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.
Cold treatment
The cold treatment consisted of relocating five clones to a 4°C-room for 24h
(starting at 6pm of day 17), where they rested on a lamp-shelf and experienced a
lower amount of light (70-90uM PAR) as compared to the control condition. The
light/dark-cycle was maintained at 16h/8h, as in the control conditions. It is
noteworthy that for the alpine B. laevigata subsp. austriaca genotype used in
these treatments, 24h of 4°C treatment represents only mild cold stress.
Drought treatment
The drought treatment was conducted in the same growth chamber as the
control and herbivory treatments, with the only difference being the watering
regime. To achieve a drought stress, we stopped watering for a total of 10 days,
which was when the first signs of wilting leaves appeared on at least three of the
five drought-treated clones. Leaves were harvested an additional 1.5 days later,
at the same time as the other treatments were terminated.
Heat treatment
For the heat treatment, five clones were relocated to a growth chamber with
initially the same temperature and light settings of 22°C and 100-120μM, as in
the growth chamber under control conditions. In order to not apply a direct heat
shock, the temperature was raised gradually for 3h until a maximum of 42°C had
been reached, where the plants remained for 6h. Larkindale and colleagues have
shown that a gradual increase in temperature, as compared to heat shock, or
large (>8°C) step-wise increments of temperature, leads to higher-fold
transcriptome changes (Larkindale et al., 20082).
Herbivory treatment
The herbivory treatment was conducted in the growth chamber under control
conditions. We applied eight larva of the Diamondback Moth (Plutella xylostella)
onto one mature leaf of each of the five clones, for 30h before onset of the
harvesting phase. The herbivory treated leaf of each clone was encapsulated with
a small transparent plastic cage, wherein the eight larvae were held. At the onset
of the harvesting phase, visible feeding damage had occurred on all the treated
leaves for about 10-20% of the encapsulated leaf area.
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.
Library preparation and RNA-sequencing was outsourced to the next-generation
sequencing platform of the University of Bern (Bern, Switzerland). For leave
tissue from the environmental stress experiment, libraries were prepared using
the "TruSeq Stranded Total RNA with Ribo-Zero Plant"-kit (Illumina, Cat. No.
20020610), including ribosomal RNA depletion and a size selection step for
300bp fragments. 50bp paired-end (PE) RNA sequencing was conducted for 17
libraries (4x control, 4x cold, 3x heat, 3x drought and 3x herbivory) on two lanes
of an S2 flow cell of the NovaSeq6000 system yielding approximately 1.1 billion
raw PE-reads.
References1. Ehlting, J. et al. Comparative transcriptome analysis of Arabidopsis thaliana
infested by diamond back moth (Plutella xylostella) larvae reveals signatures
of stress response, secondary metabolism, and signalling. BMC genomics 9,
154; 10.1186/1471-2164-9-154 (2008).
Less...