2.1 Plant material and light treatments
Wild bilberry (Vaccinium myrtillus L.) ecotype from Tromsø, Norway (69° 75′N, 19° 01′E) was used for the experiments. The bilberry bushes were collected during early July after the fruit set at stage S2 when berries were small, unripe, and green (Figure1a). The plants were collected in boxes (50 x 70 cm) with their root system and forest soil and watered well. The plants were kept in the phytotron conditions at 16°C for few days to acclimatize until they reached berry developmental stage S3 when berries were large, unripe, and green (Figure 1a)
For light treatments, the plants were placed in chambers covered from sides with photo reflective sheets and irradiated from top with blue (460 nm) or red (660 nm) light wavelength provided by the Heliospectra RX30 lamps (Heliopspectra AB, Gothenburg, Sweden; Figure 1b). In addition to the specific spectral light wavelengths, the plants received continuous ambient white light (400-800 nm) from the top. The plants under the ambient white light (400-800 nm) served as control for the experiment. All plants were kept at 16°C, and the photon fluence rate (μmol m−2 s−1) and irradiation energy flux (μW cm-2) were measured using JAZ Spectrometer (Ocean Optics Inc., Orlando, FL, USA) and used to calculate the relative light intensity expressed as arbitrary units (AU) (Figure 1c).
Berry samples were collected after 6 days from the beginning of the light treatments when berries had reached the stage S4 and started to develop red color on their skin (Figure 1a), and utilized for RNA sequencing and real-time quantitative reverse transcription PCR (qRT-PCR) analyses. For metabolite analyses, light treated S4 stage berries at 6 and 12 days and fully ripe berries after 4 weeks from the beginning of light treatments at stage S5 (Figure 1a) were collected. Approximately 20-25 berries were collected per treatment from three replicate bushes for RNA extraction and metabolite analyses. Immediately after collection, all the berry samples were frozen in liquid nitrogen and stored at -80°C until further used for analyses.