Elsevier

Phytochemistry

Volume 103, July 2014, Pages 38-49
Phytochemistry

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

https://doi.org/10.1016/j.phytochem.2014.03.024Get rights and content

Highlights

  • Anthocyanin content in grape was higher in berries from low nitrogen supplied vines.

  • Nitrogen plant status impacts anthocyanin biosynthesis regulatory and structural genes expression.

  • Coordinated regulation by positive (MYB) and negative (LBD) transcription factors.

Abstract

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 ha−1 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-3′,5′-hydroxylase (F35H), 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−1 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.

Graphical abstract

Nitrogen controls a coordinated regulation of both positive (MYB transcription factors) and negative (LBD proteins) regulators of the flavonoid pathway in grapevine.

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Introduction

The sensory properties of grapevine berries as well as their enological potential depend on the accumulation of both primary (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 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 (chalcone isomerase), F3H (flavanone 3-hydroxylase) and F35H (flavonoid 3′5′-hydroxylase) genes (Deluc et al., 2008, Deluc et al., 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, F3H (flavonoid 3-hydroxylase), F35H, 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.

Section snippets

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 (δ13C) values of grape juice were −26.2 ± 0.17, −25.8 ± 0.25 and −25.8 ± 0.29 ppm for N0, N1 and N2 modalities,

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 ha−1 added). Nevertheless, in a field experiment like the one reported here, anthocyanin biosynthesis is often affected by many environmental factors,

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.

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 ha−1 (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

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.

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