Illumina HiSeq 2500 sequencing; RNA-seq of five Populus x canescens (Grey poplar) organs/tissues during periodic or chronic drought-heat stress treatment and after recovery in comparison to untreated controls
RNA-seq of five Populus x canescens (Grey poplar) organs/tissues during periodic or chronic drought-heat stress treatment and after recovery in comparison to untreated controls
Plants were harvested at noon at the last day of stress treatment (S) and seven days later at the end of the recovery period (R). Leaves (LE1: young leaves n. 4-6, LE2: old leaves n. 9-12 counting from the apex, respectively) were immediately frozen in liquid N2. A stem segment of 10 cm was cut 10 cm above the stem base and immediately frozen in liquid N2. The roots (ROO) were washed three times in water, carefully stripped with filter paper and then also frozen in liquid N2. All material was stored at -80 °C until homogenization. Homogenization of plant materials was performed under liquid N2 with mortar and pestle. The bark containing the phloem tissue (PHL) was removed from the stem section with a scalpel. Young developing xylem tissue (XYL) was obtained by scraping off the first 1-2 mm of the hardwood section. The experiments were performed with wild-type plants of Grey poplar (Populus ✕ canescens [INRA clone 7171-B4]; syn. Populus tremula ✕ Populus alba). Plantlets were amplified by micropropagation under sterile conditions51 and raised for five weeks in 2.2-L pots on a sandy soil (1:1 [v/v] silica sand and Fruhstorfer Einheitserde, initially mixed with slow-release fertilisers: Triabon [Compo] and Osmocote [Scotts Miracle-Gro], 1:1, 10 g L-1 soil; every two weeks fertilised with 0.1% [w/v] Hakaphos Grün [Compo]) in the greenhouse (16/8 h photoperiodicity with supplemental lighting 200-240 μmol photons m-2 s-1 at the canopy level, photosynthetically active radiation [PAR]; day/ night temperature 22 °C/ 18 °C; CO2 concentration 380 μL L-1). Then the plants were moved to phytotron chambers to simulate specific climate scenarios. The control conditions (control380, control500) include ambient or elevated CO2 levels (380 μL L-1 and 500 μL L-1, respectively) with a daily maximum temperature of 27 °C. Plants were kept for 25 days under control conditions before starting the stress treatments for a duration of 22 days. The stress treatments include a periodic and a chronic drought-heat scenario at elevated CO2 levels (500 μL L-1) with a daily maximum temperature of 33 °C. The periodic stress treatment (droughtPER) included three cycles of reduced irrigation (50%, 60% and 70% reduction compared with the controls), each one lasting for six days; between the cycles, there were recovery periods with a duration of two days and a daily maximum temperature of 27 °C. In the chronic stress treatment (droughtCHR), irrigation was gradually reduced for 22 days, down to 70% reduction compared with the controls. After the stress treatment, plants recovered for seven days under control conditions. Total RNA was extracted from 50 mg frozen tissue using the Aurum Total RNA Mini kit (Bio-Rad, Germany) following the manufacturer's instructions. mRNA-focused libraries (Illumina TruSeq kit)
Experimental Factor: organism part
xylem
Experimental Factor: growth condition
carbon dioxide; 500 microliter per liter
Experimental Factor: environmental stress
chronic drought environment
Experimental Factor: sampling time point
directly after stress