Plant material, growth conditions and experimental design
For the analysis, two genotypes within each species, F. arundinacea cv. Kord (2n = 6x = 42) and F. glaucescens (F. arundinacea Schreb. subsp. Fenas (Lag.) Arcang.) (2n = 4x = 28), significantly differing with the potential of drought resistance: high drought resistant (HDR) and low drought resistant (LDR), were used (Tab. 1). The drought resistance of the selected genotypes was evaluated based on the measurements of chlorophyll fluorescence (OJIP) during short-term drought treatment (11-day water deficit and further 10-day recovery) performed on the pot-planted plants growing in the environmental chamber (hereafter termed pot-experiment) (5). The genotypes of F. arundinacea derived from single seeds originated from the collection of Institute of Plant Genetics, Polish Academy of Sciences, created by prof. Zbigniew Zwierzykowski. The seeds of F. glaucescens (ABY-Bn 354-1980) derived from the collection of the Institute of Biological, Environmental and Rural Sciences (IBERS) (UK), originated from the Centre de Recherches de Lusignan, INRA (France) and donated in 1985 to IBERS. This collection at IBERS was hold by the Genetic Resources Unit (Mr Ian D., Thomas). The analysis of genomic structure was performed for both species, F. arundinacea and F. glaucescens, to precisely confirm their identity.
The genotypes of both Festuca species were exposed for short-term drought in the pot-experiment as described previously by Kosmala et al. (2012) (5). Each genotype was represented by 15 independent clones (each three growing in a separate pot with 4 dm3 of sand : peat (1:3) mixture). The conditions of experiment were as follows: temperature – 22 °C, 16 h photoperiod, 400 μmol m-2s-1 PPFD (photosynthetic photon flux density), air humidity 50-60%. Plant material (leaf tissue) for the analysis was harvested before stress treatment when plants were fully hydrated (control, C), on the 3rd (D1), 6th (D2) and 11th (D3) day of watering cessation, and 10 days after subsequent re-watering (RH) (Fig. 12).
Fluorescent in situ hybridization (FISH) analysis
To verify the genomic status of F. arundinacea and F. glaucescens plants, FISH experiment with two highly conserved rDNA sequences (5S and 35S rDNA) as probes was applied. The wheat clone pTa794 containing 5S rDNA was labeled by PCR with tetramethyl-rhodamine-5-dUTP (Roche, Mannheim, Germany). Whereas, 35S rDNA, generated from a 2.3 kb fragment of the 25S rDNA coding region of Arabidopsis thaliana, was labeled by nick-translation with digoxigenin-11-dUTP (Roche, Mannheim, Germany). The preparation of slides, the labeling of probes and FISH experiments were performed according to protocols described in Majka et al. (2017) (50). Briefly, in FISH protocol, a hybridization mixture consisted of 50% formamide, 2 × SSC, 10% dextran sulfate and 100 ng of rDNA probes. The hybridization mixture together with the good quality of chromosome slides were denatured at 80 °C for 2 min and then incubated overnight at 37 °C. In the protocol, it was applied fluorescein isothiocyanate-conjugated (FITC) anti-digoxigenin antibody to detect digoxigenin-labeled 35S rDNA probe. After counterstaining with 4, 6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, Missouri), the slides were mounted in antifade Vectashield solution (Vector Laboratories, Burlingame, CA, USA). Slides were evaluated under an Olympus BX 61 automatic epifluorescence microscope equipped with an Olympus XM10 CCD camera. All images were captured using Olympus Cell-F imaging software (ver. 3.1; Olympus Soft Imaging Solutions GmbH, Germany) and Micrographx Picture Publisher software (ver. 10; Corel Corporation, Canada).
Physiological parameters
A relative water content (RWC), electrolyte leakage (EL), chlorophyll ‘a’ fluorescence and gas exchange (net photosynthesis (CO2 assimilation), transpiration, stomatal conductance) were measured as described previously in detail by Kosmala et al. (2012) and Perlikowski et al. (2014) (5, 8). For all the physiological measurements, the second fully expanded leaves from the top of the plant were used. The RWC was calculated according to the following formula: RWC% = (FW-DW)/(SW-DW)×100, where FW was the leaf fresh weight, DW was the leaf dry weight, and SW was the leaf turgid weight. The EL was measured using conductivity meter (Hanna Instruments EC215 Conductivity Meter) and calculated as follows: L1/L2 X 100, where L1 and L2 were electrolyte leakage of the fresh leaves and the leaves frozen in liquid nitrogen, respectively. Chlorophyll ‘a’ fluorescence was measured by the HandyPEA fluorimeter (Hansatech Instruments Ltd., King's Lynn, England) during midday. For RWC, EL, and chlorophyll ‘a’ fluorescence measurements three biological and ten technical replicates of all the analyzed genotypes at each time-points of experiment (C, D1, D2, D3, RH), were applied. Gas exchange was measured through CIRAS-2 Portable Photosynthesis System (PP SYSTEMS) at three selected time-points (C, D3, and RH) in three biological replicates.
Transcript levels of Calvin cycle enzymes under drought and recovery
RT-qPCR analyses were carried out for chloroplastic fructose-1,6-bisphosphate aldolase (pFBA), phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Total RNA was extracted from 100 mg of the leaves using the RNeasy Plant Mini Kit (Qiagen) according to the protocol. The remaining DNA was removed from the samples using the RNase-Free DNase set (Qiagen). The cDNA was synthesized with the Maxima First Strand cDNA Synthesis Kit (Thermo Scientific). The RT-qPCR assays were performed using the FastStart Essential DNA Probes Master (Roche) through the Bio-Rad CFX 96 thermal system as described by Pawłowicz et al. (2018) (7). The reaction temperature profile was as follows: initial denaturation 95 °C for 10 min, followed by 44 cycles of 95 °C for 10 s, and 60 °C for 30 s and final 50 °C for 30 s. The relative quantification method (ΔΔCq) was used. The reactions were normalized using actin and ubiquitin as reference genes. The expression stability of reference genes under drought conditions was evaluated using the BestKeeper software. Primers and TaqMan probes of analyzed genes were designed through the Beacon Designer software. All the measurements were carried out in three biological and two technical replicates.
Protein levels of Calvin cycle enzymes and antioxidant enzymes under drought and recovery
Protein accumulation profiles of three enzymes of Calvin cycle (pFBA, PGK, GAPDH) and seven antioxidant enzymes including: glutathione reductase (GR, AS06 181), chloroplastic glutathione peroxidase (GPX, AS04 055), chloroplastic Fe-dependent superoxide dismutase (FeSOD, AS06 125), chloroplastic Cu/Zn superoxide dismutase (Cu/Zn-SOD, AS06 170), manganese superoxide dismutase (Mn-SOD, AS09 524), L-ascorbate peroxidase (APX, AS08 368) and catalase (CAT, AS09 501), were analyzed. Total proteins were extracted from the leaves using Hurkman and Tanaka protocol with slight modifications (4, 51, 52). Briefly, the 200 mg of powdered tissue was homogenized with 500 µl of extraction buffer (0.7 M sucrose, 0.5 M TRIS, 30 mM HCl, 50 mM EDTA, 2% DTT, and 0.1 M KCl). An equal volume of phenol was then added, vortexed and centrifuged at 21 500 g for 15 min. The upper phenol phase was transferred to new tubes and 500 µl of extraction buffer was added. After vortexing and centrifuging in the same conditions, the proteins from the phenol phase were precipitated by the addition of 5 volumes of cold 0.1 M ammonium acetate in methanol in the new tube. After at least overnight incubation at -20 °C, the samples were centrifuged at 9 000 g at 0 °C for 30 min. The precipitate was washed once with the cold ammonium acetate in methanol and twice in cold acetone, and dried in SpeedVac (Heraeus Instruments). Dried precipitate was dissolved in 150 µl of resolving buffer (50 mM TRIS, 2% SDS, DTT) at room temperature (RT) and then denatured for 5 minutes at 99 °C. Western blot assay was performed as described by Pawłowicz et al. (2012) (53). Briefly, proteins were separated by 12% SDS-polyacrylamide gel and electroblotted (Trans-blot SD, Semi-Dry Transfer Cell, Bio-Rad) onto nitrocellulose membranes (Bio-Rad). Immunodetections of pFBA, PGK and GAPDH were performed with the use of polyclonal antibodies (Agrisera) diluted at 1:4000. The protein level of antioxidant enzymes was detected using the commercial rabbit polyclonal antibodies (Agrisera). The GR, GPX, Fe-SOD, Cu/Zn-SOD, MnSOD, and CAT antibodies were diluted at 1:4000, whereas the APX antibody was diluted at 1:2000. The membranes were incubated with the antibodies for 1 h. Antigen–antibody complexes were detected using a secondary anti-rabbit IgG–horse radish peroxidase conjugate (Sigma) in dilution 1:20000 (1 h of incubation), chemiluminescent substrates Westar Supernova (Cyanogen) and ChemiDocTMTouch Igmagin System (Bio-Rad) to visualize the results.
Chloroplast aldolase activity under drought and recovery
The activity of pFBA in the leaves was measured according to the modified method of Sibley-Lehninger (54, 55). Chloroplast proteins were extracted as described by Kosmala et al. (2012) and Perlikowski et al. (2016) with slight modifications (5, 8). The amount of 1 g of frozen material was ground in a liquid nitrogen and then suspended in 10 ml of chloroplast isolation buffer (CIB) (Sigma) with 0,1% BSA. The homogenised samples were filtered through a Sefar nitrex filter and centrifuged at 200 g at RT for 3 min. The collected supernatant was subsequently centrifuged for 15 min at 900 g at RT and then washed two times in 4 ml of CIB solution. Each time, the suspension was centrifuged at 900 g at RT for 15 min. The chloroplast pellet was dissolved in 500 µl of 0.1 M phosphate buffer (0.1 M Na2HPO4) with 3% Triton X100, shaken by vortex and centrifuged at 21 500 g in RT for 10 min. The collected supernatant was used to determine the pFBA activity. The volume of 100 µl 0.06 M fructose-1,6-bisphosphate and 140 µl of incubation buffer (0.05 M 2,4,6-trimethylpyridine, 0.08 M hydrazine sulfate, 0.3 mM sodium iodoacetate) pH 7.4 was pre-incubated in water bath for 10 min at 30 °C. Additionally, a material sample was performed for each biological replication which contained 100 µl of 0.06 M fructose-1,6-bisphosphate, 140 µl of incubation buffer and 300 µl of 10% trichloroacetic acid (TCA). The volume of 20 µl of chloroplast extract was added to the pre-incubated solutions, mixed and incubated at 30 °C for 45 min. The reaction was stopped by adding 300 µl of 10% TCA to the solution, and tubes were chilled on ice. Ice-chilled samples were centrifuged at 21 500 g for 10 min in RT to remove the precipitated proteins. The volume of 100 µl of each supernatant was incubated with 100 µl of 0.75 M NaOH at RT for 10 min and after that 100 µl of 0.1% 2,4-dinitrophenylhydrasine was added and the samples were incubated for 10 minutes in a water bath at 30 °C. The tubes were taken out and 700 µl of 0.75 M NaOH was added to them and mixed well. After 3 min of incubation, the absorbance at 540 nm was measured against the material sample. A standard curve was prepared with use of 0.01 mM D-glyceraldehyde as described in Perlikowski et al. (2016) (8). The amount of produced trioses in the pFBA assay was read according to the standard curve and after calculations of glyceraldehyde mg produced by 1 g of plant sample during 1 h.
Lipid peroxidation under drought and recovery (TBARS assay)
The level of lipid peroxidation was measured spectrophotometrically as a content in the samples of the thiobarbituric-reactive substances (TBARS) according to the method of Heath and Packer (1968) with slight modifications (56, 57, 58). Briefly, 300 mg of fresh leaves were homogenized with 2 ml of a buffer containing 0.25% TBA in 10% TCA at RT. After homogenization, samples were incubated at 100 °C for 15 min in a water bath. Next, the samples were cooled on ice and centrifuged at 10 000 g by 10 min at 4 °C. The supernatant was collected and the absorbance was measured at λ = 532 nm and at λ = 600. Amount of TBARS was calculated through the following formula: TBARS (µM) = (A532 – A600)/155, where 155 was an extinction factor.
Superoxide anion radical and hydrogen peroxide under drought and recovery
The level of superoxide anion radical (O2•−) and hydrogen peroxide (H2O2), were assayed spectrophotometrically. Superoxide anion radical measurement was performed according to Doke (1983), and Arasimowicz et al. (2009) (59, 60). Nitroblue tetrazolium (NBT) was used as a substrate which undergoes reduction by O2•− to form diformazan. Leaf discs (0.6-0.8 cm in diameter) were incubated with 3 ml of mixture containing 0.05 M potassium-phosphate buffer (pH 7.8) with 0.1 mM EDTA, 10 mM NaN3 and 0.05% NBT, for 1 h in the dark. Next, the samples were heated at 85 °C by 15 min and cooled down on ice. The absorbance was measured at λ = 580 nm. The level of O2•− was expressed as absorbance at 580 nm per 1 g of fresh weight (FW).
The concentration of hydrogen peroxide (H2O2) was assayed using the titanium (Ti4+) method (61, 62). The amount of 400 mg of plant tissue was homogenized on ice with 1.5 ml of 0.1 M potassium-phosphate buffer, pH 7.8. Obtained extracts were centrifuged at 14 000 g at 4 °C by 25 min. The volume of 1.5 ml of reaction mixture containing 400 μl of enzymatic extract, 600 μl of potassium-phosphate buffer and 500 μl of titanium reagent (0.6 mM PRL and 0.6 mM PTO in a ratio 1:1) were prepared for each samples. After 10 min of incubation, the absorbance was measured at λ = 508 nm. The standard curve was used. The level of H2O2 was determined on the basis of absorbance and expressed as µmol H2O2 per 1 g of fresh weight (FW).
Statistical analysis
All the statistical analyses were performed with the STATISTICA 10.0 software (StatSoft, Tulsa OK, USA). A two-way analyses of variance (ANOVA), with genotype and time-point as classification factors, were performed. Differences in physiological parameters, protein accumulation, pFBA activity, RT-qPCR, TBARS assay, ROS measurement between the plants during experiment duration were evaluated using Fisher’s least significant difference (LSD) test at P = 0.01. Homogeneity groups according to test were denoted by the same letters on the graphs.
