Chemicals and plant material
Authentic standards for pelargonidin and cyanidin were purchased from TransMIT PlantMetaChem (Giessen, Germany). All other chemicals were from Sigma-Aldrich (Milano, Italy), water was purified by a Milli-Q water purification system. Petunia seeds were germinated and plants were grown in peat:vermiculite (1:1) under fluorescent illumination (16 h day length) at 23 °C. Origin of the different petunia cultivars is shown in Table 1, the nontransgenic line W80 (Huits et al. 1994) was a kind gift of Dr. Ronald Koes (University of Amsterdam, Swammerdam Institute for Life Sciences, Amsterdam, The Netherlands).
RNA extraction, cDNA synthesis and RT-PCR reactions
Total RNA was isolated as described (Chang et al. 1993) from unopened flowers when anthocyanin biosynthesis was active and the petals were gaining color. A treatment with RNase-free DNase (Nucleo Spin RNA clean-up XS, Macherey–Nagel, Germany) was done to remove any residues of genomic DNA. First-strand cDNA was synthesized from 166 ng of total RNA using the Superscript III Reverse Transcriptase Kit (Invitrogen). Using the cDNA as template, the full-length maize A1 transcript was amplified with primers GER945 and GER946, and a fragment of the nptII transcript with primers GER522 and GER523 using Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Waltham, MA, USA). The thermal cycler was programmed in the following way: an initial cycle of denaturation at 98 °C for 30 s, followed by 25 (A1) or 30 (nptII) cycles of: 98 °C for 30 s, 65 °C (A1) or 56 °C (nptII) for 30 s, 72 °C for 1 min, followed by a final extension for 10 min at 72 °C.
DNA extraction and genomic PCR
Genomic DNA was isolated from leaves using the miniprep II method (Dellaporta et al. 1983). 100 ng of petunia genomic DNA was used for amplification of sequences between the bla gene and the 35S promoter (primers GER1003 and GER992), between the 35S promoter and the maize A1 gene (primers GER993 and GER984) and between the maize A1 gene and the nptII gene (primers GER981 and GER976) with Dream Taq DNA polymerase (Thermo Scientific) with the following program: an initial cycle of denaturation at 95 °C for 3 min, followed by 30 cycles of: 94 °C for 1 min, 55 °C for 30 s, 72 °C for 2 min. Amplified fragments were purified using High Pure PCR Product Purification Kit (Roche Diagnostics, Indianapolis, IN, USA) and directly sequenced using the amplification primers and, when needed, internal primers designed from the sequences. The sequences were assembled together based on their overlapping segments and deposited in GenBank with the accession number KY964325.
HPLC analysis was carried out as described (Bashandy et al. 2015) with minor modifications. Samples were collected from fully opened petunia flowers, ground in liquid nitrogen and extracted with a 2× volume of methanol with 1% HCl. After sonication for 30 min at room temperature (Finn sonic W181, Oy ULTRA sonic Finland Ltd, Lahti, Finland), the extracts were cleared from debris by centrifugation (3220g, 10 min). The supernatant was mixed 1:1 with 4 M HCl and hydrolyzed by incubating for 40 min at 95 °C. The hydrolyzed extracts were centrifuged at 17,000g for 10 min before injection into HPLC column. The standard compounds were applied at 10 µg/ml in methanol.