Plants of C. salviifolius L. were collected in the geothermal alteration field near to Sasso Pisano town (Castelnuovo Val di Cecina, Pisa province, Italy), in the site of Regional Interest (RIS B12, IT 5160103) named “Monterotondo Marittimo and Sasso Pisano geothermal fields”, hosting two types of natural habitats listed in the Natura 2000 EU directive: the “Forests of Castanea sativa Miller (cod 9260)” and the “Lava fields and natural cavities (cod 8320)” [19]. The sampling site is characterised by emissions of steam vents containing H₂S, CO₂, boric acid and water vapour from cracks in the rocks with surface soil temperature from 50 °C to 100 °C and extremely low pH values (pH = 1.13) [17].

The considered geothermal alteration field is part of the geothermoelectric basin of Larderello, in southern Tuscany. The geological structure of the Larderello geothermal area is described in Bertini et al. [20].

The sampling site is located in the upper valley of Cornia river, at an elevation of about 550 m a.s.l. The climate is Mediterranean, with a mean annual temperature of 13.3 °C [21] and a mean annual rainfall of 1107 mm [17]. At the time of the sample collection (May 2011), the mean monthly temperature recorded was 16.3 °C (min: 10.3 °C; max: 22.5 °C) and the mean monthly rainfall was 7.0 mm (data from the wheather station of Castelnuovo Val di Cecina, Pisa available at http://www.castelnuovometeo.it/) and the monthly averaged daily mean global insulation upon a normal surface was 7780.1 Wh/m² (data from “Atlante Italiano della radiazione solare”, edited by “ENEA, Agenzia nazionale per le nuove tecnologie, l’energia e lo sviluppo economico sostenibile”, available at http://www.solaritaly.enea.it/index.php).

In the sampling site, the daily temperature of the soil was recorded 30.4 °C (min: 25 °C; max: 35 °C) and soil pH was 3.8 [4].

In May 2011, ten healthy plants of C. salviifolius, similar in size, growing near the fumaroles, were screened. The selected plants were then explanted, transferred in pots filled with perlite, after the complete removal of the native soil, and kept for 30 days inside a growth chamber (BINDER, KBW 400, Germany), under controlled conditions (12:12 h light:dark photoperiods, a day/night temperature of 25 °C, an estimated irradiation of 6510 Wh/m²). Plants were weekly watered with 1/2 × Hoagland's solution (Sigma).

Mature leaves were sampled from plants before the explants (G leaves) and from the same plants transferred to controlled conditions (C leaves) for anatomical and morpho-functional analyses (fresh material or chemically fixed) and for physiological determinations (fresh materials or fixed in liquid nitrogen and stored at –20 °C until use).

Leaf portions were excised from G and C leaves and were fixed for 24 h in FAA fixative (10 % formaldehyde–5 % acetic acid–45 % ethanol), dehydrated in a graded ethanol series, and embedded in LR-White medium Grade (London Resin Company). Semi-thin sections (3 μm) were cut with the ultramicrotome Ultratome Nova LKB using glass knives. The sections were stained with toluidine blue O (0.05 % in 100 mM benzoate buffer at pH 4.4) for histological characterisation [22]. The sections were cleared in xylene, air dried, mounted in DPX Mountant (Sigma) and then observed with a LEITZ DIAPLAN light microscope. At least 100 histological sections for each experimental group were analysed. Images of each slide were taken using a Leica DFC 420.

For surface investigations on fresh leaf pieces, a Leica M165 C stereomicroscope fitted with a Leica DFC 420 was employed.

For scanning electron microscopy (SEM) observations, leaf portions from the two experimental groups were fixed in 3% glutaraldehyde in 100 mM sodium phosphate buffer (pH 7.4) for 24 h and then dehydrated in a graded ethanol series. After critical point drying, leaf portions were collected on stubs through a double adhesive tape and subsequently they were coated with gold, examined and captured with a JEOL/JSM-5410 scanning electron microscope.

Epidermal stripping of both adaxial and abaxial surfaces of fresh leaves were made to determine stomatal density (SD), expressed as the number of stomata per square millimetre leaf area [23] and hair density (HD), expressed as the number of hairs per square millimetre leaf area. For each experimental group, 20 leaves collected from the selected plants (10 individuals) were analysed. Counts were made on both surfaces of the leaves for a total of 40 counts per experimental group.

Twenty histological median cross-sections randomly collected from 10 leaves per plant accession were used for the determination of total leaf thickness (TLT), palisade parenchyma thickness (PPT), adaxial and abaxial epidermis thickness (ADE and ABE), all expressed in μm. Analyses were performed by using the open source UTHSCSA Imagetool software (available at http://ddsdx.uthscsa.edu/dig/itdesc.html).

Leaf area (LA) was the one-sided projected surface area of a fresh leaf, expressed in mm². Specific leaf area (SLA) was the one-sided area of a fresh leaf divided by its oven dry mass (DW = dry weight), expressed in mm²/mg. Leaf dry matter content (LDMC) was the oven dry mass of a leaf (DW) divided by its water-saturated fresh mass (TW = turgid weight), expressed in mg/g. Succulence index (SI) was calculated as the ratio of the difference between the oven dry mass of a leaf and its water-saturated fresh mass to the leaf surface area, expressed in mg/cm² [24,25]. The following formulae are reported:

For each accession, 20 leaves from 10 individuals were collected. Sample storing and processing followed the standardised methodologies detailed by Cornelissen et al. [26]. Leaf projected area was acquired with a CanoScan LiDE 90 (Canon) and determined by CompuEye, Leaf & Symptom Area software (available at http://www.ehabsoft.com/CompuEye/LeafSArea/). For calculating mean and standard error, the average SD, HD, TLT, PT, ADE, ABE, LA, SLA, LDMC and SI for each individual plant was one statistical observation.

Calculations of leaf fresh weight, dry weight and moisture content were based on weights determined before and after oven drying of leaf samples at 100 °C for 24 h. Water content percentage was estimated on the fresh weight basis. Leaf relative water content (RWC), [27, modified] was calculated from the formula:

Fresh weight was obtained by weighing the fresh leaves. The leaves were then immersed in water overnight, blotted dry and then weighed to get the turgid weight. The leaves were then dried overnight in an oven at 100 °C and reweighed to obtain the dry weight.

Chlorophylls (a, b and total) and carotenoids were extracted in 80% acetone and determined according to Hassanzadeh et al. [28] and Lichtenthaler [29], respectively. Then, 100 mg of fresh leaves were homogenised and the extracts were centrifuged for 10 min at 6000 g at 4 °C. The supernatants were collected and the pellets were resuspended and extracted with 80% acetone until they resulted colourless. The collected supernatants were read using spectrophotometer at 645, 663 and 470 nm. Pigment contents were expressed as mg/g DW.

H₂O₂ content of leaves was determined according to Jana and Choudhuri [30]. Leaves (250 mg) were ground in a mortar and homogenised with 15 mL of phosphate buffer 50 mM pH 6.5. The homogenate was centrifuged at 6000 g for 25 min. To determine the H₂O₂ content, 3 mL of extracted solution were mixed with 1 mL of 0.1% titanium chloride in 20% (v/v) H₂SO₄, then, the mixture was centrifuged at 6000 g for 15 min and the supernatant absorbance at 410 nm was read. The amount of H₂O₂ in the extracts was calculated from a standard curve and expressed as μmol/g DW.

The amount of lipid peroxidation products in leaves was estimated by determining the malonyldialdehyde (MDA) content in the leaves according to Hartley-Whitaker et al. [31] with minor modifications as in Spanò et al. [32]. Powder from freeze-dried leaves material (10 mg) was mixed with TBA reagent (4 mL of 10% w/v trichloroacetic acid + 0.25% w/v thiobarbituric acid), heated (95 °C for 30 min), cooled for 15 min and centrifuged at 2000 g for 15 min. The level of MDA (155 mM/cm extinction coefficient) was measured as specific absorbance at 532 nm, by subtracting the non-specific absorbance at 600 nm [33] and expressed as nmol/g DW.

Total phenols were measured according to Arezki et al. [34]. Phenolic extracts were obtained after centrifugation of frozen leaf samples homogenised in HCl 0.1 N and left at 20 °C for 3 h. Then, 300 μL of extract were added to 1.5 mL H₂O + 0.1 mL Folin–Ciocalteu reagent and left so for 3 min. After incubation at 100 °C for 1 min in the presence of 400 μL Na₂CO₃ (20%), samples were cooled and the absorbance was measured. The level of phenolic compounds was determined spectrophotometrically at 750 nm and calculated as equivalent of gallic acid (GAE, mg/g DW) referring to a standard curve.

Ascorbate (ASA) and dehydroascorbate (DHA) extraction and determination were performed according to Kampfenkel et al. [35] with minor modifications. Briefly, the leaves (500 mg) were ground in a chilled mortar and homogenised with 3.75 mL of 5% (w/v) TCA. The homogenate was centrifuged at 12 000 g for 10 min at 4 °C and the supernatant was used for the determination at 525 nm [35]. Total ascorbate was determined after reduction of DHA to ASA by dithiothreitol and DHA level was estimated on the basis of the difference between total ascorbate and ASA value. Calculations were made on the base of a standard curve and correction was made for colour development in the blank (absence of sample). Content was expressed as mg/g DW.

Glutathione was extracted and determined according to Gossett et al. [36]. The leaves (500 mg) were homogenised in 1.5 mL of ice-cold 6% (w/v) m-phosphoric acid (pH 2.8) containing 1 mM ethylenediaminetetraacetic acid (EDTA). The homogenate was centrifuged at 20 000 g for 15 min at 4 °C and the supernatant was collected and stored in liquid nitrogen until use. Total glutathione (GSH + GSSG) was determined by the 5,5′-dithio-bis-nitrobenzoic acid (DTNB)-glutathione reductase recycling procedure [37] and the reaction was monitored as the rate of change in absorbance at 412 nm. GSSG was determined after removal of GSH from the sample extract by 2-vinylpyridine derivatisation. GSH was detected by subtracting the amount of GSSG from total glutathione and calculations were made on the base of a standard curve. A blank was made in the absence of the extract and content was expressed as nmol/g DW.

Acetonic powders were made according to Saari et al. [38] with modifications as in Bartoli et al. [4]. Briefly, the leaves were homogenised in ice-cold acetone. Homogenates were filtered through a Buckner filter with Macherey–Nagel MN 618 filter paper under vacuum and washings were repeated until the powder resulted colourless. After the complete removal of acetone under vacuum, acetonic powders were extracted in 100 mM potassium phosphate buffer pH 7.5 containing 1 mM EDTA, and 1% (w/v) polyvinylpyrrolidone (PVP-40) as in Spanò et al. [39]. All the extractions were performed at 4 °C. The homogenate was then centrifuged at 15 000 g for 20 min. For ascorbate peroxidase, 2 mM ascorbate was added to the extraction medium. For glutathione reductase, the supernatant was desalted on a Sephadex G-25 column. Supernatants were collected and stored in liquid nitrogen until their use for enzymatic assays.

Ascorbate peroxidase (APX) activity was measured according to Nakano and Asada [40] with modifications. Enzyme activity was assayed from the decrease in absorbance at 290 nm (extinction coefficient 2.8 mM/cm) as ascorbate was oxidised. The reaction mixture contained 100 mm potassium phosphate pH 7.5, 0.5 mm ascorbate and enzyme extract (25 μg protein/mL). The reaction was started by adding 0.2 mM H₂O₂. Correction was made for the low, non-enzymatic oxidation of ascorbate by hydrogen peroxide (blank).

Dehydroascorbate reductase (DHAR) activity was determined as described by Nakano and Asada [40]. The activity of DHAR was determined by monitoring the glutathione-dependent reduction of dehydroascorbate. Enzymatic extract contained 12.5 μg protein/mL. The activity was determined by measuring the increase in absorbance at 265 nm for 3 min. Specific activity was calculated from the 14 mM/cm extinction coefficient. A correction for the non-enzymatic reduction of DHA by GSH was carried out in the absence of the enzyme sample (blank).

Glutathione reductase (GR) activity was determined as described by Rao et al. [41], following the oxidation of NADPH at 340 nm (extinction coefficient 6.2 mM/cm). Enzymatic extract contained 25 μg protein/mL. A correction for the non-enzymatic reduction of GSSG was carried out in the absence of the enzyme sample (blank).

Ascorbate oxidase (AO) activity was measured as described by Moser and Kanellis [42]. The activity was determined by monitoring ASA oxidation following the decrease in absorbance at 265 nm for 3 min. Enzymatic extract contained 50 μg protein/mL. Specific activity was calculated from the 14 mM/cm extinction coefficient.

Glutathione peroxidase (GPX) activity was determined according to Navari-Izzo et al. [43], following the oxidation of NADPH at 340 nm (extinction coefficient 6.2 mM/cm). Enzymatic extract contained 25 μg protein/mL.

Catalase (CAT) activity was determined as described by Aebi [44]. Enzymatic extract contained 100 μg protein/mL. A blank containing only the enzymatic solution was made. Specific activity was calculated from the 23.5 mM/cm/extinction coefficient.

All enzymatic activities were determined at 25 °C and expressed as U/g protein. Protein measurement was performed according to Bradford [45], using BSA as standard.

Morpho-functional and physiological leaf traits were compared using the non-parametric test of Wilcoxon–Mann–Whitney. All the statistical tests were performed using the software R 2.14.0 (R Development Core Team. 2011. R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria. http://cran.r-project.org/).

A crimped and partially rolled lamina, forming several abaxial crypts (Fig. 1a, c, e), was observed in G leaves that also displayed a significantly greater thickness than the C leaves, which were characterised by large, thin and flat lamina (Fig. 1b, d, f and Table 1). Stellate trichomes occurred in adaxial surfaces of both G and C leaves (Fig. 1c, d), while dendritic trichomes, significantly more abundant in G leaves, occurred in leaf abaxial surface (Fig. 1e, f and Table 1). Leaves of C. salviifolius were amphistomatic (Fig. 2a–d) and G leaves, characterised by stomata of heterogeneous size (Fig. 2b), showed the greatest adaxial and abaxial stomatal densities (Table 1). Additionally, these leaves showed significantly higher values of LDMC and SI than C ones that, on the contrary, exhibited the greatest values of both LA and SLA (Table 1).

The thickness of adaxial and abaxial epidermis did not differ significantly between G and C leaves (Table 1), but epidermal cells of G leaves were covered by an abundant and roughened cuticle, mainly on the adaxial side, and showed thick walls with abundant pectins, (Fig. 1a, c, e; Fig. 2a, b; Fig. 3a, c, d). G leaves were, moreover, characterized by a densely packed and multilayered palisade parenchyma, consisting of highly elongated cells with few and small chloroplasts, and by a spongy parenchyma with small intercellular spaces (Fig. 3a). On the contrary, C leaves showed smooth cuticle (Fig. 2c, d), thin-walled epidermal cells (Fig. 3b, e, f) and a relaxed mesophyll, consisting of a bi-layered palisade parenchyma, characterised by short cells mainly in the inner layer, and a spongy parenchyma with very large intercellular spaces (Fig. 3b). Both tissues were characterised in C leaves by many and large chloroplasts (Fig. 3b).

While water content (Table 2) was significantly higher in C leaves than in G ones, there was no significant difference in RWC (Table 2) between the two materials. The content of total chlorophyll and of carotenoids (Table 2) were about four-fold higher in C leaves than in G ones that showed also a higher Chla/Chlb ratio.

ROS production was monitored by measuring the concentration of hydrogen peroxide (Table 3). The content of this signalling molecule was significantly higher in G leaves than in C leaves, showing a much stronger stress condition in the first ones. The measurement of lipid peroxidation (Table 3) was detected as MDA nmol/g DW: damage was approximately 3.4 times higher in G leaves than in C ones.

The antioxidant level was assessed by monitoring the variation of phenols and ascorbate/glutathione cycle metabolites (Table 3). Phenol content, measured as gallic acid equivalents, was significantly higher in G than in C leaves. The same also occurred for ascorbate pool, due mainly to the higher content of the reduced form of this low molecular weight antioxidant. In accordance, ASA/DHA ratio was significantly higher in G than in C leaves. Anyway, the content of total glutathione was significantly higher in C than in G leaves that, on the other hand, showed a significantly higher GSH/GSSG ratio.

Results of enzymatic assays are shown in Table 3. The activity of APX was higher in G than in C leaves. The other enzymatic activities were not significantly different between the two materials with the exception of the low CAT activity that was about two-fold higher in G leaves than in C ones.