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First published online August 20, 2008; 10.1104/pp.108.120691 Plant Physiology 148:730-750 (2008) © 2008 American Society of Plant Biologists Reconfiguration of the Achene and Receptacle Metabolic Networks during Strawberry Fruit Development1,[C],[W]Abteilung Willmitzer, Max-Planck-Institut für Molekulare Pflanzenphysiologie, 14476 Potsdam-Golm, Germany (A.F., V.J.N., A.R.F.); Department of Biosciences, University of Kuopio, 70210 Kuopio, Finland (K.H.); CRA Cereal Research Center, 71100 Foggia, Italy (R.B.); Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel (N.D.); and Department of Plant Sciences, Weizmann Institute of Science, 76100 Rehovot, Israel (I.R., A.A.)
The anatomy of strawberry (Fragaria x ananassa) fruit, in which the achene is found on the outer part of the fruit, makes it an excellent species for studying the regulation of fruit development. It can provide a model for the cross talk between primary and secondary metabolism, whose role is of pivotal importance in the process. By combining gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry with the aim of addressing the metabolic regulation underlying fruit seed development, we simultaneously analyzed the composition of primary and secondary metabolites, separately, in achene and receptacle during fruit ripening of strawberry cultivar Herut. The results from these analyses suggest that changes in primary and secondary metabolism reflect organ and developmental specificities. For instance, the receptacle was characterized by increases in sugars and their direct derivatives, while the achene was characterized by a major decrease in the levels of carbon- and nitrogen-rich compounds, with the exception of storage-related metabolites (e.g. raffinose). Furthermore, the receptacle, and to a lesser extent the achene, exhibited dynamic fluctuations in the levels and nature of secondary metabolites across the ripening process. In the receptacle, proanthocyanidins and flavonol derivatives characterized mainly early developmental stages, while anthocyanins were abundant in the mature red stage; in the achene, ellagitannin and flavonoids were abundant during early and late development, respectively. Correlation-based network analysis suggested that metabolism is substantially coordinated during early development in either organ. Nonetheless, a higher degree of connectivity within and between metabolic pathways was measured in the achenes. The data are discussed within the context of current models both of the interaction of primary and secondary metabolism and of the metabolic interaction between the different plant organs.
In many plants, fruit development can be divided into four distinct phases (Gillaspy et al., 1993  ). The first phase commences after flower opening (anthesis) and involves fertilization and development of the ovary (in so-called true fruit) and is generally referred to as fruit set. In the second phase, fruit growth by cell division is the most prominent process and is accompanied by seed and early embryo formation. In the third phase, following cell division, fruit growth is maintained largely by increases in cell volume. During this stage of fruit expansion, the embryo passes through a maturation phase. This phase often leads to the induction of seed dormancy and is characterized by (1) the accumulation of storage products, (2) the suppression of precocious germination, (3) the acquisition of desiccation tolerance, and (4) water loss (Bewley and Black, 1994).
Strawberry (Fragaria x ananassa) fruit initiate from a single inflorescence and are actually an aggregate, composed of many ovaries, each with a single ovule (Perkins-Veazie, 1995
During the early stages of strawberry fruit development, auxin, synthesized in the achenes, promotes fruit growth (Nitsch, 1950
In the case of primary metabolism, the major soluble sugars in strawberry are Glc, Fru, and Suc, and their content is significantly enhanced during the developmental processes (Hancock, 1999). In contrast, the levels of minor soluble sugars, such as inositol, Xyl, and Gal, decrease during maturation (Moing et al., 2001
Metabolomics approaches, including those utilizing gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry, NMR, and Fourier transform ion cyclotron resonance-mass spectrometry, now allow the profiling of numerous metabolites within a single experiment (recently reviewed by Bedair and Sumner, 2008
GC-MS and UPLC-QTOF-MS Analyses of Strawberry Achene and Receptacle Reveals Primary and Secondary Metabolic Profiles That Are Characteristic to Organ and Developmental Stage To follow the repertoire of metabolic changes that occur in parallel in strawberry achene and receptacle organs, we carried out extensive metabolic profiling in which both primary and secondary metabolism were investigated during fruit development. Strawberry fruit growth and maturation can be divided into six different stages: small green (SG), medium green (MG), big green (BG), white (Wh), turning (Tu), and red (Re; Fig. 1 ). Under our growth conditions, the development of fruit from anthesis to the red ripe stage encompassed an average period of 30 d. The ripening process in strawberry is relatively rapid and typically occurs 5 to 10 d following the white stage. Fruit at any of the six stages of development were harvested five times during the season (i.e. harvested in a period of 4 months) and were separated to achenes and receptacle prior to extraction and metabolite determination (Fig. 1). GC-MS and UPLC-QTOF-MS analyses were conducted to detect mainly primary and secondary metabolites, respectively.
The data sets obtained by GC-MS and UPLC-QTOF-MS were examined by principal component analysis (Fig. 2 ). In both types of analyses, samples derived from the various stages of receptacle and achene development were clearly separated on the basis of metabolic differences. The analysis further highlighted a clear metabolic shift, in both the UPLC-QTOF-MS and GC-MS data, between the first three stages (SG, MG, BG) and the later stages (Wh, Tu, Re) in either organ. The metabolic events occurring in the achene and receptacle are best exemplified by the metabolites (primary or secondary) with highest principal component analysis scores and ANOVA P values in each organ (i.e. those metabolites with main impact on the variance of the data set; Supplemental Table S1). In the receptacle data set, sugars and sugar phosphates, ascorbate (AscA), and the shikimate precursor QuinA accounted for the main changes in primary metabolism. On the contrary, in the achene data set, amino acids such as Glu, Asp, and Asn and tricarboxylic acid (TCA) cycle intermediates had the highest impact on the metabolic shift. Furthermore, the number of primary metabolites that significantly changed during achene development (P < 0.01, following Bonferroni correction) was 1.5-fold higher than that detected in the receptacle (Supplemental Table S1).
Changes in secondary metabolism were also organ specific (Supplemental Table S2). However, as opposed to the results obtained for primary metabolism, larger changes in the levels of secondary metabolites were found in the receptacle compared with the achenes. In the receptacle, the secondary metabolites that exhibited highest variation in content across development included anthocyanins, procyanidins, phenolic acids, and two terpenoid derivatives. In the achenes, the secondary metabolites that exhibited highest fluctuations included catechin, phenylpropanoid glucosides, kaempferol coumaroyl glucoside, and several ellagitannins.
When comparing the metabolic shifts occurring throughout the development of receptacle and achenes, characteristic changes in patterns of metabolite accumulation were revealed to be common between the two different organs. However, in certain cases, such as sugar metabolism, the trends of metabolic change were even opposite. In the following sections, the changes in metabolite composition during the maturation of both organs are compared.
Metabolism of Sugar, Sugar Alcohols, and Phosphate Moieties
The accumulation patterns of the various sugars measured during achene development were diverse and in most cases different from those observed in the receptacle. Suc and hexose phosphates were measured to decrease by 50% their initial levels at the BG stage. Their level was further reduced during late achene maturation (Fig. 3). In contrast, a few important sugars and storage compounds, including Raf, iMal, sorbitol, glycerol, and the oligosaccharine gentiobiose, reached significantly higher abundance in the mature achene.
Accumulation Profiles of Proteinogenic and Nonproteinogenic Amino Acids and Polyamines In the achenes, the pattern of amino acid accumulation was fairly similar to that described for the receptacle. The most typical pattern of change was an increase in their levels toward the MG stage of development and a sharp decline when ripening commenced in the BG stage (Fig. 3). Such a characteristic pattern was particularly evident for the N-rich amino acids Lys, Arg, Asn, and Gln (Fig. 3). Among the amino-containing compounds, GABA exhibited an exceptional pattern, accumulating to 2-fold its initial content in the Re stage of achene development.
Accumulation Profiles of Organic Acids and TCA Cycle Intermediates
In the achene, a general reduction in the levels of organic acids, predominantly TCA cycle intermediates, was evident during fruit maturation (Fig. 3). In contrast to the trend displayed by this class of metabolites within the receptacle, a pronounced decrease in content characterized TCA cycle intermediates from the very early stages under study (SG), with the exception of CitA. For example, we observed a 10-fold decrease in AcoA, SuccA, and FumA and a 60-fold decrease in MalA. In contrast, CitA accumulated until mid maturation (MG stage; 4-fold higher compared with its levels in the SM stage) and exhibited a 40-fold drop in its levels in the following stages. This pattern was also evident for AscA. Early reports suggested that CitA acts as a competitive inhibitor of AscA oxidase (Gerwin et al., 1974
Strawberry fruit is a rich source of secondary metabolites, a large number of those identified to date belonging to the phenolic class (Aharoni et al., 2002b
Phenolic Acids and Aromatic Amino Acids The strawberry receptacle accumulates a range of glycosylated hydroxybenzoic and hydroxycinnamic acid derivatives (Fig. 4). The hydroxybenzoic acid derivatives were more abundant at the early to mid stages of receptacle development (SG to Wh), while the hexose sugar-containing caffeic and ferulic acids accumulated at the Wh and Tu maturation stages. The coumaric acid and sinapic acid derivatives were detected almost exclusively in the ripe stage of the receptacle (Figs. 4 and 5A). Chlorogenic acid, a different hydroxycinnamic acid derivative, showed a unique profile and accumulated specifically in the BG stage in the receptacle. Most of the phenolics detected here are derived from the aromatic amino acid Phe, which exhibited a double sigmoid curve of accumulation during receptacle development (Fig. 3). Its levels in the receptacle decreased very dramatically between the SG and MG and the Tu and Re stages. Several of the metabolites described above also accumulated in the achenes, albeit to lower levels. For example, chlorogenic acid was detected in all stages of achene development, while dihydroxy methyl benzoic acid hexose accumulated in the three latter stages of achene development. By contrast, ferulic acid hexose was detected particularly in the early, SG, stage of achene development (Fig. 4).
Flavonoids
Pigmentation of the receptacle during ripening in most strawberry cultivars is mainly due to the accumulation of glycosylated pelargonidins and a small fraction of glycosylated cyanidins, which, in our study, were both detected at the mature fruit. In addition to the hexose-sugar derivative of pelargonidin, five other differentially derivatized forms of pelargonidin were detected, while cyanidin was present solely as hexose substitutes. A relatively less studied anthocyanin derivative reported earlier in strawberry (Fossen et al., 2004
Proanthocyanidins, end products of the flavonoid pathway, were detected in our analysis as polymers, containing up to 10 units. Procyanidins accumulated mainly in the early SG and MG stages of receptacle development (Figs. 4 and 5F). In addition to procyanidin homopolymers, heteropolymers containing at least one (epi)afzelechin subunit (propelargonidins) have also been reported in strawberry fruit (Gu et al., 2003
Ellagitannins
Classes of Secondary Metabolites Previously Nondetected in Strawberry Fruit Two phenylpropanoid glycosides (eutigoisde A and grayanoisde A) were also detected in both organs and were particularly abundant in the MG stage of receptacle development (Fig. 4). The identity of these metabolites was confirmed in a different study of strawberry leaves by the use of NMR analysis (K. Hanhineva and A. Aharoni, unpublished data).
Correlation analysis was performed on the entire data set collected for the two organs. Association of metabolites across development is reflected in the degree of similarity in accumulation patterns. The correlation analysis was carried out by calculating the Pearson correlation coefficient for each and every metabolite pair (Carrari et al., 2006 In the receptacle data set, significant metabolite-metabolite correlations were less frequent and less pronounced compared with the achene data set (Fig. 6 versus Fig. 7 ; Supplemental Table S4 versus Supplemental Table S5). That said, the levels of certain metabolites, including hexose phosphate, Suc, and Mal, displayed a reasonable number of correlations with metabolites across both primary and secondary metabolism (Fig. 6, enlarged sections). However, relatively few correlations were apparent for amino acids, and those observed were only medium in strength (correlation coefficients between 0.8 and 0.9; Fig. 6; Supplemental Table S4).
During achene development, we found high correlations within the amino acids category (e.g. Asn:Asp:Gln; Fig. 7; Supplemental Table S5). Similarly, organic acids were associated significantly within their category (e.g. FumA::SuccA:MalA) but also displayed extensive similarity in the pattern of change with several amino acids (e.g. enlarged sections MalA:Ile, MalA:Ser, CitA:Val, CitA:Ala; Fig. 7; Supplemental Table S5). As might be expected, the hexose phosphates (X18 to X20 in the heat map) displayed significant correlation with other sugars and sugar derivatives, such as threonate (ThrA; X52 in the heat map) and myoinositol (Myo-Ino; X16 in the heat map), but also with a large number of metabolites across different categories, including amino acids (see enlarged sections). This suggests a coregulation of the glycolytic events and other metabolic processes such as amino acid metabolism during achene development. Metabolite-metabolite associations were positive in the vast majority of cases with the exception of those involving Raf (X1 in the heat map), a storage-related compound, which displayed significant negative correlation with hexose phosphates, amino acids, and organic acids (Fig. 7; Supplemental Table S5). In contrast to primary metabolism, among the secondary metabolite categories, negative correlations were detected between a first group composed of phenolic acids (coumaric, ferulic, and sinapic acid derivatives), eriodictyol hexose, naringenin/naringenin chalcone hexose, kaempferol hexose, and pelargonidin hexose derivative and a second group characterized by procyanidin derivatives (Fig. 7; Supplemental Table S5). The correlation analysis also highlighted links between primary and secondary metabolism, and significant metabolite-metabolite correlations were found in both receptacles and achenes. In the receptacle, significant correlations were found among metabolites across different metabolic categories in the primary metabolism (including sugars, hexose phosphates, organic acids [e.g. QuinA, CitA, and 2-oxoglutarate], and the amino acid Ala) and the secondary metabolites coumaric and ferulic acid hexoses, kaempferol hexoses, and procyanidin derivatives (Fig. 6; Supplemental Table S5). With respect to the nature of the association between primary and secondary metabolites, correlations involving Ala were opposite to those involving, for example organic acids. Similarly, correlations involving Suc were found inverse to those involving other sugars or sugar phosphates (Supplemental Table S5). In the achene, significant correlations were detected between several amino acids and secondary intermediates across different categories, for example, Glu (X34), Asp (X35) Thr (X38), Ser (X39), Pro (X40), and Ile (X41) and quercetin glucuronide (X75), phenolic derivatives (Fig. 7, enlarged section), digalloylquinic acid (X116), galloylglucosides, and ellagitannins (Fig. 7; Supplemental Table S5). The pattern of change of these secondary intermediates was shown to follow that of organic acids like QuinA, ThrA, MalA, and SuccA and Glc-6-P. Dihydroxy methyl benzoic acid hexose was exceptionally negatively correlated with the above-mentioned primary intermediates (Fig. 7, enlarged section).
In order to evaluate these data further, we examined the metabolite correlation networks in early and late stages of development in both organs. Such networks can provide insight into biochemical processes and their regulation (Camacho, 2005
Thus, this analysis agrees with the point-by-point description above in suggesting that metabolism in the receptacle is not highly coordinated either in early or late stages of development but that there is a high degree of coordination of the metabolic shifts occurring during early achene development (albeit one that breaks down at later stages of development).
The interrelation between ovary growth that is followed by fruit maturation and the parallel interdependent development of ovules and seeds is a highly complex process. Although the strawberry receptacle is not a "real" fruit and the achenes cannot strictly be defined as "real" seeds, this work along with previous extensive gene expression studies suggest that they could serve as an excellent model for studying the regulation of synchronous developmental processes in fruit and seeds (Aharoni and O'Connell, 2002 ). Phytohormones are good candidates to play a major role in the metabolic interplay between developing fruit and seeds, as exemplified in the classical experiments of Nitsch (1950). Removal of achenes or application of auxin to areas from which achenes were manually removed at various stages of fruit development could accelerate or repress ovary growth and ripening. Regardless of hormonal signaling, other signaling molecules are likely to play a role in the fruit-seed cross talk, including mRNAs, several types of small and noncoding RNAs, peptides, and proteins (Lindsey, 2001; Jones-Rhoades et al., 2006; Corbesier et al., 2007; Tamaki et al., 2007; Kalantidis et al., 2008). The spatial and temporal production and accumulation of small molecules (other than hormones) is an additional key factor that could mediate the regulation of the concerted development of fruit and seeds, with much recent work in other species highlighting the importance of sugar and amino acid transporters linking seeds to the nutrient supply of the mother plant (Borisjuk et al., 2004; Gibson, 2005; Weber et al., 2005). Here, we carried out extensive metabolic profiling experiments across defined developmental stages in dissected achenes and receptacle. Our aim was to characterize the developmental reconfigurations of the metabolic network in the two organs.
The developing receptacle exhibits considerable changes in the metabolism of sugars and sugar phosphates. The major soluble sugars in strawberry are Glc, Fru, and Suc (Hancock, 1999; Macías-Rodríguez et al., 2002
A characteristic feature of the early stages of receptacle development is the abundance of the flavonoid pathway end products, particularly the proanthocyanidins. The occurrence of proanthocyanidins in the early developmental stages of the fruit is common to several plant species (Dixon et al., 2005
The appearance, in the receptacle, of intermediates of the phenylpropanoid and flavonoid pathways in this study is compatible with previous gene expression studies (Halbwirth et al., 2006
In view of its role as a dispersal unit, primary metabolism in the achene might follow a program dedicated to the accumulation of storage compounds. During the early stages of achene development (SG–BG), we measured an increase in the content of amino acids such as Arg, Asn, and Gln, possibly supported by the import of nitrogen-rich compounds from the mother plant. This process could be mediated by the high abundance of ATP-binding cassette transporters in the achene (Aharoni and O'Connell, 2002
The similarities with the other seed systems are reflected also in the pattern of change of sugars and sugar phosphates. Indeed, Suc and Mal were shown to transiently accumulate in the early maturation events, while Fru and Glc were converted to their phosphate forms. This metabolic pattern is probably best explained by an import of sugar moieties into the young achene and the limited operation of a not yet fully active glycolytic pathway in this organ, as has been described in legumes (Borisjuk et al., 2004
During early stages of achene development, there was little alteration in the levels of secondary metabolites, with the exception of metabolites belonging to the ellagitannin group of hydrolyzable tannins (Figs. 4 and 5, H–J) and dihydroxybenzoic acid hexose as well as the flavonoid kaempferol derivatives, which showed mild fluctuations. The sugar-containing ellagitannins were found to be particularly abundant during early achene development, decreasing to 50% of their initial levels by mid maturation. In addition to this quantitative change, we observed qualitative difference in this class of metabolites, with, for example, penta-galloyl Glc being abundant during early achene development but not in the receptacle, suggesting divergent ellagitannin production that potentially functions in the two organs. While Rosaceae family plants including strawberry are a well-known source of ellagitannins (Haslam and Cai, 1994
In the receptacle, the primary metabolism of mid to late maturation (MG to Re) was characterized by an increase of several amino acids (Arg, Ser, Thr, Ala, β-Ala, and, to some extent, GABA) as well as all TCA cycle intermediates with the exception of 2-oxoglutarate. Interestingly, apart from playing myriad roles in the fruit itself, CitA and MalA are important components of the organoleptic properties of the fruit, influencing its acidity. Given the relative changes in CitA and MalA across development observed here, we suggest that pH regulation is more likely driven by the latter, at least in the strawberry variety under study. Similarly, levels of Suc, Fru, Glc, Rib, and Raf, as well as the sugar alcohols such as mannitol and glycerol, increase at this stage, and they are also important in determining the taste of the mature fruit. As observed previously in early receptacle development, a decrease in AscA and the relatively minor changes in dehydroascorbate (its oxidized form) and ThrA (a product of AscA) throughout mid development suggest continued redox activity and carbon recycling.
Among the secondary metabolites detected during the mid and late stages of receptacle development, hexose derivatives of several different aromatic acids, including benzoic, hydroxybenzoic, caffeic, coumaric, ferulic, and sinapic acids, were observed to accumulate. These aromatic acids serve as precursors for the various branches of the phenylpropanoid pathway and the metabolism of phenolics. Elevated levels of caffeic, ferulic, and sinapic acid hexoses might contribute to monolignol biosynthesis and lignin formation in the fruit vasculature (Humphrey and Chapple, 2002
An increase in specific flavinoids and their derivatives characterized mid to late maturation of the strawberry receptacle. The biosynthesis of flavonoids is initiated by the activity of chalcone synthase, and the concomitant processing by specialized flavonoid pathway enzymes determines the end products (Dana et al., 2006
From the BG stage onward, the achene undergoes a dramatic metabolic shift, with metabolite pools of the major energy pathways being heavily depleted. This presumably fuels storage compound accumulation and fatty acid elongation in a similar manner to the one postulated in seeds (Fait et al., 2006
In parallel with the changes observed in carbohydrate levels in the achene, an abrupt change in redox status at the ripening stage is apparent, following which both CitA and AscA levels are synchronously reduced. Such a link with regard to cellular redox poise is probably due to the intimate relation of both metabolites to the mitochondrial electron transport chain (Nunes-Nesi et al., 2005
The secondary metabolism of maturing achenes also exhibited considerable shifts. While, compared with the level monitored in the receptacle, hexose derivatives of several different aromatic acids were low in content, the flavonol derivatives kaempferol hexose and kaempferol coumaroyl hexose accumulated to marked levels at this stage, with an initial dramatic increase at mid maturation. Although these metabolites have been detected previously in strawberry (Aaby et al., 2007a
In general, correlation network analysis suggests that metabolism in the receptacle is not highly coordinated either in early or late stages of development. There is, however, a high degree of coordination of the metabolic changes in the achene, particularly during early development.
When looking into the patterns of change of metabolites across development, subtle associations can be highlighted. In the receptacle, the two-wave behavior of Trp, Phe, and, intriguingly, Gln suggests a synchronous regulation of primary nitrogen metabolism and secondary metabolism. The link between Trp and Gln has been described previously in Arabidopsis (Zhao et al., 1998
Auxin production by the achenes and its transport to the receptacle have been shown to sustain receptacle enlargement in early stages of development (Nitsch, 1950
With the aim of providing a global view of the metabolic events associated with fruit development, we here describe, to our knowledge, the first parallel profiling of primary and secondary metabolism of strawberry receptacle and achenes across several developmental stages. Our results reveal that both metabolic synchrony and speciation exist during the development of achenes and receptacle in strawberry fruit (Fig. 9 ). Changes in metabolite levels in the receptacle most probably reflect the metabolic activity of the fruit. As such, these are somewhat similar to those seen previously in (climacteric) tomato fruit (Carrari et al., 2006 ), although displaying clear differences, such as the mild accumulation of TCA cycle intermediates. When comparing our results with those of previous studies concerned with the analysis of gene expression (Aharoni and O'Connell, 2002), we were able to suggest that the metabolic program of the achene is largely concerned with the accumulation of storage and protective compounds as well as precursors for hormonal and secondary metabolites. These are required to sustain seed quiescence and secure future germination, to support prolonged pregermination periods, and to guide aspects of seed development, respectively. In addition, we were able to evaluate the coregulation of a vast number of metabolites both across development and between organs. These analyses revealed concerted regulation within and between metabolite categories (e.g. amino acids and TCA cycle intermediates, amino acids, and procyanidin derivatives). Correlation-based network analysis highlighted a dense degree of connectivity, building organ-specific metabolic modules, mainly during early to mid strawberry maturation. Intriguingly, the sugar module in the receptacle correlation network resembled that recently described for tomato pericarp tissue (Ursem et al., 2008). This and other evidence described here suggest a common mechanism between climacteric and nonclimacteric fruit as the basis of metabolic regulation. Taken together, the comprehensive metabolite analysis described here sheds light on the metabolic regulation during strawberry development and the different metabolic programs occurring in two functionally distinct organs. It further highlights the cross talk between primary and secondary metabolism. The conspicuous differences found between receptacle and achenes emphasize the importance of dissecting the organ-specific events in order to gain a better understanding of strawberry growth, development, and response to environmental cues.
Plant Material Frozen strawberries (Fragaria x ananassa) were ground to a fine powder using a grinding mill (analytical mill A11 basic; IKA) or a mortal and pestle. Plants of the Israeli cultivar Herut were grown in a plastic tunnel in the Sharon coastal area in Israel. Fruit were harvested at six different developmental stages (Fig. 1) five times during the season (each harvest served as a replicate). Achenes were removed and collected manually using a scalpel or shaking them frozen in a 50-mL tube (for young fruit).
Unless stated otherwise, all chemicals were purchased either from Sigma-Aldrich or from Merck. Naringenin, chlorogenic acid hemihydrate, and trans-cinnamic acid were purchased from Fluka; ferulic acid was from Aldrich;
Metabolite analysis by GC-MS was carried out essentially as described by Roessner et al. (2001)
Frozen powder (200–300 mg) was extracted with 80% (v/v) MeOH, and the solid:liquid ratio was kept at 1:3 (w/v). The mixture was sonicated for 20 min at room temperature, centrifuged (3,000g, 10 min), and filtered through a 0.22-µm PTFE membrane filter (Acrodisc CR 13 mm; PALL) before injection to a UPLC-MS system (Premier QTOF, Waters) with a UPLC column connected online to a UV light detector (Waters Acquity). Separation of metabolites was performed on a 100- x 2.1-mm (i.d.), 1.7-µm UPLC BEH C18 column (Waters Acquity). The mobile phase consisted of 0.1% formic acid in acetonitrile:water (5:95, v/v; phase A) and 0.1% formic acid in acetonitrile (phase B). The linear gradient program was as follows: 100% to 72% A over 22 min, 72% to 60% A over 0.5 min, 60% to 0% A over 0.5 min, hold at 100% B for a further 1.5 min, return to the initial conditions (100% A) in 0.5 min, and conditioning at 100% A for 1 min. The flow rate was 0.3 mL min–1, and the column temperature was kept at 35°C. The UV light trace was measured at 240 nm, and masses were detected by a Q-TOF Premier MS system equipped with an electrospray ionization source. Acquisition was performed in the electrospray ionization-positive and electrospray ionization-negative modes. The following settings were applied during the UPLC-QTOF-MS runs: capillary voltage at 3.0 kV; cone voltage at 30 eV; collision energy at 3 eV; and argon was used as a collision gas. The mass-to-charge ratio (m/z) range was 50 to 1,500 D. The following settings were applied during the UPLC-QTOF-MS/MS runs: capillary spray at 3.0 kV; cone voltage at 30 eV; collision energy at 15 and 30 eV; and argon was used as a collision gas. The MS apparatus was calibrated using sodium formate, and Leu enkaphalin was used as the lock mass. MassLynx software version 4.1 was used to control all instruments and calculate accurate mass. In some cases, the UV light spectra (recorded between 200 and 600 nm) were acquired on another UPLC Waters Acquity instrument equipped with the Acquity 2996 PDA detector and a 150- x 2.1-mm (i.d.), 1.7-µm UPLC BEH C18 column (Waters Acquity). The liquid chromatography conditions were as described for UPLC-QTOF analysis. A mixture of 15 standard compounds, injected after each 10 samples, was used for quality control.
Metabolites were identified using standard compounds by comparison of their retention times and MS/MS fragments and putatively identified applying several steps. The elemental composition was selected according to the accurate masses and the isotopic pattern using MassLynx software and searched against metabolite databases: Dictionary of Natural Products (Chapman and Hall/CRC), SciFinder (SciFinder Scholar 2007), the KNApSAcK database (http://prime.psc.riken.jp/KNApSAcK), and the MOTO database (http://appliedbioinformatics.wur.nl/moto). Predicted log D values for pH 3 (pH of the UPLC mobile phase), found using the SciFinder tool, were utilized for the retention time prediction in order to narrow the number of proposed structures. The interpretation of the observed MS/MS spectra in comparison with those found in the literature (where possible) was the main tool for putative identification of metabolites. Some compounds reported earlier in strawberry are annotated based on the fragmentation in source, and in several cases these were analyzed by MS/MS in our previous study (K. Hanhineva, I. Rogachev, H. Kokko, S. Mintz-Oron, I. Venger, S. Kärenlampi, and A. Aharoni, unpublished data). Two of the compounds, eutigoside A and grayanoside A, were identified by NMR in strawberry in our previous study (K Hanhineva and S Kärenlampi, unpublished data). The references to previous liquid chromatography-mass spectrometry analyses of strawberry for each compound are given in Supplemental Table S3.
The metabolites not previously detected in strawberry were putatively identified as follows. Eriodictyol hexose was identified based on MS/MS analysis and was distinguishable from another flavanone, dihydrokaempferol, having the same molecular formula (C15H12O6). The compound exhibited the same MS/MS fragmentation as eriodictyol aglycone as described by Es-Safi et al. (2005; Supplemental Table S3). Naringenin/naringenin chalcone hexose was identified by comparison with the naringenin chalcone standard aglycone. By following the formula [M-H+288]– or [M-2H+144]2– (Nunez et al., 2006 The quantification of the compounds is based on the relative peak response area of each signal in mass chromatograms. The five replicate samples were prepared from the same amount of frozen material, and the results were normalized to the dry weight in both achenes and receptacle. The data presented here are the direct amounts of peak area, as no quantitative standard was applied in this study. The heat map analysis was done on a Microsoft Excel-implemented macro program available at http://www.senorjosh.com/archives/2003/04/heatmap_tool_vba.shtml.
Principal component analysis was performed on the data sets obtained from metabolite profiling with the software package TMEV (Saeed et al., 2003
The following materials are available in the online version of this article.
We thank Luigi Cattivelli for his support. Received April 10, 2008; accepted August 10, 2008; published August 20, 2008.
1 This work was supported by the Alexander von Humboldt Foundation (award to A.F.), by the Saastamoinen Foundation (research visit funding to K.H.), by the Israel Ministry of Science (IMOS grant no. 3–2552 to A.A.), by the European Union project META-PHOR (contract no. FOODCT–2006–036220), by Mr. and Mrs. Mordechai Segal, and by the Henry S. and Anne Reich Family Foundation. A.A. is an incumbent of the Adolfo and Evelyn Blum Career Development Chair. 
2 These authors contributed equally to the article.
3 Present address: Ben-Gurion University of the Negev, Jacob Blaustein Insts. for Desert Research, Dept. of Dryland Biotechnology, 84990 Midreshet Ben-Gurion, Israel. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Aaron Fait (fait{at}bgu.ac.il).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.108.120691 * Corresponding author; e-mail fait{at}bgu.ac.il.
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-tomatine was from Apin Chemicals; HPLC-grade acetonitrile was from J.T. Baker; methanol was from Merck; formic acid (approximately 98%) was from Fluka. Double deionized water from a Milli-Q purification unit was from Millipore.