Electrophoresis 2014, 00, 1–8 1 Maura Ferri 1 Marina Franceschetti 1 * Michael J. Naldrett 2 ** Gerhard Saalbach 2 Annalisa Tassoni 1 1 Department of Biological, Geological and Environmental Sciences, University of Bologna, Bologna, Italy 2 Department of Biological Chemistry, Proteomics Facility, John Innes Centre, Norwich Research Park, Norwich, UK Received December 11, 2013 Revised February 6, 2014 Accepted February 24, 2014 Research Article Effects of chitosan on the protein profile of grape cell culture subcellular fractions Grapevine is a large source of healthy polyphenols for human diet, and red table-grapes and wines are the main source of stilbenes. These compounds are important both in the plant defence system and for human health. In the present study, Vitis vinifera cv. Barbera cell cultures were treated with 50 g/mL chitosan and proteomic analyses on soluble and membrane subcellular fractions were performed against suitable controls. Three soluble stilbene synthase protein spots, four stilbene synthase spots in the microsomal fraction and four spots of membrane ATPase subunits were identified, the accumulation of which was modulated in response to chitosan treatment. Present proteomic and immunolocali- sation data seem to provide evidence supporting the hypothesis that a stilbene biosynthetic multi-enzyme complex is associated with the intracellular membrane. In addition, pro- teomic analyses showed a general decrease in the accumulation of proteins belonging to different primary metabolism pathways, both in the soluble and membrane fractions. In particular, energy, sugar and amino acid metabolisms were down-regulated as a conse- quence of chitosan and acetic acid treatments. These metabolic modifications could lead to a consistent change in the profile and amount of metabolites stored in grape berries, with consequent effects on taste, flavour, organoleptic and nutraceutical properties of derived food products. Keywords: Chitosan / In vitro cell cultures / Stilbene synthase / Vitis vinifera (grape) DOI 10.1002/elps.201300624  Additional supporting information may be found in the online version of this article at the publisher’s web-site 1 Introduction Epidemiological studies have strongly suggested that dietary consumption of fruits, vegetables and grains plays a crucial role in the prevention of heart disease, cancer, diabetes and Alzheimer pathologies. This effect seems to be related to the high levels of phytochemicals present in these foods that counteract oxidative stress helping to maintain a balance be- tween oxidants and antioxidants. Grape and its derivatives, amongst them wine, are one of the largest sources of healthy antioxidant polyphenols for human diet [1]. The importance of grapevine as a crop makes Vitis vinifera a good model sys- tem for studies on secondary metabolite production. The pos- sible grape nutraceutical properties have been investigated both by in vivo food analyses and in vitro studies. In fact, Correspondence: Dr. Annalisa Tassoni, Department of Biological, Geological and Environmental Sciences, University of Bologna, via Irnerio 42 40126 Bologna, Italy E-mail: annalisa.tassoni2@unibo.it Fax: +39-051-242576 Abbreviations: PR, pathogenesis-related; RT, room tempera- ture; STS, stilbene synthase cell suspension cultures obtained from V. vinifera cv. Barbera petioles were used to study the production of stilbenes and flavonoids in response to biotic and abiotic elicitors [2–7]. Cell cultures are also suitable to study plant physiology and organ metabolic properties. In a recent paper [8], it was demon- strated that grape cell suspensions could be used to model a range of protein activities found in vivo in organs. Explants ob- tained from mature tissues, de-differentiate and form meris- tematic cells during the process of callus formation, but the total cytosolic proteome still remains significantly different among cultures originating from explants collected at several ripening stages [8]. In the last decade, proteomics-based technology has been applied successfully to study food, grape and wine [9]. Differential-expression proteomics, based on the compari- son of different proteome compositions, was typically used to ∗ Current address: Dr. Marina Franceschetti, Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK ∗∗ Current address: Dr. Michael J. Naldrett, Proteomics & Mass Spectrom- etry Facility, Donald Danforth Plant Science Center, Saint Louis, MO 63132, USA Colour Online: See the article online to view Figs. 2 and 3 in colour. C  2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com