Table 4 Basic properties of soil.
pHKCl
|
Corg
|
CEC
|
Total As
|
Water extractable As
|
Sand
|
Silt
|
Clay
|
Bulk density
|
(-)
|
(%)
|
(mmol+ kg-1)
|
(mg kg-1)
|
(mg kg-1)
|
(%)
|
(%)
|
(%)
|
(g cm-3)
|
7.1
|
1.83
|
258
|
16 ± 1.7
|
0.10 ± 0.01
|
26
|
72
|
2
|
2.57
|
Corg, organic carbon; CEC, cation exchange capacity.
Determination of arsenic and other elements
Fronds were oven-dried for three days at 40 °C. Homogenised material (0.5 ± 0.05 g) was digested with a mixture of HNO3 and H2O2 (4:1, v/v) in an Ethos 1 device (MLS GmbH, Leutkirch im Allgäu, Germany). Contents of As, Cu, Mg, Mn, and Zn were determined using inductively coupled plasma-optical emission spectrometry (ICP-OES; Agilent 720, Agilent Technologies Inc., Santa Clara, CA, USA). Certified reference material (CRM NIST 1573a Tomato leaves, Analytika®, Czech Republic) was mineralized under the same conditions for quality assurance.
Isolation of DNA and determination of relative DNA methylation status based on % 5-methylcytosine
The fronds were weighed, frozen in liquid nitrogen and stored at –80 °C prior to DNA methylation analysis. To isolate total DNA, the fronds (1 g fresh weight) were ground to a fine powder in liquid nitrogen by mortar and pestle. DNA was extracted from 100 mg of powdered tissue using a NucleoSpin Plant II molecular kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany), as instructed in the user manual. The global DNA methylation status of DNA was determined using 100 ng of isolated DNA and a MethylFlash Methylated DNA Quantification Kit (Fluorometric; Epigentek Group Inc., Farmingdale, NY, USA) according to the manufacturer´s instructions. A SpectraMax MiniMax 300 Imaging Cytometer (Molecular Devices LLC, San Jose, CA, USA) with excitation at 530 nm was used to measure the fluorescence at 590 nm.
Determination of pigments
Pigment content in the leaves was measured photometrically with an Evolution 2000 UV-Vis (Thermo Fisher Scientific Inc., Waltham, MA, USA). A vessel-free leaf segment (0.5 cm2) excised from a freshly separated frond was incubated in the dark in 1 ml dimethylformamide with shaking for 24 hours. The absorbance of the extract was measured at wavelengths 480, 646.8, and 663.8 nm. Absorbance values at 710 nm were subtracted from these measurements. Data pigment contents were calculated from these data:
Chlorophyll A (Chl A; nmol ml–1): Chl A = 12.0 × A663.8—3.11 × A646.8
Chlorophyll B: (Chl B; nmol ml–1): Chl B = 20.78 × A646.8—4.88 × A663.8
Total chlorophyll (Σ Chl; nmol ml–1): Chl A + Chl B = 7.12 × A663.8 + 17.67 × A646.8
Carotenoids (Crt; nmol ml–1): Crtx+c = (1000 × A480—1.12 Chl A—34.07 Chl B) / 245
Determination of fluorescence
The chlorophyll fluorescence [variable fluorescence (Fv)/maximal fluorescence (Fm); µmol m–2 s–1]was measured using a modulated chlorophyll fluorometer OS1-FL (Opti-Sciences, ADC, BioScientific, Ltd., Hoddesdon, UK). The fresh leaf was obscured by clipping after 20 minutes to set up a dark-adapted state. Chlorophyll fluorescence was excited by a 660 nm solid-state light source, with filters blocking radiation longer than 690 nm. Saturation of the photosystem being measured was achieved by using a filtered 35 W halogen lamp (350 - 690 nm) with a pulse of 15,000 µmol m–2 s–1 during 0.8 seconds.
Determination of water potential
Water potential (WP; MPa), a measure of the energy status of the water in a system, was measured using a dew point PotentiaMeter (Decagon Devices, Inc., Pullman, WA, USA). The leaves of the plants were placed in a disposable syringe, the air was drawn off from the syringe, and the syringe was tightly closed with parafilm. The specimen was frozen at –18°C, then thawed, and the sap flow was pushed out into the measuring chamber of the PotentiaMeter.
Determination of selected photosynthesis parameters with gas-exchange parameters (GEP)
The portable gas exchange system LCpro+ (ADC BioScientific, Ltd., Hoddesdon, UK) was used for in situ determination of the net photosynthetic rate (PN; µmol CO2–1 m–2 s–1), the rate of transpiration (E; mmol H2O m–2 s–1), and calculation of water-use efficiency parameter (WUE; WUE = PN/E). All measurements were conducted between 8:00 and 11:30 Central European Time (CET). The duration of each individual measurement was 10 min after the establishment of steady-state conditions inside the measurement chamber. The conditions in the chamber were: 25 °C, ambient CO2 concentration 550 ± 50 µl l–1, air-flow rate 205 ± 30 µmol s–1 and irradiance 650 ± 50 µmol–1 m–2 s–1 of photosynthetically active radiation [46].
Statistical analysis
All data were checked for homogeneity of variance and normality by Levene and Shapiro-Wilk tests. Collected data did not meet the conditions for the use of analysis of variance (ANOVA) and were thus evaluated by non-parametric Kruskal-Wallis test in the Statistica 12.0 program (StatSoft, Inc., Tulsa, OK, USA). A principal component analysis (PCA), in the CANOCO 4.5 program, was applied to all collected data as a single set. We used standardisation of species because data of different characters were analysed together. PCA was used to draw correlations from the complex data set. The results were visualised in the form of bi-plot ordination diagrams using the CanoDraw program [47]. Correlations were confirmed using a linear correlation (r, p < 0.05, p < 0.01, p < 0.001) by Statistica 12.0.