Table Of ContentRenouardetal.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.
Competinginterests
Theauthorsdeclarethattheyhavenocompetinginterests.
Received:19October2011 Accepted:9January2012
Published:9January2012
References
1. MoïseJA,HanS,Gudynaite-SavitchL,JohnsonDA,MikiBLA:Seedcoats:
structure,development,compositionandbiotechnology.InVitroCellDev
BiolPlant2005,41:620-644.
2. NorthH,BaudS,DebeaujonI,DubosC,DubreucqB,GrappinP,JullienM,
LepiniecL,Marion-PollA,MiquelM,RajjouL,RoutaboulJM,CabocheM:
Arabidopsisseedsecretsunravelledafteradecadeofgeneticand
omics-drivenresearch.PlantJ2010,61:971-981.
3. HanoC,MartinI,FliniauxO,LegrandB,GutierrezL,ArrooRR,MesnardF,
LamblinF,LainéE:Pinoresinol-lariciresinolreductasegeneexpression
andsecoisolariciresinoldiglucosideaccumulationindevelopingflax
(Linumusitatissimum)seeds.Planta2006,224:1291-1301.
4. RenouardS,HanoC,CorbinC,FliniauxO,LopezT,MontguillonJ,
BarakzoyE,MesnardF,LamblinF,LainéE:Cellulase-assistedreleaseof
secoisolariciresinolfromextractsofflax(Linumusitatissimum)hullsand
wholeseeds.FoodChem2010,122:679-687. Submit your next manuscript to BioMed Central
5. WuL,El-MezawyA,ShahS:Aseedcoatouterintegument-specific and take full advantage of:
promoterforBrassicanapus.PlantCellRep2011,30:75-80.
6. AusubelFM,BrentR,KingstonRE,MooreDD,SeidmanJG,SmithJA,
StruhlK:CurrentProtocolsinMolecularBiologyNewYork:GreenePublishing/ • Convenient online submission
Wiley-Interscience;1989,12.0.1-12.2.10. • Thorough peer review
7. WangW,TaiF,ChenS:Optimizingproteinextractionfromplanttissues
• No space constraints or color figure charges
forenhancedproteomicanalysis.JSepSci2008,31:2032-2039.
8. AbdallaKO,ThomsonJA,RafudeenMS:Protocolsfornucleiisolationand • Immediate publication on acceptance
nuclearproteinextractionfromtheresurrectionplantXerophytaviscosa • Inclusion in PubMed, CAS, Scopus and Google Scholar
forproteomicstudies.AnalBiochem2009,384:365-367.
• Research which is freely available for redistribution
9. WanasundaraPKJPD,ShahidiF:Removalofflaxseedmucilageby
chemicalandenzymatictreatments.FoodChem1997,59:47-55.
Submit your manuscript at
www.biomedcentral.com/submit
