Electrical Stimulation as a Potential Technique for Enlarging Table Grape Berry Size by Enhancing Cell Division

Materials and Methods

Plant materials. Shine Muscat (V. vinifera × V. labrusca) and Pione (a tetraploid hybrid of V. vinifera × V. labrusca) were cultivated in commercial vineyards located in Fuefuki City, Yamanashi Prefecture, Japan. Three-year-old Shine Muscat was cultivated in the shelf style in a greenhouse. Ten-year-old Pione was cultivated in the shelf style in an open field. For the shelf-style cultivation, two long canes (∼20 buds each) were severed as the overhead arbor and branched near the top of the trunk (1.5 m above the ground) (Figure 1).

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Figure 1

Experimental design of electrical stimulation applied to field-grown grapevines. (A) Shine Muscat grapevines were cultivated in the shelf style in a greenhouse. (B) Pione grapevines were cultivated in the shelf style in an open field. Solar panels (SPs) were positioned ∼1.5 m aboveground. Electrodes were screwed into the grapevine trunks and connected to two solar panels (－, negative electrode; +, positive electrode). Bunches of Shine Muscat and Pione are shown in insets of figures (A) and (B), respectively. Bar = 5 cm.

Grape cultured cells prepared from meristems of V. vinifera cv. Koshu were maintained on modified Gamborg’s B5 medium at 27°C in the dark (Fujita et al. 2018).

Electrical stimulation of field-grown grapevines. Ten Shine Muscat and five Pione grapevines were used for electrical stimulation. Electrical stimulation was carried out at budbreak (20 Feb 2018 and 18 Jan 2019 for Shine Muscat, and 25 April 2018 and 16 April 2019 for Pione). Two electrodes (steel screws, 40-mm length) were screwed into a grapevine trunk (5 cm aboveground for the positive electrode and 130 cm aboveground for the negative electrode) and connected to a solar panel (Figure 1). The solar panel had the following electrical characteristics: maximum voltage 11.6 V ± 5%, maximum current 100 mA ± 5%, and working temperature -35°C to 85°C. As described previously (Mikami et al. 2017), illuminance exceeding 8000 lux induced full capacity of the solar panel, whereas voltage was low when illuminance was below 8000 lux. No voltage was detected in grapevines at night. The electrodes and the solar panels were detached from the grapevines after harvest (end of May for Shine Muscat and end of August for Pione), suggesting that in our experimental design, the grapevines connected to the solar panels were stimulated in the daytime, but not at night, from budbreak to harvest. Untreated grapevines were used as control. The same treatment on the same grapevine was performed in the 2018 and 2019 growing seasons.

Berry characteristics. To compare berry characteristics of grapevines exposed to electrical stimulation with those of the control, bunches at harvest were collected and berry characteristics were measured following a previously described method (Kobayashi et al. 2020). Briefly, 40 bunches (four bunches from each grapevine) for Shine Muscat and 10 bunches (two bunches from each grapevine) for Pione were randomly collected from grapevines exposed to electrical stimulation or control on 17 May 2018 and 23 May 2019 for Shine Muscat and 21 Aug 2018 and 23 Aug 2019 for Pione. Each bunch was weighed using an electronic balance (EK2000i, A & D Co.). Ten berries (three from the top of the bunch, four from the middle of the bunch, and three from the bottom of the bunch) for Shine Muscat and seven berries (two from the top of the bunch, three from the middle of the bunch, and two from the bottom of the bunch) for Pione were collected from each bunch. The transverse diameter of each berry was measured using a digital caliper (AD-5764A-100, A & D Co.). The weights of the 10 or seven berries were measured using an electronic balance.

After measurement of berry weight, juices were obtained from the 10 or seven berries by hand-pressing. Soluble solids content (Brix) and total acid content (g/100 mL) in the juices were measured with a refractometer (PAL-BX/ACID2, Atago).

Thin slices of berry skins were prepared from the 10 Shine Muscat berries using a razor. Skin thickness (thickness of epidermal and subepidermal cell layers) was measured under a light microscope (BX51, Olympus). A small piece of skin (5-mm square) was also cut out from each of the 10 Shine Muscat berries using a razor. The skin pieces were treated with an enzymatic solution (pH 5.6) containing 2.5 g/L pectolyase (Kyowa Chemical Products), 91 g/L mannitol, and 5 g/L sodium dextran sulfate for four hours at room temperature. Then the skin pieces were washed with distilled water, and each was mounted on a glass slide and covered with a cover slip. The cell number in a 0.5 mm² area of the epidermis at four points of each piece of skin was counted under a light microscope.

Skins of the seven berries from each bunch of Pione were frozen immediately in liquid nitrogen. Extraction of anthocyanins from berry skins and measurement of total anthocyanin content were performed as described previously (Moriyama et al. 2020). Briefly, berry skins were homogenized in a mortar containing liquid nitrogen using a pestle. One gram of the pulverized sample was macerated in 10 mL of HCl-methanol [36:1 (v/v)] at room temperature in the dark overnight. The absorbance (OD₅₂₀) of the solution was measured using a spectrometer (UV-1800, Shimadzu). Total anthocyanin content was calculated according to a previously published formula (Bakker et al. 1986) and converted into malvidin-3-glucoside equivalent as mg/g of berry skin fresh weight.

Microarray analysis of grape cultured cells exposed to electrical stimulation. Microarray analysis of grape cultured cells exposed to electrical stimulation was performed according to a previously described method (Mikami et al. 2017). Briefly, grape cultured cells were grown at 27°C for two weeks on modified Gamborg’s B5 medium. Positive and negative electrodes (steel needle, 2-mm length, 0.5-mm diam) were entirely pricked on a mass of grape cultured cells and connected to a solar panel, the same as for field-grown grapevines. The solar panel was set under two fluorescent lamps (∼4000 lux each). Untreated grape cultured cells were used as a control. After electrical stimulation for four hours at room temperature, the cell mass was homogenized in a mortar containing liquid nitrogen using a pestle. Total RNA isolation from the cell mass was performed with a Fruit-mate for RNA Purification (Takara), followed by a NucleoSpin RNA Plant (Takara) according to the manufacturer’s instructions. Total RNA was subjected to microarray analysis using GeneChip V. vinifera (grape) Genome Array (Affymetrix). Briefly, biotin-labeled cRNA synthesis using GeneChip 3′IVT PLUS Reagent Kit (Affymetrix), hybridization using GeneChip Hybridization, Wash and Stain Kit (Affymetrix), and signal detection using GeneChip Scanner 3000 7G (Affymetrix) were performed according to the manufacturer’s instructions. The signal intensity of each spot, signal evaluation, and normalization were analyzed using Affymetrix GeneChip Command Console Software 4.0 and Affymetrix Expression Console Software 1.4. Genes upregulated by electrical stimulation were defined as follows: background <100, fold change electrical stimulation/control >2 (p < 0.01).

Real-time RT-PCR. First-strand cDNA was synthesized from total RNA of electrically stimulated or control grape cultured cells using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara) according to the manufacturer’s instructions. Real-time RT-PCR was performed with SYBR Premix Ex Taq II (Takara). PCR amplification was performed for 40 cycles at 95°C for 5 sec and at 60°C for 1 min after an initial denaturation at 95°C for 30 sec. The nucleotide sequences of the primers used for real-time RT-PCR were as follows: V. vinifera kinesin-like protein KIN-5C (5′-AATGGAGGCCCTTCTTGACG-3′ and 5′-ACGAGTATGGAGCTGTCCCT-3′, LOC100240753), V. vinifera transducin beta-like protein 3 (5′-TCAAAGGCCACAAAGGGGTT-3′ and 5′-AGCACTGAGCAAGGTCCATC-3′, LOC100252767), V. vinifera replication protein A 14 kDa subunit B (5′-TGGATACATCAAGCCCTGCA-3′ and 5′-ATTCCCCATTTGCAAGCAGG-3′, LOC100244193), V. vinifera nuclear pore complex protein NUP88 (5′-ATGCAATGGTGGGGAGGAAG-3′ and 5′-CTGCAATCCAGACTGGCTGA-3′, LOC100265724), and V. vinifera β-actin (5′-CAAGAGCTGGAAACTGCAAAGA-3′ and 5′-AATGAGAGATGGCTGGAAGAGG-3′, GenBank accession no. AF369524). β-Actin was used for data normalization. The dissociation curves were evaluated to verify the specificity of the amplification reaction. Using the standard curve method and Thermal Cycler Dice Real Time System Single Software ver. 3.00 (Takara), the expression level of each gene was determined as the number of amplification cycles needed to reach a fixed threshold. Data are expressed as relative values to β-actin and presented as means ± standard deviations.

Statistical analysis. Data are presented as means ± standard deviations of biological replicates. Statistical analysis was performed by analysis of variance, followed by Student’s t test, using Excel statistics software 2012. To rule out the possibility that berry characteristics might be dependent on meteorological conditions in each growing season, statistical analysis for berry characteristics in each growing season was independently carried out.