Effect of different foliar nitrogen applications on the must amino acids and glutathione composition in Cabernet Sauvignon vineyard

Accepted Manuscript Effect of different foliar nitrogen applications on the must amino acids and glutathione composition in Cabernet Sauvignon vineyard Gastón Gutiérrez-Gamboa, Teresa Garde-Cerdán, Ana Gonzalo-Diago, Yerko Moreno-Simunovic, Ana M. Martínez-Gil PII:

S0023-6438(16)30525-4

DOI:

10.1016/j.lwt.2016.08.039

Reference:

YFSTL 5688

To appear in:

LWT - Food Science and Technology

Received Date: 24 February 2016 Revised Date:

8 August 2016

Accepted Date: 18 August 2016

Please cite this article as: Gutiérrez-Gamboa, G., Garde-Cerdán, T., Gonzalo-Diago, A., MorenoSimunovic, Y., Martínez-Gil, A.M., Effect of different foliar nitrogen applications on the must amino acids and glutathione composition in Cabernet Sauvignon vineyard, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2016.08.039. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Effect of different foliar nitrogen applications on the must amino acids and

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glutathione composition in Cabernet Sauvignon vineyard

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Gastón Gutiérrez-Gamboaa, Teresa Garde-Cerdánb, Ana Gonzalo-Diagob, Yerko

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Moreno-Simunovica,*, Ana M. Martínez-Gila,* a

Centro Tecnológico de la Vid y el Vino, Facultad de Ciencias Agrarias, Universidad de Talca, Av. Lircay S/N, Talca, Chile. Tel: +56967676307. *e-mail: [email protected];

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[email protected]

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Instituto de Ciencias de la Vid y del Vino (Gobierno de La Rioja-CSIC-Universidad de La Rioja). Carretera de Burgos Km. 6. Finca La Grajera. 26007 Logroño, Spain

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Abstract

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The effect of different foliar nitrogen applications on the must amino acid and

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glutathione composition in a Cabernet Sauvignon vineyard was studied. Nitrogen

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treatments applied to the grapevines were urea (Ur), urea plus sulphur (Ur+S) and

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arginine (Arg). Also, two commercial nitrogen complexes, Basfoliar Algae (BA) and

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Nutrimyr Thiols (NT), were used. For each treatment, 2 kg N/ha was applied, divided in

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two applications. The commercial nitrogen complexes (NT and BA) improved the

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amino acid content. Ur+S treatment had a better assimilation than Ur, increasing the

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amino acid composition. Arg treatment did not increase the content of any amino acid,

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however increased the easily extractable anthocyanins, total anthocyanins and total

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polyphenol index. Organic sources treatments (Arg, NT and BA) increased glutathione

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concentration. These results can be of oenological interest to improve grape quality

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enhancing must amino acid and glutathione content in high proline accumulator variety.

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Keywords: amino acids, glutathione, foliar application, vineyard, must, Cabernet

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Sauvignon

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1. Introduction Cabernet Sauvignon is one of the most important varieties cultivated in Chile

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accounting for almost 32 % of the area planted with wine grapes. It was introduced to

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the country from Bordeaux along with other varieties around 1851, before the

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phylloxera crisis (Hernandez, 1997). The raise of temperatures and decrease of

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precipitations due to climate change in certain areas of the country, has led to a change

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in the organoleptic profile of Cabernet Sauvignon grapes, so maintaining/improving

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grape quality potential is an important challenge for the Chilean wine production. In this

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context, Acevedo-Opazo, Ortega-Farias, and Moreno (2004) studied the effect of

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different irrigation treatments in Cabernet Sauvignon grape quality, yield and vegetative

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balance. The results showed that replacing 40 and 70 % of vine evapotranspiration is

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possible to reduce water consumption, maintaining yield and grape composition. Also,

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Peña-Neira et al. (2004) studied the phenolic compounds in skins and seeds during

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ripening of low, medium and high vigor vines, showing that vigor alters the

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concentration of all phenolic compounds. However, none of these studies considered the

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potential changes in grape amino acid composition on Cabernet Sauvignon variety.

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Amino acids together with ammonium ion play an essential role as nitrogen

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sources for yeast and lactic acid bacteria, responsible for alcoholic and malolactic

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fermentations, respectively (Garde-Cerdán et al., 2009; Moreno-Arribas & Polo, 2009).

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Thus, must nitrogen concentration affects yeast and bacteria growth and fermentation

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processes, being involved indirectly in the final quality of wine (Arias-Gil, Garde-

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Cerdán, & Ancín-Azpilicueta, 2007; Garde-Cerdán & Ancín-Azpilicueta, 2008; Carrau,

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Medina, Farina, Boido, & Dellacasa, 2010; Garde-Cerdán et al., 2011; Martínez-Gil et

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al., 2012). A good parameter to estimate must fermentability is the concentration of

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amino acids in the must (Löhnertz, Prior, Bleser, & Linsenmeier, 1998). A traditional

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ACCEPTED MANUSCRIPT way to increase N must content has been soil N fertilization as reported by Conradie

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(2001) and Linsenmeir, Loos, and Löhnertz (2008) who studied the effects of N soil

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application on grape amino acid composition. Another strategy is to increase N vine

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status by means of foliar N applications, an interesting technique because of the quick

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and efficient assimilation of applied products by plants (Lasa et al., 2012). Previous

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studies have shown that urea foliar application affects grape and wine composition

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(Ancín-Azpilicueta, Nieto-Rojo, & Gómez-Cordón, 2013; Lasa et al., 2012; Garde-

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Cerdán et al., 2014). In recent years, new commercial nitrogen fertilizers, whose

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composition includes amino acids with nutritional purposes, are emerging on the

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market. Some of the products used to improve the quality of grapes through foliar

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applications are Basfoliar Algae and Nutrimyr Thiols. The first one is a concentrated

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extract of Chilean natural algae (Durvillaea antarctica) that could stimulate the

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synthesis of different compounds on grapes. For its part, Nutrimyr Thiols is a foliar

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fertilizer which is sold as a wine flavor enhancer. So, it is interesting to study the effect

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that foliar commercial products have on the grape quality.

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However, only a single publication has studied the effect of foliar vineyard

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application of amino acids on must amino acid content. In this study, phenylalanine

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foliar applications improved Tempranillo must amino acid content, however the use of

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proline had no effect (Garde-Cerdán et al., 2014). To our knowledge this is the first

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report regarding the use of arginine as a new fertilizer. Of all amino acids that may be

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present in grape juice, proline and arginine are those found in highest amounts (Bell &

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Henschke, 2005). To study the vine´s response to arginine foliar application turns out to

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be interesting because this is one of the most abundant amino acid in grape musts, being

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an important yeast nitrogen source. Another interesting molecule composed by three

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different amino acids, cysteine, glutamic acid and glycine is the glutathione. The

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ACCEPTED MANUSCRIPT function of this tripeptide in winemaking has been recently discussed and reviewed by

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Kritzinger, Bauer, and du Toit (2013). This molecule acts as an antioxidant, preventing

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the appearance of browning pigments in musts and protecting anthocyanins from

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oxidation (Gambuti, Han, Peterson, & Waterhouse, 2015). Glutathione also exerts a

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protective effect in wine volatile compounds (Ugliano et al., 2011) but until now, there

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were no studies in the literature reporting the effects of foliar N application on the

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content of glutathione in grapes. Thus, there is already a lack of information relating to

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the impact of nitrogen application, and more concretely, amino acid application in the

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vineyard, on the must amino acid and glutathione composition.

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For these reasons, the aim of this work was to study the influence of foliar

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application of arginine, urea, urea + sulphur and two commercial nitrogen fertilizers on

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the must amino acid and glutathione composition in a Cabernet Sauvignon vineyard.

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2. Materials and methods

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2.1. Study site

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A field study was conducted on a commercial vineyard in Pencahue, Maule

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Valley, Chile (35°20’S, 71°46’W; 87 m above sea level), during the 2015 growing

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season. A 4 year old Cabernet Sauvignon vineyard, grown in 1103 Paulsen rootstock,

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trained to a vertically shoot positioned system (2.3 x 1.0 m) with a plant density of

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4,347 plants/ha was used. The vineyard was equipped with a drip irrigation system

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using 4 l/h drippers, to assure the plant water needs. The vines were irrigated when the

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leaf water potential (ψl) reached 1.0 to 1.2 MPa. The vineyard plot was homogeneous

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on its vegetative expression and fruit load. The site´s annual average temperature is

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14.5°C with a minimum of -2.5°C (July) and a maximum of 36.7°C (January), and an

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average annual rainfall of 583.8 mm. The vineyard soil is clay loam classified as

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Cunculén series Vertic Haploxeralfs (CIREN, 1997).

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2.2. Grapevines treatments In this study, five treatments were carried out using several nitrogen sources:

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urea (Ur), urea plus sulphur (Ur+S), and arginine (Arg) (Sigma-Aldrich, Darmstadt,

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Germany), and two commercial products, Nutrimyr Thiols (NT) (Italpollina Spa,

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Casalmenini, Italy) and Basfoliar Algae (BA) (Compo Agro, Santiago, Chile).

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Treatments were made in triplicate and were distributed as a complete randomized

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block design. Each replication was carried out on 20 vines, so a total of 60 plants were

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used for each treatment, leaving 18 untreated plants in the same row and two rows

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between replicates to avoid contamination. Treatments consisted of applying 2 kg N/ha

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dose, divided in two moments, the first at the beginning of veraison and the second two

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weeks later. In the Ur+S treatment besides the urea nitrogen, 0.5 kg/ha of sulphur for

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each application was added. 200 ml of each formulation was applied evenly per plant by

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spraying over the full canopy. Additionally, 60 plants, distributed as the same form,

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were kept as an untreated (Control).

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2.3. Commercial products nitrogen content

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Two foliar commercial products were applied to the vines. Basfoliar Algae is a

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concentrated extract of Chilean natural algae (Durvillaea antarctica), supplemented

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with nutrients, minerals and phytohormones (auxins and cytokinins) and Nutrimyr

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Thiols is a foliar fertilizer which is sold as a wine flavor enhancer. The nitrogen content

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of both products is the following:

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ACCEPTED MANUSCRIPT Basfoliar Algae (BA): the total nitrogen content was 6.9 %, of which 0.9 % was

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from the following amino acids: 0.76 (Ala), 1.31 (Gly), 0.51 (Val), 0.29 (Thr), 0.35

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(Ser), 0.73 (Leu), 0.34 (Ile), 0.69 (Pro), 0.06 (Cys), 0.54 (Hyp), 0.23 (Met), 0.69 (Asp),

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0.45 (Phe), 0.93 (Glu), 0.57 (Lys), 0.30 (Tyr), 0.38 (Arg), and 0.09 (His), concentrations

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expressed in g/l.

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Nutrimyr Thiols (NT): the total nitrogen content was 16 %, with 1.2 % of organic nitrogen and 14.8 % of ureic nitrogen.

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2.4. Harvest and must samples

Grapes were harvested at their optimal technological maturity, when the weight

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of 200 berries remained constant, the content of soluble solids was approximately 24-25

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°Brix and the total acidity remained between 5 to 6 g/l of tartaric acid.

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After harvesting, grapes were stored in a cold chamber at 6°C during one day

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before processing and then were destemmed and crushed to obtain the must. Before

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that, a portion of the grapes were separated to measure easily extractable anthocyanins

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and total anthocyanins. The remaining must was protected by adding 50 mg SO2/kg

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grape. The must of each repetition was introduced into 20 l tank, so 18 tanks were filled

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(3 for each treatment). These deposits were stored at 6°C for pre-fermentative

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maceration during two days. Immediately after, the oenological parameters were

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determined in the obtained must (°Brix, density, pH, total acidity, total polyphenol

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index (TPI) and yeast assimilable nitrogen (YAN)). Then, aliquots of each sample were

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frozen at -20ºC in order to subsequent determinations of amino acid composition.

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2.5. Oenological parameters and YAN analysis Grape and must analysis such as °Brix, pH, titratable acidity (g/l tartaric acid),

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were determined according to the methodology established by OIV (2003). Easily

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extractable anthocyanins and total anthocyanins were determined using the

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methodology proposed by Glories and Augustin (1993) and total polyphenol index

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(TPI) was calculated with the methodology developed by Bordeu and Scarpa (1998).

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Yeast assimilable nitrogen (YAN) in must was analyzed with the Sörensen

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methodology (1907). All treatments were performed in triplicate, the results of these

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parameters are shown as the average of three analyses with their statistical difference

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and their standard deviation (n = 3).

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2.6. Analysis of amino acids and glutathione by HPLC

Must amino acid and glutathione analysis was performed by the method

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described by Garde-Cerdán et al. (2014). Free amino acids were analyzed by reversal-

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phase HPLC using an Agilent 1100 Series (Palo Alto, USA), equipped with an ALS

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automatic liquid sampler, a fluorescence detector and a diode array detector. Each

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sample was centrifuged at 4,000 rpm for 10 minutes at 20°C and then, 5 ml of the

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sample was mixed with 100 µl of norvaline, internal standard for quantify all amino

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acids except proline and 100 µl of sarcosine, internal standard for quantify proline. This

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mixture was filtered through 0.45 µm OlimPeak pore filter (Teknokroma, Barcelona,

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Spain) and submitted to an automatic pre column derivatization with o-

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phthaldialdehyde (OPA Reagent, Agilent) and with 9-fluorenylmethylchloroformate

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(FMOC Reagent, Agilent). The injected amount from the derivatized sample was 10 µl

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at 40°C. All separations were performed on a Hypersil ODS (250 x 4.0 mm, I.D. 5 µm)

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column (Agilent).

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ACCEPTED MANUSCRIPT Two eluents, previously filtered through 0.45 µm OlimPeak pore filter

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(Teknokroma, Barcelona, Spain), were used as mobile phases: eluent A: 75 mM sodium

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acetate, 0.018 % triethylamine (pH 6.9) + 0.3 % tetrahydrofuran; eluent B: water,

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methanol and acetonitrile (10:45:45, v/v/v). Identification of compounds was performed

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by comparison of their retention times with their pure reference standards. The pure

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reference compounds and internal standards were obtained from Sigma-Aldrich.

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Ammonium nitrogen was calculated as difference between YAN and amino nitrogen

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without proline. The treatments were carried out in triplicate, so the results for free

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amino acids correspond to average of three analysis (n = 3).

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2.7. Statistical analysis

A statistical analysis on oenological parameters, nitrogen fractions, amino acid

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and glutathione composition was performed using variance analysis (one-way

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ANOVA), by Statgraphics Centurion XVI.I. Differences between samples were

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compared using the Duncan test at 95 % probability level. Principal component analysis

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(PCA) was performed using InfoStat (www.infostat.com.ar).

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3. Results and discussion

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3.1. Oenological parameters and nitrogen fractions

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The oenological parameters and nitrogen fractions for Cabernet Sauvignon

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musts are summarized in Table 1. The different treatments did not affect must °Brix, pH

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or total acidity. Previous studies, where different nitrogen sources such as urea, urea +

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sulphur, phenylalanine and proline were applied, showed similar results for these

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parameters in Sauvignon blanc, Cabernet Sauvignon and Tempranillo musts (Lacroux et

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al., 2008; Lasa et al., 2012; Hannam et al., 2013; Garde-Cerdán et al., 2014).

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Nevertheless, other authors observed differences in Merlot and Tempranillo (Lasa et al.,

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2012; Ancín-Azpilicueta et al., 2013). Significant effects were found in the content of grape phenolic maturity in must

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for the arginine treatment (Arg). Easily extractable anthocyanins, total anthocyanins and

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total polyphenol index (TPI) increased respect to the control when this application was

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done (Table 1). So, this treatment may have a positive influence on the wine quality, as

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the color is one of the principal parameters on the consumer’s choice. Portu, López-

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Alfaro, Gómez-Alonso, López, and Garde-Cerdán (2015) reported that foliar application

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of urea and phenylalanine improves the synthesis of polyphenol compounds.

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Furthermore, together with the arginine, the NT treatment also increased the total

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anthocyanins content, respect to the control and the other treatments. Urea did not affect

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these parameters respect to the control, however, Ur+S decreased easily extractable

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anthocyanins content. This effect was probably due to the action of the sulphur on the

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plant as the urea did not modify this content.

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YAN must concentrations varied from 251 to 282 mg N/l, all of them considered

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“moderate” levels of nitrogen, enough to develop a correct alcoholic fermentation

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(Bely, Sablayrolles, & Barre, 1990; Bell & Henschke, 2005). All treatments increased

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YAN values respect to the control, although the application of Ur+S and BA did not

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significantly affect YAN values. Probably the increments were not very high due to the

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fact that plants had already a moderate N level. Lasa et al. (2012) showed few or no

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significant differences on YAN after the applications when control vines had already a

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sufficient nitrogen level. The must coming from urea treatment showed the higher YAN

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value.

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Ammonium ion is the most preferred nitrogen source by yeast, and it is readily

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assimilated (Jiranek, Langridge, & Henschke, 1995; Bell & Henschke, 2005). The vines

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ACCEPTED MANUSCRIPT treated with urea reached high contents of ammonium nitrogen while BA showed the

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lowest amount of ammonium even lower than control musts (Table 1). Ammonium

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accounted on average approximately for 50 % (BA), 57 % (Ur+S and NT), 63 % (Ctr),

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67 % Arg and 71 % (Ur) of the YAN in grape juices. Similar percentage of ammonium

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for YAN was observed by Huang and Ough (1989) in Cabernet Sauvignon. This variety

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is a high proline accumulator with respect to arginine, so in this variety the ammonium

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resulting in a greater contribution to YAN than amino nitrogen without proline (Bell &

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Henschke, 2005).

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Amino nitrogen is the nitrogen given by the amino acids. All treatments

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increased the concentration of amino nitrogen, although only Ur and BA treatments

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showed significant differences in relation to the control. The increase in urea treatment

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was due to an increase of nitrogen from proline, since the other amino acids had similar

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or lower concentration than control samples. Nevertheless, in the case of BA treatment,

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it increased nitrogen from most of the amino acids. Proline is not metabolized by

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Saccharomyces cerevisiae in suitable nitrogen concentrations on musts, so stuck or

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sluggish fermentations can occur when YAN amount is low (Alexandre & Charpentier,

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1998; Bisson & Butzke, 2000). By this reason, it is important to know the amino

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nitrogen content without proline. This nitrogen content was not affected by the urea and

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arginine treatment since not showed significant difference regarding the control.

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Although, the treatment with urea did not increase the amino nitrogen without proline

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content, the treatment with urea+sulphur did it. Other authors that applied urea and

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urea+sulphur have observed an increase in its wine aromas when sulphur was present,

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although they don´t study the amino acid composition (Lacroux et al., 2008). However,

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in other crops, as wheat, was observed an increase on nitrogen when urea is applied

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with sulphur respect to urea alone (Tea, Genter, Naulet, Lummerzheim & Kleiber,

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2007). Not only Ur+S showed an increase of amino nitrogen without proline, also NT

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and BA did it. These treatments probably could improve the wine quality as the amino

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acids play an important role in the formation of fermentation bouquet products (Bell &

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Henschke, 2005).

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3.2. Amino acid content in grapes

Figure 1 shows control samples and the effect of different foliar nitrogen

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applications such as urea (Ur), urea plus sulphur (Ur+S), arginine (Arg) and nitrogen

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commercial complex as Nutrimyr Thiols (NT) and Basfoliar Algae (BA) on amino acids

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concentration in Cabernet Sauvignon musts, except the proline content that was shown

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in Table 1.

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The amino acids found in higher concentrations were proline, arginine, GABA,

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glutamic acid, and alanine (Figure 1a and Table 1). The proline concentrations varied

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from 2,199 to 2,912 mg/l (Table 1), representing around 82 % to 89 % of total amino

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acids, showing that Cabernet Sauvignon is high proline accumulator. These values are

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normal in Cabernet Sauvignon variety (Huang & Ough, 1989; Huang & Ough, 1991;

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Spayd, & Andersen-Bagge, 1996). However, these concentrations are very high respect

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to other varieties as Pinot Noir, Chardonnay, Sauvignon Blanc, Merlot, Tempranillo,

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Carignan, Grenache and so on (Hannam et al., 2016; Hernández-Orte, Cacho, &

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Ferreira, 2002; Huang & Ough, 1991). Moreover, the different foliar treatments

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increased the concentration of proline although this was only significant in Ur

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treatment. Other authors have also observed an increase in proline when foliar urea is

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applied to grapevines (Hannam et al., 2016; Garde-Cerdán et al., 2014).

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Arginine was the second most abundant amino acid in the samples, being

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arginine an important nitrogen source for yeasts. The arginine concentrations varied

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ACCEPTED MANUSCRIPT from 99 to 156 mg/l (Figure 1a), lower values than those found by Huang and Ough

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(1991), but according to other authors (Spayd & Andersen-Bagge, 1996; Hernández-

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Orte et al., 2002; Bell & Henschke, 2005) in Cabernet Sauvignon samples. The urea and

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arginine treatments did not affect the arginine content, nevertheless, BA treatment

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increased the concentration of arginine in 44 % with regard to the control. Also Ur+S

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and NT increased arginine content but not with significant differences.

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The concentration of γ-aminobutyric acid (GABA) varied from 84 to 101 mg/l.

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GABA is catalyzed from glutamic acid by glutamic acid decarboxylase. Although

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significant differences were observed among the samples in the case of glutamic acid,

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none of the treatments applied modified the content of GABA (Figure 1a).

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Glutamic acid and glutamine are key components of central nitrogen metabolism

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and are preferred nitrogen source for yeasts (Watson, 1976; Bell & Henschke, 2005).

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Moreover, glutamine is the most predominant amino acid in early berry development

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and can act as a precursor of other amino acids as proline and arginine via glutamate

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(Stines et al., 2000). The commercial products, NT and BA, applied to the vineyard,

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increased must glutamine concentration, however, of these two products, only NT

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treatment affected the concentration of glutamic acid (Figure 1a). In relation to the urea

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treatment, it showed a lower content of glutamine and glutamic acid with respect to the

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control. However, the Ur+S treatment increased the glutamic acid content, having not

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effect in glutamine concentration.

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Alanine also constitutes a good source of nitrogen for yeast and its content is

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correlated with volatiles compounds and yeast metabolites such as 2-ketopropionic acid,

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acetaldehyde and ethanol (Bell & Henschke, 2005). Different effects were found in the

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alanine content after treatments applications. BA increased the alanine concentrations,

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showing an increase of 75 %. However, the Ur treatment decreased it a 39 %. Different

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Lasa et al. (2012) did not find differences when applied urea at 10 kg N/ha in Merlot

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and Sauvignon blanc vineyards. On the other hand, Hannam et al. (2016) found

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differences when urea foliar application was carried out on the vineyard, increasing this

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content compared to control and soil applications in Merlot vines.

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The concentration of serine, histidine, threonine, valine and leucine was in all

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the samples around 12 and 25 mg/l. Ur, NT and BA treatments increased the

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concentration of serine regarding the control. Histidine and valine did not show

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significant differences in relation to the control. In the case of threonine, BA treatment

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increased its content respect to the control whereas Ur treatment decreased it. In the

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case of leucine was the NT treatment which presented significant differences in relation

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to the control. The arginine foliar application did not affect the concentration of any of

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the compounds shown in the Figure 1a.

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The amino acids found in lower concentrations are shown in Figure 1b. Among

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them, aspartic acid, tyrosine, cysteine, phenylalanine and isoleucine presented

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concentrations from 5 to 9 mg/l whereas the sum of glycine, methionine, tryptophan,

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ornithine and lysine, accounted for the 2 % of all amino acid content without proline.

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These amino acids are usually in low concentrations in Cabernet Sauvignon as reported

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by Hernández-Orte et al. (2002) and Huang and Ough (1989).

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Aspartic acid is one of the preferred sources of nitrogen by yeast at the start of

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alcoholic fermentation and is quickly consumed (Arias-Gil et al., 2007). Significant

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differences were found in aspartic acid content when Ur, Ur+S, Arg and NT were

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applied to the vineyard. Ur and Arg treatments decreased its contents in musts samples

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between 23 and 25 % respectively, while Ur+S and NT increased the amount

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significantly in 42 and 51 %, respectively compared with control samples. Probably,

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Ur+S and NT wines, would have high contents of higher alcohols due to aspartic acid

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together with phenylalanine, alanine and threonine influence the production of higher

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alcohols in wines. Tyrosine, cysteine, tryptophan and lysine concentrations were not modified due

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to the foliar applications. Furthermore, no significant differences were found due to the

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treatments regarding the control in asparagine, phenylalanine and isoleucine (Figure

334

1b), although there were differences among the treatments. Arginine treatment

335

decreased the content of glycine with respect to the control and the Ur+S treatment

336

enhanced its concentration. In relation to ornithine, NT treatment was the only one that

337

increased its content with respect to the control.

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Significant differences were found in the concentration of methionine due to the

339

treatment with Ur+S and BA (Figure 1b). Sulphur has an important role in the

340

methionine and cysteine formation (Jamal, Moon, & Abdin, 2010). However, in our

341

study, only methionine was affected by the treatment Ur+S.

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It is important to compare the different effect of Ur and Ur+S application in the

343

content of amino acids. Ur+S treatment showed an increment in glutamic acid, aspartic

344

acid, glycine and methionine whereas Ur decreased some of them, as glutamic acid,

345

glutamine, threonine, alanine and aspartic acid. Thus, a different behavior was observed

346

between them. Not results have been found in the literature about urea with sulphur

347

foliar applications in amino acid grape content. However, we found two papers where

348

thiols concentration, after urea and urea+sulphur foliar application, was studied

349

(Lacroux et al., 2008; Geffroy, Dufourcq, López, Serrano, Gracia-Moreno, & Cacho,

350

2012). They observed that when sulphur is present, the thiols concentration is higher,

351

improving the aromatic expression.

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ACCEPTED MANUSCRIPT Nitrogen applications on vines improve the concentration of several amino acids,

353

total amino acids, and ammonium (Bell & Henschke, 2005). It has been found that

354

foliar application in the phenological state of veraison improves the nutrient absorption

355

in relation to soil application (Lacroux et al., 2008). However, soil fertilization cannot

356

be replaced due to technical difficulties to meet all the nutritional needs of the crop

357

through foliar applications. Hannam et al. (2016) showed that the concentrations of

358

several amino acids increased through foliar nitrogen application in relation to soil

359

nitrogen additions. Thereby, Ur+S, NT and BA applications stimulated the nitrogen

360

metabolism and therefore the synthesis of amino acids. This may lead to store nitrogen

361

on sources organs, which could be transported to sink organs, such as young leaves and

362

grapes.

363 364

3.3. Glutathione content in musts

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Figure 2 shows the foliar nitrogen effect on the glutathione amount in musts.

366

Significant differences were found in its concentration when Arg, NT and BA were

367

applied to vineyard. The NT treatment increased considerably the glutathione content in

368

grapes, folding 16 times the control value, followed by Arg and BA, in which the

369

concentration increased more than 10 times. No significant effects were found in Ur and

370

Ur+S applications with respect to the control samples. Glutathione concentration in

371

grapes is closely related to the vine nitrogen status estimated as must assimilable

372

nitrogen (Kritzinger, Bauer, and du Toit, 2013). Lacroux et al. (2008) reported an

373

increase in wine glutathione concentration, applying urea and urea plus sulphur in their

374

vines, although they applied them in major doses than in our study (10 kg N/ha of urea

375

and 5 kg /ha of sulphur). Also, these authors found that the effect was similar with or

376

without sulphur. For its part, Choné et al. (2006) reported an increase of yeast

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15

ACCEPTED MANUSCRIPT 377

assimilable nitrogen and therefore an improvement of glutathione concentration when

378

was applied nitrogen on Sauvignon blanc vines. Probably, NT wines may be less

379

sensitive to oxidation due to its high content of glutathione, which may mean an

380

improvement in the wines quality from these grapes.

382

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3.4. Treatments classification

To classify the different treatments and assess its effects on the amino acid and

384

glutathione concentration in musts, PCA was performed (Figure 3). Principal

385

component 1 (PC 1) explained 60.0 % of the variance and principal component 2 (PC2)

386

explained 18.6 %, representing a 78.6 % of all the variance. PC 1 was strongly

387

correlated with Asp, Asn, Gly, Cys, Met, Phe, Ser, Thr, Arg, Ala, Val and Leu, while

388

PC 2 was strongly correlated only with His. PC 1 allowed to separate the different

389

samples. The Ur+S and BA treatments were correlated with major content of several

390

amino acids such as Met, Ile, Ala, Gln, Thr, Phe, Arg, Cys, Val, Ser and Leu, respect to

391

the other treatments. The NT treatment was correlated with high content of other amino

392

acids such as Asp, Lys, Asn, Glu, Orn, and GSH. The Ctr and Ur treatments were

393

correlated with minor amount of Cys, Met, Phe, Ile, Ser, Gln, Thr, Arg, Ala, Val and

394

Leu. Arg treatment was correlated with minor concentrations of the same amino acids

395

than Ctr and Ur including Gly, His and GABA but with major amount of Trp, Orn and

396

GSH.

398

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4. Conclusions

399

For high proline accumulator varieties as Cabernet Sauvignon, in vineyards with

400

moderate nitrogen conditions, foliar application using two commercial nitrogen

401

complex (NT and BA) improved must amino acid content. In relation to the urea

16

ACCEPTED MANUSCRIPT treatments (Ur and Ur+S), Ur+S treatment had a significant impact in the nitrogen

403

assimilation, so the sulphur applied together with urea improved grape amino acid

404

content respect to the treatment with urea. Commercial nitrogen products and Ur+S

405

were correlated with major concentration of amino acids than Control, Ur and Arg

406

treatments. Arg treatment increased the easily extractable anthocyanins, total

407

anthocyanins and total polyphenol index. Although, Arg treatment did not increase the

408

content of any amino acid, enhaced the synthesis of glutathione. Also, NT and BA

409

increased the concentration of this compound. The present study shows the first results

410

about foliar nitrogen applications in high proline accumulating varieties such as

411

Cabernet Sauvignon, so it could have oenological interest because contributes to gain

412

knowledge about the response of amino acids and glutathione content to different

413

nitrogen foliar applications.

414

Acknowledgements

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We wish to thank the San Pedro’s commercial vineyard (Winery in the Maule

417

Valley, Region del Maule, Pencahue, Chile), for their collaboration in this research. Our

418

thanks for the financial support given by the Universidad de Talca through Vicerrectoría

419

de Innovación y Transferencia Tecnológica and the Magister en Horticultura with the

420

proyect TAL1201 (RCE 860007). Many thanks for the financial support given by

421

Gobierno de La Rioja under the project R-11-14. T. G.-C. also wishes to thank the

422

Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)-

423

Gobierno de La Rioja and European Social Fund for her doctoral contract. A. G.-D.

424

thanks the Gobierno de La Rioja for the research personal formation grant.

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ACCEPTED MANUSCRIPT Figure captions

572

Figure 1. Amino acid concentration (mg/l) in musts from untreated, control (Ctr), and

573

treated vineyards with different foliar nitrogen applications such as urea (Ur), urea with

574

sulphur (Ur+S), arginine (Arg), and two different commercial products, Nutrimyr Thiols

575

(NT) and Basfoliar Algae (BA). a) Glutamic acid (Glu), Serine (Ser), Glutamine (Gln),

576

Histidine (His), Threonine (Thr), Arginine (Arg), Alanine (Ala), γ-aminobutyric acid

577

(GABA), Valine (Va) and Leucine (Leu). b) Aspartic acid (Asp), Asparagine (Asn),

578

Glycine (Gly), Tyrosine (Tyr), Cysteine (Cys), Methionine (Met), Tryptophan (Trp),

579

Phenylalanine (Phe), Isoleucine (Ile), Ornithine (Orn), and Lysine (Lys); All parameters

580

are given with the standard deviation (n = 3). For each amino acid, different letters

581

indicate significant differences (p ≤ 0.05) between treatments.

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582

Figure 2. Glutathione (GSH) concentration (mg/l) in musts from untreated, control

584

(Ctr), and treated vineyards with different foliar nitrogen applications such as urea (Ur),

585

urea with sulphur (Ur+S), arginine (Arg), and two different commercial products,

586

Nutrimyr Thiols (NT) and Basfoliar Algae (BA). All parameters are given with the

587

standard deviation (n = 3). Different letters indicate significant differences (p ≤ 0.05)

588

between treatments.

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590

Figure 3. Principal components analysis (PCA) performed with all amino acids (mg/l)

591

and glutathione (mg/l) in Cabernet Sauvignon samples from untreated, control (Ctr),

592

and treated vineyards with different foliar nitrogen applications such as arginine (Arg),

593

urea (Ur), urea with sulphur (Ur+S) and two different commercial products, Nutrimyr

594

Thiols (NT) and Basfoliar Algae (BA).

595

24

ACCEPTED MANUSCRIPT

Ur

Ur+S

Arg

NT

BA

°Brix

24.77 ± 1.10 a

25.00 ± 0.46 a

24.33 ± 0.86 a

25.10 ± 0.44 a

24.10 ± 0.87 a

23.93 ± 0.81 a

pH

3.97 ± 0.09 a

3.88 ± 0.09 a

3.91 ± 0.08 a

4.01 ± 0.06 a

3.98 ± 0.06 a

3.94 ± 0.03 a

Total acidity (g/l)*

5.68 ± 0.01 a

5.83 ± 0.66 a

5.82 ± 0.32 a

5.87 ± 0.09 a

5.91 ± 0.02 a

5.77 ± 0.09 a

Easily extractable anthocyanins (mg/l)

519.75 ± 115.83 b

539.58 ± 14.88 b

389.08 ± 82.27 a

681.33 ± 11.65 c

535.50 ± 48.72 b

461.42 ± 17.88 ab

Total anthocyanins (mg/l)

934.50 ± 37.12 a

889.58 ± 51.63 a

824.25 ± 39.60 a

1260.00 ± 78.01 b

1162.58 ± 84.22 b

898.92 ± 119.43 a

TPI**

13.93 ± 2.90 ab

15.70 ± 1.90 bc

12.03 ± 1.16 a

17.83 ± 1.30 c

14.93 ± 0.64 abc

13.00 ± 2.63 ab

YAN (mg N/l)

251.33 ± 9.02 a

282.00 ± 5.29 d

260.67 ± 10.07 abc

266.67 ± 6.11 bc

270.67 ± 10.07 cd

253.00 ± 5.29 ab

Ammonium nitrogen (mg N/l)

158.38 ± 10.76 bc

199.71 ± 9.87 d

150.60 ± 10.90 b

178.36 ± 9.55 cd

154.38 ± 15.60 b

126.30 ± 14.64 a

Amino nitrogen (mg N/l)

360.36 ± 41.37 a

436.48 ± 8.58 bc

395.30 ± 42.78 ab

380.97 ± 12.50 ab

390.55 ± 56.03 ab

469.31 ± 31.95 c

Proline (mg/l)

2199.00 ± 291.95 a

2912.67 ± 2.06 b

2345.60 ± 317.37 ab

2406.76 ± 42.44 ab

2255.39 ± 362.75 ab

2817.42 ± 150.54ab

Amino nitrogen without proline (mg N/l)

92.95 ± 5.87 a

82.29 ± 8.33 a

110.07 ± 4.19 b

88.30 ± 7.34 a

116.29 ± 11.91 b

126.70 ± 13.65 b

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Table 1. Oenological parameters, and nitrogen fractions including proline concentration in must from untreated (Control) and treated grapevines with different nitrogen sources as foliar fertilizer: urea (Ur), urea with sulphur (Ur+S), arginine (Arg), and different commercial products Nutrimyr Thiols (NT) and Basfoliar Algae (BA).

≤ 0.05) between treatments. *Expressed as g/l tartaric acid.

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All parameters are given with their standard deviation (n = 3). Different letters in the same row indicate significant differences (p **TPI: total polyphenol index.

25

ACCEPTED MANUSCRIPT

Figure 1

RI PT

a) 180

c

160

SC

bc

140

b

a

mg/l

100

80

60

b

a a

20

b

b a

a

b

d

cd

bc a

ab

0

Ser

Gln

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His

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a

a

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c

bc

d

c

abc

b a

Thr Ctr

a a a

Arg Arg

NT

Ala

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ab

bc a c c

Val

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ab

bc a c bc

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26

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b) 12,00

c

c

a a a a a

b

a

b

8,00

a a

a

a

a

a

a

c abc bc abc ab a

4,00

b ab

c

c

ab

0,00

ab a

a

Tyr

Cys

Ctr

Ur

Ur+S

a

a

c

a

b

Met Arg

a a ab a

Trp NT

Phe

Ile

Orn

b

a a a a a

ab

Lys

BA

EP

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Asn

ab

a a a a a a

ab b a

Asp

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ab ab

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Figure 2

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EP

0

NT

BA

Glutathione

AC C

mg/l

30

28

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Figure 3

29

ACCEPTED MANUSCRIPT Study of foliar N application to Cabernet Sauvignon (Pro accumulator variety) vines Sulphur+urea treatment improved grape N assimilation respect to urea application Commercial N sprays improved must amino acid content Arginine treatment increased the content of polyphenols

AC C

EP

TE D

M AN U

SC

RI PT

Foliar N application with organic sources increased the glutathione content