Differential gene expression in Miscanthus sinensis under chilling stress
Differential gene expression in Miscanthus sinensis under chilling stress
RNA was extracted from 2 cm middle fully expanded leaf segments. Three  replicates per genotype (2) per time point (4) for each treatment and control plants were collected. Material was frozen in liquid nitrogen and stored at −80 °C.  Plants were first grown under greenhouse conditions (25°C day / 19°C night) and then subjected to either a constant control environment or a chilling treatment. Maternal plants were grown from 5 cm rhizome segments, each planted in 40 L containers filled with a 1:1 peat:sand mixture. A standardized nutrient solution (100 mg/kg N, 50 mg/kg P, 200 mg/kg K, 2 mg/kg Fe, 10 mg/kg Zn, 10 mg/kg Ca) with daily watering was implemented. Collected plantlets were initially cultivated in the greenhouse conditions under a 12-hour photoperiod using white fluorescent lighting with photosynthetically active radiation (PAR) at 750 μmol m⁻² s⁻¹. Day/night temperatures were held at 25/19°C, with 60% ± 10% of relative humidity. At the five-leaf stage, 20 individuals per genotype were relocated to a cultivation chamber at the Wielkopolska Centre for Advanced Technologies (Poznań, Poland). Light intensity, photoperiod and temperature were matched to the greenhouse setting to facilitate acclimation.  After 10 days of acclimation, a chilling stress treatment was initiated. Ten plants per genotype were subjected to the chilling treatment and 10 were designated as control plants. For treated plants a pre-hardening regime (7 days at 10°C/5°C day/night), followed by hardening (14 days at 5°C/2°C day/night) was implemented. On day 22, plants were exposed to acute chilling conditions (0.5°C/0°C) for 24 hours. Recovery was conducted under standard conditions (25°C, 12 h light, RH 60% ± 10%) for 14 days, concluding the experiment on day 36. Control plants remained under constant temperature (25°C/19°C) throughout the duration of the experiment with light and humidity regimes analogous as in treated plants. To eliminate positional bias, plants were randomized within the growth chamber every second day. Such an experimental design enabled the characterization of genotype-specific physiological responses to chilling. Samples were harvested at day 0 (baseline), day 7 (pre-hardening), day 21 (end of hardening), day 22 (after extremum), and day 36 (end of recovery).  Frozen samples were homogenized using TissueLyser II (Qiagen, Hilden, Germany). RNA was extracted using an automated method, Maxwell® RSC Plant RNA Kit. The concentration and purity of RNA was measured using the Nanodrop 1000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). RNA integrity was determined using Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Only RNA samples with 260/280 ratio higher than 1.8, 260/230 ratio higher than 2.0, and RIN (RNA integrity number) values higher than 8.0 were used for the RNA-seq analyses.  For Illumina RNA sequencing, TruSeq RNA Stranded mRNA protocol was used.