ORIGINAL ARTICLE

A gene encoding a germin-like protein, identified by a cDNA–AFLPapproach, is specifically expressed during germinationof Phaseolus vulgaris

Received: 15 October 2002 / Accepted: 25 January 2003 / Published online: 5 March 2003� Springer-Verlag 2003

Abstract In order to identify markers of germination inPhaseolus vulgaris L., a cDNA–amplified fragmentlength polymorphism (AFLP) approach was conductedon mRNAs from embryo axes and from cotyledons.Among changes observed throughout the germinationprocess, a cDNA fragment not detected 9 h afterimbibition (HAI) but present specifically in axes 24 HAIwas further studied. The complete cDNA was recoveredby rapid amplification of cDNA ends, then cloned andsequenced. It includes an open reading frame predictinga 206-amino-acid polypeptide of 21.8 kDa. Analysis ofthe nucleotide sequence and deduced amino acidsequence revealed a high homology with germin-likeproteins (GLPs), and particularly with an auxin-bindingprotein from peach, ABP19, that belongs to the GLPfamily. Thus, we propose that this cDNA encodes thefirst GLP described in P. vulgaris, designated PvGLP1.Northern blot analysis carried out on mRNAs from seedaxes showed a dramatic increase in PvGLP1 expressiona few hours before radicle emergence (17 HAI). Amongmature vegetative tissues, PvGLP1 expression was veryweak in pods and not detected in leaves, stems or roots.Immunoblot analysis using antibodies raised againstAtGER3 from Arabidopsis thaliana showed that theprotein could be detected only in axes from the dry seedstage onwards, at a steady-state level. Then, PvGLP1expression seems to be associated with the early stages ofembryo axis growth. The high homology indicated withABP19 led us to study the effect of different concentra-tions of indole-3-acetic acid (IAA) on PvGLP1 expres-sion during germination. Whereas no effect was noticedat low concentrations (1, 5, 10 lM), a marked decreasein PvGLP1 mRNA level was observed in axes of seeds

imbibed with 100 lM IAA. Thus, PvGLP1 geneexpression is not stimulated by auxin and, moreover,it might be inhibited by high concentrations of IAA.

Keywords Auxin Æ cDNA–amplified fragment lengthpolymorphism Æ Germination Æ Germin-likeprotein Æ Phaseolus

Abbreviations ABP: auxin-binding protein Æ AFLP:amplified fragment length polymorphism Æ GLP:germin-like protein Æ HAI: hours after imbibition ÆIAA: indole-3-acetic acid Æ TDF: transcript-derivedfragment

Introduction

Induction of germination following re-hydration of theseed induces an increase in both respiration and meta-bolic activity, thus allowing embryo growth and devel-opment to occur at the expense of mobilized reserves(Baskin and Baskin 1998). Several studies aiming todetermine the physiological and developmental pro-cesses that allow seed germination have been focused ongene expression in relation to reserve mobilization andbreakage of dormancy. The control of C-reserve mobi-lization during germination has been extensively studiedthrough the regulation of a-amylase expression in barleyaleurone cells (Fath et al. 2001; Gomez-Cadenas et al.2001). Expression of several major genes involved inamino acid synthesis and degradation pathways hasbeen studied in various species, improving our knowl-edge of the role of N reserves as a source of energy andmetabolites for germination (Garciarrubio et al. 1997;Limami et al. 2002). However in Arabidopsis, Pritchardet al. (2002) demonstrated that the germination processand storage-reserve mobilization are regulated inde-pendently.

In contrast, little is known about genes involved inthe early developmental processes responsible for

Planta (2003) 217: 466–475DOI 10.1007/s00425-003-1004-9

Catherine Aubry Æ Marie-Christine Morere-Le Paven

Anne-Laure Chateigner-Boutin Æ Beatrice Teulat-Merah

Claudie Ricoult Æ Didier Peltier Æ Raymond Jalouzot

Anis M. Limami

C. Aubry Æ M.-C. Morere-Le Paven Æ A.-L. Chateigner-BoutinB. Teulat-Merah Æ C. Ricoult Æ D. Peltier Æ R. JalouzotA.M. Limami (&)UMR Physiologie Moleculaire des Semences,2 bd Lavoisier, 49045 Angers Cedex, FranceE-mail: anis.limami@univ-angers.frFax: +33-2-41735352

germination completion that could be used as markersof the onset of growth in germinating embryos. Whilesearching for germination-specific proteins in wheat,Thompson and Lane (1980) pointed out the nascentsynthesis of a novel soluble protein in concert with therenewed growth of the embryo; since then related pro-teins have been found in other monocotyledons (barley,maize, rice, rye grass) and designated germins because oftheir putative role in germination, even though no pre-cise function could be attributed to these proteins, andno homology with known proteins at the time of theirdiscovery was found. They are homohexamers of 25- to30-kDa apoplastic glycoprotein monomers (Woo et al.1998). Wheat and barley germins are hydrogen perox-ide-generating oxalate oxidases (Dumas et al. 1993;Lane et al. 1993). They could be involved in the regu-lation of embryo growth by controlling the cross-linkingof plant cell wall polymers (Caliskan and Cuming 1998),and in defense against abiotic stresses and pathogenattack (Dumas et al. 1995; Hurkman and Tanaka 1996;Berna and Bernier 1999).

Germins have never been detected in dicots. However,proteins that bear three oligopeptides, named A, B andC, conserved in all germins (reviewed in Bernier andBerna 2001; see Fig. 3), and share 30–70% identity withthe wheat germin encoded by the reference gene gf-2.8have been found in all angiosperm families including theGramineae, in gymnosperms (Neutelings et al. 1998) andmosses (Yamahara et al. 1999). About 100 differentsequences related to germin are now identified; they arecalled germin-like proteins (GLPs; Bernier and Berna2001). The GLP group is characterized by high hetero-geneity, with amino acid identities ranging from 25% toalmost 100%. Phylogenetic analyses defined four GLPsubgroups distinct from the true germin subfamily(Carter et al. 1999; Carter and Thornburg 2000).Recently, systematic genome sequencing and expressedsequence tag (EST) analysis programmes revealedcomplex gene families encoding proteins of all GLPsubgroups (Bernier and Berna 2001).

Plant GLPs have been found in various organs, i.e.leaves, roots, and floral tissues, under various physio-logical conditions, i.e. stress and pathogen attack(Michalowski and Bohnert 1992; Membre et al. 1997;Wojtaszek et al. 1997), but have not reported in seedsduring germination. In general, no enzyme activity hasbeen associated with most known GLPs except an oxa-late oxidase activity reported for Silene vulgaris GLP(Bringezu et al. 1999), an ADP-glucose pyrophospha-tase/phosphodiesterase activity for barley HvGLP1(Vallelian-Bindschedler et al. 1998; Rodrıguez-Lopezet al. 2001) and a superoxide dismutase (SOD) activityfor a moss GLP (Yamahara et al. 1999) and tobaccoNectarin I (Carter and Thornburg 2000). On the basis ofthese results, several putative roles have been suggestedfor GLPs. They are suspected to be structural proteinsbecause of their localization in the extra-cellular matrix,or to have a role in defense against pathogens because ofthe finding of SOD activities (Schweizer et al. 1999).

However, the most plausible hypothesis to date proposesGLPs as cell surface receptors, being the plant coun-terpart of animal adhesion proteins (Bernier and Berna2001). A rhicadhesin receptor in pea root was identifiedby N-terminal sequencing as a GLP (Swart et al. 1994)and finally two auxin-binding proteins, ABP19 andABP20 (Ohmiya et al. 1998) purified from peach, werefound to be closely related to GLPs.

In order to identify markers of germination ofPhaseolus vulgaris seeds, we have used a cDNA–am-plified fragment length polymorphism (AFLP)approach (Bachem et al. 1996). This work led us tocharacterize a cDNA that accumulates in the embryoaxis before radicle emergence. Sequence and expressionanalyses allowed us to identify this cDNA as part of theGLP encoding sequences; thus it was called PvGLP1.Analysis of the deduced amino acid sequence ofPvGLP1 revealed 60.4% identity with the auxin-bindingprotein ABP19 from peach, suggesting that PvGLP1could play a role in auxin reception in the embryo axisduring germination. Regulation of its expression andtissue-specificity studies confirmed that PvGLP1 isexpressed in the embryo axis during germination andshowed that it is expressed neither in cotyledons nor inmature vegetative tissues. To our knowledge, this is thefirst report of a GLP gene specifically expressed duringgermination.

Materials and methods

Plant material and germination procedure

Bean (Phaseolus vulgaris L. cv. Fin de Bagnols) seeds (Vilmorin,France), were sown on moist sand (8% distilled H2O, w/v) in thedark at 22 �C. The percentage of germinated seeds was determinedat various times between 0 and 40 h after imbibition (HAI), thegermination time being characterized by radicle emergence.Figure 1 represents the seed germination time-course derived. Thetime at which 50% of the seeds germinated (T50) corresponded to25 HAI. Samples of axes and cotyledons were harvested separatelyat different stages of germination, frozen in liquid nitrogen andstored at )80 �C until used. In addition, plants were grown in agreenhouse; roots, leaves and stems were harvested after 5 weeksand stored at )80 �C.

Fig. 1 Seed germination time-course of common bean (Phaseolusvulgaris). The percentage of germinated seeds was determined fromthree batches of 30 seeds, cv. Fin de Bagnols. In our conditions,50% of the seeds had germinated 25 HAI

467

RNA extraction

Tissues were ground in liquid nitrogen. Total RNA was extractedfrom frozen powder using a hot-phenol extraction procedure and aselective precipitation with 4 M LiCl to remove traces of DNA(Verwoerd et al. 1989).

cDNA–AFLP procedure

In order to identify marker/candidate gene(s) of germination, acDNA–AFLP approach was first developed with axes recordedat 9 and 24 HAI, and from cotyledons at 24 HAI. The cDNA–AFLP fingerprints were obtained according to Bachem et al.(1996) with a few modifications. mRNAs were isolated fromtotal RNA using biotinylated oligo (dT) and streptavidin mag-netic beads (PolyAtract IV; Promega, Madison, Wis., USA).cDNA was synthesized using 200 ng of mRNAs according tostandard protocols and using MMLV Reverse transcriptase(Promega). Half of the cDNAs obtained were used as a controlon a 1% agarose gel. The remaining sample was subjected tostandard AFLP template production (Vos et al. 1995). Restric-tion enzymes used for template production were EcoRI andMseI. Fifteen cycles of pre-amplification using primers withoutextension were carried out with 1/10th volume of the restriction/ligation mix (94 �C denaturation for 30 s, 56 �C annealing for1 min, 72 �C polymerization for 1 min). The products of pre-amplification were diluted 10-fold and the final PCR was carriedout as follows: 41 cycles, including cycle touchdown, where theannealing temperature was reduced from 65 �C to 56 �C in0.7 �C steps for 11 cycles and subsequently maintained at 56 �Cfor 30 cycles.

All PCR reactions were carried out using a BioRad (Marnes-la-Coquette, France) thermocycler with Taq DNA polymerase fromPromega. Oligonucleotides were obtained from MWG-Biotech SA(Ebersberg, Germany). The adaptors ligated to the restriction frag-ments and the primers used in the pre-amplification and final PCRare given below:

– EcoRI adaptor: 5¢-CTCGTAGACTGCGTACC-3¢; 3¢-CTGACGCATGGTTAA-5¢

– EcoRI pre-amplification primer: 5¢-GTAGACTGCGTACCAATTC-3¢

– EcoRI amplification primer: 5¢-GACTGCGTACCAATTCAC-3¢

– MseI adaptor: 5¢-GACGATGAGTCCTGAG-3¢; 3¢-TACTCAGGACTCAT-5¢

– MseI pre-amplification primer: 5¢-GACGATGAGTCCTGAGTAA-3¢

– MseI amplification primer: 5¢-GATGAGTCCTGAGTAACT-3¢

PCR products were separated on a 5% sequencing gel andvisualized by silver staining.

Isolating and sequencing of a transcript-derived fragment (TDF)

Among the bands obtained, one band of interest was rehydrated in50 ll of water, excised from the gel and incubated for 15 min at37 �C for further investigation. The TDF was then re-amplified byPCR using 30 cycles and the primers used for the final amplifica-tion. The PCR product was checked on a 1.4% agarose gel. Thefragment was sequenced using the EcoRI amplification primer andABI-Prism Big Dye terminator cycle sequencing ready reaction kits(PE Biosystems) on an ABI Prism 310 DNA sequencer. The se-quence was compared with the GenBank database using FASTA(Pearson and Lipman 1988), BLASTn and BLASTx (Altschul et al.1997).

cDNA cloning

TheDNase I treatment and synthesis of single-stranded cDNAwereachieved according to the method described by Huang et al. (1996).Two degenerate primers, previously designed to isolate a GLPcDNA fromPinus caribaea (Neutelings et al. 1998), were used for thePCR. The reaction mixture contained 2 ll of total cDNAs as tem-plate, 100 lM of each dNTP, 1 lM of each primer, 2 mM MgCl2,1 unit of Taq DNA polymerase (Promega) with buffer supplied bythe manufacturer in a total volume of 50 ll. The reactions werecarried out in a Crocodile III thermal cycler (Appligene Oncor, Ill-kirch, France) programmed as follows: 94 �C for 4 min, 40 cycles of94 �C for 1 min, 33 �C for 1 min, 72 �C for 1 min and a final step at72 �C for 10 min. The 387-bp product obtained was cloned in apGEM-T Easy vector (Promega) and sequenced (MWG-BiotechSA, Ebersberg, Germany).

Rapid amplification of cDNA ends (RACE) was performed aspreviously described (Frohman et al. 1988) using PCR conditionsdescribed by Ziadi et al. (2001). All PCR fragments of interest werecloned and sequenced as mentioned above and the nucleotidesequence of the complete cDNA was checked by end-to-end PCR.

RT–PCR analysis

Expression of the target gene was studied by reverse transcription–polymerase chain reaction (RT–PCR) as described by Chateigneret al. (1999). Amplifications were performed with the primer pairGLPup (5¢-GATAAAGATGATTTACATTC-3¢, 38–57) andGLPlow (5¢-GATAAAACTTTACGAACCT-3¢, 838–820) at anannealing temperature of 52 �C. A constitutive control was carriedout using the translation elongation factor 1 alpha gene (EF-1a)using the following primer pair: EFup (5¢-CTTCAGGATGTBTACAAGATTG-3¢) and EFlow (5¢-GCAGCCTTGGTVACCTTGCWCC-3¢). Amplifications were performed with an annealingtemperature of 50 �C.

Southern and northern blotting

Genomic DNA was isolated according to the method of Dellaportaet al. (1983). Fifteen micrograms of DNA was digested with EcoRI,which cuts PvGLP1 cDNA, and with EcoRV, which does not cut it.The resulting fragments were separated on a 1% agarose gel, blottedand hybridized with a-[32P]dCTP-labeled PvGLP1 cDNA probe aspreviously described (Metais et al. 2000).

Northern blot analysis was conducted according to Sambrooket al. (1989) with 10 lg of total RNA per lane and PvGLP1 cDNAused as a probe. After hybridization, filters were washed twice with2·SSPE (saline sodium phosphate EDTA), 0.1% (w/v) SDS at60 �C for 15 min, twice with 1·SSPE, 0.1% (w/v) SDS at 60 �C for15 min, and twice with 0.1·SSPE, 0.1% (w/v) SDS at room tem-perature for 15 min.

Protein extraction and western blotting

Tissues were ground in liquid nitrogen in 1 ml WG buffer (20 mMHepes, pH 7.5; 100 mMK-acetate; 3 mMMg-acetate; 1 mMDTT)per gram of tissue. The homogenate was centrifuged at 16,000 gfor 10 min: the supernatant constituted the protein fraction.

Proteins were solubilized in a suitable volume of Laemmli(1970) sample buffer [50 mM Tris–HCl (pH 6.8), 2.0% (w/v) SDS,15% (v/v) glycerol, 5% (v/v) b-mercaptoethanol]. 10 lg of proteinper sample was boiled at 95 �C for 5 min and separated by poly-acrylamide gel electrophoresis (SDS–PAGE) according to Laemmli(1970) using a 5% polyacrylamide (w/v) stacking gel and 12%polyacrylamide (w/v) resolving gel. The separated proteins weretransferred onto polyvinylidenedifluoride (PVDF) membrane(BioRad). Western blotting and immunodetection were performedin the conditions previously described by Poupard et al. (1998),

468

except protein detection which was done with the Immun-starchemiluminescent kit (BioRad), used according to the manufac-turer’s instructions. Proteins were immunodetected using a rabbitanti-AtGER3 polyclonal antibody (kindly supplied by Prof.F. Bernier, IBMP Strasbourg, France), raised against an oligo-peptide (amino acids 29–40) specific for an A. thaliana GLP,AtGER3: DPKGPQSPSGYS (Membre et al. 2000).

Results

Molecular cloning and sequence analysisof a cDNA encoding a GLP in common bean

One of the transcript-derived fragments (TDFs) gener-ated by cDNA–AFLP that accumulated specifically inembryo axes was cut out of the acrylamide gel, clonedand sequenced. It corresponded to a cDNA fragment of241 bp (Fig. 2). The expression pattern of this TDFshowed that it was not detected in axes at 9 HAI whereasit was present in axes at 24 HAI. Sequence analysis bycomparison with GenBank databases allowed us toidentify this TDF as a part of a GLP encoding sequence.Indeed, the best FASTA scores were recovered withAtGLP3a (GenBank U75203) and ABP19 from peach(GenBank U79114) cDNA sequences with 4.2e-11 and8.4e-12, respectively. RT–PCR was then performed onmRNAs extracted from 24-HAI embryo axes, followedby RACE, and all PCR products of interest were clonedand sequenced. The nucleotide sequence of the completecDNA was called PvGLP1 (accession numberAJ276491). In parallel, the use of genomic DNA asa template in PCR reactions strongly suggested the

absence of an intron in the coding sequence (data notshown). The cDNA is 838 bp with a 5¢-untranslatedregion (UTR) of 44 bp, an open reading frame (ORF) of621 bp, and a 3¢-UTR of 173 bp. Two putative polya-denylation signals were found, respectively 55 bp and144 bp downstream from the stop codon TAG. Theamino acid sequence deduced from the ORF revealedthat PvGLP1 encodes a protein of 206 amino acids(Fig. 3) with a calculated molecular weight of 21,833 Daand a theoretical isoelectric point of 7.77 (ExPASy ProtParam Tool). The three highly conserved oligopeptides(Bernier and Berna 2001) are present in the proteinsequence. Box A is QDFCVAD, but in PvGLP1 the firstresidue is N instead of Q, as in the Pharbitis nil PnGLP(Ono et al. 1996) sequence (Fig. 3). In PvGLP1, itincludes a cysteine residue, cys-23, and a second cysteine,cys-38, was retrieved in a conserved position; these resi-dues could form disulfide bonds. Box B is G-P-H-HPRATEXXXX-G and C is GXXHFQ-N-G, in whichX corresponds to any hydrophobic amino acid. Threehistidine residues known to form a histidine cluster in apotential metal-binding site (Gane et al. 1998) werefound in the PvGLP1 sequence (His-99, His-101 andHis-144). A single potential N-glycosylation site (Asn-58)was identified in PvGLP1. Interestingly, all germins andGLPs have a glycosylation site at the same location. Likemost of the plant GLPs, a putative signal peptide(Nielsen et al. 1997) of 17 amino acids was found on thePvGLP1 sequence. PvGLP1 seems to be a proteinprecursor; the mature protein of 189 amino acids has acalculated molecular weight of 19,887 Da and a theo-retical isoelectric point of 7.99. Thus, PvGLP1 may be anapoplastic glycoprotein. A phylogenetic tree deducedfrom the sequence alignment of 70 GLPs showed thatPvGLP1 is a new member of the GLP subfamily 3(Carter and Thornburg 2000). The alignment of theamino acid sequence of PvGLP1 with members ofdifferent families of GLPs and with the wheat germinencoded by the reference gene gf-2.8 is shown in Fig. 3.The best score was recovered with the sequence ofABP19, an auxin-binding protein from peach, reachinga percentage identity of 60.4%. Moreover, inside theregion 96–113 of PvGLP1 Box B, six of the seven aminoacids highly conserved between the putative Box-A auxin-binding site of maize ABP1 (Inohara et al. 1989) andpeach ABPs (Ohmiya et al. 1998) were present (Fig. 3).

Southern blot analysis revealed that the PvGLP1 geneis present in the common bean genome as a single copy(Fig. 4). However, decreasing stringency allowed us todetect other faint bands, suggesting that other genesencoding GLP isoforms would be present in the com-mon bean genome.

Expression of PvGLP1 during germinationin embryo axes

Expression of PvGLP1 was studied by Northern blotanalysis in embryo axes throughout the germination

Fig. 2 Germination-associated changes in the embryo axes, andcotyledon transcript contents, of P. vulgaris as determined bycDNA–AFLP. mRNAs were purified from seed axes 9 and 24 HAI(lanes A; two and three lanes, respectively) and from cotyledons24 HAI (lanes C). In this experiment, the cDNAs were amplifiedwith the 5¢-GATGAGTCCTGAGTAACT-3¢ and 5¢-GACTGCGTACCAATTCAC-3¢ primer combination. The arrow indi-cates a TDF not detected at 9 HAI but present at 24 HAI.Lanes M 10-bp DNA ladder (Life Technologies)

469

process from 0 HAI up to 36 HAI (Fig. 5). Up to9 HAI, PvGLP1 expression in embryo axes was at thesame level as that detected in the dry seed (0 HAI).Between 9 and 17 HAI, before radicle emergence, ex-pression of PvGLP1 strongly increased and remainedconstant thereafter.

In order to detect the presence of the PvGLP1 proteinin embryo axes during germination, immunoblot analysiswas performed using antibodies raised specifically againstthe GLP AtGER3 from A. thaliana (Membre et al. 2000).A protein cross-reacted with the antiserum at all stagesduring embryo axis development at a steady-state level(0–24 HAI; Fig. 6, lanes a). Its molecular weight, esti-mated to be approximately 23 kDa in comparison tothe molecular weight marker (not shown) and theAtGER3monomer detected inA. thaliana (Membre et al.2000), is in agreement with the molecular weight calcu-lated from the PvGLP1 peptide sequence (19,887 kDa)considering that GLPs are glycosylated proteins.

Expression of GLPs in mature vegetative tissues

PvGLP1 expression in mature vegetative tissues (root,stems, leaves and pods) was analyzed by eitherNorthern blotting (data not shown) or RT–PCR withPvGLP1 sequence-specific primers. Except for a lowexpression detected by RT–PCR in pods, no expres-sion was found in the other organs (Fig. 7a). The useof GLP sequence-degenerate primers, however, led tocDNA amplification in all tissues showing variablelevels of expression depending on the organ; the GLPtranscripts were more abundant in roots and leavesthan in pods, and were only faintly detected in stems(Fig. 7b). PvGLP1 protein was not detected by im-munoblot analysis in roots, stems or leaves (Fig. 6,lanes b). Expression of PvGLP1 seemed to occurspecifically in embryo axis while expression of otherGLP isoforms could be detected in mature vegetativetissues.

Fig. 3 Comparison of theamino acid sequence deducedfrom the PvGLP1 cDNA withsequences of the three GLPsubfamily members, one truegermin protein and the putativeauxin-binding site Box A ofmaize ABP1 (Inohara et al.1989). The GenBank accessionnumbers of the sequencesanalyzed are: for the GLPsubfamily 3: PvGLP1,AJ276491; PpABP19, U79114;AtGLP3b, U75195; PnGLP,D45425; for the GLPsubfamily 2: NectarinI,AF132671; for the GLPsubfamily 1: AtGLP2b,X91957; for the true germinsubfamily: Ta2.8, M63223.Gaps were introduced tooptimize the alignments(Corpet 1988). The threeconserved boxes A, B and C areframed

470

Effect of indole-3-acetic acid (IAA)on PvGLP1 expression

The high sequence identity of PvGLP1 with ABP19prompted us to check out the effect of IAA on theexpression of PvGLP1 during early stages of germina-tion. Several IAA concentrations (1, 5, 10 and 100 lM)were tested and PvGLP1 expression in embryo axes wasfollowed by RT–PCR with the PvGLP1-specific primersat 4, 9 and 14 HAI. No effect of auxin was observed onPvGLP1 expression at low concentrations, while at100 lM there was a substantial decrease in expression in

IAA-treated embryo axes compared to the control,particularly at 14 HAI (Fig. 8). Expression of EF-1aused as a constitutive control was not affected in thesame conditions. Interestingly, the IAA effect onPvGLP1 expression was accompanied by a delay in thegermination process: the T50 of IAA-treated seeds(27 HAI) was 2 h longer than that in the control.

Discussion

PvGLP1 encoding a GLP is specifically expressed in theembryo axis of common bean during germination

Germins are a very homogeneous group of proteinsexhibiting oxalate oxidase activity, identified in germinating

Fig. 5 Northern blot analysis of the expression of the PvGLP1gene during and following germination of common bean. TotalRNA was extracted from axes of dry and imbibed seeds (from 0 to36 HAI). RNA samples (10 lg/lane) were separated on a 1.2%formaldehyde gel, blotted and hybridized with a32P-labeledPvGLP1 cDNA. Equal loading was checked by direct visualizationof 25S and 18S rRNAs on the membrane under UV exposure

Fig. 6 Detection of PvGLP1 by Western blot analysis. Twentymicrograms of proteins from common bean seed axes (lanes a; 0, 9,17, 24 HAI) anddifferent vegetative tissues (lanes b), roots (R), stems(S) and leaves (L), previously heat-denatured were separated on a12% SDS–PAGE gel, blotted and reacted with antibodies raisedagainst the N-terminal peptide (amino acids 29–40) of Arabidopsisthaliana AtGER3. In parallel, proteins extracted from leaves ofA. thaliana and heat-denatured were used as a control (lane At)

Fig. 4 Southern blot analysis of the PvGLP1 gene. Common beangenomic DNA (15 lg each lane) was digested with EcoRI (lane 1)and EcoRV (lane 2) and hybridized with a-32P-labeled PvGLP1cDNA under high-stringency conditions. Size markers are indicatedon the left in kb

Fig. 7a–c Search for PvGLP1 expression in mature vegetativetissues of common bean by RT–PCR. One microgram of totalRNA purified from roots (R), stems (S), leaves (L), pods (P) andaxes (A) at the 24-HAI stage was used for reverse transcription andcDNAs were submitted to 35 cycles of PCR with PvGLP1sequence-specific primers (a) and GLP sequence-degenerate prim-ers (b). In parallel, RT–PCR was performed with primers specificfor the translation elongation factor EF-1a gene as a control (c).PCR products were analyzed on a 1.4% agarose gel. Somemolecular size markers are indicated on the left (1-kb DNA ladder;Life Technologies)

471

embryos of monocots. The search for germin-relatedproteins in dicots, on the basis of sequence identity, ledto the discovery of the group of GLPs. Characterizationof this group of proteins was mainly carried out in A.thaliana (Carter et al. 1998; Membre et al. 2000) andtobacco (Carter and Thornburg 2000). It yielded severaldifferences between germins and GLPs, the most strikingamong them are as follows: while germins are specific tomonocots, GLP genes are expressed in all angiosperms,in gymnosperms and mosses; the majority of knownGLPs are not oxalate oxidases; and no GLP gene dis-covered so far has been found to be specifically ex-pressed in seeds during germination (Bernier and Berna2001). Thus not one of the already-known GLPs couldbe considered as a valuable marker gene/protein ofgermination and onset of growth of embryos, and thediscovery of this kind of marker in dicots is still awaited.

The use of the cDNA–AFLP approach allowed us toidentify a transcript-derived fragment specificallyexpressed in embryo axes of P. vulgaris during germi-nation. Comparison of the complete cDNA and deducedamino acid sequences with the available databasesrevealed a high homology with GLPs. Furthermore, thepresence of three highly conserved oligopeptides, desig-nated A, B and C (Fig. 3; Bernier and Berna 2001), anda single N-glycosylation site (Asn-58) led us to proposethat this polypeptide corresponds to a GLP that wenamed PvGLP1, the first GLP described in P. vulgaris(accession number AJ276491).

Expression studies of PvGLP1 by northern blotanalysis, carried out on RNA from embryo axes at dif-ferent times of imbibition produced three major results.First, the transcripts of PvGLP1 are stored in the dryseed, as was previously shown for gene transcriptionproducts that are needed during the early stages ofgermination, i.e. b-tubulin genes (Vassilevskaia et al.1996) and LA-ACS1, an isoform of 1-aminocyclopro-pane-1-carboxylate-synthase (Bekman et al. 2000). Sec-ond, the transcripts of PvGLP1 in the embryo axisstrongly increased a few hours before radicle emergence(Fig. 5). Third, PvGLP1 mRNAs were not detected in

mature vegetative tissues (roots, stems, leaves), exceptvery faintly in pods using RT–PCR (Fig. 7a). To ourknowledge, this is the first report on an embryo-specificGLP of a dicot species whose gene expression stronglyincreases during germination.

Genomic Southern blot analysis under low-stringencyconditions indicated the existence of several GLPisoforms in the P. vulgaris genome. This result wasconfirmed by using GLP degenerate primers in RT–PCRreactions in which GLP transcripts were detected in alltissues at various abundances (Fig. 7b). They were moreabundant in roots and leaves than in pods, and wereonly faintly detected in stems. These results suggest theexistence of a GLP multigenic family in common bean,as is the case in most plants expressing GLPs, with aspecific expression pattern for each of the members.Systematic genomic sequencing or EST analyses allowedthe identification of 27, 8 and 14 different GLP-encodinggenes in A. thaliana, rice and barley, respectively(Bernier and Berna 2001), and a recent study allowed124 different germins and GLP cDNAs, distributed intofive subfamilies, to be distinguished in barley (Drukaet al. 2002).

Immunoblot analysis using antibodies raised againstAtGER3 from A. thaliana (Fig. 6) allowed us to detectPvGLP1 in the dry seed and embryo axes at all stages ofgermination and, interestingly, the protein was presentat the same amount at all analyzed stages (Fig. 6a). Thisresult confirms that PvGLP1 is expressed during embryodevelopment, and that mRNA and protein are stored inthe dry seed, supporting the idea that PvGLP1 would beneeded during the early stages of germination. Consis-tent with the results of PvGLP1 expression, PvGLP1protein was detected neither in roots nor in stems andleaves (Fig. 6b) confirming its embryo axis specificitywith probably an important role in the germinationprocess. A variation in mRNA level not accompanied bya variation in the protein level was previously reportedfor the expression of a GLP in mustard (Heintzen et al.1994): whereas the mRNA level drastically fluctuatedduring dark/light cycles, the amount of protein wasmaintained at a fairly constant level. It is still intriguingthat the large increase in mRNA was not accompaniedby a variation in protein level. The increase in geneexpression and protein neo synthesis could be bufferedby a high protein turnover; however, we cannot rule outthat AtGER3 antibodies could recognize two GLP iso-forms in the bean embryo axis, one corresponding to anisoform stored in the dry seed and the other to PvGLP1that would be synthesized after imbibition, followingPvGLP1 mRNA synthesis.

PvGLP1 a putative auxin receptor (ABP)in germinating embryos of P. vulgaris

Several lines of evidence led us to put forward thehypothesis that PvGLP1 is an auxin receptor (ABP)operating in embryos during germination. Comparison

Fig. 8 Effect of IAA on PvGLP1 expression. One microgram oftotal RNA purified from axes of untreated or 100 lM IAA-treatedcommon bean seeds at 0, 4, 9 and 14 HAI was used for RT–PCRwith PvGLP1 sequence-specific primers. In parallel, the sameexperiment was performed with primers specific for the translationelongation factor EF-1a gene. PCR products were analyzed on a1.4% agarose gel. Some molecular size markers are indicated on theleft (1-kb DNA ladder; Life Technologies)

472

of the deduced amino acid sequence of PvGLP1 withdatabases showed the highest identity score (60.4%)with ABP19 (Ohmiya et al. 1998), a GLP that plays therole of an auxin-binding protein in shoot apices of peachtree (Ohmiya et al. 1993). Furthermore, the Box B ingermins and GLPs (Fig. 3) coincides with the Box Adescribed as a putative auxin-binding site in maize ABP1and its homologs (Jones 1994). It has been speculatedthat conserved residues of this domain would constitutethe core of the ligand-binding site whereas variableresidues would allow the specificity of the interaction(Membre et al. 2000). Between ABP19 and ABP1, 7 outof 18 amino acids constituting this domain are conserved(Ohmiya et al. 1998); it is noteworthy that 6 of these 7amino acids are also conserved in PvGLP1, strength-ening the hypothesis that PvGLP1 might play an ABProle in germinated embryos of common bean.

However, this result raises the question of the physi-ological relevance of the presence of an ABP and subse-quently the role of auxin in embryos during germination.The role of auxin in germination and seedling develop-ment is more discreet than its role in other developmentalprocesses such as apical dominance, tropisms, shootelongation, induction of cambial division and lateral rootinitiation (Tamas 1987; Lomax et al. 1995). However, itseems that auxin plays a significant role either in the earlyor late stages of germination and seedling growth. InLupinus albus, it was recently shown that auxin controlsthe production of ethylene by controlling the expressionof several 1-aminocyclopropane-1-carboxylate synthasegenes in the embryo axis at the time of radicle emergenceand later in the hypocotyl (Bekman et al. 2000). It wasshown that auxin intervenes in hypocotyl growth bypromoting cell elongation and that, later, shoot-derivedauxin is essential for early lateral root emergence (Bhal-erao et al. 2002). Auxin needed for germination and initialseedling growth is not synthesized de novo; it derives frommobilization of IAA stored in the embryo as free andester-linked conjugates of IAA and indol-3 ethanol(Epstein et al. 1980; Ljung et al. 2001). Storage in theembryo itself in a readily used form allows immediateaccess of the hormone to targeted tissues. It is then con-ceivable that an auxin receptor should also be present inthe dry seed and its level controlled throughout the ger-mination process. We have shown that the PvGLP1transcript is present in the dry seed and that the proteinlevel seems to be kept constant. Our results are consistentwith the requirement for auxin from the early stages ofgermination onwards (Bekman et al. 2000).

We were interested to test whether the synthesis ofPvGLP1, a good candidate for the role of auxin receptor(ABP), would be also regulated by auxin. The resultsobtained by RT–PCR from embryo axes of bean seedsgerminated with various IAA concentrations (1, 5, 10,100 lM) showed that, in contrast to wheat germin gf-2.8whose expression is stimulated by auxin (Berna andBernier 1997), PvGLP1 expression is not induced byIAA (Fig. 8). However, Berna and Bernier worked on agermin with an oxalate oxidase activity while PvGLP1

most probably plays a different role. Furthermore, it hasbeen shown that besides the control of the concentrationof the hormone and its ratio to other hormones, thesensitivity/responsiveness of tissues to IAA determinedby auxin receptors is a primary determinant in the con-trol of correct auxin signaling and homeostatic balancenecessary for the regulation of auxin action (Michalczuket al. 1992; Ljung et al. 2001). Then it makes sense thatsynthesis of ABP might not be induced by auxin itself;rather, it is developmentally controlled. Transcriptionalfusions of the 5¢-flanking region to a reporter gene arecurrently being used in plant transformation experimentsto help to elucidate the regulatory mechanism ofPvGLP1 gene expression.

Acknowledgments We thank Prof. F. Bernier (IBMP, Strasbourg,France) for providing anti-AtGER3 polyclonal antibodies and forcritical comments on the manuscript. C. Aubry and M.-C. Morere-Le Paven contributed equally to this work.

References

Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, MillerW, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs. Nucleic AcidsRes 25:3389–3402

Bachem CW, van der Hoeven RS, de Bruijn SM, Vreugdenhil D,Zabeau M, Visser RG (1996) Visualization of differential geneexpression using a novel method of RNA fingerprinting basedon AFLP: analysis of gene expression during potato tuberdevelopment. Plant J 9:745–753

Baskin CC, Baskin JM (1998) Seeds—ecology, biogeography, andevolution of dormancy and germination. Academic Press, SanDiego

Bekman EP, Saibo NJ, Di Cataldo A, Regalado AP, Ricardo CP,Rodrigues-Pousada C (2000) Differential expression of fourgenes encoding 1-aminocyclopropane-1-carboxylate synthase inLupinus albus during germination, and in response to indole-3-acetic acid and wounding. Planta 211: 663–672

Berna A, Bernier F (1997) Regulated expression of a wheat ger-min gene in tobacco: oxalate oxidase activity and apoplasticlocalization of the heterologous protein. Plant Mol Biol33:417–429

Berna A, Bernier F (1999) Regulation by biotic and abiotic stress ofa wheat germin gene encoding oxalate oxidase, a H2O2-pro-ducing enzyme. Plant Mol Biol 39:539–549

Bernier F, Berna A (2001) Germins and germin-like proteins: plantdo-all proteins. But what do they do exactly? Plant PhysiolBiochem 39:545–554

Bhalerao RP, Eklof J, Ljung K, Marchant A, Bennett M, SandbergG (2002) Shoot-derived auxin is essential for early lateral rootemergence in Arabidopsis seedlings. Plant J 29:325–332

Bringezu K, Lichtenberger O, Leopold I, Neumann D (1999)Heavy metal tolerance of Silene vulgaris. J Plant Physiol154:536–546

Caliskan M, Cuming AC (1998) Spatial specificity of H2O2-gen-erating oxalate oxidase gene expression during wheat embryogermination. Plant J 15:165–171

Carter C, Thornburg RW (2000) Tobacco Nectarin I. Purificationand characterization as a germin-like, manganese superoxidedismutase implicated in the defense of floral reproductive tis-sues. J Biol Chem 275:36726–36733

Carter C, Graham RA, Thornburg RW (1998) Arabidopsis thalianacontains a large family of germin-like proteins: characterizationof cDNA and genomic sequences encoding 12 unique familymembers. Plant Mol Biol 38:929–943

473

Carter C, Graham RA, Thornburg RW (1999) Nectarin I is anovel, soluble germin-like protein expressed in the nectar ofNicotiana sp. Plant Mol Biol 41:207–216

Chateigner AL, Le Deunff Y, Jalouzot R (1999) Germination-as-sociated changes in the transcript content of pea seedlings li-poxigenases. Lipoxigenase-g: a new marker of axis growthresumption. Planta 208:606–613

Corpet F (1988) Multiple sequence alignment with hierarchicalclustering. Nucleic Acids Res 16:10881–10890

Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA micro-preparation: version II. Plant Mol Biol Rep 1:19–21

Druka A, Kudrna D, Kannangara CG, von Wettstein D, KleinhofsA (2002) Physical and genetic mapping of barley (Hordeumvulgare) germin-like cDNAs. Proc Natl Acad Sci USA99:850–855

Dumas B, Sailland A, Cheviet JP, Freyssinet G, Pallett K (1993)Identification of barley oxalate oxidase as a germin-like protein.C R Acad Sci Life Sci 316:793–798

Dumas B, Freyssinet G, Pallett KE (1995) Tissue-specific expres-sion of germin-like oxalate oxidase during development andfungal infection of barley seedlings. Plant Physiol107:1091–1096

Epstein E, Cohen JD, Bandurski RS (1980) Concentration andmetabolic turnover of indoles in germinating kernels of Zeamays L. Plant Physiol 65:415–421

Fath A, Bethke PC, Jones RL (2001) Enzymes that scavenge re-active oxygen species are down-regulated prior to gibberellicacid-induced programmed cell death in barley aleurone. PlantPhysiol 126:156–166

Frohman MA, Dush MK, Martin GR (1988) Rapid production offull-length cDNAs from rare transcripts: amplification using asingle gene-specific oligonucleotide primer. Proc Natl Acad SciUSA 85:8998–9002

Gane PJ, Dunwell JM, Warwicker J (1998) Modeling based on thestructure of vicilins predicts a histidine cluster in the active siteof oxalate oxidase. J Mol Evol 46:488–493

Garciarrubio A, Legaria JP, Covarrubias AA (1997) Abscisic acidinhibits germination of mature Arabidopsis seeds by limiting theavailability of energy and nutrients. Planta 203:182–187

Gomez-Cadenas A, Zentella R, Walker-Simmons MK, Ho THD(2001) Gibberellin/abscisic acid antagonism in barley aleuronecells: site of action of the protein kinase PKABA1 in relation togibberellin signaling molecules. Plant Cell 13:667–679

Heintzen C, Fischer R, Melzer S, Kappeler S, Apel K, Staiger D(1994) Circadian oscillations of a transcript encoding a germin-like protein that is associated with cell walls in young leaves ofthe long-day plant Sinapis alba L. Plant Physiol 106:905–915

Huang Z, Fasco MJ, Kaminsky LS (1996) Optimization of DNaseIremoval of contaminating DNA from RNA for use in quanti-tative RNA–PCR. Biotechniques 20:1012–1020

Hurkman WJ, Tanaka CK (1996) Germin gene expression is in-duced in wheat leaves by powdery mildew infection. PlantPhysiol 111:735–739

Inohara N, Shimomura S, Fukui T, Futai M (1989) Auxin-bindingprotein located in the endoplasmic reticulum of maize shoots:molecular cloning and complete primary structure. Proc NatlAcad Sci USA 86:3564–3568

Jones AM (1994) Auxin-binding proteins. Annu Rev Plant PhysiolPlant Mol Biol 45:393–420

Laemmli UK (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227:680–685

Lane BG, Dunwell JM, Ray JA, Schmitt MR, Cuming AC (1993)Germin, a protein marker of early plant development, is anoxalate oxidase. J Biol Chem 268:12239–12242

Limami MA, Rouillon C, Glevarec G, Gallais A, Hirel B (2002)Genetic and physiological analysis of germination efficiency inmaize in relation to nitrogen metabolism reveals the impor-tance of cytosolic glutamine synthetase. Plant Physiol130:1860–1870

Ljung K, Ostin A, Lioussanne L, Sandberg G (2001) Develop-mental regulation of indole-3-acetic acid turnover in scots pineseedlings. Plant Physiol 125:464–75

Lomax TL, Muday GK, Rubery P (1995) Auxin transport. In:Davies PJ (ed) Plant hormones: physiology, biochemistry,and molecular biology, 2nd edn. Kluwer, Dordrecht, pp 509–530

Membre N, Berna A, Neutelings G, David A, David H, Staiger D,Vasquez JS, Raynal M, Delseny M, Bernier F (1997) cDNAsequence, genomic organization and differential expression ofthree Arabidopsis genes for germin/oxalate oxidase-like pro-teins. Plant Mol Biol 35:459–469

Membre N, Bernier F, Staiger D, Berna A (2000) Arabidopsisthaliana germin-like proteins: common and specific featurespoint to a variety of functions. Planta 211:345–354

Metais I, Aubry C, Hamon B, Jalouzot R, Peltier D (2000) De-scription and analysis of genetic diversity between commercialbean lines (Phaseolus vulgaris L.), Theor Appl Genet 101:1207–1214

Michalczuk L, Ribnicky DM, Cooke TJ, Cohen JD (1992) Regu-lation of indol-3-acetic acid pathways in carrot cell cultures.Plant Physiol 100:1346–1353

Michalowski CB, Bohnert HJ (1992) Nucleotide sequence of aroot-specific transcript encoding a germin-like protein from thehalophyte Mesembryanthemum crystallinum. Plant Physiol100:537–538

Neutelings G, Domon JM, Membre N, Bernier F, Meyer Y, DavidA, David H (1998) Characterization of a germin-like proteingene expressed in somatic and zygotic embryos of pine (Pinuscaribaea Morelet). Plant Mol Biol 38:1179–1190

Nielsen H, Engelbrecht J, Brunak S, Von Heijne G (1997) Identi-fication of prokaryotic and eucaryotic signal peptides and pre-diction of their cleavage sites. Prot Eng 10:1–6

Ohmiya A, Motoyuki K, Sakai S, Hayashi T (1993) Purificationand properties of an auxin-binding protein from the shoot apexof peach tree. Plant Cell Physiol 34:177–183

Ohmiya A, Tanaka Y, Kadowaki KI, Hayashi T (1998) Cloning ofgenes encoding auxin-binding proteins (ABP19/20) from peach;significant peptide sequence similarity with germin-like pro-teins. Plant Cell Physiol 39:492–499

Ono M, Sage-Ono K, Inoue M, Kamada H, Harada H (1996)Transient increase in the level of mRNA for a germin-likeprotein in leaves of the short-day plant Pharbitis nil during thephotoperiodic induction of flowering. Plant Cell Physiol37:855–61

Pearson WR, Lipman DJ (1988) Improved tools for biologicalsequence comparison. Proc Natl Acad Sci USA 85:2444–2448

Poupard P, Strullu DG, Simoneau P (1998) Two members of theBet v 1 gene family encoding birch pathogenesis-related pro-teins display different patterns of roots expression and wound-inducibility. Aust J Plant Physiol 25:459–464

Pritchard SL, Charlton WL, Baker A, Graham IA (2002) Germi-nation and storage reserve mobilization are regulated inde-pendently in Arabidopsis. Plant J 31:639–647

Rodrıguez-Lopez M, Baroja-Fernandez E, Zandueta-Criado A,Moreno-Bruna B, Munoz FJ, Akazawa T, Pozueta-RomeroJ (2001) Two isoforms of a nucleotide-sugar pyrophosphatase/phosphodiesterase from barley leaves (Hordeum vulgare L.) aredistinct oligomers of HvGLP1, a germin-like protein. FEBSLett 490:44–48

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: alaboratory manual, 2nd edn. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, New York

Schweizer P, Christoffel A, Dudler R (1999) Transient expressionof members of the germin-like gene family in epidermal cells ofwheat confers disease resistance. Plant J 20:541–552

Swart S, Logman TJJ, Smit G, Lugtenberg BJJ, Kijne JW (1994)Purification and partial characterization of a glycoproteinfrom pea (Pisum sativum) with receptor activity for rhicad-hesin, an attachment protein of Rhizobiaceae. Plant Mol Biol24:171–183

Tamas IA (1987) Hormonal regulation of apical dominance. In:Davies PJ (ed) Plant hormones and their role in plant growthand development. Nijhoff, Boston, pp 393–410

474

Thompson EW, Lane BG (1980) Relation of protein synthesis inimbibing wheat embryos to the cell-free translational capacitiesof bulk mRNA from dry and imbibing embryos. J Biol Chem255:5965–5970

Vallelian-Bindschedler L, Mosinger E, Metraux JP, Schweizer P(1998) Structure, expression and localization of a germin-likeprotein in barley (Hordeum vulgare L.) that is insolubilized instressed leaves. Plant Mol Biol 37:297–308

Vassilevskaia TD, Bekman E, Jackson P, Pinto Ricardo C,Rodrigues-Pousada C (1996) Developmental expression andregulation by light of two closely related beta-tubulin genes inLupinus albus. Plant Mol Biol 32:1185–1189

Verwoerd TC, Dekker BM, Hoekema A (1989) A small-scaleprocedure for the rapid isolation of plant RNAs. Nucleic AcidsRes 17:2362

Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M,Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995)AFLP: a new technique for DNA fingerprinting. Nucleic AcidsRes 23:4407–4414

Wojtaszek P, Stobiecki M, Bolwell GP (1997) Changes in thecomposition of exocellular proteins of suspension-culturedLupinus albus cells in response to fungal elicitors or CuCl2. J ExpBot 48:2015–2021

Woo EJ, Dunwell JM, Goodenough PW, Pickersgill RW (1998)Barley oxalate oxidase is a hexameric protein related to seedstorage proteins: evidence from X-ray crystallography. FEBSLett 437: 87–90

Yamahara T, Shiono T, Suzuki T, Tanaka K, Takio S, Sato K,Yamazaki S, Satoh T (1999) Isolation of a germin-likeprotein with manganese superoxide dismutase activity fromcells of a moss, Barbula unguiculata. J Biol Chem 274:33274–33278

Ziadi S, Poupard P, Brisset MN, Paulin JP, Simoneau P (2001)Characterization in apple leaves of two subclasses of PR-10transcripts inducible by acibenzolar-S-methyl, a functionalanalogue of salicylic acid. Physiol Mol Plant Pathol 59:33–43

475