Identification of potential protein markers of noble rot infected grapes

Food Chemistry 179 (2015) 170–174

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Identification of potential protein markers of noble rot infected grapes Marilinda Lorenzini a, Renato Millioni b,c, Cinzia Franchin c,d, Giacomo Zapparoli a, Giorgio Arrigoni c,d,⇑, Barbara Simonato a,⇑ a

Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy Department of Medicine, University of Padova, Via Giustiniani 2, 35128 Padova, Italy c Proteomics Center of Padova University, Via G. Orus 2/B, 35129 Padova, Italy d Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy b

a r t i c l e

i n f o

Article history: Received 31 October 2014 Accepted 20 January 2015 Available online 31 January 2015 Keywords: Botrytis cinerea 2-DE Mass spectrometry Proteomics Grape proteins Amarone wine

a b s t r a c t The evaluation of Botrytis cinerea as noble rot on withered grapes is of great importance to predict the wine sensory/organoleptic properties and to manage the winemaking process of Amarone, a passito dry red wine. This report describes the first proteomic analysis of grapes infected by noble rot under withering conditions to identify possible markers of fungal infection. 2-D gel electrophoresis revealed that protein profiles of infected and not infected grape samples are significantly different in terms of number of spots and relative abundance. Protein identification by MS analysis allowed to identify only in infected berries proteins of B. cinerea that represent potential markers of the presence of the fungus in the withered grapes. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Grape withering, a technological process used to produce the so-called ‘‘passito’’ wines, allows the partial removal of water from the grape berries, with a consequent weight loss up to 40%. During this process, Botrytis cinerea can colonize grape berries. This fungus, under favorable conditions, can develop as noble rot, inducing chemical modifications that lead to a grape composition change (Costantini, Bellincontro, De Santis, Botondi, & Mencarelli, 2006; Guarrera, Campisi, & Asmundo, 2005; Ribéreau-Gayon, Dubourdieu, Donèche, & Lonvaud-Funel, 2006; Tosi et al., 2012). The presence of the fungus affects specifically the aroma of some wines such as Tokaji Aszu, Sauternes, and Trockenbeerenauslese, called ‘‘botrytized wines’’ (Ribéreau-Gayon et al., 2006), that are intentionally produced from grapes infected by B. cinerea. On the other hand, the oxidase activity of the B. cinerea enzymes can affect the organoleptic properties of other wines, inducing a loss of color, alterations of flavor and aroma, not appreciated by many consumers (Li, Guo, & Wang, 2008) ⇑ Corresponding authors at: Department of Biomedical Sciences, Via U. Bassi 58/B 35131 Padova, Italy. Tel.: +39 049 8217449; fax: +39 049 8217468 (G. Arrigoni), Department of Biotechnology, Strada Le Grazie 15, 37134 Verona, Italy. Tel.: +39 045 8027832; fax: +39 045 8027929 (B. Simonato). E-mail addresses: [email protected] (G. Arrigoni), barbara.simonato@ univr.it (B. Simonato). http://dx.doi.org/10.1016/j.foodchem.2015.01.112 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

Amarone is the most important ‘‘passito’’ red wine mainly produced from non-botrytized withered Corvina grapes. The presence of noble rot however, cannot be excluded for sure, since B. cinerea can grow in the thermo-hygrometric conditions occurring in the withering process. Although the noble rot impact on aroma and sensory quality of Amarone has been investigated (Fedrizzi et al., 2011; Tosi et al., 2012), so far only few studies have been carried out on the effects of Botrytis infection on withered grape proteins (Vincenzi et al., 2012) Since the noble rot infection used for the Amarone production cannot be easily assessed (being the mold not visible on red berries), the possibility to detect B. cinerea on grapes before the winemaking could be of great importance for the producers in order to predict the wine sensory/organoleptic properties and to manage the winemaking process. In this study, a proteomic analysis using two-dimensional electrophoresis (2-DE) combined with mass spectrometry (MS) was carried out to compare healthy vs noble rot infected grapes. The aim of this study was to investigate the effects of noble rot infection on the protein pattern of Corvina withered grapes and to identify possible markers of infection. These could be used to assess the presence of noble rot infected berries before the process of winemaking. Such information could be very valuable for the producers to correctly manage the winemaking process.

M. Lorenzini et al. / Food Chemistry 179 (2015) 170–174

2. Materials and methods 2.1. Grape sample preparation and infection by B. cinerea Grapes (cv. Corvina) were collected from vineyards of Valpolicella area (Italy). The infection of grape berries was performed using a suspension of B. cinerea strain B2 as described by Lorenzini, Azzolini, Tosi, and Zapparoli (2013). A total of 100 infected and 100 healthy berries were incubated under withering conditions until they reached 35% of weight loss. 2.2. Chemical and enzymatic analysis of grapes The analyses were carried out on juices obtained from the berries. Organic acids, glycerol, glucose, and fructose were measured enzymatically using a commercial kit (La Roche, Basel, Switzerland). Laccase activity was determined as previously described (Tosi et al., 2012). 2.3. Protein extraction Frozen healthy and botrytized grapes berries were separately powdered by grinding in a mortar with liquid nitrogen. Proteins were extracted from 2 g of the resulting powder as described by Di Carli et al. (2011). The final dried protein pellet was dissolved at room temperature in IPG buffer (7 M urea, 2 M thiourea, 2% triton 100, 65 mM dithiothreitol). The supernatant obtained after centrifugation at 20,000g for 15 min was used for 2-DE analysis. 2.4. Protein quantification The protein content was quantified with the BCA™ Protein Assay Kit (Pierce) and bovine serum albumin (BSA) as a standard. A microplate reader (Bio-Tek Instruments) was used. Quantification was performed in triplicate. 2.5. Two-dimensional electrophoresis (2-DE) IPG strips (11 cm, 3–10 linear pH gradient, Bio-Rad) were passively rehydrated with 100 lg of protein in 200 lL of IPG buffer, for 10 h. IEF was carried out at 20 °C using a Protean IEF Cell (Bio-Rad) and the following program: 5 h linear gradient at 500 V, 4 h rapid gradient at 1000, 8000 V rapid gradient until 32,000 V/h was reached. After the first-dimension, the IPG strips were equilibrated in a buffer containing urea 6 M, glycerol 30%, SDS 2%, Tris–HCl 50 mM pH 8.8 and DTT 1%, and then in the same buffer containing iodoacetamide 2.5%, instead of DTT. The second dimension was carried out using 16% polyacrylamide precast Criterion Strain Free Gels (Bio-Rad) and a Criterion Cell system (BioRad). Separation was performed at 30 mA constant current. Gels were stained with Coomassie Brilliant Blue R250 (Sigma, St. Louis, MO). Five technical replicates for each condition (berries infected and not infected with B. cinerea) were performed. 2.6. Gel image analyses The images were recorded using an Epson Expression 1680 Pro scanner (Seiko-EPSON Corp., Japan) with 16 bit dynamic range and 300 dpi resolution. Gel image analysis was performed using the ProteomweaverÒ software (Bio-Rad, Hercules, CA). The quantitative comparison of the spots was carried out by the scanner-generated spot volume that was expressed as a numeric value of optic density after subtraction of background. Spot inten-

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sities were normalized by the software to compare the different gels. Normalization was performed by an algorithm and did not require any internal standard: the software computes an intensity factor for every gel, which makes all the normalization factors as close to one as possible. For every gel match, the ratio between the pair matched spots was calculated. The normalization factor is the median of these ratios. After completion of spot matching in 2-DE gels, the normalized intensity values of individual protein spots were used to compare the protein quantitative levels between the two groups (treated vs untreated).

2.7. Statistical analysis A paired t-test analysis was performed and significance was accepted at p < 0.05. To provide high statistical confidence in the subsequent identification of proteins affected by the treatment, MS analysis was conducted only on spots whose difference in intensity between groups reached both statistical significance and at least 1.5-fold-change. Multivariate analysis was also performed to investigate patterns in protein profiles. Principal component analysis (PCA) was performed using the statistical software Statgraphics (Statpoint Technologies, Inc., USA).

2.8. In-gel digestion and LC–MS/MS analysis Spots were excised from 2D gels and protein digested with sequencing grade-modified trypsin (Promega) as described in (Tibaldi, Arrigoni, Brunati, James, & Pinna, 2006). Samples were then dried under vacuum and suspended in 10 lL of water/0.1% formic acid (FA) for MS analysis. LC–MS/MS analyses were performed as previously described (Tolin, Pasini, Simonato, Mainente, & Arrigoni, 2012) using a 6520 ESI-Q-TOF mass spectrometer (Agilent Technologies) coupled to a nano-HPLC 1200 series through a Chip-Cube interface (Agilent Technologies). A large Capacity Chip was used and 2 lL of samples were injected into the column at a flow rate of 4 lL/min. Peptides were separated in the C18 nano-column (40 mm  75 lm) at a flow rate of 0.3 lL/min using Water/FA 0.1% (eluent A) and ACN/FA 0.1% (eluent B). Chromatographic separation was achieved by a linear gradient of eluent B from 5% to 50% in 15 min. The instrument operated in a data dependent mode: MS/MS spectra of the 3 most intense ions were acquired for each MS scan. Data files were analyzed using Proteome Discoverer Software (version 1.4, Thermo Fisher Scientific) connected to a Mascot Search Engine server version 2.2.4 (Matrix Science, London, UK). Spectra were searched against an in-house database obtained by a Vitis vinifera database (version November 2013, 80431 entries) concatenated with the protein databases of B. cinerea, and a database of the most common contaminant proteins usually found in proteomics experiments. Data were filtered to exclude MS/MS spectra containing less than 5 peaks and with a total ion count lower than 50. Enzyme specificity was set to trypsin with 1 missed cleavage, while precursor and fragment ions tolerances were set to 10 ppm and 0.05 Da, respectively. Carbamidomethylation of cysteine residues was set as fixed modification and oxidation of methionine as variable modification. A False Discovery Rate (FDR) of 5% and 1% was calculated by the software based on the search against the corresponding randomized database. Peptides were classified as high (99%) and medium (95%) confidence, according to the corresponding FDR. Only proteins identified with at least 2 independent, unique peptides were considered as positive hits. Proteins were grouped into protein families according to the principle of maximum parsimony.

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3. Results and discussion 3.1. Grape analysis The B. cinerea infection on grapes was chemically evaluated. Data reported in Table 1 show a glycerol/gluconic acid ratio and a laccase activity that confirm the presence of noble rot on infected berries (Ribéreau-Gayon et al., 2006). The infection protocol, previously described (Lorenzini et al., 2013), proved to be effective for noble rot induction on grapes under withering conditions in small and large scale assays (Tosi et al., 2013). 3.2. 2-D protein analysis Total protein extracts from healthy and infected berries were analyzed by 2-DE in order to evaluate the effect of noble rot infection on grape proteins and overall to highlight the presence of B. cinerea proteins in infected berries. The protein profiles of the two grape samples were significantly different in terms of number of spots and relative abundance (Fig. 1A and B). 2-DE maps revealed approximately fifty spots that could be matched across all gel replicates. Unsupervised PCA was performed to assess whether the two groups would segregate according to the presence or absence of infection. Score plot of the first two PCs was obtained based on 2-DE dataset after standardization. As it is possible to see in Fig. 2, PC1 (33% of total variance) is by far the most relevant for discriminating between maps obtained from healthy (A–E) and infected (F–L) berries. Notably, five spots (n° 3, 9, 10, 16, 23) whose difference in intensity between groups reached both statistical significance (t-test; p < 0.05) and at least 1.5-fold-change were also the top scoring variables on PC1. Moreover, six spots (n° 46, 47, 48, 49, 50, 51) were detected only on noble-rotten berry protein profiles, indicating that these spots are most probably due to the presence of B. cinerea proteins. 3.3. Identification of protein spots Spots from 2-DE gel that showed both a significant difference between infected and healthy samples (p < 0.05) and a fold change of at least 1.5 were analyzed by mass spectrometry. The results reported in Table 2 show the identified proteins of B. cinerea in infected grapes, while all details regarding the identification of all B. cinerea and V. vinifera proteins are reported in Table S1, supplementary material. The most important result of our study is the identification of a number of B. cinerea proteins that were found in the infected berries. These proteins are expressed only in the berries treated with the mold and could therefore represent potential markers of infection. The B. cinerea protein named major protein of Woronin body has been identified in spots 46 and 47. The Woronin body is a peroxisome-derived dense-core vesicle that is specific to several genera of filamentous ascomycete and is required for development

Table 1 Composition of Corvina grape, either healthy or infected by noble rot.

Gluconic acid (g/L) Glycerol (g/L) Glycerol/gluconic acid Glucose and fructose (g/L) L-malic

acid (g/L)

L-lactic

acid (g/L) Laccase activity (U/mL)

*

Significant for p < 0.05.

Healthy

Infected

0.07 ± 0.03 0.03 ± 0.01 0.4 ± 0.1 297.3 ± 9.4 0.93 ± 0.03

0.29 ± 0.05* 4.32 ± 0.22* 14.7 ± 0.2* 305.1 ± 13.1 1.21 ± 0.04*

0.04 ± 0.02

0.05 ± 0.01

0.2 ± 0.1

6.3 ± 0.6*

and function of appressoria during the host colonization (Chua et al., 2004). A hypothetical acid protease from B. cinerea (spot 51) was also detected on infected grapes. Recently, the use of an aspartic acid protease from B. cinerea has been proposed for the removal of haze-forming proteins, especially chitinases, in white winemaking (Van Sluyter et al., 2013). Moreover, previous studies demonstrated that B. cinerea proteases are able to hydrolyze must and wine proteins involved in sparkling wine foam formation (Cilindre, Castro, Clément, Jeandet, & Marchal, 2007; Cilindre et al., 2008; Hong et al., 2011; Marchal, Berthier, Legendre, Jeandet, & Maujean, 1998). Among B. cinerea proteins present in infected berries we could also identify peroxiredoxin-5 (spot 47), belonging to the category of defense proteins against reactive oxygen species (ROS) and associated with defense responses in plant–fungus interactions; malate dehydrogenase (spot 47), reported as virulence factor involved in host-tissue invasion (Gonzalez-Fernandez & Jorrin-Novo, 2012). This enzyme was detected also in Erbaluce withered berries infected by B. cinerea (Vincenzi et al., 2012). Moreover, other identified B. cinerea proteins were: ATP synthase beta chain (spots 48 and 49) and ATP synthase subunit alpha (spot 50), involved in energy metabolism of the fungus; 3-alpha(or 20-beta)-hydroxysteroid dehydrogenase (spot 49), an enzyme belonging to the family of oxidoreductases (Edwards & Orr, 1978); protein yjbQ (spot 50) with thiamine phosphate synthase activity (Morett et al., 2008), and a hypothetical protein similar to ribosomal protein (spot 49). Finally some hypothetical proteins of B. cinerea have been identified in infected berries: a conserved hypothetical protein (spots 47 and 48) and two predicted proteins (spot 49), all with unknown function. The noble rot infection is not easy to identify in withered grapes by visual inspection, especially in red berries. The proteins described above, being present only in the infected grapes, could represent potential specific markers for noble rot infection, so that the availability of specific tests to detect them in the drying fruit rooms and to quantify the level of infection could be very important for the producers in order to manage the process of winemaking. Antibodies against water-soluble fungal antigens, in particular against the whole mycelium of B. cinerea have been obtained by some research groups. Polyclonal (Ricker, Marois, Dlott, Bostock, & Morrison, 1991) and monoclonal antibodies (Meyer & Dewey, 2000) were used in plate-trapped antigen-enzyme-linked immunosorbent assays (PTA-ELISAs) or in Tube immunoassays (Dewey & Meyer, 2004) to detect B. cinerea infection in grape juice. However, the use of antibodies is always associated to the risk of cross-reactivity with proteins derived from other fungi, such as Aspergillus and Penicillium, and could therefore be a rather imprecise way to monitor infection due to B. cinerea. In fact, the weak growth of mycelium that occurs under the berry skin during the dehydration process, as well as the presence of other fungal pathogens could prevent a reliable detection of this type of B. cinerea infection. Moreover, ELISA tests may suffer of a lack of sensitivity due to the interference with other compounds that can be present at high concentrations in the matrix, overall when the test is performed on red wines or red grapes (Weber, Steinhart, & Paschke, 2007). The adsorption of the proteins on the solid matrices could also alter their structure, compromising the sensitivity of the assay (Kaul et al., 2007). In the present study, we identified several proteins of noble rot that are highly expressed in infected grape and that could therefore represent good specific markers of infection. Their detection and quantification in the berries during withering could be a valuable tool for the producers to correctly manage the winemaking process. However, in order to fully exploit the potential of the present study, the optimization of a mass spectrometry-based targeted

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Fig. 1. 2-DE analysis of healthy (A) and infected (B) grapes. Numbers are referred to the spots subjected to MS analysis. Spots numbered in white (n° 3, 9, 10, 16, 23) show a fold change of at least 1.5 between groups and reached statistical significance (t-test; p < 0.05). Spots numbered in black (n° 46, 47, 48, 49, 50, 51), were detected only in noble-rotten infected berry protein profiles.

Fig. 2. Principal component analysis (PCA) obtained from the 2-DE profiles of healthy (A–E) and infected berry grapes (F–L).

quantification assay, based on the application of a Selected Reaction Monitoring (SRM) approach would be advantageous. Indeed, the use of MS-based approaches that do not suffer of the typical limitations of antibodies-based approaches, would allow for a more sensitive and specific detection and quantification of target proteins compared to an immunoassay-based quantification (Tolin, Pasini, Simonato, et al., 2012; Tolin, Pasini, Curioni, et al., 2012). As could be expected, our data show also that B. cinerea infection induces the alteration of some V. vinifera proteins. Several V. vinifera proteins showing an altered expression level upon infection were identified in this study (all reported in Table S1). However the limited resolution of the 2D-gels used for our investigation often resulted in the co-migration of several proteins within a single spot (as evidenced in Table S1), therefore a

thorough quantitative analysis of the alterations of V. vinifera proteins induced by B. cinerea infection was not possible. However, it is interesting to highlight here that several proteins identified in the spots showing a significant alteration belong to the category of plants defense mechanisms. In particular three isoforms of class IV endochitinase and a 1,3-b-glucanase were observed in infected grapes. Moreover triosephosphate isomerase, enolase, two different types of proteasome subunit alpha, nucleoside diphosphate kinase, phosphoglycerate kinase, and a putative thaumatin-like protein have also been observed. 4. Conclusions To the best of our knowledge, this is the first proteomic investigation of B. cinerea protein expression in withered grapes infected

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Table 2 Proteins of B. cinerea identified in infected grapes by mass spectrometry. Spot number, database accession code, protein description, Mascot score, number of identified peptides, and sequence coverage are reported. Spot no

Accession number

Matched protein

Score

# peptides

% coverage

46

BC1G_12729

Woronin body major protein

115.52

3

16.27

47

BC1T_05133 BC1T_12729 BC1T_07409 BC1T_05503

Peroxiredoxin-5, mitochondrial precursor Woronin body major protein Malate dehydrogenase, mitochondrial precursor Conserved hypothetical protein (126 aa)

83.54 72.27 54.26 41.10

2 2 2 2

17.31 21.08 4.99 8.80

48

114,575 BC1T_05503

ATP synthase b chain, mitochondrial precursor Conserved hypothetical protein (126 aa)

73.25 46.02

2 2

4.50 29.60

49

BC1T_10819 BC1T_05004 BC1T_13687 114,575 BC1T_08322

Predicted protein 3-a-(or 20-b)-Hydroxysteroid dehydrogenase Hypothetical protein similar to ribosomal protein ATP synthase b chain, mitochondrial precursor Predicted protein

885.10 100.04 93.43 86.79 46.31

6 3 3 2 2

33.00 7.00 6.91 4.89 8.39

50

BC1T_10900 BC1T_07780

Protein yjbQ ATP synthase subunit a, mitochondrial precursor

89.37 55.42

2 2

13.19 4.10

51

BC1T_14153

Hypothetical protein similar to acid protease

321.73

2

7.78

by noble rot. The most straightforward result of this study is the detection and identification of several proteins present only in infected grapes. These proteins may represent potential markers of the presence of fungus in the grapes. Since noble rot can influence the physico-chemical characteristics and the stability of wine, the development of an assay to detect these specific markers of noble rot infection in drying fruit rooms could be of great interest for winemakers. The development and application of MS-based targeted quantitative assays based on the proteins identified in this study could represent a promising approach for the sensitive and selective detection and quantification of noble rot in infected grapes. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015. 01.112. References Chua, N. H., Soundararajan, S., Jedd, G., Li, X., Ramos-pamplon, M., & Naqvi, N. I. (2004). Woronin body function in Magnaporthe grisea is essential for efficient pathogenesis and for survival during nitrogen starvation stress. The Plant Cell, 16, 1564–1574. Cilindre, C., Castro, A. J., Clément, C., Jeandet, P., & Marchal, R. (2007). Influence of Botrytis cinerea infection on champagne wine proteins (characterized by twodimensional electrophoresis/immunodetection) and wine foaming properties. Food Chemistry, 103(1), 139–149. Cilindre, C., Jégou, S., Hovasse, A., Schaeffer, C., Castro, A. J., Clément, C., Van Dorsselaer, A., Jeandet, P., & Marchal, R. (2008). Proteomic approach to identify champagne wine proteins as modified by Botrytis cinerea infection. Journal of Proteome Research, 7, 1199–1208. Costantini, V., Bellincontro, A., De Santis, D., Botondi, R., & Mencarelli, F. (2006). Metabolic changes of Malvasia grapes for wine production during postharvest drying. Journal of Agricultural and Food Chemistry, 54(9), 3334–3340. Dewey, F., & Meyer, U. (2004). Rapid, quantitative tube immunoassays for on-site detection of Botrytis, Aspergillus and Penicillium antigens in grape juice. Analytica Chimica Acta, 513(1), 11–19. Di Carli, M., Zamboni, A., Pè, M. E., Pezzotti, M., Lilley, K. S., Benvenuto, E., & Desiderio, A. (2011). Two-dimensional differential in gel electrophoresis (2DDIGE) analysis of grape berry proteome during postharvest withering. Journal of Proteome Research, 10, 429–446. Edwards, C. A., & Orr, J. C. (1978). Comparison of the 3alpha-and 20betahydroxysteroid dehydrogenase activities of the cortisone reductase of Streptomyces hydrogenans. Biochemistry, 17(21), 4370–4376. Fedrizzi, B., Tosi, E., Simonato, B., Finato, F., Cipriani, M., Caramia, G., et al. (2011). Changes in Wine Aroma Composition According to Botrytized Berry Percentage: A Preliminary Study on Amarone Wine. Food Technology and Biotechnology, 49(4), 529–535. Gonzalez-Fernandez, R., & Jorrin-Novo, J. V. (2012). Contribution of proteomics to the study of plant pathogenic fungi. Journal of Proteome Research, 11(1), 3–16.

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