Journal of Heredity Advance Access published online on March 15, 2008
Journal of Heredity, doi:10.1093/jhered/esn007
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Protein Polymorphism between 2 Picea abies Populations Revealed by 2-Dimensional Gel Electrophoresis and Tandem Mass Spectrometry
From the Section of Forest Genetics, Technische Universität München, Am Hochanger 13, D-85354 Freising-Weihenstephan, Germany (Valcu, Müller-Starck, and Schlink); and the UMR Biogeco 1202, INRA, Equipe de Génétique, Cestas, Cedex, France (Lalanne and Plomion)
Address correspondence to C.-M. Valcu at the address above, or e-mail: valcu{at}wzw.tum.de.
In species with high gene flow and consequent low interpopulation differentiation over wide geographic ranges, differential gene expression along ecological gradients often reveals adaptive significance. We investigated potential differences in protein expression between Picea abies ecotypes adapted to contrasting altitude conditions. Protein expression patterns were compared between needles and roots of 2-month-old P. abies seedlings by means of 2-dimensional electrophoresis. Proteins exhibiting differential expression between the 2 ecotypes were analyzed by tandem mass spectrometry. A total of 19 proteins exhibited qualitative or quantitative polymorphism between the 2 populations. These proteins exhibited organ-specific expression, and the level of interpopulation protein polymorphism was organ dependent. Among differentially expressed proteins, we identified proteins involved in photosynthesis, photorespiration, root tracheary element differentiation, and transmitochondrial membrane transport. Our results show that P. abies seedlings from locally adapted ecotypes exhibit consistent differences in protein expression. The expression polymorphism of some of these proteins has potential adaptive significance.
Trees' response to environmental selective pressure in terms of nucleotide substitution rates and speciation rates is relatively slow compared with other species, due to the high age of reproductive maturity and the long generation interval (Jump and Penuelas 2005; Petit and Hampe 2006). Compensatory mechanisms have therefore developed that allow tree species to rapidly adapt to changing environmental conditions (Saxe et al. 2001). As most tree species, conifers maintain unusual high intrapopulation levels of genetic diversity, due to specific life-history characteristics (long-lived woody species with outcrossing breeding systems spread over large geographic ranges) (Hamrick 2004). They have also acquired high levels of phenotypic plasticity adding to their adaptive potential and increasing their chances of surviving extreme climatic events. In spite of the low genetic differentiation between populations (reviewed by Hamrick 2004), conifers exhibit rather strong interpopulation variations not only of phenotypic characters including morphologic and physiologic features (Oleksyn et al. 1998) but also of phenotypic traits relevant for climatic adaptation such as frost hardiness (Skroppa et al. 1994; Daehlen and Johnsen 1995). Parental effects (i.e., influence of the environmental conditions during the sexual reproduction on the phenotype of the progeny) act synergistically with phenotypic plasticity, contributing to the maintenance of a high genetic variability in population, while exhibiting a phenotype "tuned" to the local environmental conditions (Skroppa and Johnsen 2000). Thus, phenotypic differences between populations are a result of not only genotype selection but also the environmental influence on gene expression through imprinting (Johnsen and Skroppa 1996) and phenotypic plasticity.
Besides assessing between- and within-species genetic variation based on molecular markers, targeting the protein level can bring valuable information on the mechanisms behind adaptation and evolution of populations. Although genetic markers are extremely useful for establishing genetic relationships and history of populations (e.g., Achere et al. 2005), they only account for the presence/absence of the given marker at individual level, ignoring quantitative traits. A more complete picture of within- and between-population variability can be acquired at gene expression level, that is, transcriptome and proteome level. This type of information is highly relevant for the processes of adaptation and evolution of natural populations. Qualitative polymorphism of a protein (e.g., presence/absence variation) most often mirrors variation of the structural genes, whereas quantitative variation (i.e., polymorphism of protein amounts) is the result of a complex network of regulatory mechanisms subject themselves to genetic variation and interaction with the environment (Thiellement et al. 2002; Rockman and Kruglyak 2006). Patterns of protein expression integrate thus the genetic background of the individual (structural and regulatory variation), with the environmental influence.
Two-dimensional gel electrophoresis (2DE) is an efficient tool for such investigations allowing the separation, simultaneous display, and quantification of a large number of proteins. This method has a large yet underexploited potential to address scientific hypothesis specific to population ecology and phylogenetic and evolutionary studies (Navas and Albar 2004; Biron et al. 2006; Karr 2008). Proteomic-like approaches have so far been successfully applied for investigating variation or establishing genetic relationships both at species or above-species level (Barreneche et al. 1996; Marquès et al. 2001; Lum et al. 2002) and below-species level (Bahrman et al. 1994; Jacobsen et al. 2001; Chevalier et al. 2004; Jorge et al. 2006; Rocco et al. 2006).
For species characterized by strong genetic flow over wide geographical ranges like trees, genetic variation with potential adaptive significance often develops in response to ecological gradients (Lexer et al. 2007). Genes whose expressions deviate under the pressure of selection in spite of the strong gene flow might underlie characters that confer adaptive advantage under the given environmental conditions (Lexer et al. 2007). We therefore used a proteomic approach (2DE followed by tandem mass spectrometric identification of proteins) to investigate the level of differentiation between 2 Picea abies ecotypes adapted to different environmental conditions. As most conifers, P. abies exhibits a low level of population genetic differentiation across its geographical range (Achere et al. 2005). However, differences in allelic distribution have been described between populations for several isozyme loci (e.g., Puglisi et al. 1999; Mitton and Duran 2004). In the present study, our objective was to verify if populations adapted to different environmental conditions also exhibit more consistent differences in protein expression, in addition to the rather subtle differences in allelic distribution previously described. For this, we compared 2DE patterns obtained for needles and root samples from 2-month-old seedlings from a typical low-altitude P. abies ecotype and a population adapted to high-altitude conditions. Proteins displaying consistent variation between the 2 ecotypes were identified by means of tandem mass spectrometry.
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Plant Material
Two different autochthonous P. abies populations were selected: a subalpine population (Forestry Office Füssen, at 1300–1600 m above sea level, certified "selected reproductive material") and a submontane ecotype recommended as particularly well-adapted low-elevation climatic conditions (Monastery Forestry Office Westerhof, at 300 m above sea level, certified "tested reproductive material"). The 2 locations have different temperature and precipitation conditions (average annual temperatures [1961–1990]: Füssen 4.5 °C, Westerhof 8.5 °C; average annual precipitations: Füssen 1750 mm, Westerhof 800 mm; climatic data available at www.klimadiagramme.de) (Enders 1996).
Seed lots from the 2 P. abies ecotypes, obtained by stand collection of seed from 21.8 ha (of the 41.7 ha stand at Füssen) and over 3 ha (of the 418.2 ha stand at Westerhof), were supplied by the Staatliche Samenklenge Laufen and Nordwestdeutsche Forstliche Versuchsanstalt (Abteilung Waldgenressourcen, Hann. Münden, Germany), respectively. Both seed lots were collected after mast seeding with extraordinary fructification rates ensuring a high genetic diversity of the reproductive material. After vernalization, seeds were soaked overnight in sterile demineralized water and surface sterilized for 30 min with 30% hydrogen peroxide. Floating seeds were discarded, and the remaining seeds were sown in sterile Phytatray boxes (Sigma-Aldrich, Taufkirchen, Germany) on sterilized vermiculite soaked with sterile nutritive solution (Ingestad 1979). Seeds germinated and seedlings grew in growth chambers at 16 °C, 100% humidity, and 16 h photoperiod (30 µmol m–2 s–1; Osram True light T8, 5500 K). After 2 months, needle, stem, and root length were measured to the nearest millimeter for 40 seedlings per ecotype.
For 2DE, needles and roots were harvested, frozen in liquid nitrogen within 30 s to avoid proteolysis, and stored at –76 °C until protein extraction. Equal amounts of biological material (first crowns of needles and whole roots) from 30 to 40 seedlings were pooled per sample. A total of 20 pools were obtained per sample in 4 different experiments. The pooling procedure is not only known to be useful for the comparison of natural populations by means of DNA markers (e.g., Randomly Amplified Polymorphic DNA [RAPD] patterns for P. abies populations; Scheepers et al. 1997) but has also been successfully used in proteomic studies (Thiellement et al. 1989; David et al. 1997; Rocco et al. 2006; Weinkauf et al. 2006).
The choice of young seedlings as biological material was based on the need to minimize environmental and individual life history–related bias of gene expression patterns because it is recognized that gene expression depends not only on the genotype but also on the particular environmental conditions of germination (Johnsen et al. 2005) and ontogeny (Gion et al. 2005).
Protein Extraction
Proteins were precipitated with trichloroacetic acid/acetone and extracted in buffer containing 5 M urea, 2 M thiourea, 100 mM dithiothreitol (DTT), 2% [(3-cholamidopropyl)-dimethyl-ammonio]1-propanesulfonate, 2% SB3-10, and 0.5% Pharmalyte 3–10 for the needle samples and 7 M urea, 2 M thiourea, 2 mM tris(2-carboxyethyl)phosphine, 50 mM dithiothreitol DTT, 2% dodecyl maltoside DM, and 0.5% Pharmalyte 3–10 for the root samples. Protein concentration was measured using the RC-DC Protein Assay (Bio-Rad, München, Germany). A detailed description of the protocol is presented elsewhere (Vâlcu and Schlink 2006a, 2006b).
Two-Dimensional Gel Electrophoresis
For optimal resolution, acidic and basic proteins were focused on separate gradients, following previously optimized protocols (Vâlcu and Schlink 2006a, 2006b). Samples were cup loaded near the cathode for the 4–7 IPGs and near the anode for the 6–11 immobilised pH gradients IPGs, on 24-cm-long IPG strips (GE Healthcare, Freiburg, Germany) previously rehydrated for at least 12 h. The rehydration buffer contained 100 mM hydroxyethyldisulphide HED, except for the focusing of root proteins, where it contained the same reducing agents as the extraction buffer. The rehydration buffer was supplemented with 10% isopropanol for the focusing of basic gradients. Focusing was performed in an Ettan IPGPhor Cup Loading Manifold at 20 °C and a 50-mA/IPG gel strip for a total of 55 kVh in case of acid IPGs and 60 kVh in the case of basic ones. After a 2-step equilibration of the strips with buffer containing 2% dithiothreitol DTT and 4% iodoacetamide, respectively, the second dimension was performed on 12.5% polyacrylamide 1-mm thick gels in an Ettan Dalt6 electrophoresis chamber (GE Healthcare, Freiburg, Germany). Gels were silver stained according to Heukeshoven and Dernick (1988), using 1% glycine in the stop solution as a substitute for acetic acid.
Image and Data Analysis
Silver-stained gels were scanned under blue light, at 300 dpi, with Image Scanner (GE Healthcare). ImageMaster 2D Platinum (GE Healthcare) was used for spot identification, quantitation, and matching. A Wacom digital plate was used for a more accurate manual editing of spots. Mismatches were manually corrected. Relative spot volumes (normalized per gel) were compared for each organ/ecotype/IPG between 5 biological replicates by means of Welch t-tests. Statistical analyses were performed with R2.2.1 (R Development Core Team 2006). In order to avoid type I errors, only spots showing very significant (P < 0.01) differences between the 2 ecotypes were selected. Among those, spots whose relative volumes differed by a factor of at least 2 were kept for further characterization. The differential expression between ecotypes of the spots thus selected was validated by comparison with a set of further 15 samples.
Protein Identification by Mass Spectrometry
Preparative electrophoresis was performed similarly to the analytic one, except for the higher protein amount (between 500 and 800 µg per gel) loaded by paper bridge loading and the prolonged focusing (65 kVhr for acidic gradient and 72 kVhr for basic gradient). Colloidal Coomassie G-250 staining was performed according to Anderson (Anderson et al. 1991). Weaker spots were cut from gels stained with MS-compatible silver staining (with glutaraldehyde and formaldehyde excluded from the sensitizing and silver solution, respectively). Spots were cut from gels using a scalpel, destained with 40% ethanol/50 mM ammonium bicarbonate (Coomassie-stained spots) or in a 30 mM potassium hexacyanoferrate/100 mM sodium thiosulphate 1:1 mixture (silver-stained spots), then dehydrated with acetonitrile and dried for 30 min under vacuum. After reduction and alkylation (Shevchenko et al. 2006), spots were washed and dehydrated with acetonitrile, dried under vacuum, and stored at –20 °C until analysis. In-gel digestion with trypsin was performed overnight at 37 °C. The resulting digests were analyzed with a nanospray LCQ ion trap mass spectrometer (Thermo-Finnigan, San Jose, CA), and peptide identification was performed with SEQUEST against all Picea ESTs available at dbEST in January 2007. Validation filters were set for Xcorr values higher than 1.90 for +1 peptides, 2.20 for +2 peptides, and 3.75 for +3 peptides and Delta CN higher than 0.1.
SEQtools software package by Søren W. Rasmussen (www.seqtools.dk) was used for DNA sequence translation and protein sequence alignments. If necessary, CLUSTALW alignments (Thompson et al. 1994) were optimized using T-COFFEE (Notredame et al. 2000). Protein pattern and motif searches were performed using INTERPROSCAN (http://www.ebi.ac.uk/InterProScan/) (Zdobnov and Apweiler 2001), Motif Scan of the Swiss Institute for Experimental Cancer Research provided by the Swiss Institute of Bioinformatics website (http://myhits.isb-sib.ch/cgi-bin/motif_scan), and PFAM Protein Search (http://pfam.janelia.org/hmmsearch.shtml) (Sonnhammer et al. 1998).
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Morphometric Comparison
Two-month-old seedlings from Füssen and Westerhof populations (hereafter, high-elevation and low-elevation populations, respectively) grown under identical conditions displayed clear morphometric differences (see pictures in Supplementary Material 1). An analysis of variance with organ length as dependent variable and the organ and the ecotype as factors revealed highly significant differences between the seedlings from the 2 ecotypes (F3,371 = 378.9, P < 0.001). The length of aboveground organs was higher for seedlings from the lower elevation population, and the differences were highly significant for both needles (t1,123 = –3.2899, P = 0.001) and stems (t1,123 = –13.1852, P < 0.001) (Figure 1). Roots of the high-elevation population were longer in comparison with the low-elevation population, though the difference was not significant (t1,123 = 0.107, P = 0.915). A Wilcoxon rank sum test with continuity correction was used to compare the root-to-stem length ratio. The seedlings from the high-elevation population were characterized by higher values of the ratio as compared with seedlings from the lower elevation population (W = 2673, P < 0.001) (Figure 1).
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Needle and Root 2DE Patterns
Highly reproducible 2DE patterns were obtained for needles and roots of P. abies seedlings from both ecotypes. A detailed investigation of the biological and technical reproducibility is presented elsewhere (Vâlcu and Schlink 2006a, 2006b; Vâlcu CM and Vâlcu M 2007). Over 1150 spots were reproducibly detected on acidic gradients and over 650 on the basic gradients for the root samples. Needle 2DE patterns were richer in spots with approximately 40% in the acidic gradient and 20% in the basic gradient. Typical 2DE separations of root and needle proteins are exemplified in Supplementary Material 1.
Following the criteria defined in the Materials and Methods, a total of 19 protein spots were detected, with estimated pH values ranging between 5 and 7.3 and molecular weight between 17.5 and 104.2, exhibiting differential constitutive expression between the 2 ecotypes. Ten proteins had a higher abundance in seedlings of high-elevation plants (4 in needles and 6 in roots; see Figures 2 and 3 and Table 1), and 7 proteins were overexpressed in low-elevation plants (all in root samples; see Figure 3 and Table 1). We also identified 2 proteins (spots #9 and #18) that appeared to be uniquely expressed in seedlings of the high-elevation ecotype or else their abundance in the low-elevation ecotype was below the level of detection.
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The ratios between the abundances of these proteins in the 2 ecotypes are presented in Table 1 together with the associated bootstrapped 95% confidence intervals (CIs) obtained after 10 000 iterations. The lower limit of the bootstrapped 95% CIs was in all cases higher than 1, supporting the consistency of the differences in the corresponding proteins’ abundance between the 2 ecotypes.
Mass Spectrometric Identification of Proteins
Of the 19 spots with different abundance between the 2 ecotypes, 7 different protein functions were identified by tandem mass spectrometry corresponding to 8 spots (Table 1). Peptides were mapped on the sequences of the Picea EST clones used for protein identification (Supplementary Material 2), and alignments of these clones with the best GenBank matches were build to verify function assignments (e values in Table 1). Four of the identified proteins had higher abundance in the seedlings of the high-elevation ecotype: glycine cleavage system T protein (spot #3) and protochlorophyllide reductase (spot #4) in the needles and glyoxysomal malate synthase (spot #16) and a homologue of the r40c1 protein (spot #9) in the roots. The remaining 4 spots were expressed at higher levels in the roots of seedlings of the low-elevation ecotype and were identified as a tracheary element differentiation protein (TED2, spot #7), a voltage-dependent anion-selective channel (VDAC) (Porin, spot #14), and 2 isoforms of a nicotinamide adenine dinucleotide phosphate (NAD(P))–binding nonmetallo dehydrogenase (spots #12 and #19) sharing 5 peptides.
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Discussion |
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2DE is known as a powerful technology for the investigation of qualitative and quantitative gene expression variation between different genetic units. Comparison of the 2DE patterns between populations on an individual basis can nevertheless be a laborious process especially when large numbers of individuals are to be investigated. The use of pooled samples allows for larger sample sizes and results in a more representative 2DE pattern for the compared populations (David et al. 1997) when the interest lies in identifying major differences in protein expression at the population level and not in comparing levels of polymorphism between individuals.
In our study, protein expression patterns were compared in a differential display experiment between pooled samples of needles and roots of P. abies seedlings from 2 ecotypes adapted to contrasting elevations. This approach revealed both qualitative and quantitative protein polymorphism between the 2 ecotypes. The level of interpopulation protein polymorphism was organ dependent. The significance of these findings is discussed below.
Morphometric Comparison
The differences in the morphometric characteristics of seedlings from the 2 ecotypes are consistent with known altitudinal and latitudinal trends. Plant height declines with the altitude or latitude (Oleksyn et al. 1998; Danusevicius and Gabrilavicius 2001), whereas plants from the higher altitude allocate comparatively more biomass to roots than plants from lower elevations (Oleksyn et al. 1998). These differences could result from genetic differentiation between the 2 populations, environmental effects on plant growth (Lindgren and Wei 1994), or both. The longer stems and primary needles of low-elevation plants as well as the higher values of the root to stem ratios for the high-elevation seedlings confirm that this phenotypic differentiation appears in P. abies as early as 2 months of age.
Qualitative and Quantitative Interpopulation Protein Polymorphism
Differences in protein expression detected in differential display 2DE experiments are of course limited by the resolution and sensitivity of protein separation and detection, respectively. The level of interecotype protein expression differentiation found in the present study parallels similar studies on forest tree ecotypes (e.g., Jorge et al. 2006).
Most of the between-ecotype variation found in the present study was quantitative (17 of 19 spots). Only 2 cases of qualitative variation were identified between the 2 ecotypes, both proteins being specifically expressed in the roots of seedlings from the high-elevation ecotype.
Qualitative protein polymorphism among individuals (i.e., presence/absence or shift in the position of a protein spot) is often attributed to allelic variation but can also reflect quantitative variation close to the level of detection or posttranslational modification of proteins. Quantitative variation (i.e., variation in spot intensity) on the other hand may result from the concerted action of several factors including the stability of the protein or its level of expression, which can be under the control of multiple loci (Damerval 1994). Levels of qualitative and quantitative variation estimated from 2DE patterns of single individuals vary between species. Zea mays lines, for example, have comparable levels of qualitative and quantitative protein polymorphism (de Vienne et al. 1988; Leonardi et al. 1988). The polymorphism of protein amount between P. abies ecotypes on the other hand seems to be larger than the variation in gene structure measured as presence/absence or shift in the spot position (Sieffert 1988).
By pooling several individuals per sample, the information concerning allele frequency and interindividual variations in the protein expression level is discarded and differences relevant at between-population level become apparent. Quantitative polymorphism between pooled samples can therefore reflect differences in the proportion of individuals that express a given protein, differences in the actual protein abundance at individual level (rates of synthesis and degradation, stability), or both. The number of proteins that discriminate the populations will be correspondingly reduced as compared with polymorphism assessed at individual level (Sieffert 1988).
Such quantitative variation of protein abundances at population level is essential when comparing plants under different environmental pressure. Regulation of gene expression is a multilevel complex process exhibiting a wide range of genetic variability, probably reflecting the large basis it offers for mutations (de Vienne et al. 1988), and is likely to be more involved in adaptive or morphological evolution than structural variations of proteins (MacIntyre 1982). This is supported by the fact that, at least in some species (i.e., Z. mays), morphological distances are correlated with polymorphism of protein amounts rather than with qualitative distances between lines (Damerval et al. 1987).
Variability of Organ-Specific and Organ-Nonspecific Peptides
All spots found to consistently and reproducibly distinguish the 2 ecotypes were organ specific. Most of the proteins differentially expressed between the 2 ecotypes (79%) were root-specific proteins as compared with only 21% needle-specific proteins. Previous studies on plant proteome demonstrated that the variability of protein abundance is under genetic control (reviewed by Thiellement et al. 1999) and depends on the organ or type of tissue. Pinus pinaster needle-, pollen-, and bud-specific proteins, for example, were found to exhibit levels of polymorphism of 44%, 58.1%, and 70.4%, respectively (Bahrman and Petit 1995). Similar differences in the level of variability were described between Z. mays second leaf blade, mesocotyl, and sheath (7.5%, 12.6%, and 13.2%, respectively) (Leonardi et al. 1988). Moreover, the latter study also showed that the dominant inheritance, that is, protein abundance in hybrid individuals similar to one of the parental spots (Leonardi et al. 1988), of most polypeptides is organ specific. These findings are not surprising because it has been repeatedly shown that organ-specific proteins also exhibit a larger genetic variation than organ-nonspecific ones (Klose 1982; de Vienne et al. 1988; Leonardi et al. 1988; Bahrman and Petit 1995). It has been hypothesized (Klose 1982) that proteins expressed in all organs are more critical for the individual's metabolism and therefore under higher selective pressure than organ-specific proteins. This would explain why they are less variable than the latter. Alternatively, de Vienne et al. (1988) proposed that the higher variability of proteins displaying organ-specific level of expression might reflect the higher number and complexity of regulatory mechanisms needed for the control of their abundance, each of them subject to genetic variability.
Function of Proteins Differentially Expressed between P. abies Ecotypes
It is, at least for some of the identified proteins, difficult to speculate whether their differential expression has any adaptive significance for P. abies ecotypes located at different elevations. A closer inspection of their functions will nevertheless offer some insight into the putative significance of their expression patterns in the 2 populations investigated. Detection of molecular signature of natural selection at the nucleotide level could constitute a further step toward the validation of the adaptive significance of the detected proteins (Wright and Gaut 2004; Ehrenreich and Purugganan 2006).
Isoforms of protochlorophyllide reductase and glycine cleavage system T protein were more abundant in the needles of the high-elevation ecotype. Protochlorophyllide oxidoreductase (spot #4, POR) catalyzes a late light-dependent step in the synthesis of chlorophyll (Heyes and Hunter 2005). In gymnosperms are known 2 por gene subfamilies (A and B) involved in different developmental stages of the green tissue and with different (light dependent/independent) expression regulation (Skinner and Timko 1998). In Pinus taeda, mature needles only express PORB but both isoforms are detectable in cotyledons and primary needles (Skinner and Timko 1999). First crown needles in P. abies might also express both POR genes, but we could not distinguish whether the protein differentially expressed in the needles of seedlings from the 2 spruce ecotypes is a PORA or a PORB ortholog.
Glycine cleavage system T protein (spot #3) is one of the 4 components of the mitochondrial glycine decarboxylase multienzyme system (Douce et al. 2001) that catalyzes the oxidative decarboxylation and deamination of glycine produced in the peroxisomes during photorespiration. T protein is light induced (Vauclare et al. 1998) and developmentally regulated (Vauclare et al. 1996; Thompson et al. 1998), and its expression was shown to be under posttranscriptional control (Vauclare et al. 1996). A higher abundance of the T protein in high-elevation seedlings needles might indicate higher photorespiration rates. Similarly, elevated photorespiration rates have been described both in herbaceous (Streb et al. 1998, 2005; Kumar et al. 2006) and woody plants (Oleksyn et al. 1998) as adaptation to high-altitude conditions.
Two of the proteins showing specific or higher expression in high-elevation seedlings roots were identified as a homologue of the r40c1 protein carrying a lectin domain (spot #9) and glyoxysomal malate synthase (spot #16), respectively.
Glyoxysomes are found in cells of storage organs like endosperm (Cooper and Beevers 1969) and during postgerminative growth of oilseed plants. Malate synthase is part of the glyoxylate cycle, which converts storage lipids into sucrose (Beevers 1961). The sucrose is then transferred to shoot and root apical meristems and provides the carbon source necessary for growth before the plants start photosynthesis (Hayashi 2000). In roots, malate synthase expression was shown to be activated in response to carbohydrate deprivation (Ismail et al. 1997) and may confer adaptive advantage to plants grown in low-carbohydrate conditions.
From the proteins overexpressed in the roots of the low-elevation seedlings, we identified 4 spots as a TED2, a VDAC, and 2 isoforms of a NAD(P)-binding nonmetallo dehydrogenase belonging to the short-chain dehydrogenase/reductase family (SDR).
TED2 (spot #7) is a quinone reductase involved in differentiation of tracheary elements and considered a marker for the development of the root vascular system (Demura and Fukuda 1994). The protein is under a strict temporal and spatial regulation (Demura and Fukuda 1994) and is expressed in early stages of differentiation of procambial cells into immature xylem or phloem cells. Given its narrow time and space frame of expression, the differential expression of TED2 in roots from the 2 ecotypes might indicate asynchrony in their root systems development.
VDAC (spot #14) is a 30-kDa protein that forms homodimers in the mitochondrial outer membrane (Thomas et al. 1991). It is believed to provide the major pathway for metabolite flux through the outer membrane (Manella and Colombini 1984; Manella et al. 1992; Colombini 1997), and reduction of its permeability can regulate mitochondrial respiration (Liu and Colombini 1992).
Two spots (#12 and #19) were identified as isoforms of a member of the SDRs. Over 3000 members of these superfamily of enzymes with reduced nicotinamide adenine dinucleotide phosphate–dependent (Rossmann-fold domains) oxidation/reduction activities have been described. They have a broad substrate spectrum, ranging from alcohols, sugars, steroids, aromatic compounds to xenobiotics (Kallberg et al. 2002). For the 2 proteins shown here to be more abundant in the low-elevation plants, the matches to the PANTHER PTHR19410 extension indicate that the proteins are not alcohol dehydrogenases but another type of dehydrogenase or reductase.
Significance of between-Population Protein Polymorphism in P. abies
Picea abies covers a large natural range across Europe. The large-scale geographical patterns of genetic variation over its natural range confirm the 3 major genetic domains resulting from postglacial recolonization (Collignon et al. 2002; Achere et al. 2005). Similarly, to other woody plant species, P. abies exhibits high levels of within-population variation in contrast to a low between-population differentiation (Achere et al. 2005). Both ecotypes used in the present study belong to the alpine domain (Achere et al. 2005) and can therefore be assumed to show low genetic differentiation.
However, even in genetically similar coniferous populations, genetic structure is known to be influenced by environmental conditions such as water or nutrient availability (Stutz and Mitton 1988; Cobb et al. 1994; Mitton and Duran 2004). Clinal variations of isozymes’ allele frequency along latitudinal (Bergmann and Gregorius 1993) or altitudinal gradients have been described in several coniferous species (Grant and Mitton 1977; Mitton et al. 1980; Puglisi et al. 1999) including P. abies (Bergmann 1978; Lundkvist 1979).
Protein expression polymorphism between ecotypes locally adapted to different environmental conditions can be the result of genetic differentiation (at structural and/or regulatory loci) between the 2 populations but can also reflect environmental influence on gene expression through imprinting. Recent work on the same P. abies ecotypes showed differences in DNA methylation levels, providing evidence for the possible contribution of imprinting to ecotype differentiation (Baumann 2004). Irrespective of the underlying mechanisms, these characters are potentially under selective pressure and might constitute the material for subsequent population divergence.
Overall, our results show that 2DE performed on pooled samples can reveal qualitative and quantitative between-population protein polymorphism with putative adaptive significance. Picea abies ecotypes adapted to contrasting elevations exhibit differential expression of proteins potentially conferring adaptive advantage under selective pressure.
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Supplementary Material |
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Supplementary materials 1 and 2 can be found at http://www.jhered.oxfordjournals.org/.
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Acknowledgments |
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We would like to thank Eliane Escher for excellent technical assistance. The characterization of proteins was performed at the Proteomic facility of Bordeaux. We thank Stéphane Claverol and Aurélien Barré for their help in mass spectrometry and bioinformatics analysis of mass spectrometry data. Comments of 3 anonymous referees helped improve the manuscript.
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Corresponding Editor: David Wagner
Received May 20, 2007
Accepted December 18, 2007
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