Photoperiod proteomics

Plant material The same plant material used for transcriptome analysis in (Flis et al., 2016) was the basis of our proteome study. Briefly, Arabidopsis thaliana Col-0 plants were grown on GS 90 soil mixed in a ratio 2:1 (v/v) with vermiculite. Plants were grown for 1 week in a 16 h light (250 μmol m−2 s−1, 20 °C)/8 h dark (6 °C) regime followed by an 8 h light (160 μmol m−2 s−1, 20 °C)/16 h dark (16 °C) regime for one week. Plants were then replanted with five seedlings per pot, transferred for 1 week to growth cabinets with an 8 h photoperiod (160 μmol m−2s−1, 20 °C throughout the day/night cycle) and then distributed into small growth cabinets with an 18, 12, 8 or 6 h photoperiod (160 μmol m−2s−1 and 20/18 °C in the day/night). This growth protocol was used to decrease differences in size between plants at the time of harvest, and to prevent an early transition to flowering that would otherwise occur if plants were grown from germination in long photoperiods. Plant material was harvested 9 days after transfer, at the end of the day (end-of-day samples were taken prior to lights switching off). Plant material was homogenized using a Ball-Mill (Retch, Germany). Approximately 50 mg of material per sample was aliquoted into 2 mL Eppendorf tubes while frozen and distributed for analysis to consortium partners in three biological and two technical replicates.

Protein Extraction and Digestion Frozen plant material was suspended in 100 µL SDS extraction medium (4% w/v SDS, 40 mM Tris, 60 μL ml-1 protease inhibitor cocktail (Roche)) and mixed vigorously. The extract was cleared by centrifuged for 10 min at 16,000 g followed by ultracentrifugation at 100,000 g for 45 min. The resulting supernatant was diluted 4:1 (v/v) in Laemmli sample buffer and incubated at 65°C for 5 min. For each sample, 400 μg protein was subjected to electrophoresis overnight on a 10% SDS-polyacrylamide gel at 60 V. Samples were loaded randomized on the gels to minimize positional effects. Gels were stained in Coomassie Blue solution (20% v/v methanol, 10% v/v acetic acid, 0.1% m/v Coomassie Brilliant Blue R) for 45 min then de-stained twice in 10% v/v methanol, 5% v/v acetic acid for 1 h at room temperature. Each lane of the gel was cut into 7 fractions and transferred to a 96-deep well plate. Gel pieces were fully de-stained by three rounds of 50% v/v methanol, 100 mM ammonium bicarbonate, incubating each time for 1 h at 37 °C. In-gel digestion of proteins using trypsin was performed as previously reported (Shevchenko et al., 1996). Volumes of solutions were adjusted to ensure that the gel pieces were fully covered during the reduction, alkylation and washing steps. Following in-gel tryptic digestion peptides were purified by reversed-phase chromatography on Finisterre C18 SPE columns (Teknokroma, Barcelona, Spain) and dried in a vacuum centrifuge at 45°C.

Mass Spectrometry Analysis Peptides were re-suspended in 40 μL 3% v/v acetonitrile, 0.1% v/v formic acid. Measurements were performed on a LTQ-Orbitrap Velos (Thermo Scientific) coupled with a NanoLC 1D HPLC (Eksigent). Samples were loaded onto a laboratory-made capillary column (9 cm long, 75 μm inner diameter), packed with Magic C18 AQ beads (3 μm, 100 Å, Microm) and eluted with a 5% to 40% v/v acetonitrile concentration gradient over 70 min, followed by 80% v/v acetonitrile for 10 min, at a flow rate of 0.25 μL min-1. Peptide ions were detected in a full MS1 scan for mass-to-charge ratios between 300 and 2000. MS2 scans were performed for the ten peptides with the highest MS signal (minimal signal strength 500 hits, isolation width mass-to-charge ratio 3 m/z, relative collision energy 35%). Peptide masses for which MS/MS spectra had been recorded were excluded from further MS/MS scans for 30 seconds.