MATERIALS AND METHODS
Plant material and growth
conditions
The Columbia-0 (Col-0) ecotype of Arabidopsis thaliana was used
as the wild type. The following mutant and transgenic Arabidopsisplants were used in this study: abcg14-2 (Ko et al., 2014; kindly
provided by Youngsook Lee); cyp735a1-2 cyp735a2-2 (cypDM ;
Kiba et al., 2013; kindly provided by Hitoshi Sakakibara); ahp2-1
ahp3 ahp5-2 and respective double mutants (Hutchison et al., 2006);arr2 (GK-269G01; Nitschke et al., 2016); arr10-5 arr12-1and the respective arr10-5 and arr12-1 single mutants
(Argyros et al., 2008; Mason et al., 2005). If not mentioned otherwise,
seeds were obtained from The European Arabidopsis Stock Centre (NASC;
http://arabidopsis.info/). The arr2 arr10-5 arr12-1 , arr2
arr10-5 , arr2 arr12-1 mutants were generated by genetic crossing
and the genotypes were confirmed by PCR analysis. Arabidopsisplants were grown on soil in a growth chamber under SD conditions (8 h
light/16 h dark) as described in Nitschke et al. (2016). For photoperiod
stress treatment, plants were exposed to a light period of 32 h. For CK
treatment, plants were watered daily from below (ca. 150 mL/tray
corresponding to ca. 4 mL/plant) with either 10 µM t Z (dissolved
in 0.01 % DMSO), 10 µM t ZR (dissolved in 0.01 % DMSO) or 0.01
% DMSO (control) dissolved in water.
Quantification of lesions
Water-soaked lesions were quantified three to four hours after the night
following PLP treatment. First, the total number of fully expanded
leaves (except for leaf 1 and 2 as well as cotyledons) of a plant was
counted. Afterwards, the total number of limp leaves was determined (0 =
no water-soaked lesion, 0.5 = less than 50 % of leaf surface
water-soaked, 1 = more than 50 % of leaf surface water-soaked) and the
percentage was calculated for each plant by dividing the number of limp
leaves by the total number of fully expanded leaves.
Chlorophyll fluorometry
As a measure of the response to photoperiod stress the photosystem II
maximum quantum efficiency (Fv/Fm ratio;
Baker, 2008) was determined six to seven hours after the night following
the PLP. First, healthy and lesioned leaves of several plants (three
leaves per plant) were detached in a ratio reflecting the determined
lesion percentage of the respective genotype in the same experiment.
Detached leaves were placed in Petri dishes filled with water with the
abaxial part of the leaf directly facing the water. After 20 min of
incubation in darkness, pulse-amplitude-modulated (PAM) measurements
were performed with the chlorophyll fluorometer FluorCam (Photon Systems
Instruments). The minimum fluorescence emission signal
F0 was recorded first and then the maximum fluorescence
yield Fm (induced by a saturating light pulse of 1500
μmol m-2 s-1).
RNA isolation and quantitative
RT-PCR
Ca. 100 mg of leaf material was harvested into 2 mL Eppendorf tubes and
shock-frozen in liquid nitrogen under white light (0 h time point) or
green safety light (7.5, 15 h time points). RNA isolation was performed
as described by Sokolovsky et al. (1990) with a few alterations.
Briefly, frozen samples (100 mg fresh weight) were ground using a Retsch
mill in pre-cooled adapters. Afterwards, samples were solved in 750 μL
extraction buffer (0.6 M NaCl, 10 mM EDTA, 4 % (w/v) SDS, 100 mM
Tris/HCl pH 8) and 750 μL phenol/chloroform/isoamyl alcohol (PCI;
25:24:1) solution was added. Samples were vortexed, shaken for 20 min at
room temperature and centrifuged at 19.000 g for 5 min at 4 °C.
The supernatants were transferred into fresh 1.5 mL Eppendorf tubes and
CI solution was added in a 1:1 ratio. Samples were vortexed briefly and
centrifuged at 19.000 g for 5 min at 4 °C.
Supernatants were transferred into fresh tubes and RNA was precipitated
for 2 h on ice by adding 0.75 volumes of 8 M LiCl. After centrifugation
at 19.000 g for 15 min at 4 °C, supernatants were removed and
resolved in 300 μL RNase-free water. RNA was precipitated again by the
addition of 30 μL 3 M sodium acetate and 750 μL absolute ethanol and
incubation at -70 °C for 30 min. Samples were centrifuged at 19.000g for 10 min at 4 °C and the supernatant was discarded. Pellets
were washed with 200 μL 70 % ethanol and after centrifugation, pellets
were dried at room temperature and resolved in 40 μL RNase-free water.
cDNA synthesis and qRT-PCR analysis were performed as described in
Cortleven et al. (2016) using 500 ng of total RNA and a
CFX96TM Real-Time Touch System (Bio-Rad Laboratories
GmbH; Feldkirchen, Germany). All primers used in this study can be found
in Supplemental Table 1 of Nitschke et al. (2016). Gene expression data
were normalized against reference genes according to Vandesompele et
al., 2002. PROTEIN PHOSPHATASE2A SUBUNIT A2 (PP2AA2 ,
AT3G25800), UBIQUITIN-CONJUGATING ENZYME10 (UBC10 ,
AT5G53300) and METACASPASE 2D (MCP2D , AT1G79340) served as
reference genes.
Determination of CK
concentrations
For CK measurements, 100 mg fresh weight of leaf tissue per sample was
collected and shock-frozen in liquid nitrogen under white light (time
points during light exposure) or green safety light (time points during
night). CK quantification was performed according to the method
described by Svačinová et al. (2012), including modifications described
by Antoniadi et al. (2015). Using 15 mg per technical or biological
replicate, samples were homogenized and extracted in 1 ml of modified
Bieleski buffer (60% MeOH, 10% HCOOH and 30% H2O)
together with a cocktail of stable isotope-labeled internal standards
(0.25 pmol of CK bases, ribosides, N -glucosides, and 0.5 pmol of
CK O -glucosides, nucleotides per sample added). The extracts were
applied onto an Oasis MCX column (30 mg/1 ml, Waters), eluted by
two-step elution using 1 ml of 0.35M NH4OH aqueous
solution and 2 ml of 0.35M NH4OH in 60% (v/v) MeOH
solution and then evaporated to dryness in vacuo . CK analysis was
carried out using ultra-high performance liquid
chromatography-electrospray tandem mass spectrometry using stable
isotope-labelled internal standards as a reference. All samples were
measured in quintuplicate for each genotype and each time point.
Statistical analysis
For CK measurements, the significance of differences between control and
PLP samples was calculated with a paired Student’s t-test in Microsoft
Excel®. For statistical analysis of all other data
SAS®Studio (https://odamid.oda.sas.com/SASStudio) was
used. Homogeneity and homoscedasticity were tested by Shapiro-Wilk and
Levene tests (p ≥ 0.01) before ANOVA testing was performed followed by
Tukey post hoc test. If assumptions were not met, transformations
(log2, log10, sqrt,
n0.1, n0.4, n1.5,
n7, n25) were performed. Paired
Wilcoxon test was performed if assumptions were still not met after
transformation.