Nitrogen supply affects anthocyanin biosynthetic and regulatory genes in grapevine cv. Cabernet-Sauvignon berries

Phytochemistry 103 (2014) 38–49

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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 b c

Univ. Bordeaux, ISVV, EGFV, UMR 1287, F-33140 Villenave d’Ornon, France INRA, ISVV, EGFV, UMR 1287, F-33140 Villenave d’Ornon, France Bordeaux Sciences Agro, ISVV, EGFV, UMR 1287, F-33140 Villenave d’Ornon, France

a r t i c l e

i n f o

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

a b s t r a c t Accumulation of anthocyanins in grape berries is influenced by environmental factors (such as temperature 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 varieties. 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 ha1 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 synthase (CHS), flavonoid-30 ,50 -hydroxylase (F30 50 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 ha1 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 enological potential depend on the accumulation of both primary Abbreviations: AOMT, anthocyanin O-methyltransferase; CHI, chalcone isomerase; CHS, chalcone synthase; Cy, cyanidin; DAV, days after veraison; Dp, delphinidin; DFR, dihydroflavonol-4-reductase; F3H, flavanone 3-hydroxylase; F30 50 H, flavonoid-30 50 -hydroxylase; F30 H, flavonoid-30 -hydroxylase; HPLC, high performance 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-Oglucosyltransferase; YAN, Yeast Available Nitrogen. ⇑ Corresponding author. Tel.: +33 (0) 557 575910; fax: +33 (0) 557 575903. E-mail addresses: [email protected] (E. Soubeyrand), cyril. [email protected] (C. Basteau), [email protected] (G. Hilbert), vanleeuwen@ agro-bordeaux.fr (C. van Leeuwen), [email protected] (S. Delrot), [email protected] (E. Gomès). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.phytochem.2014.03.024 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

(sugar, organic acids) and secondary metabolites (anthocyanins, tannins, aromas, etc.) (Coombe and McCarthy, 2000). Anthocyanins, 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 functions 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

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pathway, starting with phenylalanine as a precursor (Fig. 1) (He et al., 2010; Tanaka et al., 2008). The accumulation and the proportion of these compounds in the berry skin depends on genetic (Dai et al., 2011; He et al., 2010; Río Segade et al., 2008), and environmental factors as well as on viticultural practices (Downey et al., 2006). Light, temperature, irrigation and nitrogen (N) supply have been shown to impact grape berry anthocyanin content (Keller, 2010). In several crops and model plants, N deficiency increases the concentration of phenolics (Feyissa et al., 2009; Fritz et al., 2006; Hilbert et al., 2003; Løvdal et al., 2010). In vineyards, the content and availability of N in the soil can be modified by viticultural practices such as fertilization or cover cropping (Gouthu et al., 2012; Lopes et al., 2008; Tesic et al., 2007). Limited nitrogen supply before bloom favors the accumulation in berries of total polyphenols, including anthocyanins (Keller and Hrazdina, 1998); whereas it is decreased by an excessive N supply (Keller et al., 1999). Agronomic and phenotypic descriptions of the impact of nitrogen nutrition on anthocyanin accumulation in grapevine berries have been thoroughly reported (Gouthu et al., 2012; Hilbert et al., 2003; Keller

et al., 1999). However, to our best knowledge, little has been published about the molecular regulation of the anthocyanin biosynthetic pathway by nitrogen, particularly in terms of gene expression regulation. Transcriptional regulators from different protein families control the expression of the structural genes of the flavonoid pathway. In grapevine, as in model plants, the transcription factors R2R3MYB, basic helix-loop-helix (bHLH), and tryptophan–aspartic acid repeat (WDR) proteins appear to control the expression of several structural genes of the anthocyanin biosynthetic pathway (Hichri et al., 2010; Jeong et al., 2006). In grape, several MYB-family proteins controlling various points of the flavonoid pathway have been identified (Fig. 1). MYBA1 and MYBA2 transcription factors activate the last biosynthetic steps of anthocyanin synthesis, a glycosylation reaction catalyzed by the UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) and a methylation catalyzed by the Anthocyanin O-methyltransferase (AOMT) (Bogs et al., 2007; Cutanda-Perez et al., 2009; Kobayashi et al., 2004, 2002; Walker et al., 1999). MYB5a and MYB5b trans-activate the upstream part of the pathway, especially the CHS (chalcone synthase), CHI

Phenylalanine PAL

4-Coumaroyl-CoA CHS1 CHS2 CHS3

MYB5a/b

Naringenin chalcone

CHI

MYB5a/b MYBPA1-2

F3íH Eriodictyol F3H1 F3H2

F3í5íH Naringenin

MYB5a/b

F3H1 F3H2

Pentahydroxy-flavonone MYB5a/b MYBPA1-2

F3í5íH

F3íH Dihydroquercetin

DFR

MYBPA1-2

Dihydromyricetin

Dihydrokaempferol

MYBPA1-2

MYBPA1-2

MYBPA1-2

MYBA1-2

Cyanidin-3-O-glucoside AOMT

MYBA1-2

LDOX

Delphinidins

Cyanidins

UFGT

DFR

Leucodelphinidin

Leucocyanidin

LDOX

F3H1 F3H2

MYB5a/b

MYBA1-2

UFGT

Delphinidin-3-O-glucoside MYBA1-2

AOMT

Petunidin-3-O-glucoside Peonidin-3-O-glucoside Malvidin-3-O-glucoside Fig. 1. Simplified diagram of the anthocyanin biosynthetic pathway and its regulation by MYB genes in grape. PAL, phenylalanine ammonia-lyase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3b-hydroxylase; F30 H, flavonoid 30 -hydroxylase; F30 50 H, flavonoid 30 50 -hydroxylase; DFR, dihydroflavonol-4-reductase; LDOX, leucoanthocyanidin dioxygenase; UFGT, UDP glucose:flavonoid-3-O-glucosyltransferase; and AOMT, anthocyanin O-methyltransferase.

E. Soubeyrand et al. / Phytochemistry 103 (2014) 38–49

(chalcone isomerase), F3H (flavanone 3-hydroxylase) and F30 50 H (flavonoid 30 50 -hydroxylase) genes (Deluc et al., 2008, 2006). Moreover, MYBPA1 and MYBPA2 activate catalytic steps mediated by the dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase/leucoanthocyanidin dioxygenase (ANS/LDOX), leading to both anthocyanins and proanthocyanidins biosynthesis (Bogs et al., 2007; Terrier et al., 2009). In grapevine, transcripts levels of the above mentioned MYB genes are largely influenced by environmental factors, including light (Koyama et al., 2012; Matus et al., 2009), water supply (Deis et al., 2011; Deluc et al., 2009) and temperature (Tarara et al., 2008; Yamane et al., 2006). The molecular regulations of anthocyanin biosynthesis in response to nitrogen supply have been studied essentially in model plants such as Arabidopsis thaliana, tomato or tobacco, mostly in controlled conditions (greenhouse) and/or in simplified in vitro systems (cell suspensions) (Larbat et al., 2012; Scheible et al., 2004; Zhou et al., 2012). In Arabidopsis, low N supply significantly increases the transcript levels of numerous flavonoid biosynthetic genes including PAL (phenylalanine ammonia-lyase), CHS, F3H, F30 H (flavonoid 30 -hydroxylase), F30 5H, ANS and DFR genes. Accordingly, transcript levels of MYB transcription factors PAP1 and PAP2, reported to be positive regulators of anthocyanin biosynthetic genes, are also increased by low nitrogen supply to the plant (Scheible et al., 2004; Lea et al., 2007; Lillo et al., 2008; Zhou et al., 2012). Other transcription factors, LBD37, 38 and 39, members of the class II Lateral Organ Boundary (LOB) Domain (LBD) protein family have recently been demonstrated to be negative regulators of anthocyanins biosynthesis, in A. thaliana (Rubin et al., 2009). Their expression is induced by high nitrogen supply, which represses anthocyanin biosynthesis. In tobacco plants, the levels of transcript of the first gene of the phenylpropanoid pathway, PAL, is induced in N deficient plants (Fritz et al., 2006). In leaves from greenhouse grown tomato, similar results have been reported: an increase of the structural genes in the phenylpropanoid pathways, PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), F3H (flavanone 3-hydroxylase) was observed in response to N depletion. This effect of N depletion were apparently mediated through the overall regulators of the pathway by the MYB transcription factor (Larbat et al., 2012; Løvdal et al., 2010). In contrast, there is very little knowledge about the expression of anthocyanin biosynthetic and regulatory genes in response to N supply in field conditions, particularly on non-model plants such as grapevine. The present work was designed to study the effects of variable N fertilization levels on anthocyanin biosynthesis regulation, under real field conditions (i.e., in a production vineyard, with well established vines). Special emphasis was put on its impact on the expression levels of regulatory and structural genes of the flavonoid pathway. The results indicate that nitrogen supply has a coordinated effect on both positive (MYB proteins) and negative (LBD39) regulators of the structural genes of the pathway. Results Berry weight and berry maturity parameter at harvest Different fertilization treatments did not induce any significant difference in berries weight, pH, sugar/titratable acidity ratio and °Brix (total soluble solids) in berry juice (Table 1), suggesting that the nitrogen supply did not significantly affect the phenology, the growth, the ripening or the technological maturity of the berries. Water stress assessment by carbon isotope discrimination The photosynthetic carbon isotopic composition (d13C) values of grape juice were 26.2 ± 0.17, 25.8 ± 0.25 and 25.8 ± 0.29 ppm

Table 1 Physical and chemical analyses of Cabernet-Sauvignon berries and grape juice sampled from each nitrogen supply treatment, at 49 days after véraison. TA = Titratable Acidity (expressed in gram equivalent H2SO4 L1). No significant difference were found in any of these measurement using Tukey’s HSD post-hoc test (mean ± se; n = 3). Nitrogen treatment

N0

N1

N2

Berry mass (g) Must pH °Brix Sugar/TA ratio

1.25 ± 0.07 3.35 ± 0.06 11.98 ± 0.2 47.1 ± 2.4

1.29 ± 0.09 3.53 ± 0.32 11.9 ± 0.32 47.6 ± 2.28

1.28 ± 0.03 3.4 ± 0.05 11.73 ± 0.23 46.9 ± 4.25

for N0, N1 and N2 modalities, respectively. This corresponds to a moderate water deficit (Gaudillère et al., 2002). No significant difference in vine water status was shown between N0, N1 and N2 modality or between any of the experimental blocks. Plant and berry nitrogen status Berry total nitrogen content significantly differed at two stages of the berry development (26 and 49 days after véraison) between N0 (no nitrogen fertilization) and N2 (+120 kg ha1 nitrogen) treatments (Fig. 2). During the whole experiment, there was no significant difference between N1 (+60 kg ha1) and N2 treatments. In addition, other plant N status indicators also measured (N-tester measures, total nitrogen in leaves blades and petiols, YAN, total amino acids and arginine content) showed similarly results (Table S1). This confirmed that the fertilization treatment N2 led to an increase in nitrogen uptake and assimilation by the vines. Effect of different nitrogen levels on berry anthocyanin composition Total anthocyanin content of berry skin increased rapidly until the second sampling date 26 days after véraison (DAV) for all treatments and then stabilizes (Fig. 3A). Analysis of anthocyanin composition clearly showed significant differences between the low (N0) and high N (N2) treatments, but not between N2 and N1 (intermediate) treatments. Berries sampled from plant submitted to N0 treatment exhibited a significantly higher anthocyanin levels in ripe berries. Total anthocyanin content was significantly higher in low nitrogen treatment (9.56 ± 1.0 mg g1 of dry wt), than N1 and N2 treatments (6.79 ± 1.0 and 6.62 ± 1.5 mg g1 of dry wt, respectively) including the glycosylated, acylated and p-coumaroy-

Berry total nitrogen content (% of dry mass)

40

Days relative to véraison Fig. 2. Total nitrogen content evolution in Cabernet Sauvignon berries during fruit development, under three different nitrogen supply conditions. N0, no added N (control); N1, +60 kg ha1 N; N2, +120 kg ha1 N (mean ± se; n = 3). The zero day indicates véraison. Letters (a, b) indicate significant differences between treatments as calculated by Tukey’s HSD post-hoc test (p < 0.05).

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(E)

Petunidin derivatives (mg g-1 dry wt)

(D)

(F)

Peonidin derivatives (mg g-1 dry wt)

(C)

Days after véraison

Malvidin derivatives (mg g-1 dry wt)

Total anthocyanins (mg g-1 dry wt)

(B)

Delphinidin derivatives (mg g-1 dry wt)

(A)

Cyanidin derivatives (mg g-1 dry wt)

E. Soubeyrand et al. / Phytochemistry 103 (2014) 38–49

Days after véraison

Fig. 3. Effect of nitrogen supply on the amounts of total anthocyanins (A), cyanidin (B), petunidin (C), malvidin (D), peonidin (E) and delphinidin (F) derivatives compounds. Anthocyanin amounts are calculated in malvidin equivalents (mean ± se; n = 3). Letters (a, b) indicate significant differences between treatments as calculated by Tukey’s HSD post-hoc test (p < 0.05).

lated derivatives of all anthocyanins (Fig. 3B–F). In grapes, the anthocyanin glycosides are organized in di-hydroxylated derivatives cyanidin (Cy) and peonidin (Pn), and tri-hydroxylated derivatives delphinidin (Dp), petunidin (Pt) and malvidin (Mv). In Cabernet-Sauvignon cultivar, the tri-hydroxylated derivatives represent more of 80% of total anthocyanins, malvidin being the most abundant with more 60% (Ali et al., 2011). Fourteen anthocyanin molecules were quantified by HPLC-DAD and the amount of each individual anthocyanin was always significantly lower in N2 treatment compared to the N0 control treatment (Table S2). However, no significant effect of the nitrogen treatments was found on the relative percentage of the glycosylated, acylated and p-coumaroylated derivatives of each anthocyanins at harvest (+49 DAV). At harvest stage (+49 DAV, low N supply (N0) increased the levels of all forms of delphinidin derivatives (Dp), cyanidin (Cy), petunidin (Pt), peonidin (Pn) and malvidin derivatives (Mv), although differences were not significant for peonidine (Fig. 3B–F). In low N treatment (N0), the levels of precursors of anthocyanin (Cy and Dp) were significantly higher from +26 and +49 DAV in comparison with medium (N1) and high N supply (N2) treatments (Fig. 3B and F). A higher increase of petunidin derivatives accumulation was also observed (Fig. 3C). Mv and Pn derivatives slightly increased

in low N supply treatment, but differences were not statistically significant (Fig. 3D and E). No significant difference of anthocyanins content appeared in skins among the three treatments N at the véraison stage, and for N2 berries compared to N1 berries throughout all ripening. No significant differences were observed between the three nitrogen supply treatments for the flavonols, which were also analyzed on the same samples (data not shown). Expression of the genes of the flavonoid pathway Anthocyanin profiling indicated that the most significant differences were found between the N0 and N2 treatment, whereas in some cases (i.e., peonidine and malvidine derivatives at 26 DAV) the N1 treatment did not lead to a significant difference in anthocyanin accumulation in berries. Therefore, only the N0 and N2 modalities were chosen to proceed with transcript level analysis. Expression levels of several structural genes of the flavonoid pathway were affected by nitrogen fertilization. Two genes of the early steps of this pathway, VvPAL2 and VvCHS2 were up-regulated in N0 as compared to N2 modality (Fig. 4). VvPAL2 transcript level was 1.9 and 2.1 fold higher in N0 treated berries at 26 DAV and 49

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DAV, respectively; whereas VvCHS2 transcript only increased by 1.2 and 1.5 fold in the same samples. Transcript levels of other isoforms of chalcone synthase (VvCHS1 and VvCHS3), as well as chalcone isomerase (VvCHI), were not affected by nitrogen fertilization levels, at all sampling points (data not shown). From naringenin chalcone, the product of the chalcone isomerase, the flavonoid pathway splits in two branches producing the di-hydroxylated and the tri-hydroxylated flavonoids, respectively. The first step of the dihydroxylated branch is catalyzed by F30 H, and the first one of the tri-hydroxylated branch by F30 50 H (Fig. 1). Only VvF30 50 H transcript level was increased by 1.66 fold by low N supply as compared to high N supply, at 49 DAV (Fig. 4). The VvF3H1 transcript levels, which code for the flavanone 3b-hydroxylase isoform 1 that catalyzes the next downstream step in both the di- and tri-hydroxylated branch of the pathway, increase by 1.3 (26 DAV) and 1.7 (49 DAV) fold in the berries of the N0 modality, compared to N2 modality (Fig. 4). The second isoform expressed in berries, VvF3H2, was unaffected by the nitrogen treatment (data not shown). In the late steps of anthocyanin biosynthesis, transcripts levels of dihydroflavonol-4-reductase (VvDFR), leucoanthocyanidin dioxygenase (VvLDOX) and anthocyanin O-methyltransferase (VvAOMT) were all up-regulated in response to low N supply, particularly at 49 DAV, with induction of 1.5 (VvLDOX), 1.6 (VvDFR) and 2.1 fold (VvAMOT) (Fig. 5). The transcripts of UDP-glucose: flavonoid 3-O-glucosyltransferase (VvUFGT) were more abundant at the end of the berry development, but no significant difference between the N0 and N2 treatments was observed (Fig. 5). Besides structural genes, several flavonoid and anthocyaninspecific regulatory genes were also affected by nitrogen treatment. Low N supply doubled VvMYBA1 (the master positive regulator of the anthocyanin biosynthesis; (Cutanda-Perez et al., 2009) expression levels at 26 and 49 DAV (Fig. 6). VvMYB5a and b, two general

activators of the flavonoid pathway (Deluc et al., 2006, 2008) were not significantly affected by nitrogen supply, with the exception of VvMyb5b at 26 DAV, even though a clear trend of lower transcript levels at harvest in the N2 treatment could be observed (Fig. 6). Two other transcriptional factors, VvMYBPA1 and VvMYBPA2, regulate the last steps of proanthocyanidins biosynthesis (Fig. 1; Bogs et al., 2007; Terrier et al., 2009). Neither MYBPA1 nor MYBPA2 were induced in response to low N supply (N0) (Fig. 6).

VvLBD39: a putative grape transcriptional factor of the LBD protein family positively regulated by nitrogen In A. thaliana, three members (AtLBD37, 38 and 39) of the class II Lateral Organ Boundary Domain protein family were recently reported to be negative regulators of anthocyanins biosynthesis, in response to high nitrogen supply (Rubin et al., 2009; Shi and Xie, 2011). Using AtLBD39 sequence to probe the grape genome database, a BLAST analysis allowed us to identify its closest grapevine homolog, that we named VvLBD39 (Grape Genome ID VIT_07s0129g00330, Refseq ID: XP_002284296.1). At the aminoacid level, VvLBD39 shares 58% of identity and 70% of similarity with AtLBD39, and sequence analysis revealed the presence of the canonical C-block (CX2CX6CX3C), conserved in all LBD proteins, in the N-terminus of the protein (Fig. 7A). In a phylogenic tree constructed with the 6 class II LBD proteins from A. Thaliana, VvLBD39 appear closer to AtLBD39 than any of the 5 other A. thaliana sequences (Fig. 7B). According to qPCR results, VvLBD39 transcript was detected in all three stages of grapes berry development analyzed; and its expression level was increased by high nitrogen supply (N2) at 49 d after véraison, with a 3-fold increase compared to control (N0) plants (Fig. 6).

VvPAL2

VvCHS2

VvF3’H

VvF3’5’H VvF3’5’H

Days after véraison

VvF3H1

Days after véraison

VvDFR Days after véraison

Fig. 4. Changes in transcript levels of genes of VvPAL2, VvCHS2, VvF3H1, VvF30 H and VvF30 50 H in grape berries throughout development. N0; low nitrogen (black bars), N1; high nitrogen (grey bars). Transcript levels were analyzed by real-time PCR and are shown relative to expression of VvEf1c and VvGAPDH in each sample (mean ± se; n = 3). Letters (a, b) indicate significant differences between treatments as calculated by Tukey’s HSD post-hoc test (p < 0.05).

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VvDFR

VvUFGT

Days after véraison

VvLDOX

VvAOMT

Days after véraison

Fig. 5. Effects of nitrogen supply on transcript levels of VvDFR, VvLDOX, VvUFGT and VvAOMT in grape berries. N0; low nitrogen (black bars), N2; high nitrogen (grey bars). Transcript levels were determined by real-time PCR and are shown relative to expression of VvEf1c and VvGAPDH in each sample. Results are expressed as means ± se (n = 3). Letters (a, b) indicate significant differences between treatments as calculated by Tukey’s HSD post-hoc test (p < 0.05).

Discussion In this work, all plant N status indicators that were measured (YAN, N-tester, total amino acids and arginine content, total N content of grape juice, leaf blades and petiol) concluded to a significant difference in vine nitrogen status between the plant from the N0 (no nitrogen fertilization) and the high fertilization modality (N2, 120 kg ha1 added). Nevertheless, in a field experiment like the one reported here, anthocyanin biosynthesis is often affected by many environmental factors, including light, temperature, water supply and soil nitrogen content (Dai et al., 2011; Deis et al., 2011; He et al., 2010; Hilbert et al., 2003; Keller and Hrazdina, 1998; Tarara et al., 2008). Such multifactorial control can easily blur the results of a field study of the impact of a specific single factor, e.g. nitrogen fertilization. Thus, in this work, it was of paramount importance to check that in the experimental plot no other factors than vine N status imported on the anthocyanin biosynthesis. Water stress has been reported to enhance the accumulation of anthocyanins (Castellarin et al., 2007; Kennedy, 2002; Ollé et al., 2011; Tregoat et al., 2002), but in this experimental plot, d13C analysis on grape juice at harvest demonstrated that no differences in vine water status existed between blocks or nitrogen modalities, ruling out that it could account for the observed differences in anthocyanin accumulation. Viticultural practices (hedging, soil work and fruit zone leaf removal) were conducted according to commercial vineyard standard and were not different from one block to another, all blocks being in the same estate plot, with the same row orientation. Therefore, one can be confident that the decrease in anthocyanin content of the berries from the high nitrogen N2 modalities are indeed due to nitrogen fertilization

treatment. High nitrogen vine status can affect anthocyanin content in several ways. It can directly affect anthocyanin metabolism, delaying berry development and ripening and/or increasing the pulp to skin ratio, thereby decreasing berry anthocyanin content by a simple dilution effect (Hilbert et al., 2003; Keller and Hrazdina, 1998). Here, the later effects can be excluded since all indicators of berry ripening (i.e. °Brix index, grape juice pH value, sugar to acidity ratio), as well as the average berry weight at harvest, were not significantly affected. Therefore, the observed effects of nitrogen on anthocyanin berry content are most likely due to changes in anthocyanin biosynthetic pathway regulation. Fritz et al. (2006) identified the nitrate tissue concentration as a signal triggering phenolic accumulation in nitrogen-deficient tobacco. In our study, the nitrogen fertilization (N2 modality) led to an increase in the total nitrogen content of berry juice, (Fig. 2) as well as a significant decrease of total anthocyanin levels in ripe berries. Conversely, total anthocyanin content in berries was significantly higher at harvest in low nitrogen treatment (9.56 ± 1.0 mg per g of dry wt), than in N1 and N2 modalities (6.79 ± 1.0 mg and 6.62 ± 1.5 mg per g of dry wt, respectively); including the glycosylated, acylated and p-coumaroylated derivates of all anthocyanins (Fig. 3A). Cyanidin and delphinidin are the precursors for peonidin and malvidin and petunidin, respectively (He et al., 2010). We observed a 8–10% increase of the proportion of both Cy and Dp in the total anthocyanin berry content in N0 vines, compared N2 ones, at both 26 and 49 DAV. These results are consistent with an increase of the upstream metabolic flux and a possible limitation of enzymatic capacity by UFGT and AOMT steps, in low nitrogen supply conditions, and are in agreement with previous work (Hilbert et al., 2003; Keller and Hrazdina,

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VvMYBA1

VvLBD39

VvMYB5a

VvMYB5b

VvMYBPA1

Days after véraison

VvMYBPA2

Days after véraison

Fig. 6. Effects of nitrogen supply on transcript levels of the transcription factors that induces (MYB genes) or repress (VvLBD39) the flavonoid pathway in grape berries. N0; Low nitrogen (black bars), N1; high nitrogen (grey bars). Transcript levels were determined by real-time PCR and are shown relative to expression of VvEf1c and VvGAPDH in each sample (mean ± se; n = 3). Letters (a, b) indicate significant differences between treatments as calculated by Tukey’s HSD post-hoc test (p < 0.05).

1998). No differences in the ratio between di- and trihydroxylated anthocyanins were observed, at both 26 and 46 DAV. This is in contrast to what has been reported for the impact of other environmental factors such as water stress (Castellarin et al., 2007; Ollé et al., 2011), or temperature and light (Spayd et al., 2002). This prompted us to investigate the transcriptional regulation of the flavonoid pathway by nitrogen, for which, to the best of our best knowledge, little knowledge, if any, is currently available in grapevine. The molecular basis of anthocyanins biosynthetic pathway in grapevine and in other plants is well documented (Ageorges et al., 2006; Gutha et al., 2010; He et al., 2010). The structural genes encoding the enzymes responsible for each catalytic step have been characterized and the understanding to their molecular regulation has greatly progressed over the last 15 years, with the identification of R2R3-MYB, MYC and WD40-type transcription factors (Boss and Davies, 2009; Deluc et al., 2008, 2006; He et al., 2010; Hichri et al., 2010; Winkel-Shirley, 2001). In Arabidopsis and in tomato, the effects of nitrogen supply on the regulation biosynthetic pathway were recently investigated in detail (Larbat et al., 2012; Scheible et al., 2004; Zhou et al., 2012). In grapevine, however, only very scarce limited knowledge of this regulation is available, particularly in field conditions. Here, we have shown that in grape

berries, the evolution of the anthocyanins compounds is tightly correlated with the expression of the biosynthetic genes (Figs. 4 and 5). Seven out of the nine structural involved in flavonoid metabolism genes tested here (VvPAL2, VvCHS2, VvF3H1, VvF30 50 H, VvDFR, VvLDOX, VvAMOT) were up-regulated in low nitrogen supply conditions. Only the F30 H and UFGT were not significantly enhanced in response to N depletion. Possibly, these genes were already expressed to a sufficient level in control (N0) plants and further increase in transcript level by low nitrogen supply was not necessary to sustain higher metabolic fluxes throughout the pathway. For the F30 H gene, this hypothesis was confirmed by the proportion of the two branches di-hydroxylated and trihydroxylated anthocyanins which did not change between the N0 and N2 modalities, supporting the idea that F0 3H was probably not a point of transcriptional regulation in our experiment. This is consistent with results obtained in A. thaliana, where PAL, CHS, F3H, F30 5H and DFR were the genes the most affected by nitrogen depletion, while the F30 H was not (Lillo et al., 2008; Scheible et al., 2004). This, however, clearly differs with the regulation by sunlight or water stress where VvUFGT and VvF0 3H1 are induced by high light (Matus et al., 2009) and water deficit (Castellarin et al., 2007), respectively. Finally, the sequestration of anthocyanin into the vacuoles by GST (Glutathione-S-Transferase) and MATE family

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Fig. 7. Comparison of VvLBD39 protein sequence with the 6 class II LBD proteins from Arabidopsis thaliana. (A) Clustal W-generated alignment of the sequences. Asterisks, colons and periods under the alignments indicate strictly conserved, strongly similar or weakly similar amino-acid residues, respectively. Arrows above alignments indicate invariant residues of the LOB domain of the LBD proteins: a CX2CX6CX3C in the N-terminal part of the protein and an invariant Phe in position 80. (B) Clustal W unrooted phylogenetic tree showing the proximity of VvLBD39 and AtLBD39 protein sequences. NCBI accession numbers: XP_002284296.1 (VvLBD39); NP_201543 (AtLBD37); NP_190563 (AtLBD38); NP_195470 (AtLBD39); NP_176881 (AtLBD40); NP_566175 (AtLBD41); NP_177018 (AtLBD42).

transporters (Gomez et al., 2011), which may constitute a limiting step for anthocyanin accumulation, and were not taken into account in our transcriptomic study, could also be affected by nitrogen levels, even though that remains to be demonstrated. The expression of various transcription factors involved in the regulation of flavonoid biosynthesis was also affected by nitrogen supply. The expression level of VvMYBA1, a positive regulator of

VvUFGT and VvAOMT expression (Cutanda-Perez et al., 2009; Kobayashi et al., 2002), was significantly increased by low (N0) nitrogen supply treatment (Fig. 6). This is in agreement with results obtained in A. thaliana and tomato, where VvMYBA1 homologues, PAP1/PAP2 and ANT1, respectively, are up-regulated by low nitrogen, as well as their main target gene UFGT (Lea et al., 2007; Løvdal et al., 2010; Zhou et al., 2012). In our experiments,

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transcriptional levels of VvUFGT did not show any significant difference in response to nitrogen supply. However, VvAOMT1, another target gene for VvMYBA1 (Fournier-Level et al., 2011) was found to be up-regulated in low nitrogen supply conditions, and could participate in the global increase in anthocyanin accumulation. VvMYB5b, a general activator of the flavonoid pathway (Deluc et al., 2008, , 2006), was also slightly up-regulated by low nitrogen at 26 DAV. So, besides VvMYBA1, VvMYB5b could also participate in the transcriptional activation of the anthocyanin biosynthesis. In the N2 low nitrogen modality, the increase in transcript level of three genes that are trans-activated by VvMYB5b, VvCHS2, VvF30 50 H and VvLDOX (Matus et al., 2008) support this hypothesis. No differences in transcript levels were found for two other positive regulators, VvMYBPA1 and VvMYBPA2, that regulate the last steps of the pathway for the production of proanthocyanidins (Bogs et al., 2007; Terrier et al., 2009). Besides, the expression levels of both genes were extremely low in all three analyzed developmental stages (0, 26 and 49 DAV), in agreement with the fact that proanthocyanidin monomers and oligomers are synthesized early during berry development (Downey et al., 2003). These results may suggest that proanthocyanidin accumulation was not affected, at least at these three stages. However, since anthocyandin and proanthocyanidin biosynthesis share a large number of common enzymatic steps (up to the LDOX), there is still a chance that proanthocyanidin biosynthesis could indeed be affected by low nitrogen, but that remains to be assessed by further experiment, taking into account pre-véraison developmental stages. Finally, a homologue of AtLBD39, a negative regulator of anthocyanin biosynthesis in Arabidopsis (Rubin et al., 2009), was identified in grape genome sequence: VvLBD39. Recently, AtLBD39, and to two other ortholog genes, AtLBD37 and AtLBD38, have emerged as key negative regulators of anthocyanin biosynthesis in response to high nitrogen supply in A. thaliana (Rubin et al., 2009; Zhou et al., 2012). In our experiment, VvLBD39 transcripts levels were strongly decreased by low nitrogen supply, and could participate with VvMYBA1 and VvMYB5b in a coordinated network of transcriptional regulation of the anthocyanin pathway by nitrogen supply. We were unable to detect the expression of other LBD encoding (i.e., homologs of AtLBD37 and AtLBD38) transcripts in the berries. Future work will aim to analyze in more details the potential role of VvLBD39 in berry ripening. Conclusions In conclusion, the present work gives the first report of the molecular regulation of the anthocyanin biosynthesis in grape berry in response to nitrogen fertilization, in field conditions. It reveals a coordinated regulation of some of the structural genes of the pathway, by both positive (MYB transcription factors) and negative (LBD proteins) regulatory genes.

sampling. All viticultural practices (hedging, soil work) were carried out according to standard commercial vineyard practices and were identical for all experimental modalities. Three N fertilization levels were used: 0 (N0), 60 (N1), and 120 (N2) kg ha1 of N. The fertilizer used was NH4NO3, which contains 33% of N. Each N treatment was replicated 3 times in a randomized block design. Each replicate was composed of 40 individual vines. The nitrogen fertilization was applied in two times, a first half-dose of nitrogen was applied at budbreak stage, and the second halfdose was applied just after fruit set. Berry sampling and processing Three replicate of forty berries were sampled on eight different vines for each treatment at 5 different developmental stages: pea-size, 41 days before véraison; bunch closure, 25 days before véraison; véraison; mid-ripe, 26 days after véraison (DAV); and harvest-ripe berries, 49 days after véraison (Coombe, 1995). Berries were selected randomly and weighed, the seeds were removed manually then the berries were frozen at 80 °C. The frozen berries were powdered in a ball grinder MM200 (Retsch, Haan, Germany). Frozen powder extractions were performed in triplicate anthocyanin and amino-acid analysis and gene expression. An aliquot of frozen powder was dried at 60 °C for 48 h for total nitrogen content analysis. 200 berries for each treatment were sampled in parallel to perform Yeast Available Nitrogen (YAN). Analysis of berry nitrogen (N) content and estimation of plant nitrogen status Total nitrogen berries content was measured on a 8 mg aliquot of the dried berry samples from the three N treatments obtained as described above. Dry mass of total nitrogen content was determined according to the Dumas method with an elemental auto-analyzer (Flash EA 1112 series, Thermo Fisher Scientific, Courtaboeuf, France). In parallel, Yeast Available Nitrogen (YAN) was measured in grape juice extracted from fresh berries by Sørensen formol titration method, to validate the treatment (Table S1). This indicator is widely used in viticulture to assess vine nitrogen status during the season (Tregoat et al., 2002). The plant nitrogen status was also estimated by measuring the chlorophyll content of the leaf blade, which is closely related to the N status of the plant, using the ‘‘N-tester’’ (Norsk Hydro, Oslo, Norway). Thirty random measurements were made across each experimental block to give an average value used to indicate the vine N status for each N modality. N-tester has been validated to give an accurate estimation of the nitrogenous status of vineyard grapevines (Spring and Zufferey, 2000; Spring, 1999). In our study, the chlorophyll content was measured at +26 DAV. The leaf blades and the petiole total nitrogen content were measured by the Kjeldahl method (Bergmeyer and Beutler, 1985).

Experimental Amino acids content Plant material and experimental conditions The field experiment was conducted during the 2011 growing season, using 30 years-old Vitis Vinifera L. cv. Cabernet-Sauvignon grafted on 420A rootstock in a production vineyard at the Mission Haut-Brion estate (Pessac-Léognan, France) (44.82° N, 0.61° W). Planting density was equal to 10,000 vines ha1 (1.5 m between rows, 1 m between vines) north–south row orientation. This vineyard was not irrigated and was selected for its natural low nitrogen soil availability (estimated by the determination of the Yeast Available Nitrogen content in grape juice in the preceding vintage: 60 mg L1) soil was checked for being homogeneous by hand auger

The amino acid content in skin and pulp extracts was determined and quantified according to Pereira et al., 2006 with minor modifications. Analysis were performed using a Waters 2695 HPLC system equipped with Waters 474 fluorescence detector (Waters, Milford, MA, USA). Chromatograms corresponding to excitation at 250 nm and emission at 395 nm were recorded. The compounds were identified by their retention time and quantified by their peak area with Chromeleon software, version 6.60 (Dionex Corporation, Sunnyvale, CA, USA) using external standards. Arginine and total amino acids were expressed in nmol per gram (nmol g1) of dry berry weight (dry wt).

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Maturations parameters Two hundred berries were randomly harvested in each experimental block just before commercial harvest, defined by the sugar content and titrable acidity of the grape juice. Berry juice was obtained by hand pressing. Total soluble solids (°Brix) were determined using a hand refractometer with temperature compensation. The sugar and AT were measured respectively by an enzymatic method and a colorimetric method (Ribéreau-Gayon and Dubourdieu, 2012). Carbon isotope analysis Carbon isotope discrimination (d13C) measured on grape-juice at harvest was used to assess and compare water status of the vines, among the different experimental blocks of the vineyard plot (Gaudillère et al., 2002). Anthocyanin profiling Anthocyanin content and composition of grape skins were analyzed according to Acevedo De la Cruz et al. (2011) with minor modifications. Analyses were performed with a Summit HPLC System consisting of P680 pump, ASI-100T™ autosampler and UVD 340U UV–Vis detector operating at 520 nm (Dionex Corporation, Sunnyvale, CA, USA). After injecting 20 ll, separation was achieved on a reverse-phase Ultrasphere ODS column 25 cm  4.6 mm, 5 lm particle size with an Ultrasphere ODS guard column 4.5 cm  4.6 mm obtained from Beckman Instruments Inc. (Fullerton, CA, USA), at ambient temperature. All reagents were of analytical grade. Water was purified (18 M) with an ELGA (Bucks, UK) UHQ water purification system. Acetonitrile (HPLC grade) was obtained from Baker (Mallinckrodt Baker France, Noisy-Le-Sec, France) and formic acid (99%) from Merck (Merck Eurolab, Fontenay-sous-Bois, France). The binary gradient elution (70 min) with a 0.6 ml min1 flow rate started with 75% eluant A (10% formic acid in water (v/v)) and ended with 90% eluant B (10% formic acid and 30% acetonitrile in water (v/v)). The column temperature was 25 °C. Malvidin 3-O-glucoside chloride (Extrasynthese, Genay, France) was used as external standard for quantification on the basis of peak area. Chromeleon software, version 6.60 (Dionex Corporation, Sunnyvale, CA, USA) was used to calculate peak area. Identification and peak assignment of phenolic compounds were based on comparison of their retention times and UV–Vis spectrometric data with that of pure standards. The concentrations of individual anthocyanins were calculated in milligrams of malvidin equivalents per gram of berry dry weight (mg g1 dry wt). RNA extraction and gene expression analysis Total RNA were extracted and purified from 1 g of pooled berries powder according to the procedure described by Reid et al. (2006), quantified using a Nanodrop 2000c spectrophotometer (Thermo Scientific) and checked for integrity on an 1.2% agarose gel. RNAs were treated by DNase I to remove any trace of genomic DNA. For cDNA synthesis, 2 lg of purified, DNase I-treated RNA were reverse transcribed with 0.5 lM of oligodT primer (18 mers) and M-MLV reverse transcriptase (Promega), according to the manufacturer’s instructions. Gene expression analysis Quantitative real-time RT-PCR (qRT-PCR) expression analysis was carried out using a CFX96 Real-Time PCR Detection system (Bio-Rad). Each reaction (10 ll) contained 5 ll of iQ™ SYBR Green Supermix (Bio-Rad), 250 nM of each primer, and 2 ll of 1:10 diluted cDNA. Real-time Q-PCR was performed using the following conditions: 3 min. at 95 °C followed by 40 cycles altering between 10 s at

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95 °C at the annealing (60 °C) temperature for 1 min, and 50 s at 95 °C. Specific oligonucleotide primer pairs used in qRT-PCR experiments are listed in (Table S3). Specific annealing of the oligonucleotides was controlled by dissociation kinetics performed at the end of each PCR run. The efficiency of each primer pair was measured on a pooled of cDNA product serial dilution. All experiments were performed with three biological replicates and two technical replicates. The transcript levels of each gene were normalized with VvEF1c (GenBank accession AF176496) (Deluc et al., 2006) and VvGAPDH (GenBank accession XM_002263109) (Reid et al., 2006) as reference genes. The difference between the cycle threshold (Ct) of the target gene and the geometric average of the reference genes EF1 and GAPDH was used to obtain the normalized expression of the target gene, calculated as 2 –(Ctarget  CtRef. genes). Statistical analysis Statistical analyses were done using the statistical package of the ‘‘R’’ software (R Development Core team, 2010). A one-way analysis of variance (ANOVA) was used. The mean of the 3 biological replicate treatments was used in data analysis. Comparisons of means were performed using HSD.r multiple comparisons function of Tukey’s post-hoc test at p < 0.05. Sequences analysis tools Searches of the grapevine genome were performed using the BLAST and Genome Browser tools at the grapevine genome annotation website of the CRIBI biotechnology center (University of Padua, Italy, http://www.genomes.cribi.unipd.it/grape/). Sequence alignment and phylogenic tree were generated with the ClustalW2 package available at the European Bioinformatic Institute (http:// www.ebi.ac.uk/Tools/msa/clustalw2/), using standard parameters. Acknowledgements E. Soubeyrand was supported by a PhD Grant from the French Ministry of Research and Higher Education. Experimental cost were partly supported by the Mission Haut-Brion Estate, Pessac, France, where the experimental plots were established. 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.phytochem.2014. 03.024. References Acevedo De la Cruz, A., Hilbert, G., Rivière, C., Mengin, V., Ollat, N., Bordenave, L., Decroocq, S., Delaunay, J.-C., Delrot, S., Mérillon, J.-M., Monti, J.-P., Gomès, E., Richard, T., 2011. Anthocyanin identification and composition of wild Vitis spp. accessions by using LC–MS and LC–NMR. Anal. Chim. Acta 732, 145–152. Ageorges, A., Fernandez, L., Vialet, S., Merdinoglu, D., Terrier, N., Romieu, C., 2006. Four specific isogenes of the anthocyanin metabolic pathway are systematically co-expressed with the red colour of grape berries. Plant Sci. 170, 372–383. Ali, M.B., Howard, S., Chen, S., Wang, Y., Yu, O., Kovacs, L.G., 2011. Berry skin development in Norton grape: distinct patterns of transcriptional regulation and flavonoid biosynthesis. BMC Plant Biol. 11, 7. Bergmeyer, H.U., Beutle, H.O., 1985. Method of enzymatic analysis, metabolites, Ammonia. In: H.U.Bergmeyer (Ed.), VCH Weinheim, Deerfield Beach, pp. 454461. Bitsch, R., Netzel, M., Frank, T., Strass, G., Bitsch, I., 2004. Bioavailability and biokinetics of anthocyanins from red grape juice and red wine. J. Biomed. Biotechnol. 5, 293–298. Bogs, J., Jaffé, F.W., Takos, A.M., Walker, A.R., Robinson, S.P., 2007. The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiol. 143, 1347–1361.

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