Plant and soil materials
In previous studies, natural variations in A. thaliana populations from Catalonia were tested in multiyear small-scale common gardens under saline and carbonated conditions (Busoms et al. 2015; Terés et al. 2019). Seeds from reciprocal transplant experiments performed in 2015 were stored under cold (4 °C) and dry conditions until the beginning of the experiments. Col-0 seeds were included as a reference genome and were purchased from Nottingham Arabidopsis Stock Centre NASC (Scholl et al. 2000). Prior to use, seeds were surface sterilized by soaking in 70% (v/v) ethanol for 1 min, suspended in 30% (v/v) commercial Clorox bleach and 1 drop of Tween-20 for 5 min and rinsed 5 times in sterile 18 MΩ Milli-Q water. Seeds were stratified for 4 days at 4 °C to synchronize germination. The population coordinates and lines used in each experiment are detailed in Supplementary Dataset S1.
Soils from the native Catalan populations of A. thaliana were sampled, excavated at 10 cm depth and transported to the laboratory for further analysis. For the greenhouse experiments, soils from two different locations along the Catalan coast were excavated and transported to the greenhouse at the Universitat Autonòma de Barcelona (UAB). The saline siliceous soil was excavated at Blanes (41°53′42”N 3°01′11″E), while the saline-alkaline soil was obtained from l’Escala (42°13′03”N 3°11′30″E).
GIS data extrapolation
To estimate the geologic, edaphic and climatic parameters of each habitat of the A. thaliana natural populations, native soil, coordinate locations, and public maps from the Institut Cartogràfic i Geològic de Catalunya (ICGC) and the European Soil Data Centre (ESDAC) database (Panagos et al. 2012) were combined using Miramon v8 (Pons 2004) and Q-GIS (http://qgis.osgeo.org). Maps of soil properties at the European scale, based on Lucas 2009/2012 topsoil data, were used to extract the following variables: pH (measured in H2O), cation exchange capacity (CEC), and calcium carbonate (CaCO3) content (Ballabio et al. 2019). To determine whether the distribution based on these soil characteristics of A. thaliana in Catalonia matched the A. thaliana distribution on a larger scale, the HapMap population coordinates were used (https://1001genomes.org) to combine our data with the European maps (Supplementary Dataset S2).
Soil physical and chemical analyses
Six independent samples of each soil type were used for the analyses. The soil characterization was performed on air-dried 2-mm fraction samples. For the measurement of soil pH and electric conductivity (EC), 25 mL of 18 MΩ water was added to 5 g of soil in a Falcon tube. After mixing at constant rotation for 30 min, the pH was measured using a pH metre (Basic 20+, Crison, Barcelona. Spain), and CE was determined with a conductometer (Hanna, Woonsocket, Rhode Island, USA) (Sonmez et al. 2008). Texture, water holding capacity (WHC) and organic matter were determined following the methods described by Porta et al. (1986). The calcium carbonate content (%) was measured according to Loeppert and Suarez (1996).
To determine the available mineral nutrient concentrations, 5 g of soil was dried at 60 °C for 48 h in 50-mL Falcon tubes. Each sample was diluted to 6.0 mL with DTPA-NH4 and analysed for B, Ca, Co, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, S, and Zn on an ELAN-DRCe ICP–MS instrument (PerkinElmer, SCIEX). National Institute of Standards and Technology (NIST) traceable calibration standards (ULTRA Scientific) were used for calibration (Soltanpour and Schwab 1977).
The plant material was dried for 4 days at 60 °C. Approximately 0.1 g was used to perform open-air digestion in Pyrex tubes using 0.7 mL concentrated HNO3 at 110 °C for 5 h in a hot-block digestion system (SC154–54-Well Hot Block™, Environmental Express, SC, Charleston, USA). The concentrations of the following elements (Ca, K, Mg, Na, P, S, B, Mo, Cu, Fe, Mn and Zn) were determined by inductively coupled plasma optical emission spectroscopy ICP–OES (Thermo Jarrell-Ash, Model 61E Polyscan, England) (Soltanpour and Schwab 1977).
In January 2019, 19 A. thaliana demes and Col-0 were sown in contrasting saline and saline-alkaline soils. Five seeds of each deme were sown in 30 pots (15 of each soil type) and distributed randomly in the greenhouse. Two weeks after germination, seedlings were thinned out so that only one plant per pot was left. Irrigation was applied twice a week. Every week, photographs of the entire rosette were taken. The number of siliques was counted at plant maturity. Air temperature, humidity and sun incidence were monitored throughout the experiment.
Salinity-alkalinity tolerance assays
For germination assays, sterilized seeds from each deme were sown in plates under a flow cabinet with sterile material. Plates contained 4 treatments: control (½ Murashige & Skoog media M5524 (MS), Sigma–Aldrich, pH 5.9), neutral salinity (½ MS NaCl 50 mM, pH 5.9), alkalinity (½ MS 10 mM NaHCO3, pH 8.3), and alkaline-saline treatment (½ MS NaCl 40 mM + 10 mM NaHCO3, pH 8.3). For each deme and treatment, a total of 60 seeds were divided among 4 plates. All plates contained 0.6% Phyto-agar (Duchefa, Haarlem, The Netherlands), and solutions were buffered using different proportions of MES and BTP depending on the final pH. Plates with seeds were kept at 4 °C for synchronizing germination. After 4 days under stratification treatment, plates were moved to a growth chamber (12 h light/12 h dark, 150 μmol cm−2·s−1, 40% humidity and 25 °C). Germination and radicle emergence were checked daily using a stereomicroscope (Zoom 2000, Leica, Wetzlar, Germany) during the following 10 days.
Sterilized seeds of demes from 6 coastal (HS), 4 intermediate (MSA), and 4 inland (HA) demes were sown in 0.2 mL tubes containing 0.6% agar prepared with ½ strength Hoagland nutrient solution (½ Hoagland, pH 5.9). Seeds were kept at 4 °C for 4 days in the dark to synchronize germination and placed in the growth chamber (12 h light/12 h dark, 150 μmol cm−2·s−1, 40% humidity and 25 °C). After root emergence (approximately 48 h), the bottoms of the tubes containing seedlings were removed, and the tubes were placed in 150 mL hydroponic containers with aerated nutrient solution (½ Hoagland, pH 5.9). The nutrient solution was replaced every 2 to 3 days to maintain a constant concentration of nutrients in the solution. When 15 days old, the seedlings were separated into different sets. To avoid osmotic shock, the treatment solutions were gradually increased in each set to achieve the final treatment conditions after one week. The following treatments were applied: control (½ Hoagland, pH 5.9) and two alkaline-saline treatments (½ Hoagland + NaCl 40 mM + 10 mM NaHCO3, pH 8.3 and ½ Hoagland + NaCl 60 mM + 15 mM NaHCO3, pH 8.3). Neutral pH solutions were buffered with 1:2 MES-BTP, while for high pH solution, 2:1 MES-BTP was used. The control and treatment solutions were replaced every 3 days. Plants remained under these conditions for two weeks. Every third day, photographs of the entire plant were taken. Thirty-seven-day-old plants were harvested, and leaves and roots were photographed, weighed and stored at −80 °C. The rosette diameters were scored, and the length of the largest root of each plant was measured using ImageJ software (Bourne 2010; Schneider et al. 2012).
Plants from selected demes of all groups (HA, inland; MSA intermediate habitat; HS coast) were cultivated individually in square pots of 10-cm diameter in sterilized quartz sand. The selected demes were A1 (HA), T6 (HS), LG5 (MSA), V1 (MSA), V3 (MSA) and Col-0 as a reference. Sterilized seeds were sown on wet soil, and the pots were covered with polyvinyl chloride film until the seedlings had germinated. Pots with germinated seedlings were placed in a growth chamber with a 12-h light/12-h dark photoperiod, an irradiance of 150 mmol m−2 s−1, and a constant temperature of 22 °C. Plants were watered with ¼ strength Hoagland solution at neutral pH 5.9 every 2 to 3 days. After 2 weeks, seedlings of each deme were split into 4 groups, and different treatments were applied (n = 8). To avoid osmotic shock, the treatment solutions were gradually increased to achieve the final treatment conditions after one week. The applied treatments were control (½ Hoagland, pH 5.9), salt (½ Hoagland +75 mM NaCl, pH 5.9), alkaline (½ Hoagland +15 mM NaHCO3, pH 8.3), and alkaline-saline conditions (½ Hoagland +60 mM NaCl +15 mM NaHCO3, pH 8.3). Solutions at pH 5.9 were buffered with 1:2 MES-BTP, while for high pH solutions, 2:1 MES-BTP was used. After 2 weeks under treatment conditions, chlorophyll content was measured (see below), and plant material was collected and separated into roots and shoots. The roots were carefully rinsed with deionized water, and the fresh weights of the roots and shoots were measured prior to storage at −80 °C. Chlorophyll contents were measured in leaves under different treatments. Leaf chlorophyll concentrations were obtained using a SPAD chlorophyll metre (CCM-200, Opti-Science, Hudson, USA). To measure the osmotic potential, two full leaves were thawed and inserted into a 1 mL syringe stuffed with fibreglass. Sap samples (50 μL) were collected, and their osmolality was measured with a freezing-point depression osmometer (Osmoat 3000, Gonotec). Proline content was determined colorimetrically using a method adapted from Bates et al. (1973). Briefly, leaf material (50 mg) was homogenized in 1 mL 3% (w/v) sulfosalicylic acid and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was supplemented with ninhydrin (250 μL) and glacial acetic acid (250 μL) in a test tube. The mixture was heated in a water bath at 100 °C for 60 min, and then the reaction was stopped with ice. The mixture was extracted with a 0.3 volume of toluene, and the absorbance was read at 520 nm using a TECAN-Spark Reader (n = 4 per deme per treatment).
A detailed outline of the experimental designs is shown in Supplementary Fig. S1; the demes used in each experiment are detailed in Supplementary Dataset S1.
Gene expression analysis
Frozen leaf material of plants from the irrigation experiments was used for RNA extraction. Total RNA was extracted using a PROMEGA RNA plant kit following the manufacturer’s instructions. Total RNA was used to produce cDNAs using the iScriptTM cDNA Synthesis Kit (Bio–Rad, Hercules, CA, USA) with 1 μL iScript Reverse Transcriptase +4 μL 5x iScript Reaction Mix + Sample + Molecular Water to obtain a 20 μL volume. Samples were run in a thermocycler (48-well MJ MiniTM, Bio–Rad, Hercules, CA, USA) for 5 min at 25 °C, 30 s at 42 °C, and 5 s at 85 °C. Fifty-fold dilution of the cDNAs was performed with water (Molecular Biology Reagent, Sigma–Aldrich, St. Louis, MO, USA). Diluted cDNA (1:50) was used as a template for quantitative PCRs using iTaq Universal SYBR Green Supermix (Bio–Rad, Hercules, CA, USA). Real-time detection of fluorescence emission was performed on a CFX384 Real-Time System (Bio–Rad, Hercules, CA, USA) using the following conditions: denaturation step for 10″ at 95 °C followed by annealing and extension for 30″ at 60 °C. A total of 40 cycles were run. A melt curve was performed, increasing from 65.0 °C to 95.0 °C by 0.5 °C every 5 s. Plates were edited using CFX manager version 3.1 software. Primers from selected genes were designed using the NCBI Primer-BLAST tool (Ye et al. 2012). The sequences of the primers used are detailed in Supplementary Dataset S10. The expression of target genes was normalized to the expression levels of the Actin2 and Tubulin genes of A. thaliana (Dekkers et al. 2012). The relative expression (RE) of each gene was calculated in comparison to the control treatment. The expression of the target gene relative to the expression of the reference gene was calculated using the 2−ΔΔCt method (Livak and Schmittgen 2001).
PCAs were performed on the genome-wide SNP data using the glPCA function of the adegenet R package (Jombart and Ahmed 2011). PCAs were visualized using ggplot2 (Wickham et al. 2016). The genome-wide SNP dataset was linkage disequilibrium-pruned using custom scripts and further filtered to include only putatively neutral fourfold degenerate sites (37,574 SNPs in total). PCAs were performed using 75 samples (4 plants of 19 demes except LLO2.1, which was excluded) from Busoms et al. (2018).
Data normality was checked for all phenotypes, and nonnormal data were transformed before applying any parametric tests. Mean-standardized values ( 1 < value >1) of elemental contents of soil and leaf material were used to represent the radar plots and compare each group. One-way or multivariate ANOVA was used to test for significant differences (p value < 0.05) between means of data with respect to fitness, elemental contents of soil and leaf material, and gene expression. To test for correlations between two variables, a bivariate fit was applied. To perform multiple comparisons of group means, we used Tukey’s HSD. The phenotypic plastic responses of different A. thaliana groups were expressed by their slopes of growth (rosette diameter) in the soil reciprocal transplant, and their plasticity was shown by the absolute values of the slopes (Gao et al. 2018). All statistical analyses were performed using SAS Software JMP v.16.0 (https://www.jmp.com/es_es/home.html).