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Influence of methyl jasmonate and benzothiadiazole on the composition of grape skin cell walls and wines
Accepted Manuscript Influence of Methyl Jasmonate and Benzothiadiazole on the composition of grape skin cell walls and wines D.F. Paladines-Quezada, J.D. Moreno-Olivares, J.I. Fernández-Fernández, A.B. Bautista-Ortín, R. Gil-Muñoz PII: DOI: Reference:
S0308-8146(18)31968-X https://doi.org/10.1016/j.foodchem.2018.11.029 FOCH 23838
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
8 January 2018 30 October 2018 5 November 2018
Please cite this article as: Paladines-Quezada, D.F., Moreno-Olivares, J.D., Fernández-Fernández, J.I., BautistaOrtín, A.B., Gil-Muñoz, R., Influence of Methyl Jasmonate and Benzothiadiazole on the composition of grape skin cell walls and wines, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.11.029
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Influence of Methyl Jasmonate and Benzothiadiazole on the composition of grape skin cell walls and wines.
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Paladines-Quezada, D.F a*; Moreno-Olivares, J. D a; Fernández-Fernández, J. I. a; BautistaOrtín, A.B b and Gil-Muñoz, R* a.
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a
b
Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario, Ctra. La Alberca s/n, 30150, Murcia, Spain.
Departamento de Tecnología de Alimentos, Nutrición y Bromatología, Facultad de Veterinaria, Universidad de Murcia, 30100 Murcia, Spain.
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* Corresponding author. Tel.: +34 968 757580. E-mail address: [email protected]
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Abstract.
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Phenolic compounds are very important in crop plants, particularly in grapes. The different
16
strategies to increase their levels include the use of elicitors such as methyl jasmonate (MeJ) and
17
benzothiadiazole (BTH). In an attempt to improve the quality of wines, our aim was to evaluate the
18
effect of preharvest application of these elicitors on the composition and structure of the skin cell
19
walls of Monastrell, Merlot and Cabernet Sauvignon grapes, and to ascertain any relationship with
20
the extractability of phenolic compounds during winemaking. The results indicated that the
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exogenous application of MeJ and BTH during veraison caused significant changes in several
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components of the skin cell walls, such as phenolic compounds, proteins and structural sugars.
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However these changes manifested themselves in different proportions in each variety and year,
24
pointing to the varietal and meteorological dependence of the response to the application of these
25
elicitors. The treatments delayed the maturation process in all varieties when rainfall was low. This
26
observation, together with the observed increase in proteins and phenols in the skin cell wall of
27
Monastrell and Cabernet Sauvignon, could contribute to the strength necessary to maintain the
28
integrity of berries and to increasing resistance to fungal pathogens as the phenolic compounds
29
evolve, thus improving the phenolic profile. However, the structural integrity of Merlot variety
30
tended to decrease in the same conditions.
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Keywords: methyl jasmonate, benzothiadiazole, cell wall, grapes, wines.
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1. Introduction.
34
Phenolic compoundshave been the subject of a large num ber of studies due to their
35
importance in crop plants. Three main reasons can be cited for optimizing the level of phenolic
36
compounds in such plants: their physiological role in the plant, their technological significance for
37
food processing, and their nutritional characteristics (Ruiz-García et al., 2012).
38
Several strategies exist to increase the phenolic compound content of grapes, including
39
cultural practices (pruning, deficit irrigation, thinning of clusters) (Pérez-Lamela, García-Falcón,
40
Simal-Gándara, & Orriols-Fernández, 2007), genetic breeding programmes, clonal selection of
41
varieties (Gómez-Plaza et al., 2008), or the use of elicitors - exogenously applied chemical
42
substances that trigger the activation of certain metabolic pathways, causing the biosynthesis of
43
phenolic and volatile compounds (Gómez-Plaza, Bautista-Ortín, Ruiz-García, Fernández-
44
Fernández, & Gil-Muñoz, 2017). Among these substances methyl jJasmonate (MeJ) and
45
Benzothiadiazole (BTH) have been used to improve the phenolic content of fruits, particularly
46
grapes, while also being considered useful as agrochemicals to improve resistance against plant
47
pathogens (Gil-Muñoz, Bautista-Ortín, Ruiz-García, Fernández-Fernández, & Gómez-Plaza,
48
2017). This resistance may be based on previously existing physical or chemical barriers (such as
49
thick cell walls or high amounts of lignin or tannins) or inducible defense mechanisms (Gozzo,
50
2003).
51
Methyl jasmonate (MeJ) is a potent resistance regulator derived from jasmonic acid that
52
triggers a large number of defense responses, including the synthesis of flavonoid compounds and
53
stilbenes (Beckers & Spoel, 2006). This elicitor is able to activate the enzymes responsible for the
54
biosynthesis of polyphenols, such as the enzyme, phenylalanine ammonia-lyase (PAL). The
55
activation of PAL following postharvest application of the elicitor has been confirmed in many
56
studies in fruits such as lychees, peaches, apples, plums, table grapes, strawberries, accompanied
57
by an increase in total phenols (Ruiz-García & Gómez-Plaza, 2013). Benzothiadiazole (BTH), a
58
photostable functional analogue of the plant signal molecule salicylic acid (SA), has been shown to 2
59
have a double action in plant protection; on the one hand, it inhibits the development of decay-
60
causing fungi through its direct toxicity, and, on the other hand, it also indirectly induces
61
pathogenesis-related (PR) genes, leading to the establishment of systemic acquired resistance
62
(SAR) in a variety of plants, providing broad-spectrum protection against various pathogens
63
(Wendehenne, Durner, Chen, & Klessig, 1998). There are many studies on the effect of pre-
64
harvest application of MeJ and BTH to wine grapes. Such studies have pointed to increased levels
65
of phenolic compounds in the treated grapes and corresponding wines for many varieties (Gil-
66
Muñoz et al., 2017).
67
On the other hand, although skins represent a small percentage of total berry weight, they
68
are fundamental in wine quality, since most of the aromatic and phenolic compounds are located
69
therein. Therefore, it is necessary to know the composition and structural properties of the skins of
70
different varieties, since thse factors can determine the mechanical resistance and texture of
71
berries, and the ease with which they can be processed (Barnavon et al., 2000). Thus, the cell
72
walls of grape skins acquire great relevance, since they are highly complex and dynamic, being
73
composed of polysaccharides, phenolic compounds and proteins, and stabilized by ionic and
74
covalent linkages, all of which may differ between varieties (Ortega-Regules, Romero-Cascales,
75
Ros-García, López-Roca, & Gómez-Plaza, 2006) and even within the same variety grown in
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different terroirs (Apolinar-Valiente, Romero-Cascales, Gómez-Plaza, López-Roca, & Ros-García,
77
2015b)
78
In order to provide new tools to improve wine quality, the objective of this work was to
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evaluate whether the application of two pre-harvest elicitors (MeJ and BTH) to Monastrell, Merlot
80
and Cabernet Sauvignon grapes affects the composition and structure of their skin cell walls, and
81
to check whether they are related to the extractability of phenolic compounds during winemaking.
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2. Materials and methods.
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2.1 Reagents and standards.
3
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All solvents (acetone, ethanol) were of HPLC quality, and all chemicals were of analytical
86
grade (>99%). Water was of Milli-Q quality. BTH ([benzo-(1, 2, 3)-thiadiazole-7- carbothioic acid S-
87
methyl ester]); MeJ (methyl jasmonate); Tween 80; 3, 5-dimethylphenol, were from Sigma Aldrich
88
(St. Louis, USA). For glucose determination, an enzymatic analysis kit from R-biopharm
89
(Darmstadt, Germany) was used. As standards, pure galacturonic acid and gallic acid were
90
purchased from Sigma Aldrich (St. Louis, USA) and Bovine serum albumin (BSA) from J.T. Baker
91
(Deventer, The Netherlands).
92 93
2.2 Experimental design.
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The experiment was carried out in two consecutive years (2015-2016) using three grape
95
varieties (Monastrell, Merlot and Cabernet Sauvignon) grown in Jumilla, Murcia (south-eastern
96
Spain). The study was performed on 14 year old Vitis vinifera (syn. Mourvedre) red wine
97
grapevines grafted onto 1103-Paulsen (clon 249) rootstock and trained to a three-vine vertical
98
trellis system. Vine rows were arranged in N-NW to S-SE with between-row and within-row spacing
99
of 3 x 1.25 m (x: 636.099; y: 4.249.299).
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All treatments (spray application of the elicitors MeJ and BTH on vine clusters) were
101
performed in triplicate, with ten vines per replicate. The protocol used to apply the different
102
treatments was described previously (Gil-Muñoz et al., 2017). To carry out the treatments,
103
aqueous solutions were prepared with Tween 80 as wetting agent (0.1% v/v). BTH was used at a
104
concentration of 0.3 mM, and MeJ was used at a concentration of 10 mM. Control plants were
105
sprayed with aqueous solution of Tween 80 alone. In all cases, 200 mL of aqueous solution was
106
applied per plant. The treatments were carried out twice, at veraison and 1 week later. When
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grapes reached optimum maturity, they were harvested and transported in boxes for
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physicochemical analysis and vinification. Samples of grape berries (ca. 800 g) were taken to
109
isolate their skin cell wall. The skins were totally separated from the pulp, and stored at -80 ℃ until
110
the cell wall material (CWM) was isolated.
111
4
112
2.3 Physico- chemical analysis in grapes at harvest.
113
Total soluble solids (°Brix) were measured using an Abbé-type refractometer (Atago RX-
114
5000). The methodology for carrying out these analyses is described in ECC. Commission
115
Regulation No. 2676/90.
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2.4 Vinifications.
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All the vinifications were made in triplicate in 50 L stainless steel tanks using 50 kg of
119
grapes. Grapes were destemmed, crushed and sulfited (8 g SO2/ 100 kg). Total acidity was
120
corrected to 5.5 g/L with tartaric acid, and selected yeasts were added (Uvaferm VRB, Lallemand,
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25 g/hL). All vinifications were conducted at 25±1°C. The fermentative pomace contact period was
122
10 days. Throughout the fermentation pomace contact period, the cap was punched down twice a
123
day, and the temperature and must density were recorded. At the end of this period, wines were
124
pressed at 1.5 bar in a 75 L tank membrane press. Free-run and press wines were combined and
125
stored at room temperature. The analyses were carried out at the end of alcoholic fermentation
126
(AF) in triplicate.
127 128
2.5 Spectrophotometric parameters in wine.
129
The wines (free of CO2) were first centrifuged. Colour intensity (CI), tone, CIELab
130
parameters, total anthocyanins (TA) and total phenols (TP(wine)) were analysed. The measurements
131
were performed on a Shimadzu UV/visible spectrophotometer, model 1600PC (Shimadzu,
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Duisburg, Germany). CI was calculated as the sum of absorbance at 620 (blue component), 520
133
(red component) and 420 nm (yellow component) in undiluted wine (Glories, 1984), and tone as
134
the ratio between absorbance at 420 nm and absorbance at 520 nm (Sudraud, 1958). The CIELab
135
parameter L* (lightness) was determined by measuring the transmittance of the wine every 10 nm
136
from 380 to 770 nm, using the D65 illuminant and a 10° observer angle. TP(wine) was calculated by
137
measuring wine absorbance at 280 nm, according to Ribéreau‐Gayon, Pontallier, & Glories (1983)
138
and TA by the method proposed by Ho, Silva, & Hogg (2001). 5
139 140
2.6 Isolation of cell wall material (CWM).
141
Cell wall material was isolated using the procedure described by Apolinar-Valiente et al
142
(2015b). For this, 10 g of grape tissue was suspended in 15 mL of boiling water for 5 min and then
143
homogenized. The homogenized material was mixed with two parts of 96% ethanol and extracted
144
for 30 min at 40 ºC. The raw alcohol insoluble solids were separated by filtration through a filter
145
paper and extracted again with fresh 70% ethanol for 30 min at 40 ºC. A sample from the liquid
146
phase was taken for soluble sugar analysis (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The
147
washing process with fresh 70% ethanol was repeated several times until the Dubois test indicated
148
no sugars remained in the 70% ethanol phase. Then, the alcohol insoluble solids (AIS) were rinsed
149
twice with 96% ethanol and once with acetone, and finally dried overnight under an air stream at
150
20 ºC.
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2.7 Analysis of grape skin cell wall composition.
153
The cell wall composition was analysed according to Castro-López, Gómez-Plaza, Ortega-
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Regules, Lozada, & Bautista-Ortín (2016). Uronic acids were determined in the sulfuric acid cell
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wall hydrosylate by the colorimetric 3,5-dimethylphenol assay after pre-treating the cell walls (30
156
℃, 1 h) with aqueous 72% sulfuric acid, followed by hydrolysis with 1 M sulfuric acid (100 ℃, 3 h).
157
Pure galacturonic acid was used as standard. The proteins and total phenolic compound content of
158
the cell wall material was determined after extraction with 1M NaOH (100 ℃, 10 min) by the
159
colorimetric Coomassie Brilliant Blue assay and by the colorimetric Folin–Ciocalteau reagent
160
assay, respectively. Bovine serum albumin (BSA) fraction V and pure gallic acid were used as
161
standards, respectively. Total glucose was determined using a kit for glucose enzymatic analysis
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after pre-treatment (30 ℃, 1 h) with aqueous 72% sulfuric acid, followed by hydrolysis using 1 M
163
sulfuric acid (100 ℃, 3 h) to determine non-cellulosic glucose. Cellulosic glucose was obtained by
164
difference between the total glucose and non-cellulosic glucose content. Klason lignin was
6
165
determined gravimetrically after sulfuric acid hydrolysis (Theander & Aman, 1979) lignin content
166
was expressed as mg g-1 of cell wall.
167
2.8 Statistical analysis.
168
Significant differences among wines and grapes and for each variable were assessed by
169
analysis of variance (ANOVA) and multifactorial analysis of variance (MANOVA) using
170
Statgraphics 5.0 Plus package (Statpoint Technologies, Inc., Warrenton, VA, USA). The Duncan
171
test was used to separate the means (p < 0.05) when the ANOVA or MANOVA test was significant.
172
Pearson correlations were used to test for relationships between different measurements.
173
3. Results and discussion.
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3.1 Physicochemical characteristics of the grapes.
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The physicochemical data at the moment of harvest are shown in Table 1. As can be
176
observed, Monastrell berries were the largest, followed by Merlot and Cabernet Sauvignon grapes.
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No differences were found between the two seasons studied, except that Monastrell had larger
178
berries and with a lower sugar content in 2015, which may be related to the higher precipitation
179
recorded between June and September 2015 (Figure 1), leading to a dilution of the sugars. The
180
weather differences between years could have influenced grape physicochemical composition as
181
suggested by Ruiz-García et al. (2012).
182
With regard to Merlot, the grapes treated with MeJ were heavier than those of the
183
corresponding control in 2015. Cabernet Sauvignon had the largest berries in treated grapes (MeJ
184
and BTH treatments) in 2016 and were the only ones that significantly increased the skin/berry
185
ratio (94.2 and 85.9 mg skin/berry, respectively) during this vintage. The skin/berry ratio is a
186
fundamental value to take into account during winemaking, since the higher the proportion, the
187
greater the amount of compounds of potential interest synthesized in their skins, which can then be
188
released during the vinification process (Apolinar-Valiente, Romero-Cascales, Gómez-Plaza,
189
López-Roca, & Ros-García, 2015a).
190
As regards °Brix during 2015, the only significant differences were found in Merlot (MeJ
191
treatment), with values lower than those of the control, and Cabernet Sauvignon (BTH treatment) 7
192
in which the values increased over control values. In 2016, all three varieties treated with MeJ and
193
BTH showed a decreased °Brix with respect to their respective controls, suggesting that the
194
treatments delayed the maturation process. Similar results were found by D’Onofrio, Matarese, &
195
Cuzzola, (2018), when they applied MeJ to Sangiovese vines; and Fernandez-Marin et al. (2013)
196
when they applied BTH to Syrah vines. It might well be that the extent of the response to
197
treatments with MeJ and BTH is related to the weather conditions since, in our case, the greatest
198
response to the treatments was found in 2016, when rainfall was lower (Figure 1).
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3.2 Cell wall components.
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The cell walls are responsible for several characteristics of the berries, including firmness
202
and mechanical properties. However, they may also act as a barrier in the extraction of phenolic
203
compounds during winemaking (Chardonnet, Gomez, & Doneche, 1994). Table 2 shows the
204
results from the analysis of the grape skin cell wall components.
205 206
3.2.1 Total phenol, proteins and lignin in cell walls.
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Phenols and proteins are important compounds in the cell wall. Phenolic compounds such
208
as ferulic acid play an important role in resistance to fungal pathogens (Schnitzler, Madlung, Rose,
209
& Seitz, 1992). In our study, treatment with MeJ increased the phenol concentration in the cell
210
walls of Monastrell and Cabernet Sauvignon varieties during 2015 and 2016, when there were
211
significant increases in TP(c-w). However in Merlot variety, MeJ and BTH caused a decrease in
212
these compounds during both vintages.
213
The protein content of the cell wall was also influenced by the different treatments. Thus,
214
the highest protein levels during 2015 were found in Monastrell (BTH treatment) and Cabernet
215
Sauvignon (MeJ treatment), while in 2016 the protein levels only increased in Monastrell (MeJ
216
treatment). However, as occurred in TP(c-w), Merlot variety showed the lowest protein values during
217
two consecutive years when treated with both elicitors. Several proteins such as extensins and
218
proline-rich proteins are involved in the cell protection processes induced in response to wounding, 8
219
pathogen invasion or light (Showalter, 1993). It has also been reported that an increase in
220
structural proteins contributes to the strength necessary to maintain the integrity of the berry
221
(Huang, Huang, & Wang, 2005).
222
The lignin content was hardly affected by the treatments, except for a significant decrease
223
in Cabernet Sauvignon (MeJ treatment) in 2016. Depending on the method used, the amount of
224
lignin can be affected by the presence of proteins and phenolic compounds (Femenia, Sánchez,
225
Simal, & Rosselló, 1998); however, the data referring to lignin data barely changed in our
226
conditions, even when differences were found in the amounts of proteins and phenolic compounds.
227
Although lignin has also been associated with mechanical support, sap conduction and defence
228
mechanisms (Boudet, 2000), this effect was not observed in our samples.
229 230
3.2.2 Carbohydrate composition of cell walls (Cellulosic glucose, glucose and uronic acids).
231
The type and amount of the carbohydrates found as the main components of the skin cell
232
wall indicated the presence of pectic polysaccharides, hemicelluloses and cellulose. The presence
233
of pectic polysaccharides could be inferred from the large amounts of uronic acids. The cellulose
234
was inferred from the fact that the bulk of the glucose could be released only after a Seaman
235
hydrolysis. The presence of non-cellulosic glucose was indicative of the occurrence of
236
hemicellulosic polysaccharides (Ortega-Regules et al., 2006).
237
Cellulosic glucose and uronic acids represented the highest percentage of sugars in the cell
238
walls of skin, as described in the same varieties by Nunan, Sims, Bacic, Robinson, & Fincher
239
(1997) and Ortega-Regules, Ros-García, Bautista-Ortín, López-Roca, & Gómez-Plaza (2008),
240
although in a different proportion, probably due to the use of a different isolation technique from
241
that used in our work. The uronic acid concentrations in Merlot were higher in both treatments
242
(MeJ and BTH) than in the corresponding control in 2015. By contrast, Cabernet Sauvignon grapes
243
showed lower concentrations in the BTH treatment (in 2015) and in both treatments in the following
244
vintage than in the corresponding control.
9
245
The only changes observed in the amount of cellulose in the cell walls were found in MeJ-
246
treated grapes from Merlot, which showed lower values in both seasons compared with the
247
corresponding controls. Also, the cellulose content in Monastrell cell walls during the 2015 vintage
248
was higher compared with the rest of the varieties during the two vintages. Romero-Cascales,
249
Ortega-Regules, Lopez-Roca, Fernandez-Fernandez, & Gomez-Plaza (2005) reported that an
250
increase in the amount of cellulose, together with a thicker skin, may explain the difficulties usually
251
observed in the extraction of anthocyanins from Monastrell grapes during winemaking.
252 253
3.3 Wine chromatic characteristics.
254
The chromatic parameters of the wines analysed at the end of alcoholic fermentation are
255
shown in Table 3. As can be seen, the values found for the parameters differed between seasons.
256
In general, there were no differences from the control wines in the parameters measured in 2015,
257
except in the MeJ-treated wines from Merlot, in which the TP(wine) values were higher.
258
However in 2016 the TA content increased significantly in Monastrell (Mej and BTH
259
treatment) and Merlot (Mej and BTH treatment) but not in Cabernet Sauvignon wines; in addition,
260
the wines obtained from Monastrell (Mej and BTH treatment) and Merlot (MeJ treatment) showed a
261
higher CI than the control. Ruiz-García et al. (2012) also reported increases in CI and TP(wine) in
262
wines from Monastrell grapes treated with MeJ and BTH, which agrees with our results for 2016.
263
This is an advantage from the oenological point of view, since the grape skins are the major source
264
of colour and aroma compounds (Ortega-Regules et al., 2008).With respect to the tone and L*
265
parameter during the two vintages studied, as the results show, they were barely affected by the
266
treatments.
267
The more pronounced differences from the respective controls found for TA and CI during
268
2016 might be due to the fact that rainfall from June to September in this year (Figure 2) was lower
269
than in 2015 (Figure 1). It has been widely reported that the meteorological conditions have a great
270
influence on the concentrations of various components in berries (Gil-Muñoz, Fernández-
271
Fernández, Vila-López, & Martinez-Cutillas, 2010); indeed, an increase in rainfall or volume of
10
272
irrigation water during the months prior to harvesting affect berry development, leading to an
273
increase in size and the dilution of some cellular components, such as sugars or phenolic
274
compounds (Romero et al., 2016). These effects are especially important in the case of flavonoids,
275
which play an important role in the chromatic characteristics and long-term stability of red wines, as
276
well as for some organoleptic properties such as astringency, bitterness and body (Ruiz-García et
277
al., 2012).
278 279
3.4 Multivariate analysis
280
Table 4 shows a multifactor analysis of the variance in cell wall composition, using year,
281
treatment and variety as factors. With regard to the year effect, significant differences were found
282
for some of the cell wall components. For example, in 2015, higher values were registered for all
283
the components, except UA, glucose and Cell-Glu, which may be related with the greater rainfall
284
recorded that year, and the higher probability of fungal diseases, which would have triggered
285
different defence mechanisms to reinforce the cell wall, as explained above. In the multifactor
286
analysis, the treatment applied also influenced the cell wall, although to differing extents; MeJ and
287
BTH reduced the proportion of these components, with the exception of lignin and Cell-Glu.
288
Variety is another factor that influenced these components; thus we found that Monastrell
289
wines had a higher content of proteins, UA and Cell-Glu, but lower lignin content than Merlot and
290
Cabernet Sauvignon. As suggested by the interactions observed in Table 4, most of the
291
components of the cell wall were influenced by meteorological conditions, the applied treatment
292
and the variety. The TP(c-w), UA and glucose showed significant differences in all the interactions
293
performed. However, lignin was hardly affected, except in the year-variety interaction. The strong
294
interaction between year and variety (Y x V) was of note, since this had the strongest influence on
295
the variability of the different components of the cell wall.
296 297
3.5 Correlation between cell wall components and wine chromatic characteristics
11
298
High correlation coefficients were found between the cell wall proteins of grapes treated
299
with MeJ and the wine chromatic characteristics (Table 5); it was found that an increase in the
300
protein content of the cell wall caused a decrease in the TA and TP(wine), accompanied by a
301
decrease in CI. This partly confirms what was previously explained - that an increase in structural
302
proteins contributes to the strength necessary to maintain the integrity of the berry (Huang et al.,
303
2005), thus hindering the extraction of phenolic compounds.
304
A high correlation was also found between the UA and the TP of the grapes treated with
305
MeJ and BTH. These results indicated that the higher the percentage of UA in cell walls, the lower
306
the amount of phenolic compounds such as TP(wine) that can be extracted, as described by Ortega
307
Regules (2006) in different grape varieties, the same author also relating this problem to the
308
presence of pectic polysaccharides. Likewise, Rosli, Civello, & Martínez (2004) found
309
presence of UA to be related with firmer strawberry cultivars. According to these correlations, the
310
extractability of phenolic compounds can be partly explained by the components of the cell wall.
the
311 312
4. Conclusions
313
The results indicate that the exogenous application of MeJ and BTH during veraison
314
causesd significant changes in several components of the skin cell walls, such as phenolic
315
compounds, proteins and structural sugars. However, the extent of these changes differed
316
between varieties and each year, indicating that the response to the application of these elicitors
317
has a varietal and meteorological dependence. Likewise, the treatments delayed the maturation
318
process (lower concentration of sugar) in all grape varieties when rainfall was low. This fact,
319
together with the increase in proteins and phenols observed in the skin cell wall of Monastrell and
320
Cabernet Sauvignon, may contribute to the strength necessary to maintain the integrity of the berry
321
and resistance to fungal pathogens, and thus be able to improve the phenolic profile. However, in
322
the Merlot variety the treatments reduced the content of proteins, phenols and cellulose in the cell
323
wall, the reduction in these three components leading to a decrease in structural integrity.
12
324
On the other hand, wines made from Monastrell and Merlot grapes treated with MeJ and
325
BTH showed significant increases in total anthocyanins in 2016, although not to the extent
326
expected. In this case, the higher percentages of phenolic compounds reached following the
327
application of elicitors may have been accompanied by a greater consistency of the cell wall, thus
328
hindering extraction. Therefore, a more exhaustive study of the total phenolic compounds of fresh
329
grape skins is necessary in order to determine the real increase achieved by the use of MeJ and
330
BTH.
331
Acknowledgments: This work was made possible by financial assistance from the Instituto
332
Nacional de Investigación y Tecnología Agraria y Alimentaria (RTA2013-00053-C03-02). Diego F.
333
Paladines-Quezada is the holder of an FPI fellowship from the Spanish Government.
334 335
5. References
336 337
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S0308-8146(18)31968-X https://doi.org/10.1016/j.foodchem.2018.11.029 FOCH 23838
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
8 January 2018 30 October 2018 5 November 2018
Please cite this article as: Paladines-Quezada, D.F., Moreno-Olivares, J.D., Fernández-Fernández, J.I., BautistaOrtín, A.B., Gil-Muñoz, R., Influence of Methyl Jasmonate and Benzothiadiazole on the composition of grape skin cell walls and wines, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.11.029
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1 2
Influence of Methyl Jasmonate and Benzothiadiazole on the composition of grape skin cell walls and wines.
3 4 5
Paladines-Quezada, D.F a*; Moreno-Olivares, J. D a; Fernández-Fernández, J. I. a; BautistaOrtín, A.B b and Gil-Muñoz, R* a.
6 7 8 9 10
a
b
Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario, Ctra. La Alberca s/n, 30150, Murcia, Spain.
Departamento de Tecnología de Alimentos, Nutrición y Bromatología, Facultad de Veterinaria, Universidad de Murcia, 30100 Murcia, Spain.
11 12
* Corresponding author. Tel.: +34 968 757580. E-mail address: [email protected]
13 14
Abstract.
15
Phenolic compounds are very important in crop plants, particularly in grapes. The different
16
strategies to increase their levels include the use of elicitors such as methyl jasmonate (MeJ) and
17
benzothiadiazole (BTH). In an attempt to improve the quality of wines, our aim was to evaluate the
18
effect of preharvest application of these elicitors on the composition and structure of the skin cell
19
walls of Monastrell, Merlot and Cabernet Sauvignon grapes, and to ascertain any relationship with
20
the extractability of phenolic compounds during winemaking. The results indicated that the
21
exogenous application of MeJ and BTH during veraison caused significant changes in several
22
components of the skin cell walls, such as phenolic compounds, proteins and structural sugars.
23
However these changes manifested themselves in different proportions in each variety and year,
24
pointing to the varietal and meteorological dependence of the response to the application of these
25
elicitors. The treatments delayed the maturation process in all varieties when rainfall was low. This
26
observation, together with the observed increase in proteins and phenols in the skin cell wall of
27
Monastrell and Cabernet Sauvignon, could contribute to the strength necessary to maintain the
28
integrity of berries and to increasing resistance to fungal pathogens as the phenolic compounds
29
evolve, thus improving the phenolic profile. However, the structural integrity of Merlot variety
30
tended to decrease in the same conditions.
1
31
Keywords: methyl jasmonate, benzothiadiazole, cell wall, grapes, wines.
32 33
1. Introduction.
34
Phenolic compoundshave been the subject of a large num ber of studies due to their
35
importance in crop plants. Three main reasons can be cited for optimizing the level of phenolic
36
compounds in such plants: their physiological role in the plant, their technological significance for
37
food processing, and their nutritional characteristics (Ruiz-García et al., 2012).
38
Several strategies exist to increase the phenolic compound content of grapes, including
39
cultural practices (pruning, deficit irrigation, thinning of clusters) (Pérez-Lamela, García-Falcón,
40
Simal-Gándara, & Orriols-Fernández, 2007), genetic breeding programmes, clonal selection of
41
varieties (Gómez-Plaza et al., 2008), or the use of elicitors - exogenously applied chemical
42
substances that trigger the activation of certain metabolic pathways, causing the biosynthesis of
43
phenolic and volatile compounds (Gómez-Plaza, Bautista-Ortín, Ruiz-García, Fernández-
44
Fernández, & Gil-Muñoz, 2017). Among these substances methyl jJasmonate (MeJ) and
45
Benzothiadiazole (BTH) have been used to improve the phenolic content of fruits, particularly
46
grapes, while also being considered useful as agrochemicals to improve resistance against plant
47
pathogens (Gil-Muñoz, Bautista-Ortín, Ruiz-García, Fernández-Fernández, & Gómez-Plaza,
48
2017). This resistance may be based on previously existing physical or chemical barriers (such as
49
thick cell walls or high amounts of lignin or tannins) or inducible defense mechanisms (Gozzo,
50
2003).
51
Methyl jasmonate (MeJ) is a potent resistance regulator derived from jasmonic acid that
52
triggers a large number of defense responses, including the synthesis of flavonoid compounds and
53
stilbenes (Beckers & Spoel, 2006). This elicitor is able to activate the enzymes responsible for the
54
biosynthesis of polyphenols, such as the enzyme, phenylalanine ammonia-lyase (PAL). The
55
activation of PAL following postharvest application of the elicitor has been confirmed in many
56
studies in fruits such as lychees, peaches, apples, plums, table grapes, strawberries, accompanied
57
by an increase in total phenols (Ruiz-García & Gómez-Plaza, 2013). Benzothiadiazole (BTH), a
58
photostable functional analogue of the plant signal molecule salicylic acid (SA), has been shown to 2
59
have a double action in plant protection; on the one hand, it inhibits the development of decay-
60
causing fungi through its direct toxicity, and, on the other hand, it also indirectly induces
61
pathogenesis-related (PR) genes, leading to the establishment of systemic acquired resistance
62
(SAR) in a variety of plants, providing broad-spectrum protection against various pathogens
63
(Wendehenne, Durner, Chen, & Klessig, 1998). There are many studies on the effect of pre-
64
harvest application of MeJ and BTH to wine grapes. Such studies have pointed to increased levels
65
of phenolic compounds in the treated grapes and corresponding wines for many varieties (Gil-
66
Muñoz et al., 2017).
67
On the other hand, although skins represent a small percentage of total berry weight, they
68
are fundamental in wine quality, since most of the aromatic and phenolic compounds are located
69
therein. Therefore, it is necessary to know the composition and structural properties of the skins of
70
different varieties, since thse factors can determine the mechanical resistance and texture of
71
berries, and the ease with which they can be processed (Barnavon et al., 2000). Thus, the cell
72
walls of grape skins acquire great relevance, since they are highly complex and dynamic, being
73
composed of polysaccharides, phenolic compounds and proteins, and stabilized by ionic and
74
covalent linkages, all of which may differ between varieties (Ortega-Regules, Romero-Cascales,
75
Ros-García, López-Roca, & Gómez-Plaza, 2006) and even within the same variety grown in
76
different terroirs (Apolinar-Valiente, Romero-Cascales, Gómez-Plaza, López-Roca, & Ros-García,
77
2015b)
78
In order to provide new tools to improve wine quality, the objective of this work was to
79
evaluate whether the application of two pre-harvest elicitors (MeJ and BTH) to Monastrell, Merlot
80
and Cabernet Sauvignon grapes affects the composition and structure of their skin cell walls, and
81
to check whether they are related to the extractability of phenolic compounds during winemaking.
82 83
2. Materials and methods.
84
2.1 Reagents and standards.
3
85
All solvents (acetone, ethanol) were of HPLC quality, and all chemicals were of analytical
86
grade (>99%). Water was of Milli-Q quality. BTH ([benzo-(1, 2, 3)-thiadiazole-7- carbothioic acid S-
87
methyl ester]); MeJ (methyl jasmonate); Tween 80; 3, 5-dimethylphenol, were from Sigma Aldrich
88
(St. Louis, USA). For glucose determination, an enzymatic analysis kit from R-biopharm
89
(Darmstadt, Germany) was used. As standards, pure galacturonic acid and gallic acid were
90
purchased from Sigma Aldrich (St. Louis, USA) and Bovine serum albumin (BSA) from J.T. Baker
91
(Deventer, The Netherlands).
92 93
2.2 Experimental design.
94
The experiment was carried out in two consecutive years (2015-2016) using three grape
95
varieties (Monastrell, Merlot and Cabernet Sauvignon) grown in Jumilla, Murcia (south-eastern
96
Spain). The study was performed on 14 year old Vitis vinifera (syn. Mourvedre) red wine
97
grapevines grafted onto 1103-Paulsen (clon 249) rootstock and trained to a three-vine vertical
98
trellis system. Vine rows were arranged in N-NW to S-SE with between-row and within-row spacing
99
of 3 x 1.25 m (x: 636.099; y: 4.249.299).
100
All treatments (spray application of the elicitors MeJ and BTH on vine clusters) were
101
performed in triplicate, with ten vines per replicate. The protocol used to apply the different
102
treatments was described previously (Gil-Muñoz et al., 2017). To carry out the treatments,
103
aqueous solutions were prepared with Tween 80 as wetting agent (0.1% v/v). BTH was used at a
104
concentration of 0.3 mM, and MeJ was used at a concentration of 10 mM. Control plants were
105
sprayed with aqueous solution of Tween 80 alone. In all cases, 200 mL of aqueous solution was
106
applied per plant. The treatments were carried out twice, at veraison and 1 week later. When
107
grapes reached optimum maturity, they were harvested and transported in boxes for
108
physicochemical analysis and vinification. Samples of grape berries (ca. 800 g) were taken to
109
isolate their skin cell wall. The skins were totally separated from the pulp, and stored at -80 ℃ until
110
the cell wall material (CWM) was isolated.
111
4
112
2.3 Physico- chemical analysis in grapes at harvest.
113
Total soluble solids (°Brix) were measured using an Abbé-type refractometer (Atago RX-
114
5000). The methodology for carrying out these analyses is described in ECC. Commission
115
Regulation No. 2676/90.
116 117
2.4 Vinifications.
118
All the vinifications were made in triplicate in 50 L stainless steel tanks using 50 kg of
119
grapes. Grapes were destemmed, crushed and sulfited (8 g SO2/ 100 kg). Total acidity was
120
corrected to 5.5 g/L with tartaric acid, and selected yeasts were added (Uvaferm VRB, Lallemand,
121
25 g/hL). All vinifications were conducted at 25±1°C. The fermentative pomace contact period was
122
10 days. Throughout the fermentation pomace contact period, the cap was punched down twice a
123
day, and the temperature and must density were recorded. At the end of this period, wines were
124
pressed at 1.5 bar in a 75 L tank membrane press. Free-run and press wines were combined and
125
stored at room temperature. The analyses were carried out at the end of alcoholic fermentation
126
(AF) in triplicate.
127 128
2.5 Spectrophotometric parameters in wine.
129
The wines (free of CO2) were first centrifuged. Colour intensity (CI), tone, CIELab
130
parameters, total anthocyanins (TA) and total phenols (TP(wine)) were analysed. The measurements
131
were performed on a Shimadzu UV/visible spectrophotometer, model 1600PC (Shimadzu,
132
Duisburg, Germany). CI was calculated as the sum of absorbance at 620 (blue component), 520
133
(red component) and 420 nm (yellow component) in undiluted wine (Glories, 1984), and tone as
134
the ratio between absorbance at 420 nm and absorbance at 520 nm (Sudraud, 1958). The CIELab
135
parameter L* (lightness) was determined by measuring the transmittance of the wine every 10 nm
136
from 380 to 770 nm, using the D65 illuminant and a 10° observer angle. TP(wine) was calculated by
137
measuring wine absorbance at 280 nm, according to Ribéreau‐Gayon, Pontallier, & Glories (1983)
138
and TA by the method proposed by Ho, Silva, & Hogg (2001). 5
139 140
2.6 Isolation of cell wall material (CWM).
141
Cell wall material was isolated using the procedure described by Apolinar-Valiente et al
142
(2015b). For this, 10 g of grape tissue was suspended in 15 mL of boiling water for 5 min and then
143
homogenized. The homogenized material was mixed with two parts of 96% ethanol and extracted
144
for 30 min at 40 ºC. The raw alcohol insoluble solids were separated by filtration through a filter
145
paper and extracted again with fresh 70% ethanol for 30 min at 40 ºC. A sample from the liquid
146
phase was taken for soluble sugar analysis (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The
147
washing process with fresh 70% ethanol was repeated several times until the Dubois test indicated
148
no sugars remained in the 70% ethanol phase. Then, the alcohol insoluble solids (AIS) were rinsed
149
twice with 96% ethanol and once with acetone, and finally dried overnight under an air stream at
150
20 ºC.
151 152
2.7 Analysis of grape skin cell wall composition.
153
The cell wall composition was analysed according to Castro-López, Gómez-Plaza, Ortega-
154
Regules, Lozada, & Bautista-Ortín (2016). Uronic acids were determined in the sulfuric acid cell
155
wall hydrosylate by the colorimetric 3,5-dimethylphenol assay after pre-treating the cell walls (30
156
℃, 1 h) with aqueous 72% sulfuric acid, followed by hydrolysis with 1 M sulfuric acid (100 ℃, 3 h).
157
Pure galacturonic acid was used as standard. The proteins and total phenolic compound content of
158
the cell wall material was determined after extraction with 1M NaOH (100 ℃, 10 min) by the
159
colorimetric Coomassie Brilliant Blue assay and by the colorimetric Folin–Ciocalteau reagent
160
assay, respectively. Bovine serum albumin (BSA) fraction V and pure gallic acid were used as
161
standards, respectively. Total glucose was determined using a kit for glucose enzymatic analysis
162
after pre-treatment (30 ℃, 1 h) with aqueous 72% sulfuric acid, followed by hydrolysis using 1 M
163
sulfuric acid (100 ℃, 3 h) to determine non-cellulosic glucose. Cellulosic glucose was obtained by
164
difference between the total glucose and non-cellulosic glucose content. Klason lignin was
6
165
determined gravimetrically after sulfuric acid hydrolysis (Theander & Aman, 1979) lignin content
166
was expressed as mg g-1 of cell wall.
167
2.8 Statistical analysis.
168
Significant differences among wines and grapes and for each variable were assessed by
169
analysis of variance (ANOVA) and multifactorial analysis of variance (MANOVA) using
170
Statgraphics 5.0 Plus package (Statpoint Technologies, Inc., Warrenton, VA, USA). The Duncan
171
test was used to separate the means (p < 0.05) when the ANOVA or MANOVA test was significant.
172
Pearson correlations were used to test for relationships between different measurements.
173
3. Results and discussion.
174
3.1 Physicochemical characteristics of the grapes.
175
The physicochemical data at the moment of harvest are shown in Table 1. As can be
176
observed, Monastrell berries were the largest, followed by Merlot and Cabernet Sauvignon grapes.
177
No differences were found between the two seasons studied, except that Monastrell had larger
178
berries and with a lower sugar content in 2015, which may be related to the higher precipitation
179
recorded between June and September 2015 (Figure 1), leading to a dilution of the sugars. The
180
weather differences between years could have influenced grape physicochemical composition as
181
suggested by Ruiz-García et al. (2012).
182
With regard to Merlot, the grapes treated with MeJ were heavier than those of the
183
corresponding control in 2015. Cabernet Sauvignon had the largest berries in treated grapes (MeJ
184
and BTH treatments) in 2016 and were the only ones that significantly increased the skin/berry
185
ratio (94.2 and 85.9 mg skin/berry, respectively) during this vintage. The skin/berry ratio is a
186
fundamental value to take into account during winemaking, since the higher the proportion, the
187
greater the amount of compounds of potential interest synthesized in their skins, which can then be
188
released during the vinification process (Apolinar-Valiente, Romero-Cascales, Gómez-Plaza,
189
López-Roca, & Ros-García, 2015a).
190
As regards °Brix during 2015, the only significant differences were found in Merlot (MeJ
191
treatment), with values lower than those of the control, and Cabernet Sauvignon (BTH treatment) 7
192
in which the values increased over control values. In 2016, all three varieties treated with MeJ and
193
BTH showed a decreased °Brix with respect to their respective controls, suggesting that the
194
treatments delayed the maturation process. Similar results were found by D’Onofrio, Matarese, &
195
Cuzzola, (2018), when they applied MeJ to Sangiovese vines; and Fernandez-Marin et al. (2013)
196
when they applied BTH to Syrah vines. It might well be that the extent of the response to
197
treatments with MeJ and BTH is related to the weather conditions since, in our case, the greatest
198
response to the treatments was found in 2016, when rainfall was lower (Figure 1).
199 200
3.2 Cell wall components.
201
The cell walls are responsible for several characteristics of the berries, including firmness
202
and mechanical properties. However, they may also act as a barrier in the extraction of phenolic
203
compounds during winemaking (Chardonnet, Gomez, & Doneche, 1994). Table 2 shows the
204
results from the analysis of the grape skin cell wall components.
205 206
3.2.1 Total phenol, proteins and lignin in cell walls.
207
Phenols and proteins are important compounds in the cell wall. Phenolic compounds such
208
as ferulic acid play an important role in resistance to fungal pathogens (Schnitzler, Madlung, Rose,
209
& Seitz, 1992). In our study, treatment with MeJ increased the phenol concentration in the cell
210
walls of Monastrell and Cabernet Sauvignon varieties during 2015 and 2016, when there were
211
significant increases in TP(c-w). However in Merlot variety, MeJ and BTH caused a decrease in
212
these compounds during both vintages.
213
The protein content of the cell wall was also influenced by the different treatments. Thus,
214
the highest protein levels during 2015 were found in Monastrell (BTH treatment) and Cabernet
215
Sauvignon (MeJ treatment), while in 2016 the protein levels only increased in Monastrell (MeJ
216
treatment). However, as occurred in TP(c-w), Merlot variety showed the lowest protein values during
217
two consecutive years when treated with both elicitors. Several proteins such as extensins and
218
proline-rich proteins are involved in the cell protection processes induced in response to wounding, 8
219
pathogen invasion or light (Showalter, 1993). It has also been reported that an increase in
220
structural proteins contributes to the strength necessary to maintain the integrity of the berry
221
(Huang, Huang, & Wang, 2005).
222
The lignin content was hardly affected by the treatments, except for a significant decrease
223
in Cabernet Sauvignon (MeJ treatment) in 2016. Depending on the method used, the amount of
224
lignin can be affected by the presence of proteins and phenolic compounds (Femenia, Sánchez,
225
Simal, & Rosselló, 1998); however, the data referring to lignin data barely changed in our
226
conditions, even when differences were found in the amounts of proteins and phenolic compounds.
227
Although lignin has also been associated with mechanical support, sap conduction and defence
228
mechanisms (Boudet, 2000), this effect was not observed in our samples.
229 230
3.2.2 Carbohydrate composition of cell walls (Cellulosic glucose, glucose and uronic acids).
231
The type and amount of the carbohydrates found as the main components of the skin cell
232
wall indicated the presence of pectic polysaccharides, hemicelluloses and cellulose. The presence
233
of pectic polysaccharides could be inferred from the large amounts of uronic acids. The cellulose
234
was inferred from the fact that the bulk of the glucose could be released only after a Seaman
235
hydrolysis. The presence of non-cellulosic glucose was indicative of the occurrence of
236
hemicellulosic polysaccharides (Ortega-Regules et al., 2006).
237
Cellulosic glucose and uronic acids represented the highest percentage of sugars in the cell
238
walls of skin, as described in the same varieties by Nunan, Sims, Bacic, Robinson, & Fincher
239
(1997) and Ortega-Regules, Ros-García, Bautista-Ortín, López-Roca, & Gómez-Plaza (2008),
240
although in a different proportion, probably due to the use of a different isolation technique from
241
that used in our work. The uronic acid concentrations in Merlot were higher in both treatments
242
(MeJ and BTH) than in the corresponding control in 2015. By contrast, Cabernet Sauvignon grapes
243
showed lower concentrations in the BTH treatment (in 2015) and in both treatments in the following
244
vintage than in the corresponding control.
9
245
The only changes observed in the amount of cellulose in the cell walls were found in MeJ-
246
treated grapes from Merlot, which showed lower values in both seasons compared with the
247
corresponding controls. Also, the cellulose content in Monastrell cell walls during the 2015 vintage
248
was higher compared with the rest of the varieties during the two vintages. Romero-Cascales,
249
Ortega-Regules, Lopez-Roca, Fernandez-Fernandez, & Gomez-Plaza (2005) reported that an
250
increase in the amount of cellulose, together with a thicker skin, may explain the difficulties usually
251
observed in the extraction of anthocyanins from Monastrell grapes during winemaking.
252 253
3.3 Wine chromatic characteristics.
254
The chromatic parameters of the wines analysed at the end of alcoholic fermentation are
255
shown in Table 3. As can be seen, the values found for the parameters differed between seasons.
256
In general, there were no differences from the control wines in the parameters measured in 2015,
257
except in the MeJ-treated wines from Merlot, in which the TP(wine) values were higher.
258
However in 2016 the TA content increased significantly in Monastrell (Mej and BTH
259
treatment) and Merlot (Mej and BTH treatment) but not in Cabernet Sauvignon wines; in addition,
260
the wines obtained from Monastrell (Mej and BTH treatment) and Merlot (MeJ treatment) showed a
261
higher CI than the control. Ruiz-García et al. (2012) also reported increases in CI and TP(wine) in
262
wines from Monastrell grapes treated with MeJ and BTH, which agrees with our results for 2016.
263
This is an advantage from the oenological point of view, since the grape skins are the major source
264
of colour and aroma compounds (Ortega-Regules et al., 2008).With respect to the tone and L*
265
parameter during the two vintages studied, as the results show, they were barely affected by the
266
treatments.
267
The more pronounced differences from the respective controls found for TA and CI during
268
2016 might be due to the fact that rainfall from June to September in this year (Figure 2) was lower
269
than in 2015 (Figure 1). It has been widely reported that the meteorological conditions have a great
270
influence on the concentrations of various components in berries (Gil-Muñoz, Fernández-
271
Fernández, Vila-López, & Martinez-Cutillas, 2010); indeed, an increase in rainfall or volume of
10
272
irrigation water during the months prior to harvesting affect berry development, leading to an
273
increase in size and the dilution of some cellular components, such as sugars or phenolic
274
compounds (Romero et al., 2016). These effects are especially important in the case of flavonoids,
275
which play an important role in the chromatic characteristics and long-term stability of red wines, as
276
well as for some organoleptic properties such as astringency, bitterness and body (Ruiz-García et
277
al., 2012).
278 279
3.4 Multivariate analysis
280
Table 4 shows a multifactor analysis of the variance in cell wall composition, using year,
281
treatment and variety as factors. With regard to the year effect, significant differences were found
282
for some of the cell wall components. For example, in 2015, higher values were registered for all
283
the components, except UA, glucose and Cell-Glu, which may be related with the greater rainfall
284
recorded that year, and the higher probability of fungal diseases, which would have triggered
285
different defence mechanisms to reinforce the cell wall, as explained above. In the multifactor
286
analysis, the treatment applied also influenced the cell wall, although to differing extents; MeJ and
287
BTH reduced the proportion of these components, with the exception of lignin and Cell-Glu.
288
Variety is another factor that influenced these components; thus we found that Monastrell
289
wines had a higher content of proteins, UA and Cell-Glu, but lower lignin content than Merlot and
290
Cabernet Sauvignon. As suggested by the interactions observed in Table 4, most of the
291
components of the cell wall were influenced by meteorological conditions, the applied treatment
292
and the variety. The TP(c-w), UA and glucose showed significant differences in all the interactions
293
performed. However, lignin was hardly affected, except in the year-variety interaction. The strong
294
interaction between year and variety (Y x V) was of note, since this had the strongest influence on
295
the variability of the different components of the cell wall.
296 297
3.5 Correlation between cell wall components and wine chromatic characteristics
11
298
High correlation coefficients were found between the cell wall proteins of grapes treated
299
with MeJ and the wine chromatic characteristics (Table 5); it was found that an increase in the
300
protein content of the cell wall caused a decrease in the TA and TP(wine), accompanied by a
301
decrease in CI. This partly confirms what was previously explained - that an increase in structural
302
proteins contributes to the strength necessary to maintain the integrity of the berry (Huang et al.,
303
2005), thus hindering the extraction of phenolic compounds.
304
A high correlation was also found between the UA and the TP of the grapes treated with
305
MeJ and BTH. These results indicated that the higher the percentage of UA in cell walls, the lower
306
the amount of phenolic compounds such as TP(wine) that can be extracted, as described by Ortega
307
Regules (2006) in different grape varieties, the same author also relating this problem to the
308
presence of pectic polysaccharides. Likewise, Rosli, Civello, & Martínez (2004) found
309
presence of UA to be related with firmer strawberry cultivars. According to these correlations, the
310
extractability of phenolic compounds can be partly explained by the components of the cell wall.
the
311 312
4. Conclusions
313
The results indicate that the exogenous application of MeJ and BTH during veraison
314
causesd significant changes in several components of the skin cell walls, such as phenolic
315
compounds, proteins and structural sugars. However, the extent of these changes differed
316
between varieties and each year, indicating that the response to the application of these elicitors
317
has a varietal and meteorological dependence. Likewise, the treatments delayed the maturation
318
process (lower concentration of sugar) in all grape varieties when rainfall was low. This fact,
319
together with the increase in proteins and phenols observed in the skin cell wall of Monastrell and
320
Cabernet Sauvignon, may contribute to the strength necessary to maintain the integrity of the berry
321
and resistance to fungal pathogens, and thus be able to improve the phenolic profile. However, in
322
the Merlot variety the treatments reduced the content of proteins, phenols and cellulose in the cell
323
wall, the reduction in these three components leading to a decrease in structural integrity.
12
324
On the other hand, wines made from Monastrell and Merlot grapes treated with MeJ and
325
BTH showed significant increases in total anthocyanins in 2016, although not to the extent
326
expected. In this case, the higher percentages of phenolic compounds reached following the
327
application of elicitors may have been accompanied by a greater consistency of the cell wall, thus
328
hindering extraction. Therefore, a more exhaustive study of the total phenolic compounds of fresh
329
grape skins is necessary in order to determine the real increase achieved by the use of MeJ and
330
BTH.
331
Acknowledgments: This work was made possible by financial assistance from the Instituto
332
Nacional de Investigación y Tecnología Agraria y Alimentaria (RTA2013-00053-C03-02). Diego F.
333
Paladines-Quezada is the holder of an FPI fellowship from the Spanish Government.
334 335
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