Plant material
One individual (termed “original individual” henceforth) of Biscutella laevigata
subsp. austriaca (Brassicacea) (2n = 2x = 18) was grown under standardized
greenhouse conditions (16h/8h light/dark, 22-26°C/16-18°C, 65% relative air
humidity, 20-40kLux) for over a year. This individual stems from the alpine
population of Schneeberg in Austria (ca. 1800m a.s.l.; GPS: 47.697209,
15.609734). From this individual a number of cuttings, including root and several
leaves, where made and regenerated for four weeks at 12h/12h light/dark, 24°C
day and 18°C night temp. and 65% relative air humidity. These clones were then
repotted into larger pots, transferred back to the greenhouse described above
and kept there until they formed new leaves after an additional 6 weeks. This
way one genotype can be cloned and multiplied with a survival rate of 50%-75%
of all cuttings. All plants involved in the stress experiment were such 10-week old
clones.
Experiment design
The time-frame of the experiment can be divided into three phases, acclimation,
treatment and harvest. Acclimation started when all clones involved in the
experiments were relocated to the growth chamber under control conditions. This
phase lasted for seven days (until day 7 of the experiment). The treatment phase
started after seven days of acclimation, when the watering for the drought
treated clones was stopped. 10 days later (day 17), the drought treated clones
started to show signs of drought stress (wilting leaves on at least three clones).
The same day at around noon (day 17), after first signs of drought stress had
been observed, the second longest treatment (herbivory, 30h) was started. By
placing eight Plutella xylostella third- to fifth-instar larvae (6-9mm in length, after
Ehlting et al. 20081) onto one mature leaf per clone. The cold treatment was
initiated at the same day (day 17) at 6pm, by relocating clones to the cold room
onto a lamp shelf. The Cold treatment was ended at 6pm of day 18 at the onset
of the harvesting phase. The Heat treatment was started at 9am the next day
(day 18), by relocating the clones to a Percival initially set to control conditions.
Over 3h the temperature was raised to 42°C, where the clones remained for 6h
until 6pm of day 18 when the harvest phase began.
Harvest phase started at 6pm of day 18, when plants were harvested within half
an hour. Harvesting comprised of sampling stressed leaves (wilting and fed-on
leaves for drought and herbivory respectively, fully expanded leaves for control,
cold and heat) and snap freezing them in liquid nitrogen, still under the
corresponding treatment conditions. The experiment was terminated by
relocating all clones back to the growth chamber under control conditions, where
they were regenerated.
Control treatment
After stabilizing the plants at standard greenhouse conditions (16h/8h light/dark,
22-26°C/16-18°C, 65% relative air humidity, 20-40kLux), they were relocated to a
growth chamber, where they acclimatized to control conditions for 7 days.
Conditions in this growth chamber were 16h/8h, 22°C, 45% relative air humidity,
100-120uM PAR and daily watering, no fertilizer and no pesticides were applied.
RNA extractions and sequencing
RNA was extracted from 50-100mg of snap frozen leaf tissue, using the RNAeasy mini kit (QIAGEN, cat. no. 74104) following the manufacturers protocol. RNA was quantified with the Qubit 4 fluorometer (Invitrogen) and then treated with DNAse 1 (Thermo Fisher, cat. no. EN0525). Subsequently RNA integrity was assessed using the Bioanalyzer 2100 system (Agilent), whereupon RNA extracts with a ribosomal RNA integrity number (RIN) over 7.0 or higher were considered for library preparation.
Another round of RNA-sequencing was conducted for seven different Biscutella tissues. Libraries were prepared using the "TruSeq Stranded Total RNA Library Prep Human/Mouse/Rat"-kit (Illumina, cat. No.20020596), including ribosomal RNA depletion and a size selection step for 300bp fragments. 150bp paired-end (PE) RNA sequencing was done for seven libraries (leaf, senescent leaf, root, stem, closed flower bud, open flower and meristem on one lane of the HiSeq3000 system yielding approximately 386 million raw PE-reads.
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