Mid-winter de-acclimation triggers stress-like response in cold-acclimated wheat (Triticum aestivum L.)
Mid-winter de-acclimation triggers stress-like response in cold-acclimated wheat (Triticum aestivum L.)
The 0.05-0.09 g of leaves were sampled in three biological replicates (three different plants of the same accession) in three timepoints: just before cold-acclimation (PH), after cold-acclimation (H), and after de-acclimation (RH), and immediately frozen in liquid nitrogen. The plants were transferred to controlled conditions after sowing (growth chamber, darkness, 25/17 °C), the light (irradiance of 400 μmol m-2s-1 (HPS lamps, SON-T+ AGRO, Philips, Brussels, Belgium), photoperiod of 10/14 h, day/night) was turned on after the plantlets started to emerge. The temperature was lowered to 12°C (day and night) eight days after sowing. 21 days after sowing, the plants were cold acclimated (3 weeks, 4/2 °C, day/night temperature; photoperiod of 10/14 h and irradiance of 250 μmol m-2s-1). Cold acclimation was followed by 7 days of de-acclimating conditions, mimicking a mid-winter warm spell: 12/5°C, day/night temperature; photoperiod of 10/14 h and irradiance of 250 μmol m-2s-1). The 72 frozen leaf samples from 8 accessions, in 3 biological replicates and 3 time-points were subjected to RNA isolation using the combination of Pure Link RNA Mini Kit (Invitrogen, Thermofisher Scientific, Waltham, MA, USA) and spin columns from RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Qualified RNA were processed for library construction using the following kits: - NEBNExt PolyA mRNA magnetic isolation module, - NEBNext Ultra II Directional RNA library prep kit - NEBNext UDIs mRNA was isolated by Oligo(dT)-attached magnetic beads. First-strand cDNA was synthesized with fragmented mRNA as template and random hexamers as primers, followed by second-strand synthesis with addition of PCR buffer, dNTPs, RNase H and DNA polymerase I. Purification of cDNA was processed with magnetic beads. Double-strand cDNA was subjected to end repair. Adenosine was added to the end and ligated to adapters. AMPure XP beads were applied here to select fragments within size range of 300-400 bp.