Effects of kaolin particle film on berry histological properties in two table grape cultivars ( V. vinifera L.)

Abstract

BACKGROUND:

Berry skin tissue is important in viticulture and enology due to the storage of phenolic compounds. Changes in the berry metabolism and environmental factors may lead to changes in skin structure and composition.

OBJECTIVE:

We aimed to determine the effects of kaolin-based particle film (PF) treatment on histological properties of anthocyanin accumulation in the berries of cv. Beauty Seedless and Tekirdag Seedless in two growing seasons.

METHODS:

PF treatment was applied 3 times beginning from the berry set. Histological structure of berries was observed with optical and transmission electron microscopy. Accumulation of anthocyanins in berry skin cells was determined microscopically and spectrophotometrically.

RESULTS:

In Beauty Seedless, total anthocyanin content was 512.93–605.90 mgkg^–1 in control (C) and it raised to 606.98–666.44 mgkg^–1 in PF treated vines. Total anthocyanin content was determined as 72.50–81.43 mgkg^–1 (C) and 79.55–84.72 mgkg^–1 (PF) in Tekirdag Seedless. 3 cell types were observed regarding anthocyanin localization: cells with no anthocyanin (type 1), cells with vacuolar storage (type 2) and cells with granular storage (type 3).

CONCLUSION:

We concluded that the PF treatment could be used for increasing anthocyanin concentration with cultivar-specific skin color in Beauty Seedless and Tekirdag Seedless grown in the semi-arid regions.

Keywords

anthocyanin concentration anthocyanic vacuolar inclusions (AVI)berry skin histochemistry

1. Introduction

Berry skin tissue has great importance in viticulture and enology due to the storage of phenolic compounds that gives the basic characteristics to grapes and wines. A significant amount of color, taste, and aroma compounds in grapes are located in the epidermis and hypodermis layers of the skin tissue. Berry texture, one of the important quality criteria for table grapes, also depends on the nature of the skin tissue. The structural characteristics of the berry skin, such as thickness and cell wall structure, are cultivar-specific and genetically influenced [1]. However, changes in berry metabolism and environmental factors may lead to changes in skin structure and composition [2]. It has been reported that skin thickness is particularly sensitive to climatic conditions and canopy microclimates [3]. Studies have shown that foliar applications of particle films can contribute to improving berry composition and wine quality by reducing heat and radiation stress in arid regions [4, 5].

In this context, evaluation of the accumulation of phenolic compounds in berry skin is important in table grapes for sensory properties and in wine grapes for anthocyanin and tannin extraction. Although the biosynthesis mechanism of phenolic compounds has been studied for many years, studies on the accumulation and transportation steps in tissues and their behavior under different environmental conditions have been a recent study area [6, 7]. Most of these studies focused on the accumulation and extraction of anthocyanins and tannins in wine grapes, but there is a lack of knowledge of these regarding table grapes.

In this study, it was aimed to evaluate the effects of PF treatment on histological accumulation of anthocyanins in berry skin of two seedless table grape cultivars grown in semi-arid conditions.

2. Material and methods

2.1. Plant material

The study was performed during the growing seasons of 2014–2016 on cv. Beauty Seedless and Tekirdag Seedless (released from the national breeding program, crossing combination: Alphonse Lavallée×Sultana). The grapevines were cultivated in vineyards of the Research Station for Viticulture in Kalecik, Ankara (40°06^′N 33°25^′E, 670 m a.s.l.). Both cultivars have a black skin color and are used for fresh consumption. The vineyards were planted in 2005 with 1.5×3 m row spacing. Grapevines were grafted on 1103P rootstock and trained to bilateral cordon. The vineyard soil was clay loam with a pH of 7.5. The research region has a continental climate.

2.2. Kaolin-based particle film (PF) treatment

Kaolin applications (Screenduo, CMM Inc.) were performed in the early morning period from 08.00–10.00 a.m. with a backpack sprayer. The product was only applied to the leaves, avoiding spray onto clusters. Three periodical sprayings were made weekly beginning from the berry set in randomly selected 3 vine per replicate. The concentrations of all applications were same (3% w/v). Untreated vines were left as the control (C). In 2015 severe frost damage occurred in Beauty Seedless cultivar (– 19°C on January 9 and – 2.13°C on April 24), for this reason, the second year data were obtained from 2016. Grapes were hand-harvested at approximately 17-18°Bx. Harvest dates for PF treatment and control vines were August 26 (2014), and August 24 (2016) in Beauty Seedless and September 15 (2014 and 2015) in Tekirdag Seedless.

2.3. Fruit zone temperature

Fruit zone temperatures were measured using a digital LCD infrared thermometer. Five measurements were taken per vine far from 30–40 cm to the east and west sides of leaf/cluster zone between 11:00 and 15:00 h.

2.4. Histological observations

Right after the harvest, 10 berries per replicate were randomly selected for histological observations. Skin pieces were removed from the berries and fixed in 3% glutaraldehyde buffered with 0.1 M phosphate (pH 7.2) for 2 h. Post-fixation was carried out in 1% osmium tetroxide for 3 h at room temperature. Samples were dehydrated in a graded ethanol series. They were then transferred to 100% propylene oxide and embedded in an epoxy resin according to Luft [8]. Semi-thin and ultra-thin sections were obtained with an ultramicrotome (Reichert OMU-4). Semi-thin sections were stained with 1% toluidine blue and observed with a Leica CME DFC290 optical microscope whereas ultra-thin sections were stained with uranyl acetate and lead citrate [9] and examined with a Jeol CXII transmission electron microscope at 80 kV. Histological accumulation type of anthocyanins in the berry skin cells was examined by toluidine blue staining method according to Cadot et al. [10].

2.5. Measurements of berry parts

Berry weight, size, and berry skin thickness were measured and relative proportions of skin and flesh were determined. All the measurements were made in 50 berries/replicate.

The weight of the berries was determined with a scale.

The measurements of berry width and length were done using a digital caliper.

The thickness (μm) of berry skins (including epidermal and hypodermal tissues) was measured by an ocular micrometer under the binocular microscope (Leica S6D). For this aim, berries were cut equatorially and measurements were taken from four sides in two berry pieces.

The proportions of skin and flesh were calculated using the differences between berry and skin/flesh weight [11]. Berry skins were weighted after the skin part carefully removed from the flesh using a razor blade.

2.6. Berry skin color

Parameters of L^*, a^*, and b^* (CIELab) were measured by a Minolta CR200 chroma meter according to Carreño et al. [12]. Then, Hue and Chroma values were calculated and converted to the CIRG index.

2.7. Total anthocyanin content

Fifty berries/replicate were homogenized using a blender and allowed to extract in 2 vol (w/v) acidified methanol at 4°C overnight in the dark. Then, the samples were filtered through Whatman no. 1 filter paper by vacuum suction. Extraction was repeated until the extract was colorless. Then the filtrates were pooled and the methanol was evaporated in a rotary evaporator at 40°C under vacuum. The remaining extract was brought to a volume of 100 ml with distilled water containing hydrochloric acid 1%. The total anthocyanin content was determined by the pH differential method: 0.025 M potassium chloride (pH 1) and 0.4 M sodium acetate (pH 4.5) buffers were used for this purpose. The extracts were diluted with each buffer, and the absorbance was measured by a spectrophotometer (Shimadzu UV-1201) at 520 and 700 nm. Results were expressed as milligram malvidin-3-glucoside chloride [13].

2.8. Statistical analysis

Descriptive statistics for studied characteristics were presented as mean and standard error of mean. For each characteristics, year and cultivars; ANOVA (Analysis of Variance) was used to compare Control and PF group means. Statistical significance level were considered as 5% and SPSS (ver: 21) statistical program was used for all statistical computations.

3. Results and discussion

3.1. Effects of PF treatment on histological properties and anthocyanin localization of the berry skin

In both cultivars, it was observed that the skin tissue mainly consists of two sub-layers; the epidermis and the hypodermis (Fig. 1) The outermost layer, the epidermis was seen to be cover with cuticle and consists of narrow, elongated, and thick-walled cells forming a single-cell layer. The hypodermis, which is the closest layer to the pulp, was observed to contain most of the phenolic compounds in the berries according to toluidine blue coloration. Anthocyanin granules were determined inside vacuoles in skin cell layers.

Fig. 1

Semi-thin sections of berry skin. (A) Beauty Seedless (B) Tekirdag Seedless. ep: epidermis, hyp: hypodermis, me: mesocarp, cw: cell wall, ant: anthocyanins.

In this study, anthocyanin localization type in the cells did not vary between the control and PF treatment in both cultivars. Mainly three cell types were observed in terms of anthocyanin localization in the skin sections (Figs. 2-3) The cell types were as follows: cells without any anthocyanin (type 1), cells with vacuolar storage (type 2), and cells with granular storage (type 3). However, distribution of cell types in the skin tissues and anthocyanin density in cells were different between cultivars.

Fig. 2

Anthocyanin distribution in semi-thin sections of Beauty Seedless. (A) Section of berry skin. (B) Type 1 cells: colorless, anthocyanin-free, (C) Type 2 cells: distorted bodies into the large central vacuole, (D) Type 2 cells: small spherical bodies, (E) Type 3 cells: small granules, (F) Type 3 cells: fine granules homogenously distributed.

Fig. 3

Anthocyanin distribution in semi-thin sections of Tekirdag Seedless. (A) Section of berry skin. (B) Type 1 cells: colorless, anthocyanin-free, (C) Type 2 cells: small spherical bodies, (D) Type 3 cells: small granules, (E) Type 3 cells: fine granules homogenously distributed, (F) Type 3 cells: completely homogenous coloration.

In Beauty Seedless, type 1 cells were fewer than the other two cell types, and they were present only in the hypodermis near the mesocarp. In microscopic examinations, these cells were observed as transparent cells and only cell walls were visible (Fig. 2B) In type 2 cells, anthocyanin accumulation was observed in two different shapes. (1) Anthocyanins were visualized in the large central vacuole, which sometimes covered the whole cell (Fig. 2C) These spherical phenol deposits in vacuoles are called anthocyanic vacuolar inclusions (AVI) [14]. The dark blue/purple spherical bodies were abundant in the epidermal layer. (2) Anthocyanins were observed as round bodies of different sizes that were free in the cells or stuck to the cell wall (Figs. 2D and 4A) These small vesicles were mainly located in the outer hypodermis. Type 3 cells were identified as small anthocyanin granules that distributed in the cells (Figs. 2E and 4B) These cells were abundant in the inner hypodermis. In the layers close to the mesocarp, the granules became smaller and the cells gained a cloud-like appearance and homogeneous color (Fig. 2F).

Fig. 4

Ultrastructure of anthocyanins in berry skin cells. (A) Type 2 and (B) Type 3 cells in Beauty Seedless, (C) Type 2 and (D) Type 3 cells in Tekirdag Seedless.

In Tekirdag Seedless, the anthocyanin accumulation in each cell type had different appearance microscopically. The anthocyanin-free cells (type 1) was mainly located in the layers close to the mesocarp (Fig. 3B) In type 2 cells, anthocyanins were observed as AVIs in the small and numerous vacuoles (Figs. 3C and 4C) The cells containing AVIs was less than other cell types, and they were observed in the epidermis and outer hypodermis. Type 3 cells had a different size, shape, and density of anthocyanin granules. In some cells, anthocyanins were seen as small to medium-sized granules (Figs. 3D and 4D) while others had very fine and homogeneously distributed granules (Fig. 3E and 3F).

3.2 Effects of PF treatment on berry characteristics

There was no important effect of PF treatment on berry weight and size in both cultivars. Berry weight was determined as 1.40–1.80 g (C) and 1.36–1.88 g (PF) in Beauty Seedless, and 4.03–4.81 g (C) and 4.01–4.72 g (PF) in Tekirdag Seedless (Table 1). Measurements of berry width and length were also similar between the control and PF treatment in both cultivars. The only significant difference was seen in the berry length of Tekirdag Seedless in 2015. Since berry weight data were similar between treatments, the difference could be considered negligible. Previous studies also indicated that PF treatment had no significant effect on the berry’s physical characteristics [4, 15].

Table 1
The effects of PF treatment on berry characteristics

Cultivars Years Treatments Berry weight (mm) Berry width (mm) Berry length (mm)

Beauty Seedless 2014 PF 1.88±0.05 13.32±0.13 15.16±0.15

C 1.80±0.04 13.09±0.14 15.10±0.24

2016 PF 1.36±0.02 12.79±0.14 13.62±0.16

C 1.40±0.04 12.99±0.18 13.97±0.22

Tekirdag Seedless 2014 PF 4.01±0.18 18.57±0.26 18.90±0.23

C 4.03±0.18 18.99±0.21 18.90±0.28

2015 PF 4.72±0.18 19.45±0.18 20.76±0.18

C 4.81±0.14 19.86±0.16 19.75±0.25^#

Cultivars	Years	Treatments	Berry weight (mm)	Berry width (mm)	Berry length (mm)
Beauty Seedless	2014	PF	1.88±0.05	13.32±0.13	15.16±0.15
		C	1.80±0.04	13.09±0.14	15.10±0.24
2016	PF	1.36±0.02	12.79±0.14	13.62±0.16
		C	1.40±0.04	12.99±0.18	13.97±0.22
Tekirdag Seedless	2014	PF	4.01±0.18	18.57±0.26	18.90±0.23
		C	4.03±0.18	18.99±0.21	18.90±0.28
2015	PF	4.72±0.18	19.45±0.18	20.76±0.18
		C	4.81±0.14	19.86±0.16	19.75±0.25^#

^#For each characteristics; differences from Control group is statistically significant (p≤0.05) for each cultivar and year.

As shown in Table 2, in the control group of Beauty Seedless, berry skin thickness ranged from 79.5 to 83.5μm. The higher number of cell layers in the hypodermis led to the thicker skin of this variety. This relationship has also been demonstrated by Considine and Knox [16] and Muganu et al. [17]. Measurements of skin thickness in the PF treatment did not differ from the control in both years. The values were between 81.2–82.9μm. The skin thickness measurements of Tekirdag Seedless varied between 53.7–61.3μm in the control and 56.1–57.0μm in the PF treatment. The effects of PF treatment on skin thickness were found to be statistically important in 2015 while insignificant in 2014. In the year 2015, PF treatment led to a thinner berry skin (Table 2).

Table 2

The effects of PF treatment on berry parts

Cultivars	Years	Treatments	Skin thickness (μm)	Skin (%)	Flesh (%)
Beauty Seedless	2014	PF	82.9±1.44	7.7±0.29	92.3±0.29
		C	83.5±1.17	7.6±0.22	92.4±0.22
2016	PF	81.2±0.91	6.0±0.19	94.0±0.19
		C	79.5±1.40	5.7±0.13	94.3±0.13
Tekirdag Seedless	2014	PF	56.1±1.76	5.5±0.23	94.5±0.23
		C	53.7±0.92	5.8±0.25	94.2±0.24
2015	PF	57.0±0.84^#	5.7±0.09^#	94.3±0.10
		C	61.3±1.06	6.3±0.12	93.7±0.12^#

^#For each characteristics; differences from Control group is statistically significant (p≤0.05) for each cultivar and year.

In previous studies, berry skin thickness was determined as 152–173μm in Corvina grapes [1] and 198–243μm in Nebbiolo grapes [3]. It is expected that wine grape varieties have thicker skin from an enological point of view. However, in table grape varieties, thin skin is a desirable trait. In the study by Kök and Çelik [18], the skin thickness of 13 table grape varieties ranged from 27.50 to 89.38μm. Beauty Seedless and Tekirdag Seedless can be classified as thin-skinned cultivars compared to those in the above-mentioned studies. Regarding skin thickness measurements, Tekirdag Seedless grapes were thinner skinned than Beauty Seedless grapes.

In Beauty Seedless, the proportion of skin and flesh did not differ between the PF treatment and control. The skin/berry and flesh/berry ratios ranged from 5.7% to 7.7% and 92.3% to 94.3%, respectively. In Tekirdag Seedless, the proportion of berry parts in the PF treatment did not differ from the control in 2014. In 2015, however, the skin/berry ratio decreased due to the thinner skin tissue while the flesh/berry ratio increased (Table 2). The skin/berry ratio decreased from 6.3% (C) to 5.7% (PF) and the flesh/berry ratio increased from 93.7% (C) to 94.3% (PF) in that year. The proportional distribution of berry parts is important in terms of the enological potential of wine grapes [19]. The proportion of skin, flesh, and seeds in berry affect the extraction of anthocyanins and tannins during winemaking. It has been reported by several authors that in small-sized berries with a high skin/flesh ratio, solubility and diffusion of phenolic compounds increases during maceration [11]. In previous studies, percentages of skin, flesh, and seed were determined, respectively, as 10% – 12%, 83% – 87%, and 3.3% in Cabernet Sauvignon; 8% – 12%, 83% – 87%, and 4% – 5% in Tannat; 6.5% – 10%, 87% – 90%, and 3% – 4% in Merlot; 20%, 76%, and 4% in Syrah; and 11.5% – 13.4%, 82% – 84%, and 4% – 5% in Sangiovese [11 , 21]. As far as we know, the proportional distribution of berry parts in table grape varieties has not been determined. This study presented the findings for table grapes.

3.3. Effects of PF treatment on anthocyanin content of berry

PF treatment was found to be effective on the total anthocyanin content of both cultivars. In Beauty Seedless, PF treatment increased total anthocyanin content in both years. In control vines, total anthocyanin content was determined as 512.93–605.90 mgkg^–1; this increased to 606.98 and 666.44 mgkg^–1 in PF-treated vines in 2014 and 2016, respectively (Table 3). In Tekirdag Seedless, likely in Beauty Seedless, PF treatment increased the total anthocyanin content in both years. The findings varied from 72.50 to 81.43 mgkg^–1 in the control and from 79.55 to 84.72 mgkg^–1 in the PF treatment (Table 3). However, the increase in 2014 was not of statistical importance. It has also been reported that PF treatment has favorable effects on anthocyanin and phenol biosynthesis under excessive solar radiation and high-temperature conditions, which is due to reduced canopy temperature [4 , 23]. In this study, the fruit zone temperature of both cultivars was significantly low in PF treatment compared to control (Fig. 5) This decrease was thought to stimulate anthocyanin accumulation in the berries. Previous studies also reported that low temperatures increase anthocyanin accumulation whereas high temperatures (>30–35°C) inhibit biosynthesis and promote degradation of existing anthocyanin via chemical and/or enzymatic reactions in berries [24 –26].

Fig. 5

The effects of PF treatment on mean fruit zone temperature (°C) (p≤0.05).

Numerical values obtained from the Minolta CR200 chroma meter were around 6 in Beauty Seedless and 5 in Tekirdag Seedless. According to the measurements, berry skin color was defined as “blue-black” in Beauty Seedless and “dark red-violet” in Tekirdag Seedless. In this study, CIRG data confirmed that there was no problem in terms of the development of skin color in the cultivars as a result of PF treatment (Table 3).

Table 3

The effects of PF treatment on anthocyanin content and skin color

Cultivars	Years	Treatments	Total anthocyanin	CIRG
(mgkg^–1)
Beauty Seedless	2014	PF	606.98±8.23	6.88±0.07
		C	512.93±18.41^#	6.74±0.07
2016	PF	666.44±3.07	6.54±0.05
		C	605.90±5.76^#	6.57±0.06
Tekirdag Seedless	2014	PF	84.72±1.69	5.26±0.04
		C	81.43±1.55	5.16±0.02
2015	PF	79.55±1.40	5.19±0.04
		C	72.50±1.05^#	5.11±0.02

^#For each characteristics; differences from Control group is statistically significant (p≤0.05) for each cultivar and year.

4. Conclusion

Grape berries are a complex of tissues that reflect the sensory and chemical qualities of a genotype. Practices performed on canopies in order to control radiation and heat effects on grapevines cause metabolic changes that influence the physical and phytochemical characteristics of berries. According to our findings, PF treatment is a useful method for increasing anthocyanin content in Beauty Seedless and Tekirdag Seedless cultivars grown in semi-arid regions by means of favorable temperature conditions on fruit zone. Histochemical observations suggested that there may be a close relationship between the accumulation type and content of anthocyanins. In this study, main accumulation characteristics of anthocyanins in berry skin cells were determined to be vacuolar storage (AVI’s) in Beauty Seedless and granular storage in Tekirdag Seedless. Beauty Seedless which was characterized by vacuolar storage showed a high total anthocyanin content (512.93–666.44 mgkg^–1), meanwhile, granular storage was mainly observed in Tekirdag Seedless which had lower anthocyanin content (72.50–84.72 mgkg^–1). Additionally, it has been reported that the accumulation of phenolic substances in berry skin cells showed different anthocyanin localization, and there was a strong correlation between the number and size of AVIs in skin cells and anthocyanin content [10 , 27]. Furthermore, CIRG data of the cultivars showed that cultivar-specific skin color was gained with PF treatment. Developing advanced measurement techniques to understand cellular accumulation of anthocyanins could be a subject of new studies.

Funding

The authors report no funding.

Conflict of interest

The authors have no conflict of interest to report.

Footnotes

Acknowledgments

The authors are grateful to Ankara University Coordinatorship of Scientific Research Projects for financial support (Project no: 15L0447001) and TUBITAK (The Scientific and Technological Research Council of Turkey) for the funding of National Graduate Scholarship Programme (BIDEB 2211C).

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