Real-time PCR analyses: RNA extraction and RNA evaluation

Real-time PCR analyses were performed for four and five selected stages of berry ripening, in 2010 and 2012 respectively, from beginning of veraison, to the end of harvest. Total RNA was isolated from 200 mg and 500 mg, in 2010 and 2012 respectively, of the powdered berry skin tissue using spectrum^TM Plant Total RNA kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturers protocol. One µL of each RNA sample was measured spectrophotometrically to determine RNA quantity and 260/280 and 260/230 ratio, using a NanoDrop1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Total RNA was treated with 1 U µg-1 RQ1 DNase (Promega,

Milan, Italy). First-strand cDNA was synthesized using 1 µg of RNA, 1µL (dT)15 primer (500 µg/mL) and 1 U of GoScript™ reverse transcriptase (Promega). Quantitative RT-PCR was carried out on a Step One Plus™ (Applied Biosystems, Foster City, California, USA). Each reaction (10 µL) contained 188 nM of each primer, 2µL of diluted cDNA (1:50), 5µL U of Power SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, California, USA). Thermal cycling conditions were 95 °C for 10 min followed by 95 °C for 15 s, 55 °C for 1 min for 40 cycles, followed by a melting cycle which contains 95 °C for 15 sec, 60 °C for 1 min and 95 °C for 15 sec. Each cDNA sample was analyzed in triplicate. Primers pairs for Dihydroflavonol 4-reductase (DFR), Leucoanthocyanidin dioxygenase (LDOX) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) were retrieved from literature (Goto-Yamamoto et al., 2002); primers for VvMYBA1 were from Jeong et al., (2004); for Leucocyanidin reductase (LAR) and Anthocyanidin reductase (ANR or BAN) from Bogs et al., (2005) and for Peroxidase (POD6 ) from Mori et al., (2007). Primers for Flavanone 3-hydroxylase (F3’Hb), Flavonoid-3’5’-hydroxylase (F3’5’Hk and F3’5’Hi), Phenylalanine ammonium lyase (PAL), Flavonol synthase (FLS) and Peroxidases (POD1 to POD5) were newly designed on the original DNA sequences to amplify 150–250 bp gene fragments (Table.

1).

Table. 1: Sequence of primers used for Real time PCR amplification

Primer Forward Primer Reverse

DFR 5'-GAAACCTGTAGATGGCAGGA-3' 5'-GGCCAAATCAAACTACCAGA-3'

LDOX 5'-AGGCTCTACTCTCCAAATGA-3' 5'-GAAGCTTGAAACACAGACCA-3'

UFGT 5'-AATCTGAGAGCCCTAAGAGA-3' 5'-GGTGGTACAAGCAACAGTTC-3'

MYBA 1 5'-TAGTCACCACTTCAAAAAGG-3' 5'-GAATGTGTTTGGGGTTTATC-3'

LAR 5'-TCTCGACATAAATGATGATGTG-3' 5'-TGCAGTTTCTTTGATTGAGTTC-3'

BAN 5'-CAATACCAGTGTTCCTGAGC-3' 5'-AAACTGAACCCCTCTTTCAC-3'

PAL 5'-ACATACCTAGTGGCACTCTG-3' 5'-TCGCAGAATCTCGATGGATG-3'

F3'Hb 5'-TCCATCACCAAGTCTTCTAC-3' 5'-GCGGAGAAGCTGAACATGGA-3'

F3'5'Hi 5'-CAGACTGCATACGCACTCTG-3' 5'-ATGCCGGATGGAGTTGAGAT-3'

F3'5'Hk 5'-CCAGGCCTAGCTACTATATG-3' 5'-GCATATGGCATGGTGGTAGA-3'

FLS 5'-CTGCACTATCCAGAGATGAG-3' 5'-TTGCCGCTTCAACAACAACA-3'

POD 1 5'-TCTGAGTAAGAGCCCACACACTCT-3' 5'-GAGACCTGAGCAGATAAATACAAAAATAAA-3'

POD 2 5'-AAAGTTGTTTGGCTGTGG-3' 5'-AATCCCTCCAAACAATCA-3'

POD 3 5'-TGCTCTAAATGCCTGAAAAATG-3' 5'-ATCATATAGAAAAGTGACAGCAACCT-3'

POD 4 5'-CAACTTCACACCAACGGGAAA-3' 5'-GCAGCTGAGCAAGAAATGCTT-3'

POD 5 5'-TCGGTGAGGAACTCCGATA-3' 5'-AGCTTCCTTGTCCTCTTCCA-3'

POD 6 5'-AACTTGAGACACAACAGCATAAATAAATC-3' 5'-AGGACCAAATCACAGGATGATAAAG-3'

ACT 5'-ATTCCTCACCATCATCAGCA-3' 5'-GACGAGCCCCTCCTACTAAAACT-3'

2.7. Enzyme assay containing 0.075 g of PolyVinyl-PolyPyrrolidone (PVPP) and 100 µL of a 200 mM Dithiothreitol.

After incubation in ice for 20 minutes, centrifugation was done at (4° C, 15 min, 10,000 x g).

Then 1mL of the supernatant was desalted by passing through a NAP-10 column (Sephadex G-25, Amersham Biotech) equilibrated with 1.5 mL of the same buffer.

For the analysis of PAL activity, the reaction mixture consisted of 0.5 mL of Phenylalanine and 0.5 mL of the enzyme solution. The assay mixture was incubated in 37° C for 60 minutes. The reaction was terminated by adding 0.5 mL of HCl acid (18%). The quantity of the product, namely Trans-cinnamic acid, was calculated using its extinction coefficient of 9630 M^-1 cm^-1 at 290 nm. One unit of PAL was expressed as synthesis of 1 mol of Trans-cinnamic acid per minute, and PAL activity was expressed as µkat g-1 protein.

2.7. 2. UFGT activity assay

The extraction was performed according to the method of Mori et al., (2007) with some modifications. The following procedures for protein extraction were conducted at 4 oC. For each replication, (0.2 g) of berry skin was ground with a mortar and pestle in liquid nitrogen until a fine powder was obtained. The skin powder was homogenized with 1.3 mL of a 250 mM TRIS–HCl buffer (pH 7.5) containing 10 mM Polyethylene glycol 3400, 150 µL of 10 mM Na-diethyldithiocarbamate, 15 µL of 200 mM Dithiothreitol, and 2 µL of 2-mercaptoethanol and was incubated for 20 min on ice. After centrifugation of the homogenate at 10,000 x g for 20 min, The supernatant was passed through a NAP-10 column (Sephadex G-25, Amersham Biotech) equilibrated with a 1.5 mM TRIS–HCl buffer (pH 7.5).

The desalted crude extract was used as the enzyme solution in the following enzyme assay, using delphinidin and cyanidin as substrates. The method of Mori et al., (2007) was employed with some modifications for the analysis of UFGT activity. For the first assay, the reaction mixture consisted of 0.2 mL of the previous extraction solution (containing TRIS–HCl buffer (pH 7.5), Polyethylene glycol 3400, Na-diethyldithiocarbamate, Dithiothreitol, and 2-mercaptoethanol), 50 µL of 1mM

Cyanidin, 50 µL of 100mM UDP-glucose, and 0.2 mL of the enzyme solution. The assay mixture was incubated for 5 min at 37° C. The reaction was terminated by adding 150 µL of 5% HCl. The quantity of the product, namely Cyanidin-3-glucoside was measured using its extinction coefficient of 26900 M^-1 cm^-1 (at 520 nm / pH: 1).

Therefore, one unit of UFGT was defined as the production of 1 mol of Cyanidin-3-glucoside per second, and UFGT activity was expressed as pkat g-1 protein.

For the second assay, the reaction mixture consisted of 0.2 mL of the same extraction solution (containing TRIS–HCl buffer (pH 7.5), Polyethylene glycol 3400, Na-diethyldithiocarbamate, Dithiothreitol, and 2-mercaptoethanol), 50 µL of 1mM Delphinidin, 50 µL of 100mM UDP-glucose, and 0.2 mL of the enzyme solution. The assay mixture was incubated for 5 min at 37° C. The reaction was terminated by adding 150 µL of 5% HCl. The quantity of the product, namely Delphinidin-3-glucoside was measured using its extinction coefficient of 26000 M-1 cm-1 (at 520 nm / pH: 1).

Therefore, one unit of UFGT was defined as the production of 1 mol of Delphinidin-3-glucoside per second, and UFGT activity was expressed as pkat g-1 protein.

2.7. 3. Guaiacol PODs activity assay

Guaiacol PODs were assayed as described by Ushimaru et al., (1997), using pyrogallol as the electron donor for the reaction. The plant material (skin) was extracted in cold buffer (200 mM sodium phosphate, 5 mM sodium EDTA, 1% PVPP, pH 7.0) and incubated on ice for 30 min. After centrifugation at (10,000 × g, 30 min, 4 °C), the desalted supernatant was used for the assay. The reaction mixture (2.5 mL) included 2.2 mL of the 10 mM sodium phosphate buffer, pH 7.0, 100 µL of the 2.5 mM H₂O₂, 50 mM pyrogallol, and 100 μl extract; H₂O₂ and pyrogallol were prepared fresh just before use. Absorbance (λ=430 nm) was taken after 5 min incubation at room temperature, and referred to a blank with no extract added. One unit of Guaiacol peroxidases is defined as the amount of enzyme that catalyzes the oxidation of 1 μmol of pyrogallol per minute, under the conditions described by Chen and Asada (1989) and PODs activity was expressed as kat g-1 skin fresh weight. An absorbance coefficient of 2.47 mM-1 cm-1 was assumed for calculations.

2.7. 4. PPOs activity assay

PPOs were assayed as described by Ushimaru et al., (1997), using pyrogallol as the electron donor for the reaction. The plant material (skin) was extracted in cold buffer (200 mM sodium phosphate,

(10,000 × g, 30 min, 4 °C), the desalted supernatant was used for the assay. The reaction mixture included 2.8 mL of the 100 mM sodium phosphate buffer, pH 7.0, 1 mM pyrogallol, and 100 μl extract; the pyrogallol was prepared fresh just before use. Absorbance (λ=430 nm) was taken after 5 min incubation at room temperature, and referred to a blank with no extract added. One unit of Polyphenol oxidases is defined as the amount of enzyme that catalyzes the oxidation of 1 μmol of pyrogallol per minute, under the conditions described by Chen and Asada (1989) and PPOs activity was expressed as kat g-1 skin fresh weight. An absorbance coefficient of 2.47 mM-1 cm-1 was assumed for calculations.