Heat Acclimation under Drought Stress Induces Antioxidant Enzyme Activity in the Alpine Plant Primula minima

Heat and drought stresses are increasingly relevant topics in the context of climate change, particularly in the Alps, which are warming faster than the global average. Previously, we have shown that alpine plants, including Primula minima, can be gradually heat hardened under field conditions in situ to achieve maximum tolerance within a week. Here, we investigated the antioxidant mechanisms of P. minima leaves that had been heat hardened (H) without or with (H+D) additional drought stress. Lower free-radical scavenging and ascorbate concentrations were found in H and H+D leaves, while concentrations of glutathione disulphide (GSSG) were higher under both treatments without any change in glutathione (GSH) and little change in glutathione reductase activity. In contrast, ascorbate peroxidase activity in H leaves was increased, and H+D leaves had >two-fold higher catalase, ascorbate peroxidase and glucose-6-phosphate dehydrogenase activities compared with the control. In addition, the glutathione reductase activity was higher in H+D compared with H leaves. Our results highlight that the stress load from heat acclimation to maximum tolerance is associated with a weakened low-molecular-weight antioxidant defence, which may be compensated for by an increased activity of antioxidant enzymes, particularly under drought conditions.


Introduction
Small-stature alpine plants maintain their temperature well above the cool ambient air simply by having a stable aerodynamic boundary layer that is resistant to heat exchange [1]. This morphological strategy provides thermal benefits in a cold environment [2][3][4][5], but is counterproductive during heatwaves. As such, intentional overheating can become disadvantageous, leading to heat damage, which has been recurrently observed in smallgrowing alpine plants on hot days [6][7][8][9][10]. Climate change increases the intensity and frequency of heatwaves, which have tripled across Europe since 1958 [11]. The European heatwave in the summer of 2003 [12] was likely the warmest event since 1540 [13]. At a high elevation in the European Alps, temperature increases doubled the mean global surface increase (+0.55 K) [14], and high elevation sites seem to be particularly affected by global warming [15]. Hence, heat stress must be considered significant for alpine species and the progressive upward migration of alpine plant species is already considered to be a response to the warming climate [16].
Heatwaves are regular climatic components that can occur anywhere in the world and usually consist of a series of consecutive days of high temperatures, clear skies, and a dry atmosphere that successively leads to soil drought [17][18][19]. Drought leads to an increase in heat stress at the plant level as reduced or impaired stomatal conductance lowers transpiration cooling. At alpine sites, overheating is radiation-related, and combines with full solar radiation input, which can be exacerbated by elevation [20]. Together, these environmental factors produce a toxic mix that can pose serious problems to photosynthesis, namely strong irradiation under heat with concomitant drought-induced stomatal closure. Previously, using a purpose-built heat tolerance testing syst control the heat exposure of whole plants under nearly natural fi able to increase the critical high temperature threshold of PSII in to 46.7 °C under well-watered conditions (H), and up to 48.0 °C w combined with drought (H+D). Moreover, leaf tissue (50% s increased from 45.2 °C to 54.9 °C (H) and 51.6 °C (H+D), after 6 hardening, which was accompanied in both cases by increased [40].
While heat-stress responses were often conducted by placi elevated temperatures with no time to acclimate beforehand, th [40] allowed plants to acclimate to elevated temperatures under fi physiologically relevant to plant responses to heatwaves. In thi investigate leaf antioxidant mechanisms in heat-acclimated P. m help to explain how plants achieve maximal heat tolerance and this, we quantified LMW antioxidant concentrations, total a antioxidant enzyme kinetics in leaves that had been heat har drought, compared with non-heat-hardened leaves.

Plant Material and Heat Hardening
Growth and treatment conditions of the plants are fully des [40]. Briefly, the plants were excavated just beneath the summit 47°12′35″/E 11°27′45″), Tyrol, Austria, potted and transported

Plant Material and Heat Hardening
Growth and treatment conditions of the plants are fully described by Buchner et al. [40]. Briefly, the plants were excavated just beneath the summit of Mt. Patscherkofel (N 47 • 12 35 / E 11 • 27 45 ), Tyrol, Austria, potted and transported to the Alpine Garden Laboratory of the University of Innsbruck on Mt. Patscherkofel at an elevation of 1950 m (a.s.l.), during the summer of 2015. For heat-hardening treatments, plants were placed in a small cold frame made out of transparent Plexiglas panels (Biostar 1500, Juwel H. Wüster GmbH, Imst, Austria). The cold frames were fully temperature regulated (CR1000, AM 25 T, Campbell Scientific, Loughborough, UK). Heating was provided by ceramic heaters (12 V/150 W; AT858, Conrad Electronics, Hirschau, Germany) and cooling by ventilating fresh air via axial fans (120 mm × 120 mm × 38 mm; RD12038B12H, Conrad Electronics, Hirschau, Germany) from outside. Leaf temperatures were measured with fine-wire thermocouple sensors (TT-Ti-40, Omega Engineering Inc., Stamford, CT, USA). Mean leaf temperatures were increased from 32 • C to 38 • C over 8 days prior to the experiments. For treatment without additional drought stress (i.e., H), plants were watered every morning, and for additional drought stress (i.e., H+D) only when actual leaf water potentials, determined in 12 mm × 5 mm discs using a PSΨPRO TM water potential system (Wescor Inc., Logan, UT, USA), had approached a pre-dawn critical value of −3.3 MPa (Ψ act ). In a pre-test, leaves dehydrated to −3.3 MPa fully survived, whereas below −3.3 MPa, drought damage occurred. The drought-treated plants had significantly lower midday Ψ act on the third day and pre-dawn Ψ act on the fourth day of heat hardening [40]. The chambers were regularly calibrated by osmolality standards of 100, 290 and 1000 mmol kg −1 .

Biochemical Analyses
Leaves from untreated and from heat-hardened (H, H+D) individuals (n~25 for each treatment) were removed at midday, frozen in liquid nitrogen before freeze-drying for 3 days. Leaves from each replicate (n = 3-4, as indicated in figure legends) were ground to a fine powder in 2 mL reaction tubes (Eppendorf) with two 2 mm glass using a TissueLyser II (Qiagen, Düsseldorf, Germany) at 30 Hz for 6 min at freezing temperatures. Ground material was stored at −80 • C for 6-12 weeks before conducting measurements.

Total Free Radical Scavenging Activity
The total free radical scavenging activity was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) according to the method described by Brand-Williams et al. (1995). For each sample, 10 mg of lyophilized leaf powder were extracted in 1 mL 50% (v/v) methanol, shaken at 30 Hz for 2 min before centrifugation at 26,000× g for 10 min at 4 • C. The supernatant from each sample was transferred into new 2 mL reaction tubes (Eppendorf Hamburg, Germany). Incremental volumes of the supernatant up to 6 µL were pipetted into a 96-well microplate (Bio-Rad, Hercules, CA, USA), made up to 20 µL with 50% (v/v) methanol, and 200 µL DPPH solution was added (120 µM DPPH in 100% (v/v) methanol). The decrease in absorbance at 520 nm (i.e., radical scavenging) was followed for 30 min (Synergy HTX, Biotek, Winooski, VT, USA) until the linear decrease in absorbance ceased. Free radical scavenging activity for each sample volume was normalised to its protein content and calculated against a standard curve of ascorbate (0-8 nmols), and averaged.

Spectrophotometric Analysis of Ascorbate
Ascorbate and dehydroascorbate were measured using a colorimetric assay as previously described [41]. For each sample, 25 mg of lyophilized leaf powder was extracted in 2 mL of 6% (w/v) trichloroacetic acid and centrifuged at 26,000× g for 10 min at 4 • C. For the measurement of total ascorbate, 10 µL of supernatant was incubated with 90 µL of 66.7 mM Sørensen's buffer, pH 7.4, containing 5 mM dithiothreitol (DTT) for 15 min at 25 • C and then with 0.5% (w/v) N-ethylmaleimide (NEM), after which 100 µL of the working reagent was added. This consisted of reagent A [4.6% (w/v) trichloroacetic acid, 15.3% (v/v) orthophosphoric acid, 0.6% (w/v) FeCl 3 ] and reagent B [4% (w/v) 2,2 -dipyridyl in 70% (v/v) ethanol] combined in a 11:4 (v/v) ratio. It was incubated for 5 min at 42 • C before measuring the absorbance at 520 nm. For the measurement of reduced ascorbate, the samples were incubated as for total ascorbate, but without DTT or NEM. For each assay, three technical replicates were measured and averaged for each biological replicate. Amounts were calculated using a standard curve in the range of 0-1 µmol ascorbate, and the amount of dehydroascorbate was calculated by subtracting the amount of reduced ascorbate from total ascorbate.

HPLC Analysis of LMW Thiols and Disulphides
Low-molecular-weight thiols and disulphides were measured with HPLC as previously described [41]. For each sample, 50 mg of lyophilized leaf powder were extracted in 1.5 mL of 0.1 M ice-cold HCl at 30 Hz for 1 min before centrifugation at 26,000× g for 45 min at 4 • C. A total of 120 µL of the supernatant was used for the determination of total LMW thiols and disulphides, and 400 µL for disulphides only. Briefly, the pH was adjusted in both cases to 8.0 by adding 1.5× the sample volume of 200 mM bicine buffer. Total thiol and disulphide concentrations (e.g., GSH + GSSG) were determined after reduction of disulphides by 30 µL of 3 mM DTT, after which thiols were fluorescently labelled with monobromobimane (mBBr) by adding 20 µL of 15 mM mBBr dissolved in acetonitrile. For determination of disulphides only, thiol groups were blocked with 30 µL of 50 mM NEM dissolved in 2-propanol. Excess NEM was removed in a volume of toluene equal to the sample by vortexing, brief centrifugation and removal of the upper phase, and repeated four times. The remaining disulphides in the lower phase were reduced with DTT and labelled by mBBr as above. Labelled thiols were separated using a reverse-phase ODS-Hypersil column (250 mm × 4.6 mm, 3 µm particle size, Bischoff Chromatography, Leonberg, Germany) and detected by fluorescence (excitation: 380 nm; emission: 480 nm), using an Agilent 1100 HPLC system. The concentration of each thiol species was calculated by subtracting the disulphide concentration from the respective total thiols/disulphide amount. The glutathione half-cell reduction potential (E GSSG/2GSH ) was calculated from the molar concentrations of thiols and disulphides considering the leaf water content, and taking into account deviations from standard conditions in terms of pH (7.3) and temperature using the Nernst equation, as described in Schafer and Buettner [42] Antioxidants 2023, 12, 1093 5 of 12 and shown in Equation (1) below, where R is the gas constant (8.314 J K −1 mol −1 ), T the temperature in K, n the number of transferred electrons (i.e., 2) and F the Faraday constant (9.6485 × 10 4 C mol −1 ).

Antioxidant Enzyme Activities
For each sample, 30 mg of lyophilized leaf powder were extracted in 1.5 mL of 50 mM Sørensen's buffer, pH 7.5, at 30 Hz for 30 s. The suspension was centrifuged at 4 • C for 5 min at 26,000× g and 1 mL of the supernatant was diluted with 1 mL of extraction buffer, avoiding the upper lipid layer, and re-centrifuged for 5 min at 26,000× g. Enzymes were purified from interfering LMW compounds with PD10 Sephadex ® G-25 desalting columns (GE Healthcare, Chalfont St Giles, UK) using 1.6 mL of the extract and the centrifugation method at 4 • C according to the manufacturer's protocol. The resulting purified extract was measured immediately.
Catalase, ascorbate peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase (G6PDH) activities were measured spectrophotometrically (Synergy HTX, Biotek, Winooski, VT, USA) at 30 • C in 96-well UV-transparent plates (Corning, New York, NY, USA), using a 0.5 cm pathway correction for the Beer-Lambert law. All substrates were dissolved in the extraction buffer and the total reaction volume was 200 µL per well. Catalase activity was measured in 20 µL of extract with 10 mM H 2 O 2 and following H 2 O 2 breakdown via the absorbance decrease at 240 nm (ε = 43.6 M −1 cm −1 ) between 0.5 to 2 min after combining H 2 O 2 with the extract. Ascorbate peroxidase activity was measured in 30 µL of extract with 0.5 mM ascorbate and 0.5 mM H 2 O 2 , by following ascorbate consumption via the absorbance decrease at 265 nm (ε = 14,000 M −1 cm −1 ) between 0.5 to 2 min after combining the extract with reagents. Glutathione reductase activity was measured in 50 µL extract with 1 mM GSSG and 0.2 mM NADPH, by following NADPH oxidation via the absorbance decrease at 340 nm (ε = 6220 M −1 cm −1 ) between 1 to 10 min after combining the extract with reagents. Glucose-6-phosphate dehydrogenase activity was measured in 50 µL extract with 3 mM glucose-6-phosphate and 0.2 mM NADP + , by following NADPH formation via the increase in absorption at 340 nm (ε = 6220 M −1 cm −1 ) between 1 to 10 min after combining the extract with reagents. Catalytic rates were normalised to protein content of the extract, as measured with the Bradford assay (Bio-Rad, Hercules, CA, USA) in 2 µL of extract. For each assay, three technical replicates were averaged for each biological replicate.

Statistical Analyses
If homogeneity of variance between replicates was confirmed with Levene's test, oneway-ANOVA followed by Tukey's post-hoc test was used for determining significance differences between mean values at p < 0.05. When the data were not normally distributed, significance was tested via the Kruskal-Wallis H test, with post-hoc tests between each treatment performed using a Wilcoxon test. Analyses were performed in SPSS Statistics v.21 (IBM, New York, NY, USA).

Heat Hardening Led to a Loss of Free Radical Scavenging Activity, Irrespective of Drought
To assess the free radical scavenging activity of the leaves, the DPPH test was used, which has been described as an indicator of total LMW antioxidant activity [43]. Leaf extracts of H and H+D plants quenched the DPPH radical by ca. 30% less than leaf extracts from control plants (Figure 2), indicating that the heat-hardening process led to a loss of LMW antioxidants able to scavenge radicals.

Heat Hardening Led to a Loss of Free Radical Scavenging Activity, Irrespective of Drought
To assess the free radical scavenging activity of the leaves, the DPPH test was used, which has been described as an indicator of total LMW antioxidant activity [43]. Leaf extracts of H and H+D plants quenched the DPPH radical by ca. 30% less than leaf extracts from control plants (Figure 2), indicating that the heat-hardening process led to a loss of LMW antioxidants able to scavenge radicals. Free radical scavenging activity of leaves. Plants were exposed to increased heat (H) or to increased heat and drought (H+D) and leaves taken for analysis after 8 d of heat hardening, or from non-heat-hardened plants (Con). Free radical scavenging activity was measured using the DPPH assay, normalised to sample dry weight (DW) and expressed as ascorbate equivalents (eq.). Different letter denote significant differences (p < 0.05), n = 3 ± SD.

Heat Hardening Led to a Loss of Ascorbate, Irrespective of Drought
Leaf concentrations of reduced ascorbate and dehydroascorbate without heat hardening were 0.88 and 0.18 µmol g −1 DW, respectively. In H and H+D leaves, concentrations of reduced ascorbate significantly dropped to <0.6 µmol g −1 DW, while dehydroascorbate concentrations were insignificantly different (Figure 3). Free radical scavenging activity of leaves. Plants were exposed to increased heat (H) or to increased heat and drought (H+D) and leaves taken for analysis after 8 d of heat hardening, or from non-heat-hardened plants (Con). Free radical scavenging activity was measured using the DPPH assay, normalised to sample dry weight (DW) and expressed as ascorbate equivalents (eq.). Different letters denote significant differences (p < 0.05), n = 3 ± SD.

Heat Hardening Led to a Loss of Ascorbate, Irrespective of Drought
Leaf concentrations of reduced ascorbate and dehydroascorbate without heat hardening were 0.88 and 0.18 µmol g −1 DW, respectively. In H and H+D leaves, concentrations of reduced ascorbate significantly dropped to <0.6 µmol g −1 DW, while dehydroascorbate concentrations were insignificantly different (Figure 3). . Leaf ascorbate concentrations. Plants were exposed to increased heat (H) or to increased heat and drought (H+D) and leaves taken for analysis after 8 d of heat hardening, or from nonheat-hardened plants (Con). The stacked bar chart shows reduced ascorbate (white bars) above dehydroascorbate (black bars), normalised to sample dry weight (DW). Different letters denote significant differences (p < 0.05), n = 3 ± SD.

Drought Had a Modulating Effect on Glutathione Concentrations during Heat Hardening
Without controlled heat hardening, leaves of P. minima contained 0.50 µmol g −1 DW total glutathione, of which 0.16 µmol g −1 DW was present as GSSG and 0.34 µmol g −1 DW as GSH ( Figure 4A). Heat hardening increased the concentrations of GSSG, particularly in H+D leaves, which also had lower GSH concentrations, indicative of GSH oxidation to GSSG. This led to H+D leaves having more oxidising (less negative) values of E GSSG/2GSH relative to control and H leaves ( Figure 4A). The induction of GSH synthesis in response to heat hardening was observed by the increased concentrations of γ-glutamyl-cysteine under drought ( Figure 4B), conditions under which GSH concentrations were lowest ( Figure 4A). As the E GSSG/2GSH is calculated via the Nernst equation, a rise in temperature of 28 • C (i.e., typical difference between night and day) positively shifted the E GSSG/2GSH by 10.6-11.7 mV towards more oxidising values ( Figure 4B). hardened plants (Con). The stacked bar chart shows reduced ascorbate (white bars) above dehydroascorbate (black bars), normalised to sample dry weight (DW). Different letters denote significant differences (p < 0.05), n = 3 ± SD.

Drought Had a Modulating Effect on Glutathione Concentrations during Heat Hardening
Without controlled heat hardening, leaves of P. minima contained 0.50 µmol g −1 DW total glutathione, of which 0.16µmol g −1 DW was present as GSSG and 0.34 µmol g −1 DW as GSH ( Figure 4A). Heat hardening increased the concentrations of GSSG, particularly in H+D leaves, which also had lower GSH concentrations, indicative of GSH oxidation to GSSG. This led to H+D leaves having more oxidising (less negative) values of EGSSG/2GSH relative to control and H leaves ( Figure 4A). The induction of GSH synthesis in response to heat hardening was observed by the increased concentrations of γ-glutamyl-cysteine under drought ( Figure 4B), conditions under which GSH concentrations were lowest ( Figure 4A). As the EGSSG/2GSH is calculated via the Nernst equation, a rise in temperature of 28 °C (i.e., typical difference between night and day) positively shifted the EGSSG/2GSH by 10.6-11.7 mV towards more oxidising values ( Figure 4B). . LMW thiol and disulphide concentrations, normalised to sample dry weight (DW), and cellular glutathione redox potential (EGSSG/2GSH) of leaves in response to heat hardening. Plants were exposed to increased heat (H) or to increased heat and drought (H+D), and leaves taken for analysis after 8 d of heat hardening, or from non-heat-hardened plants (Con). (A) The stacked bar chart shows GSH (white bars) and GSSG (black bars), with EGSSG/2GSH values above (right y axis), considering daytime (38 °C, red) or night-time (10 °C, blue) temperatures (see methods). (B) γglutamyl-cysteine concentrations. Significant differences (p < 0.05) are denoted by different capital, lower-case and lower-case italic letters for thiols, GSSG and EGSSG/2GSH, respectively, n = 4 ± SD.

Heat Hardening Increased Antioxidant Enzyme Activities, Especially under Drought
Non-heat-hardened plants had catalase, ascorbate peroxidase, glutathione reductase and G6PDH activities of 6.0 ± 0.6, 12.6 ± 1.5, 0.40 ± 0.03 and 0.03 ± 0.01 µkat mg −1 protein, respectively. Ascorbate peroxidase activity was 2.2-fold higher in H leaves, and 2.4-fold higher H+D leaves, compared to non-heat-hardened plants ( Figure 5). Catalase and G6PDH activities were two-fold higher in H+D leaves, but insignificantly different in H leaves, compared with non-heat-hardened plants ( Figure 5). Glutathione reductase activity was significantly higher in H+D leaves compared with H leaves, but after H+D, they were indifferent to the activity in non-heat-hardened plants ( Figure 5). . LMW thiol and disulphide concentrations, normalised to sample dry weight (DW), and cellular glutathione redox potential (E GSSG/2GSH ) of leaves in response to heat hardening. Plants were exposed to increased heat (H) or to increased heat and drought (H+D), and leaves taken for analysis after 8 d of heat hardening, or from non-heat-hardened plants (Con). (A) The stacked bar chart shows GSH (white bars) and GSSG (black bars), with E GSSG/2GSH values above (right y axis), considering daytime (38 • C, red) or night-time (10 • C, blue) temperatures (see methods). (B) γ-glutamyl-cysteine concentrations. Significant differences (p < 0.05) are denoted by different capital, lower-case and lower-case italic letters for thiols, GSSG and E GSSG/2GSH , respectively, n = 4 ± SD.

Heat Hardening Increased Antioxidant Enzyme Activities, Especially under Drought
Non-heat-hardened plants had catalase, ascorbate peroxidase, glutathione reductase and G6PDH activities of 6.0 ± 0.6, 12.6 ± 1.5, 0.40 ± 0.03 and 0.03 ± 0.01 µkat mg −1 protein, respectively. Ascorbate peroxidase activity was 2.2-fold higher in H leaves, and 2.4-fold higher H+D leaves, compared to non-heat-hardened plants ( Figure 5). Catalase and G6PDH activities were two-fold higher in H+D leaves, but insignificantly different in H leaves, compared with non-heat-hardened plants ( Figure 5). Glutathione reductase activity was significantly higher in H+D leaves compared with H leaves, but after H+D, they were indifferent to the activity in non-heat-hardened plants ( Figure 5).

of 12
Antioxidants 2023, 12, x FOR PEER REVIEW 8 of 12 Figure 5. Changes in leaf antioxidant enzyme activities in response to heat hardening. Plants were exposed to increased heat (H; grey) or to increased heat and drought (H+D; white), and leaves taken for analysis after 8 d of heat hardening, or from non-heat-hardened plants (Con; black). Activities of catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH) are shown as fold difference relative to Con levels. Significant differences (p < 0.05) calculated individually for each enzyme are denoted by different letters, n = 3 ± SD.

Acclimation during Heat Hardening Involves Redox Signalling
As sessile organisms, plants have remarkable abilities to tolerate highly contrasting conditions. This 'plasticity' occurs via acclimation to the changed environment and alters many aspects of physiology and metabolism. Specifically regarding heat tolerance, some examples include the production of heat-shock proteins and changes in membrane composition to counteract the differences in membrane fluidity [44]. Thus, for plant tissue to achieve maximum tolerance towards adverse conditions, this requires time for acclimation. In this study, we slowly increased temperatures over days that allowed P. minima acclimation time to achieve maximal thermal tolerance [40]. Furthermore, in natural environments, plants almost always encounter combined environmental stresses. For example, due to climate change, summer heatwaves are an increasing phenomenon [18], and accompanying elevated temperatures lead to rapid drying of the soil and onset of drought. In several model plants, combined heat and drought have been shown to induce unique responses that differ from either heat or drought stress alone [23,[45][46][47]. Some of our data show that the response in H+D leaves was different to those in H leaves (e.g., Figures 4 and 5), as discussed below. Nonetheless, responses to one stress factor can accelerate response time and acclimation in local and distal ('systemic') tissue to other stresses, in a signal transduction process that involves H2O2 [23]. Previously, we showed that in P. minima and another alpine plant, Senecio incanus, leaves respond to heat hardening by drastically lowering jasmonic acid (JA) levels [40]. Zandalinas et al. [23] showed that the allene oxide synthase (aos) mutant of Arabidopsis thaliana deficient in JA signalling had accelerated H2O2 systemic signal propagation in response to heat stress. We therefore presume that H2O2 signalling in P. minima and S. incanus is enhanced during acclimation to heat stress, for which antioxidants also play a direct regulatory role.

Heat-Hardened Leaves had Weakened LMW Antioxidant Defences
The association of ROS production and redox signalling during heat [23,26] and drought stresses [33] indicates that redox regulation is particularly critical when both stresses combine. Maintaining redox homeostasis central to basal metabolic processes Figure 5. Changes in leaf antioxidant enzyme activities in response to heat hardening. Plants were exposed to increased heat (H; grey) or to increased heat and drought (H+D; white), and leaves taken for analysis after 8 d of heat hardening, or from non-heat-hardened plants (Con; black). Activities of catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH) are shown as fold difference relative to Con levels. Significant differences (p < 0.05) calculated individually for each enzyme are denoted by different letters, n = 3 ± SD.

Acclimation during Heat Hardening Involves Redox Signalling
As sessile organisms, plants have remarkable abilities to tolerate highly contrasting conditions. This 'plasticity' occurs via acclimation to the changed environment and alters many aspects of physiology and metabolism. Specifically regarding heat tolerance, some examples include the production of heat-shock proteins and changes in membrane composition to counteract the differences in membrane fluidity [44]. Thus, for plant tissue to achieve maximum tolerance towards adverse conditions, this requires time for acclimation. In this study, we slowly increased temperatures over days that allowed P. minima acclimation time to achieve maximal thermal tolerance [40]. Furthermore, in natural environments, plants almost always encounter combined environmental stresses. For example, due to climate change, summer heatwaves are an increasing phenomenon [18], and accompanying elevated temperatures lead to rapid drying of the soil and onset of drought. In several model plants, combined heat and drought have been shown to induce unique responses that differ from either heat or drought stress alone [23,[45][46][47]. Some of our data show that the response in H+D leaves was different to those in H leaves (e.g., Figures 4 and 5), as discussed below. Nonetheless, responses to one stress factor can accelerate response time and acclimation in local and distal ('systemic') tissue to other stresses, in a signal transduction process that involves H 2 O 2 [23]. Previously, we showed that in P. minima and another alpine plant, Senecio incanus, leaves respond to heat hardening by drastically lowering jasmonic acid (JA) levels [40]. Zandalinas et al. [23] showed that the allene oxide synthase (aos) mutant of Arabidopsis thaliana deficient in JA signalling had accelerated H 2 O 2 systemic signal propagation in response to heat stress. We therefore presume that H 2 O 2 signalling in P. minima and S. incanus is enhanced during acclimation to heat stress, for which antioxidants also play a direct regulatory role.

Heat-Hardened Leaves Had Weakened LMW Antioxidant Defences
The association of ROS production and redox signalling during heat [23,26] and drought stresses [33] indicates that redox regulation is particularly critical when both stresses combine. Maintaining redox homeostasis central to basal metabolic processes involves the LMW compounds ascorbate and glutathione [31]. As antioxidants, they further Antioxidants 2023, 12, 1093 9 of 12 provide tolerance to stress factors that would otherwise lead to critical imbalances in the cellular redox state [48,49]. Reacting with ROS converts antioxidants to their oxidised forms, and in response to heat hardening, decreases were observed in both ascorbate and GSH (Figures 3 and 4), indicative of a high ROS load in leaves. This likely explains the equal relative decrease in free-radical scavenging of the H and H+D leaves (Figure 2). Ascorbate and GSH partially function together since GSH is a substrate for dehydroascorbate reductase to recycle dehydroascorbate back to ascorbate. Thus, a limitation of GSH may influence ascorbate recycling, while ascorbate may also have been consumed by violaxanthin de-epoxidase in the xanthophyll cycle, which was active during heat hardening [40], and for recycling oxidised α-tocopherol [50,51]. Despite some loss of ascorbate, there was no evidence for a build-up of dehydroascorbate during heat hardening, indicating efficient recycling. However, heat-hardened leaves did accumulate GSSG, particularly under drought ( Figure 4B), inducing an oxidative shift in thiol-based cellular redox state (i.e., E 2GSH/GSSG ) and reflecting the oxidative load in leaves.
Modulations of the redox state of glutathione (i.e., E 2GSH/GSSG ) are part of the decision process of cell fate, on the one hand regulating growth and differentiation, while on the other hand inducing cell death when the E 2GSH/GSSG shifts to severely oxidising conditions [42,48,52,53]. Relevant in the context of heat tolerance is the influence of temperature on E 2GSH/GSSG , which becomes more oxidising at higher temperatures ( [42]; Figure 4A). This could lead to cell fate changing due to a shift in the cellular redox environment towards more oxidizing values under heat stress. In comparison to other studies [48,54,55], E 2GSH/GSSG was more positive (oxidised), even in the control plants. Due to the harsh environmental conditions (i.e., high light intensity and contrasting day/night temperatures) this may be a reflection of the stressful habitat of such plants. An alternative explanation is that leaves of P. minima were compartmentalising GSSG in the vacuole [56]. The E 2GSH/GSSG would then be lower (more reducing) in other cellular compartments in accordance with their lower GSSG concentrations. Nevertheless, the accumulation of GSSG in H+D leaves indicates that P. minima was particularly suffering from heat hardening under drought. In the alpine species Soldanella alpina and Ranunculus glacialis depletion of GSH rendered leaves sensitive to heat stress, especially under high light [57]. Indeed, H+D leaves were synthesising GSH, as evident by the increased concentration of γ-glutamyl-cysteine ( Figure 4B). It has been shown that the plant hormone salicylic acid (SA) is positively associated with glutathione levels [58]. Therefore, activation of glutathione synthesis could be linked to the increase in SA of heat-hardened leaves of P. minima [40].

Heat-Hardened Leaves had Enhanced Enzyme Antioxidant Defences
In contrast to the weakened LMW antioxidant defences, heat hardening increased antioxidant enzyme activity. The increased ascorbate peroxidase activity in H+D and H leaves ( Figure 5), indicated that heat tolerance required elevated control of H 2 O 2 production. Due to limited CO 2 availability under drought, the oxidase activity of RUBISCO increases, leading to glycolate accumulation (photorespiration) that is broken down by glycolate oxidase in the peroxisome, releasing H 2 O 2 that is broken down in the peroxisome by catalase [59], explaining why catalase activity only increased in H+D leaves ( Figure 5). Glutathione reductase activity was the least affected by heat hardening, despite the accumulation of GSSG. This could further indicate sequestration of GSSG in the vacuole, since levels of GSH remained stable. The increase in G6PDH activity in H+D leaves further supports an enhanced need for this pathway in supplying NADPH, as required by glutathione reductase.

Conclusions
This study has highlighted that specific antioxidant enzymes increased in activity in response to heat hardening, indicating they have a prominent role in heat stress tolerance. Increasing the abundance of ascorbate peroxidase may facilitate increasing heat tolerance in crops, while catalase and G6PDH can be considered for additional drought tolerance that