Functional & Integrative Genomics

, Volume 11, Issue 2, pp 341–355 | Cite as

Proteins involved in biotic and abiotic stress responses as the most significant biomarkers in the ripening of Pinot Noir skins

  • Alfredo Simone Negri
  • Elisa Robotti
  • Bhakti Prinsi
  • Luca Espen
  • Emilio MarengoEmail author
Original Paper


We propose an integrated approach, obtained by the combination of multivariate statistics and proteomics, useful to isolate candidate biomarkers for the evaluation of grape ripening. We carried out a comparative 2-DE analysis of grape skins collected in three moments of ripening and analyzed the spot volume dataset through the application of principal component analysis followed by forward stepwise-linear discriminant analysis. This technique allowed to discriminate véraison, quite mature and mature samples, and to sort the matched spots according to their significance. We identified 36 spots showing high discriminating coefficients through liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS). Most of them were involved in biotic and abiotic stress responses indicating these enzymes as good candidate markers of berry ripening. These evidences hint at a likely developmental role of these proteins, in addition to their reported activity in stress events. Restricting the same statistical analysis to the samples belonging to the two last stages, it was indicated that this approach can clearly distinguish these close and similar phases of berry development. Taken all together, these results bear out that the employment of the combination of 2-DE and multivariate statistics is a reliable tool in the identification of new protein markers for describing the ripening phases and to assess the overall quality of the fruit.


Grape ripening Markers Linear discriminant analysis Principal component analysis 

Supplementary material

10142_2010_205_MOESM1_ESM.pdf (26 kb)
Online Resources 1 Biochemical changes occurring during the ripening of Pinot Noir berries. Changes in the physiological parameters were measured during three different ripening stages (RS35, RS37 and RS38) of cultivar Pinot grape berries from véraison until full ripening. The véraison stage was considered as the moment when 50% of the berries started to change color. a Total soluble solids, b titratable acidity, c berry juice pH, d total anthocyanin contents. The data are the means ± SE of three experiments run in triplicate (n = 9; PDF 25 kb)
10142_2010_205_MOESM2_ESM.pdf (4 mb)
Online Resources 2 2-DE maps of three stages through the ripening of Pinot. 2-DE maps of three different ripening stages (RS35, RS37 and RS38) from véraison until full ripeness of cultivar Pinot berry skins. The véraison stage (RS35) was considered as the moment when 50% of the berries started to change color. Proteins (200 μg) were separated by IEF at pH 3–10, followed by 12.5% SDS-PAGE and visualized by cCBB-staining (PDF 3.95 mb)
10142_2010_205_MOESM3_ESM.pdf (82 kb)
Online Resources 3 Identified proteins are indicated in a 2-DE gel representative of RS38, the third ripening stage, with spot name abbreviations corresponding to those reported in Table 2. Proteins (200 μg) were analyzed by IEF at pH 3–10, followed by 12.5% SDS-PAGE and visualized by cCBB staining (PDF 81.7 kb)
10142_2010_205_MOESM4_ESM.pdf (99 kb)
Online Resources 4 List of the identified proteins by LC-ESI-MS/MS and bioinformatics analyses. The table shows the sequence of all the peptides identified by MS/MS fragmentation and the associated statistical information obtained from the search conducted by BioworksBrowser using TurboSEQUEST® software against the subset of V. vinifera protein sequences downloaded from the NCBI (56266 entries). ID spot identifier number, Protein A.N. protein NCBI accession number (version), n pep. number of unique peptides used to identify the protein, cov. (%) sequence coverage%, Sf pro protein SEQUEST Sf score, FASTS E value FASTS expectation (E) values of the entry resulting from the alignment of peptides against NCBI nr database, Hom. Protein A.N. homologous protein accession number (version), Peptide sequence of the identified peptide (the symbol M* indicates oxidized methionine), MH+ molecular mass of the peptide; z: charge state of the peptide, Sf pep SEQUEST Sf score of the peptide, Xcorr SEQUEST cross-correlation value, _Cn delta correlation value; Sp: SEQUEST preliminary score (PDF 99 kb)
10142_2010_205_MOESM5_ESM.pdf (7 kb)
Online Resources 4 PCA on the last two ripening stages: score plot of the second PC. The samples under investigation are coded by a six-characters label: the first two are letters (pn) corresponding to the genotype (Pinot Noir); the third is a number related to the ripening stage (1 = RS35; 2 = RS37; 3 = RS38), followed by a point; the fifth is a number corresponding to the biological replication (1, 2 or 3), while the last is a letter corresponding to the instrumental replication (a or b; PDF 6 kb)


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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Alfredo Simone Negri
    • 1
  • Elisa Robotti
    • 2
  • Bhakti Prinsi
    • 1
  • Luca Espen
    • 1
  • Emilio Marengo
    • 2
    Email author
  1. 1.Dipartimento di Produzione VegetaleUniversità degli Studi di MilanoMilanItaly
  2. 2.Department of Environmental and Life SciencesUniversity of Eastern PiedmontAlessandriaItaly

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