A novel system for in situ determination of heat tolerance of plants: first results on alpine dwarf shrubs
© Buchner et al.; licensee BioMed Central Ltd. 2013
Received: 3 December 2012
Accepted: 7 March 2013
Published: 14 March 2013
Heat stress and heat damage to plants gain globally increasing importance for crop production and plant survival in endangered habitats. Therefore the knowledge of heat tolerance of plants is of great interest. As many heat tolerance measurement procedures require detachment of plants and protocols expose samples to various heat temperatures in darkness, the ecological relevance of such results may be doubted. To overcome these constraints we designed a novel field compatible Heat Tolerance Testing System (HTTS) that opens the opportunity to induce controlled heat stress on plants in situ under full natural solar irradiation. Subsequently, heat tolerance can be evaluated by a variety of standard viability assays like the electrolyte leakage test, chlorophyll fluorescence measurements and visual assessment methods. Furthermore, recuperation can be studied under natural environmental conditions which is impossible when detached plant material is used. First results obtained on three alpine dwarf - shrubs are presented.
When heat tolerance of Vaccinium gaultherioides Bigelow was tested with the HTTS in situ, the visual assessment of leaves showed 50% heat injury (LT50) at 48.3°C, while on detached leaves where heat exposure took place in small heat chambers this already happened at 45.8°C. Natural solar irradiation being applied during heat exposure in the HTTS had significantly protective effects: In Loiseleuria procumbens L. (Desv.), if heat exposure (in situ) took place in darkness, leaf heat tolerance was 50.6°C. In contrast, when heat exposure was conducted under full natural solar irradiation heat tolerance was increased to 53.1°C. In Rhododendron ferrugineum L. heat tolerance of leaves was 42.5°C if the exposure took place ex situ and in darkness, while it was significantly increased to 45.8°C when this happened in situ under natural solar irradiation.
The results obtained with the HTTS tested in the field indicate a mitigating effect of natural solar irradiation during heat exposure. Commonly used laboratory based measurement procedures expose samples in darkness and seem to underestimate leaf heat tolerance. Avoidance of detachment by the use of the HTTS allows studying heat tolerance and recuperation processes in the presence of interacting external abiotic, biotic and genetic factors under field conditions. The investigation of combined effects of heat exposure under full solar irradiation, of recuperation and repair processes but also of possible damage amplification into the results with the HTTS appears to be particularly useful as it allows determining heat tolerance of plants with a considerably high ecological significance.
KeywordsChlorophyll fluorescence Electrolyte leakage Heat stress Rhododendron Loiseleuria Vaccinium Viability assessment
High temperature is an important abiotic stress factor for plants that is not restricted to tropical areas and desert belts but also may play an important role in colder regions and at high altitudes . The main reason for heat as an important stress factor in alpine sites are growth forms like rosettes and cushions that plants may have evolved to favor a decoupling from ambient cold air temperature . This improvement of thermal conditions may in turn cause critical overheating as the heat trapping nature can get fatal in situations with high solar irradiation input, combined to restricted transpiration and calm winds causing situations where the thermal high temperature thresholds can get exceeded.
As high altitudes are not excepted from climate change  and high mountain vegetation is particularly vulnerable to it  especially prostrate species and plants over bare and quickly desiccating soils like many pioneer plants and their seedlings may be affected by heat stress [5–7]. This particularly, if the projected increase in global mean temperature  in fact should come true.
Besides natural vegetation the same applies to crop plants. Food production especially in hot and arid regions due to increased temperature has already become an agricultural problem in many areas of the world .
Such forecasts emphasize the urgency and the importance of a comprehensive knowledge about heat tolerance and heat stress responses  of plants and their capacity to adapt to an increasingly warmer climate. However, this presupposes the availability of practicable and ecologically relevant methods for the assessment of heat tolerance of plants. As is known, under natural environmental conditions heat tolerance of plants may change significantly within short time spans [11–13]. Therefore, in most cases transportation of plants from a distant natural growing site to a laboratory for conducting the heat tolerance test is not meaningful. Many heat tolerance data have been collected during the last centuries using laboratory based test procedures e.g. [14–19], however, ecologically significant data on heat tolerance of plants obtained in in situ measurements on plants at their specific growing site are widely missing and mechanisms of recuperation and repair appear severely understudied.
The classical method for the determination of heat tolerance is a laboratory based test procedure [20, 21]. This procedure normally consists of two consecutive phases: (1) the exposure of the samples to controlled heat stress in darkness and (2) the viability assessment based on visual damage or thermal stress dependent cellular functions like cell membrane stability (electrolyte leakage) or chlorophyll fluorescence parameters (non - invasive) [22, 23]. Until now only two field - portable instrument systems for the determination of heat tolerance on detached plant parts have been described [24, 25]. Although with these instruments long transportation times and possible short - term alterations of heat tolerance can be avoided, they still do not allow studying after - effects and recuperation, as the samples have to be detached similar to laboratory tests. Another disadvantage of the existing field - portable systems is that heat exposure has to be conducted in darkness or under weak irradiation. This does not take into account that particularly at high altitudes midday heat usually coincides with high irradiation levels. This, however, may have significant effects on photosynthetic tissues and their ability to withstand heat stress, an aspect that has been totally neglected in the majority of earlier studies. Until today only a few studies on the determination of heat tolerance have been done, where heat stress was applied in the presence of natural solar irradiation using light transmissible tents or plastic bags e.g. [26, 27] with the restriction that either temperature control was very limited or movement of plants into growth chambers  was necessary.
To overcome these disadvantages we designed and constructed a field compatible Heat Tolerance Testing System (HTTS) which allows in situ determinations of heat tolerance without the necessity to detach plant parts and with the possibility of conducting controlled heat treatments in the presence of natural solar irradiation (light - mode) and - for comparative studies - also in darkness (dark - mode). Heat treatments can be conducted under water vapor saturated air conditions that eliminate unpredictable transpirational cooling effects and ensure best possible isothermics between air and plant material. It is known  that heat exposure within an unsaturated atmosphere can have marked effects on the viability, because if transpirational cooling can take place, leaf temperatures will not reach the desired exposure temperature and therefore heat tolerance obtained under such conditions will possibly deviate substantially from those that are determined at 100% relative humidity.
The inclusion of recuperation and repair studies in the presence of interacting external abiotic, biotic and genetic factors under natural environmental conditions subsequent to controlled heat stress exposure powerfully enhances the ecological significance of the heat tolerance data determined with the HTTS, as they represent the de facto heat tolerance of a certain species at a certain developmental stage at the natural growing site. This will increase the accuracy of risk assessments related to future survival of spontaneous vegetation and will allow optimizing cultivar testing in breeding programs.
We hypothesize (1) that the presence of natural solar irradiation during short – term heat stress events may principally affect heat tolerance of leaves and (2) that heat tolerance determined on excised leaves (ex situ) may significantly differ from results derived from attached leaves at the natural growing site (in situ). Therefore, the aim of this study was to test whether heat stress in darkness or heat stress under full solar irradiation is survived better, and to observe possible influence of detachment on the measured heat tolerance. Additionally, the practical feasibility and the main features of the HTTS (in situ heat exposure at natural solar irradiation within a water saturated atmosphere) should be demonstrated under alpine field conditions on the basis of plant species being especially suitable for this purpose. In the following, preliminary results on selected alpine dwarf shrubs are shown obtained with the HTTS in comparison to those achieved with classical tests.
Effects of relative air humidity on leaf temperatures
Determination of heat tolerance: classical methods in comparison to the HTTS
First experiments on alpine dwarf - shrubs demonstrate that both the heat exposure mode and the viability assay may have marked effects on the measured heat tolerance values. In the following results from three representative experiments are shown:
Experiment (1) - V. gaultherioides
Leaf heat tolerance (LT50 ± SD) of V. gaultherioides when determined on detached leaves (ex situ) two days after a heat exposure that took place on 24 Jul 2012 in complete darkness using the exposure chambers from Buchner and Neuner (2001)  was 45.8°C ± 0.9. If - at the same time - the heat exposure was conducted in situ using the exposure chambers of the HTTS, heat tolerance was significantly (P < 0.01) higher: 48.3°C ± 0.8. This also applies to heat tolerance values carried out on the same leaves but at a later time: 48.1°C ± 1.0 (28 Jul 2012), 48.5°C ± 1.9 (2 Aug 2012).
Experiment (2) - L. procumbens
Experiment (3) - R. ferrugineum
If viability assessment was done by chlorophyll fluorescence measurements (Fv/Fm) the differences between LT50 values were not as clear. However, apart from the first assessment date (30 Jul 2012) the trend was similar with the addition that in the case of dark - mode LT50 of detached leaves (42.6°C ± 0.8) and LT50 determined in situ (42.6°C ± 0.5; 30 Jul 2012, 43.9°C ± 1.4; 4 Aug 2012 and 43.6°C ± 1.4; 9 Aug 2012) were not significantly different (P < 0.05). Leaf heat tolerance based on heat exposure under natural solar irradiation (in situ, mean PPFD = 650 μMol photons · m-2 · s-1) was slightly higher (43.0°C ± 1.3; 30 Jul 2012), (44.5°C ± 1.9; 4 Aug 2012) and (45.0°C ± 1.6; 9 Aug 2012) with the restriction that only the last result (9 Aug 2012) showed a significant (P < 0.05) difference. The chlorophyll fluorescence method underestimated heat tolerance if the time span between heat exposure and viability assessment was not sufficiently long. It took eight recuperation days (4 Aug 2012) till LT50 values assessed by both methods had converged (Figure 4B).
Although a lot of literature concerning the determination of stress tolerance of plants such as heat, frost and drought is available e.g. [10, 11, 13, 18, 20–22, 24, 25, 31–34] a complete and detailed comparison of different methods especially for determining heat tolerance of plants is still missing. As both the heat exposure mode and the method of the following viability assessment have the potential to influence the outcome, results from one and the same sample may differ significantly. We have to state that there is not only one single method that could be appropriate under all circumstances. It depends on the scientific research question and the practical feasibility which method will be preferred. For the majority of scientific questions e.g. in the fields of plant breeding, genetic engineering and crop science appropriate and well established methods may exist. This is, however, not the case for ecological issues, where heat tolerance is understood as the potential to survive heat stress on the organ - or the whole plant - level under natural conditions. Primarily, for these special purposes the HTTS has been designed. The results from the present study underline the importance of conducting heat tolerance tests in situ and shall increase the experimenter’s sensitivity for that issue.
Effects of relative humidity on leaf temperature during heat exposure
When relative humidity of the air inside the exposure chambers was increased to between 95 and 100%, the difference between air and mean leaf temperature got clearly reduced. This is because of the physical laws governing leaf transpiration rate . If the heat exposure chambers were operated in the dark - mode, individual leaf temperatures usually were lower than or equal to air temperature. Leaf transpiration and transpirational cooling gets quenched within a water saturated atmosphere when leaf and air temperature are the same. As transpiration rate and transpiration cooling usually is different for each individual leaf, also the differences within leaf temperatures are greater in non - saturated atmosphere than under water saturation.
Effects of solar irradiation on leaf temperature
In light - mode transpirational cooling is superimposed by the thermal load of absorbed solar irradiation depending on irradiation intensity, individual leaf angle, leaf orientation and shading. This is why leaf temperature may exceed air temperature despite transpirational cooling. A water vapour saturated atmosphere may additionally promote overheating and therefore, convective cooling inside the exposure chamber is particularly important. Furthermore, particularly at high altitudes the impact of solar irradiation on leaf temperature can be changed immediately due to fast passing clouds. The effects of rapidly altering solar irradiation on leaf temperature places great demands on the control unit of the HTTS that needs to combine a short response time with minimal overshooting to prevent deviation of leaf temperatures from target temperature. In the case of extremely adverse environmental conditions the accuracy of the system can be improved significantly by mounting thermocouple sensors on a couple of leaves and by setting the set - point temperature to the resulting mean leaf temperature that is continuously determined by the control software.
Effects of detachment on leaf heat tolerance
If leaves were detached (ex situ) this had significant effects on the determined heat tolerance value. LT50 of leaves from V. gaultherioides that had been detached before they were subjected to the controlled heat exposure was lower by -2.5 K in comparison to leaves that were exposed without detachment (see Figure 2). This applied also to L. procumbens where this difference was -2.2 K. Further for detached leaves the results from ELT corresponded well with the results from VAC (see Figure 3). Also for R. ferrugineum a significant difference in heat tolerance (-2.0 K) due to leaf detachment was detectable three days after the heat exposure (see Figure 4A).
One reason for the mainly negative effect of detachment on heat tolerance may be that recuperation and repair processes at the cellular and molecular level can run more successfully if leaves remain attached to the plant, while in the case of detachment leaves are exposed mainly to degrading processes that may amplify the pattern of heat damage. Principally, countervailing effects are also conceivable, especially if the environmental conditions subsequent to the in situ heat exposure and during recovery are unfavourable for recuperation (e.g. environmental stresses such as drought, frost or excessive solar irradiation). Detached leaves are commonly exposed to standard laboratory conditions with high humidity, moderate, constant temperature and irradiation intensity that largely deviate from the natural situation. Hence, the de facto heat tolerance of a certain plant at a certain location will be better reflected by measurements that are conducted in situ than under artificial laboratory conditions using detached leaves.
Effects of irradiation mode during heat exposure on heat tolerance
Natural solar irradiation during in situ heat exposure increased leaf heat tolerance significantly in L. procumbens (+2.7 K) and in R. ferrugineum (+1.3 K) when compared to the results yielded after heat exposure in darkness. It could be demonstrated that solar irradiation may have a significant protective effect on heat stressed leaves. The reverse effect, a decline of heat tolerance due to the combined occurrence of heat and irradiation stress would principally also be conceivable, although our measurements do not give evidence for this.
Combined effects of irradiation and heat stress on leaf tissue are known for a long time [36–38] but the physiological mechanisms and constraints for possible protective effects of solar irradiation during heat stress are still widely unknown. As it is known that photosynthetic processes are more heat sensitive than the plasma membrane  in green tissue this effect possibly can be explained by photoinhibition which under certain conditions may act as a protective mechanism against heat stress .
However, our results demonstrate that the presence of natural solar irradiation during heat exposure is necessary for a realistic determination of heat tolerance of leaves in order to obtain results being ecologically more relevant.
Further in this experiment during the whole latent period heat tolerance based on the results of VEM (Figure 4A) differed distinctly from heat tolerance values obtained by the chlorophyll fluorescence method (CHFL) (Figure 4B). The reason for this may be that a reduction of Fv/Fm needs not to be linked exclusively to heat damage but also to a just temporary and reversible reduction of the maximum efficiency of photosystem II (PSII) - due to light, heat and other kind of stresses [23, 41, 42], which may considerably affect the calculation of lethal parameters such as LT50. Thus, CHFL, although it can be applied easily and in a non - invasive manner, cannot be recommended for determining heat tolerance without any restrictions, because it does not distinguish between photoinhibition and photodestruction. However, this shortcoming can be minimized by applying a sufficiently long recovery period between the heat exposure and the Fv/Fm measurements.
Importance of the re - growth test
Often it may be sufficient to determine heat tolerance ex situ and based on detached plant parts or organs. Such methods have the advantage, that heat exposure can be conducted conveniently in the lab by avoiding natural weather conditions which often - especially at high altitudes - can be very challenging. However, if heat tolerance data with high ecological relevance are needed, heat stress must be applied under natural environmental conditions directly at the natural growing site of a plant. The advantage is that there is no need to detach the organs of interests. By this recuperation under fully natural conditions including external abiotic and biotic factors and their interactions can be studied. In frost experiments on conifers [31, 32] it could be demonstrated that frost damage produced in winter took several weeks until frost damage had fully developed. If viability would have been assessed before the end of this extended latent period, frost hardiness would have been seriously overestimated. In the case of heat tolerance a similar influence of the time factor is conceivable although in summer environmental conditions, particularly temperature may be more favourable for a more rapid development of visible injuries. For example if viability is assessed by CHFL as shown for R. ferrugineum leaves, it can take several days (Figure 4B) or even weeks (data not shown) till PS II of heat stressed leaves is fully recovered which can have significant consequences on the calculated LT - values. In that case the assessed heat tolerance value differed by 2 K depending on the timing of the measurement after heat exposure.
With respect to the considerable amount of heat tolerance data on arctic and alpine plants that is already available e.g. [18, 19] which have almost exclusively been derived from detached plant material, it can be expected that future results which will be obtained with the HTTS may significantly deviate from the existing data. Alpine vegetation is particularly prone to global climate change  because in alpine environments, abiotic factors, especially climate, dominate biotic interactions . Changes in plant growth and reproduction  and upward shifts of vegetation belts are natural consequences and can already now be observed [44, 45]. High mountain vegetation is at risk of drastic area losses, extinction and of disintegration of the current vegetation patterns which impacts the stability of high mountain ecosystems . Especially for many pioneer plants the effects of the temperature increase may be severe . Taking into account these facts, prediction models concerning alpine plant species in a global warming world will become increasingly important. Heat tolerance data determined by the HTTS may contribute to their accuracy and will sensitively affect their outcome.
This work does not claim to be a comparative study of all kinds of known viability assays that are currently in use, because there is not only one ideal method that can be applied to all cases. However, being aware that viability assessment can be very difficult and resultant tolerance levels can deviate to a large extent, various methods were employed in this study. The most appropriate method concerning heat tolerance testing depends on the research question, plant species, plant organ and the level on which the heat tolerance shall be determined (cellular level, tissue level or whole plant level or with respect to certain functions such as photosynthesis, growth, fruit production or reproduction). Furthermore, the time dimension has to be considered: Is it sufficient to get a snapshot of actual heat tolerance or shall the effects of recuperation or time dependent changes of heat injuries are taken into account? Before a specific heat tolerance test assembly can be chosen, the experimenter in any case has to clarify these questions in advance before the experiments may start.
We presented a novel system for determining heat tolerance of plants in situ without the necessity of detachment of plant organs. It allows controlled heat exposure of plants optionally in darkness or under full natural solar irradiation. Heat exposure is conducted within a water saturated atmosphere to keep the cooling effect of leaf transpiration on individual leaf temperature minimal which ensures that the leaves have virtually the same temperature as the air in the exposure chamber. Heat tolerance exemplarily obtained for leaves of three different alpine dwarf shrubs was shown to be strongly dependent on the fact whether heat exposure was performed in situ or on detached leaves. Additionally, application of natural irradiation during heat exposure affected leaf heat tolerance significantly. Heat tolerance was highest when heat stress was applied in situ under the presence of natural solar irradiation. Incorporating damage amplification, recuperation and repair into the results, heat tolerance data determined by the HTTS are highly ecologically significant and will increase the accuracy of prediction models related to the future destiny of high alpine plants considerably.
Heat tolerance testing system
With the Heat Tolerance Testing System (HTTS) it is possible to determine heat tolerance of plants without the necessity of detachment. The controlled exposure to pre - defined temperatures for a unique time span can be performed in situ, directly at the natural growing site and under largely undisturbed natural conditions particularly with regard to solar irradiation. The system consists of a field portable supply unit, 8 exposure chambers, a netbook and external sensors for measuring leaf temperatures, relative humidity (Rh), ambient air temperature and photon flux density (PPFD). It is operated by special software providing a virtual front panel which contains all necessary control and display elements.
The relative humidity inside the exposure chamber is measured by a combined humidity - temperature sensor (DKRF400-10-5000, Driesen und Kern, Bad Bramstedt, Germany). The inside of the exposure chamber is covered with heating filaments (Diameter: 0.2 mm, RD 100/02, Isabellenhütte Heusler, Dillenburg, Germany) to avoid fogging that could influence spectral transmission of the Plexiglas®.
Plant material and experimental site
Experiments were performed on leaves of the dwarf shrubs Vaccinium gaultherioides Bigelow, Rhododendron ferrugineum L. and Loiseleuria procumbens L. (Desv.), growing on a north facing slope at the timberline of Mt. Patscherkofel (1960 m a.s.l., 47°12’N/11°27’E, Innsbruck, Austria) in September 2011 and July and August 2012.
Effect of relative humidity on leaf temperature
Controlled exposure of plants to a series of defined setup temperatures, which is a prerequisite for determining heat tolerance, can be disturbed by uncontrollable leaf transpiration. A simple method to prevent transpiration is to conduct heat exposure in a vapor saturated atmosphere. For illustration of the effect of vapor saturation on leaf temperature, twigs of R. ferrugineum were continuously (90 min) heated inside two exposure chambers of the HTTS (chamber 1: light - mode, chamber 2: dark - mode, set - point temperature: 40°C, relative humidity: ca. 50%) while leaf temperatures were recorded. After 45 min in both chambers relative humidity was started to increase to up to 100% (45 min). Now representative periods (duration: 10 min) at unaffected and increased relative humidity were selected and leaf temperatures were analyzed and compared using statistical methods.
Comparative determination of heat tolerance
Basic data concerning the comparative experiments focused on heat tolerance of alpine dwarf - shrubs
24 Jul 2012
ex situ 1
26 Jul 2012
24 Jul 2012
26, 28 Jul/2 Aug 2012
26 Sep 2011
ex situ 2
27, 28 Sep 2011
26 Sep 2011
28 Sep 2011
27 Jul 2012
ex situ 1
30 Jul 2012
27 Jul 2012
30 Jul/4, 9 Aug 2012
Controlled heat treatment
Ex situ - using temperature controlled water baths
Detached leaves (10 per sample) of L. procumbens were inserted into small plastic bags and these, in turn, were immersed into temperature controlled water baths (CC1, Huber, Offenburg, Germany) and remained there at the pre - heated target temperature for 30 min. The target temperatures were 20°C (control), 44°C, 46°C, 48°C, 50°C, 52°C, 54°C and 60°C (100% damage). To prevent transpiration of leaves during heat exposure, air was drawn out of the bags. After the heat treatment the leaves were placed on wet filter paper inside plastic bags and were exposed to room temperature and moderate irradiation conditions till the viability assessment took place.
Ex situ - using the equipment following Buchner and Neuner (2001)
This method is a further development of (1) and it works basically similar to it. The difference is that detached leaves can be measured immediately after excision directly in the field avoiding transportation times to the lab. By this, alterations of actual heat tolerance during transportation can be effectively avoided. Leaves were fixed to heat stable transparencies with adhesive tape (3M™ Transpore™, 3M Österreich GmbH, Perchtoldsdorf, Austria) which were sprayed with water to prevent transpirational cooling and sandwiched between small heating plates that are housed in a transportable suit case. This method was applied to V. gaultherioides and R. ferrugineum. In both cases target temperatures were set to 20°C (control), 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C and 60°C (100% damage).
In situ - using the Heat Tolerance Testing System HTTS
Heat exposure took place within the exposure chambers of the HTTS either in darkness (dark - mode) or under natural solar irradiation (light - mode) in a water saturated atmosphere to eliminate transpiration. Target temperatures ranged from 45°C to 51°C (V. gaultherioides, R. ferrugineum) and from 44°C to 54°C (L. procumbens) and were arranged in steps of 2 K to cover the whole range (0% to 100%) of damages induced by the heat treatment. The leaves could remain on the plant for the whole measurement procedure and also afterwards during the latent period before viability assessment. Viability assessment could be performed repeatedly, allowing for the inclusion of possible after effects like recuperation and stress induced acceleration of leaf senescence. With this method during the application of heat stress and in the whole latent period plants remained exposed under the natural environmental conditions.
(1) Electrolyte leakage test (ELT)
Principle: The ELT is a widely used and in numerous variants existing method based on the fact that heat stress affects cell membrane stability and semi - permeability causing a temperature dependent leaking of ions and solutes into the ambient solution that can be measured by an increase of electrical conductivity [20, 46–49].
Experimental protocol: For L. procumbens subsequently to the controlled heat exposure leaves were cut in a right angle into four individual pieces and transferred into small vials (six parallels, twelve pieces per vial) which were filled with 3 ml of distilled water and 800 μg l-1 of dissolved gentamicin, an aminoglycoside antibiotic, to prevent microbial contamination. Subsequently these samples were infiltrated by application of a vacuum with a vacuum pump (LKC 251 E, Saskia, Ilmenau, Germany) to remove air from the intercellular spaces in the mesophyll which otherwise would have hindered the leakage of ions into the solution. After 24 h the specific electrical conductivity (G) of the leachates was determined with a conductivity meter (HD 9213, Delta Ohm, Hungen, Germany). After setting G = P, XT% was calculated for each sample using formula (1).
(2) Chlorophyll fluorescence (CHFL)
Principle: Chlorophyll fluorescence parameters are particularly sensitive to heat stress [34, 50–55]. A possible criterion for the assessment of injuries on the primary photosynthetic apparatus is Fv/Fm, a stress sensitive fluorescence parameter describing the maximum photochemical efficiency of Photosystem II (PS II). The stress related reduction of Fv/Fm can be used as a simple tool for the assessment of leaf damage [22, 33].
Experimental protocol: We determined Fv/Fm on ten dark adapted leaves (30 min) before and after heat treatment (PEA MK2, Hansatech Instruments Ltd, Norfolk, UK). As a result of heat stress Fv/Fm was reduced, depending on the exposure temperature. In this protocol again, XT% was calculated for each sample by setting Fv/Fm = − P and using formula (1).
(3) Visual assessment (VAC, VEM)
Experimental protocol: For the visual assessment of heat injuries the relative proportion of damaged leaf area per leaf was determined (V. gaulterhioides: 32 leaves, L. procumbens and R. ferrugineum: 10 leaves). This happened by surveying leaf areas (injured areas divided to total leaf area) of scanned leaves using special graphical analyzing software (Optimas 6.5, Optimas Corp., Seattle, USA) (VAC) or by visual estimation (VEM) of percentage leaf area that was damaged. For leaves VAC normally is the method of choice. As it is not generally applicable to leaves still attached to the plant, for in situ measurements it was replaced by the visual estimation method (VEM) that produced similar results. The results from the visual assessment methods (VAC, VEM) were ratios between 0 and 1, which were set into formula (1) to calculate percentage damage XT [%].
For descriptive statistical data analysis and comparisons of mean values SPSS - software (SPSS Version 18.0, SPSS Inc., Chicago, US) was used. Mean values were compared either by Student’s t - test or by analysis of variance (ANOVA) followed by post hoc tests as the Bonferroni - test or the Duncan - test.
This work was enabled by the Austrian Science Fund (FWF - project 22158 - B16 to O. Buchner).
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