Application of heat stress in situ demonstrates a protective role of irradiation on photosynthetic performance in alpine plants

Plant material

Three alpine plant species were selected, representing different growth forms and environmental conditions. Rhododendron ferrugineum L. (Ericaceae) is a woody dwarf-shrub, up to 130 cm in height, with evergreen and leathery leaves. It is a typical representative of dwarf-shrub communities in the alpine timberline ecotone (approximately 1600–2100 m.a.s.l.). S. incanus L. subsp. carniolicus (Willd.) Braun-Blanq. (Asteraceae) is a herbacous species, 5–15 cm high, with leaves that are densely hairy and form rosettes close to the ground. It is often found in dry and stony grasslands, as well as eroded areas in the sub-alpine and alpine zone (approximately 1800–3000 m.a.s.l.). Ra. glacialis L. (Ranunculaceae) is also an herbaceous species (height: 5–15 cm) with fleshy leaves, typically found in the subnival and nival zone from 2300 up to >4000 m.a.s.l. It prefers humid and wet sites within scree material and moraines.

Study sites

Study site 1 (Rh. ferrugineum) was located on a north-facing slope at the timberline of Mt. Patscherkofel (1960 m.a.s.l.; 47°12'N/11°27'E), an outpost of the Tuxer Alps, near Innsbruck, Austria. Study site 2 (S. incanus) was located within a south-exposed erosion plane slightly beneath the summit of Mt. Patscherkofel (2165 m.a.s.l.; 47°12'N/11°27'E). Study site 3 (Ra. glacialis) was located within a slightly inclined, extended stony moraine in the Ötztal Alps near the Timmelsjoch (2560 m.a.s.l.; 46°54'N/11°09'E).

Micrometeorology

Micrometeorological data were recorded at 1 min intervals by data loggers (CR10X, CR1000, Campbell Scientific, Loughborough, UK) at each study site. Photosynthetic photon flux density (PPFD) was measured by cosine-corrected quantum sensors (SKP 215, Skye Instruments Ltd., Llandrindod Wells, UK). Thermocouple sensors (Type T; GG-Ti-28, Omega Engineering Inc., Stamford, CT, USA) were placed 2 m above the ground and shielded against direct sunlight for determination of air temperature. Small thermocouple sensors (Type T; TT-Ti-36, Omega Engineering Inc.; solder junction diameter: 0.3 mm) were used to measure leaf temperature and were placed on the lower leaf surface with special leaf clamps to allow unrestricted solar irradiation and leaf transpiration (see Buchner et al. 2013).

Exposure to heat stress

The HTTS was used to expose selected plants to defined levels of heat stress in situ in the field in the presence of full natural solar irradiation (light mode) or in darkness (dark mode) (Fig. 1a). The device and method are described in detail in Buchner et al. (2013). The system consists of a software-controlled (based on LabView 2012, National Instruments, Austin, TX, USA) central supply unit in which up to eight exposure chambers can be connected. Each exposure chamber is made of cylindrical highly transparent Plexiglas (XT 29070, Röhm, Darmstadt, Germany) into which the samples are inserted. When the exposure chambers are operated in the light mode, the ambient solar irradiation may pass freely through the Plexiglas illuminating the plant samples inside. But it is also possible to keep the plant samples in darkness during the heat treatment (dark mode) which is realized by putting special steel tubes over the Plexiglas window of the exposure chambers. Four leaf temperatures are recorded continuously in each chamber and mean leaf temperature is automatically controlled by the system. In order to ensure homogenous leaf temperatures, relative humidity within each exposure chamber can be increased to create water vapour-saturated air, thereby restricting leaf cooling by transpiration. In addition, 12 small fans ensure sufficient convection inside the chamber to minimize temperature fluctuations of leaves exposed to strong solar irradiation.

Figure 1

Controlled application of heat to whole twigs of Rhododendron ferrugineum. (a) The heat tolerance testing system (HTTS) was used to apply controlled heat to plants in situ under full solar irradiation or in darkness on Mt. Patscherkofel (1960 m.a.s.l.). The exposure chambers of the HTTS were operated in the light mode (1) and in the dark mode (2). (b) Examples of temperatures recorded during the time course of the controlled heat treatment in the exposure chambers. Three different phases can be distinguished: (1) stabilization; (2) heating; and (3) exposure. Solid lines, leaf temperature inside the exposure chambers (black, dark mode; blue, light mode); red line, solar irradiation (PPFD) during the heat treatment.

The heat treatment of plants always started before sunrise and was divided into three phases (Table 1, Fig. 1b): (1) stabilization phase – after plants were inserted into the exposure chambers, mean leaf temperature was held at a start temperature until the temperature within the exposure chambers stabilized. This phase lasted approximately 15–30 min depending on the environmental conditions; (2) heating phase – plant temperatures were increased over a 2 h period using a constant warming rate until designated target temperatures were reached. The warming rates were similar to natural leaf heating rates measured in natural stands of alpine plants (Neuner & Buchner 2012); and (3) exposure phase – subsequent to the heating phase, plants within the chambers were exposed to a predetermined target temperature for 30 min, a time span that is commonly used for the determination of heat tolerance (Kreeb 1990). The exposure chambers were then opened and removed. The duration (2.5 h) of the entire heat treatment was similar for all of the defined target temperatures. Plants used in the study were exposed to each specific heating regime both in the light mode (abbreviated ‘46L’) and also in the dark mode (‘46D’). This was done in order to study the effect of the presence or absence of natural solar irradiation on photosynthetic gas exchange at each of the selected temperatures (e.g. 46 °C).

Selection of high temperatures

Two different exposure temperatures, within 2 K of each other, were selected for each species that would induce a severe but sublethal heat stress. The selected temperatures were 3 and 1 K below the temperature that induced the first evidence of heat injury to the leaves (LT₅), and as close to the critical high temperature threshold of PSII (T_c) as possible. Prior to the heat exposure, an LT₅ and T_c under dark conditions were calculated to determine the exposure temperatures for each species. Leaf heat tolerance (LT₅) was determined as described by Buchner & Neuner (2001). Leaf samples (10 samples per temperature) were fixed to overhead transparencies with adhesive tape (3M™ Transpore™, 3M Österreich GmbH, Perchtoldsdorf, Austria). The transparencies were then mounted inside a series of small exposure chambers, which had been preheated to a range of incrementally increasing temperatures. After exposure (30 min; Kreeb 1990), samples were immediately removed and placed into plastic bags, and kept at high humidity under moderate illumination for 20 h, until heat damage to the leaf blade could be assessed by determining the relative proportion of the damaged leaf area to the total leaf area using graphical analysis software (Optimas 6.5, Optimas Corp., Seattle, WA, USA) (Buchner et al. 2013). The temperature threshold, defined as the temperature inducing damage to 5% of the total leaf area (LT₅), was estimated from this data and used to select the target exposure chamber temperatures for each of the species examined.

Determination of the critical high temperature threshold of F₀ in darkness and F_s at different levels of irradiation

For S. incanus and Ra. glacialis, the critical high temperature threshold of PSII in darkness (T_c) was determined in situ using the laboratory-based T-F₀ technique (Schreiber & Berry 1977; Braun et al. 2002). Leaves were inserted into the fully darkened standard cuvette of a gas exchange-fluorescence system GFS-3000 (Walz, Effeltrich, Germany) with the temperature range extended up to +60 °C. Leaf surfaces were irradiated with a low pulse-modulated measuring light (470 nm) from light emitting diodes (LED array PAM fluorometer 3055-FL, Walz) that was fixed to the cuvette. Basic fluorescence F₀ was measured at intervals of 1 s, while leaf temperature was progressively increased at a rate of 1 K min⁻¹ up to +55 °C. T_c was defined as the temperature at which F₀ exhibited a distinct increase (Neuner & Pramsohler 2006), indicating the onset of the inactivation of PSII (Larcher et al. 1997).

This procedure was also conducted at different levels (10, 30, 100, 200, 650, 1000, 1500, 1650, 1800 μmol photons·m⁻² s⁻¹) of irradiation in order to determine the critical high temperature threshold T_c′ of F_s as a function of irradiation intensity. Leaves were irradiated with actinic light emitted from 90% red (640 nm) and 10% blue (470 nm) LEDs, and steady-state fluorescence F_s was measured at intervals of 1 s. The critical temperature (T_c^′) was defined as the temperature at which F_s showed a sharp increase under the respective level of irradiation.

Determination of the potential efficiency of PSII

Chlorophyll fluorescence is commonly used for monitoring photosynthetic performance (reviewed by Baker 2008) and has been widely reported to be sensitive to heat stress (e.g. Bilger et al. 1984, Weis & Berry 1988, Weng & Lai 2005). Therefore, the maximum quantum yield of PSII (F_v/F_m) can be used as a simple indicator of heat stress (Willits & Peet 2001) and for assessing the temperature causing permanent tissue damage (Krause et al. 2010). F_v/F_m was determined in situ in dark adapted leaves (30 min) using a portable chlorophyll fluorometer (PEA MK2, Hansatech Instruments Ltd., Norfolk, UK) before and repeatedly after controlled in situ heat exposure of whole plants in darkness and under natural solar irradiation.

In situ photosynthetic light response curves

The aftereffects of heat stress on photosynthetic gas exchange were assessed using parameters derived from photosynthetic light response curves: (1) dark respiration rate R_d (μmol CO₂ m⁻² s⁻¹), which describes the rate of CO₂ released by a certain leaf area in darkness; (2) A₂₀₀₀, the assimilation rate at high irradiation (PPFD = 2000 μmol photons·m⁻² s⁻¹); (3) Φ, the maximum quantum efficiency defined as the positive linear slope of the curve (μmol CO₂ mmol⁻¹ photons); and (4) the diffusion conductivity for water G_H2O (mmol m⁻² s⁻¹), which provides information on stomatal opening or closure. (1) and (3) were calculated by fitting a linear function to the linear part of the light response curve under weak irradiation (0 to 50 μmol photons·m⁻² s⁻¹). (2) and (4) were extrapolated from the gas exchange data by fitting a linear or a polynomial function (Eqn. 1) to the data using NI DIAdem software (2012, National Instruments, Austin, TX, USA).

(1)

Measurements were conducted in situ at 380 ppm CO₂ and 10 000 ppm H₂O at a flow rate of 750 μmol s⁻¹. Leaf temperature was held at 20 °C and PPFD was incrementally increased (0, 5, 10, 15, 50, 100, 150, 500, 1000, 1500 and 1800 μmol photons·m⁻² s⁻¹). Each step lasted for 3 min and gas exchange measurements were taken at 1 min intervals.

The low water vapour content of the measuring gas [e.g. 10 000 ppm corresponds to 32% relative humidity (Rh) at an ambient temperature 20 °C and air pressure 750 kPa] was essential, when the ambient air temperature was low, to avoid water condensation inside the measurement cuvette when opening it to insert new leaf samples. Individual samples were always taken at the same time of day in order to minimize diurnal effects on stomatal conductance measurements. All measurements were taken in situ on healthy leaves 1 d before conducting the controlled heat exposure experiments on previously untreated leaves and at several time intervals (1, 2, 6, 13 d) after the heat treatment.

Radical scavenging activity and pigment analysis

Statistics

Differences between mean values were tested either by analysis of variance (anova) in combination with Duncan's multiple range test (P < 0.05) or by the t-test (P < 0.05) and were conducted using SPSS (IBM SPSS-Statistics 21, New York, NY, USA).

Micrometeorology

Rh. ferrugineum (summer 2012)

June 2012 was the sixth warmest June in the last 250 years (ZAMG 2013a) in Austria. At Mt. Patscherkofel (1960 m.a.s.l.; study site 1), elevated leaf temperature maxima (half-hour means; HHM) were detectable in Rh. ferrugineum only for short periods at the beginning, in the middle and at the end of June (e.g. 2 June 2012: 46.2 °C, 15 June 2012: 45.3 °C; Fig. 2a). Maximum leaf temperatures were much lower (36.3 °C) in July and this was followed by another episode of high temperatures, with HHMs of up to 44 °C, from August 19 to 23. The experimentally applied temperatures of 43 and 45 °C also occurred naturally in the summer of 2012. HHMs over 43 °C occurred for 5 d and over 45 °C for 2 d during the period of 1 June 2012–1 September 2012 (Table 2).

Figure 2

Leaf temperatures recorded in the alpine and nival plant species investigated during the summers of 2012 and 2013. (a) Rhododendron ferrugineum (1960 m.a.s.l.; 2012), (b) Senecio incanus (2165 m.a.s.l.; 2013), (c) Ranunculus glacialis (2560 m.a.s.l.; 2013). Data were collected in 1 min intervals using thermocouple sensors mounted onto the lower surface of leaves (n = 5 per species). The curves represent half-hour mean values (HHM) based on calculations of high resolution data (taken each minute). Bold lines, daily maximum HHM; regular lines, daily mean HHM; dashed lines, daily minimum HHM. The temperature thresholds of 0 °C (solid horizontal bar) and LT₅, the temperature at which first heat-induced leaf injuries (5% of the total leaf area) were observed (dashed horizontal bar), were also shown. Vertical lines indicate the dates of the controlled heat treatment (bold) and days on which gas exchange measurements were recorded.

Table 2

Days with leaf temperature maxima >35 °C in three alpine plant species recorded in summer 2012 or 2013

Rhododendron ferrugineum	Tmax [°C]	35	36	37	38	39	40	41	42	43	45	46
1960 m a.s.l; 2012; jd 153–243	f [%]	38	32	26	20	16	13	9	6	5	2	1
Senecio incanus	Tmax [°C]	35	36	37	38	39	40
2165 m.a.s.l.; 2013; jd 152–243	f [%]	39	28	17	13	5	1
Ranunculus glacialis	Tmax [°C]	35	36	37
2560 m.a.s.l.; 2013; jd 188–243	f [%]	11	5	4

Altitude at the study site and the investigation period (year; julian days) are given for each studied species. T_max are maximum half-hourly taken mean values (HHM) of temperatures in 1 °C classes. The frequency (f) describes the frequency of days at which a HHM was at least once higher than the corresponding temperature class.

The impact of heat on the photosynthetic parameters F_v/F_m, A₂₀₀₀, R_d and Φ

The selected heat treatments did not cause any visible leaf damage in S. incanus and Ra. glacialis whereas a minimal level of injury occurred in Rh. ferrugineum. Despite the lack of visible injury, photosynthetic parameters were significantly affected by increasing temperatures in all of the investigated species. The effects of the heat treatments are summarized in Fig. 3. It should be noted that the first measurements were conducted 1 d after the applied heat stress. In addition to effects of the heat stress per se, exposure to natural environmental conditions (including solar irradiation) during the recovery period may have made an impact on the various photosynthetic parameters. Generally, when heat stress occurred under irradiation, the impact was mitigated compared with when it was applied in the dark. The percentage change in the investigated parameters in relation to reference values obtained for each leaf before the heat treatment is discussed in the succeeding text for ease of comparison between temperature and light and dark treatments. A comprehensive table of the data, including statistics, is provided as Supporting Information Appendix S1.

Figure 3

Impact of sublethal heat treatment applied in situ on gas exchange and various photosynthetic parameters. Parameters were derived in situ from photosynthetic light response curves before and after the controlled heat exposure in the heat tolerance testing system (HTTS). Different heat exposure modes are seperated by vertical dotted lines. Left column (a–c) Rhododendron ferrugineum (Mt. Patscherkofel, 1960 m.a.s.l., 9 August 2012), middle column (d–f) Senecio incanus (Mt. Patscherkofel, 2165 m.a.s.l., 11 July 2013) and right column (g–i) Ranunculus glacialis (Ötztal Alps, 2560 m.a.s.l., 11 August 2013). Boxplots show the time courses of the potential PSII efficiency, F_v/F_m (a, d, g), of the photosynthetic CO₂ assimilation rate at a PPFD of 2000 μmol photons·m⁻² s⁻¹, A₂₀₀₀ (b, e, h), and of the dark respiration rate, R_d (c, f, i). The four boxes within each heat exposure mode (from left to right) represent the control value 1 d before (−1) the heat treatment followed by the values during recovery from the heat treatment. Different colours (orange, red) of boxes indicate different exposure temperatures. White background, heat treatment in the light mode; grey background, heat treatment in the dark mode.

Diffusive conductance and PSII

During the recovery period following the heat stress, A₂₀₀₀ was reduced, particularly in leaves that were treated in the dark mode. In plants exposed to heat stress in the light mode, however, all of the investigated species showed a clear and highly significant correlation (Spearman's Rho; see Fig. 4) between A₂₀₀₀ and G_H2O, indicating that the reduction in A₂₀₀₀ was primarily due to reduced G_H2O during the recovery period. No such correlation was found in plants treated in the dark mode, with the exception of Ra. glacialis (Fig. 4e). A₂₀₀₀ and F_v/F_m were significantly correlated in plants of Rh. ferrugineum and S. incanus treated in the dark mode, indicating that the reduced A₂₀₀₀ during the recovery period was mainly caused by a reduction in F_v/F_m (Fig. 4b,d). No significant correlation was detected between A₂₀₀₀ and F_v/F_m (Fig. 4f) in Ra. glacialis and A₂₀₀₀ remained reduced even when F_v/F_m was nearly completely recovered.

Figure 4

Photosynthetic performance following sublethal heat stress. Correlation diagram between the assimilation rate at 2000 μmol photons·m⁻² s⁻¹, A₂₀₀₀, and (left column) the diffusive conductance G_H2O at PPFD 2000 μmol photons·m⁻² s⁻¹ and (right column) F_v/F_m for 1, 2 and 6 d after controlled heat treatment at two different sublethal temperatures in the dark mode (solid circles) or in the light mode (open circles). (a, b) Rhododendron ferrugineum, (c, d) Senecio incanus, (e, f) Ranunculus glacialis. Numbers: Correlation coefficient (Spearman's Rho) and significance of the correlation (values in parentheses). Significant correlations are indicated in bold letters.

T_c and T_c′

During progressive heating (1 K min⁻¹) of S. incanus and Ra. glacialis plants, a rise in F₀ was observed at a lower temperature than the rise in F_s (Fig. 5a). Furthermore, T_c was found to be 45.0 ± 1.2 °C (n = 5) in S. incanus, whereas T_c′ was 4 K higher (49.2 °C) when the irradiation was low (10 μmol photons·m⁻² s⁻¹). Above a PPFD of 100 μmol photons·m⁻² s⁻¹, T_c′ was elevated, reaching a maximum value of 53.3 °C at 1500 μmol photons·m⁻² s⁻¹. The maximum difference between T_c and T_c′ observed in plants of S. incanus was 8.3 K (Fig. 5b).

Figure 5

Critical high temperature threshold of PSII. (a) Typical T-F₀ plot (temperature-basic fluorescence) for determination of T_c determined during controlled heating in darkness (solid line) compared with steady-state fluorescence (dotted line) recorded during controlled heating in the light (1600 μmol photons·m⁻² s⁻¹). At critical temperatures (T_c in darkness, T_c′ in the light), the chlorophyll fluorescence signal shows a distinct increase. (b) T_c and T_c′ determined on leaves of Senecio incanus (circles) and Ranunculus glacialis (triangles) as a function of irradiation intensity. T_c is lowest (solid symbols, n = 5). T_c′ (open symbols) increases with irradiation intensities, initially steeply in both species, then plateaus and after reaching approximately 1800 μmol photons·m⁻² s⁻¹ drops again. Boxplots of T_c (grey boxes) and T_c′ (white boxes; PPFD from 30 to 1650 μmol photons·m⁻² s⁻¹) in leaves of (c) S. incanus and (d) Ra. glacialis. Significant differences between mean values based on the Student's t-test (P < 0.01) are indicated by different letters.

Similar results were obtained for Ra. glacialis with a T_c of 42.9 ± 0.7 °C (n = 5) compared with a T_c′ of 45.4 °C at a PPFD of 10 μmol photons·m⁻² s⁻¹. T_c′ reached maximum values ranging from 47.1 to 49.2 °C, when PPFD was between 100 and 1650 μmol photons·m⁻² s⁻¹. A decrease to 45.1 °C was observed, however, at a PPFD of 1800 μmol photons·m⁻² s⁻¹. The maximum difference between T_c and T_c′ observed was 6.3 K. In both species, T_c and mean T_c′ differed significantly (P < 0.01) between 100 and 1650 μmol photons·m⁻² s⁻¹ (Fig. 5c,d).

Free radical scavenging activity and xanthophyll cycle pigment levels

Free radical scavenging activity

Free radical scavenging activity was lowest in the non-heat-stressed control group and significantly (P < 0.05) increased by 34% and 46% in S. incanus and Ra. glacialis, respectively, when plants were exposed to heat stress in the dark. In contrast, when the heat treatments were administered in the light, no significant effect on free radical scavenging activity was observed (Fig. 6).

Figure 6

Free radical scavenging activity following controlled sublethal heat stress. Free radical scavenging activity in Senecio incanus (white bars) and Ranunculus glacialis (grey bars), expressed as trolox equivalents per mg dry mass (nmol TE·mg⁻¹DM; means ± SD) immediately after the termination of an in situ heat stress treatment (S. incanus: 44 °C, R. glacialis: 41 °C) in the dark and light mode is shown relative to untreated plants (control). Significant differences between species (superscript numbers) are indicated by different letters (one-way anova, Duncan's test, P < 0.05).

Xanthophyll cycle pigments

The xanthophyll cycle pigments violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z) were significantly (P < 0.05) affected by heat stress under both light and dark conditions (Fig. 7). However, the effect was most pronounced in plants exposed to heat stress in the light. The de-epoxidation status of the xanthophyll cycle pigments (V + A)/(V + A + Z) after the heat treatment was significantly (P < 0.05) higher in both S. incanus (dark mode: 41.6% ± 2.6; light mode: 60.6% ± 3.9) and Ra. glacialis (dark mode: 24.7% ± 1.6; light mode: 71.5% ± 2.8) relative to the non-stressed control plants (S. incanus: 32.0% ± 5.9; Ra. glacialis: 15.4% ± 6.2). Other photosynthetic pigments (chlorophyll a and b, β-carotene, neoxanthin and lutein) did not change significantly in response to the heat treatment, with the exception of β-carotene and lutein, which significantly (P < 0.05) increased by 15% and 28%, respectively, in S. incanus after heat treatment in the dark compared with heat treatment in the light. These data are in agreement with the finding that free radical scavenging activity increased for S. incanus in response to the heat treatment when it was administered in the dark.

Figure 7

Xanthophyll cycle pigments following controlled sublethal heat stress. Violoxanthin (hatched bars), antheraxanthin (dotted bars) and zeaxanthin (white bars) in (a) Senecio incanus and (b) Ranunculus glacialis, expressed as a percentage of total xanthophyll cycle pigments, and their de-epoxidation state (A + Z)/(V + A + Z)·100% (black bars) immediately following the termination of controlled, in situ heat treatments (S. incanus: 44 °C, Ra. glacialis: 41 °C) in the dark mode and in the light mode are shown in comparison with untreated plants (control). Significant differences between the individual pigments (superscript numbers) are indicated by different letters (one-way anova, Duncan's test, P < 0.05)

Residual effects of sublethal heat stress on photosynthesis

The effect of global warming on the climate of the European Alps is rather evident, however, few studies have been conducted on the effects of heat stress on alpine plants (e.g. Larcher & Wagner 1976, Gauslaa 1984, Neuner et al. 1999, Buchner & Neuner 2001, 2003; Marcante et al. 2014). It is important to note that, in these few studies, heat treatments were typically applied in the dark to detached plant material in a laboratory setting. Such experimental conditions do not provide information on: (1) the effect of solar irradiation on the response of plants to heat stress, and; (2) the ability of plants in their natural habitat to recover from the deleterious impacts of the heat stress. In the current study, the HTTS was used, which allowed for the in situ evaluation of the impacts of sublethal heat stress on photosynthetic performance, photosynthetic pigments and free radical scavenging activity in three high mountain species. Results indicated that the alpine plant species studied tolerated heat stress much better in the light, at levels that corresponded to natural solar irradiation. By contrast, after the termination of the heat stress, the residual effects on F_v/F_m, A₂₀₀₀, Φ and R_d were greater in plants that had been exposed to the heat stress in the dark. In this context, it again must be pointed out that the presented residual effects may not only be due to the heat stress per se but may show also impacts made by exposure to natural environmental conditions during recovery. Further, it was demonstrated that the heat response of the investigated plant species also has species-specific components.

The heat stress-induced decrease in A₂₀₀₀ in the light in all three species can be primarily attributed to a reduction in G_H2O, which lasted for a long time (Fig. 4). Abiotic stress factors, such as salinity or drought, are known to alter photosynthetic gas exchange via their effect on stomatal and non-stomatal parameters (see Ashraf & Harris 2013). The strong correlation between A₂₀₀₀ and G_H2O after exposure to heat stress in the light mode indicates that the reduction in A₂₀₀₀ during the recovery period was mainly due to stomatal parameters.

In contrast, the effect of heat stress in the dark on A₂₀₀₀ was primarily due to the reduced efficiency of PSII. It is evident that stomatal conductance, photosynthesis and transpiration interact in a complex manner (Farquhar & Sharkey 1982; Tuzet et al. 2003) and that stomatal conductance may be correlated with actual photosynthetic capacity (Wong et al. 1979). However, the long-term effect of heat stress, when applied in situ in the light, on G_H2O with a concomitant decrease in photosynthetic performance, does not appear to have been previously reported in the literature. Our results demonstrated that within either 6 or 13 d, depending on species, F_v/F_m recovered slowly in all three species, when the heat stress was administered in the dark, whereas A₂₀₀₀ recovered fully in S. incanus, slowly in Rh. ferrugineum and hardly at all in Ra. glacialis. Hence, the HTTS allowed us to gain deeper insights into the remarkable and species-specific ability of high mountain plants to recover photosynthetic performance following a severe heat stress.

The data in Figs 4 and 5 offer a possible explanation for the contribution of PSII to the protective effect of light in heat-stressed plants. T_c^′, the critical high temperature threshold for PSII, increased to higher temperatures during heat exposure with increasing irradiation. The increase in T_c^′ supports the observation that PSII was more stable when heat stress occurred in the light, resulting in smaller residual effects on photosynthesis after the heat stress was terminated. Nevertheless, the heat treatments in situ were done at lower temperatures than the critical temperature thresholds, potential aftereffects of critically high temperatures remain to be examined. As observed in Rh. ferrugineum, even low irradiation during heat stress (e.g. mean PPFD 261 μmol photons·m⁻² s⁻¹) was sufficient to elicit a protective effect. These findings also corroborate earlier results obtained in laboratory experiments which indicated the potential protective role of irradiation, even at very low intensities, on the stability of PSII during heat stress (Weis 1982). The mechanisms responsible for the increased heat stability of PSII under irradiation are complex and still not fully understood. The stability of PSII function at high temperatures may not only be affected by light but also by osmotic potential and sugar concentration (Hüve et al. 2006), as well as by any process leading to an impairment of the electron transport chain.

Importantly, the observed protective effect of irradiation during heat stress on photosynthesis need not only be due to the increased stability of PSII but could also be explained by other mechanisms. It is conceivable that the heat treatment may have reduced the activation status of rubisco because of the heat sensitivity of rubisco activase (Salvucci et al. 2001), which is known to play a key role in limiting photosynthesis during heat stress (Salvucci & Crafts-Brandner 2004) and during recovery from exposure to high temperature (Kim & Portis 2005). As the activation state of rubisco activase is also affected by light-mediated changes of the stromal ATP/ADP ratio (Portis et al. 2008), it can be assumed that heat stress applied in the dark mode could result in a stronger decrease in rubisco activase activity compared with heat stress applied in the light mode. The exposure of the plants from darkness to high irradiation intensities after the termination of the heat treatment could have induced photoinhibition and photodamage because of at least a temporary partial inactivation of the Calvin cycle. In turn, this partial inactivation may have resulted in a reduction in F_v/F_m and A₂₀₀₀. In this scenario, the observed reduction in F_v/F_m could be interpreted as a consequence resulting from the impairment of the Calvin cycle, rather than because of the limited heat stability of PSII as a primary cause. The relevance of rubisco activase and non-photochemical fluorescence quenching (NPQ) in relation to rapidly increasing irradiation was recently demonstrated by Yamori et al. (2012). When rice plants were suddenly subjected to high irradiation, the electron transport rate (ETR) increased much faster in genetically modified plants overexpressing rubisco activase compared with wild-type plants, while NPQ was lower. Unfortunately, our field measurements did not provide the data necessary to perform a similar analysis.

During the first hour after the heat treatment was terminated, leaves of Rh. ferrugineum were exposed to moderate irradiation (PPFD approximately 500 μmol photons·m⁻² s⁻¹ with maxima approximately 1000 μmol photons·m⁻² s⁻¹), while S. incanus was continuously exposed to approximately 1000 μmol photons·m⁻² s⁻¹ and Ra. glacialis was exposed to approximately 2000 μmol photons·m⁻² s⁻¹. This may also have potentially affected the results obtained in the present study. High mountain plants have the potential to cope with the rapidly changing environmental conditions, including irradiation and temperature. PPFD can rapidly fluctuate between very low levels (<50 μmol photons·m⁻² s⁻¹) when the sky is clouded, and >2500 μmol photons·m⁻² s⁻¹ in full sunlight. Photosynthetic carbon assimilation in some species is often not saturated at a PPFD of 2000 μmol photons·m⁻² s⁻¹ (Streb & Cornic 2012). Therefore, such effects of the sudden exposure to high levels of irradiation immediately after the heat treatment seem rather unlikely but cannot be ruled out entirely, and may have to some minor degree influenced the results. Also, the accumulation of heat shock proteins (HSP) which may be induced in response to a combination of high temperature and light, as demonstrated in Solidago altissima (Barua & Heckathorn 2006), could have contributed to the minor residual effects of heat stress in the light mode. Regardless of the mechanism, irradiation alters the effect of heat stress on PSII. When the heat stress is applied in the light, PSII appears to play a pivotal role in the response of photosynthesis to heat stress. This is due to (1) photoinhibition, which is increased at higher temperatures (see Streb et al. 2003, Dongsansuk et al. 2005) and (2) a shift in T_c^′ towards higher temperatures.

Protective effect of natural solar irradiation

The protective effect of irradiation in plants subjected to heat stress has fascinated plant scientists for a long time, and yet the mechanistic basis of the protection still remains to be elucidated. As previously stated, the protective role of irradiation is not likely the result of a single mechanism but rather results from a combination of several factors, including the activation state of rubisco, ROS-scavenging activity and HSP accumulation. ROS levels often increase in response to heat stress and ROS-scavenging mechanisms are important in protecting plants against high temperature stress (Allakhverdiev et al. 2008). In addition, ROS are also intricately involved in oxidative signalling (Foyer & Noctor 2009). In the present study, free radical scavenging activity was used as an overall indicator of the level of stress. The increase of free radical scavenging activity was significant when heat was applied in the dark, confirming that the heat treatment caused more stress in the dark than in the light. It is also conceivable that an earlier onset of ROS signalling may have taken place in dark-treated leaves than in the light-mode leaves, and led to a more rapid stress response, resulting in a more extensive up-regulation or activation of ROS-scavenging molecules. The stable free radical DPPH reacts with many antioxidants, albeit with different kinetics (Mishra et al. 2012), and so the DPPH assay does not allow one to draw conclusions about the composition of the ROS-scavenging components. It will be interesting to characterize the role of specific ROS-scavenging compounds in the response to heat stress applied in darkness versus in lightness (especially at different levels of irradiation), but this was not an aim of the present study.

Havaux et al. (1999) reported a potential connection between the photoprotection of PSII and irradiation-induced inter-thylakoid acidification, resulting in the stabilization of the thylakoid membrane and PSII reaction centres. Additionally, acidification of the thylakoid lumen also activates violaxanthin de-epoxidase, thus activating the xanthophyll cycle. The formation of ROS can be partly avoided by non-photochemical quenching (NPQ) of excess light and the xanthophyll cycle is an important part of NPQ. In accordance with earlier results (Yin et al. 2010; Dongsansuk et al. 2005), heat treatment applied in the light led to a significant increase of xanthophyll cycle activity in both species investigated. Streb et al. (2003) also reported increased xanthophyll cycle activity in Ra. glacialis leaves at 38 °C under strong irradiation. Increased xanthophyll cycle activity in the heat-exposed plants likely contributed to the protection of the photosynthetic apparatus from heat damage and may at least partly explain the better survival of leaves exposed to heat stress in the light. Nevertheless, the xanthophyll cycle was also activated when heat stress was applied in the dark, although to a much lesser extent. Investigating the mechanism underlying the activation of the xanthophyll cycle in the dark was not a key objective in the present study. However, it is interesting to note that Fernández-Marín et al. (2010, 2011) also found that the enzyme violaxanthin de-epoxidase was activated in the lichen, Lobularia pulmonaria, and in the brown seaweed, Pelvetia canaliculata, in the dark in response to desiccation or high temperature. Furthermore, violaxanthin de-epoxidation was also induced in response to heat stress at very low irradiation (12 μmol photons m⁻¹·s⁻¹) in wheat seedlings (Ilik et al. 2010). In contrast, Abramchik et al. (2013) reported that a 3 h heat treatment at 44 °C and a PPFD of 64 μmol photons·m⁻² s⁻¹ did not affect xanthophyll cycle activity in seedlings of different Triticale cultivars. Zeaxanthin may also protect thylakoid membranes from lipid peroxidation not only by quenching of ¹Chl but also by NPQ-independent mechanisms (see Havaux & Niyogi 1999; Johnson et al. 2007; Jahns & Holzwarth 2012). In summary, heat stress applied in the light, and to a lesser extent also in the dark, led to violaxanthin de-epoxidation, resulting in the formation of antheraxanthin and zeaxanthin, which may have contributed to the protection of the photosynthetic apparatus.

Finally, Yamauchi et al. (2011) showed that in the light, heat stress can cause preferential excitation of PSI and enhance thermal dissipation and light-driven cyclic electron flow around PSI. This can protect PSII from damage because of a backflow of reducing power from the stroma to PSII. In turn, this backflow may cause an overreduction of plastoquinone and result in damage to the D1 protein of PSII reaction centres (Marutani et al. 2012).

It is generally accepted that two or more stress factors are more stressful to an organism than one factor alone (Kranner et al. 2010). On their own, light and heat both have the potential to cause photoinhibition and photodestruction. Therefore, one could intuitively assume that the combination of high light and heat stress will cause more damage than heat stress in the dark. However, this assumption is fraught with difficulties because for a plant, the absence of light is stressful. Few authors have considered the potential protective effects of irradiation on photosynthetic functions, including the reduced inactivation of CO₂-fixation in Spinacia oleracea chloroplasts (Weis 1982) and the enhanced PSII function in barley leaves (Havaux & Tardy 1997; Kalituho et al. 2003), when plants were subjected to heat stress. Havaux et al. (1999) suggested that light may protect photochemical activity from inactivation by heat. Our results largely support these observations, but they also demonstrate that recovery of heat-treated leaves under field conditions is highly dynamic and species-specific.

Relevance of the study to current and future microclimate conditions of alpine plants

In the European Alps, particularly at high elevation, the increase in atmospheric temperature was found to be doubled than what was estimated for global warming in general (1890–1998, +1.1 °C versus +0.55 °C; Böhm et al. 2001). If this trend continues, heat stress will become an increasingly significant factor for alpine plants.

The present study illustrates that under current climatic conditions, photosynthesis of individual alpine plant species can already be temporarily reduced by naturally occurring heat waves. Global temperatures are predicted to increase erratically (IPCC 2013), with more frequent heat waves in Europe (Schär et al. 2004), which will affect photosynthetic performance of high mountain plants accordingly. However, this will not necessarily lead to a widespread extinction of alpine plants, because due to the dense thermal microhabitat mosaics that are characteristic of high elevation sites, most plant species can readily ‘escape’ the detrimental thermal regime (Scherrer & Körner 2011). Furthermore, global climate change will undoubtedly promote an upward shift of certain species to higher elevations (Grabherr et al. 1994; Holzinger et al. 2008; Pauli et al. 2012). We demonstrated that the temperatures applied to alpine plants in the current study are realistic and may occur under present climate conditions. Alpine dwarf-shrub heaths can exhibit canopy temperatures that are significantly higher than air temperature (Larcher & Wagner 2010), and plants in the sub-alpine zone may be at a higher risk of being injured by heat stress than nival species. Based on the available data, it seems likely that Ra. glacialis but not S. incanus will be negatively affected if leaf temperatures rise to between 41 and 46 °C (as in our experiments). It is expected that mean air temperature will increase by 0.3–0.4 K per decade in the European Alps from the present through 2100 (Gobiet et al. 2014). Therefore, it can be assumed that any further increases in temperature, or more frequent heat waves, will have the potential to reduce photosynthetic performance for considerable periods of time in a very species-specific manner. In the long term, such a changing thermal environment will further increase the pressure on heat-sensitive and less-competitive species and favour species migration, which can already be observed in the European Alps today.