Nitrogen supply affects anthocyanin biosynthetic and regulatory genes in grapevine cv. Cabernet-Sauvignon berries Eric Soubeyrand a,1 , Cyril Basteau b,1 , Ghislaine Hilbert b , Cornelis van Leeuwen a,c , Serge Delrot a , Eric Gomès a,⇑ a Univ. Bordeaux, ISVV, EGFV, UMR 1287, F-33140 Villenave d’Ornon, France b INRA, ISVV, EGFV, UMR 1287, F-33140 Villenave d’Ornon, France c Bordeaux Sciences Agro, ISVV, EGFV, UMR 1287, F-33140 Villenave d’Ornon, France article info Article history: Received 12 November 2013 Received in revised form 11 March 2014 Available online 12 April 2014 Keywords: Flavonoid pathway Grape berry Nitrogen MYB transcription factor Lateral Organ Boundary Domain proteins abstract Accumulation of anthocyanins in grape berries is influenced by environmental factors (such as temper- ature and light) and supply of nutrients, i.e., fluxes of carbon and nitrogen feeding the berry cells. It is established that low nitrogen supply stimulates anthocyanin production in berry skin cells of red varie- ties. The present works aims to gain a better understanding of the molecular mechanisms involved in the response of anthocyanin accumulation to nitrogen supply in berries from field grown-plants. To this end, we developed an integrated approach combining monitoring of plant nitrogen status, metabolite measurements and transcript analysis. Grapevines (cv. Cabernet-Sauvignon) were cultivated in a vineyard with three nitrogen fertilization levels (0, 60 and 120 kg ha À1 of nitrogen applied on the soil). Anthocyanin profiles were analyzed and compared with gene expression levels. Low nitrogen supply caused a significant increase in anthocyanin levels at two ripening stages (26 days post-véraison and maturity). Delphinidin and petunidin derivatives were the most affected compounds. Transcript levels of both structural and regulatory genes involved in anthocyanin synthesis confirmed the stimulation of the phenylpropanoid pathway. Genes encoding phenylalanine ammonia-lyase (PAL), chalcone syn- thase (CHS), flavonoid-3 0 ,5 0 -hydroxylase (F3 0 5 0 H), dihydroflavonol-4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX) exhibited higher transcript levels in berries from plant cultivated without nitrogen compared to the ones cultivated with 120 kg ha À1 nitrogen fertilization. The results indicate that nitrogen controls a coordinated regulation of both positive (MYB transcription factors) and negative (LBD proteins) regulators of the flavonoid pathway in grapevine. Ó 2014 Elsevier Ltd. All rights reserved. Introduction The sensory properties of grapevine berries as well as their eno- logical potential depend on the accumulation of both primary (sugar, organic acids) and secondary metabolites (anthocyanins, tannins, aromas, etc.) (Coombe and McCarthy, 2000). Anthocya- nins, which are key compounds for red wine making, are present in the skin (epicarp) of the red grape berries, and sometimes, in the case of the so-called ‘‘teinturier’’ cultivars also in the pulp (mesocarp). Anthocyanins exert a wide range of biological func- tions in plants such as antioxidant capacity, protection against UV-light and pathogen attack (Chalker-Scott, 1999; Takahama, 2004). They also have been reported to be beneficial to human health by contributing to protection against cardiovascular diseases and cancer (Bitsch et al., 2004; De Pascual-teresa and Sanchez-ballesta, 2008; Wang et al., 1997). Red fruits, red grapes and red wines constitute an important source of anthocyanins for human diet. Hence, in order to optimize anthocyanin content in the berries, it is of importance to fully understand the regulation of the anthocyanin pathway by environmental factors and cultural practices. Anthocyanins are synthesized through the flavonoid http://dx.doi.org/10.1016/j.phytochem.2014.03.024 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved. Abbreviations: AOMT, anthocyanin O-methyltransferase; CHI, chalcone isomer- ase; CHS, chalcone synthase; Cy, cyanidin; DAV, days after veraison; Dp, delphin- idin; DFR, dihydroflavonol-4-reductase; F3H, flavanone 3-hydroxylase; F3 0 5 0 H, flavonoid-3 0 5 0 -hydroxylase; F3 0 H, flavonoid-3 0 -hydroxylase; HPLC, high perfor- mance liquid chromatography; LBD, Lateral Organ Boundary Domain; LDOX, leucoanthocyanidin dioxygenase; Mv, malvidin; N, nitrogen; PAL, phenylalanine ammonia-lyase; Pn, peonidin; Pt, petunidin; UFGT, UDPglucose: flavonoid 3-O- glucosyltransferase; YAN, Yeast Available Nitrogen. ⇑ Corresponding author. Tel.: +33 (0) 557 575910; fax: +33 (0) 557 575903. E-mail addresses: eric.soubeyrand@bordeaux.inra.fr (E. Soubeyrand), cyril. basteau@gmail.com (C. Basteau), hilbert@bordeaux.inra.fr (G. Hilbert), vanleeuwen@ agro-bordeaux.fr (C. van Leeuwen), serge.delrot@bordeaux.inra.fr (S. Delrot), eric.gomes@bordeaux.inra.fr (E. Gomès). 1 Both authors contributed equally to this work. Phytochemistry 103 (2014) 38–49 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem