A possible approach for gel-based proteomic studies in recalcitrant woody plants

  • Mónica Sebastiana1,

    Affiliated with

    • Andreia Figueiredo1Email author,

      Affiliated with

      • Filipa Monteiro1,

        Affiliated with

        • Joana Martins2,

          Affiliated with

          • Catarina Franco2,

            Affiliated with

            • Ana Varela Coelho2,

              Affiliated with

              • Fátima Vaz3,

                Affiliated with

                • Tânia Simões3,

                  Affiliated with

                  • Deborah Penque3,

                    Affiliated with

                    • Maria Salomé Pais1 and

                      Affiliated with

                      • Sílvia Ferreira1

                        Affiliated with

                        Contributed equally
                        SpringerPlus20132:210

                        DOI: 10.1186/2193-1801-2-210

                        Received: 4 February 2013

                        Accepted: 4 April 2013

                        Published: 8 May 2013

                        Abstract

                        Woody plants are particularly difficult to investigate due to high phenolic, resin, and tannin contents and laborious sample preparation. In particular, protein isolation from woody plants for two-dimensional gel electrophoresis (2-DE) is challenging as secondary metabolites negatively interfere with protein extraction and separation. In this study, three protein extraction protocols, using TCA, phenol and ethanol as precipitation or extraction agents, were tested in order to select the more efficient for woody recalcitrant plant gel-based proteomics. Grapevine leaves, pine needles and cork oak ectomycorrhizal roots were used to represent woody plant species and tissues. The phenol protocol produced higher quality 2-DE gels, with increased number of resolved spots, better spot focusing and representation of all molecular mass and isoelectric point ranges tested. In order to test the compatibility of the phenol extracted proteomes with protein identification several spots were excised from the phenol gels and analyzed by mass spectrometry (MALDI-TOF/TOF). Regardless the incomplete genome/protein databases for the plant species under analysis, 49 proteins were identified by Peptide Mass Fingerprint (PMF). Proteomic data have been deposited to the ProteomeXchange with identifier PXD000224. Our results demonstrate the complexity of protein extraction from woody plant tissues and the suitability of the phenol protocol for obtaining high quality protein extracts for efficient 2-DE separation and downstream applications such as protein identification by mass spectrometry.

                        Keywords

                        Grapevine Pine Oak Ectomycorrhizal roots Protein extraction 2-DE Mass spectrometry

                        Background

                        Nowadays, proteomics constitutes one of the priority research areas in biological sciences. Knowledge generated in the past years has shown that dynamism, variability and behaviour of proteins are more complex than what was thought (Abril et al. 2011). Unlike model biological systems, the full potential of proteomics is far from being completely exploited in plant biology research. Thus, only a low number of plant species have been investigated at the proteomics level and, mainly, by using strategies based on 2-DE coupled to MS, resulting in low proteome coverage (Carpentier et al. 2008). On proteomics, most of the biological research has been carried on model plants such as Arabidopsis thaliana, Solanum tuberosum or Medicago truncatula. Yet, knowledge generated from these and other model plants need to be applied to other plant species. Within the plant group, woody species are the most difficult to investigate due to high phenolic, resin, and tannin contents, as well as, very often, an incompletely sequenced genomes. In the plant kingdom, woody species are found within both Angiosperms and Gymnosperms. On the Gymnosperm group, much research has been conducted on the genus Pinus (Wu et al. 2008;Valledor et al. 20082010;Wang et al. 2013), with Maritime pine (Pinus pinaster Ait.) being one of the most representative species used for reforestation in South-western Europe. Angiosperm considers a large variety of broad-leaved trees and shrubs including oak and grapevine. Grapevine (Vitis vinifera) is considered the most important fruit plant throughout the world, thus much proteomic research has been conducted in the last decade on this species (reviewed in Giribaldi and Giuffrida 2010). Cork Oak (Quercus suber L.) is a Mediterranean forest species with a remarkable ecological, social and economic value. Cork production from cork-oak supports an industry of economic and social relevance in Mediterranean countries, but few proteomic studies have been conducted (Gómez et al. 2009;Ricardo et al. 2011).

                        For proteomic studies, particularly in woody species, sample preparation and protein separation are of extreme importance for optimal results as most problems associated with 2-DE can be traced down to the co-extraction of non protein cellular components that affect protein gel migration. Plant tissues are very rich in proteases and interfering compounds such as secondary metabolites (Wang et al. 2008), thus comparatively to other organisms, extraction of proteins is of great challenge (Görg et al. 2004;Isaacson et al. 2006). Two protocols, TCA-acetone and phenol, are generally used with some optimization related to the specific tissue, in function of the amounts of indigenous contaminants (organic acids, lipids, polyphenols, pigments or terpenes among others). The TCA-acetone protocol was initially developed by Damerval et al. (1986) and is based on protein denaturation and precipitation under acidic/hydrophobic conditions, which help to concentrate proteins and remove contaminants (Wang et al. 2008). Up to date, this is the most used protocol for protein extraction from plant tissues for proteomic analysis (Jorrín et al. 2007;Jorrín-Novo et al. 2009). For recalcitrant tissues, the phenol-based method has the potential to generate samples of higher purity than TCA-acetone, as compounds such as polysaccharides and other water-soluble contaminants are separated from the proteins that are solubilized in the phenolic layer (Hurkman and Tanaka 1986).

                        Until now studies comparing protein extraction protocols for plant proteomics have been focused on herbaceous plants, mainly on fruit tissues (Saravanan and Rose 2004;Carpentier et al. 2005;Song et al. 2006;Zheng et al. 2007), with few being conducted on woody plant tissues (Jellouli et al. 2010;Dziedzic and McDonald 2012). With this study we aimed to compare three previously published protein extraction protocols and to evaluate their performance for the extraction of high-quality protein extracts suitable for 2-DE and MS analysis using woody recalcitrant plant tissues (leaves and roots). We have used pine needles representing a tissue that is highly rich in terpene metabolites (Wang et al. 2008); grapevine mature leaves, typically more problematic during 2-DE analysis than young leaves due to high levels of polyphenols and organic acids (Wang et al. 2008), and cork oak roots, a highly vacuolated with low protein content and high level of secondary metabolites such as lignin (Chatterjee et al. 2012). Moreover, cork oak roots typically establishes ectomycorrhizal (ECM) symbiosis and the symbiotic fungus may present triterpenoids and pigments (Baumert et al. 1997) that can also interfere with 2-DE. We have tested the two most commonly used protein extraction methods in plants, TCA-acetone (Damerval et al. 1986) and phenol (Hurkman and Tanaka 1986), as well as a single-step ethanol precipitation-based protocol that was successfully applied to poplar proteome isolation (Ferreira et al. 2006), in order to select the best extraction method for woody recalcitrant plant species/tissues. As mass spectrometry is one of the most used techniques for protein identification, compatibility of the best protein extraction method with mass spectrometry was tested.

                        Results

                        Considering the protein yield obtained with the different protocols, a similar trend was observed in the different species/tissues analysed: ethanol-acetone precipitation allowed obtaining higher amounts of protein (3.6 – 21.9 mg/g FW) than TCA-acetone precipitation (2.8 – 16.6 mg/g FW) and phenol-based extraction protocol (0.6 – 5.8 mg/g FW) (Table 1). Considering the amount of protein extracted from each plant material with the different extraction protocols, ECM oak roots produced the lowest protein yields (Table 1) with all the extraction protocols. For pine needles and grapevine leaves, the three protein isolation methods produced equivalent amounts of total protein. Representative 2-DE gels for each species/method are shown in Figure 1. Both qualitative and quantitative differences were found among 2-DE patterns for the three protein extraction protocols. For pine needles, all three extraction protocols resulted in good quality well-resolved gels (Figure 1D,E,F). However, when compared with the phenol protocol, TCA-acetone and ethanol-acetone have resulted in lower number of spots as well as reduced in several areas of the gels especially at the high molecular weight region particularly for the highest pI range. For grapevine leaves, the phenol protocol resulted in good quality gels with efficient protein separation and good spot focusing (Figure 1G). TCA and ethanol produced inferior quality gels, when compared to phenol, with decreased spot focusing and under representation of proteins in the high molecular mass area of the gels (Figure 1H,I). For ECM oak roots, the phenol protocol was the only producing high quality gels (Figure 1A), with TCA-acetone and ethanol extraction methods producing atypical gels with deficient protein separation, low number of protein spots and bad spot focusing (Figure 1B,C). The highest number of protein spots observed in gels was using the phenol extraction for all the three species/tissues analysed (532 – 904 spots) (Table 1). For grapevine leaves and pine needles, TCA-acetone resulted in an intermediate number of spots (657 and 362, respectively) and ethanol precipitation produced the lowest number of spots (166 and 392, respectively). In ECM oak roots, both TCA-acetone and ethanol produced a significantly lower amount of spots when compared with the phenol protocol (904), with ethanol producing 111 spots and TCA-acetone only 36 spots. To characterize quantitative differences between the protocols assayed, spot distribution by molecular mass and pI were compared for the three extraction methods (Figure 2). For all the plant tissues/species analysed the phenol protocol permitted to obtain a more evenly spot distribution across all M r and pI regions. On the contrary, with the TCA-acetone and ethanol extraction protocols spots were located preferentially at the lower M r and acidic pI regions of the gels, especially in ECM oak roots. The phenol extraction protocol permitted to obtain more spots within the high molecular mass range when compared with the other two precipitation methods.
                        http://static-content.springer.com/image/art%3A10.1186%2F2193-1801-2-210/MediaObjects/40064_2013_Article_276_Fig1_HTML.jpg
                        Figure 1

                        Maritime pine ( Pinus pinaster ) needles and grapevine (A, D, G), ethanol-acetone (B, E, H) and TCA-acetone (C, F, I) extraction methods from cork oak ECM roots, Martime Pine ( Pinus pinaster ) needles and Grapevine ( Vitis vinifera cv Regent) leaves. Proteins were separated on a 4–7 linear pH gradient in the first dimension (IEF) and 15% polyacrylamide gels in the second dimension.

                        Table 1

                        Protein yields and total number of 2-DE protein spots, from grape leaves, pine needles, and cork oak ectomycorrhizal ECM roots after phenol, ethanol and TCA-acetone extraction protocols

                        Plant species

                        Protocol

                        Protein yield (mg/g FW)a

                        Total number of spots

                        Pine

                        Phenol

                        5.81 ± 0.46

                        805

                        Ethanol

                        21.88 ± 4.00

                        392

                        TCA-acetone

                        13.86 ± 1.14

                        657

                        Grapevine

                        Phenol

                        3.78 ± 0.61

                        532

                        Ethanol

                        20.55 ± 1.79

                        166

                        TCA-acetone

                        16.57 ± 1.31

                        362

                        Oak

                        Phenol

                        0.61 ± 0.14

                        904

                        Ethanol

                        3.57 ± 0.20

                        111

                         

                        TCA-acetone

                        2.77 ± 0.14

                        36

                        a Mean and standard deviation from three technical replicates.

                        FW, fresh weight.

                        http://static-content.springer.com/image/art%3A10.1186%2F2193-1801-2-210/MediaObjects/40064_2013_Article_276_Fig2_HTML.jpg
                        Figure 2

                        2-DE distribution of protein spots from grapevine fully developed leaves, pine needles and cork oak ectomycorrhizal roots proteomes extracted in the three protocols tested, according to their M r (A) and p I (B).

                        As the phenol protocol was found to be the most adequate to extract proteins from the three species/tissues analysed, its compatibility with MS for protein identification was investigated. Several protein spots from the phenol 2-DE gels from each species/tissue were excised and identified by MS. Protein spots were chosen from different gels regions in order to include acidic, basic, high and low molecular mass proteins and also different spot intensities. MALDI-TOF/TOF analysis showed that excised protein spots lead to good quality spectra (Figure 3A,B,C). Results of protein identification by MALDI-TOF/TOF are presented in Table 2 and Additional file 1: Table S1, Additional file 2: Table S2, Additional file 3: Table S3. Of the 52 total spots analysed in the three species, all were identified with significant MOWSE/ProteinPilot scores (i.e., a score greater than 50/2, respectively, at p < 0.05) confirming the compatibility of the phenol extraction method with MS analysis.
                        http://static-content.springer.com/image/art%3A10.1186%2F2193-1801-2-210/MediaObjects/40064_2013_Article_276_Fig3_HTML.jpg
                        Figure 3

                        Examples of tandem MS spectra of protein spots excised from a 2-DE gel, trypsin-digested and analyzed by MALDI-TOF/TOF. (A) Spot S6 MS/MS spectrum of the parent ion [MH] + 1 868.39 identified as ATGDDYAR; (B) Spot P1 MS/MS spectrum of the parent ion [MH] + 1 1000.53 identified as AHASTEGVTK; (C) Spot V6 MS/MS spectrum of the parent ion [MH] + 1 1069.57 identified as LESEHLAQIAK.

                        Table 2

                        Protein annotation in the grapevine fully developed leaves (V1-V15), cork oak ectomycorrhizal roots (S1-S20) and pine needles (P1-P14) spots excised from 2-DE gels and trypsin-digested

                        Spot

                        Protein ID

                        Annotation

                        Score

                        Search engine

                        Protein score

                        Sequence of the distinct fragmented peptides (p < 0,05)

                        V1

                        8615601

                        cyclase [Vitis pseudoreticulata]

                        532

                        ProteinPilot

                        14

                        EFESDYAGFTEDGAR

                        EVILVESLK

                        KEFESDYAGFTEDGAR

                        LDDVPAGMYNVHCLHLR

                        LPGAEGAPIR

                        SEAYPSAYGSGSCNVELIPVKR

                        WLVENTDIK

                        EFESDYAGFTEDGAR

                        GPALLVDAPR

                        LPGAEGAPIR

                        V2

                        49388156

                        putative chlorophyll a/b-binding protein type III precursor [Oryza sativa Japonica Group]

                        270

                        ProteinPilot

                        10.67

                        FQDWANPGSMGK

                        QGADRPLWFASK

                        QSLTYLDGSLPGDYGFDPLGLSDPEGTGGFIEPR

                        QYFLGLEK

                        WLAYGEVINGR

                        RFQDWANPGSMGK

                        LKEVKNGR

                        QGADRPLWFASK

                        QYFLGLEK

                        RFQDWANPGSMGK

                        WLAYGEVINGR

                        V3

                        225446775

                        oxygen-evolving enhancer protein 2, chloroplastic [Vitis vinifera]

                        449

                        ProteinPilot

                        2.13

                        SITDYGSPEEFLSK

                        TNTDFLPYNGEGFK

                        EFPGQVLR

                        V4

                        73647738

                        ascorbate peroxidase [Vitis pseudoreticulata]

                         

                        ProteinPilot

                        9.32

                        ALLSDPAFRPLVEK

                        EDKPEPPPEGR

                        NCAPIMLR

                        SYPTVSEEYKK

                        TGGPFGTMK

                        EDKPEPPPEGR

                        NCAPIMLR

                        V5

                        349048

                        ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, partial (chloroplast) [Pogostemon cablin]

                         

                        MASCOT

                        121.49

                        TFKGPPHGIQVER

                        V6

                        225460496

                        PREDICTED: ATP synthase delta chain, chloroplastic [Vitis vinifera]

                         

                        MASCOT

                        154.41

                        LESEHLAQIAK

                        TAIDPSLVAGFTIR

                        EIAKEFELVYNR

                        V7

                        225461287

                        PREDICTED: cytochrome b6-f complex iron-sulfur subunit, chloroplastic isoform 1 [Vitis vinifera]

                         

                        MASCOT

                        234.97

                        GDPTYLVVENDK

                        DALGNDVIADEWLK

                        FICPCHGSQYNNQGR

                        V8

                        22797822

                        ATP synthase epsilon subunit [Vitis vinifera]

                         

                        MASCOT

                        258.01

                        TRVEAINVTS

                        QIIEANLALR

                        IGNNEITVLVNDAEK

                        LNDQWLTMALMGGFAR

                        V9

                        359475330

                        PREDICTED: glycine-rich RNA-binding protein GRP1A-like [Vitis vinifera]

                         

                        MASCOT

                        313.75

                        DRGYGDGGSR

                        NITVNEAQSR

                        AFSQFGEILESK

                        GGGGGYGGGGGGYGGGSR

                        GFGFVTFSSEQSMR

                        CFVGGLAWATDDQSLER

                        V10

                        225468761

                        oxygen-evolving enhancer protein 1, chloroplastic [Vitis vinifera]

                        610

                        MASCOT

                        984.17

                        VPFLFTIK

                        RLTYDEIQSK

                        FGGEFLVPSYR

                        FCLEPTSFTVK

                        KFCLEPTSFTVK

                        DGIDYAAVTVQLPGGER

                        GTGTANQCPTIDGGVDSFAFK

                        FEEKDGIDYAAVTVQLPGGER

                        SKPETGEVIGVFESIQPSDTDLGAK

                        V11

                        225459768

                        plastocyanin, chloroplastic isoform 1 [Vitis vinifera]

                        331

                        MASCOT

                        453.18

                        GTYSFYCSPHQGAGMVGK

                        ISMSEEDLLNAPGEVYSVTLTEK

                        NNAGFPHNVVFDEDEVPSGVDVSK

                        V12

                        30687535

                        Quinone reductase family protein [Arabidopsis thaliana]

                        393

                        MASCOT

                        62.96

                        AFLDATGGLWR

                        V13

                        225456238

                        PREDICTED: glutamine synthetase cytosolic isozyme 1[Vitis vinifera]

                         

                        MASCOT

                        183.79

                        VIVEYIWVGGSGMDLR

                        GNNILVMCDTYTPAGEPIPTNKR

                        V14

                        359473178

                        Quinone oxidoreductase-like protein At1g23740, chloroplastic-like [Vitis vinifera]

                        612

                        MASCOT

                        460.41

                        VKPVVDPK

                        LNPYLESGK

                        KLNPYLESGK

                        VVAAALNPVDAK

                        AWVYGDYGGVDVLK

                        QFGSFAEYTAVEEK

                        EGGSVVALTGAVTPPGFR

                        ELKEGDEVYGDINEK

                        ATDSPLPTVPGYDVAGVVVK

                        V15

                        225432496

                        PREDICTED: glutamine synthetase leaf isozyme, chloroplastic [Vitis vinifera]

                         

                        MASCOT

                        349.06

                        DISDAHYK

                        AAEIFGNKK

                        EHISAYGEGNER

                        TISKPVEHPSELPK

                        HKEHISAYGEGNER

                        HETANINTFSWGVANR

                        GGNNILVICDSYTPAGEPIPTNKR

                        S1

                        4838443

                        symbiosis regulated acidic polypeptide SRAP32-3 [Pisolithus tinctorius]

                         

                        Protein Pilot

                        4

                        DKLEAKLDKAAGDYIDGVDI

                        TDVANSLEFASR

                        S2

                        160897637

                        hypothetical protein Daci_2194 [Delftia acidovorans SPH-1]

                         

                        MASCOT

                        59.58

                        ERAQSAAAIER

                        S3

                        71659717

                        hypothetical protein [Trypanosoma cruzi strain CL Brener]

                         

                        MASCOT

                        58.93

                        KDIAEEVLER

                        S4

                        20162432

                        AF493154_1 32 kDa-cell wall symbiosis regulated acidic polypeptide [Pisolithus microcarpus]

                         

                        MASCOT

                        80.35

                        NDPLYSEAEK

                        S5

                        71659717

                        hypothetical protein [Trypanosoma cruzi strain CL Brener]

                         

                        MASCOT

                        57

                        KDIAEEVLER

                        S6

                        20162434

                        32 kDa-cell wall symbiosis regulated acidic polypeptide precursor [Pisolithus microcarpus]

                        358

                        MASCOT

                        210.62

                        ATGDDYAR

                        NSLEFAAR

                        FQLAVCSEK

                        AADKATGDDYAR

                        S7

                        390601324

                        cysteine peroxiredoxin [Punctularia strigosozonata HHB-11173SS5]

                        391

                        MASCOT

                        334.84

                        NFDEVLR

                        TVFVIDPK

                        LTISYPASTGR

                        VVDSLQLGDKYR

                        LGSIAPDFEAETTAGPIK

                        ISTLYDMLDEQDATNR

                        S8

                        225461209

                        PREDICTED: flavoprotein wrbA isoform 1 [Vitis vinifera]

                         

                        MASCOT

                        383.52

                        GAASVEGVEAK

                        KGAASVEGVEAK

                        AFLDATGGLWR

                        GGSPYGAGTFAGDGSR

                        VKGGSPYGAGTFAGDGSR

                        VYIVYYSMYGHVEK

                        S9

                        20097

                        jgi|Pisti1|20097|gm1.2716_g

                         

                        MASCOT

                        54.44

                        NPDIQAPR

                        S10

                        218533914

                        serine proteinase inhibitor [Clitocybe nebularis]

                        50.1

                        MASCOT

                        119.09

                        AQEWVIR

                        YRELQDAYTIVK

                        S11

                        20097

                        jgi|Pisti1|20097|gm1.2716_g

                         

                        MASCOT

                        302.72

                        VFAVMEGR

                        LDEPGEIGWIAPTDGSSQIR

                        RLDEPGEIGWIAPTDGSSQIR

                        EIPTAPPGQYRPEELYNLAFPLE

                        S12

                        218533914

                        serine proteinase inhibitor [Clitocybe nebularis]

                        50.1

                        MASCOT

                        89.1

                        AQEWVIR

                        ELQDAYTIVK

                        YRELQDAYTIVK

                        S13

                        20097

                        jgi|Pisti1|20097|gm1.2716_g

                         

                        MASCOT

                        343.22

                        LDEPGEIGWIAPTDGSSQIR

                        RLDEPGEIGWIAPTDGSSQIR

                        EIPTAPPGQYRPEELYNLAFPLE

                        S14

                        33323059

                        major latex protein [Ficus pumila var. awkeotsang]

                        187

                        MASCOT

                        485.65

                        GIDEHITKA

                        LREDVPAPDK

                        EKVEYDDANR

                        SPPEKYYNIFK

                        SATLIGVDGDIMQEYK

                        GQAYHVPNAAPDHIQGVDVHEGDWETHGSVK

                        S15

                        3164115

                        major latex-like protein [Rubus idaeus]

                         

                        MASCOT

                        68.2

                        EKVELDDVNK

                        S16

                        Q9S1X8

                        Na(+)/H(+) antiporter NhaA 1/4[Streptomyces coelicolor strain ATCC BAA-471/A3(2)/M145]

                         

                        MASCOT

                        483.67

                        NDAYVIAK

                        EEREEER

                        GVGWVAPSPENK

                        VGECTYVISAR

                        SVTEPPTFNMEK

                        KSVTEPPTFNMEK

                        GVGWVAPSPENKEER

                        S17

                        375333787

                        lectin 2 [Agrocybe aegerita]

                        565

                        MASCOT

                        647.19

                        FLGEATGDGR

                        FVVDLTGDGR

                        DFAYSAGGWR

                        DGFSIQPFVAIK

                        ADIVGFGDGGVLVSK

                        SVIDNFTYSAGGWR

                        FVLNNFGVQQGWQVNK

                        NTGGGNFSPASLALNDFGYNAGGWR

                        S18

                        392590852

                        phosphoglycerate mutase-like protein [Coniophora puteana]

                        540

                        MASCOT

                        431.54

                        VYASPEFK

                        DIGGIGNLPGR

                        TAQPFFGAIR

                        LPPTLIEQAR

                        GPAPEDRDFLR

                        ADIPLTEFFYR

                        SVYLSPSSPSYITNMK

                        S19

                        160184939

                        Serine protease inhibitor [Lentinula edodes (Shiitake mushroom)]

                        58.9

                        MASCOT

                        106.97

                        WCIQYTER

                        VGDCTYVISAR

                        S20

                        1001331

                        jgi|Pisti1|1001331|fgenesh1_kg.33_#_73_#_Locus10529v3rpkm0.40_PRE

                         

                        MASCOT

                        205.69

                        YYINYLIER

                        WIITFVPQPGR

                        NNLLYEQVTAPQK

                        P1

                        332591479

                        phosphoglycerate kinase 1 [Pinus pinaster]

                         

                        MASCOT

                        260.9

                        AHASTEGVTK

                        LTELLGVNVVK

                        ELDYLVGAVSNPK

                        ADLNVPLDENQNITDDTR

                        P2

                        396547

                        glutamate-ammonia ligase [Pinus sylvestris]

                         

                        MASCOT

                        134.75

                        SLSGPVSSVK

                        VIAEYIWIGGSGMDMR

                        P3

                        218155

                        chloroplastic aldolase [Oryza sativa Japonica Group]

                         

                        MASCOT

                        129.98

                        EAAWGLAR

                        AKANSLAQLGK

                        LASIGLENTEANR

                        P4

                        3415126

                        phenylcoumaran benzylic ether reductase [Pinus taeda]

                         

                        MASCOT

                        497.86

                        VVILGDGNAR

                        SLAQAGLTAPPR

                        ILLIGATGYIGR

                        DKVVILGDGNAR

                        ASLDLGHPTFLLVR

                        FFPSEFGNDVDNVHAVEPAK

                        GDQTNFEIGPAGVEASQLYPDVK

                        AIEAEGIPYTYVSSNCFAGYFLR

                        P5

                        413951269

                        ferredoxin-NADP reductase, leaf isozyme [Zea mays]

                        768

                        MASCOT

                        388.09

                        KDNTYVYMCGLK

                        RLVYTNDQGEIVK

                        LYSIASSALGDFGDSK

                        ITGDDAPGETWHMVFSTEGEIPYR

                        P6

                        359473184

                        carbonic anhydrase, chloroplastic-like isoform 2 [Vitis vinifera]

                        299

                        MASCOT

                        109.34

                        FMVVACADSR

                        QTAFIEDWIK

                        P7

                        359473184

                        carbonic anhydrase, chloroplastic-like isoform 2 [Vitis vinifera]

                        299

                        MASCOT

                        107.77

                        FMVVACADSR

                        QTAFIEDWIK

                        P8

                        14719331

                        putative 3-beta hydroxysteroid dehydrogenase/isomerase protein [Oryza sativa]

                        496

                        MASCOT

                        245.13

                        MKPGFDPSK

                        IGGGDDVFVGDIR

                        AEQYLADSGLPYTIIR

                        KAEQYLADSGLPYTIIR

                        P9

                        116790330

                        unknown [Picea sitchensis]

                         

                        MASCOT

                        104.32

                        TTFLSDSEVK

                        TTFLSDSEVKR

                        P10

                        116782111

                        unknown [Picea sitchensis]

                         

                        MASCOT

                        220.45

                        EYYNISVLTR

                        YEDNGDTVSNVSVMVIPTDKK

                        P11

                        16798638

                        AF434186_1 Cu-Zn-superoxide dismutase precursor [Pinus pinaster]

                         

                        MASCOT

                        234.71

                        LTHGAPEDDVR

                        KLTHGAPEDDVR

                        GGHELSLTTGNAGGR

                        GNSQVEGVVNLSQEDNGPTTVK

                        P12

                        2911276

                        LMW heat shock protein [Fragaria x ananassa]

                        103

                        MASCOT

                        105.95

                        QPEPQPPQPK

                        ASMEDGVLTVTVPK

                        P13

                        413946843

                        Putative peptidyl-prolyl cis-trans isomerase family protein [Zea mays]

                        307

                        MASCOT

                        138.86

                        TFEDENFK

                        KLESEETNR

                        IVLGLFGEDVPK

                        P14

                        20794

                        Type III chlorophyll a/b-binding protein [Pinus sylvestris]

                        259

                        MASCOT

                        268.1

                        LQDYRNPGSMGK

                        YLGGSGNPAYPGGPLFNPLGFGK

                         

                        YLGGSGNPAYPGGPLFNPLGFGKDEK

                        (Protein annotations retrieved from NCBI protein database restricted to Viridiplantae, to Vitis , to Agaricomycotina, JGI Pisolithus tinctorius manual and NCBI Blastp).

                        Discussion

                        Woody plant tissues contain significant amounts of secondary metabolites with different roles ranging from structural functions to defence against pathogens (Rhodes 1994). Most plant secondary metabolites belong to the class of phenolics including phenols, flavonoids, stilbenes, terpenes, tannins and lignins (Rhodes 1994) and can negatively interfere with protein extraction and 2-DE protein separation. For example, phenolics can build irreversible complexes with proteins, and the oxidation of phenolics by phenoloxidases and peroxidases can cause streaking and generate artifactual spots on gels (Vâlcu and Schlink 2006). Carbohydrates can block gel pores causing precipitation and extended focusing times, resulting in streaking and resolution loss (Carpentier et al. 2005). Also terpenoids, pigments, lipids and waxes produce streaking and charge heterogeneity (Carpentier et al. 2005). Secondary metabolites accumulate as soluble forms in the vacuoles and are more abundant in adult mature tissues than in young etiolated tissues (Granier 1988). Thus, sample preparation becomes a critical step for a proteomic approach focused on mature woody plants tissues. In the context of proteomic studies, comparison of 2-DE gels requires well-resolved proteomes. For total proteome extraction, an ideal protocol should reproducibly capture all the protein species composing the proteome with low contamination from other molecules. In the present study, the protocols based on ethanol-acetone (Ferreira et al. 2006), TCA-acetone (Damerval et al. 1986), and phenol (Hurkman and Tanaka 1986) were evaluated for proteome isolation, on three different woody recalcitrant plant tissues: grapevine leaves, pine needles and ECM oak roots. To compare the effects of ethanol, phenol and TCA protein extraction methods on the 2-DE maps, equal amounts of protein extracted from the different plant materials, were separated by 2-DE under identical conditions. Comparison of the extraction methods was done based on protein yield, spot focusing and resolution. Additionally, several 2-DE protein spots from each of the species/tissues analyzed were selected from gels of the best performing method, phenol extraction, to evaluate its compatibility and quality for protein identification by MS-based techniques.

                        Considering protein yield, TCA-acetone and ethanol precipitation methods produced higher yields than the phenol method for all the species/tissues analyzed. Studies comparing the performance of TCA and phenol protocols have been conducted earlier by Saravanan and Rose (2004) and Carpentier et al. (2005), that reported the same protein yield by the two methods in several recalcitrant fruit tissues (tomato, orange, banana and avocado), leaves and roots. However, the tissues analyzed in our study are much more lignified than the ones used by these authors and this could have contributed to the observed difference in protein yield between the two extraction protocols. Leaves and roots of woody plants are very rich in lignin, an aromatic polymer that results from the oxidative combinatorial coupling of 4-hydroxyphenylpropanoids which accumulates in the walls of secondary thickened cells, causing rigidness (Vanholme et al. 2010). We hypothesize that these compounds, present in our samples, could have co-precipitate with proteins in the TCA and ethanol protocols leading to an overestimation of protein yield using the Bradford assay. The Coomassie blue dye in this assay binds primarily to aromatic amino acid residues (Bio-Rad Protein Assay Manual), possibly also binding to the aromatic compounds of lignin leading to false positive results in woody plant tissues. This is corroborated by the observation in our samples of a lower spot number in 2-DE gels from the TCA and ethanol protocols, when compared with the phenol protocol (Figure 1). A similar result was also reported in a study comparing TCA and phenol protein extraction of Douglas fir needles, a woody plant tissue like the ones hereby analysed, with TCA showing lower intensity spots when compared to gels from a phenol protocol (Dziedzic and McDonald 2012). TCA has been reported as a suitable extraction method for soft/young plant tissues but it was found unsuitable for more complex plant tissues due to the co-extraction of polymeric contaminants (Saravanan and Rose 2004;Carpentier et al. 2005). Using the phenol protocol, similar protein yields were obtained to the ones reported for other woody plant tissues (Wang et al. 20032006;Dziedzic and McDonald 2012) extracted with a phenol based protocol, corroborating our results. As expected, protein recovery from roots was substantially lower than from leaves/needles, for the three protocols used, highlighting the cellular structural differences between the two tissues. Roots are highly vacuolated tissues containing lower protein amounts when compared to aerial parts, which makes them one of the most recalcitrant plant tissues for protein purification.

                        For the three species/tissues analyzed, the phenol extraction protocol produced the best quality gels despite presenting the lowest protein yields. The phenol 2-DE gels showed higher number of spots, increased resolution and spot focusing, increased number of high molecular weight spots, and lower background when compared with TCA-acetone and ethanol-acetone methods. Using the phenol extraction, up to 904, 805 and 532 spots were resolved from ECM oak roots, pine needles and grapevine leaves, respectively. These values are in agreement with the number of spots obtained in the same species/tissues previously reported (Burgess et al. 1995;Jellouli et al. 2010;Liu et al. 2012).

                        Phenol has been reported as the most suitable protein extraction protocol for tissues containing low concentrations of protein and high content of interfering compounds that inhibit electrophoresis (Saravanan and Rose 2004;Wang et al. 2008). It has been widely used to extract proteins from difficult plants like olive and cotton (Wang et al. 2003;Yao et al. 2006), or fruits including banana, strawberry, apple or grape (Saravanan and Rose 2004;Vincent et al. 2006;Wang et al. 2008). Its superior performance has been attributed to a higher capacity to physically separate proteins from contaminating substances like nucleic acids, carbohydrates and cellular debris. Therefore, a great amount of the 2-DE interfering substances are immediately eliminated in the aqueous phase through phase separation, which is increased by the presence of added sucrose. Proteins, which remain solubilized and mostly purified in the phenolic phase, can then be precipitated with methanol and ammonium acetate (Faurobert et al. 2007). In addition to its selectivity as a solvent, phenol is one of the strongest dissociating agents known to decrease molecular interactions between proteins and other materials (Carpentier et al. 2005).

                        In order to determine the compatibility of the phenol isolated proteome from the species/tissues analysed with protein identification methods, several protein spots were excised from 2-DE gels and subjected to MS analysis. Identification of all the excised spots confirmed the compatibility of the phenol extraction protocol with MS protein identification. This is in agreement with previous studies on protein extraction from recalcitrant fruit tissues (Carpentier et al. 2005;Zheng et al. 2007) and woody plant tissues (Wang et al. 20032006;Dziedzic and McDonald 2012). Some of the proteins identified, such as SRAP32 from P. tinctorius identified in oak ECM roots, were previously described (Burgess et al. 1995;Laurent et al. 1999) in the symbiotic roots of other forest tree species. These acidic cell wall symbiosis regulated proteins (SRAPS) are induced by ECM development and are thought to be involved in the attachment of fungal hyphae to the root surface during symbiosis formation. In our 2-DE gels, SRAP32 molecular mass and isoelectric point is in accordance to those reported earlier (Burgess et al. 1995;Laurent et al. 1999). Also, for ECM cork oak roots only 3 out of the 20 protein spots analysed match plant proteins, which is in accordance to Burgess et al. (1994) and Zeppa et al. (2005), which report a marked inhibition of the plant polypeptide synthesis and an enhanced accumulation of fungal peptides during ECM development. For grapevine leaves and pine needles, several photosynthesis/energy related proteins, such as ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, chloroplastic aldolase or ATP synthase delta chain chloroplastic, among others were identified, which is in agreement with the photosynthetic and carbon fixation primary function of foliar tissues. Photosynthesis and energy related proteins were also the major group of proteins identified by ESI-MS/MS in Douglas-fir needles (Dziedzic and McDonald 2012).

                        Conclusions

                        The phenol extraction protocol allowed an efficient proteome isolation and 2-DE separation of the woody recalcitrant plants used in this study. Also, the resulting protein spots were found to be compatible with identification by MALDI-TOF/TOF. This study illustrates the need to establish a proper protein extraction method when preparing plant tissues for proteomic analysis, particularly when working with woody recalcitrant plant tissues containing high levels of interfering compounds.

                        Methods

                        Plant material

                        Grapevine

                        V. vinifera ‘Regent’ grapevine wood cuttings were harvested at Quinta da Plansel (Montemor, Portugal) and grown in 12 cm ø pots under greenhouse conditions (natural day/night rhythm and a temperature range between 5 and 28°C) for ten weeks. Leaves were harvested, frozen and grounded in liquid nitrogen using a mortar and pestle and stored at −80°C until protein extraction.

                        Pine

                        Pinus pinaster trees with breast height diameter (BHD) classes > 20 cm were selected in mid-end June (Comporta, Portugal). Samples were collected from one branch of the lower canopy at a height of at least 8 m. Needles were harvested, frozen in liquid nitrogen and stored at −80°C.

                        Cork oak

                        The Pisolithus tinctorius (Pers.) Couker & Couch isolate Pt23 from the collection of the Center of Biodiversity, Functional & Integrative Genomics (BioFIG), Sciences Faculty of Lisbon University, was grown on a peat/vermiculite (v/v) mixture moistened with liquid BAF medium (Moser 1960), for two months in the dark at 25°C, and then used as ECM inoculum. Quercus suber L. seeds were surface disinfected by shaking in 30% commercial bleach for 30 min and washing in four changes of distilled water. Seeds were sown on soil in plastic trays, and seedlings were grown in a greenhouse under natural light and temperature and watered as needed. Four months old seedlings were transferred from the sowing beds to 1,5 L pots containing soil, and inoculated with the fungal inoculum by depositing 350 mL of peat-vermiculite grown mycelium (previously rinsed with water to remove excess nutrients) in the plantation hole, in direct contact with the roots. Four months after inoculation, ten cork oak ectomycorrhizal seedlings were sampled. Roots were rinsed to eliminate soil particles, first with tap water and after with deionized water. Excess water was removed with filter paper. Secondary roots presenting ECM root tips were sampled and immediately frozen in liquid nitrogen, grounded and stored at −80°C.

                        Proteome extraction

                        Ethanol-acetone method

                        Plant tissue (1 g) was dispersed in 4 vol of ethanol (Merck). After 1 h at −20°C, the same volume of cold acetone (Merck) was added and proteins were allowed to precipitate overnight, at −20°C. Proteins were collected through centrifugation at 26000 g (−10°C, 15 min), followed by a washing step with ethanol:acetone:triple distilled water 4:4:1 (v/v/v) with 9 sample volume for 6 h at −20°C. Proteins were recovered by centrifugation at 26000g (−10°C, 40 min), followed by two additional washing steps. The final pellet was dried overnight at room temperature and solubilized in lysis buffer [7 M urea, 2 M thiourea, 0.25% (v/v) of Pharmalyte 3–10 and 0.5% (v/v) of Pharmalyte 4–7 (Amersham Pharmacia Biotech, Uppsala, Sweden), 2% (w/v) 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and 25 mM dithiothreitol (DTT)] for 24 h at room temperature. Protein quantification was performed with Bradford reagent (Bradford 1976) using Bovine Serum Albumin (BSA) as standard (Bio-Rad protein assay, BioRAD, USA). Solubilized proteomes were kept at −20°C until further use. Three technical replicates of each extraction were performed for each species.

                        TCA-acetone method

                        Plant tissue (1 g) was suspended in 10% TCA (w/v) (Sigma) in acetone (Merck) at −20°C, with 0.1% (w/v) of DTT (Sigma). Proteins were precipitated overnight at −20°C and recovered through centrifugation at 26000 g for 1 h at −10°C. Pellet was resuspended in 90% (v/v) acetone at −20°C with 0.1% (w/v) DTT and precipitated for 2 h at −20°C, followed by centrifugation at 26000 g for 45 min at −10°C. This washing procedure was repeated twice. Final protein solubilisation and quantification procedures were done as described above. Three technical replicates of each extraction were performed.

                        Phenol extraction method

                        Plant tissue (1 g) was suspended in 10 mL of extraction buffer [5 mL of Tris pH 8.8 buffered phenol and 5 mL of extraction media (0.1 M Tris–HCl pH 8.8, 10 mM EDTA, 0.4% (w/v) 2-mercaptoethanol and 0.9 M sucrose)]. Samples were homogenized and incubated for 30 min at 4°C with agitation and then centrifuged 10 min at 5000 g, 4°C. The phenol phase was recovered and proteins were precipitated by addition of 5 vol of 0.1 M ammonium acetate in 100% methanol (pre-chilled to −20°C) and incubated overnight at −20°C. The precipitate was collected by centrifugation (30 min, 4000 g, -10°C) washed twice with the ammonium acetate solution in methanol, twice with ice-cold 80% (v/v) acetone and one time with cold 70% (v/v) ethanol. Between each washing step, the resuspended sample was kept at −20°C for 20 min. Final protein solubilization and quantification procedures were done as described above. Three technical replicates of each extraction were performed for each species.

                        Two-dimensional electrophoresis

                        Analytical gels were performed using 18 cm IPG strips of linear 4–7 pH gradient (GE Healthcare). Prior proteins isoelectric focusing (IEF), strips were passively rehydrated overnight with lysis buffer containing 300 μg of protein per sample in an IEF Rehydration Tray (GE Healthcare). IEF was performed using an IPGphor™ Isoelectric Focusing System (Amersham-Pharmacia Biotech Pharmacia Biotech) with the IPGPhor Manifold. IEF was performed for 26 h at 20°C to a total of 86000 Vh. Subsequently, focused IPG strips were immediately equilibrated for 15 min in equilibration buffer [2% (w/v) sodium dodecyl sulfate (SDS), 10% (v/v) glycerol, 50mM Tris–HCl pH 6.8 and 1% (v/v) DTT], followed by immediate storage at −80°C until use, as previously described (Ferreira et al. 2006). IPG strips were thawed and reequilibrated for 15 min using fresh equilibration buffer (Ferreira et al. 2006), and immediately loaded onto 26 × 20 × 0.1 cm3 15% polyacrylamide gels (acrylamide:bisacrylamide at 200:1). The top of the gel was sealed using agarose sealing solution (0.5% (w/v) agarose in running buffer with bromophenol blue). Electrophoresis was performed in recirculating running buffer for 16 h at 10°C, under constant power settings (80 mA). The three replicates prepared per extraction protocol were resolved on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 2D-PAGE was allowed to run until the dye front reached the lower end of the gels. Protein isoelectric points were determined by the use of Isoelectric Focusing Calibration kit Broad pI (pH 4–7), while their molecular masses were determined using PageRuler™ unstained protein ladder (Thermo Fisher Scientific). Gels were stained with Oriole™ fluorescence gel stain (Bio-Rad), following manufacturer’s instructions. Given the broad UV excitation of Oriole™, image acquisition was done on the UV-based image equipment ChemiDoc™ XRS+ (BioRad) using the software Image Lab™ 2.0. Gels exposure times to UV excitation were always set below the limit of spot saturation.

                        Image analysis

                        The 2-DE gel images were analyzed using REDFIN software v. 3.3 (http://​www.​ludesi.​com). Each protein extraction method (TCA-acetone, phenol and ethanol-acetone) was represented by three 2-DE gels images matching three technical replicates. For each protocol, gel images were warped after setting vector points to construct a composite image (i.e. raw master gel). This fusion gel image, i.e. normalized image, was created to eliminate noise and minor discrepancies between gels. The spots were detected and quantified as the cumulative intensity of optical density of each spot, proportional to spot volume. Normalization of spot volumes was automatically done by REDFIN 3 software (Ludesi, Lund, Sweden, http://​www.​ludesi.​com) using the total spot volume methods, by removing technical differences in staining, scanning and sample volume. Spot-by-spot visual validation of automated analysis was done thereafter to increase the reliability of the matching (Chich et al. 2007). Experimental pI was determined using a 4–7 linear scale over the total length of the IPG strip (18 cm). M r values were calculated by mobility comparisons with the PageRuler™ protein ladder (Thermo Fisher Scientific). Total number of spots was calculated as spots present in three technical replicate gels.

                        MS analysis and protein identification

                        Preparative 2-DE gels loaded with 600 μg of protein extracted with the phenol-based method, for each plant were used for spot picking. After 2-DE, the gel was colloidally CBB-stained (Neuhoff et al. 1988) and around 2% (52 spots) of total spots present per plant material (15 spots on grapevive leaves, 15 spots for pine needles and 22 for oak ECM roots) were randomly excised and trypsin-digested as described by da Costa et al. (da Costa et al. 2008). Sample peptides were acidified with formic acid, desalted, and concentrated with POROS R2 microcolumns (Applied Biosystems, Foster City, CA) and co-crystallised in MALDI-TOF/TOF sample plates according to da Costa et al. (da Costa et al. 2008) using the matrix α-cyano-4-hydroxycinnamic acid (CHCA). Tandem MS/MS was performed using a MALDI-TOF/TOF 4800 plus MS/MS (Applied Biosystems, Foster City, CA, USA). The MS/MS was externally calibrated using des-Arg-Bradykinin (904.468 Da), angiotensin 1 (1296.685 Da), Glu-Fibrinopeptide B (1570.677 Da), ACTH (1–17) (2093.087 Da), and ACTH (18–39) (2465.199 Da) (4700 Calibration Mix, Applied Biosystems, Foster City, CA, USA). Each reflectron MS spectrum was collected in a result-independent acquisition mode, typically using 1000 laser shots per spectra and a fixed laser intensity of 3500V. The fifteen strongest precursors were selected for MS/MS, the weakest precursors being fragmented first. MS/MS analyses were performed using CID (Collision Induced Dissociation) assisted with air, with a collision energy of 1 kV and a gas pressure of 1 × 10-6 torr and the PRIDE Team for all the support during data submission to the public data repository PRoteomics IDEntifications database PRIDE. Two thousand laser shots were collected for each MS/MS spectrum using a fixed laser intensity of 4500V.

                        Protein identification was performed by homology search on different protein databases using the Mascot and Protein Pilot (Applied Biosystems, Foster City, CA, USA) search engines. Searches in MASCOT (v. 2.2; Matrix Science, Boston, MA, USA) were performed without taxonomical restrictions, a minimum mass accuracy of 30 ppm for the parent ions, an error of 0.3 Da for the fragments, trypsin as digesting enzyme with one missed cleavage allowed, and carbamidomethylation of Cys and oxidation of Met as fixed and variable amino acid modifications, respectively. ProteinPilot (Protein Pilot software v. 3.0, rev. 114732; Applied Biosystems, Foster City, CA, USA) searches were performed without taxonomic restrictions and search parameters set as follows: enzyme, trypsin; Cys alkylation, iodoacetamide; special factor, gel-based ID; and ID focus, biological modification and amino acid substitution. Peptide sequences belonging to the different plant species, i.e. grapevine and pine leaves, and ECM oak roots, were queried against NCBI’s Viridiplantae protein database available on both in-house Mascot and ProteinPilot servers. The NCBI proteins from Vitis (102484 entries, July 2012) and Agaricomycotina (334526 entries, July 2012), and the proteins from P. tinctorius Marx 270 v1.0 at the JGI portal (BestModels v1.0, release date April 10, 2012; http://​genome.​jgi-psf.​org/​Pisti1/​Pisti1.​home.​html) were also queried for annotation. Protein sequences that were identified as “unknown” or as “hypothetical protein”, were further annotated by using the protein homologs sequences for an additional query using BLASTP algorithm (http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi), searching first the UniProtKB/Swiss-Prot database, and then the NCBI non redundant database. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://​proteomecentral.​proteomexchange.​org) via the PRIDE partner repository (Vizcaíno et al., 2013) with the dataset identifier PXD000224.

                        Declarations

                        Acknowledgements

                        This work was developed and supported within the frame of the projects “Unravelling grapevine defense mechanism against downy mildew through O’mics (transcriptomics, metabolomics and proteomics) networking” - PTDC/AGR-GPL/119753/2010; “Deciphering ectomycorrhizal symbiosis through O’mics (transcriptome, metabolome and proteome profiling) networking” – PTDC/AGR-AAM/105531/2008 of the Portuguese Foundation for Science and Technology; BIOFIG PEst-OE/BIA/UI4046/2011 and by the fellowships SFRH/BPD/25661/2005, SFRH/BPD/63641/2009 and SFRH/BPD/79271/2011. The authors would like to acknowledge Dr. Regina Freitas and the Disease and Stress Biology group from Instituto Superior de Agronomia (ISA, Lisbon) for the acquisition of 2-DE gels images.

                        Authors’ Affiliations

                        (1)
                        Plant Systems Biology Lab, Center of Biodiversity, Functional & Integrative Genomics (BioFIG), Science Faculty of Lisbon University
                        (2)
                        Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa
                        (3)
                        Laboratório de Proteómica, Departamento de Genética, Instituto Nacional de Saúde Dr. Ricardo Jorge INSA I.P

                        References

                        1. Abril N, Gion JM, Kerner R, Muller-Starck G, Cerrillo RM, Plomion C, Renaut J, Valledor L, Jorrín-Novo JV: Proteomics research on forest trees, the most recalcitrant and orphan plant species. Phytochem 2011, 72(10):1219-1242. 10.1016/j.phytochem.2011.01.005View Article
                        2. Baumert A, Schumann B, Porzel A, Schmidt J, Strack D: Triterpenoids from pisolithus tinctorius isolates and ectomycorrhizas. Phytochem 1997, 45(3):499-504. 10.1016/S0031-9422(97)00007-1View Article
                        3. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72: 248-254. 10.1016/0003-2697(76)90527-3View Article
                        4. Burgess T, Dell B, Malajczuk N: Variation in mycorrhizal development and growth stimulation by 20 pisolithus isolates inoculated on to eucalyptus grandis W. Hill ex Maiden. New Phytol 1994, 127(4):731-739. 10.1111/j.1469-8137.1994.tb02977.xView Article
                        5. Burgess T, Laurent P, Dell B, Malajczuk N, Martin F: Effect of fungal-isolate aggressivity on the biosynthesis of symbiosis-related polypeptides in differentiating eucalypt ectomycorrhizas. Planta 1995, 195: 408-417.View Article
                        6. Carpentier SC, Witters E, Laukens K, Deckers P, Swennen R, Panis B: Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. Proteomics 2005, 5(10):2497-2507. 10.1002/pmic.200401222View Article
                        7. Carpentier SC, Panis B, Vertommen A, Swennen R, Sergeant K, Renaut J, Laukens K, Witters E, Samyn B, Devreese B: Proteome analysis of non-model plants: a challenging but powerful approach. Mass Spectrom Rev 2008, 27(4):354-377. 10.1002/mas.20170View Article
                        8. Chatterjee M, Gupta S, Bhar A, Das S: Optimization of an efficient protein extraction protocol compatible with two-dimensional electrophoresis and mass spectrometry from recalcitrant phenolic rich roots of chickpea (cicer arietinum L.). Int J Proteomics 2012. 10.1155/2012/536963
                        9. Chich JF, David O, Villers F, Schaeffer B, Lutomski D, Huet S: Statistics for proteomics: experimental design and 2-DE differential analysis. J Chromatogr B Analyt Technol Biomed Life Sci 2007, 849(1–2):261-272.View Article
                        10. Da Costa G, Lamy E, Capela e Silva F, Andersen J, Sales Baptista E, Coelho AV: Salivary amylase induction by tannin-enriched diets as a possible countermeasure against tannins. J Chem Ecol 2008, 34: 376-387. 10.1007/s10886-007-9413-zView Article
                        11. Damerval C, De Vienne D, Zivy M, Thiellement H: Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis 1986, 7: 52-54. 10.1002/elps.1150070108View Article
                        12. Dziedzic JA, McDonald AG: A comparative survey of proteins from recalcitrant tissues of a non-model gymnosperm, douglas-fir. Electrophoresis 2012, 33(7):1102-1112. 10.1002/elps.201100526View Article
                        13. Faurobert M, Mihr C, Bertin N, Pawlowski T, Negroni L, Sommerer N, Causse M: Major proteome variations associated with cherry tomato pericarp development and ripening. Plant Physiol 2007, 143(3):1327-1346. 10.1104/pp.106.092817View Article
                        14. Ferreira S, Hjernø K, Larsen M, Wingsle G, Larsen P, Fey S, Roepstorff P, Pais MS: Proteome profiling of populus euphratica oliv. Upon heat stress. Ann Bot 2006, 98(2):361-377. 10.1093/aob/mcl106View Article
                        15. Giribaldi M, Giuffrida MG: Heard it through the grapevine: proteomic perspective on grape and wine. J Proteomics 2010, 73: 1647-1655. 10.1016/j.jprot.2010.05.002View Article
                        16. Gómez A, López JA, Pintos B, Camafeita E, Bueno MA: Proteomic analysis from haploid and diploid embryos of quercus suber L. Identifies qualitative and quantitative differential expression patterns. Proteomics 2009, 9(18):4355-4367. 10.1002/pmic.200900179View Article
                        17. Görg A, Weiss W, Dunn MJ: Current two-dimensional electrophoresis technology for proteomics. Proteomics 2004, 4(12):3665-3685. 10.1002/pmic.200401031View Article
                        18. Granier F: Extraction of plant proteins for two-dimensional electrophoresis. Electrophoresis 1988, 9: 712-718. 10.1002/elps.1150091106View Article
                        19. Hurkman WJ, Tanaka CK: Solubilization of plant membrane proteins for analysis by two-dimensional gel. Plant Physiol 1986, 81: 802-806. 10.1104/pp.81.3.802View Article
                        20. Isaacson T, Damasceno CM, Saravanan RS, He Y, Catalá C, Saladié M, Rose JK: Sample extraction techniques for enhanced proteomic analysis of plant tissues. Nat Protoc 2006, 1(2):769-774. 10.1038/nprot.2006.102View Article
                        21. Jellouli N, Salem AB, Ghorbel A, Jouira HB: Evaluation of protein extraction methods for vitis vinifera leaf and root. J Integr Plant Biol 2010, 52(10):933-940. 10.1111/j.1744-7909.2010.00973.xView Article
                        22. Jorrín JV, Maldonado AM, Castillejo MA: Plant proteome analysis: a 2006 update. Proteomics 2007, 7(16):2947-2962. 10.1002/pmic.200700135View Article
                        23. Jorrín-Novo JV, Maldonado AM, Echevarría-Zomeño S, Valledor L, Castillejo MA, Curto M, Valero J, Sghaier B, Donoso G, Redondo I: Plant proteomics update (2007–2008): second-generation proteomic techniques, an appropriate experimental design, and data analysis to fulfill MIAPE standards, increase plant proteome coverage and expand biological knowledge. J Proteomics 2009, 72(3):285-314. 10.1016/j.jprot.2009.01.026View Article
                        24. Laurent P, Voiblet C, Tagu D, de Carvalho D, Nehls U, De Bellis R, Balestrini R, Bauw G, Bonfante P, Martin F: A novel class of ectomycorrhiza-regulated cell wall polypeptides in pisolithus . Mol Plant Microbe Interact 1999, 12(10):862-871. 10.1094/MPMI.1999.12.10.862View Article
                        25. Liu D, Shi L, Han C, Yu J, Li D, Zhang Y: Validation of reference genes for gene expression studies in virus-infected nicotiana benthamiana using quantitative real-time PCR. PLoS One 2012, 7(9):e46451. 10.1371/journal.pone.0046451View Article
                        26. Moser M: Die gattung Phlegmacium, Schleimköpfe. Bad Heilbrunn: Verlag Julius Klinkhardt; 1960:440.
                        27. Neuhoff V, Arold N, Taube D, Ehrhardt W: Improved staining of proteins in polyacrylamide gels including isoelectric. Electrophoresis 1988, 9(6):255-262. 10.1002/elps.1150090603View Article
                        28. Rhodes MJ: Physiological roles for secondary metabolites in plants: some progress, many outstanding problems. Plant Mol Biol 1994, 24(1):1-20. 10.1007/BF00040570View Article
                        29. Ricardo CP, Martins I, Francisco R, Sergeant K, Pinheiro C, Campos A, Renaut J, Fevereiro P: Proteins associated with cork formation in quercus suber L. Stem tissues. J Proteomics 2011, 74(8):1266-1278. 10.1016/j.jprot.2011.02.003View Article
                        30. Saravanan RS, Rose JK: A critical evaluation of sample extraction techniques for enhanced proteomic. Proteomics 2004, 4(9):2522-2532. 10.1002/pmic.200300789View Article
                        31. Song J, Braun G, Bevis E, Doncaster K: A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis. Electrophoresis 2006, 27(15):3144-3151. 10.1002/elps.200500921View Article
                        32. Vâlcu CM, Schlink K: Efficient extraction of proteins from woody plant samples for two-dimensional. Proteomics 2006, 6(14):4166-4175. 10.1002/pmic.200500660View Article
                        33. Valledor L, Castillejo MA, Lenz C, Rodríguez R, Cañal MJ, Jorrín J: Proteomic analysis of pinus radiata needles: 2-DE map and protein identification. J Proteome Res 2008, 7(7):2616-2631. 10.1021/pr7006285View Article
                        34. Valledor L, Jorrín JV, Rodríguez JL, Lenz C, Meijón M, Rodríguez R, Cañal MJ: Combined proteomic and transcriptomic analysis identifies differentially expressed pathways associated to pinus radiata needle maturation. J Proteome Res 2010, 9(8):3954-3979. 10.1021/pr1001669View Article
                        35. Vanholme R, Demedts B, Morreel K, Ralph J, Boerjan W: Lignin biosynthesis and structure. Plant Physiol 2010, 153(3):895-905. 10.1104/pp.110.155119View Article
                        36. Vincent D, Wheatley MD, Cramer GR: Optimization of protein extraction and solubilization for mature grape berry. Electrophoresis 2006, 27(9):1853-1865. 10.1002/elps.200500698View Article
                        37. Vizcaíno JA, Côté RG, Csordas A, Dianes JA, Fabregat A, Foster JM, Griss J, Alpi E, Birim M, Contell J, O’Kelly G, Schoenegger A, Ovelheiro D, Pérez-Riverol Y, Reisinger F, Ríos D, Wang R, Hermjakob H: The Proteomics Identifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res 2013, 41(D1):D1063-D1069. 10.1093/nar/gks1262View Article
                        38. Wang W, Scali M, Vignani R, Spadafora A, Sensi E, Mazzuca S, Cresti M: Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds. Electrophoresis 2003, 24(14):2369-2375. 10.1002/elps.200305500View Article
                        39. Wang W, Vignani R, Scali M, Cresti M: A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis. Electrophoresis 2006, 27(13):2782-2786. 10.1002/elps.200500722View Article
                        40. Wang W, Tai F, Chen S: Optimizing protein extraction from plant tissues for enhanced proteomics. J Sep Sci 2008, 31(11):2032-2039. 10.1002/jssc.200800087View Article
                        41. Wang X, Liu Z, Niu L, Fu N: Long-term effects of simulated acid rain stress on a staple forest plant, pinus massoniana lamb: a proteomic analysis. Trees 2013, 27: 297-309. 10.1007/s00468-012-0799-zView Article
                        42. Wu X, Chen T, Zheng M, Chen Y, Teng N, Samaj J, Baluska F, Lin J: Integrative proteomic and cytological analysis of the effects of extracellular Ca(2+) influx on pinus bungeana pollen tube development. J Proteome Res 2008, 7(10):4299-4312. 10.1021/pr800241uView Article
                        43. Yao Y, Yang YW, Liu JY: An efficient protein preparation for proteomic analysis of developing cotton. Electrophoresis 2006, 27(22):4559-4569. 10.1002/elps.200600111View Article
                        44. Zeppa S, Sisti D, Pierleoni R, Potenza L, Guescini M, Vallorani L, Stocchi V: Tilia platyphyllos scop.- tuber brumale vittad. vs. T. Platyphyllos scop.- T. Borchii vittad. Ectomycorrhizal systems: a comparison of structural and functional traits. Plant Physiol Biochem 2005, 43(7):709-716. 10.1016/j.plaphy.2005.06.008View Article
                        45. Zheng Q, Song J, Doncaster K, Rowland E, Byers DM: Qualitative and quantitative evaluation of protein extraction protocols for apple and strawberry fruit suitable for two-dimensional electrophoresis and mass spectrometry analysis. J Agric Food Chem 2007, 55(5):1663-1673. 10.1021/jf062850pView Article

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                        © Sebastiana et al.; licensee Springer. 2013

                        This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.