Renouardetal.BMCResearchNotes2012,5:15 http://www.biomedcentral.com/1756-0500/5/15 TECHNICAL NOTE Open Access Linum Isolation of nuclear proteins from flax ( usitatissimum L.) seed coats for gene expression regulation studies Sullivan Renouard1,2, Corbin Cyrielle1, Tatiana Lopez1, Frédéric Lamblin1, Eric Lainé1 and Christophe Hano1* Abstract Background: While seed biology is well characterized and numerous studies have focused on this subject over the past years, the regulation of seed coat development and metabolism is for the most part still non-elucidated. It is well known that the seed coat has an essential role in seed development and its features are associated with important agronomical traits. It also constitutes a rich source of valuable compounds such as pharmaceuticals. Most of the cell genetic material is contained in the nucleus; therefore nuclear proteins constitute a major actor for gene expression regulation. Isolation of nuclear proteins responsible for specific seed coat expression is an important prerequisite for understanding seed coat metabolism and development. The extraction of nuclear proteins may be problematic due to the presence of specific components that can interfere with the extraction process. The seed coat is a rich source of mucilage and phenolics, which are good examples of these hindering compounds. Findings: In the present study, we propose an optimized nuclear protein extraction protocol able to provide nuclear proteins from flax seed coat without contaminants and sufficient yield and quality for their use in transcriptional gene expression regulation by gel shift experiments. Conclusions: Routinely, around 250 μg of nuclear proteins per gram of fresh weight were extracted from immature flax seed coats. The isolation protocol described hereafter may serve as an effective tool for gene expression regulation and seed coat-focused proteomics studies. Keywords: Flax, Gene expression, Mucilage, Nuclear proteins, Phenolics, Seed coat Background seed coat development and metabolism has taken The seed coat plays a crucial role for seed protection advantage of this acceleration of knowledge but has not against biotic and abiotic stress and has an impact on provided enough information about specific biosynthetic embryo development, seed dormancy and germination pathways to many of our crops [1]. For instance, flax [1]. The seed coat also constitutes a rich source of valu- seed coat constitutes a model for the biosynthesis of lig- able compounds such as pharmaceuticals, and their fea- nans (diphenolic compounds with high potential for tures are associated with important agronomical traits pharmaceutical or cosmetic industries [3,4]) while A. [1,2]. Obviously, during the last decade, technologies thaliana seeds are not known to produce these com- such as genomics, proteomics and metabolomics applied pounds. Another important example is the accumulation to the Arabidopsis thaliana model have allowed deeper of specific anti-nutritional factors in canola seed coats understanding in seed biology [2]. Our understanding of that require modifications to improve meal quality [5]. In both examples, our knowledge of seed coat biology is still too limited to take advantage of valuable com- *Correspondence:[email protected] 1LaboratoiredeBiologiedesLigneuxetdesGrandesCulturesUPRESEA pounds or to improve agronomical quality [1]. 1207,Universitéd’Orléans,EquipeLignanesdesLinacées,Antenne Isolation and identification of transcription factors ScientifiqueUniversitairedeChartres,21ruedeLoignylaBatailleF-28000 responsible for seed coat specific expression are pre- Chartres,France Fulllistofauthorinformationisavailableattheendofthearticle ©2012Renouardetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited. Renouardetal.BMCResearchNotes2012,5:15 Page2of7 http://www.biomedcentral.com/1756-0500/5/15 requisites for the understanding of seed coat develop- Methods ment and metabolism regulation. Transcription factors Plant material represent only 0.001 to 0.01% of the total cellular pro- Linum usitatissimum L. (linseed cultivar Barbara) imma- tein content and their extraction could be a great chal- ture seed coats (development stage S2 [3] (torpedo lenge [6]. Moreover, compared to other organisms, stage, 16 days after flowering), Figure 1a) were used as plants are generally more problematic for protein starting material. extraction because they contain high levels of proteases and interfering compounds that can both hinder extrac- Commercial or published methods for nuclear proteins tion itself, DNA binding experiments or gel-based extraction separation [7]. Despite the availability of commercial One commercial kit (CelLytic PN for plant nuclei iso- kits and published protocols it is well known that in lation kit from leaves, Sigma) and two published meth- most cases the extraction procedure must be optimized ods [10,11] were tested as described by manufacturer for each plant species, tissue, or cell compartment [7,8]. or following author’s recommendations for the extrac- The seed coat generally harbors high quantities of inter- tion of nuclear protein from immature flax seed coats. fering compounds such as polyphenols, mucilage, starch The commercial kit (Sigma) was designed for nuclear and lipid derivatives [1] that can severely affect the per- protein extraction from Arabidopsis thaliana leaves formance of protein extraction. Phenolic compounds whereas the two published methods were designed and can build irreversible complexes with proteins and it has used respectively for nuclear protein extraction from been shown that oxidation of phenolics by oxidases and Phaseolus vulgaris whole seeds [10] and maize embryos peroxidases can cause streaking and generate artefactual [11]. spots on 2D electrophoresis gels [7]. The presence of mucilage may also hinder the separation of proteins due Optimized nuclear protein extraction protocol to their swelling in aqueous medium [9]. So far, no pro- The workflow of the optimized protein extraction proto- tocol published has been designed for the extraction of col is summarized in Figure 1b. nuclear proteins from seed coat. The aim of this study was to extract DNA binding nuclear proteins suitable for gene expression study by gel shift experiments using seed coats of immature flax- a. seeds as starting material. Gel shift assay is the first step of transcription factor study and this method has been widely used in the study of sequence-specific DNA- sc binding proteins such as transcription factors [6]. The em assay relies on the ability for a protein to bind a labeled DNA fragment in vitro, followed by electrophoretic separation of DNA-proteins complexes that migrate b. Seed coat tissue (e.g. flax) more slowly than free DNA fragments. The key to suc- cess of gel shift assay relies on the availability of a suffi- CELL LYSIS cient amount of high-quality nuclear protein extracts 1) Tissue disruption in liquid nitrogen with mortar and pestle 2) Cell wall and mucilage digestion by a 1 u.mL-1 Macerozymesolution (containing PIC) containing native transcription factors. Flax seed coat is 3) Centrifugation NUCLEI ISOLATION rich in both mucilage and phenolics and therefore con- 1) Pellet resuspensionin homogenization buffer (containing PVPP, PEG and PIC) stitutes a good example of the presence of these com- 2) Filtrations through a double 100 μm filter mesh (twice) 3) Centrifugation pounds that can interfere with the extraction process. NUCLEAR LYSIS Established methods (commercial and published proto- 1) Pellet resuspensionin high salt lysisbuffer (containing PIC) 2) Centrifugation cols) have failed to provide suitable nuclear proteins 3) Pellet resuspensionin low salt buffer (containing PIC) NUCLEAR PROTEIN PURIFICATION from seeds for gel shift assay. In the present study, we 1) Trizol-based phase separation describe an optimized and efficient protocol for the 2) Centrifugation 3) DNA precipitation by absolute ethanol extraction of nuclear proteins from flax seed coat, com- 4) Centrifugation 5) Nuclear protein precipitation by isopropanol pare it to other methods and discuss the critical steps. 66)) NGuecl lfeilatrra tpioront eoinn Sexetprahcatd reexsucsopluemnsni on Given the fact that, in a number of plant species, seed NUCLEAR PROTEIN EXTRACT coats contain either high mucilage or phenolic contents Figure 1 a Morphology of dissected flax seedcoat (sc) and (e.g. in Brassicaceae, Solanaceae and Linaceae species), embryo(em)atdevelopmentalstageS2(Torpedostage),16 the isolation protocol described below may serve as an daysafterflowering,squarerepresenting1mm2;b.Workflow effective tool for gene expression regulation and proteo- oftheoptimizedprotocolforextractionofanactivenuclearprotein fractionfromflaxseedcoat.(FormoredetailsseeMethods). mics focusing on seed coat in these species. Renouardetal.BMCResearchNotes2012,5:15 Page3of7 http://www.biomedcentral.com/1756-0500/5/15 Cell lysis Estimation of contamination bycytosolic proteins Seed coats were ground in liquid nitrogen and incubated The presence of contaminating proteins was estimated in a 100 mM citrate-phosphate pH 4.8 buffer containing using alcohol dehydrogenase (ADH) activity assay [12] 1 unit.mL-1 Macerozyme for 1 h at 25°C. as an indication of cytosolic protein presence. Nuclei isolation The ADH specific activity was assayed spectrophoto- After centrifugation at 8,000 g for 10 min at 4°C, the metrically with ethanol as substrate. NADH production pellet was resuspended in homogenization buffer pH from NAD was measured by increase in absorbance at 8.5 containing 0.44 M sucrose, 2.5% (w/v) Ficoll 400, 340 nm at 25°C [12]. The reaction mixture contained 5.0% Dextran 40, 25 mM Tris-HCl (pH7.6) 10 mM 0.1 M Tris-HCl (pH 9.0), 2 mmol NAD and 1% (v/v) MgCl , 10 mM b-mercaptoethanol, 0.5% (v/v) Triton ethanol in a final volume of 5 ml. 2 X100 and 1% (w/v) PVPP, 2% (w/v) PEG-8000 and 1% (v/v) protease inhibitor cocktail for plants (PIC; Sigma Protein concentration determination P9599), filtered twice through a double 100 μm filter The protein concentration was determined using a fluo- mesh (Sigma) and centrifuged at 2,000 g for 5 min at rometer and the Quant-iT Protein Assay Kit (Invitro- 4°C. gen) adapted for the Qubit fluorometer according to the Nuclear lysis manufacturer’s protocol. The pellet was washed twice in a solution containing 25 mM Tris (pH 8.5), 5 mM MgCl , 2% (v/v) glycerol, 10 Protein western blot and dot blot 2 mM b-mercaptoethanol and 1% (v/v) PIC) and resus- Nitrocellulose membranes (BioRad) were prepared and pended in high salt buffer (20 mM HEPES (pH 7.5), nuclear proteins were applied on the membrane as a dot 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl , 0.2 following manufacturer’s recommendation. Nitrocellu- 2 mM EDTA, 0.5 mM DTT and 1% (v/v) PIC), the mix- lose membranes were blocked with 10% nonfat milk in ture was left at 4°C for 40 min and occasionally vor- TBS containing 0.1% Tween-20 for 1 h, incubated with texed. Following centrifugation at 8,000 g for 10 min at a 1:1000 dilution of the commercial antibodies, pre- 4°C, the pellet was resuspended in a low salt buffer (20 viously used for plant cellular compartments characteri- mM HEPES (pH7.8), 20 mM KCl, 1.5 mM MgCl , 0.5 zation [8], anti-histone H1 (Sigma, H7665) or anti-actin 2 mM DTT, 25% glycerol and 1% (v/v) PIC). 2 (Sigma, A2066) antibodies produced in rabbit for 1 h Nuclear protein purification at room temperature and then washed in PBS contain- Then the resuspended pellet was submitted to a phe- ing 0.1% Tween-20 three times for 5 min. Blots were nol-based purification step: 1 ml of Trizol (guanidium incubated with a 1:4000 dilution of the commercial anti- isothiocyanate-phenol-chloroform solution) was added rabbit secondary antibody peroxidase-conjugated and mixed by pipetting. This solution was then vigor- (Roche) for 1 h at room temperature, and developed ously vortexed for 20 min at 4°C for further nuclear using Lumi-Light Plus POD substrate (Roche). The lysis and nuclear proteins solubilization. Following a 5 color intensity was analyzed and quantified by densito- min incubation at room temperature, 0.2 ml chloro- metry using a digital camera (AlphaImager 1220; Alpha form was added and the mixture was vortexed for 15 s Innotech Corporation) and densitometry software and incubated 5 min to allow phase separation by cen- (Alpha Innotech Corporation). trifugation at 12,000 g for 15 min at 4°C. The upper phase containing RNA was removed and the DNA was Gel shift assay precipitated by addition of 0.3 ml absolute ethanol. The band shift assay was performed using the non- The phenol/ethanol phase was carefully mixed, incu- radioactive Roche Gel Shift assay kit according to manu- bated 3 min at room temperature and centrifuged at facturer’s instructions with 10 μg nuclear protein pre- 2,000 g for 5 min at 4°C to pellet the remaining DNA. paration and 10 nM of 5’-Digoxigenin-labeled double Supernatant was transferred into a new tube for stranded oligonucleotide 5’-GAGCATGCTAAC- further precipitation of the nuclear proteins by addi- CAAAATGT-3’ containing a MYB binding site (under- tion of 1.5 ml 100% isopropanol. Following 15 min lined and bold) present in the promoter region of incubation at room temperature, the nuclear protein LuPLR1 gene [3]. This oligonucleotide was synthesized were pelleted by centrifugation at 15,000 g for 15 min by Eurofins MWG Operon. The assay was carried out at at 4°C and then resuspended in 1 mL of storage buffer room temperature for 30 min in a final volume of 25 μL (20 mM Tris pH 7.5, 1 mM EDTA, 1 mM MgCl , 10 in binding buffer (Roche). The binding reaction pro- 2 mM KCl, 5% glycerol). Residual phenol and salts were ducts were separated on a nondenaturating 6% acryla- removed by gel filtration onto a G-25 Sephadex desalt- mide:bis-acrylamide gel (37.5:1) in a low ionic strength ing column. buffer (6.7 mM Tris, pH 7.5; 1 mM EDTA, pH 7.5 and Renouardetal.BMCResearchNotes2012,5:15 Page4of7 http://www.biomedcentral.com/1756-0500/5/15 3.5 mM sodium acetate) at 4°C. The next steps were a carried out according to manufacturer’s instructions for Anti-Histone H1 Anti-Actin the Roche Gel Shift assay kit. kDa MW Ø NPE Cyt Ø NPE Cyt Treatment of data 34.3 Alldatapresentedinthisstudywerethemeansofatleast three independent replicates ± standard error of the mean. Comparative statistical analyses of groups were 28.8 performed using Student’s t-test or one-way analysis of varianceaccordingtothedata.Infigures,thesameletter 20.7 indicates that values are not significantly different. All statisticaltestswereconsideredsignificantatP<0.05. b Results and discussion Evaluation of published methods for nuclear protein a extraction b Attempts to use published or commercial protocols on c immature flax seed coat for nuclear protein extraction for gene expression regulation were unproductive. d The CelLytic PN extraction kit from Sigma, a com- mercial adaptation of a protocol described to isolate nuclear proteins from A. thaliana leaves [12], yielded up to 168.5 μg proteins per gram fresh weight (Table 1). The presence of nuclear proteins in this fraction was Anti-Histone H1 confirmed by protein blot using an anti-histone H1 anti- Commercial kit Published method 1 Published method 2 Optimized method body (Figure 2), suggesting that the preparation is Figure2ProteinblotsdetectionofhistoneH1andactin 2a. enriched in nuclear proteins. But this final extract also WesternblotanalysisforchemiluminescentdetectionofhistoneH1 appeared contaminated by a non negligible amount of (around35kDa)andactin2(around42kDa).Nuclear(obtained non-nuclear proteins, evidenced by the presence of withtheoptimizedprotocol)andcytosolicproteinfractions(50μg) extractedfromimmatureflaxseedcoatswereseparatedona12% ADH activity in the final extract (Table 1). The presence SDS-PAGE,transferredtonitrocellulosemembrane,andprobedwith of these contaminants could partly explain the fact that eachoftheabove-notedantibodies.Equalloadingofthegelswas this extract failed to produce active DNA binding verifiedbyproteinquantization(usingfluorescentprobe,see nuclear protein for gel shift assay. Only a very faint Methods)beforeloadingandCoomassiebluestainingofthe retardation signal was observed (Figure 3). membraneafterproteintransfer.MW:molecularweight;Ø:Loading bufferwithoutprotein(negativecontrol);NPE:nuclearproteins Similar results were obtained with two published pro- extract(optimizedmethod);Cyt:cytosolicextract(firstsupernatant, tocols designed specifically for nuclear protein extrac- nucleiisolationstep).b.ProteinDotblotforchemoluminescent tion from seed. The first protocol was designed for detectionofhistoneH1wasperformedusingnuclearfraction extraction of nuclear protein from Phaseolus vulgaris obtainedwiththedifferentmethods.Thecolorintensitywas seed ([10], published method 1) and for the second, analyzedandquantifiedbydensitometryusingadigitalcameraand densitometrysoftware.Thesameletterindicatesthatvaluesarenot from maize embryos ([11], published method 2). Using significantlydifferent(P>0.05).Valueismean±standarderror. immature flax seed coat as starting material, these two protocols yielded higher amounts of protein 188.2 and 173.8 μg per gram fresh weight as compared to the extracts (Figure 2), these two extracts were also con- result obtained with the commercial kit (Table 1). But taminated by cytoplasmic fraction (Table 1). Gel shift as already observed with the commercial protocol, assay with these two extracts produced detectable but despite the clear presence of nuclear proteins in these very weak retardation signal (Figure 3). Table 1Nuclearprotein extract concentration andquality estimated by the contamination by non nuclear (cytosolic) fraction nd: notdetected; the same letter indicates that valuesare notsignificantly different (P>0.05).Value ismean ± standard error Commercial Publishedmethod1 Publishedmethod2 Optimizedprotocol(present kit [10] [11] study) Proteinconcentration(μg.mg-1 168.5±7.2a 188.2±6.6b 173.8±9.0b 247.8±6.1c FW) AdHspecificactivity(AU.mg-1FW) 20.7±3.0c 8.8±1.2bc 15.9±3.8c nda Renouardetal.BMCResearchNotes2012,5:15 Page5of7 http://www.biomedcentral.com/1756-0500/5/15 general scheme but several critical steps were added or modified to address specific problems encountered dur- a ing extraction. The general scheme of this optimized protocol for nuclear protein extraction from seed coat is presented in Figure 1b and described in detail in the Methods. It consists of the following steps: 1) cell lysis, migration 2) nuclei isolation (Additional file 1), 3) nuclear lysis and 4) nuclear protein purification. The critical steps are discussed hereafter. In the first part of this work, we observed that what- ever the protocol used, the final extract had high viscos- b c d ity and a pale brown coloration (data not shown) that [Protein]: 10 5 1 μg ml-1 Comp: 0 20 40 100 fold wild mutated led us to suspect the presence of both mucilage and oxi- dized phenolics. Seed coats generally contain high quan- tities of phenolics and mucilage, associated with starch migration migration migration annodliclsipaindddemriuvcaitliavgeesc[a1n].sIetvheareslybeaefnfecotbtsheervpederftohramt apnhcee- of the protein extraction and separation [7,9]. For exam- ple, oxidized phenolics can build complexes with pro- teins and interfere with gel separation but also affect Figure3GelshiftassayfornuclearproteinbindingonMYB2 DNA binding capacity of the extracted proteins [7,14]. siteTheanalysisoftheDNA-proteincomplexbygelshift The use of seed coats as starting material results in a assaywasperformedwithnuclearextractsusing10nMDIG- concentration effect of these interfering compounds. labeledprobefragmentwithnuclearproteinsextractedfrom immatureseedcoatsfromflax(developmentalstage3).Thegel Flax seed coats contain huge amounts of phenolic com- shiftassaywasperformedina6%polyacrylamidegel.White pounds [3] and mucilage [4,9] that are able to hinder arrowheadsshowtheretardedDNAsignals.ThisFigureis the protein extraction. Adaptations described below representativeofatleast5independentexperimentsperformedin were performed to minimize problems generated by the thesameconditions.a.ComparativeanalysisoftheDNA-protein presence of these compounds found in high amounts in complexformationbygelshiftassayperformedusing10nMDIG- labeledprobefragmentwith10μgofnuclearproteinextracted the seed coats. withthedifferentnuclearproteinextractionmethods.b.Effectof Following tissue disruption in liquid nitrogen, a pre- nuclearproteins(obtainedwiththeoptimizedprotocol) treatment with Macerozyme was performed. Macero- concentrationsonDNA-bindingcapacityontheputativeMYB2site. zyme is an extract of Rhizopus sp., which displays Theamountofnuclearproteinsaddedperlaneisindicated.c. pectinase, cellulase and hemicellulase activities. Indeed, Titrationofnuclearproteins(obtainedwiththeoptimizedprotocol) specificaffinityfortheDig-labeledMYB2containingfragmentusing we have observed that Macerozyme (an enzyme mixture thesameunlabelledfragmentattheindicatedfold-molar-excessin commonly used for protoplasts isolation) was able to thepresenceof10μgnuclearproteinsextractfromimmatureflax efficiently degrade the mucilage layer covering flaxseeds seedcoat.d.EffectofmutationoftheputativeMYB2bindingsite (ruthenium red staining disappearance and the reducing onDNA-bindingcapacityof10μgnuclearproteins(obtainedwith sugars release were observed, Additional file 2). This theoptimizedprotocol). enzymatic mucilage removal allowed the recovery of a less viscous extract and has been previously proved to It was frequently observed that in most cases extrac- be beneficial for both proteins [9] and lignans [4] recov- tion procedure has to be optimized for each plant tissue ery from flaxseed. or cellular compartments [7,13]. The results confirmed To address the problems of viscosity and coloration of this observation. Nuclear protein extraction requires the final extract, due the presence of cell wall polysac- improvements and adaptations for their use with tegu- charides and phenolics, PEG 8000 and Polyvinylpolypyr- ment as starting material. The absence of protocols rolidone (PVPP) were added in the homogenization designed for extraction of nuclear proteins from this buffer. Mucilage is abundant in a number of major valuable cell compartment has motivated this study. crops seed coats belonging to the Brassicaceae, Solana- ceae and Linaceae species [1] and in the aerial parts of Optimized protocol for nuclear protein extraction from plants belonging to families such as Crassulaceae, Cac- seed coats taceae or Malvaceae as well as around the root tips of The optimized protocol for nuclear proteins extraction every plant. PEG 8000 have previously been used to from seed coats described in the present study shares avoid problems linked to the presence of polysacchar- common features with established protocols in its ides during RNA isolation [3,15]. PVPP is able to trap Renouardetal.BMCResearchNotes2012,5:15 Page6of7 http://www.biomedcentral.com/1756-0500/5/15 the phenolic compounds and prevent the formation of (Table 1). The absence of contamination by non nuclear complexes with proteins [7,14]. proteins of the nuclear protein extract obtained with the Subsequent to high salt nuclear lysis and protein pre- optimized protocol was further accessed by western blot cipitation, a phenol-based protein purification step using using an anti-actin 2 antibody: a signal was detected Trizol was essential. It allowed complete removal of the only in the cytosolic fraction and not in the nuclear pro- nucleic acids that can interfere during gel shift assay tein extract (Figure 2a). Finally gel mobility shift experi- and also of remaining starch granules still present in the ments were performed using a labeled double stranded extract at this stage. Trizol phase separation has been DNA fragment containing the binding site and nuclear previously used for nuclear protein extraction from Xer- proteins extracted from maturing flax seed coat (Figure ophyta viscose [8]. Following this purification step, phe- 3). Gel shift experiments clearly evidenced a high and nol and salts were then efficiently eliminated by gel reproducible (n = 6) mobility shift of the DNA fragment filtration performed by Sephadex G25 column. This last containing a MYB2 binding site with nuclear proteins purification step was required because phenol has to be obtained with the optimized protocol (Figure 3a). This totally removed to obtain better DNA-binding capacity interaction was even more pronounced at a higher con- of the nuclear protein extract. centration of nuclear proteins (Figure 3b). Specific com- It is noteworthy that the protease inhibitor cocktail for petition with the same but unlabelled DNA fragment plants (PIC) was added in every buffer used. It has the containing the box fully out-competed the DNA-protein advantage of a wider range of inhibition as compared to interaction at a 100-fold molar excess (Figure 3c). More- the frequently used PMSF (phenylmethanesulfonylfluor- over, mutation of the putative MYB2 binding site ide, a serine protease inhibitor). Addition of PIC appears resulted in a complete loss of the retarded signal (Figure to be essential during Macerozyme digestion, as this cell 3d). These notable gains were also observed in other wall degrading mixture, not being a purified enzyme experiments using probes containing other fixation sites preparation, can as well display some protease activity. for seed coat specific transcription factor such as ABRE boxes (abscisic acid responsive element; data not Comparison of the optimized protocol with other shown). These results highlight the effective binding affi- published methods nity of the nuclear proteins extracted from flax seed The addition of several critical steps for optimal extrac- coats using our optimized protocol. tion of nuclear protein from seed coat was not detri- mental to the protein yield since the 247.8 μg.mg-1 FW Conclusion and potential uses of nuclear protein in the final extract was significantly To the best of our knowledge this study describes the higher than those obtained with the other tested meth- first nuclear protein extraction protocol optimized for ods (Table 1; a one dimension SDS-PAGE analysis of seed coat. This protocol allows an efficient removal of the nuclear proteins obtained with this optimized interfering compounds from the final protein samples, method is shown in Additional file 3). This could be thus providing high quality nuclear protein extracts, sui- due to a better release of protein entrapped into or table for transcriptional gene regulation studies. It was linked with co-extracted soluble phenolics and/or muci- used to obtain nuclear protein from flax seed coat with lage, as well as to a less important loss during filtration minimal contamination and sufficient yield and quality and centrifugation steps as a consequence of the for subsequent use in gel shift experiments. Given the reduced viscosity of the extract. The presence of nuclear fact that a number of plant species harbor either high protein was evidenced by protein western and dot blot- mucilage or phenolic contents (or both) in their seed ting performed using an anti-histone H1 antibody (Fig- coat (e.g. in Brassicaceae, Solanaceae and Linaceae spe- ure 2a, b), previously used to characterize nuclear cies), this optimized protocol could be of great interest protein extracts from the resurrection plant Xerophyta for workers involved in DNA-protein interaction or pro- viscose [8]. As shown in Figure 2a, histone H1 was pre- teomics studies in these species to obtain information sented only in the nuclear protein extract (obtained with on specific seed coat development and metabolism. the optimized protocol) and not in the cytoplasmic frac- tion. If all methods resulted in an effective nuclear pro- Additional material tein extraction, the yield varied considerably according to the method used and better results were obtained Additionalfile1:Structureoftheisolatednucleifromimmature with our optimized protocol (Figure 2b). Beside these flaxseedcoatsa.Nucleimicrographunderthelightmicroscope.b. Detailofonenucleusmicrographunderthelightmicroscope.c.RNA quantitative variations, qualitative differences were also visualizationofRibogreen-stainednucleusmicrographunder observed since ADH activity was undetectable in the fluorescence(excitation480nm,emission520nm).Nucleiwere final nuclear protein extracts of the optimized protocol, visualizedundertheoilimmersionlenswithoutandwithfluorescence, respectively,usingaLeitz-invertedmicroscopeDIAVERT.Thebar assessing a high purity of the purified nuclear proteins Renouardetal.BMCResearchNotes2012,5:15 Page7of7 http://www.biomedcentral.com/1756-0500/5/15 10. RiggsCD,VoelkerTA,ChrispeelsMJ:Cotyledonnuclearproteinsbindto representaround15μm(picturea)andsquaresrepresentaround5μm2 DNAfragmentsharboringregulatoryelementsofphytohemagglutinin (picturesbandc). genes.PlantCell1989,1:609-621. Additionalfile2:Rutheniumredstainingofthemucilagelayerof 11. BuskPK,PagèsM:Microextractionofnuclearproteinsfromsinglemaize controlandMacerozyme-treatedflaxseedcoatsa.Controlseeds embryos.PlantMolBiolRep1997,15:371-376. wereincubatedinwater.b.Seedsweresubmittedtodigestionby 12. YamaguchiJ,LimPY,AkazawaT:Isolationandcharacterizationofnuclei Macerozymesolution(1unit.ml-1).Afterincubation,seedswerestained fromriceembryo.CellStructFunct1992,17:87-92. by0.5%(w/v)rutheniumredandthenrinsed3timeswithdistilled 13. ConleyTR,ParkSC,KwonHB,PengHP,ShihMC:Characterizationofcis- water.Arrowindicatemucilagelayer(m). actingelementsinlightregulationofthenucleargeneencodingtheA Additionalfile3:10%SDS-PAGEofnuclearproteinsextractedfrom subunitofchloroplastisozymesofglyceraldehydes-3-phosphate immatureflaxseedcoatsstainedwithCoomassieblue.Theresults dehydrogenasefromArabidopsisthaliana.MolCellBiol1994, shownarerepresentativeofthreeindependentbiologicalreplicates.MW: 14:2525-2533. molecularweight;NPE:Nuclearproteinsextractobtainedusingthe 14. ValcuCM,SchlinkK:Reductionofproteinsduringsamplepreparation hereinpresentedoptimizedmethod. andtwodimensionalgelelectrophoresisofwoodyplantsamples. Proteomics2006,6:1599-1605. 15. GehrigHH,WinterK,CushmanJ,BorlandA,TaybiT:AnimprovedRNA isolationmethodforsucculentplantspeciesrichinpolyphenolsand polysaccharides.PlantMolBiolRep2000,18:369-376. Acknowledgements SRobtainedagrantfromtheFrenchMinistryofResearchandTechnology. doi:10.1186/1756-0500-5-15 Thisprojectwasfundedby“ConseilGénérald’EureetLoir”and“Ligue Citethisarticleas:Renouardetal.:Isolationofnuclearproteinsfrom ContreleCancer,Comitéd’EureetLoir”.WethankMichelBonoraforEnglish flax(LinumusitatissimumL.)seedcoatsforgeneexpressionregulation editingofthemanuscript. studies.BMCResearchNotes20125:15. Authordetails 1LaboratoiredeBiologiedesLigneuxetdesGrandesCulturesUPRESEA 1207,Universitéd’Orléans,EquipeLignanesdesLinacées,Antenne ScientifiqueUniversitairedeChartres,21ruedeLoignylaBatailleF-28000 Chartres,France.2LaboratoiredePhytotechnologieEA3900BioPI,Facultéde Pharmacie,UniversitédePicardieJulesVerne,1ruedesLouvelsF80037 Amiens,France. Authors’contributions SR,CC,TLandCHperformedtheresearch.FL,ELandCHparticipatedinthe experimentaldesignandcoordination,anddraftedthemanuscript.All authorsreadandapprovedthefinalmanuscript. 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