Differentially Accumulated Proteins in Coffea

Article pubs.acs.org/JAFC

Differentially Accumulated Proteins in Coffea arabica Seeds during Perisperm Tissue Development and Their Relationship to Coffee Grain Size Leonardo Cardoso Alves,†,Δ Diogo Maciel De Magalhaẽ s,† Mônica Teresa Veneziano Labate,# Simone Guidetti-Gonzalez,# Carlos Alberto Labate,# Douglas Silva Domingues,† Tumoru Sera,† Luiz Gonzaga Esteves Vieira,† and Luiz Filipe Protasio Pereira*,†,§ †

Biotechnology Laboratory, Instituto Agronomico do Parana, Londrina, Parana 86047-902, Brazil Department of Biochemistry and Biotechnology, Universidade Estadual de Londrina, P.O. Box 6001, Londrina, Parana 86051-990, Brazil # Max Feffer Plant Genetics Laboratory, ESALQ, Universidade de Sao Paulo, Piracicaba, Sao Paulo, Brazil § EMBRAPA Café, Brasilia, DF, Brazil Δ

S Supporting Information *

ABSTRACT: Coffee is one of the most important crops for developing countries. Coffee classification for trading is related to several factors, including grain size. Larger grains have higher market value then smaller ones. Coffee grain size is determined by the development of the perisperm, a transient tissue with a higly active metabolism, which is replaced by the endosperm during seed development. In this study, a proteomics approach was used to identify differentially accumulated proteins during perisperm development in two genotypes with regular (IPR59) and large grain sizes (IPR59-Graudo) in three developmental stages. Twenty-four spots were identified by MALDI-TOF/TOF-MS, corresponding to 15 proteins. We grouped them into categories as follows: storage (11S), methionine metabolism, cell division and elongation, metabolic processes (mainly redox), and energy. Our data enabled us to show that perisperm metabolism in IPR59 occurs at a higher rate than in IPR59-Graudo, which is supported by the accumulation of energy and detoxification-related proteins. We hypothesized that grain and fruit size divergences between the two coffee genotypes may be due to the comparatively earlier triggering of seed development processes in IPR59. We also demonstrated for the first time that the 11S protein is accumulated in the coffee perisperm. KEYWORDS: perisperm metabolism, grain size, seed development, proteomics, quality

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the final size of the coffee bean.8 By 90−120 DAF, the perisperm starts an involution process until it becomes a thin layer covering the expanding endosperm near the final maturation stage at 150−210 DAF.9 Transcriptomics studies using the C. arabica cultivar IAPAR 59 and a closely related coffee genotype with distinct difference in fruit size (IAPAR 59-Graudo) have shown that α-expansin isoforms are differentially expressed at the earlier stages of fruit development. A time-prolonged expression of α-expansins in IAPAR 59-Graudo was suggested as one of the reasons for its larger fruits.10 Perisperm development is crucial not only for grain size but also for the biosynthesis of compounds that will contribute to the final biochemical characteristics of the coffee grain, including sucrose, diterpenes, chlorogenic acids, and caffeine.7 Those compounds are produced during perisperm formation and transferred to endosperm. Proteins, peptides, and free amino acids are strongly related to beverage quality because they serve as precursors to aromas and flavors.2 A number of proteomic studies analyzing fruits

INTRODUCTION Coffee is one of the most appreciated beverages worldwide and is an important commodity. Coffea arabica, which represents 60% of the market share,1 has a better beverage quality than Coffea canephora, which is used mainly for instant coffee.2 Interest from consumers for drinking better quality coffee has increased during recent years mainly due to its health benefits, better agricultural practices for high-quality coffee production, and advances in the understanding of relationships between components and sensory properties.2,3 The quality of commercial coffee beans is evaluated considering the percentage of physical defects, color, density, and size. Large coffee grain size samples allow a more uniform and steadier roasting and are the preferable choice for special and gourmet coffee, reaching higher prices for the producers.4 Endosperm, also known as “real grain”, is the commercial part of the coffee fruit. This tissue is covered by the perisperm, a thin and green pellicle5 remaining at the end of the fruit ripening stage. However, at the beginning of fruit development (from 60 to 90 days after flowering (DAF)), the perisperm faces intense cell division and expansion, occupying the entire volume of the locule while the endosperm is still not apparent.6,7 This is a critical stage to the quality and value of grain, because the space occupied by the perisperm will define © XXXX American Chemical Society

Received: September 7, 2015 Revised: January 11, 2016 Accepted: January 25, 2016

A

DOI: 10.1021/acs.jafc.5b04376 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

and 70% ethanol. After drying, pellets were stored at −80 °C until further analyses. Protein Solubilization and Two-Dimensional Electrophoresis. Pellets were solubilized as described in ref 21 with modifications. Thiourea was added in combination with urea to increase solubilization of the proteins. Solubilization buffer contained 7 M urea (Sigma-Aldrich), 2 M thiourea (GE Healthcare, Uppsala, Sweden), and 4% (w/v) 3-[(cholamidopropyl)demethylammonio]-1propanesulfonate (CHAPS, GE Healthcare) at room temperature. Protein content was determined according to the Bradford assay.22 For the first-dimension separation IPG strips (13 cm, pH 4−7, GE Healthcare) were rehydrated overnight in the Immobiline Dry Strip Reswelling Tray (GE Healthcare) at room temperature with 250 μL of a rehydration solution containing 600 μg of extracted proteins, 4 μL of 50 mM dithiotreitol (DTT; Fischer Scientific, Nepean, Canada), 2% (v/v) IPG buffer (13 cm, pH range 4−7) (GE Healthcare), and DeStreak buffer (GE Healthcare). Isoelectrofocalization was carried out in the Ettan IPGphor II (Amershan Biosciences) at 20 °C and maximum amperage of 55 μA, applying the following conditions according to a previously described method 17 with minor modifications in voltages: phase 1, step 200 V for 1 h; phase 2, step 500 V for 1 h; phase 3, gradient 1000 V until accumulation of 1000 Vh; phase 4, gradient 7000 V until accumulation of 11300 Vh; phase 5, gradient 8000 V until accumulation of 35000 Vh. IPG strips were incubated for protein reduction during 15 min (6 M urea, 75 mM TrisHCl, pH 8.0, 29.3% (v/v) glycerol (Ge Healthcare), 2% (w/v) SDS, a trace of bromophenol blue (Sigma-Aldrich), and 10% (w/v) DTT) and a second time for alkylation for 15 min with iodoacetamide (IOD; Ge Healthcare, Buckinghamshire, UK). Second-dimension electrophoresis was carried out in 11% (w/v) polyacrylamide gels with SDS (gel size 18 × 16 cm) in two steps: step 1, 45 min and 15 mA each gel; and step 2, 3 h and 40 min and 30 mA each gel. Both were run at 10 °C, 50 W, and 250 V. Gels were fixed in solution containing 10% (v/v) acetic acid (J. T. Baker) and 40% (v/v) ethanol (J. T. Baker) overnight and stained with Colloidal Coomassie Blue G-250 solution (USB, Cleveland, OH, USA). Two gels from each of the three bulks of plants were produced, resulting in a total of six gels from each sample in each stage of fruit development. Image Analysis. Gel images were captured using a scanner (ImageScanner UMAX, GE Healthcare), and Image Master 2D Platinum 6.0 software (Ge Healthcare) was used for spot detection, gel matching, and detection of changes in protein abundance. Detected spots were refined manually when necessary, and spots presenting area and intensity lower than 0.8 and 0.01, respectively, were discarded. Each spot had its abundance estimated by the percentage of volume (% vol), which means that each spot volume was divided by the total volume from all gel spots. Only the spots that were present in at least two gels of each stage of fruit development or genotype were considered for abundance variation analysis. Only spots with an average ratio calculated by Image Master as increasing or decreasing >1.5-fold and with a p value ≤0.05 (Student’s t test) were considered as significantly changing and considered for further analysis. Seven comparative analyses were performed in the search for differentially expressed proteins: (1) IPR59 60 × 90 DAF; (2) IPR59-GDO 60 × 90 DAF; (3) IPR59 90 × 180 DAF; (4) IPR59-GDO 90 × 180 DAF; (5) IPR59 × IPR59-GDO 60 DAF; (6) IPR59 × IPR59-GDO 90 DAF; (7) IPR59 × IPR59-GDO 180 DAF. In-Gel Digestion and Protein Identification by MS/MS. Spots were cut from the gels, and protein cleavage was carried out according to ref 23 with modifications in the concentrations of solutions. For the discoloration step the spots were washed repeatedly with a solution containing 50% (v/v) acetonitrile (ACN; J. T. Baker) and 25 mM (w/ v) ammonium bicarbonate buffer (AMBIC; Sigma-Aldrich) for 10 min under agitation. After discoloration, spots were dehydrated by pure ACN. Proteins were reduced (20 mM DTT and 50 mM AMBIC) for 40 min at 56 °C and alkyled (55 mM IOD and 50 mM AMBIC) for 30 min at room temperature and then cleaved by 20 ng/μL trypsin (Trypsin Gold Mass Spectrometry, Promega, Madison, WI, USA) in 50 mM AMBIC for 14 h at 37 °C. Sequential elution of peptides was accomplished using solution A (60% (v/v) methanol and 1% (v/v)

from coffee cultivars in different maturation stages have identified proteins related to carbohydrate biosynthesis, peroxide reduction, RNA splicing, ATP synthesis, photosynthesis, defense and disease resistance, cold tolerance, and nitrogen source.11−15 However, most of those studies were performed at the final stages of fruit maturation or with the whole fruit instead of isolated tissues. Despite recent advances in coffee transcriptomics and genomics,16−18 to date, there have been relatively few studies examining proteomes of seeds in early developmental stages, when perisperm is the predominant tissue. Taking into account that fruits are the commercial part of diverse crops and therefore require special attention, recent studies19,20 have shown differential proteomics is a powerful tool to analyze molecular changes in fruits and seeds from different cultivars, aiming at the identification of proteins associated with quality and development processes. The objective of this study was to identify proteins differentially changing in abundance in perisperm of coffee seeds at early development stages in two genotypes of C. arabica with contrasting bean sizes as well as to discuss the role of these proteins on the development of the coffee beans. For this, we performed a two-dimensional gel electrophoresis (2DE) analysis coupled with mass spectrometry (MS) protein sequencing using perisperm tissue in three seed developmental stages. This approach allowed us to identify 24 differentially accumulated spots corresponding to 15 proteins. We discuss the roles of these proteins on perisperm development and their relationships with coffee grain quality.

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MATERIALS AND METHODS

Plant Material. C. arabica cvs. IAPAR 59 (IPR59) and IAPAR 59Graudo (IPR59-GDO), a genotype that produces large fruits and seeds,10 were cultivated in field conditions at the Institute Agronomic of Parana, Londrina, Brazil (23°21′26″ S; 51°09′43″ W). Fruits were harvested from 30 plants divided into three bulks of 10 plants each on November 28, 2007, December 27, 2007, and March, 19, 2008, correspondig to 60, 90, and 180 DAF, respectively. Perisperm tissue was carefully separated by hand, avoiding endosperm contamination, and ground in liquid nitrogen. Samples were stored at −80 °C until analysis. Protein Extraction. Proteins were extracted from 10 g of ground perisperm as previously described,21 with modifications. Polyvinylpolypyrrolidone (PVPP) and β-mercaptoethanol were added at the first steps of the protocol to avoid oxidations. Samples were washed with a chilled solution (20 mL) containing 15% (w/v) PVPP (Sigma-Aldrich, St. Louis, MO, USA) in acetone, 2% (v/v) β-mercaptoetanol (Acros Organics, Morris Plains, NJ, USA), 1 mM phenylmethanesulfonyl fluoride (PMSF; Fluka, Buchs, Switzerland), and 90% acetone (J. T. Baker, Xalostoc, Mexico). Samples were maintained for 40 min on ice, and then the material was sonicated and centrifuged at 25000g for 7 min. Pellets were resuspended and centrifuged at 25000g for 7 min sequentially in the following chilled solutions (15 mL): 100% acetone, 10% TCA (Sigma-Aldrich) in acetone, 10% (w/v) TCA in water, 80% acetone, and 80% ethanol (J. T. Baker). After drying at room temperature, a solution (20 mL) containing 30% (w/v) sucrose (Sigma-Aldrich), 2% (w/v) SDS (Invitrogen, Carlsbad, CA, USA), 0,1 M Tris-HCl buffer (Invitrogen), 2% (w/v) 2-mercaptoethanol, and 1 mM PMSF plus 12 mL of buffered phenol (pH 7.3) (Invitrogen) was added. After incubation for 15 min, samples were centrifuged at 25000g for 10 min. Phenolic supernatants were carefully collected and incubated overnight with 0.1 M ammonium acetate (Merck, Darmstadt, Germany). Supernatants were removed and pellets resuspended and centrifuged at 19500g for 10 min with chilled solutions (15 mL) containing 0.1 M ammonium acetate, 80% acetone, B


Article


Figure 1. Two-dimensional gels from perispermal proteins from IPR59 and IPR59-GDO at 60, 90, and 180 DAF. Most of the proteins ranged between pI 4.5 and 7 and between MW 16 and 97 kDa. Arrows indicate the differentially accumulated spots identified by mass spectrometry. Numbers inside parentheses refer the analysis where spots were considered to be differentially accumulated (p ≤ 0.05). Proteins were extracted, separated by two-dimensional gel electrophoresis, and colored by Coomassie Brilliant Blue. Six gels were produced for each genotype at each stage of fruit development. After scanning, Image Master Platinum 6.0 detected the spots and normalized its abundances (% vol). Comparisons were made for detecting differences in protein abundance between IPR59 and IPR59-GDO at each stage of fruit development and within each genotype (between the stages of fruit development). Differentially accumulated spots were selected for further MALDI-TOF-MS/MS analysis. formic acid (J. T. Baker)) for 15 min at 40 °C and solution B (50% (v/ v) ACN and 1% (v/v) formic acid) and by pure ACN. Peptide solutions were purified using Zip-Tip C18 microcolumns (Millipore, Bedford, MA, USA). Mass spectrometry analyses were performed on a MALDI-TOF-TOF instrument (AB Sciex 5800, Framingham, MA, USA) by applying 1 μL of peptides mixed with 1 μL of α-cyano-4hydroxycinnamic acid in a MALDI plate using automatic method. MS and MS/MS mass spectra were obtained by the positive reflector method at 400 Hz (MS) and 1000 Hz (MS/MS) and ion source voltage of 3400 kV (MS) and 4700 kV (MS/MS). Peptide mass fingerprints were obtained through Collision Induced Dissociation method. Mass/charge data were obtained by AB Sciex MALDI-TOFTOF Series Explorer 4.1 software. Data analyses were carried out using Protein Pilot 4 software (AB Sciex) through MASCOT platform (Matrix Science) using coffee ESTs from NCBI as a filter. The searches for homologous sequences were made on April 20, 2014, and the following parameters were applied: monoisotopic ions; carbamidomethyl (C) as fixed and oxidation (M) as variable modifications; two missed cleavages; 0.5 Da for both peptide and MS/MS tolerances. MASCOT protein scores >72 were considered significant (p ≤ 0.05). The EST sequences obtained were analyzed by the BLASTn tool against a database containing 35153 C. arabica unigenes downloaded at April 20, 2014, from the Brazilian Genome Consortium databank (http://bioinfo03.ibi.unicamp.br/cafe/). The obtained unigenes were then analyzed by BLASTx tool against the Arabidopsis genome

database TAIR (http://www.arabidopsis.org/Blast/index.jsp) or NCBI database for functional identification.

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RESULTS AND DISCUSSION Protein Extraction and 2D Electrophoresis. Extractions employing precipitation with TCA, solubilization with phenol/ SDS, and precipitation with ammonium acetate have yielded an average of 1.8 mg/g protein/fresh perisperm. The well-resolved and reproducible spots have shown the efficiency of this method in minimizing the presence of other cell components, which are intrinsic to coffee extracts, such as polyphenols and pigments (Figure 1). Fruits were harvested from three pools of plants at each fruit developmental stages (60, 90, and 180 DAF) from each genotype (IPR59 and IPR59-GDO). Two gels were produced for each pool, resulting in six 2D gels with good quality and reproducibility per genotype at each stage of fruit development. Gels from IPR59 had an average amount of spots higher than IPR59-GDO, except at 180 DAF when the same amount was detected (Table S1). Overlaps from all gels were >59% in all analyses, showing that there were matches between analyzed spots. Figure 1 demonstrates gels from each genotype and stage C


Article


more abundant in IPR59 at 90 DAF (spot 9 (6), Table 1 and Figure 2). There was no MeSe spot detected at 180 DAF, which supports the indication that MeSe accumulation in the perisperm occurs at the beginning of C. arabica seed development. In C. arabica cultivars Mocca and Catimor, the expression of MeSe and other alkaloid biosynthesis genes suggested the active synthesis of caffeine occurs in the perisperm and pericarp at the beginning of the development (around 60 DAF) and in the endosperm at 150−180 DAF.29 The caffeine biosynthesis pathway is characterized by a sequence of methylation reactions where methyl groups are transferred from S-adenosylmethionine (SAM), which is the product of the conversion of methionine and ATP by SAM-synthase (EC 2.5.1.6).30 Interestingly, methionine synthase genes are highly expressed in early fruit stage,27 and the transcriptional profile of the caffeine synthase gene in coffee fruits showed a pattern of accumulation according to the perisperm development.26 Another possible explanation for the higher expression of MeSe in the initial stages of seed development is related with ethylene synthesis. Methionine is the ethylene precursor, being converted to SAM by SAM-synthase. Then, ACC-synthase catalyzes the conversion of SAM into 1-aminocyclopropane-1carboxylic acid (ACC), which, in turn, is converted into ethylene by ACC-oxidase.31 In coffee, it has been shown that ethylene concentration rose from a basal level at 140 DAF to a 6 times increase at 200 DAF.32 Recent study also demonstrated this pattern and suggested that ethylene evolution is strongly related to the fruit maturation time in coffee cultivars.33 MeSe accumulation at 60 DAF was detected only in IPR59 (spot 5 (1)) and was more abundant in IPR59 at 90 DAF compared to IPR59-GDO (spot 9 (6)). The differential accumulation of proteins involved with methionine metabolism between both genotypes may imply an earlier start of ethylene synthesis in the perisperm of IPR59 seeds. In IPR59-GDO, a MeSe decrease in abundance together with SAM-synthase presence only at 90 DAF (spot 22 (4)) might represent a longer period of the growing stage of perisperm, which may be the cause of the increased seed and fruit sizes due to the delay of the final steps of maturation. Similarly, in IPR59-GDO fruits, genes encoding expansins had a prolonged expression until 90 DAF, whereas in IPR59 it stopped at 60 DAF.10 The expression of genes involved in ethylene biosynthesis also has been reported in early stages of coffee fruit development.26,27 Understanding the accumulation dynamics of proteins related to ethylene biosynthesis can be of interest for further studies not only on the mechanisms controlling grain size but also on ripening uniformity, two characteristics related to coffee quality and, consequently, higher commercial coffee value. Cell Division and Elongation. Three spots were identified as coffee chitinase-like xylanase inhibitor protein (CLXIP), a member of class III chitinases (spots 2 (1), 3 (5), and 21 (6), Table 1 and Figure 2). In IPR59 60 DAF spot 2 (1) was 1.75 times more abundant than at 90 DAF and spot 3 (5) was positively accumulated 2.17 times more than in IPR59-GDO 60 DAF. Spot 21 (6) was 1.57 times more abundant in IPR59GDO 90 DAF than in IPR59. This class of proteins was abundant in coffee tissues under significant cellular division activity such as during the induction of somatic embryogenesis34 and developing endosperm,11 suggesting also involvement in plant growth, cell division, and developmental processes.6 Interestingly, in our observations spots 2 (1) and 21 (6) were part of a high-volume group in gels from both

of fruit maturation. A higher amount of differentially accumulated spots was detected in comparisons between 90 against 180 DAF instead of 60 against 90 DAF, possibly because the larger time interval comprised in 90 × 180 DAF results in greater differences in the metabolic processes. In addition, there were more spots present only at 90 DAF when compared to 180 DAF (Table S2). Comparisons between IPR59 against IPR59-GDO in all developmental stages have shown a higher amount of more abundant and present spots in IPR59 (Table S2). Spots from comparison 2, IPR59-GDO 60 × 90 DAF, were not significant at Student’s t test (p ≤ 0.05) and were thus discarded from the analysis. Protein Identification. Spots differentially accumulated by at least 1.5-fold were cut and the proteins sequenced by a MALDI-TOF-MS/MS system. After MASCOT search against the Cof fea EST database, we identified 24 spots, which corresponded to 15 proteins (Table 1 and Figure 2). Further data such as images from in-gel triplicates of each identified spot, as well as its abundances and MS data, are available in Figure S1 and Tables S3 and S4, respectively. Some proteins had different MASCOT suggested values for pI and MW than the observed in gel and were present in more than one spot. This may occur due to post-translational modifications (PTMs), alternative splicing, and protein degradation.24 This may also occur due to disulfide bond disruption and subunit fragmentation, as observed for the 11S protein.25 Storage Proteins: 11S Is Highly Accumulated in Perisperm. We observed the presence of 11S in perisperm on both genotypes by identifying five spots (Table 1 and Figure 3). Whereas at 60 DAF there was no detectable spot with this protein, the presence of 11S spots was noticeable at 90 and 180 DAF. Spot 12 (1) was detected in IPR59 90 DAF and remained stable in % vol, whereas the other spots have rose in abundance from 90 to 180 DAF or were detected only at the late stage (Table 2). At 180 DAF, spot 17 (3) and spot 13 (3) accumulated 2.6- and 8.5-fold respectively, in cultivar IPR59. In IPR59-GDO, two spots were identified as 11S: spot 23 (4), present only at 180 DAF, and spot 24 (7), accumulated by 1.58-fold relative to IPR59. Seeds utilize globulins, such as 11S, as nitrogen source for germination.25 The presence of the 11S proteins was initially observed during the exponential growth phase of the endosperm25 as well as in mature fruits, seeds, coffee embryos11,14 and even in initial stages of somatic embryogenesis.13 Transcriptional studies with fruits at the initial stages of development reported 11S transcripts at the early stages of fruit development, when the main fruit tissue is the perisperm.26,27 Here, we provide the first evidence of the presence of the 11S protein in the isolated perisperm of coffee seeds. As perisperm was isolated, there was no pericarp or endosperm contamination that could be the sources of 11S at 90 DAF. Our data demonstrate that 11S synthesis starts at the beginning of fruit development (around 90 DAF) when perisperm comprises the main tissue of the coffee seed. Methionine Metabolism: Differences in Grain Size Can Be Involved with Earlier Ethylene Synthesis. Methionine synthase (MeSe) (EC 2.1.1.13) catalyzes the last step of methionine synthesis using homocysteine as substrate.28 Our results showed IPR59 accumulated MeSe earlier than IPR59-GDO. Spot 5 (1) was 2.43 times more abundant in IPR59 at 60 DAF than at 90 DAF (Table 1 and Figure 2), whereas in IPR59-GDO it was not present at 60 DAF. Comparing both cultivars shows that MeSe was almost 2 times D


DAYVGDEAQSKR YPIEHGIVSNWDDMEK + oxidation (M) IWHHTFYNELR VAPEEHPVLLTEAPLNPK TTGIVLDSGDGVSHTVPIYEGYALPHAILR ILTERGYMFTTSAER + oxidation (M) GYMFTTSAER + O LAYVALDFEQELETAK NYELPDGQVITIGNER

AGIATYWGQNTDEGSLEDACR SACSSLSSEIK GIQVLLSLGGAPNLSSR RDFLDDLAK RVHLSAAPQCSYPDYYLDAAIR

SACSSLSSEIK RDFLDDLAK VHLSAAPQCSYPDYYLDAAIR LFLGLPASPEAAPSGGFIPHR

GGFLEKVK LVSGLIPDAGTLHAHGSETVK IPLTLIYDDIK STYDDIKPGSIIPYR

PMKGMLTGPVTILNWSFVR + oxidation (M) AGINVIQIDEAALR YGAGIGPGVYDIHSPR

VAPEEHPVLLTEAPLNPK TTGIVLDSGDGVSHTVPIYEGYALPHAILR GYSFTTTAER SYELPDGQVITIGSER

DIVFYSHHANVDR VFPGPLNK FDVFINDEDDNPNDFAK

AGGLGDLKYPLISDITR YPLISDITR

2 (1)

3 (5)

4 (1)

5 (1)

6 (5)

7 (5)

8 (5)

peptidec

1 (5)

spot IDb

E

171

124

124

120

127

123

229

290

scored protein name

access-EST (NCBI)f

29

15

38

24

28

26

33

2-cysteine peroxiredoxin

polyphenol oxidase

actin 7

methionine synthase 2

late embryonegesis abundant protein 2

chitinase-like xylanase inhibitor protein


GT667766

GT664927

GW429264

GW472309

GT714152

GW447671

GW447448

Proteins for Which Accumulation Was Induced in Gels from IPR59 60 DAF 55 actin 7 GT714384

sequence coveragee (%)

15/4.90

67/6.35

42/5.50

18/5.29

29/4.90

55/5.30

19/4.99

36/5.53

MW (kDa)/pI observedg

24.9/5.16

27.7/7.94

21.5/4.81

22.5/5.22

21/4.79

26/5.48

27/5.32

27.2/5.39

MW (kDa)/pI expectedh

Table 1. Proteins Identified in Coffee Perisperm from IPR59 and IPR59-GDO Cultivars at 60, 90, and 180 DAF by MALDI-TOF/TOF-MS/MSa

presence

1.70

1.52

2.43

1.50

2.17

1.75

1.50

% vol changei

0.04375

0.0073

0.00993

0.00718

0.00131

0.00994

0.0025

Student’s t test

Journal of Agricultural and Food Chemistry Article


DILTVNGLFPGPTLHLR VIITEEIGTLWWHAHSDWSR ATVHGAIIIYPK YLSGEAQYVDWSK LNGGLGTTMGCTGPKSVIEVR + oxidation (M) SNIEIHTFNQSQYPR RFHATSI YWKGCMVSS + oxidation (M) LNAQEPSFR LPHYSNVPK QRFPDR KFFLAGNPQQGGGK KFFLAGNPQQGGGKEGHQGQQQQHR EGHQGQQQQHR FFLAGNPQQGGGK

LSENIGLPQEADVFNPR ITTVNSQKIPILSSLQLSAER IPILSSLQLSAER VFDDEVK QGQLIIVPQYFAVIK KAGNQGFEYVAFK AGNQGFEYVAFK TNDNAMINPLVGR + oxidation (M) AIPEEVLR SSFQISSEEAEELKYGR YGRQEALLLSEQSQQGK QEALLLSEQSQQGK

RLTYDEIQSK

10 (6)

13 (3)

14 (3)

12 (1)

11 (3)

AGINVIQIDEAALR KAEHAFYLDWAVHSFR YGAGIGPGVYDIHSPR IPSTEEIADR

SISAAYNVLIPDQGIALR GLFIIDKEGVIQHATINNLAIGR EGVIQHATINNLAIGR SVDETLR

peptidec

9 (6)

spot IDb

Table 1. continued protein name

access-EST (NCBI)f

Proteins for Which Accumulation Was Induced in Gels from IPR59 60 DAF


F

275

372

174

85

117

11 S

UDP-glucose pyrophosphorylase 2 (UGPase)

laccase 14

GW489458

GT728999

GT707363

56

photosystem II subunit O-2

GT697882

Proteins for Which Accumulation Was Induced in Gels from IPR59 180 DAF 51 11 S GW468619

20

28

23

Proteins for Which Accumulation Was Induced in Gels from IPR59 90 DAF 193 29 methionine synthase GT664389

scored

20/5.50

77/6.30

27/6.09

61/6.23

110/6.50

96/6.81


26.7/5.86

27.4/7.92

26.9/6.51

25.8/8.23

24/6.46

20.2/6.71


presence

8.50

presence

2.98

1.88

1.56

% vol changei

0.001

0.00144

0.00468

0.00273

Student’s t test



IILELSAR TYVHER VYDEFVEK SVGDPFRQEIEQGPQVDSEQFEK EEIFGPVQSILKFK FKDLDEVIR DLDEVIR YGLAAGVFTQNLDTANR

FSVAFWHTFR IRPLWGTAQLFLHPR KAIEVTHYLGGENYVFWGGR AIEVTHYLGGENYVFWGGR EGYQTLVNTDMER + oxidation (M) HQYDWDAATSANFLR

LNAQEPSFR FPSEAGLTEFWDSNNPEFGCAGVEFER LPHYSNVPK QRFPDR KFFLAGNPQQGGGK KFFLAGNPQQGGGKEGHQGQQQQHR FFLAGNPQQGGGK FFLAGNPQQGGGKEGHQGQQQQHR EGHQGQQQQHR

VFDQAKRK

16 (7)

17 (3)

18 (5)

LTYDEIQSK GTGTANQCPTIDGGVDK GTGTANQCPTIDGGVDKFAFKPGK KLCLEPTSFTVK NSPPEFQK FVEKDGIDYAAVTVQLPGGER DGIDYAAVTVQLPGGER VPFLFTIK QLVASGKPESFSGEFLVPSYR GSSFLDPK GRGGSTGYDSAVALPAGGR GGSTGYDSAVALPAGGRGDEEELVK

peptidec

15 (7)

spot IDb

Table 1. continued

G

protein name

access-EST (NCBI)f

35

29

31

11 S

xylose isomerase family protein

aldehyde dehydrogenase 2B7

GW489432

GT733807

GT706082

Proteins for Which Accumulation Was Induced in Gels from IPR59 180 DAF


Proteins for Which Accumulation Was Induced in Gels from IPR59-GDO 60 DAF 125 29 ascorbate peroxidase 1 GW441078

377

107

102

scored

23/5.70

25/6.32

46/5.70

50/6.10


26.3/5.39

23.7/5.14

28.9/6.28

28.8/5.25


1.51

2.60

1.62

1.53

% vol changei

0.0104

0.0021

0.00682

0.01135

Student’s t test



VNQIGSVTESIEAVR YNQLLR IEEELGADAIYAGASFR FVEDKHLLFLLLLFR

AKMLCLEPTSFTVKAEGANK + oxidation (M) NAPPEFQK FVEKDGIDYAAVTVQLPGGER DGIDYAAVTVQLPGGER VPFLFTIK QLVASGKPESFSGEFLVPSYR GSSFLDPK GGSTGYDSAVALPAGGRGDEEELVKENIK GDEEELVKENIK SKPETGEVIGVFESVQPSDTDLGSK IQGIWYAQLE

LEDACRR SACSSLSSEIK GIQVLLSLGGAPNLSSR VHLSAAPQCSYPDYYLDAAIR LFLGLPASPEAAPSGGFIPHR

IVRDTCR GIGFTSADVGLDADNCK VLVNIEQQSPDIAQGVHGHLTK TCPWLRPDGK TIFHLNPSGR FVIGGPHGDAGLTGR

LSENIGLPQEADVFNPR IPILSSLQLSAER VFDDEVK

20 (4)

21 (6)

22 (4)

23 (4)

LAWHSAGTFDQGSK TGGPFGTMR + oxidation (M) TEPPVEGR ALLSDPAFRPLVEK YAADEDAFFADYAVAHQK

peptidec

19 (5)

spot IDb

Table 1. continued sequence coveragee (%) protein name

access-EST (NCBI)f

27

enolase 2

GT661493

H

39

33

GW447671

GW481550


S-adenosylmethionine synthase 2

Proteins for Which Accumulation Was Induced in Gels from IPR59-GDO 180 DAF 359 42 11 S GW490912

105

204

Proteins for Which Accumulation Was Induced in Gels from IPR59-GDO 90 DAF 334 62 photosystem II subunit O-2 GT703947

110

Proteins for Which Accumulation Was Induced in Gels from IPR59-GDO 60 DAF

scored

16/6.04

44/6.60

20/5.16

29/5.88

60/5.60


24.3/7.11

22.5/5.98

25.6/5.48

26.1/6.03

21.2/5.61


presence

presence

1.57

2.91

presence

% vol changei

0.01894

0.00014

Student’s t test



Article

Proteins are organized according to cultivar and ripening stage where they were found to be up-regulated. bNumbers within parentheses refer to the performed analyses as follows: 1, IPR59 60 × 90 DAF; 3, IPR59 90 × 180 DAF; 4, IPR59-GDO 90 × 180 DAF; 5, IPR59 × IPR59-GDO 60 DAF; 6, IPR59 × IPR59-GDO 90 DAF; 7, IPR59 × IPR59-GDO 180 DAF. cPeptide sequences identified by MS. d MASCOT score calculated by the MOWSE tool. Significance is presented by the score calculated by −10 × Log(P), where P is the probability that the alignment obtained was a random event (p ≤ 0.05). Scores >72 were considered significant. eIdentified peptides coverage in relation to the complete sequence protein. fNCBI access reference number to the corresponding C. arabica EST. gMolecular weight (MW) and isoelectric point (pI) observed in gel by using Image Master 2D. hMolecular weight (MW) and isoelectric point (pI) predicted by the MASCOT tool. iSome spots are considered as “presence”. In these cases the spots were observed and statistically confirmed to be accumulated only in one maturation stage or genotype according to the analyses performed.

genotypes at 60 and 90 DAF, but faced a drastic reduction in % vol at 180 DAF (Figure S2). Results suggest that these proteins are necessary at the first stages of perisperm development, when intense cell division and expansion processes are in progress.6 Although chitinase genes were most expressed in leaves, a high number of transcripts were also observed in early fruit development of C. canephora.27 Two spots were identified as actin 7 (ACT7; spot 1 (5) and spot 6 (5)); both were 1.5 times more abundant in IPR59 than IPR59-GDO at 60 DAF (Table 1 and Figure 2). Actin microfilaments, together with microtubules, are involved on mitotic apparatus, cytokinesis, and cell elongation. Accumulation of actin ligand proteins is associated with the rising of cell division rates.35 Additionally, in apricot fruits ACT7 was upregulated in the first stage of maturation.20 The differential accumulation of actins detected on our study suggests that IPR59 undergoes perisperm cell division earlier or faster than the large-grain genotype IPR59-GDO, similarly as previously discussed for ethylene-related proteins. Metabolic Processes. Polyphenol oxidase (PPO; EC 1.10.3.1) accumulation was increased in IPR59 at 60 DAF (spot 7 (5)). In vivo PPO has high affinity to CGAs, specifically the isomer 5-CQA.36 PPO oxidizes phenolic compounds to quinones, and it has been associated with ROS detoxification and oxygen regulation.6,36 Interestingly, at 90 DAF, laccase 14 (LAC14; EC 1.10.3.2) was highly abundant in IPR59 compared to IPR59-GDO (spot 10 (6)). LACs are multicopper enzymes that catalyze the oxidation of phenolic compounds, producing semiquinones and water.37 LAC acts similarly to PPO, and its spectrum of activity can involve lignin metabolism, pollutents tolerance, and, specifically for LAC 15, flavonoid polymerization, which collaborates in seed maturation.38 At 60 DAF, 2-cysteine peroxirredoxin (2-CYS-PRX) was present only in IPR59 (spot 8 (5)) when compared to IPR59GDO. One feature of 2-CYS-PRX is the presence of two cysteine residues, which are essential for its activity in H2O2 detoxification.39 2-CYS-PRX can also act as a chaperone avoiding oxidation-induced unfolding, ROS scavenging, or also preventing photoinhibition at the photosystem by allowing the dissipation of energy in excess.40 Late embryogenesis abundant protein 2 (LEA2) was more abundant in IPR59 at 60 DAF than at 90 DAF (spot 4 (1)). There are variable functions suggested for the LEA group such as reduction of electrolytes escaping from cell and ROS scavenging activity in dehydration cells.41 Spot 18 (5) increased its abundance 1.5 times in IPR59GDO relative to IPR59 at 60 DAF and corresponded to ascorbate peroxidase 1 (APX1; EC 1.11.1.11). APX is the major player removing H2O2 from cell compartments, although it was suggested its levels are modulated in a species-specific manner.20 The increase in abundance of those proteins at 60 DAF when compared to 90 DAF may represent an important role in ROS detoxification in a scenario of intense growth and later senescence of the perisperm. At 60 DAF, the perisperm is in intense cell division and is probably subjected to high ROS concentrations. Indeed, PPO and LEA2 were more abundant at this time in IPR59, whereas 2-CYS-PRX was absent in IPR59GDO and only APX1 was increased in abundance in IPR59GDO compared to IPR59. This observation reinforces the suggestion that IPR59 presents a faster and earlier perisperm development than IPR59-GDO.

a

15/5.81 121

24

11 S

GW468125

Proteins for Which Accumulation Was Induced in Gels from IPR59-GDO 180 DAF QGQLIIVPQYFAVIK KAGNEGFEYVAFK AIPEEVLR SSFQISSEEAEELKYGR SSFQISSEEAEELKYGR

LSENIGLPQEADVFNPR IPILSSLQLSAER AGNEGFEYVAFK AIPEEVLR 24 (7)

spot IDb

Table 1. continued

peptidec

scored


protein name

access-EST (NCBI)f


23.1/5.97


1.58

% vol changei

0.00637

Student’s t test


I


Article


Figure 2. Images from identified spots showing the decrease/increase in abundance (% vol) by at least 1.5-fold in (A) IPR59 60 × 90 DAF, (B) IPR59 90 × 180 DAF, (C) IPR59-GDO 90 × 180 DAF, (D) IPR59 × IPR59-GDO 60 DAF, (E) IPR59 × IPR59-GDO 90 DAF, and (F) IPR59 × IPR59-GDO 180 DAF. Means of the normalized protein abundances from each identified spot are shown in the histograms.

Although laccases can be involved in lignification, early perisperm is an aqueus tissue.6,7,9 This could mean that LAC14 could be acting on direct detoxification of ROS produced by apoptosis or for signalizing apoptosis events by using phenolic compounds as substrates, because plant LAC has been reported as highly active in catalyzing oxidation of such compounds.49 As already mentioned, the perisperm involution (with massive cell death) initiates around 90 DAF in IPR59, when this tissue ceases its intense growth stage at the same time that the endosperm starts to fill its space. Thus, the higher accumulation of LAC14 could be involved in degradation of phenolics in a higher intensity than in IPR59-GDO. Aldehyde dehydrogenase 2B7 (ALDH2B7; EC 1.2.1.3) is a mitochondrial isoform from ALDH that catalyzes the conversion of acetaldehyde to glycoaldehyde.42 It has been suggested its accumulation is induced in defense against desiccation43 and at final maturation stages.44 In accordance with these suggestions, we have observed an increase in abundance in IPR59 against IPR59-GDO at 180 DAF. As perisperm at this developmental stage has already ceased its growth and started shifting to a dehydrated condition,6

ALDH2B7 might be collaborating to protect cells against acetaldehydes in IPR59. Energy: Supply for an Intensively Growing Grain. Proteins involved in carbohydrate synthesis and catabolism are frequently reported in fruit proteomics studies, such as in peach,45 citrus,19,46 apricot,20 and coffee.14 In glycolysis, the enzyme enolase catalyzes the removal of water from 2-phosphoglycerate to produce phosphoenolpyruvate.47 In our study, enolase 2 (ENO2; EC 4.2.1.11) was present only in IPR59-GDO at 60 DAF (spot 19 (5); Table 1 and Figure 2). In peach endocarp, ENO was also more abundant in the initial ripening stage.45 Meanwhile, in Citrus sinensis this protein was accumulated constitutively at the beginning of ripening and became more abundant during the transition from intermediary to the final stages of this process.46 An increased accumulation of UDP-glucose pyrophosphorylase 2 (UGPase2; EC 2.7.7.9) was observed in IPR59 at 90 DAF (spot 11 (3)), in accordance with previous data where there was a maximum expression of UGPase gene at 105 DAF in C. arabica cv. Laurina.7 In plants, glycolysis is able to shift between reactions involving ATP and pyrophosphate (PPi) as energy donors in response to environmental factors, tissue type, J


Article


Figure 3. Spots identified as protein 11S by mass spectrometry. (A) 3D views from spots 13 (3) and 17 (3) demonstrate examples of the evolution in accumulation during fruit maturation in IPR59; also spot 24 (7) was more abundant in IPR59-GDO than in IPR59 at 180 DAF. (B) In-gel images of spots 12 (1) and 17 (3) demonstrating the absence of these 11S spots at 60 DAF and presence at 90 and 180 DAF.

Photosystem II subunit O-2 (PSBO2) was present in IPR59 180 DAF (spot 14 (3); Figure 1 and Table 1) when compared to 90 DAF, whereas in IPR59-GDO it was already accumulated at 90 DAF and differentially accumulated in comparison to 180 DAF (spot 20 (4)). This shows IPR59 fruits are still photosynthetically active at 180 DAF and that photosystem subunits could be synthesized in perisperm and translocated to pericarp, as both are connected by vascular tissues.8 At 180 DAF, there was a higher accumulation of xylose isomerase (XI; EC 5.3.1.5) in IPR59 compared to IPR59-GDO (spot 16 (7)). In green coffee beans, the hemicellulosic fraction of cell wall is composed mainly by xylose arabinose, galactose, and mannose.49 In addition, plants have a pentose metabolism pathway that employs XI.50 This enzyme catalyzes the interconversion of aldoses (xylose or glucose) to ketoses (xylulose or fructose) so they can be assimilated in different steps of the pentose phosphate pathway (PPP).47 XI is possibly involved in xylose conversion to xylulose collaborating to PPP and photosynthesis, which is in accordance with the presence of PSBO2 at 180 DAF in IPR59. This study examined the changes of proteins in two C. arabica genotypes differing in seed size during the formation of

Table 2. Evolution in Abundance (% vol) from 90 to 180 DAF of 11S Protein Spotsa genotype IPR59 spot 12 13 17 23 24 a

(1) (3) (3) (4) (7)

IPR59-GDO

abundance at 90 DAF

abundance at 180 DAF

spot

0.66 0.17 0.36

0.46 1.83 1.63 0.97 0.65

12 13 17 23 24

0.15

abundance at 90 DAF

abundance at 180 DAF

0.30 0.13 0.16

0.29 1.55 5.19 2.44 1.35

There were no 11S spots at 60 DAF.

and developmental stage.48 UGPase mediates the glycolytic steps dependent on PPi by catalyzing the reversible reaction between UDP-glucose and glucose-1-phosphate, which can go to glycolysis, cellulose, or sucrose synthesis. It has been reported the perisperm of IPR59 has its peak of sucrose production at 89 DAF,8 which can explain the induced abundance in this seed developmental stage. K


Article


assisted time of flight tandem mass sprectrometry; % vol, percentage of volume; CGA, chlorogenic acid; ROS, reactive oxygen species; SAM, S-adenosyl-methionine-synthase; MeSe, methionine synthase; CLXIP, chitinase-like xylanase inhibitor protein; ACT, actin; APX1, ascorbate peroxidase 1; LEA2, late embryogenesis abundant protein 2; 2-CYS-PRX, 2-cysteine peroxiredoxin; ALDH2B7, aldehyde dehydrogenase 2B7; PPO, polyphenol oxidase; LAC14, laccase 14; ENO2, enolase 2; UGPase2, UDP-glucose phosphorilase; PSBO2, photosystem II subunit O-2; XI, xylose isomerase

the perisperm. We identified 15 proteins and highlighted their importance in perisperm development. We also described how the temporal accumulation of these proteins influences the duration of the perisperm development, which will ultimately directly affect the size of the coffee grain. Earlier accumulation of proteins involved in methionine biosynthesis and in ROS detoxification appeared to serve as a marker for the fast development of the perisperm in the smaller seed-sized genotype IPR59 compared to IPR59-GDO.

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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.5b04376. Table S1, average number of spots detected in perisperm gels from IPR59 and IPR59-GDO; Table S2, amount of spots differentially accumulated in each analysis; supporting file 3, evolution in abundance (% vol) of spots from chitinase-like xylanase inhibitor protein; Table S3, abundances (% vol) for differentially accumulated proteins from the comparisons between IPR59 and IPR59-GDO; Table S4, MS data obtained for identification of the differentially accumulated proteins listed in Table 1 (PDF) Figure S1, in-gel triplicates from the differentially accumulated spots; Figure S2, evolution in abundance (% vol) of spots from chitinase-like xylanase inhibitor protein (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*(L.F.P.P.) Mail: Biotechnology Laboratory − IAPAR, Rodovia Celso Garcia Cid, Km 375, CEP 86047-902, Londrina, PR, Brazil. Phone: +55 43 3376-2399. E-mail: filipe.pereira@ embrapa.br. Present Addresses ‡

(L.C.A.) Center of Molecular Biology and Genetic Engineering, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil. ⊥ (D.S.D.) Department of Botany, Instituto de Biociencias, UNESP, Rio Claro, SP, Brazil. Funding

This project was supported by Financiadora de Estudos e Projetos (FINEP Qualicafé); INCT-Café and Consorcio Pesquisa Café. L.C.A. was supported by Coordenaçaõ de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES). S.G.G thanks Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo (FAPESP Process 2010/15417-6). L.G.E.V and L.F.P.P.P. received a research fellowship from Conselho Nacional de Desenvolvimento Cientıfí co e Tecnológico (CNPq). Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We thank Professor Guilherme T. Valente (UNESP, Brazil) for valuable discussions. ABBREVIATIONS USED DAF, days after flowering; IPR59, IAPAR 59; IPR59-GDO, IAPAR 59 Graudo; MALDI-TOF/TOF-MS/MS, matrixL


Article

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M
