieldingthe plasmid pBI-OsNPSNs. These constructs were intro-duced into Agrobacterium tumefaciens strain LBA4404.The Agrobacterium culture for transient expression inonion epidermal cells was prepared as described by Yanget al. (2000). The onion epidermal cells were immersed inthe Agrobacterium solution for 40 min, transferred toMurashige and Skoog (MS) plates, and incubated at 25°Cunder light for 24–48 h. The localization of OsNPSNs:GUS fusion proteins were observed by white lightmicroscopy (XS-213, JNOEC). The onion epidermal cellswere mounted in 0.85 M NaCl for 5 min to achieveplasmolysis.

Yeast strain and plasmids

Saccharmyces cerevisiae strain DY1455 (MAT� ade2can1 his3 leu2 trp1 ura3) was used as the host cell. Theplasmid pFL61 with the PGK promoter was used as anexpression vector in yeast using uracil as an auxotrophicmarker. The complete cDNAs of OsNPSN11, OsNPSN12and OsNPSN13 were each ampliWed by PCR, with thesame primers used for cloning of OsNPSNs, cloned intothe vector pGEM-T, digested with SpeI and SacIIrestriction endonucleases, and then ligated to the expres-sion vector pFL61 to yield the plasmids pFL-OsNPSN11,pFL-OsNPSN12 and pFL-OsNPSN13. These recon-structed plasmids, and empty plasmid pFL61, wererespectively transformed into S. cerevisiae DY1455 usingthe lithium acetate method (Ito et al. 1983) to yield four

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yeast transformants separately harboring pFL61, pFL-OsNPSN11, pFL-OsNPSN12 and pFL-OsNPSN13 .

Growth of yeast transformants

In order to compare the diVerence in growth rate betweenthree genes transformants and empty vector transformantunder various abiotic stresses, the four yeast transformantswere inoculated in liquid synthetic medium with dextroselacking uracil (SD-Ura) at 30°C with 200 rpm shaking for2 days until the culture OD600 levels reached 1.0. Then,50 �l cultures were transferred to 50 ml SD-Ura mediumfor further growth. According to the method of Lee et al(1999) and Yu et al (2005), two experiments for growthrate were conducted. For growth in solid medium, the cul-tures were made serially diluted of 1:10. A volume of 10 �lof the cultures, or the serial dilutions, were spotted on thesolid SD-Ura medium in Petri dish (diameter 120 mm) con-taining 1 mM H2O2, 0.7 M NaCl, or 0.5 M mannitol andincubated at 30°C for 2 days. For growth in liquid medium,2.5 ml of culture solution was transferred into 80 ml of liq-uid SD-Ura medium containing diVerent concentrations ofH2O2, NaCl or mannitol, and grown at 30°C with 200 rpmshaking for 2 days. The growth rates of these yeast cells at30°C were monitored by measuring absorbance at 600 nmevery 5 h. The experiments were repeated three times andthe standard errors (§SE) were measured.

Generation of OsNPSN11 transgenic tobacco plants

The full-length OsNPSN11 was ampliWed by PCR contain-ing each Xba I sites at the 5� end and the 3� end, with theprimers: sense: 5-ATTCTAGATGGATTTGGAGTCGGTCA-3, antisense: 5-TGCTCTAGATTGATTTGTTCACATGT-3, conWrmed by sequencing and inserted into the XbaI sites of the plant binary vector pBI121. Then theOsNPSN11 gene driven under the cauliXower mosaic virus(CaMV) 35S promoter was introduced into tobacco plants(Nicotiana tabacum cv. Xanthi) via Agrobacterium-medi-ated transformation as described (Horsch et al. 1988).

Growth of transgenic tobacco plants under stresses

To evaluate the performance of the transgenic tobaccoplants under stresses, the seeds of T1 transgenic lines weregerminated and grown in 1/2 Murashige and Skoog (MS)medium for 3 days, then transferred to the 1/2 MS mediumplus 0, 10 mM H2O2, 20 mM H2O2, 100 mM NaCl,200 mM NaCl, 300 mM mannitol or 500 mM mannitol.After 15 days, the root length and chlorophyll contents ofeach OsNPSN11 transgenic or non-transgenic plant weremeasured. The measurement method of chlorophyll con-tents was according to Zhang et al. (2004). The experiment

was independently repeated thrice and the standard errors(§SE) were measured.

Results

Cloning of OsNPSNs

Three target cDNA fragments, each containing a completeORF, were cloned by RT-PCR from rice seedlings. Theirfull lengths were 852, 986 and 921 bp and they were desig-nated as sNPSN11, OsNPSN12 and OsNPSN13, respec-tively. The complete cDNAs and deduced amino acidsequences have been submitted to the DDBJ/EMBL/GenBank databases under accession numbers AY740014and AAU94635, AY740015 and AAU94636, AY740016and AAU94637, respectively.

The three OsNPSNs are highly conserved over theirentire coding regions, with about 70% nucleotide similar-ity. The gDNA sequence of each gene comprises ten exonsand nine introns (Fig. 1). The lengths of the exons in orderand exon–exon junction position in their mRNAs are con-served, although their intron lengths are obviously diverse.The sequences of the cloned cDNAs were consistent withthe prediction of the exon–intron structures, based on therice genome-sequencing consortium. In addition, the 5�-UTR of each OsNPSN contains the stop codon sequenceTGA or TAG in the same reading frame with the initiatorcodon ATG, and no other ATG codons are present beyondthe Wrst. The ORFs cloned here may be the complete openreading frames. The cDNA sequences were used as queryprobes to search the rice NR database of Genbank (http://www.ncbi.nlm.nih.gov/) in order to in silica localize theirpositions on rice chromosomes. Three BAC clonesP0481E08 (GenBank accession no. AP003614),OSJNBa0013M12 (GenBank accession no. AC082644),and OJ1136_D11 (GenBank accession no: AP003749) con-tained OsNPSN11, OsNPSN12 and OsNPSN13, respec-tively. According to the positions of BAC clones searchedfrom the Rice Genome Project database (RGP, http://rgp.dna.affrc.go.jp), OsNPSN11 was mapped in silica to aposition between RFLP markers R2614 and E12186S onthe short arm of chromosome 6, OsNPSN12 between RFLPmarkers C12845S and C1186 on the long arm of chromo-some 3, and OsNPSN13 between RFLP markers E20959Sand C60933 on the long arm of chromosome 7.

Analysis of deduced amino acid sequences

The predicted protein products of OsNPSN11, OsNPSN11and OsNPSN11 comprise 261, 275 and 266 amino acidresidues with molecular weights of 29.3, 31.0 and 30.0 kD,respectively. It was predicted that there was one Qb-SNARE

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domain from Thr-147 to Ile-203 of OsNPSN11, Thr-153 toIle-209 of OsNPSN12, and Thr-150 to Ile-206 ofOsNPSN13, respectively, and one membrane-spanningdomain from Met-214 to Pro-235 of OsNPSN11, Met-220to Pro-241 of OsNPSN12, and Met-217 to Pro-238 ofOsNPSN13 in the C-terminal regions, respectively. TheQb-SNARE and membrane-spanning domains were highlyconserved in all the known t-SNARE genes encodingQb-SNARE proteins (Salaün et al. 2004). Multiplesequence alignment showed that the OsNPSNs were exten-sively homologous to AtNPSNs proteins, with 57–80%amino acid identities (Fig. 2a).

We constructed a molecular phylogenetic tree of riceOsNPSNs and other 21 Qb-SNARE proteins in the

Genbank databases from various organisms. It revealsthat the OsNPSN proteins are closest to the AtNPSN pro-teins and seem to be unique to the plant kingdom(Fig. 2b).

Subcellular localization

Agrobacterium-mediated transient expression in onionepidermal cells revealed that the 35S:OsNPSN-GUS fusionproteins were localized on the plasma membranes of onionepidermal cells, whereas the control GUS protein wasspread over the entire cytoplast (Fig. 3). AtNPSNs weresimilarly located in plasma membranes of Arabidopsisprotoplasts (Uemura et al. 2004).

Fig. 1 Schematic diagrams of the OsNPSNs genes. Exons are depicted as gray boxes and introns indicated by wedges in proportion to their size. Solid thick lines represent 5�-UTR and 3�-UTR in proportion to the region size. ATG initiator codon; TAG or TGA stop codons

Fig. 2 Multiple sequence alignment of amino acid sequences ofOsNPSNs with the AtNPSNs from Arabidopsis and phylogenetic treeanalysis based on an alignment of rice OsNPSN11 » 13 with otherQb-SNARE proteins. a Multiple sequence alignment of amino acidsequences of OsNPSNs with the AtNPSNs from Arabidopsis. Blackboxes indicate positions at which the residues are identical and grayboxes highlight residues that are similar. AtNPSN12 is included toshow positions in the Qb-SNARE domain that contribute to stabilizing

ionic (*) or hydrophobic (¥) interactions with other SNARE proteins.b Phylogenetic tree analysis based on an alignment of riceOsNPSN11 » 13 with other Qb-SNARE proteins. Protein sequencesfrom GenBank (Arath, Arabidopsis; Sacce, budding yeast; Homsa, hu-man; Musmu, mouse; see “Materials and methods” for accession no.)were aligned using the ClustalW. Distance tree was calculated usingthe neighbor-joining method. The lengths of the branches are propor-tional to the degree of divergence

AB

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Characterization of OsNPSNs expression in rice

The expression of OsNPSNs in various rice tissues at theadult plant stage was investigated by semi-quantitative RT-PCR. The three OsNPSN genes were expressed in diVerenttissues, but they had diverse expression proWles (Fig. 4a).OsNPSN11 was highly expressed in the leaves and imma-ture spikes, OsNPSN12 highly expressed in the culms andleaves, and OsNPSN13 was moderately expressed in roots,culms and leaves. All three were much less expressed inother tissues.

The expression of OsNPSNs in rice seedlings under oxi-dative, salt and osmotic stresses also was detected. Thetranscript levels of all three OsNPSNs were obviouslyincreased at 3 h after H2O2 treatment, after which expres-sion of OsNPSN11 remained stable until 48 h, whereasthose of OsNPSN12 and OsNPSN13 slightly declined afterreaching peaks at 12 h (Fig. 4b). Under salt stress, theexpressions of OsNPSN11 and OsNPSN13 slightlydecreased, but that of OsNPSN12 dramatically and

continuously declined after 3 h of treatment (Fig. 4c). Theexpression of OsNPSN11 had obviously declined after12 hof PEG6000 treatment, whereas that of OsNPSN12 rapidlydeclined after 3 h of treatment. Both genes were hardlydetectable after 48 h of treatment. The transcript levels ofthe OsNPSN13 were not obviously changed by PEG6000treatment (Fig. 4d).

Tolerance of transformed yeast cells to stresses

The expression of OsNPSNs in the transformed yeast cellswith pFL-OsNPSN11, pFL-OsNPSN12 and pFL-OsNPSN13 was examined by semi-quantitative RT-PCRanalysis, and the transformed yeast cells with vector pFL61were used as control. The OsNPSN genes were expressed inthe transformed yeast cells and no expression was detectedin the controls (Supplementary Fig. 1).

There were obviously diVerent growth rates betweentransformed yeast cells with pFL-OsNPSN11, pFL-OsNPSN12 or pFL-OsNPSN13, and the transformant with

Fig. 3 Subcelluar localization of OsNPSNs-GUS fusion proteins and 35S:GUS proteins in onion epidermal cells by transient expression. (a), (b), (c) indicate the location of 35S:OsNPSN11-GUS fusion proteins, 35S:OsNPSN12-GUS fusion proteins and 35S:Os-NPSN13-GUS fusion proteins are attached to the plasma membrane in the plasmolyzed epidermal cells, respectively. d 35S:GUS proteins are spread all over the cytoplast. Bars 40 �m

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vector pFL61 under oxidative, salt and osmotic stresseseither in solid or liquid growth conditions (Figs. 5, 6).When serial dilutions were spotted on solid SD-Uramedium and cultured for 48 h, there were no diVerences inthe densities of colonies (Fig. 5a). The three transfor-mants with pFL-OsNPSNs had denser growth than thevector pFL61 transformant as dilutions spotted in SD-Uramedium containing 1 mM H2O2 (Fig. 5b). However, thegrowths of transformants with pFL-OsNPSNs were moreinhibited than control in SD-Ura medium containing0.7 M NaCl (Fig. 5c) and 0.5 M mannitol (Fig. 5d). Theresults suggested that the expression of OsNPSNs inyeasts increased their tolerances to oxidative stress,but decreased their tolerances to the salt and osmotictreatments.

In liquid SD-Ura medium containing various concentra-tions of H2O2, NaCl or mannitol, the transformed yeastcells with pFL-OsNPSNs showed diVerent growth ratescompared with the vector pFL61 transformant (Fig. 6). Thetransformed yeast cells with pFL-OsNPSN11, pFL-OsNPSN12 and pFL-OsNPSN13 showed better growth thanthe control under various concentrations of H2O2 (Fig. 6a).The optical densites (OD600) of the transformants with pFL-OsNPSNs were 20–29-fold higher than those of the controlsup to 20 h of growth with 1 mM H2O2 (Fig. 6b). The trans-formants with pFL-OsNPSNs grew more slowly than thecontrol under NaCl treatments ranging from 100 to1,000 mM (Fig. 6c), or mannitol from 100 mM to 500 mM(Fig. 6e) as well as in solid medium. The OD600 of the threetransformants with pFL-OsNPSNs were 19.81–66.48% of

control within 15 h of growth at 500 mM of NaCl treatment(Fig. 6d) and 300 mM of mannitol treatment (Fig. 6f),respectively.

Fig. 4 Expression pattern of OsNPSNs genes in rice (cv. Heikezijing). a Expression of OsNPSNs in rice roots(1), culms(2), leaves(3), immature spikes(4) and Xowering spikes(5). b–d Expression of OsNPSNs in the rice seedlings after H2O2 (20 mM), NaCl (250 mM) and 20% PEG6000 treatments, respectively. The actin gene Rac1 was used as the internal control

Fig. 5 Growth of transformed yeasts on the solid SD-Ura medium.a Growth of the transformed yeasts with vector pFL61(1), pFL-OsNPSN11(2), pFL-OsNPSN12 (3) and pFL-OsNPSN13(4), respec-tively, on SD-Ura medium. b–d Growth of the transformed yeasts onSD-Ura medium with 1 mM H2O2, 0.7 M NaCI and 0.5 M mannitol,respectively

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Tolerance of 35S:OsNPSN11 transgenic tobacco to abiotic stresses

To analyze the biological function of OsNPSN11, we gen-erated transgenic tobacco plants expressing OsNPSN11.The RT-PCR analysis indicated that OsNPSN11 was mark-edly transcribed in 5#, 15# and 32# T0 transgenic tobacco(Supplementary Fig. 2). As the expression of OsNPSN11gene was induced by oxidative stresses and down-regulatedby salt stresses and osmotic stresses in rice (Fig. 4), the oxi-dative, salt and osmotic tolerance of OsNPSN11 transgenictobacco plants were analyzed. The 35S:OsNPSN11 trans-genic seedlings showed more tolerance to oxidative stressthan the non-transgenic tobacco, which agreed with themeasurement of root length and chlorophyll contents(Fig. 7a and Supplementary Fig. 3). However, the growthsof 35S:OsNPSN11 transgenic seedlings were moreinhibited than non-transgenic tobacco on 1/2 MS mediumcontaining NaCl (Fig. 7b) and mannitol (Fig. 7c). The rootlength of the 35S:OsNPSN11 transgenic seedlings was

smaller than the root length of non-transgenic tobacco(Fig. 7b, c), but with no diVerence in the chlorophyll con-tents (data not shown). Thus, expression of OsNPSN11 intransgenic tobacco plants might increase plant tolerance tooxidative stress.

Discussion

The machinery of vesicle traYcking appears to be con-served among eukaryotes including yeasts, mammals,insects and plants (Beervers and Raikhel 1998; Basshamet al. 2000). The plant SNARE family is a superfamily withhundreds of members (McNew et al. 2000; Sanderfootet al. 2000). Increasing evidence suggests that plantSNAREs play a variety of roles in growth and developmentof the whole organism (Collins et al. 2003; Mayer andJürgens 2004; Pratelli et al. 2004). In this work, we clonedand characterized OsNPSNs in rice. The three OsNPSNgenes have about 70% nucleotide identity over their entire

Fig. 6 Growth of transformed yeasts on the liquid SD-Ura medium. a Growth of transformed yeasts in SD-Ura containing various concentrations of H2O2 for 20 h. b Growth of transformed yeasts in SD-Ura containing 1 mM H2O2 for various times. c Growth of transformed yeasts in SD-Ura containing various concentrations of NaCl for 15 h. d Growth of transformed yeasts in SD-Ura containing 500 mM NaCl for various times. e Growth of transformed yeasts in SD-Ura containing various concentrations of mannitol for 15 h. f Growth of transformed yeasts in SD-Ura containing 300 mM mannitol for various times. Data represent the mean values § SE of three replicates

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Mol Genet Genomics (2008) 279:291–301 299

coding regions and share similar genomic structures withten exons and nine introns. Since the predicted products ofOsNPSNs contain Qb-SNARE and membrane-spanningdomains in the C-terminal region and share high homologywith AtNPSN proteins in Arabidopsis, they may belong toa three-member gene family of plant-speciWc Qb-SNAREs(Zheng et al. 2002). Transient expression method in onionepidermal cells revealed that OsNPSNs were located in theplasma membrane. Uemura et al. (2004) showed thatAtNPSNs were located in the plasma membrane using tran-sient expression method in Arabidopsis suspension culturedcells. Zheng et al. (2002) found using immunoXuorescencemicroscopy that AtNPSNs can interact with KNOLLE andwere located in the cell plate of dividing Arabidopsis cells.

Preliminary results in this paper obtained on the over-expression of the fusion OsNPSN proteins in a heterolo-gous system, and 35S-driven expression, can be misleadingas an example of KNOLLE (Volker et al. 2001). So immu-noXuorescence microscopy should be used to analyze theendogenous OsNPSN proteins localizations in rice cells,and membrane fusion assays in vivo or in vitro also shouldbe carried out in order to obtain deWnitive evidence thatOsNPSNs are involved in membrane fusions between vesi-cles and target membranes in rice cells.

The OsNPSNs were ubiquitously expressed in roots,culms, leaves, immature spikes and Xowering spikes ofadult rice plants, but with diverse expression levels. Similarubiquitous expression patterns of AtNPSN11 and

Fig. 7 Root growth of trans-genic and wild-type tobacco plants under osmotic and salt stresses. Seven-day-old seed-lings were transferred and grown for 15 days vertically on 1/2 MS plate or 1/2 MS plates containing 10 and 20 mM H2O2 (a), 100 and 200 mM NaCl (b), 300 and 500 mM mannitol (c), respec-tively. Results are the mean § SE of the three individ-ual measurements 0

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300 Mol Genet Genomics (2008) 279:291–301

AtNPSN12 occurred in Arabidopsis, although no expressionof AtNPSN13 was detected (Zheng et al. 2002). This sug-gests that the three genes may play important but diVerentroles in rice growth and development. The expressions ofOsNPSNs in rice seedlings were up-regulated by H2O2

treatment and down-regulated by NaCl and PEG6000. Thisis the Wrst report that the expression of OsNPSNs in rice canbe induced by oxidative stress and down-regulated with saltand drought treatments. The products of OsNPSN genesmight be involved in diVerent aspects of signal transductioncascades that occur when plants respond to abiotic stresses.

Yeasts have been used as model systems for eukaryoticcells to address functions of heterologous genes (Gellissenet al. 1992). To determine the physiological roles ofOsNPSNs, the OsNPSNs genes were transformed andexpressed in yeast cells. The three transformants with pFL-OsNPSNs grew much better than the empty vector pFL61transformant under H2O2 treatment, but poorer under saltand osmotic stresses. The 35S:OsNPSN11 transgenictobacco also showed more tolerance to H2O2 treatment, andsensitive to NaCl and mannitol stresses than the wild-typetobacco. Together with the changes in expression ofOsNPSNs observed in rice plants under equivalent stressconditions, these data suggest that rice plants might up-reg-ulate OsNPSNs to counteract oxidative stress and down-regulate their expression to avoid more damage under saltand drought stress conditions. H2O2 has established itselfas a key eVector in stress and programmed cell deathresponses (Desikan et al. 2001; Gechev and Hille 2005).Levine et al. (2001) reported that the Arabidopsis v-SNAREAtVAMP7 could prevent H2O2-induced apoptosis inyeast and Arabidopsis cells by improved membranerepairing. In this report, the expression of Qb-SNARE fam-ily genes OsNPSNs in yeast cells and tobacco couldenhance their tolerance to oxidative stress. This can beexplained by the same mechanism as AtVAMP7. Leshamet al. (2006) reported that suppression of AtVAMP7 geneexpression in Arabidopsis increased salt tolerance. Expo-sure to high-salt conditions causes immediate ionic andosmotic stresses, followed by the production of reactiveoxygen species. The mechanism for this is that failure ofthe H2O2-containing vesicles to fuse with the tonoplastcould improve vacuolar functions and increase plant salttolerance (Leshem et al. 2006). In our case, expression ofthe OsNPSNs in yeast cells and tobacco reduced their toler-ances to salt and osmotic stresses. One possible explanationfor this is that the H2O2-containing vesicles might easilyfuse with the tonoplast, and then H2O2 or other reactiveoxygen species might congregate in the vacuole. It couldfurther cause the yeast cells and Arabidopsis to becomemore sensitive to salt or osmotic stresses. However, the reg-ulation of OsNPSNs in rice under abiotic stresses may bemore complex than currently understood. Rice mutants

with impaired OsNPSNs are necessary to gain an under-standing of the roles of vesicle traYcking and functions ofOsNPSNs in rice subjected to abiotic stresses. The applica-tion of genomics and proteomics approaches, as well ascytological methods, will also accelerate our understandingof vesicle traYcking and the functions of the OsNPSNgenes in rice.

Acknowledgments We wish to thank Dr. S. Q. Guo, Ms. L. Y. Dingand Mr. X. Huang for their experimental help and we are indebted toDr. David Eide (Nutritional Science Program, University of Missouri,Columbia, USA) for providing yeast strain DY1455 and yeast expres-sion vector pFL61. This work was supported by grants from theNational Science Foundation of China (30470921 and 30571141),Natural Science Foundation of Jiangsu Province (BK2005090), thePh.D program foundation of the Ministry of Education (20060307035),and the Program for Changjiang Scholars and Innovative ResearchTeams in Universities.

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