A Stress-Associated Protein Containing A20/AN1 Zing-Finger Domains

Plant Physiology and Biochemistry 49 (2011) 303e310

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Plant Physiology and Biochemistry

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Research article A stress-associated protein containing A20/AN1 zing-ﬁnger domains expressed in Medicago truncatula seeds

Christine Gimeno-Gilles 1, Marie-Laure Gervais 1, Elisabeth Planchet*, Pascale Satour, Anis M. Limami, Eric Lelievre

University of Angers, UMR-1191 Physiologie Moléculaire des Semences, IFR 149 Quasav, 2 Boulevard Lavoisier, 49045 Angers Cedex 01, France article info abstract

Article history: MtSAP1 (Medicago truncatula stress-associated protein 1) was revealed as a down-regulated gene by Received 15 July 2010 suppressive subtractive hybridization between two mRNA populations of embryo axes harvested before Accepted 2 January 2011 and after radicle emergence. MtSAP1 is the first gene encoding a SAP with A20 and AN1 zinc-finger Available online 13 January 2011 domains characterized in M. truncatula. MtSAP1 protein shares 54% and 62% homology with AtSAP7 (Arabidopsis thaliana) and OsiSAP8 (Oryza sativa) respectively, with in particular a strong homology in the Keywords: A20 and AN1 conserved domains. MtSAP1 gene expression increased in the embryos during the acqui- A20/AN1 zinc-finger domain sition of tolerance to desiccation, reached its maximum in dry seed and decreased dramatically during Abiotic stress fi Desiccation the rst hours of imbibition. Abiotic stresses (cold and hypoxia), abscisic acid and desiccation treatments Medicago truncatula induced MtSAP1 gene expression and protein accumulation in embryo axis, while mild drought stress did SAP not affect significantly its expression. This profile of expression along with the presence of anaerobic Seed response elements and ABRE sequences in the upstream region of the gene is consistent with a role of MtSAP1 in the tolerance of low oxygen availability and desiccation during late stages of seed maturation. Silencing of MtSAP1 by RNA interference (RNAi) showed that the function of the encoded protein is required for adequate accumulation of storage globulin proteins, vicilin and legumin, and for the development of embryos able to achieve successful germination. Ó 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction environmental challenges such as drought, salt, hypoxia, cold stress and pathogen attacks [2,3]. The tolerance to low oxygen availability Seed maturation is an important phase of seed development. in plants includes the induction of the so called ‘anaerobic’ poly- According to several plant models, this process can be divided into peptides [4], which the best characterized are enzymes involved in two stages, morphogenesis and maturation [1]. Seed maturation metabolic pathways related to oxidative catabolism of sugars begins when developing embryos cease their cell division, and start (glycolysis, ethanol and lactic fermentation) and carbon skeleton growing by cell enlargement and accumulating storage reserves. storage as amino acids (alanine fermentation). Maturation ends with a desiccation phase after which the embryo Stress-associated protein (SAP) families were characterized by enters into a quiescent state, thereby permitting its maintenance the presence of A20/AN1 domains. The A20 zinc-finger (ZnF) and survival under a range of environmental conditions. In domain was first identified in a TNF-a inducible protein in human maturing seeds, the embryo is challenged by two adverse condi- cells and is characterized by multiple Cys2/Cys2 finger motifs. The tions that are the low oxygen availability and the desiccation of the AN1 ZnF domain was first identified in the C-terminus of the tissues. Several genes regulated by the phytohormone abscisic acid ubiquitin-like protein coded by the Xenopus laevis animal hemi- (ABA) are induced in the embryo, besides to those required for the sphere 1 (AN1) maternal RNA [5] and is usually found associated synthesis of storage reserves they include those involved in the with the A20 ZnF domain. The AN1-type ZnF contains six conserved acquisition of desiccation tolerance. Actually, in most vegetative cysteines and two histidines that could potentially coordinate two tissues, ABA mediates several aspects of physiological responses to zinc atoms [5]. The role of the A20/AN1 proteins has been well studied in animal immune systems [6,7]. In plants, very few information is known about A20/AN1 proteins. Vij and Tyagi [8],by screening public databases, have conducted a survey of A20/AN1 * Corresponding author. Tel.: þ33 2 41 73 53 83; fax: þ33241735456. E-mail address: [email protected] (E. Planchet). ZnF protein across diverse organisms with a special emphasis 1 These authors contributed equally to this work. on plants (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa,

Sorghum bicolor and Vitis vinifera). All the species contain between (62%) and Arabidopsis AtSAP7 (54%) (Fig. 2B), but also with virtually one to nineteen A20/AN1 proteins. From A. thaliana and O. sativa translated ESTs of Lotus japonicus (TC14575, 87%) and soya bean genomes, fourteen and eighteen genes code for SAP respectively, (ACU14538, 77%), and presents identical ZnF domains (Fig. 2B). In and most of them encode proteins with A20/AN1 domains. comparison to some human proteins containing A20/AN1 domains, A member of SAP gene family from rice OsiSAP1 (O. sativa stress- MtSAP1 shares 46% and 47% homology with human ZnF216 and associated protein 1) was the first plant protein identified as a ZnF AWP1 proteins respectively, and shows identities in the ZnF protein and has been shown to be induced in rice seedlings domains (data not shown). in response to environmental challenges and abscisic acid (ABA) treatment, and to confer abiotic stress tolerance to transgenic 2.2. MtSAP1 gene expression in embryo axis during maturation, tobacco over-expressing the gene [9]. Recently, the over-expression germination and post-germination of M. truncatula of OsiSAP8 has been shown to confer tolerance to salt, drought and cold stress [10] suggesting that protein encoded by this gene is The expression of MtSAP1 in embryo axis was investigated by likely to intervene in the stress signaling pathway. The mechanism real RT-PCR during seed development, germination and post- by which the over-expression of OsiSAP8 conferred stress tolerance germination growth (Fig. 3). During seed development, gene is still unknown. The authors demonstrated that A20 and AN1-type transcription exhibited a low level during the first 20 days after ZnF domains from the protein OsiSAP8 interact with each other but pollination (DAP). A strong increase in MtSAP1 gene expression was only A20 interacts with itself in a yeast two-hybrid system. observed after 28 and 35 DAP when desiccation was complete in Our strategy for improving knowledge of crucial events of the dry seed. Five hours after imbibition, amounts of mRNA germination was based on a suppressive subtractive hybridization decreased and picked up a low background expression after 25 h fi (SSH) transcriptomic analysis of embryo axis for the identi cation and remained constant even after 96 h. These data show of genes involved in germination completion. The subtraction was that MtSAP1 gene expression kinetic is correlated with a progres- carried out between two mRNA populations extracted at two sive tissue dehydration occurring in the last 10 days of seed imbibition stages, at the end of the phase of passive and massive development. uptake of water and after germination completion characterized by radicle protrusion. In our previous studies, our attention was focused on up-regulated genes with the attempt to unravel their 2.3. Analysis of RNAi MtSAP1 lines involvement in the control and the completion of germination process [11,12]. At the opposite, in the present work we focused our Plants disrupted in the expression of MtSAP1 gene were attention on a down-regulated gene that encodes a SAP protein. obtained by RNAi technology. These MtSAP1 silencing plants dis- Only little work was dedicated to SAP proteins in plants, and to our played fewer leaves than the control plants, and furthermore, the best of knowledge this is the first study dedicated to a SAP protein leaves became chlorotic in comparison to the WT (data not shown). expressed in seed. We have shown that MtSAP1 gene expression From seven transgenic lines selected by PCR, three lines were increased in embryos during maturation reaching its maximum further analysed by immunoblotting with anti-MtSAP1 antibody on during tolerance of desiccation phase and declining after few hours the mature dry seeds. The MtSAP1 protein was highly abundant of seed imbibition. The expression of the gene and the quantity of in the mature WT seeds and faintly detectable in the mature seeds the protein were re-induced after germination in embryo axes by of the transgenic line (Fig. 4A). The disruption of the expression of ABA treatment and hypoxia and desiccation stresses. Seeds of RNAi MtSAP1 resulted in a severe seed phenotype. The transgenic seeds transgenic plants in which MtSAP1 gene expression was disrupted were smaller in terms of size and weight compared to the WT showed lower level of storage globulin proteins, vicilin and legu- (Fig. 4B and C). The analysis of the seed protein content showed that min, and deficient germination. the total protein content was not affected, while the accumulation of legumin and vicilin, storage globulin proteins, was specifically 2. Results reduced in RNAi seeds in comparison to the WT seeds, even if the reduction of vicilin content in RNAi lines appeared to less signifi- 2.1. Cloning of MtSAP1 and in silico analysis cant (Fig. 4D). A germination test run on seeds from WT and three RNAi lines An SSH library was constructed from embryo axis between two was carried out. The disruption of the expression of MtSAP1 early steps of Medicago truncatula seed germination (6 h and 23 h severely affected the germination capacity of the transgenic lines. after imbibition). Sequencing was performed on 900 clones, and The percentage of germinated seeds in WT was 78% after 48 h of sequence analysis and comparison revealed 355 unique ESTs. imbibition, while it hardly reached 12% in the RNAi seeds during Among them, 68 ESTs were down-regulated between 6 h and 23 h, the same period (Fig. 5). with in particular MtSAP1 (TC95263) for which the full-length sequence has already been cloned [12]. The genomic sequence of 2.4. Induction of MtSAP1 gene expression by ABA MtSAP1 (UniProtKB/TrEMBL entry A2Q3D5) is located on chromo- during seed imbibition some 7. The coding sequence of MtSAP1 (Fig. 1A) is intronless, except two introns of 1137 bp and 92 bp in the 50UTR region. The MtSAP1 gene expression was strongly re-induced in embryo protein sequence is constituted of 172 amino acids with a predicted axes treated with ABA (100 mM). The expression was six to eightfold molecular mass of 18.8 kDa and contains two ZnF domains, A20 and higher than in the control germinated on water (Fig. 6A). The gene AN1 (Fig. 1B). The upstream region (2.5 kb) of the MtSAP1 gene was expression was induced in embryo axes by ABA at concentrations used to search the database for regulatory motif using PLACE ranging between 5 mM and 100 mM (data not shown). MtSAP1 database. Anaerobic-responsive elements and ABA responsive protein strongly accumulated in WT embryo axes treated with ABA element (ABRE) were identified (Fig. 1C). and failed to accumulate in RNAi embryo axes (Fig. 6B). The These A20/AN1 domains are found alone or together in eight expression of MtSAP1 was not modulated in embryo axes by other putative proteins of M. truncatula, as shown in the sequence treatments with hormones known to stimulate germination, alignments in Fig. 2A. The MtSAP1 protein sequence shares gibberrelin (100 mM) and the ethylene precursor 1-Amino- homologies not only with other plant proteins like rice OsiSAP8 Cyclopropane-1-Carboxilic acid (ACC; 100 mM) (data not shown). C. Gimeno-Gilles et al. / Plant Physiology and Biochemistry 49 (2011) 303e310 305

Fig. 1. Coding sequence (A) and deducted protein sequence (B) of MtSAP1. Shadowed amino acids represent ZnF A20 and AN1 domains. Amino acids implicated in the interaction with zinc atoms are represented in black boxes. Upstream region of the MtSAP1 gene exhibited anaerobic response element sequences and ABRE-related sequences (C).

2.5. Response of MtSAP1 to desiccation condition (Fig. 8A). A very signiﬁcant effect on the gene expression was observed under hypoxia, in which MtSAP1 level of expression The increase in MtSAP1 gene expression in the embryos during was ﬁvefold higher than in the control (Fig. 8A). In agreement with the late stages of maturation could be related to desiccation. To test gene expression data, the amount of MtSAP1 protein was largely this hypothesis, M. truncatula embryo axes were submitted after induced by cold and hypoxia in comparison to water condition, 15 h of imbibition to a treatment with high concentration of poly- whereas drought (PEG) and saline stress failed to induce MtSAP1 ethylene glycol (PEG, 3 MPa). This treatment has been shown to protein accumulation (Fig. 8B). induce desiccation in M. truncatula embryo axes [13]. In comparison with the control, MtSAP1 was over-expressed approximately 3. Discussion sixfold in desiccation condition (Fig. 7). The over-expression of the gene was accompanied by an accumulation of the protein (Fig. 7, 3.1. Expression of MtSAP1 is required for adequate storage e globulins inset). accumulation and seed germination

2.6. Response of MtSAP1 to abiotic stresses, drought, salt, Unlike the A20/AN1 ZnF proteins in humans and other model cold and hypoxia animal systems, very little information is available on such proteins in plants. In animal systems, these proteins play an important role in In order to test MtSAP1 abiotic stress-responsiveness and vali- regulating the immune response, inflammatory responses and anti- date in silico analysis of the upstream region, seedlings were apoptosis [6,17]. A transcriptomic approach on the model legume M. submitted to various stress conditions: drought stress mimicked by truncatula allowed us to isolate the first gene coding for an A20/AN1 mild PEG treatment (0.25 MPa), osmotic stress (NaCl, 200 mM), ZnF stress-associated protein expressed in an embryo during the cold (4 C) and hypoxia induced by submergence [14,15]. Proline maturation phase. MtSAP1 is strongly induced at the end of seed and alanine, commonly used as indicators of mild drought stress maturation with the maximum of expression during the period of and hypoxia respectively, were accumulated in these stressed acquisition of tolerance to desiccation and in the dry seed. The tissues (data not shown; [16]). expression of MtSAP1 declined dramatically in the embryo axis Expression analysis showed that MtSAP1 gene was not sensitive during the first hours of imbibition. This profile of expression to drought stress while a drastic saline stress (NaCl) induced suggests that the protein encoded by MtSAP1 might play a role in the a twofold increase of the gene expression, similarly to the cold embryo during maturation and acquisition of tolerance to 306 C. Gimeno-Gilles et al. / Plant Physiology and Biochemistry 49 (2011) 303e310 C. Gimeno-Gilles et al. / Plant Physiology and Biochemistry 49 (2011) 303e310 307

5 involved in proteineprotein interaction. The disruption of the expression of MtSAP1 by RNAi strategy resulted in a severe seed phenotype i.e. the absence of MtSAP1 led to a reduction of seed 4 weight and size and in particular a strong reduction (almost 70%) of ) 6 the content of storage globulin proteins, vicilin and legumin. The 3 reduction in the content of storage globulins may explain the reduction of the rate of germination in RNAi seeds. It has been shown that vicilin represents the initial source of amino acids for early 2 growth and differentiation processes in the legume Vicia sativa, and legumin serves as a bulk amino acid source for subsequent seedling growth during post-germinative growth [18]. Prevascular strands of Copy numbersCopy (10 1 embryo axis, the radicle and epidermal layers of embryo axis organs contain nearly exclusively vicilin; thus before differentiation of 0 conductive tissue and mobilization of globulins in the cotyledons (up to two days after the beginning of imbibition), embryo axis is h ed 5 h autonomous in amino acid provisionviavicilin mobilization [18e20]. 15 h 25 h 50 h 75 96 h 16 DAP18DAP20 DAP28 DAP36 DAP Dry se 3.2. MtSAP1 gene expression and protein accumulation Fig. 3. Expression proﬁle of MtSAP1 in seeds and in embryo axes during maturation are up-regulated by ABA, hypoxia and desiccation and germination. MtSAP1 gene expression was determined by quantitative real time RT-PCR. Each measure was carried out with at least two biological repeats using In order to learn more about the role of MtSAP1 in the maturing a duplicated PCR reaction for determining Ct values. Data are means SD of three replicates (DAP: days after pollination). embryo, we put forward the hypotheses that the expression of the gene might be regulated by ABA and that the protein might play a role in relation to the tolerance of either desiccation or low desiccation. Most of ZnF domain proteins act as transcriptional oxygen stress (two adverse conditions encountered in the maturing factors, however in silico analysis of MtSAP1 gene product did not seed). These hypotheses were based on (i) the expression proﬁling predict any potential nuclear localization signal. MtSAP1 might not be during maturation, (ii) the presence of ABRE and ‘anaerobic a transcription factor alternatively with its ZnF domain; it may be response’ elements in the promoter sequence of MtSAP1 and (iii)

Fig. 4. Characterization of RNAi MtSAP1 lines. (A) Western blot on protein extracts from mature seeds of WT and RNAi MtSAP1 line. SAP protein band is expected at the arrow level. The speciﬁc antibody directed against Medicago truncatula SAP1 protein was the speciﬁc peptide VSPEVPENPISNESC. (B) Comparison of seed size in WT and RNAi line. (C) Seed weight of transgenic and wild-type lines (mg seed 1). (D) Effect of RNAi-mediated silencing of MtSAP1 on protein content in mature seeds. Total amount of proteins, vicilin and legumin were measured in mature seeds of WT and transgenic RNAi MtSAP1 lines (expressed in mg protein g 1 DW). Data of RNAi line are representative of three independent transgenic lines. Data are means SD of three replicates, each one containing ten seeds.

Fig. 2. Protein sequence alignments of MtSAP1 with eight other Medicago truncatula proteins containing A20 and/or AN1 ZnF domains (A), and with A20/AN1 proteins from various plant species (B). Shadowed amino acids represent ZnF A20 and AN1 domains. Amino acids implicated in the interaction with zinc atoms are represented in red boxes. AtSAP7: Arabidopsis thaliana stress-associated protein 7; OsiSAP8: Oryza sativa stress-associated protein 8; VIRT_Gm: deducted amino acid sequence of Glycine max (ACU14538); VIRT_Lj: deducted amino acid sequence of Lotus japonicus TC14575 (VIRT ¼ virtual). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) 308 C. Gimeno-Gilles et al. / Plant Physiology and Biochemistry 49 (2011) 303e310

100 WT RNAi

20 Seed germination rate (%) 0 012243648 Time (h)

Fig. 5. Effect of absence of MtSAP1 protein on seed germination. Germination rate is Fig. 7. Effect of a dehydration inducer (PEG) on MtSAP1 gene expression in embryo the mean of ﬁfty seeds germinated under normal conditions (water) from WT or RNAi axis. Transcript copy numbers are monitored in dry seed and at different times after line seeds. Data are means SD of three independent experiments. imbibition. In order to re-induce desiccation, PEG ( 3 MPa) was added after 15 h of imbibition. Data are means SD of three replicates. The inset shows the MtSAP1 protein expression in embryo axes of Medicago truncatula seedlings under normal conditions (H O) or after PEG treatment (3 MPa). the literature in which recent works on rice and Arabidopsis showed 2 the involvement of members of the family of the A20/AN1 ZnF proteins in abiotic stresses [21]. OsiSAP1 and OsiSAP8 have been stresses and ABA treatments, and to confer tolerance to salt, shown to be induced in rice seedlings in response to various abiotic drought and cold stress to transgenic tobacco over-expressing the gene [9,10]. Several evidences are in favor of our hypotheses. The ABA-induction of the expression and protein accumulation of MtSAP1 in germinating embryo axis and the absence of accumulation of MtSAP1 in the RNAi embryo axis by ABA treatment showed clearly that MtSAP1 is up-regulated by ABA. The involvement of MtSAP1 in the tolerance of low oxygen availability in seeds during

Fig. 8. Effects of different abiotic stress conditions on MtSAP1 gene expression in Fig. 6. MtSAP1 production in embryo axes is sensitive to ABA treatment. (A) The embryo axis. (A) Seedlings were grown under drought stress (PEG, 0.25 MPa), salt MtSAP1 gene expression was represented as a ratio, comparing ABA treatment (10 mM) stress (NaCl, 200 mM), cold stress (þ4 C) and hypoxia for 24 h. MtSAP1 gene versus control condition (H2O) at different time points. Data are means SD of three expression was expressed in ratio in comparison with control (H2O). Data are replicates. (B) MtSAP1 protein expression in WT and RNAi lines under ABA treatment. means SD of three replicates. (B) MtSAP1 protein expression was determined in MtSAP1 immunoblot was realized on protein extract from embryo axes imbibed for embryo axes of Medicago truncatula seedlings by immunoblotting. Seeds were incu-

48 h in water or on ABA (10 mM). Expression under water condition in WT line was bated in H2O for 24 h before being transferred to abiotic stress (PEG, salt, cold and used as negative control. SAP protein band is expected at the arrow level. Data of RNAi hypoxia) for additional 24 h. The data are representative examples out of two inde- line are representative examples out of two independent transgenic lines. pendent experiments. C. Gimeno-Gilles et al. / Plant Physiology and Biochemistry 49 (2011) 303e310 309 late stages of maturation is supported by the induction of the structure was compared to known proteins with BLAST tool from the expression of MtSAP1 and the accumulation of the protein in ExPASy website. Clustal program has been used for multiple sequence germinating embryo axis submitted to submergence treatment alignments. (hypoxia). Gas exchanges are considerably reduced in seeds because of their cutinized cell layers and low stomatal frequency on 4.4. RNA isolation and reverse transcription pods. Concomitantly with seed desiccation, tissues lose their green color and photosynthetic activity, leading to very low oxygen For total RNA isolation, frozen embryos were crushed with content in the dry seeds [22]. The regulation of MtSAP1 by desic- Ò a mortar and pestle. The powder was treated with a TriReagent cation seemed specific to this situation of extreme dehydration of (Ambion, Austin, TX, USA) following the manufacturer instructions. the tissues, neither the messengers nor the protein accumulated in cDNAs were obtained by retrotranscription of 2 mg of total RNA germinating embryo axis submitted to mild drought stress. This using 200 units of RT-MMLV (Invitrogen, Breda, The Netherlands), difference suggests that, although MtSAP1 is responsive to ABA, its 2 mg of random primer (Invitrogen) in the presence of 40 units of regulation by desiccation might be dependent upon a signaling a Recombinant Rnasin Ribonuclease Inhibitor (Promega, Madison, pathway different from that activated by drought stress. WI, USA). Reaction occurred for 1 h at 37 C in total volume of 50 ml. In conclusion, MtSAP1 a gene encoding a protein of the ‘stress- associated proteins’ family was isolated in M. truncatula embryos. The up-regulation of the expression of MtSAP1 and the accumula- 4.5. Real-time quantitative PCR tion of the encoded protein under ABA treatment, desiccation and hypoxia suggest the involvement of MtSAP1 in the tolerance of Reactions took place on the light cycler ABI Prism 7000 SDS these two challenging constraints encountered in maturing (Applied Biosystems, Foster city, CA, USA). Each reaction was per- embryos. Extinction of the expression of MtSAP1 in RNAi transgenic formed with a 3 ml of a 1/2 (v/v) dilution of the first cDNA strands plants showed that the function of MtSAP1 is required for the using an SYBR Green PCR Master Mix (Applied Biosystems), accumulation of storage globulin proteins, vicilin and legumin, following the manufacturer instructions, with 200 nM of each and for the development of embryos able to achieve successful primer in a total reaction of 25 ml. The reaction was incubated for germination. 2 min at 50 C and 10 min at 95 C followed by 40 cycles of 15 s at 95 C and 1 min at 58 C. The specificity of the PCR amplification 4. Materials and methods procedure was checked with a heat-dissociation protocol (from 65 Cto95C) after the final cycle of PCR. Each measure was carried 4.1. Plant material and experimental treatments out with at least two biological repeats using a duplicated PCR reaction for determining Ct values. mRNA amounts were calculated Seeds of M. truncatula cv Parragio were germinated in 9 cm in copy numbers, referring to a standard curve made from MtSAP1 diameter Petri dishes on Whatman paper soaked with 3.5 ml qPCR amplicon cloned in plasmid vector. solution with ultra-pure water (control) or with abscisic acid (ABA; 10e100 mM) (Sigma, Saint Quentin Fallavier, France). For abiotic 4.6. MtSAP1 protein analysis by immunoblotting stresses, seeds were germinated for 48 h on water. Afterwards, seedlings were subjected to various treatments: saline stress (NaCl, To raise a specific antibody directed against M. truncatula SAP1 200 mM), drought stress mimicked by mild polyethylene glycol protein, the specific peptide VSPEVPENPISNESC was used to treatment (PEG 6000, 0.25 MPa), dehydration stress (PEG was immunize rabbits. Antibodies were affinity-purified (GenScript, adjusted to concentration for an osmotic pressure of 3 MPa), and Piscataway, NJ, USA). Soluble proteins were extracted from frozen water (control). For hypoxia condition, seeds were immersed with material in 25 mM TriseHcl buffer (pH 7.6) with 1 mM MgCl2,1mM ultra-pure water in hermetically closed polypropylene flask. All 1 EDTA and a cocktail of protease inhibitors (aprotinin 5 mgml , experiments were made in darkness and maintained at 20 C, leupeptin 2 mgml 1, pepstatin 0.1 mgml 1, PMSF 1 mM, Na VO except for cold stress where embryo axes were placed at 4 C. After 3 4 1 mM, NaF 5 mM). After denaturation, equal amounts of protein various imbibition times, embryo axes were collected and imme- (30 mg) were separated on an SDSepolyacrylamide gel (14% (v/w) diately frozen in liquid nitrogen. polyacrylamide). Proteins were then transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). After incubation with 4.2. Cloning of MtSAP1 a rabbit polyclonal anti-SAP1 antibody (1/2000), proteins were detected using a goat peroxidase-conjugated anti-rabbit antibody Reverse library of SSH between 6 h and 23 h after M. truncatula (1/4000; Sigma) and visualized using ECL chemiluminescence (Bio- cv Parragio seed imbibitions, revealed full-length MtSAP1 cDNA. Rad Hercules, CA, USA). The library was constructed using a Clontech PCR-select cDNA subtraction Kit (Clontech, Mountain View, CA, USA) following the manufacturer instructions. Finally, MtSAP1 was cloned in pGEMT 4.7. Storage protein analysis vector (Promega, Madison, Wisconsin, USA) flanked by a sequence of nested primers, and propagated in a TOP10 Escherichia coli strain Ten mature seeds from either wild-type (WT) or transgenic (Invitrogen, Breda, The Netherlands). plants were harvested and grinded in liquid nitrogen with a tissue- lyser (Qiagen). Resulting powder was then dissolved in 500 ml 4.3. In silico analysis extraction buffer (50 mM HEPES, 1 mM EDTA and 1 mM PMSF) and the lysate was stirred for 30 min and centrifuged for 15 min at MtSAP1 cDNA sequence and M. truncatula homologues were found 13,000 g at 4 C. Subsequently, the pellet was resuspended with using TGIP BLAST software available online (http://compbio.dfci. extraction buffer supplemented with 0.2 M NaCl to extract vicilin harvard.edu/tgi/cgi-bin/tgi/Blast/index.cgi) and browsing genomic fraction. After 15 min centrifugation at 13,000 g at 4 C, supernatant data from Information System revealed genomic sequences and was removed and pellet was resuspended a last time with extraction chromosome locations. Cis-acting elements in the upstream region buffer supplemented with 1 M NaCl to extract legumin fraction. were discovered using the PLACE database [23]. A deducted protein Protein concentration was determined using Bradford assay. 310 C. Gimeno-Gilles et al. / Plant Physiology and Biochemistry 49 (2011) 303e310

4.8. Plasmid construction and transformation procedure [11] S. Bouton, L. Viau, E. Lelièvre, A.M. Limami, A gene encoding a protein with a proline-rich domain (MtPPRD1), revealed by suppressive subtractive hybridization (SSH), is specifically expressed in the Medicago truncatula fi To construct the MtSAP1 RNAi plasmid a speci c region 600-bp embryo axis during germination, J. Exp. Bot. 56 (2005) 825e832. portion of MtSAP1 was amplified. The product of the amplification [12] C. Gimeno-Gilles, E. Lelièvre, L. Viau, M. Malik-Ghulam, C. Ricoult, A. Niebel, was transferred by recombination using the Gateway Cloning N. Leduc, A.M. Limami, ABA-mediated inhibition of germination is related to the inhibition of genes encoding cell-wall biosynthetic and architecture: Technology (Invitrogen) into the binary vector pFCGC5941. This modifying enzymes and structural proteins in Medicago truncatula embryo plasmid contains sense and antisense Gateway cassette, separated axis, Mol. Plant 2 (2009) 108e119. by a chalcone synthase A intron and driven by a 35S promoter. [13] J. Buitink, J.J. Leger, I. Guisle, B.L. Vu, S. Wuillème, G. Lamirault, A. Le Bars, N. Le Meur, A. Becker, H. Küster, O. Leprince, Transcriptome profiling uncovers Transformation of the plasmid into R108 and in vivo culture was metabolic and regulatory processes occurring during the transition from performed according to Trinh et al. [24] using Agrobacterium desiccation-sensitive to desiccation-tolerant stages in Medicago truncatula tumefaciens strain EHA105. Selection of positive transgenic lines seeds, Plant J. 47 (2006) 735e750. [14] C. Ricoult, J.B. Cliquet, A.M. Limami, Stimulation of alanine amino transferase was performed by PCR. (AlaAT) gene expression and alanine accumulation in embryo axis of the model legume Medicago truncatula contribute to anoxia stress tolerance, Funding Physiol. Plantarum. 123 (2005) 30e39. This work was funded by grants from the EU FP6 project FOOD- [15] C. 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