Effect of two exogenous plant growth regulators on the color and quality parameters of seedless table grape berries

Journal Pre-proof Effect of two exogenous plant growth regulators on the color and quality parameters of seedless table grape berries

Pasquale Crupi, Vittorio Alba, Gianvito Masi, Angelo Raffaele Caputo, Luigi Tarricone PII:

S0963-9969(19)30553-8

DOI:

https://doi.org/10.1016/j.foodres.2019.108667

Reference:

FRIN 108667

To appear in:

Food Research International

Received date:

11 May 2019

Revised date:

4 August 2019

Accepted date:

9 September 2019

Please cite this article as: P. Crupi, V. Alba, G. Masi, et al., Effect of two exogenous plant growth regulators on the color and quality parameters of seedless table grape berries, Food Research International (2018), https://doi.org/10.1016/j.foodres.2019.108667

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© 2018 Published by Elsevier.

Journal Pre-proof EFFECT OF TWO EXOGENOUS PLANT GROWTH REGULATORS ON THE COLOR AND QUALITY PARAMETERS OF SEEDLESS TABLE GRAPE BERRIES

Pasquale Crupi1 *, Vittorio Alba 1 , Gianvito Masi, Angelo Raffaele Caputo, Luigi Tarricone

CREA-VE, Council for Agricultural Research and Economics – Research Centre for Viticulture and Enology. Via

These authors contributed equally to the article

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1

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Casamassima, 148 – 70010 Turi (BA), Italy

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Running title: Harpin proteins versus ABA in enhancing the color of grapes

*corresponding author: CREA-VE - Council for Agricultural Research and Economics – Research Centre for Viticulture and Enology, Via Casamassima 148 - 70010 Turi (BA) – Italy. Phone: +39-080-8915711 Fax: +39-080-4512925;

ORCID: 0000-0001-6457-8619

Journal Pre-proof Abstract Some red-pink table grape varieties, cultivated in warm climates, can fail in achieving the right level of anthocyanins responsible for the intense and uniform red color of berries. Nowadays, this is becoming an important technological issue in the Mediterranean area, which may result in decreasing market acceptance and potential economic value of table grape. Usually, plant growth regulators or phytohormones, such as S-ABA, can overcome this problem because they drive the accumulation of anthocyanins over the ripening season. Harpin proteins (HrP), which enhance the

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plant disease resistance, may be supposed to stimulate the anthocyanins biosynthesis in grape skin if applied close to veraison. Therefore, this research aimed at comparing the effect of HrP and S-ABA

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over the anthocyanin and color improvement of Crimson Seedless table grape grown in Southern

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Italy. For the first time, the exogenous treatment with HrP showed as effective as the less

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sustainable S-ABA one in favoring the anthocyanin accumulation, leading to peonidin-3-Oglucoside, cyanidin-3-O-glucoside, and malvidin-3-O-glucoside values up to 4 folds higher than

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control grapes and giving rise to a greater concentration of the more stable acylated anthocyanins.

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Overall, the color of berries was improved but keeping high the other quality characteristics.

Keywords: anthocyanins; Crimson Seedless; harpin proteins; HPLC-DAD; ripening; S-abscisic acid; veraison.

1. Introduction

Journal Pre-proof Grapes are among the most widespread fruit in the world, with a total production of hundreds million tons per year, partly (~ 30%) consumed as fresh table grape (Medouni-Adrar et al., 2015). In particular, in the last decades, seedless table grapes have taken over the consumer thanks to their suitability for the fresh market due to their quality parameters associated with the absence of seeds. The quality and value of the table grapes are strongly affected by size, texture, and color (intensity and uniformity) of the individual berries, as well as the overall appearance of the cluster (Vox et al., 2012; Perniola, Crupi, Genghi, & Antonacci, 2016). Mechanical parameters of the whole berry

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(flesh texture profile, berry firmness, and berry skin characteristics), as well as pulp texture characteristics (defined as hardness, springiness, cohesiveness, and chewiness) and rachis status are

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the main properties related to freshness and shelf life of table grape from the field to the market, and

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have a high impact on consumer acceptability (Commission Implementing Regulation (EU) No

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543/2011).

Anthocyanins content primarily determines the color of black, red, and pink grape berries; grapes

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with high levels of these compounds in their skin appear darker and more red-colored than grapes

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with low ones, even though a real linear relationship between pigments content and berry color does

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not exist (Carreño, Luis Almela, & Fernández‐López, 1996; Peppi, Fidelibus, Dokoozlian, 2007). Anthocyanin structure contains a benzopyrilium moiety substituted by different groups (i.e., methoxyls and/or hydroxyls). Usually, they are present in the grapes skin as 3-O- glycosides derivatives eventually acylated (by aliphatic or aromatic groups) at the C6-position of the glucose molecule (Guidoni & Hunter, 2012). Aside from having a crucial role for the commercial and organoleptic quality of grapes, nowadays, there is a growing interest in anthocyanins as bioactive compounds, basically because of their human wellbeing impacts, overall influenced by their chemical structures (Carrieri et al., 2013; Phan, Bucknall, & Arcot, 2019). Anthocyanin biosynthesis and accumulation in the skin cells start from veraison (the onset of maturation) until the harvest, and are mainly under genetic control (Gagné, Cluzet, Merillon, & Geny, 2011; Costantini et al., 2015); however, gene expression and activation of the biosynthetic

Journal Pre-proof enzymes are also influenced by climatic conditions and cultural practices, including the use of exogenous plant growth regulators (PGRs) (Lurie et al., 2009; Yamamoto et al., 2015; Crupi et al., 2016; Basile, Alba, Gentilesco, Savino, & Tarricone, 2018). In this context, some varieties (i.e., Crimson Seedless) can fail in achieving the desired level of red skin color at harvesting, especially in very warm climates, maybe due to high summer daily temperature together with the narrow day/night temperature range during ripening (Mori, Goto-Yamamoto, Kitayama, & Hashizume, 2007; Crupi et al., 2015; Koyama et al., 2018). This poor coloration may result in decreasing market

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acceptance and potential economic value of the commodity (Roberto et al., 2012). Commonly, the issue of inadequate grapes color is overcome in the vineyard by using various PGRs

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or plant hormones, which can significantly increase the activity of a wide range of genes involved

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in anthocyanin biosynthesis, such as ethephon (2-chloroethylphosphonic acid), abscisic acid (S-

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ABA), and sucrose (Ferrara et al., 2015; Olivares et al., 2017; Ferrero et al., 2018). Many studies exist in the literature about the influence of ABA in the up-regulation of both upstream (VvPAL,

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VvCHS, and VvCHI) and downstream genes (VvMYBA1 and VvUFGT) involved in the

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phenylpropanoid pathway (Gagné, Cluzet, Merillon, & Geny, 2011; Leng, Yuan, & Guo, 2014;

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Pilati et al, 2017). Azuma et al. (2009) report that these genes normally contain one or more conserved ABA-responsive element and coupling-element in their promoter regions and, due to the conservative and homologous nature of these sequences, Olivares et al. (2017) hypothesize their presence in the promoter regions of the genes of the anthocyanin pathway in Crimson Seedless, too. Anyway, as several authors reported (Peppi, Fidelibus, & Dokoozlian, 2008; Koyama et al., 2018), the use of exogenous ABA on grape show some limitations as a PGR, such as few commercial products availability on the market, short half-life in plants, its rapid conversion to inactive metabolites, sensitivity to light and consequent inactivation by sunlight, as well as its high cost. Alternatively, harpin proteins (HrP), encoded by hpr (hypersensitive response and pathogenicity) genes from Gram-negative plant pathogenic bacteria, may be useful for the purpose; indeed, they are non-host-specific elicitors of the hypersensitive response (Alfano & Collmer, 1997) and can

Journal Pre-proof work as PGR, in the sense that, if applied on plants during growing period, they trigger the expression of hundreds of genes including those engaged with the biosynthesis of phytochemicals (such as anthocyanins) (Akbudak, Tezcan, Akbudak, & Seniz, 2006; Liu et al., 2010; Li et al., 2013). Chuang et al. (2014) reported that HrP enhances the plant disease resistance by activating pathogen-associated molecular patterns (PAMPs), inducing the expression of different members of the jasmonic acid resistance family involved in plant resistance mechanisms. This can have a role in the promotion of anthocyanin accumulation (Li et al., 2014). The correlation between jasmonate

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pathway and anthocyanins accumulation was investigated, showing that an F-box protein COI1, which has a function of jasmonate receptor (Sheard et al. 2010), regulates the expression of some

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transcription factors which regulate the downstream anthocyanin biosynthetic pathway, thereby

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modulating JA-induced anthocyanin biosynthesis in Arabidopsis (Shan, Zhang, Peng, Wang, & Xie,

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2009).

Thus, even though to the best of our knowledge no reports exist about HrP application on grapes for

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improving berry skin color, their effective use in triggering the anthocyanin biosynthesis and

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enhancing their content could be hypothesized in grape skin, too. Therefore, the aim of this work

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was to assess the effect of a HrP experimental formulation (named PHCO 2 ) compared to commercial S-ABA phytohormone, applied onto Crimson Seedless grapes by spraying, towards the color improvement and the anthocyanin content in grape skin for outweighing the technological issue related to the coloration defect but keeping a high level of the other quality parameters of this variety.

2. Material and methods 2.1. Plant materials The trial was conducted in 2017 on a 12-year-old commercial vineyard of ‘Crimson Seedless’ table grape (Vitis vinifera L.), grafted onto 1103 Paulsen rootstock (Vitis berlandieri x Vitis rupestris),

Journal Pre-proof located at Lizzano (40°22'3" N; 17°22'46" E, 21 m a.s.l.) in Apulia region, Southern Italy. Vines were spaced 2.5 x 2.5 m and trained to an overhead trellis system (‘tendone’). Table grapes grew following the regional viticulture practice; moreover, at the beginning of veraison 600 mg L-1 of PHCO2 experimental formulate (SIPCAM Italia S.p.A.) and 400 mg L (active ingredient concentration of 104 g L-1 S-cis-abscisic acid,

-1

of commercial S-ABA

Excelero®, Sumitomo Chemical

Agro Europe s.a.s.) were applied by spraying in two times (from 18th July to 17th August 2017). In particular, S-ABA treatment was directly sprayed onto the clusters while PHCO2 onto the whole

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vine canopy.

A randomized blocks design was employed, consisting of six rows of 30 vines divided into three

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sections and each section was treated with PHCO2 (T2), S-ABA (T3), or untreated as control (T1).

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Finally, 4 replicates for treatment were obtained. From veraison to harvest, 6 samplings of 3

were

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bunches from each replicate were carried out; besides, from each replicate, 10 berries for bunch homogenously removed (from the top, middle, and bottom of the bunch), with their pedicel

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still attached, and stored at -20 °C until anthocyanins extraction. Then, other samples of 60 berries

2.2. Chemicals

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the latter ones).

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(20 per bunch) were collected for chemical, physical, and color analyses (just at the harvest time for

Formic acid, acetonitrile and water HPLC grade were purchased from J. T. Baker (Deventer, Holland). Ethanol, hydrochloric acid, potassium chloride, and sodium acetate were purchased from Carlo Erba (Milano, Italy). Sodium hydroxide (NaOH) 0.1N and bromothymol blue were purchased from Sigma Aldrich (Milano, Italy). Delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin3-O-glucoside, peonidin-3-O-glucoside and malvidin-3-O-glucoside chlorides were purchased from Extrasynthese (Genay, France) and used as HPLC and UV-Vis spectrophotometer reference standards.

Journal Pre-proof

2.3. Physical analysis on grapes Just after the pickling, compression and tensile tests were performed on the berries using a Zwick Roell ver. Z 0.5 Materials Testing Machine (Woonsocket, Rhode Island, USA). All acquisitions were carried out using TestXpert® II software ver. 3.31 working in the MS Windows environment. A 2-cycle compression test was carried out on each whole berry in the equatorial position under a deformation of the berry of 20% with waiting time between the two bites of 1 s using a crosshead

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speed of 3.334 mm s-1 , with a standard force of 0.1 N and a 0.02 m diameter cylindrical probe. Figure S1 showed a two peaks graph for each measure. Typical texture parameters were determined

gumminess-G

(N,

as

hardness

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(E21+E22)/(E11+E12)),

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and calculated by the software: firmness-H (N, as F13), cohesiveness-Coh (adimensional, as ×

cohesiveness),

springiness-S

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(adimensional, as (S23-S20) / (S13)), chewiness-Chw (adimensional, as gumminess × springiness). The tensile test was performed by scoring the maximum load application force-TF (N) at which the

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detachment of the pedicel took place, with a crosshead speed of 3.334 mm s-1 and a standard force

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of 0.05 N. These parameters were replicated on 20 berries for each treatment (T1, T2, and T3).

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Besides, the penetration test was performed by the Digital Fruit Firmness Tester (TR Turioni S.R.L. Forlì) which scored the penetration index-PI (N), that is the resistance opposed by the berry to be penetrated by a 1 mm diameter plunger.

2.4. Chemical and color analyses on grapes Total soluble solids (TSS), titratable acidity (TA), and pH were measured according to protocols established by the OIV, 1990. Berries were crushed to determine TSS (expressed as ° Brix) of the juice using a portable refractometer (ATAGO PR32). Even TA (as g L-1 of tartaric acid equivalents) was determined on the juice by titrating with 0,1 N sodium hydroxide to the bromothymol blue end point. Finally, the juice pH was measured too throughpH meter (CRISON BASIC 20). Berries color was determined by a chromameter CM-5 (Konica Minolta, Chiyoda, Tokyo, Japan) using the

Journal Pre-proof Commision Internationale de l’Eclairage Lab (CIELAB) color system, evaluating lightness, L* (0, black – 100, white), chroma, C* (0, achromatic), and hue angle on the color wheel, h (0, red – 90, yellow – 180, green – 270, blue), as previously described (Ferrara et al., 2013).

2.5. Extraction of anthocyanins from grapes From the frozen 10 berry samples, the skins were manually separated and cleaned by the pulp; they

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were dried at 30 °C for 24h, grounded by an analytical mill (IKA A11 basic, WERKE GMBH & CO.KG, Germany) and then ~ 0.4 g of powder (with particle size < 1 mm, selected by a standard

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sieve) were extracted by water/ethanol/ hydrochloricacid 30:70:1 at 0.07 (g mL-1 ) skins/extraction

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solvent ratio. The extraction procedure was carried out in a capped ultrasonic bath (SONICA 2200

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EP, SOLTEC, Milano, Italy); the mixtures, placed into closed polypropylene tube (15 mL), were sonicated for 21 min at a controlled temperature 50 °C. All the extracts were centrifuged at 4000g

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for 3 min, filtered through a 0.45 μm syringe cellulose filter, and analyzed by UV-Vis and HPLC-

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2.6. UV-Vis analyses

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DAD.

Determination of total monomeric anthocyanin pigment content was performed according to a validated pH differential method (Lee, Durst, & Wrolstd, 2005). Briefly, sets of 2 solutions in 10 mL volumetric flasks were obtained by diluting 2 mL of skin extracts with 8 mL of freshly prepared pH 1.0 buffer (potassium chloride, 0.025 M) or pH 4.5 buffer (sodium acetate, 0.4 M), respectively. The absorbance (A) of the solutions were measured within 20 – 50 min of preparation at both 520 and 700 nm by an UV-Vis spectrophotometer Agilent 8453 (Agilent Technologies, Palo Alto, USA).

Finally,

anthocyanin

pigment

concentration

(expressed

equivalents) was calculated as follows: Total monomeric anthocyanins (g g-1 ) =

𝐴 × 𝑀𝑊 × 𝐷𝐹 × 106 × 𝑉 𝜀×𝑙×𝑚

as

cyanidin-3-O-glucoside

Journal Pre-proof where A = (A520nm – A700nm )pH 1.0 – (A520nm – A700nm)pH 4.5 ; MW (molecular weight) = 449.2 g mol-1 ; DF (dilution factor) = 4; l = pathlenght in cm;  (molar extinction coefficient) = 26900 L mol-1 cm-1 ; 106 = factor for conversion from g to g; V = extract volume in L; m = mass of dry skin samples in g.

2.7. HPLC-DAD-MS analyses

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HPLC-DAD-MS analysis was performed by an HPLC 1100 (Agilent Technologies, Palo Alto,

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USA) equipped with a degasser, quaternary pump solvent delivery, thermostated column

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compartment, diode array detector, and XCT-trap Plus mass detector coupled with an ESI interface. Chromatographic separation was performed by using Zorbax SB C18 5 µm (250 x 4.6 mm i.d.,

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Agilent Technologies) with a pre-column Gemini C18 5 µm (4 x 2 mmi.d., Phenomenex) as

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reversed stationary phase, and a binary solvent system (solvent A: 10% formic acid in water; solvent B: acetonitrile) as mobile phase. The following gradient elution program was used: 0 – 10

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min, 5 – 13% B; 10 – 20 min, 13 – 15% B; 20 – 30 min, 15 – 22% B; 30 – 50 min, held at 22% B;

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50 – 55 min, 22 – 95% B; 55 – 56 min, held at 95% B; 56 – 60 min, 95 – 5% B; held for 5 min at

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5% B to re-equilibrate the column. Flow rate, column temperature, and injection volume were set up at 0.7 mL min-1 , 22 °C, and 5 µL. Diode array detection was between 220 and 700 nm, and absorbance were recorded at 520 nm. Positive electrospray mode was used for ionization of molecules with capillary voltage at 4000 V and skimmer voltage at 30 V. The nebulizer pressure was 40 psi and the nitrogen flow rate was 9 L min-1 . Temperature of drying gas was 350 °C. The monitored mass range was from m/z 100 to 1000. Anthocyanins were tentatively identified by matching the elution pattern and mass spectra with those of reference standards and data reported in the literature (Crupi et al., 2012; Ferrara et al., 2015); then, they were quantified according to the external standard method. Five standard solutions at different concentrations (ranging from 50 to 300 g mL-1 ) of cyanidin-3-O-glucoside were used to obtain the calibration curve (R2 = 0.9996). The detection limit (LOD = 0.26 g mL-1 ) and

Journal Pre-proof quantification limit (LOQ = 0.79 g mL-1 ) were calculated on the basis of chromatograms and defined as signal to noise (6*SD of baseline) ratio of 3 and 10, respectively. Results were expressed as g cyanidin-3-O-glucoside equivalents per g of dry skins.

2.8. Sensorial evaluation Four blind replicates for each treatment (T1 – T3) were subjected to 10 trained official judges of the

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Council for Agricultural Research and Economics – Research Centre for Viticulture and Enology

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(CREA-VE). Four visual and six gustative descriptors were selected; a schedule dependent on a

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non-structured scale was adopted in which each descriptor was measured by putting a cross on a bar

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10 cm long based on its perceived level.

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2.9. Statistical analysis

Some collected data were analyzed by STATISTICA 8.0 (StatSoft Inc., Tulxa, OK) software

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package. Specifically, after testing their normal distribution by Shapiro-Wilk’s W test together with

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their homoscedasticity by means of Levene test, a two-way analysis of variance (ANOVA)

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followed by a Tukey HSD post hoc test was performed for the HPLC-DAD identified anthocyanins and total anthocyanins determined by UV-Vis spectrophotometric analyses, in order to evaluate the effects of ripening and harpin proteins treatment and their interactions. While, a one-way ANOVA for mean differences was applied to L*, C*, and h color parameters, and graphically discussed by bar graphs. Finally, histogram graphics were constructed by using Microsoft Office Excel 2007. Moreover, texture data were subjected to one-way ANOVA and the means separated by Tukey HSD post hoc test. A mean value on the 4 replicates per treatment was considered as a single value for each of 10 judges, therefore each descriptor had 10 replicates. Each of 10 sensorial descriptors scored were processed by non parametric Kruskall-Wallis test. These last statistical analyses were performed with Statgraphics 300 Centurion XV ver. 15.1.02.

Journal Pre-proof 3. Results and discussion

3.1. Variation of anthocyanin profile and color in Crimson Seedless as affected by PGRs application The evolution of the total monomeric anthocyanins (expressed as g g-1 ) extracted from Crimson Seedless grape skins during the maturation period (2 nd August – 12nd October) and affected by PHCO2 (T2) and S-ABA (T3) treatments was depicted in Figure 1. Both the two factors “treatment” and “ripening” were statistically significant

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(p < 0.001; F = 42.37 and 25.32,

respectively), as well as their interaction (p < 0.001; F = 4.05); it means that the concentration of

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anthocyanins increased from veraison to harvest, as expected from the increasing maturation index

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(TSS/TA; Table 1), more intensely in grapes treated with harpin proteins (T2) and exogenous S-

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ABA (T3) than control samples (T1). Although having been demonstrated how the physiological events leading to the onset of grape ripening are not all coincident, generally, sugars and

anthocyanin

biosynthesis by activating the protein kinases and

protein phosphatases

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the

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anthocyanins accumulations have been recognized strictly correlated, because sugars can enhance

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(Lecourieux et al., 2014; Castellarin et al., 2016). However, in our study, a good relationship between TSS and total anthocyanins was just observed in T2 and T3 (r = 0.8229 and 0.8308, respectively) but not in T1 (r = 0.5197) grapes (Figure 2). Considering that the expression of anthocyanin biosynthetic genes are also heavily influenced by temperature, the described behavior could be ascribed to the high temperatures, registered during the ripening period in our experimental vineyard (data not shown), causing a decrease in the transcript levels of the genes and/or the loss of anthocyanins by chemical and enzymatic oxidation in the control Crimson Seedless grape berries (T1). Indeed, several works in the literature have reported how high temperatures reduced the endogenous ABA level, which led to diminished expression of VvMYBA1 and VvUFGT, encoding the overall activation of the anthocyanin biosynthesis mainly into the red than black grapes; moreover, the red varieties are characterized by a high content of

Journal Pre-proof anthocyanins (such as cyanidin-3-O-glucoside) structural less stable towards degradation reactions (Yamane et al., 2006; Mori, Goto-Yamamoto, Kitayama, & Hashizume, 2007; Ferrero et al., 2018). The application of PHCO2 and S-ABA to the grapes overcome the negative effect of temperature (Figures 1 and 2). Thereby, exogenous HrP and ABA treatments strongly affected the color development in the grape skin, indeed the level of anthocyanins at the harvest time was from 2.5 to 3 folds higher (5000  500 and 6300  1000 g g-1 of dry skins in T2 and T3, respectively) than that

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of control fruit (2100  400 g g-1 of dry skins in); even though the significant influence of the

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treatment was just observed from the first sampling date (Figure 1). This finding was in agreement

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with previous researches, which demonstrated the positive effect of exogenous applied PGRs (such as S-ABA and strigolactone) in favoring the biosynthesis of pigments and overall improving the

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berry color in Crimson Seedless grapes (Lurie et al., 2009; Ferrara et al., 2015; Ferrero et al., 2018).

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Thus, it can be speculated that, as already shown for S-ABA (Berli, Fanzone, Piccoli, & Bottini, 2011; Koyama et al., 2018), the exogenous HrP treatment close to veraison is more effective in

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increasing anthocyanin accumulation, too.

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To gauge the effect of HrP and S-ABA PGRs on the main anthocyanins singularly, the mean values

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of the pigments identified and quantified (by HPLC-DAD-MS analyses) in Crimson Seedless skins, as affected by the two experimental factors, were reported in Table 1. As typically found in pink and red-colored table grape varieties (Liang, Owens, Zhon, & Cheng, 2011; Crupi et al., 2012; Crupi et al., 2016), the cultivar was prevalently characterized by dihydroxylated-type anthocyanins, in particular peonidin-3-O-glucoside, whilst lower concentrations of threehydroxilated-type, namely delphinidin, petunidin, and malvidin-3-O-glucoside, were found; as regards acylated pigments, acetyl and p-coumaroyl peonidin and malvidin glucosides were just revealed (Table 1). Individual anthocyanins were identified (Figure S2) using low resolution mass spectrometry and comparing the m/z of each molecular ion (M+), its aglicon (M+ - X), and elution order (retention time) to data previously published

(Crupi et al., 2012). “Ripening” and “treatment” factors significantly

influenced (p < 0.001; F = 5.23 and 9.81, respectively) the accumulation of all identified

Journal Pre-proof anthocyanins, whose values reached the maximum at the final two sampling dates and in T2 and T3 samples (Figures S3 and S4). In particular, except for delphinidin-3-O-glucoside, the treatment with PHCO2 and S-ABA positively interacted with ripening (p < 0.001; F = 2.58), enhancing the concentrations of the compounds, as especially evident in the case of peonidin-3-O-glucoside, cyanidin-3-O-glucoside, and malvidin-3-O-glucoside at the last two samplings and with contents ranging from 2 to 4 folds higher in T2 and T3 than T1 grapes (Table 1). Because the extracts were from grounded dry skins of constant weight, these findings allow us to

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infer that the different anthocyanins levels were due to the modulation of their biosynthetic pathway (He at al., 2010). The mere change of berry dimensions and skin/flesh proportion could not explain

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this variation (Roby, Harbertson, Adams, & Matthews, 2004; Perniola, Crupi, Genghi, &

(diOH/diCH3 O)

and

methoxylated and

anthocyanins,

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hydroxylated

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Antonacci, 2016)., . Indeed, for instance, considering the ratios at the harvest time between cyanidin-3-O-glucoside/peonidin-3-O-glucoside

delphinidin-3-O-glucoside/(petunidin-3-O-glucoside

+

malvidin-3-O-

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glucoside) (threeOH/threeCH3 O), as well as between acylated (acetyl and p-coumaroyl peonidin

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and malvidin) and non-acylated (peonidin and malvidin glucosides) compounds (acylGl/Gl), the

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treatment by the two PGRs could be supposed to fine-tuning the genes which encode for Omethyltransferases and acyltransferases (He et al., 2010; Costantini et al., 2015), inhibiting the methoxylation of OH-groups, mostly of delphinidin, but favoring the esterification (acetylation and p-coumaroylation) of the glucoside moieties of peonidin and malvidin (Figure 3). Instead, both such treatments did not reducethe difference between the di- and threehydroxylated anthocyanins in the grapes during ripening (Figure S5), unlike Koyama et al. (2018) and Katayama-Ikegami et al. (2016) who stated how the application of S-ABA was able to mostly stimulate the gene expression of F3’,5’H relative to F3’H. The highlighted changes also impacted on both the hue and color intensity of berry skins, which are specifically promoted by acylation, because acylated anthocyanins have been proposed to be preferentially trapped in anthocyanin vacuolar inclusions resulting in dark coloration (Mizuno,

Journal Pre-proof Hirano, & Okamoto, 2006); precisely, T2 and T3 Crimson Seedless grapes looked like darker red than T1 ones (Figure 4). To confirm the visual appraisal, the color indices – lightness (L*), hue angle (h), and chroma (C*) – obtained as a linear combination of CIELAB coordinates, were measured and discussed on the grape samples at the harvest time; as shown in Figure 5, except for the case of h values, which appeared not significantly different, the PHCO2 and S-ABA treatments (T2 and T3) similarly provoked the expected decrease of L* and C* when the color of the fruits

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became darker (Carreño, Luis Almela, & Fernández‐López, 1996).

3.2. Variation of physical parameters and sensorial characteristics of Crimson Seedless as affected

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by PGRs application

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For what concerns texture parameters the one-way ANOVA revealed no significant differences

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among the samples T1-T3 at the harvest; in particular, treatments with either S-ABA or PHCO2 did not influence berry firmness as well as the other mechanical parameters at harvest time, mostly

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because of the high oscillation of these data around the mean values (CV%) for all the scored traits

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(Table 2). This behavior was frequently observed relating to the phenotypic characteristics of

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Crimson Seedless berries (Lurie et al., 2009; Ferrara et al., 2013). Since ABA application is acknowledged to cause loosening rigidity of cell wall, resulting in fruit softening and a higher probability of berry cracking as proved in foregoing researches on Crimson Seedless, too (Lurie et al., 2009; Gambetta, Matthews, Shaghasi, Mcelrone, & Castellarin, 2010), our findings were in accordance to that obtain in Puglia (Ferrara et al., 2013) and California (Cantin, Fidelibus, & Crisosto, 2007). Anyway, that PHCO2 use did not affect either firmness or the other parameters correlated to fruit freshness, which are relevant features for the successful post-harvest handling of grapes for the fresh fruit market because they influence transportability and shelf-life (Koyama et al., 2018), can be considered a really valuable result. Our statements were further confirmed by sensorial analyses; Figure 6 depicted the sensory profile of the three grape samples based on 10 visual and gustative descriptors scored by 10 judges. As reported before, the

Journal Pre-proof statistical approach was based on the non-parametric Kruskall-Wallis test since the non-structured scale of values for each descriptor was adopted. Significant differences emerged only for the two visual descriptors related to skin color components, i.e., Berry Color Uniformity (BCU) and Skin Color (SC). In particular, the PGR treated grapes (T2 and T3) were judged more uniformly and intensely colored than the control (T1), even though no difference among the two treatments emerged, describing a substantial equivalent effect of PGRs on color components. This aspect assumes great importance since consumers tend to select foods mostly based on their sensory

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perception, in particular for what concerns fruit and vegetables (Chironi et al., 2017); in the case of table grape consumers usually associate color to other appreciable market qualities such as pleasant

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flavor (Pathare, Opara, &, Al-Said, 2013).

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4. Conclusions

In this work, the exogenous treatment with harpin proteins close to veraison demonstrated as

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effective as S-ABA one in increasing the anthocyanins level of Crimson Seedless. In particular,

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PHCO2 and S-ABA positively interacted with ripening, enhancing the peonidin-3-O-glucoside,

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cyanidin-3-O-glucoside, and malvidin-3-O-glucoside concentrations up to 4 folds higher than control grapes. Moreover, the esterification of the glucose moieties to give rise to the more stable acylated peonidin and malvidin was also favored. Overall, the color of berry skins actually improved at harvest. Therefore, for the first time, a HrP experimental formulation has been proved as a real alternative to the less sustainable S-ABA phytohormone for overcoming the typical coloration defect of Crimson Seedless grown in a warm climate but keeping high its other quality characteristics.

Journal Pre-proof

Acknowledgements We would like to thank SIPCAM Italia S.p.A. for supplying the HrP experimental formulation,

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PHCO2, used in this work. We also express our gratitude to Dr. Giambattista Debiase for technical

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assistance.

Journal Pre-proof FIGURE CAPTIONS Figure 1. Changes in total monomeric anthocyanins concentration (g g-1 of dry skins) of control (T1), PHCO2 (T2), and S-ABA (T3) treated Crimson Seedless grapes during ripening. Values are means of four replicates and vertical bars denote 0.95 confidence intervals. Figure 2. Pearson correlation between total soluble solids (TSS) and total monomeric anthocyanins in a) control (T1; r = 0.5197), b) PHCO2 (T2; r = 0.8229), and c) S-ABA (T3; r = 0.8308) treated

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grapes.

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Figure 3. Ratios between hydroxylated and methoxylated anthocyanins, and acylated and nonacylated anthocyanins of control (T1), PHCO2 (T2), and S-ABA (T3) treated Crimson Seedless

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grapes at the harvest time (12nd October). Values are means of four replicates  S.E.

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Figure 4. Representative clusters of Crimson Seedless subjected to the two exogenous PGRs

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treatments. Control (T1), PHCO2 (T2), and S-ABA (T3) at the harvest time (12nd October).

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Figure 5. Effect of the two exogenous PGRs application to Crimson Seedless on the colour

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CIELAB parameters of berries at the harvest time (12 nd October). Control (T1), PHCO2 (T2), and S-ABA (T3) treated samples.

Figure 6. Sensory profile of Crimson Seedless table grapes treated with different Plant Growth Regulators. Control (T1), PHCO2 (T2), and S-ABA (T3). Footnotes: BS: Berry Size; BCU: Berry Colour Uniformity; SC: Skin Colour; PC: Pulp Colour; BC: Berry Crunchiness; Pcon: Pulp Consistency ; S: Sweetness; A: Acidity; SAB: Sweet/Acid Balance; PA: Pulp Aroma. Different letters indicate significant difference at p<0.05.

Supplementary data Figure S1. Two peaks graph generated for each measure by TestXpert® II software ver. 3.31 by a two cycle compression test.

Journal Pre-proof Figure S2. HPLC-DAD chromatogram (at 520 nm) of skin extracts of S-ABA treated Crimson Seedless at harvest. (1) delphinidin-3-O-glucoside (RT = 12.52 min, M+ = 465 m/z, M+ - X = 303 m/z); (2) cyanidin-3-O-glucoside (RT = 14.69 min, M+ = 449 m/z, M+ - X = 287 m/z); (3) petunidin3-O-glucoside (RT = 16.05 min, M+ = 479 m/z, M+ - X = 317 m/z); (4) peonidin-3-O-glucoside (RT = 18.88 min, M+ = 463 m/z, M+ - X = 301 m/z); (5) malvidin-3-O-glucoside (RT = 20.47 min, M+ = 493 m/z, M+ - X = 331 m/z); (6) peonidin-3-O-acetyl-glucoside (RT = 33.46 min, M+ = 505 m/z, M+ - X = 301 m/z); (7) malvidin-3-O-acetyl-glucoside (RT = 35.40 min, M+ = 535 m/z, M+ - X = 331

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m/z); (8) peonidin-3-O-p-coumaroyl-glucoside (RT = 42.40 min, M+ = 609 m/z, M+ - X = 301 m/z); (9) malvidin-3-O-p-coumaroyl-glucoside (RT = 43.70 min, M+ = 639 m/z, M+ - X = 331 m/z).

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RT: retention time; M+, M+ - X: molecular ion and aglicon in positive ionization mode.

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Figure S3. Effect of the “ripening” factor on the identified anthocyanins content in Crimson

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Seedless berries skin.

Figure S4. Effect of the “treatment” factor on the identified anthocyanins content in Crimson

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Seedless berries skin.

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Figure S5. Ratios between di- and threehydroxylated anthocyanins of control (T1), PHCO2 (T2),

replicates  S.E.

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and S-ABA (T3) treated Crimson Seedless grapes during ripening. Values are means of four

Journal Pre-proof References Akbudak, N., Tezcan, H., Akbudak, B., & Seniz, V. (2006). The effect of harpin protein on plant growth parameters, leaf chlorophyll, leaf colour and percentage rotten fruit of pepper plants inoculated with Botrytis cinerea. Scientia Horticulturae, 109, 107-112. Alfano, J.R., & Collmer, A. (1997). The type III (HrP) secretion pathway of plant pathogenic bacteria: trafficking harpins, Avr proteins, and death. Journal of Bacteriology, 179, 5655-5662.

f

Azuma, A., Kobayashi, S., Goto-Yamamoto, N., Shiraishi, M., Mitani, N., Yakushiji, H., &

oo

Koshita, Y. (2009). Color recovery in berries of grape (Vitis vinifera L.) ‘Benitaka’, a bud sport of

pr

‘Italia’, is caused by a novel allele at the VvmybA1 locus. Plant Science, 176, 470-478. Basile, T., Alba, V., Gentilesco, G., Savino, M., & Tarricone, L. (2018). Anthocyanins pattern

e-

variation in relation to thinning and girdling in commercial Sugrathirteen® table grape. Scientia

Pr

Horticulturae, 227C, 202-206.

Berli, F.J., Fanzone, M., Piccoli, P., & Bottini, R. (2011). Solar UV-B and ABA are involved in

al

phenol metabolism of Vitis vinifera L. increasing biosynthesis of berry skin polyphenols. of

rn

Agricultural and Food Chemistry, 59, 4874-4884.

Jo u

Cantin, C.M., Fidelibus, M.W., & Crisosto, C.H. (2007). Application of abscisic acid (ABA) advanced red color development and maintained postharvest quality of ‘Crimson Seedless’ grapes. Postharvest Biology and Technology, 46, 237-241. Carreño, J.,

Luis Almela, A.M., & Fernández‐López, J.A. (1996). Measuring the color of table

grapes. Color Research and Application, 21, 50-54. Carrieri, C., Milella, R.A., Incampo, F., Crupi, P., Antonacci, D., Semeraro, N., & Colucci, M. (2013). Antithrombotic activity of 12 table grape varieties. Relationship with polyphenolic profile. Food Chemistry, 140, 647–653. Castellarin, S.D., Gambetta, G.A., Wada, H., Krasnow, M.N., Cramer, G.R., Peterlunger, E., Shackel, K.A., & Matthews, M.A. (2016). Characterization of major ripening events during

Journal Pre-proof softening in grape: turgor, sugar accumulation, abscisic acid metabolism, colour development, and their relationship with growth. Journal of Experimental Botany, 67, 709-722. Chironi, S., Sortino, G., Allegra, A., Saletta, F., Caviglia, V., & Ingrassia, M. (2017). Consumer assessment on sensory attributes of fresh table grapes Cv 'Italia' and 'Red Globe' after long cold storage treatment. Chemical Engineering Transactions, 58, 421-426. Chuang, H.W., Chang, P.Y., & Syu, Y.T. (2014). Harpin Protein, an Elicitor of Disease Resistance, Acts as a Growth Promoter in Phalaenopsis Orchids. Journal of Plant Growth Regulation, 33, 788–

oo

f

797.

Commission Implementing Regulation (EU) No 543/2011. Marketing Standard for table grapes. 7 2011.

Available

pr

June

at

e-

https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/2

Pr

99241/Table_Grapes.pdf. Retrieved on 19th March 2019.

Costantini, L., Malacarne, G., Lorenzi, S., Troggio, M., Mattivi, F., Moser, C., & Grando, M.S.

al

(2015). New candidate genes for the fine regulation of the colour of grapes. Journal of

rn

Experimental Botany, 66, 4427-4440.

Jo u

Crupi, P., Antonacci, D., Savino, M., Genghi, R., Perniola, R., & Coletta, A. (2016). Girdling and gibberellic acid effects on yield and quality of a seedless red table grape for saving irrigation water supply. European Journal of Agronomy, 80, 21–31. Crupi, P., Coletta, A., Milella, R.A., Perniola, R., Gasparro, M., Genghi, R., & Antonacci, D. (2012). HPLC-DAD-ESI-MS analysis of flavonoid compounds in 5 seedless table grapes grown in Apulian region. Journal of Food Science, 77, C174 – C181. Ferrara, G., Mazzeo, A., Matarrese, A.M.S., Pacucci, C., Pacifico, A., Gambacorta, G., Faccia, M., Trani, A., Gallo, V., Cafagna, I., & Mastrolilli, P. (2013). Application of abscisic acid (S-ABA) to ‘Crimson Seedless’ grape berries in a Mediterranean climate: effects on color, chemical characteristics, metabolic profile, and S-ABA concentration. Journal of Plant Growth Regulation, 32, 491-505.

Journal Pre-proof Ferrara, G., Mazzeo, A., Matarrese, A.M.S., Pacucci, C., Punzi, R., Faccia, M., Trani, A., & Gambacorta, G. (2015). Application of abscisic acid (S-ABA) and sucrose to improve colour, anthocyanin content and antioxidant activity of cv. Crimson Seedless grape berries. Australian Journal of Grape Wine Research, 21, 18-29. Ferrero, M., Pagliarini, C., Novák, O., Ferrandino, A., Cardinale, F., Visentin, I., & Schubert, A. (2018).

Exogenous

strigolactone

interacts

with

abscisic

acid-mediated

accumulation

of

anthocyanins in grapevine berries. Journal of Experimental Botany, 69, 2391-2402.

oo

f

Gambetta, G.A., Matthews, M.A., Shaghasi, T.H., Mcelrone, A.J., & Castellarin, S.D. (2010). Sugar and abscisic acid signaling orthologs are activated at the onset of ripening in grape. Planta,

pr

232, 219-234.

e-

Gagné, S., Cluzet, S., Merillon, J.M., & Geny, L. (2011). ABA initiates anthocyanin production in

Pr

grape cell cultures. Journal of Plant Growth Regulation, 30, 1-10. Guidoni, S., & Hunter, J.J. (2012). Anthocyanin profile in berry skins and fermenting must/wine, as

rn

Technology, 235, 397-408.

al

affected by grape ripeness level of Vitis vinifera cv. Shiraz/R99. European Food Research and

Jo u

He, F., Mu, L., Yan, G.L., Liang, N.N., Pan, Q.H., Wang, J., Reeves, M.J., & Duan, C.Q. (2010). Biosynthesis of anthocyanins and their regulation in colored grapes. Molecules, 15, 9057-9091. Katayama-Ikegami, A., Sakamoto, T., Shibuya, K., & Katayama, T. (2016). Effects of abscisic acid treatment on berry coloration and expression of flavonoid biosynthesis genes in grape. American Journal of Plant Science, 7, 1325-1336. Koyama, R., Roberto, S.R., de Souza, R.T., Borges, W., Anderson, M., Waterhouse, A.L., Cantu, D., Fidelibus, M.W., & Blanco-Ulate, B. (2018). Exogenous abscisic acid promotes anthocyanin biosynthesis and increased expression of flavonoid synthesis genes in Vitis vinifera×Vitis labrusca table grapes in a subtropical region. Frontiers in Plant Science, 9, 323. Yamamoto, L.Y., Marinho de Assis, A., Ruffo Roberto, S., Bovolenta, Y.R., Nixdorf, S.L., GarcíaRomero, E., Gómez-Alonso, S., & Hermosín-Gutiérrez, I. (2015). Application of abscisic acid (S-

Journal Pre-proof ABA) to cv. Isabel grapes (Vitis vinifera × Vitis labrusca) for color improvement: Effects on color, phenolic composition and antioxidant capacity of their grape juice. Food Research International, 77, 572-583. Lecourieux, F., Kappel, C., Lecourieux, D., Serrano, A., Torres, E., Arce-Johnson, P., & Delrot, S. (2014). An updated on sugar transport and signalling in grapevine. Journal of Experimental Botany, 65, 821-832. Lee, J., Durst Robert, W., & Wrolstad Ronald, E. (2005). Determination of total monomeric

oo

f

anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: collaborative study. Journal of AOAC International, 88, 1269-1278.

pr

Leng, P., Yuan, B., & Guo, Y. (2014). The role of abscisic acid in fruit ripening and responses to

e-

abiotic stress. Journal of Experimental Botany, 65, 4577-4588.

Pr

Li, M., Yu, M.L., Zhang, Z.Q., Liu, Q.N., Wu, Y.C., & Liu, Z.G. (2013). Effects of Harpin protein on fruit quality and shelf life of winter jujube (Ziziphus jujube Mill. Cv. Dongzao). Biological

al

Agriculture & Horticulture, 29, 58-68.

rn

Li, T., Jia, K.P., Lian, H.L., Yang, X., Li, L., & Yang, H.Q. (2014). Jasmonic acid enhancement of

Jo u

anthocyanin accumulation is dependent on phytochrome A signaling pathway under far-red light in Arabidopsis. Biochemical Biophysical Research Communications, 454, 78-83. Liang, Z., Owens, C.L., Zhon, G., & Cheng, L. (2011). Polyphenolic profiles detected in the ripe berries of Vitis vinifera germplasm. Food Chemistry, 129, 940-950. Liu, R., Lu, B., Wang, X., Zhang, C., Zhang, S., Qian, J., Chen, L., Shi, H., & Dong, H. (2010). Thirty-seven transcription factor genes differentially respond to a harpin protein and affect resistance to the green peach aphid in Arabidopsis. Journal of Biosciences, 35, 435-450. Lurie, S., Ovadia, R., Nissim-Levi, A., Oren-Sharim, M., Kaplunov, T., Zutahy, Y., Weksler, H., & Lichter, A. (2009). Abscisic acid improves colour development in ‘Crimson Seedless’ grapes in the vineyard and on detached berries. Journal of Horticultural Science and Biotechnology, 84, 639-644.

Journal Pre-proof Medouni-Adrar, S., Boulekbache-Makhlouf , L., Cadot, Y., Medouni-Haroune, L., Dahmoune, F., Makhoukhe, A., & Madani, K., 2015. Optimization of the recovery of phenolic compounds from Algerian grape by-products. Industrial Crops and Products, 77, 123 – 132. Mizuno, H., Hirano, K., & Okamoto, G. (2006). Effect of anthocyanin composition in grape skin on anthocyanin vacuolar inclusion development and skin coloration. Vitis, 45, 173-177. Mori, K., Goto-Yamamoto, N., Kitayama, M., & Hashizume, K. (2007). Loss of anthocyanins in red-wine grape under high temperature. Journal of Experimental Botany, 58, 1935-1945.

oo

f

Office International de la Vigne et du Vin (OIV). 1990. Recueil des Methodes Internationales d’Analyse des Vins et des Mouts : Office International de la Vigne et du Vin. Paris.

pr

Olivares, D., Contreras, C., Munoz, V., Rivera, S., Gonzalez-Agüero, M., Retamales, J., &

e-

Defilippi, B.G. (2017). Relationship among color development, anthocyanin and pigment-related

Pr

gene expression in ‘Crimson Seedless’ grapes treated with abscisic acid and sucrose. Plant Physiology and Biochemistry, 115, 286-297.

al

Pathare, P.B., Opara, U.L., & Al-Said, F.A.J. (2013). Colour measurement and analysis in fresh and

rn

processed foods: A Review. Food Bioprocess Technology, 6,36-60.

Jo u

Peppi, M.C., Fidelibus, M.W., & Dokoozlian, N. (2007). Application timing and concentration of abscisic acid affect the quality of ‘Redglobe’ grapes. Journal of Horticultural Science and Biotechnology, 82, 304-310.

Peppi, M.C., Walker, M.A., & Fidelibus, M.W. (2008). Application of abscisic acid rapidly upregulated UFGT gene expression and improved color of grape berries. Vitis, 47, 11-14. Perniola, R., Crupi, P., Genghi, R., & Antonacci, D. (2016). Cultivar and rootstock interaction affects the physiology and fruit quality of table grape with different water management – Preliminary results. Acta Horticolturae, 1136, 129-136.

Journal Pre-proof Phan, M.A.T., Bucknall, M.P., & Arcot, J. (2019). Interferences of anthocyanins with the uptake of lycopene in Caco-2 cells, and their interactive effects on anti-oxidation and anti-inflammation in vitro and ex vivo. Food Chemistry, 276, 402–409. Pilati, S., Bagagli, G., Sonego, P., Moretto, M., Brazzale, D., Castorina, G., Simoni, L., Tonelli, C., Guella, G., Engelen, K., Galbiati, M., & Moser, C. (2017). Abscisic acid is a major regulator of grape berry ripening onset: New Insights into ABA Signaling Network. Frontiers in Plant Science, 8, 1093.

oo

f

Roberto, R.S., Assis, A.M., Yamamoto, L.Y., Miotto, L.C.V., Sato, A.J., Koyama, R., & Genta, W. (2012). Application timing and concentration of abscisic acid improve color of ‘Benitaka’ table

pr

grape. Scientia Horticulturae, 142, 44-48.

e-

Roby, G., Harbertson, J.F., Adams, D.A., & Matthews, M.A. (2004). Berry size and vinewater

Grape and Wine Research, 10, 100-107.

Pr

deficits as factors in winegrape composition: anthocyanins and tannins. Australian Journal of

al

Shan, X., Zhang, Y., Peng, W., Wang, Z., & Xie, D. (2009). Molecular mechanism for jasmonate-

rn

induction of anthocyanin accumulation in Arabidopsis. Journal of Experimental Botany, 60, 3849-

Jo u

3860.

Sheard, L.B., Tan, X., Mao, H., Withers, J., Ben-Nissan, G., Hinds, T.R., Kobayashi, Y., Hsu, F.F., Sharon, M., Browse, J., Yang He, S., Rizo, J., Howe, G.A., & Zheng, N. (2010). Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor. Nature, 468, 400–405. Yamane, K., Jeong, S.T., Goto-Yamamoto, N., Koshita, Y., & Kobayashi, S. (2006). Effects of temperature on anthocyanin biosynthesis in grape berry skins. American Journal of Enology and Viticulture, 57, 54-59. Vox, G., Scarascia Mugnozza, G., Schettini, E., de Palma, L., Tarricone, L., Gentilesco, G., & Vitali, M. (2012). Radiometric properties of plastic films for vineyard covering and their influence on vine physiology and production. Acta Horticolturae, 956, 465-472.

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Journal Pre-proof Table 1. Changes in anthocyanins content (expressed as g g-1 of dry skins) in Crimson Seedless grapes from véraison to maturity. 2017

Sam pling date

2nd August

8th August

21st August

19th September

6th October

12nd October

T1

T2

T3

T1

T2

T3

T1

T2

T3

T1

T2

T3

T1

T2

T3

T1

T2

T3

12. 0b

14

13. 8

15. 0

16

15. 4

16. 6

18

17. 8

21. 3

20. 4

20

20. 6

21. 1

21. 2

19. 8

21. 6

21. 3

TSSa (1. 2) c

(2)

(1. 0)

(1. 8)

(2)

(0. 5)

(1. 8)

(2)

(0. 9)

(0. 7)

(0. 9)

(3)

(1. 0)

(0. 3)

(0.8 )

(1. 0)

(0. 9)

(0.5 )

2.7 3

2.7 9

2.7 9

3.0 0

2.9 9

3.0 7

3.2 5

3.3 1

3.3 3

3.3 8

3.4 2

3.4 8

3.4 4

3.5 2

3.5 5

3.4 4

3.3 8

3.4 7

(0. 05)

(0. 11)

(0. 07)

(0. 10)

(0. 07)

(0. 04)

(0. 02)

(0. 06)

(0. 02)

(0. 04)

(0. 07)

(0. 05)

(0. 07)

(0. 05)

(0.0 4)

(0. 05)

(0. 04)

(0.0 3)

16. 4

14

13. 4

11. 4

10. 4

9.7

9.6

8.6

8.1

(1. 2)

(1. 6)

(1. 2)

(0. 4)

(1. 0)

(1. 1)

(0. 8)

10. 4

13

15

15. 9

17

21

(3)

(3)

7.3 (1. 9)

10 (4)

(1. 6)

0.8

0.9

(0. 7)

4.4 Cyanidi n-3-Oglucosi de

(1. 9) eg 1.3

Petunid in-3-Oglucosi de

(0. 5) d

Peonidi n-3-Oglucosi de

21 0 (90 ) e

Malvidi n-3-Oglucosi

11

oo

6.3

5.7

5.6

6.6

5.6

5.9

(0. 2)

(0. 4)

(0. 2)

(0. 3)

(0. 4)

(0.2 )

(0. 5)

(0. 4)

(0.3 )

21. 8

30. 6

30. 6

31. 6

32. 9

36. 8

38. 1

30. 2

38. 3

36. 2

(3)

(0. 7)

(0. 6)

(1. 1)

(1. 7)

(1. 0)

(0. 7)

(0.8 )

(1. 3)

(1. 1)

(0.4 )

(0. 2)

1.0

0.9

0.8

2.8

1.6

1.4

2.4

3.3

2.8

3.2

4.2

3.0

3.3

5.2

(0. 5)

(0. 3)

(0. 3)

(0. 8)

(0. 5)

(0. 5)

(0. 9)

(1. 2)

(0. 8)

(0. 7)

(1.7 )

(0. 6)

(1. 1)

(1.4 )

60

70

34

13 0

190

49

16 0

200

(30 )

(30 )

(40 )

(19 )

cd e

cd e

ab

d

3.9

13

13

17

28

8.1

(1. 5)

(6)

(3)

(7)

(13 )

(1. 9)

bc d

bc d

cd

ab d

a

cd

22 00

23 00

11 00

24 00

460 0

(50 0)

(80 0)

(30 0)

(90 0)

bc d

bc d

cd e

61

10 0

rn

(0. 3)

n.d .

Jo u

n.d .

al

Com po unds f

Delphin idin-3Oglucosi de

(2)

6.2

Pr

TSS/ TAe

(3)

6.7

pr

(1. 5)

7.0

e-

TA

d

f

pH

27

13

11

(8)

(5)

(6)

de

e

e

2.8

2.7

(1. 2)

(1. 1)

d

d

45 0

46 0

(19 0)

(16 0)

cd e

de

de

60

67

(30

(33

8 (3) cd 11 00 (40 0)

19

35

24

(15 )

(10 )

de

de

70 23 (7) de

(30 )

90 (30 )

cd e

cd

10

9

(3)

(3)

cd

cd

4.0

3.8

(1. 5)

(1. 6)

cd

cd

12 00

12 00

82 0

18 00

23 00

(60 0)

(40 0)

(30 0)

(60 0)

(70 0)

cd e

cd e

cd e

bc de

bc d

46

32

30

64

54

(19

(11

(13

(18

(19

8 (3) cd

28 (9) de

cd 47 0 (13 0) de

17

(19

(11 ) de

7

(40 ) bc

(40 ) ab

(60 ) a

25

29

(7)

(9)

ab

a

15 00

33 00

470 0

(18 00)

(50 0)

(70 0)

(14 00)

bc

a

bc

ab

a

40

90

180

57

10 0

190

(14

(30

(80

(19

(2)

(50

Journal Pre-proof )

)

)

)

)

(7)

)

e

cd e

cd e

de

cd e

cd e

cd e

cd e

cd e

de

cd e

bc

2.8

2.5

6

8

13

12

4.2

11

14

(0. 8)

4.9 (1. 7)

(1. 0)

(0. 8)

(2)

(3) cd ef

7

f

cd ef

ef

ef

cd ef

1.2

3.6

1.4

1.6

6

(0. 4)

(0. 9)

(0. 2)

(0. 5)

d

cd

d

d

1.8

5.4

3.0

3.1

(0. 8)

(1. 4)

(1. 0)

(0. 7)

f

f

f

f

1.5

0.7

(0. 3)

(0. 2)

c

c

n.d .

n.d .

4.4 (1. 5) cd ef

3.6

(3)

(3)

(1. 2)

bc d

bc d

cd

11

11

11

(3)

(4)

(4)

ef

ef

ef

1.6

1.5

1.2

(0. 6)

(0. 4)

(0. 5)

c

c

c

(5)

(5)

bc de

bc de

11

12

(4)

(5)

bc d

bc d

39 (11 ) bc d

23

(1. 5)

(3)

(5)

def

bc de

bc d

3.7

12

(1. 4) cd

5

(2) ab

(3) bc d

9

47

(8)

(2)

(12 )

cd ef

ef

2.6

1.2

5.8

(1. 0)

(0. 4)

(1. 7)

bc

c

ab

bc

)

)

)

)

cd e

cd

a

cd e

6.4 (1. 6) cd ef

15

27

(4)

(9)

bc

6.2 16 (4)

f

1.7

(20 )

bc

19 (1. 9)

(5)

bc d

b

17

48

(6)

(9)

def

bc

1.5

5.8

(0. 4)

(1. 9)

c

ab

33

(15 ) bc de 5 (2) ab

8 (2)

a

cd ef

34

7

(13 )

(3)

a

56 (14 ) ab

5 (2) ab

bc d

20 (4) def

(30 )

) a

bc

19

29

(4)

(7)

ab

a

19 (6) b

35 (13 ) a

46

70

(16 )

(20 )

bc

a

3.6

5.8

(1. 1)

(1. 5)

bc

ab

8 (2) a

rn

al

Malvidi n-3-Opcoumar oylglucosi de

(6)

oo

Peonidi n-3-Opcoumar oylglucosi de

)

pr

Malvidi n-3-Oacetylglucosi de

)

e-

Peonidi n-3-Oacetylglucosi de

(5)

Pr

de

a

b

c

d

Jo u

Total soluble solid are expressed in °Brix; Means of three replicates; Standard deviation at p  0.05; Total acidity e f g expressed in g/L as tartaric acid; Maturation index; Expressed as cyanidin-3-O-glucoside equivalents; Different letters in the same raw are significantly different (Tukey HSD test). T1: untreated samples (control).

T2: treated samples with PHCO2. T3: treated samples with S-ABA n.d.: not detected.

Journal Pre-proof Table 2. Physical parameters of Crimson Seedless grapes at the harvest time as affected by the different Plant Growth Regulators.

T1 a

10(3)

Springiness

c

2.4(0.9)

36

2.1(1.0)

48

29

12(4)

35

11(5)

42

0.57(0.05)

9

0.58(0.04)

7

0.56(0.04)

8

Gumminess (N)

2.1(0.5)

25

2.2(0.7)

31

2.4(0.7)

29

Chewiness (N)

1.2(0.3)

23

1.3(0.3)

26

1.4(0.3)

25

Cohesion

0.21(0.03)

15

0.21(0.03)

14

0.20(0.03)

13

Detachment (N)

7(2)

33

6(3)

47

8(3)

32

b

c

Means of three replicates; Standard deviation at p  0.05; CV%.

e-

a

45

f

Firmness (N)

b

oo

2.4 (1.1)

T3

pr

Penetration (N)

T2

Pr

T1: untreated samples (control). T2: treated samples with PHCO2.

Jo u

rn

al

T3: treated samples with S-ABA.

Journal Pre-proof Graphical abstract

Research Highlights  Harpin proteins treatment favored the anthocyanin accumulation in grape  Some glycosylated anthocyanins values increased up to 4 folds in treated grape  Greater content of the more stable acylated anthocyanins was found in treated grape  Color of berries and the overall grape quality was actually improved

Jo u

rn

al

Pr

e-

pr

oo

f

 Harpin proteins as alternative to S-ABA to overcome coloration defect of red grape

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11