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.