Sweet Cherry - ACS Publications - American Chemical Society

Article pubs.acs.org/JAFC

Proteomic Comparison of Fruit Ripening between ‘Hedelfinger’ Sweet Cherry (Prunus avium L.) and Its Somaclonal Variant ‘HS’ Bhakti Prinsi,* Alfredo S. Negri, Luca Espen, and M. Claudia Piagnani Department of Agricultural and Environmental Sciences − Production, Landscape, Agroenergy (DISAA), Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy S Supporting Information *

ABSTRACT: The somaclonal variant HS, from sweet cherry (Prunus avium L.) ‘Hedelfinger’ (H), was previously selected for reduced tree vegetative vigor and lesser canopy density. In this work, we compared H and HS fruits at early unripe (green) and full ripe (dark red) stages by biochemical and proteomic approaches. The main biochemical parameters showed that fruit quality was not affected by somaclonal variation. The proteomic analysis identified 39 proteins differentially accumulated between H and HS fruits at the two ripening stages, embracing enzymes involved in several pathways, such as carbon metabolism, cell wall modification, stress response, and secondary metabolism. The evaluation of fruit phenolic composition by mass spectrometry showed that HS sweet cherries have higher levels of procyanidin, flavonol, and anthocyanin compounds. This work provides the first proteomic characterization of fruit ripening in sweet cherry, revealing new positive traits of the HS somaclonal variant. KEYWORDS: sweet cherry, Prunus avium, fruit ripening, proteomics, somaclonal variation, phenols, anthocyanin

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INTRODUCTION Sweet cherry (Prunus avium L.) is largely produced worldwide, accounting for >2 million metric tonnes per year. It is one of the most appreciated fruits for its organoleptic properties and high content of nutrients and healthy phytochemicals, such as anthocyanins.1,2 Attractive skin color, fruit size and firmness, balance between sweetness and sourness, and flavor are the main characteristics defining cherry fruit quality,3,4 and they are attained and influenced by fruit ripening. Sweet cherry is a nonclimacteric stone fruit characterized by a rapid development through a biphasic growth pattern, in which it is possible to discern a distinct ripening period.5 For these reasons, it was proposed as a valuable model for the study of ripening in nonclimacteric fruits.5 In particular, sweet cherry ripening occurs during the second phase of fruit development, and its beginning is associated with changes in skin color from green to red, due to chlorophyll degradation and anthocyanin accumulation.5,6 During this phase many biochemical changes lead to fruit weight increase, loss of flesh firmness, changes in organic acid composition, accumulation of soluble sugars, and volatile emission.5−8 From a molecular point of view, the regulation of fruit maturation in sweet cherry is still poorly characterized, and the plethora of physiological and biochemical events interplaying during fruit ripening remain to be fully elucidated. Beyond intrinsic differences among cultivars,3,9 sweet cherry quality is also influenced by agronomic practices modifying tree vigor.10,11 Overall, one of the main strategies proposed to improve productivity consists of adopting cultivars able to maintain fruit quality in high-density orchard systems. Together with traditional breeding methods, somaclonal variation is one of the resources to improve desirable polygenetic traits in crops.12 We recently selected the sweet cherry somaclonal variant HS (HS), obtained from leaf explants of the ‘Hedelfinger’ sweet cherry (H),13,14 the trees of which are © 2016 American Chemical Society

characterized by lower vegetative vigor and a less crowded canopy.15 Along with these positive agronomic traits, the main parameters of fruit quality in HS were very similar to those of the wild-type.15 Considering that some carpometric differences suggested possible effects on fruit-ripening physiology,15 we investigated these aspects by biochemical and proteomic approaches. Nowadays, proteomics is widely applied to study fruit ripening in several tree crops. For instance, in Prunus spp. such as apricot (Prunus armeniaca L.)16 and peach (Prunus persica L.),17 proteomics provided new information about biochemical aspects related to fruit physiology and ripening as well as fruit technological properties. In sweet cherry, to our knowledge, proteomic approaches were applied only for clinical18 or phytopathological19 purposes, but no studies are available about proteomic changes related to fruit ripening. To improve this kind of knowledge and to individuate possible differences induced by somaclonal variation, this work aimed for a proteomic comparison among sweet cherries of the cultivar H and those of its somaclonal variant HS at two ripening stages: the onset of ripening (green fruits) and full ripeness (dark red fruits). Some biochemical parameters in fruits (chlorophyll, sugar, and amino acid contents, total antioxidant capacity, and phenolic speciation) were also evaluated to define fruit properties and to support the interpretation of the physiological roles of the proteomic changes observed between the two ripening stages. Received: Revised: Accepted: Published: 4171

March 3, 2016 April 26, 2016 May 4, 2016 May 4, 2016 DOI: 10.1021/acs.jafc.6b01039 J. Agric. Food Chem. 2016, 64, 4171−4181

Article

Journal of Agricultural and Food Chemistry

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80% (v/v) acetone. The pellet was dried under vacuum and dissolved in IEF buffer [7 M urea, 2 M thiourea, 3% (w/v) CHAPS, 1% (v/v) Nonidet P-40, 50 mg mL−1 dithiothreitol, and 2% (v/v) IPG buffer pH 4−7 (GE Healthcare, Little Chalfont, UK)] by vortexing and incubating for 1 h at room temperature. The sample was centrifuged at 10000g for 10 min and the supernatant stored at −80 °C until further use. The protein concentration was determined by using a 2-D Quant Kit (GE Healthcare). Each protein sample (400 μg) was loaded onto the pH 4−7, 24 cm IPG strips (GE Healthcare), and the following electrophoretic analyses were conducted as previously described.23 For each sample, three biological replicates were extracted and quantified independently. Two 2-DE were obtained for each of them (n = 6). Image and Proteomic Data Analysis. The gels were stained with colloidal Coomassie Brilliant Blue G-250 (cCBB), acquired by an Epson Expression 1680 Pro Scanner (Seiko Epson Corp., Suwa, Nagano, Japan) and analyzed with ImageMaster 2-D Platinum software v.6.0 (GE Healthcare) as previously described.23 Spot quantification was expressed as spot relative volume (%Vol), and the matching procedure was manually implemented for the first most abundant 1000 spots in each gel, among replicates and experimental conditions. To assess the differences among genotypes and ripening stages, the %Vol data set was analyzed through the application of the principal component analysis (PCA) and by partial least squares− discriminant analysis (PLS-DA) to select the spots (loadings) distinguishing between these comparisons: U versus R; HU versus HR; HSU versus HSR; H versus HS; HU versus HSU; HR versus HSR. The %Vol of the selected spots was further analyzed by the twoway ANOVA test (Tukey’s post hoc test, p ≤ 0.05). Protein Identification by LC-ESI-MS/MS. The selected spots were digested with trypsin and analyzed on an Agilent 6520 Q-TOF mass spectrometer with an HPLC Chip Cube source (Agilent Technologies, Santa Clara, CA, USA), as previously described.24 Analysis of MS/MS spectra was performed by protein database searching with Spectrum Mill MS Proteomics Workbench (Rev B.04.00.127; Agilent Technologies). Carbamidomethylation of cysteine and oxidation of methionine were set as fixed and variable modifications, respectively, accepting two missed cleavages per peptide. The search was conducted against the subset of Prunus genus (ID tax: 3754, Aug 2015, 93802 entries) downloaded from the National Center for Biotechnology Information (NCBI, http://www. ncbi.nlm.nih.gov) and concatenated with its reversed one. The thresholds used for peptide identification were peptide Local FDR ≤ 1%, Score Peak Intensity% ≥ 70%, difference of forward and reverse scores ≥ 2, and precursor mass error ≤ ± 10 ppm. Protein identification was accepted only if confirmed by at least three unique peptides. Physical properties of the proteins were predicted by in silico tools at ExPASy (http://www.expasy.org/). Phenolic Composition. Frozen samples of H and HS cherries at the two ripening stages were finely powdered by pestle and mortar in liquid N2 and dissolved in 3 volumes of 90% (v/v) methanol and 0.1% (v/v) FA. After an incubation of 3 h at 4 °C under shaking, the samples were centrifuged at 5000g at 4 °C for 20 min. The supernatants were filtered onto a sterilized PVDF hydrophilic membrane with pores of 0.45 μm. After dilution, the samples were analyzed by an Agilent Technologies 1200 series capillary pump coupled with a dual ESI source on a 6520 Q-TOF mass spectrometer. Briefly, LC runs were done on an XDB-C18 column (2.1 × 50 mm, 1.8 μm, Agilent Technologies) in acidic condition [FA 0.1% (v/v)] applying a 12 min linear gradient from 5 to 40% of acetonitrile with a flow rate of 200 μL min−1. The ESI source was set at 350 °C at 3500 V. Data acquisition was performed in positive mode within a range from m/z 125 to 1200. Chromatographic peak interpretation was performed with MassHunter Workstation software (version B.03.01, Agilent Technologies). The search was conducted against a database consisting of the main molecules of the phenylpropanoid pathways (maps from 00940 to 00944) of KEGG pathways Web site (http:// www.genome.jp/kegg/pathway.html), identifying the compounds as positive ions (+1 z) with a tolerance of ±10 ppm. The relative quantification of compounds was done on MS spectra by extracting

MATERIALS AND METHODS

Plant Material. Cherries were randomly handpicked from at least five self-rooted trees of cultivar ‘Hedelfinger’ (H) and somaclone HS (HS), 6 years after planting in the CLIFOF Foundation orchard (Minoprio, Italy). The first picking was done in May when fruits were unripe (green), and the second one was done the first week of June. In both cases, H and HS fruits were harvested on the same date. At the second picking, fruit skin color was assessed by a Minolta reflectance colorimeter CR200 (Minolta Co Ltd., Osaka, Japan) measuring the a*, representing the change in color from green to red, and b* value, representing the change in color from blue to yellow, on the L*a*b* CIELAB scale. As previously described for both H and HS,15 only fruits corresponding to the full ripening stage (very dark red) were selected. Because splitting the skin (epicarp) from the flesh (mesocarp) would have seriously damaged green fruits, for both ripening stages, fruit pedicel and pit were removed and pulp and skin were jointly collected. Three independent biological samples, each made up of pulp and skin from at least 30 fruits, were obtained for each condition (n = 3). The samples were frozen in liquid N2 and stored at −80 °C until further uses. Conditions were named combining genotype and ripening stage of the fruits: HU, HR, HSU, and HSR. Sugars, Free Amino Acids, and Chlorophyll Content. Reducing sugars, sucrose, free amino acids, and chlorophyll were determined in frozen samples of H and HS cherries at the two ripening stages as previously described.20 In particular, reducing sugars were evaluated by the colorimetric assay proposed by Nelson.21 After an aliquot of the perchloric acid extract had been boiled for 1 h and neutralized, total soluble sugars were determined according to the same method. Finally, sucrose content was estimated as the difference between the total and reducing fractions. Three biological samples were analyzed for each condition (n = 3). Data were analyzed by a two-way ANOVA (Tukey’s post hoc test, p ≤ 0.01). Determination of Total Antioxidant Capacity and Total Anthocyanin Content. Frozen samples (about 0.5 g) were finely powdered in liquid N2 and extracted in 4 volumes of methanol with shaking for 1 h at 4 °C. After centrifugation at 14000g for 10 min at 4 °C, the Trolox equivalent antioxidant capacity (TEAC) was measured by the method using 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, as described by Huang and co-workers.22 Total anthocyanins were extracted with the same procedure but using acidified methanol with 3% (v/v) formic acid (FA). The absorbance at 520 nm was referred to a calibration curve as milligrams of cyanidin-3glucoside equivalents (CGE). Three biological samples were analyzed for each condition (n = 3). Data were analyzed by the two-way ANOVA (Tukey’s post hoc test, p ≤ 0.01). Protein Extraction and Two-Dimensional Electrophoresis (2DE). Frozen samples were finely powdered in liquid N2 using a pestle and mortar. Two grams of the powder was transferred to a 50 mL tube, resuspended in 40 mL of the ice-cold “cleaning solution” [0.1% (v/v) HCl, 2% (v/v) β-mercaptoethanol (β-ME), 10% (w/v) polyvinylpyrrolidone-40, in methanol], and incubated overnight at −20 °C. After a centrifugation for 30 min at 5000g, the pellet was dissolved in the same volume of cleaning solution and incubated at −20 °C for 1 h. The centrifugation step was repeated, and then the pellet was dried under vacuum and resuspended in 15 mL of phenol with shaking for 30 min in ice. The same volume of extraction buffer [0.7 M sucrose, 0.1 M KCl, 50 mM Na2-EDTA salt, 4 mM ascorbic acid, 2 mM phenylmethanesulfonyl fluoride, 0.1 mg mL−1 Pefabloc SC (Sigma-Aldrich, St. Louis, MO, USA), 0.2% (v/v) Triton X-100, 2% (v/v) β-ME, 1% (w/v) polyvinylpolypyrrolidone] was added, and then the sample was incubated for 2 h at 4 °C and finally centrifuged at 5000g for 20 min at 4 °C. The upper phenol phase was collected, whereas the aqueous phase at the bottom was back-extracted with an equal volume of phenol. Proteins were precipitated by the addition of 5 volumes of ice-cold 0.1 M ammonium acetate in methanol to the phenol phase, then vortexed briefly, and finally incubated at −20 °C overnight. Precipitated proteins were recovered by centrifuging at 13000g for 30 min, then washed again with cold methanolic ammonium acetate twice, and subsequently washed twice with cold 4172

DOI: 10.1021/acs.jafc.6b01039 J. Agric. Food Chem. 2016, 64, 4171−4181

Article


Table 1. Metabolic Profiles in Sweet Cherries of ‘Hedelfinger’ and Its Somaclonal Variant HS at Unripe and Ripe Stagesa chlorophyll (μg/g FW) reducing sugars (μmol glc/g FW) sucrose (μmol glc/g FW) amino acids (μmol leu/g FW) TEAC (mM Trolox/100 g FW) total anthocyanins (mg CGE/100 g FW) a

H unripe

HS unripe

H ripe

HS ripe

11.75 ± 0.27 c 432.3 ± 14.4 a 26.27 ± 1.54 a 73.57 ± 1.15 b 1.59 ± 0.01 b 0.33 ± 0.03 a

8.33 ± 0.34 b 417.2 ± 7.7 a 31.05 ± 1.23 a 77.06 ± 4.66 b 1.79 ± 0.01 b 0.37 ± 0.02 a

1.88 ± 0.11 a 1025.1 ± 11.9 b 33.14 ± 4.59 a 32.86 ± 1.06 a 0.95 ± 0.08 a 11.47 ± 0.28 b

2.53 ± 0.19 a 1124.8 ± 49.6 b 20.44 ± 3.73 a 36.30 ± 0.97 a 1.03 ± 0.08 a 14.38 ± 0.76 c

Values are the mean ± SE (n = 3). The letters were assigned according to Tukey’s test (p < 0.01).

the EIC for [MH+]. The compound assignment was refined by targeted MS/MS analyses with an isolation width of m/z 4, a RT ± 0.5 min, and a fixed collision energy of 30 V, accepting a mass error of ±0.01 Da. The assignment was verified by the MS and MS/MS analyses of standard compounds and by comparison with the literature. The RT and the fragmentation profiles are reported in Table S2. Three samples were analyzed for each condition (n = 3). The amount of each compound was expressed as the relative percentage abundance with respect to the average value among all of the samples. Data were log10 transformed and analyzed by the twoway ANOVA (Tukey’s post hoc test, p ≤ 0.01).

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quality of H seems to be not altered by the somaclonal variation in HS, if not even improved by a higher content of total anthocyanins in ripe sweet cherries (Table 1). Proteomic Analysis of Sweet Cherry Ripening. Fruits are considered recalcitrant material for the 2-DE proteomic analyses because of low protein concentration, high content of proteases, and accumulation of a broad spectrum of peculiar metabolites.25 Moreover, changes in metabolite composition of fruits at different ripening stages may hamper a proper comparison among fruit proteomic profiles. To overcome this technical problem in sweet cherry fruits, which are typically rich in hydroxycinnamic acids, catechins, and anthocyanins,2 we modified common extraction procedures based on the use of organic solvents.25 The use of the “cleaning solution” as first treatment of samples (described under Materials and Methods) was chosen to ensure favorable conditions (i.e., hydrophobic, acidic, and reducing) to avoid polyphenol/protein interactions that could limit protein extraction yield and provoke electrophoretic distortions. This procedure permitted us to obtain similar protein extraction yields between H and HS fruits as well as between the unripe and ripe stages, showing no interferences linked to the different fruit chemical compositions (Figure 1). Moreover, the 2-DE patterns were well comparable among the four conditions, consisting of an average of about 1300 spots with similarly good resolution (Figure 1 and Figure S1). To evaluate the differences among experimental conditions, we applied multivariate analyses based on PCA and PLS-DA. The PCA score plot is reported in Figure 2. The first component (PC1), explaining 36% of the total variance, separated unripe and ripe fruits, whereas H and HS genotypes were distinguishable along PC2, which accounted for a lower but significant percentage of variance (25%, Figure 2). These results showed that the main proteomic differences were ascribable to the ripening stage, as expected considering the deep biochemical changes occurring during this process. Interestingly, an explorative technique such as PCA could distinguish the two genotypes. PLS-DA was then applied to individuate the spots discriminating the proteomic profiles of the experimental classes (Table S1; Figures S2−S7). Among them, it was possible to characterize 39 proteins by LC-ESIMS/MS (Table 2). Detailed statistical data about protein characterization are reported in Table S3. The spot electrophoretic positions and the spot abundances in unripe and ripe sweet cherries of H and HS are reported in Figures 3 and 4, respectively. The classification of the identified proteins according to their functional roles highlighted eight main categories (Table 2), embracing different percentages of identified proteins: carbon metabolism (26%); lipid metabolism (15%); cell wall modification (8%); protein translation (8%) and cytoskeleton organization (8%); stress-related proteins (15%); secondary metabolism (15%); other proteins (5%).

RESULTS AND DISCUSSION

Biochemical Comparison between H and HS Sweet Cherries. Sweet cherries of the cultivar ‘Hedelfinger’ (H) and its somaclonal variant HS were sampled at early unripe stage (green fruits) and at full ripening, selecting the fruits with very dark red skin. The evaluation of some ripening-related parameters showed that the chlorophyll content was slightly lower in the unripe fruits of somaclone but, decreasing during fruit maturation, it accounted for comparable amounts in ripe sweet cherries of H and HS (Table 1). Similarly, H and HS fruits showed no differences in the levels of reducing sugars, which more than doubled during ripening. Therefore, reducing sugars were the main sugar compounds in the fully ripe sweet cherries of both genotypes, as previously indicated for several other cultivars.9 Conversely, sucrose concentration was low, and it did not change during fruit development, according to what observed in the cultivar ‘Marvin-Niram’.6 In addition, H and HS fruits contained equal amounts of free amino acids that decreased by half during fruit maturation (Table 1). The total antioxidant capacity of fruit tissues, evaluated as TEAC, was similar in both genotypes, and it was slightly higher at the unripe stage with respect to that at full ripening (Table 1). This trend agrees well with the observation that in sweet cherry the fruit antioxidant activity follows a parabolic trend, reaching high values at the beginning and at the end of development, probably with different contributions of hydroxycinnamic acids and anthocyanins.6 In this respect, the fruit anthocyanin content, starting from almost negligible amounts, greatly increased during ripening in both genotypes. Interestingly, at full maturation HS fruits showed a higher amount of total anthocyanins with respect to those of H (ca. +25%, Table 1). Overall, this evaluation revealed only small differences between H and HS fruits. The lower level of chlorophyll in unripe fruits of HS may be attributable to the different responses to light by which the somaclone was selected.13,14 Even more intriguingly, it is conceivable that the reduction of vegetative vigor in HS trees,15 by modifying the organ source/ sink relationship, might trigger a slight precocity of the fruitripening process. However, the other parameters indicate that this difference has no effects on the fruit biochemical composition, especially at full ripening. Therefore, the fruit 4173


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wall modification and cytoskeleton organization, respectively (Figure 5). Carbon Metabolism. Carbon metabolism was the largest functional class (Table 2), including enzymes the abundance of which both changed during fruit ripening and was different between H and HS proteomes (Figure 5). The most recognizable trend related to fruit ripening depicted a general decline in photoautotrophic activities (Figure 4). In both genotypes, the drops of oxygen-evolving enhancer proteins (OEE1 and OEE2), required for the photosystem II core stability, were consistent with the lower chlorophyll content in ripe fruits with respect to the unripe ones (Table 1). Moreover, the decreases of RuBisCO, transketolase (TK), and malate dehydrogenase (NADP) (MDH) confirmed a general decline of chloroplastic functionality (Figure 4). Overall, this decline is in agreement with the lessening of gross photosynthesis and the increment in sink strength observed in fruits of cherry spp. at the last phases of maturation.26 Interestingly, sucrose synthase 3 (SUS3) is the only enzyme of carbon metabolism that greatly increased during maturation in both genotypes (Figure 4). At first glance, this trend may seem to be in contrast with the observation that sucrose content did not differ in the unripe and ripe H and HS fruits (Table 1). However, considering the pivotal role of SUS in determining the skin strength in plant organs,27 it is conceivable that its increment reflected the physiological transition that fruits undergo through ripening. Namely, the photosynthetic decay occurring in fruit tissues might be counterbalanced by an increase in usage of the photoassimilates translocated from leaves. At the same time, several differences in enzymes involved in carbon metabolism were related to the sweet cherry genotype (Figure 5). We included in this category aspartate aminotransferase (AspAT) and the soluble inorganic pyrophosphatase (PPase; Table 2 and Figure 4) because of their strict relationship with the Krebs cycle, glycolysis, and energy metabolism in plant cells.28 The majority of the genotyperelated differences were detected at the unripe stage (Table 2). In particular, HS unripe fruits were characterized by higher levels of RuBisCO, TK, and succinate dehydrogenase (SDH), whereas glucose-6-phosphate 1-epimerase (G6PE), AspAT, and PPase showed lower abundance in HS with respect to H unripe fruits. At the same time, HS sweet cherries were also characterized by higher abundance of SUS3 at full ripening stage (Figure 4). These results allowed hypothesizing some relationships with the modification of the tree vigor and canopy density of the somaclonal variant HS.15 It is possible that the higher exposure to light of leaves and/or fruits in HS trees supports an increment in fruit carbon metabolism. However, the greater similarity between H and HS fruits at full maturation suggests that this aspect loses relevance during fruit development. Overall, the results showed a similar trend in enzymes involved in carbon metabolism during fruit ripening in both genotypes, confirming that the somaclonal variation did not affect main fruit characteristics, such as carpometric parameters15 and sugar composition (Table 1). At the same time, the proteomic analysis suggests new relationships between the modulation of pivotal enzymes of carbon metabolism in fruits and some tree morphological traits, such as the canopy light interception. Lipid Metabolism. Albeit with some genotypic differences, the enzymes belonging to lipid metabolism were primarily

Figure 1. (a) Protein yield (mg protein g−1 FW) of the protein extraction protocol (n = 3) and (b) average number of spots visualized in the 2-DE profiles (n = 6) from sweet cherry samples of ‘Hedelfinger’ (H, plain bars) and its somaclonal variant HS (HS, slashed bars) at unripe (U, white bars) and ripe (R, gray bars) stages. Values are the means ± SE. The letters were assigned according to Tukey’s test (p < 0.05).

Figure 2. PCA score plot performed on the overall data set considering the first two PCs from the 2-DE profiles of sweet cherries of ‘Hedelfinger’ (H, triangles) and its somaclonal variant HS (circles) at unripe (white symbols) and ripe (gray symbols) stages.

Interestingly, the arrangements of spots distinguishing between the ripening stages and/or the genotypes in the eight functional classes were dissimilar. In general, the functional categories were heterogeneous, even if some of them were more specifically influenced by ripening or genotype, such as cell 4174


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Table 2. Identified Proteins in Sweet Cherry Proteome Discriminating between the Two Ripening Stages (RS) and/or H and HS Genotypes (G)a differencef

pI/MW spot IDb

acronymb

NCBI accession

Prunus species

1355

OEE1

XP_008241598

mume

1552

OEE2

EMJ17026

persica

1737 330 1099

RuBisCO SUS3 G6PE

AKC99613 XP_008235123 XP_008223738

maximowiczii mume mume

419 1002

TK MDH

EMJ21439 XP_008232320

persica mume

499

SDH

XP_008241667

mume

1058 1524

AspAT PPase

AGF95102 EMJ05931

persica persica

227

LOX2−1

XP_008231380

mume

319 565 914

PLDα HACL 5βStR

XP_008243731 XP_008229283 EMJ24225

mume mume persica

1130 1159

EOa EOb

EMJ16888 EMJ16888

persica persica

1340

XTH

XP_007205666

persica

1734 506

XYL1 XYL2

EMJ26514 EMJ26444

persica persica

1301 851 482

eIF3F eIF4A NRS

EMJ19975 EMJ03367 EMJ12582

persica persica persica

1714 901 760

ARF2 ACT TUB

XP_008221835 AJB28509 XP_008220065

mume avium mume

426 1654

BiP5 sHSP

XP_008239207 XP_008219986

mume mume

535 485

PDI NRX1

EMJ23316 XP_008221828

persica mume

1262

LGL

EMJ12953

persica

1715

USPA

EMJ24798

persica

494 1049

βGLC COMT

AAA91166 XP_008241354

avium mume

1741

CAD

BAE48658

mume

1564 951 1062

CHI DFR ANS

AEJ88218 AHL45016 AJO67970

persica avium avium

protein description

exptl

Carbon Metabolism oxygen-evolving enhancer prot 5.16/29.4 1 chl oxygen-evolving enhancer prot 6.01/24.8 2 chlg RuBisCO large subunit 5.95/50.3 sucrose synthase 3 6.04/93.5 put. glc-6-phosphate 16.38/38.9 epimerase 5.73/77.9 transketolase, chlg malate dehydrogenase 5.57/42.6 [NADP], chl succinate dehydrogenase 5.57/63.7 [ubiq] flavoprotein sub 1, mit. aspartate aminotransferase 5.86/40.1 5.32/25.5 soluble inorganic pyrophosphataseg Lipid Metabolism linoleate 13S-lipoxygenase 2-1, 5.43/104.3 chl like phospholipase D α 1 5.96/96.1 2-hydroxyacyl-CoA lyase 5.79/59.9 3-oxo-δ(4,5)-steroid 5-β5.39/45.1 reductase-likeg enone oxidoreductaseg 5.24/37.6 5.26/36.7 enone oxidoreductaseg Cell Wall Modification 6.38/29.8 xyloglucan endotransglucosylase/ g hydrolase α-xylosidase 1g 5.75/97.9 5.63/63.8 β-xylosidase/α-Larabinofuranosidaseg Protein Translation eukaryotic initiation factor 3F 4.81/31.6 eukaryotic initiation factor 4Ag 5.26/47.2 5.70/64.3 asparagine–tRNA ligase, cyt 1-likeg Cytoskeleton Organization ADP-ribosylation factor 2 6.18/19.3 actin V1 5.20/45.7 tubulin β chain 4.95/50.6 Stress-Related Proteins luminal-binding protein 5 5.15/76.8 18.5 kDa class I heat shock 5.33/21.7 protein 4.86/61.6 protein disulfide-isomeraseg probable nucleoredoxin 1 4.74/64.6 isoform X1 putative lactoylglutathione 5.19/33.0 lyaseg universal stress protein A-like 5.92/19.2 proteing Secondary Metabolism β-glucosidase, partialh 5.11/63.0 caffeic acid 3-O5.24/40.7 methyltransferase cinnamyl alcohol 6.01/35.4 dehydrogenase chalcone flavonone isomerase 4.99/24.5 dihydroflavonol 4-reductase 5.80/44.0 anthocyanidin synthase 5.53/40.2

4175

theor

p scorec

% aad

P U(T)e

RS

6.09/35.4

249.2

38

15 (19)

H, HS

8.52/28.6

89.9

18

5 (5)

H, HS

6.13/53.0 5.91/92.8 6.47/38.6

205.1 76.4 82.4

24 7 16

12 (17) 6 (6) 5 (6)

H, HS H, HS HS

U R U

6.37/81.2 8.45/48.6

232.2 246.0

22 24

14 (16) 14 (18)

H, HS H, HS

U

6.01/70.4

115.0

14

7 (8)

H

U

5.90/44.0 5.41/24.0

140.6 157.5

23 41

8 (8) 9 (12)

6.12/94.8

248.5

16

15 (20)

H, HS

5.86/92.7 5.80/62.1 5.26/44.6

153.5 250.4 64.1

13 32 13

9 (13) 16 (20) 5 (5)

H H, HS H, HS

5.44/34.4 5.44/34.4

215.7 127.9

41 31

12 (15) 8 (8)

7.88/33.7

90.4

19

5 (7)

H, HS

5.74/99.0 7.33/84.6

131.0 148.0

9 12

8 (11) 9 (9)

H, HS H

4.85/31.7 5.38/47.0 6.68/68.4

177.0 90.4 173.5

44 14 17

11 (12) 6 (6) 11 (14)

H, HS H

6.43/20.7 5.31/41.9 4.79/50.9

128.8 39.6 133.9

39 7 20

7 (12) 3 (4) 8 (14)

5.16/73.7 5.57/18.1

352.1 50.7

32 17

22 (27) 4 (4)

H, HS HS

4.95/56.3 5.02/66.1

166.3 90.7

22 11

11 (11) 6 (9)

H

R R

5.27/32.7

133.8

30

8 (8)

H

U

6.61/18.1

109.0

33

6 (6)

5.68/60.8 5.20/40.7

249.9 200.4

23 34

13 (20) 12 (16)

H, HS H, HS

R R

5.83/35.8

221.6

48

15 (18)

HS

U, R

4.91/23.4 5.80/39.2 5.46/40.6

150.9 183.0 193.9

34 29 32

8 (10) 12 (14) 10 (11)

H, HS H, HS

G

U, R U, R

U

U, R R

R

R

H

U

R R U, R R

U, R

U R


Article

Journal of Agricultural and Food Chemistry Table 2. continued

differencef

pI/MW spot IDb 856 1022

acronymb ACY-1 WWP

NCBI accession XP_008235828 EMJ19969

Prunus species mume persica

protein description

exptl

Other Proteins aminoacylase-1 isoform X1 5.36/46.9 WW domain-containing prot 4.84/41.8 C11B10.08

theor

p scorec

% aad

P U(T)e

RS

5.59/49.2 4.81/30.9

155.6 76.3

20 14

8 (11) 4 (5)

H H, HS

G R R

a

The proteins are grouped in functional classes according to GenBank and the literature. bSpot ID and acronyms refer to Table S2 and Figures 3 and 4. cp score: protein score consisting of distinct summed MS/MS search score. d% aa, percentage of amino acid coverage. eP U(T), number of peptide, U, unique; (T), total. fRS, differences related to ripening stage in H and/or HS; G, differences detected between the two genotypes at unripe (U) and/or ripe (R) stage. gAnnotation by BLAST alignment against the Viridiplantae protein subset. hPartial sequence.

Hence, it is possible that these EOs are involved in the production of sweet cherry aromas. At the same time, a phospholipase D α 1 (PLDα) and a 2hydroxyacyl-CoA lyase (HACL) almost doubled in abundance from unripe to ripe fruits, without any genotype-related differences (Table 2; Figure 4). PLDα is a pivotal enzyme of phospholipid catabolism in the senescing system, and its activity is developmentally regulated during ripening in tomato (Lycopersicon esculentum Mill.) and strawberry fruits.33,34 Otherwise, HACL participates in peroxisomal fatty acid degradation in mammals,35 but it is scarcely characterized in plants. Interestingly, a similar high increase in abundance of HACL was previously observed during fruit ripening in apricot.16 Overall, these results suggest that fatty acid catabolism has valuable relevance during fruit development in sweet cherry. In addition, the high degree of similarity between the two genotypes seems to indicate that the somaclonal variation has had a lower impact on this metabolism in fruits. Cell Wall Modification. A xyloglucan endotransglucosylase/hydrolase (XTH, formerly abbreviated XET) and an αxylosidase 1 (XYL1) were highly accumulated in unripe fruits and sharply decreased at full maturation without any genotypic differences, whereas a β-xylosidase/α-L-arabinofuranosidase (XYL2) only declined in ripe H fruits (Table 2; Figure 4). All three of these enzymes are plant glycoside hydrolases (families 16, 31, and 3, respectively) implicated in the biogenesis and structure remodeling of primary cell wall.36 Among them, XTH is the better-characterized enzyme, known to catalyze hydrolysis and transglycosilation reactions of xyloglucans. Hence, XTH might be implicated in cell division and expansion in green fruits as well as in cell wall loosening during fruit ripening.37 From their higher abundances in green fruits, the XTH and XYL1 enzymes identified in this work seem to have prominent roles during the first phase of fruit development in sweet cherry. Interestingly, in tomato the XET activity in green fruits strongly affects the final size of fruits;37 as well as in sweet cherry the content of total cell wall materials seems to be related with the fruit firmness.38 Although the difference in the XYL2 profile might deserve further investigations, the similar proteomic trends of XTH and XYL1 support, from a biochemical point of view, the observation that H and HS genotypes produce fruits with similar firmness and similar final size.15 Protein Translation and Cytoskeleton Organization. The proteomic comparison between H and HS fruits at unripe and ripe stages revealed that two eukaryotic initiation factors (eIF3F and eIF4A, Table 2), an asparagine-tRNA ligase (NRS, Table 2), and three proteins involved in cytoskeleton organization showed very heterogeneous profiles (Table 2;

Figure 3. 2-DE profile of the proteins differentially accumulated in ‘Hedelfinger’ (H) and its somaclonal variant HS sweet cherries at unripe and ripe stages. The figure shows one of the electrophoretic maps of the ripe fruits of H. Total proteins were analyzed by IEF at pH 4−7, followed by SDS-PAGE and visualized by cCBB. Acronyms refer to Table 2 and Figure 4. Standard molecular mass range in kDa (Mr) and pI range are reported on the left and at the bottom, respectively.

influenced by the ripening process (Figure 5), showing similar trends in H and HS fruits (Figure 4). A linoleate 13Slipoxygenase 2-1 chloroplastic (LOX2-1, Table 2) was more abundant in the unripe fruits of HS than in H, but it markedly decreased to similar levels in ripe fruits of both genotypes. It is possible that the profile of LOX2-1 reflected the influences of light exposure and the lessening of chloroplastic functionality during ripening. However, it is important to note that in strawberry (Fragaria × ananassa Duch) fruits the total LOX activity reaches the maximum at the fruit-turning-color phase and decreases during ripening.29 This behavior strictly correlates with the pivotal roles played by LOXs in the production of volatile fatty acid derivatives.30 This class of volatiles includes main components of the sweet cherry aroma, such as hexanal and (E)-2-hexenal, the fruit content of which follows a similar curve trend.31 Therefore, it is possible that the LOX2-1 identified in this work participates in volatile biosynthesis in H and HS sweet cherries. In this view, it is interesting to highlight the identification of three enone oxidoreductases (Table 2), the EOa and EOb and the 3-oxoδ(4,5)-steroid 5-β-reductase (5βStR). In particular, EOa and EOb (GenBank EMJ16888) have 85% identity with the strawberry FaOE (UniProtKB Q84V25) involved in the biosynthesis of furaneol,32 a key flavor, in strawberry fruits. 4176


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Figure 4. Abundance levels %Vol of the identified proteins differently accumulated in sweet cherries of H (plain bars) and HS (slashed bars) at unripe (U, white bars) and ripe (R, gray bars) stage. Acronyms refer to Table 2 and Figure 3. The proteins are grouped in the functional classes reported in Table 2: (a) carbon metabolism; (b) lipid metabolism; (c) cell wall modification; (d) protein translation; (e) cytoskeleton organization; (f) stress-related proteins; (g) secondary metabolism; (h) other proteins. Values are the means ± SE (n = 6). Letters were assigned according to Tukey’s test (p < 0.05).

enhancing cell expansion.40 Because the morphological comparison between H and HS sweet cherry showed that the fruits of the wild-type (H) reach more quickly their final weight and have higher longitudinal diameter,15 this follow-up proteomic investigation allows proposing the novel hypothesis that ARF2 may play some roles in determining cell expansion in sweet cherry fruits. Stress-Related Proteins. The proteomic analysis revealed that six proteins involved in stress responses changed in abundance among experimental conditions. A luminal-binding protein 5 (BiP5) and an 18.5 kDa class I heat shock protein (sHSP) increased in abundance in ripe fruits of both genotypes, even if to different extents (Table 2; Figure 4). On the contrary, a protein disulfide-isomerase (PDI), a nucleoredoxin 1 (NRX1), a lactoylglutathione lyase (LGL), and a universal stress protein A (USPA) were differently modulated between H

Figure 4). In particular, the functional class related to cytoskeleton appeared the most divergent between the two genotypes (Figure 5). Probably, its modulation reflects the numerous physiological events occurring during fruit maturation. Interestingly, ADP ribosylation factor 2 (ARF2; Table 2) is accumulated to a very higher abundance in H fruits with respect to those of HS (Figure 4). The ARF family embraces several GTP-binding proteins involved in vesicle transport, and in mammal systems, several pieces of evidence indicate that ARFs are also implicated in the regulation of actin cytoskeleton dynamics.39 Hence, it is possible that the different modulations between H and HS fruits observed for actin and tubulin β chain (ACT, TUB; Table 2; Figure 4) are somehow related to the different ARF2 abundances. Moreover, a recent study conducted in Arabidopsis thaliana L. suggests that the Zea mays L. ZmARF2 has pivotal roles in determining organ size by 4177


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Secondary Metabolism. All of the identified enzymes involved in plant secondary metabolism belong to biochemical pathways related to phenolic compounds (Table 2). In particular, the identified β-glucosidase (βGLC) shows >90% identity with several Prunus spp. protein sequences annotated as prunasin hydrolases (BLAST alignment against Viridiplantae NCBI database) and, therefore, it is probably involved in the hydrolysis of cyanogenic glycosides typical of rosaceous stone fruits. Interestingly, Gerardi and co-workers showed that a similar enzyme is accumulated in fruit tissues during ripening in sweet cherry, and they proposed possible roles in cell wall modification.43 Moreover, caffeic acid 3-O-methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD) act in the cinnamate/monolignol pathway, which supplies precursors of several phenylpropanoids44 (i.e., lignin, hydroxycinnamate esters, and flavonoids), whereas chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) are pivotal enzymes in catechin and/or anthocyanin biosynthesis.45 Most of these enzymes followed an upward trend during ripening in both H and HS fruits, although the extents of their increment showed genotypic differences (Figure 4). In particular, βGLC and COMT were almost twice in amount in the ripe H fruits with respect to the ripe HS ones (Figure 4), whereas CAD was accumulated only in the somaclone fruits. On the contrary, CHI was more abundant in H unripe fruits, but it was slightly modulated by ripening only in HS (Figure 4). Finally, DFR and ANS were the two enzymes with the highest increment in ripe sweet cherries, and whereas DFR reached a higher amount in HS fruits, ANS levels were not affected by genotype (Figure 4). These enzymatic trends are well in agreement with the great increase of anthocyanin levels in H and HS during fruit ripening (Table 1). However, further investigations are needed to verify if the higher level of DFR in HS ripe fruits is related to and/or is able to sustain the higher extent of anthocyanin accumulation observed in the somaclone sweet cherries (Table 1). From a proteomic point of view, these results confirm the relevance of the metabolism of phenolic compounds in sweet cherry fruits and provide new information about the enzymes involved in the anthocyanin accumulation. On the other hand, the proteomic differences observed between H and HS, together with the difference in anthocyanin content (Table 1), raise the question of whether the somaclonal variation provoked some effects on phenolic metabolism in HS fruits. To gain more information about this issue, a comparison of the

Figure 5. Number of proteins assigned to the different functional classes (Table 2) whose trends discriminated between ripening stages (black bars) or genotypes (white bars).

and HS fruits (Table 2; Figure 4). The first four proteins (BiP5, sHSP, PDI, and NRX1) have roles in protein homeostasis, acting as molecular chaperones or participating in regulation of protein structure and/or activity. Our results well agree with previous proteomic works highlighting that these proteins are modulated during fruit ripening in several species, such as tomato,41 peach,17 grape,23 and strawberry.42 Although their regulation depends on protein family and plant species, altogether these results provide evidence that these proteins are developmentally regulated to cope with stress conditions inherent to fruit maturation. At the same time, this work revealed that PDI, NRX, and USPA were more abundant in H fruits, especially at full ripening (Figure 4). Considering that PDI and NRX1 belong to the thioredoxin superfamily, it is possible that these genotypic differences reflected metabolic traits. However, the observation that the tissue TEAC values were similar in H and HS fruits (Table 1) indicates that the somaclonal variation did not deeply modify the cell redox status. Therefore, further studies are needed to elucidate the metabolic roles of these proteins. These considerations can be extended to the aminoacylase-1 (ACY-1) and to the WW domain-containing protein (WWP; Table 2; Figure 4), the functionalities of which in plant cell are not yet characterized.

Table 3. Relative Percent Abundance of the Identified Phenolic Compounds in Sweet Cherries of ‘Hedelfinger’ and of Its Somaclonal Variant HS at Unripe and Ripe Stagesa H unripe coumaroylquinic acid neochlorogenic acid chlorogenic acid catechin epicatechin procyanidin B quercetin-3-O-rutinoside kaempferol-3-O-rutinosideb cyanidin-3-O-glucoside cyanidin-3-O-rutinoside a

148.8 139.8 145.8 158.9 138.8 90.3 75.2 52.3 ndc nd

± ± ± ± ± ± ± ±

6.8 6.2 9.3 5.7 8.3 5.1 2.8 2.6

HS unripe b b b b b a a a

153.4 139.2 141.6 152.5 154.0 133.6 85.0 63.1 nd nd

± ± ± ± ± ± ± ±

6.1 4.9 4.6 4.3 6.2 7.0 0.4 1.2

H ripe b b b b b b a b

46.3 59.4 56.9 40.3 50.7 81.2 114.0 127.5 108.3 89.9

± ± ± ± ± ± ± ± ± ±

0.7 0.6 0.8 0.7 0.3 0.5 3.9 5.5 4.6 2.5

HS ripe a a a a a a b c b a

51.4 61.6 55.7 48.3 56.6 94.9 125.8 157.1 91.7 110.1

± ± ± ± ± ± ± ± ± ±

1.4 1.9 1.2 1.6 1.5 2.4 2.6 2.1 1.0 2.8

a a a a a a b d a b

Values are the mean ± SE (n = 3). Letters were assigned according to Tukey’s test (p < 0.01). bTentatively assigned. cNot detected. 4178


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Moreover, the somaclone fruits contained higher amounts of kaempferol-3-O-rutinoside, showing a higher abundance of about +10% in green fruits that increased to +30% in dark red sweet cherries. In addition, the anthocyanin composition of ripe fruits also differed between the two genotypes. Whereas H fruits possessed a higher level of cyanidin-3-O-glucoside, HS fruits contained a higher amount of cyanidin-3-O-rutinoside (ca. +20%) (Table 3). Considering that this compound was the most abundant flavonoid in ripe sweet cherries, this difference was very consistent with the higher amount of total anthocyanins (ca. +25%) detected in HS red fruits (Table 1). Taken together these results revealed that, even if the phenolic profiles in H and HS fruits were very similar, the sweet cherries of the somaclone HS were characterized by higher contents of phenolic compounds at both the unripe and ripe stages. Given that the phenolic composition of fruits is not deeply affected, it is unlikely that this trait is a direct consequence of the genetic differences between the two clones. Indeed, it is probably more appropriate to take into account that these compounds participate in the protection of tissues from UV radiation and oxidative stress51 and that their accumulation in fruits is deeply influenced by climate conditions.47 Hence, similar to what previously stated about carbon metabolism, it is possible to hypothesize that the reduction of vegetative vigor in HS trees, and the resulting higher exposure of fruits to sunlight, could result in a higher accumulation of phenolic compounds and, in particular, anthocyanins at full ripening. In conclusion, this work revealed that the somaclonal variation between H and HS genotypes does not deeply modify the main fruit biochemical characteristics or the fruitripening process, except for suggesting that the reduction of tree vigor in the HS somaclone sustains a higher accumulation of flavonoids in fruits. Future studies will allow understanding how these positive traits of the new somaclone HS could be influenced by different pedoclimatic conditions and orchard managements.

phenolic compound composition in H and HS sweet cherries at the two ripening stages was performed. Comparison among the Phenolic Profiles of Cherries of H and HS at the Two Ripening Stages. To investigate possible effects of the somaclonal variation on fruit secondary metabolism, an evaluation of the phenolic profiles in H and HS fruits as well as of their percent relative changes at unripe and ripe stages was performed. The HPLC-MS/MS analyses of the methanolic extracts from samples (skin plus pulp) allowed identifying 10 main metabolites (Table 3). The phenolic profiles of H and HS fruits were very similar and consistent with what had been previously observed in other sweet cherry cultivars,46−49 as well as specifically in ‘Hedelfinger’.50 In H and HS fruits, the coumaroylquinic acid, neochlorogenic acid, and chlorogenic acid were the main hydroxycinnamic acids, whereas the flavonol-3-ol derivatives mainly consisted of catechin and epicatechin (Table 3). Interestingly, the analysis revealed the presence of a procyanidin B-type compound. Even if the discrimination among isomers was not performed, it is likely that this compound corresponds to the procyanidin B2, according to what was recently indicated in ‘Kordia’ sweet cherries.49 Moreover, two major flavonol derivatives, quercetin3-O-rutinoside and kaempferol-3-O-rutinoside, and two major anthocyanins, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside, were identified (Table 3). According to previous works,46−50 the evaluation of chromatographic profiles indicated that cyanidin-3-O-rutinoside was the most abundant compound among flavonols and anthocyanins (data not shown). In this respect, it is important to note that the goal of this analysis was to compare the whole composition of H and HS fruits, and the trace compounds were not considered. This selection probably excluded peonidin and pelargonidin derivatives, which are minority anthocyanins in sweet cherry.46−50 Interestingly, most of the identified phenolic classes deeply changed during ripening, with trends very similar in H and HS fruits (Table 3). In particular, hydroxycinnamic acids and catechins were the major compounds in unripe sweet cherries and sharply decreased at full ripening (Table 3). Concomitantly, it was possible to observe an increase of flavonols, especially of kaempferol-3-O-rutinoside, and an accumulation of anthocyanins, that, starting from undetectable levels in green fruits, became two of the major phenolic compounds in red ripe fruits (Table 3). This accumulation was consistent with the increase of total anthocyanins detected by spectrophotometric analysis (Table 1) as well as with the levels of DFR and ANS enzymes (Table 2; Figure 4). Moreover, the changes observed in the phenolic profiles are in agreement with what was previously reported for ‘Petrovka’48 cultivar, supporting the hypothesis that hydroxycinnamic acids (and probably catechins) are channeled to sustain the synthesis of anthocyanins throughout fruit maturation in sweet cherry. In addition, considering the TEAC did not significantly change during ripening (Table 1), it is conceivable that these classes of phenolic compounds both contribute, to different extents in green and red fruits, to protect tissues from oxidative stress, as previously suggested by Serrano and co-workers.6 At the same time, this analysis revealed differences in phenolic composition between H and HS fruits at both ripening stages (Table 3), confirming that some changes in secondary metabolism occurred. In particular, procyanidin B was more abundant by about +40% in HS with respect to H fruits, and it decreased to similar levels at full maturation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b01039. 2-DE profiles of sweet cherries of H and HS at unripe and ripe stages; performance of PLS-DA models; PLSDA score plots; technical parameters for phenolic compound identification by HPLC-MS/MS; statistical data about protein identification by LC-ESI-MS/MS analysis (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(B.P.) E-mail: [email protected]. Phone: ++39 02 503 16610. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful to Remo Chiozzotto for kindly helping during the collection and evaluation of the sweet cherry fruits. ABBREVIATIONS USED 2-DE, two-dimensional electrophoresis; β-ME, β-mercaptoethanol; cCBB, colloidal Coomassie Brilliant Blue G-250; 4179


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CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CGE, cyanidin-3-glucoside equivalents; FA, formic acid; H, ‘Hedelfinger’; HR, ‘Hedelfinger’ ripe fruits; HS, somaclonal variant of ‘Hedelfinger’; HSR, somaclonal variant of ‘Hedelfinger’ ripe fruits; HSU, somaclonal variant of ‘Hedelfinger’ unripe fruits; HU, ‘Hedelfinger’ unripe fruits; LCESI-MS/MS, liquid chromatography−electrospray ionization− tandem mass spectrometry; NCBI, National Center for Biotechnology Information; PCA, principal component analysis; PSL-DA, partial least squares−discriminant analysis; TEAC, Trolox equivalent antioxidant capacity

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Sweet Cherry - ACS Publications - American Chemical Society

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