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Article

Characterization of Heat Tolerance in Two Apple Rootstocks Using Chlorophyll Fluorescence as a Screening Method

by
Ines Mihaljević
*,
Marija Viljevac Vuletić
,
Vesna Tomaš
,
Dominik Vuković
and
Zvonimir Zdunić
Agricultural Institute Osijek, Južno Predgrađe 17, 31000 Osijek, Croatia
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(6), 1442; https://doi.org/10.3390/agronomy15061442
Submission received: 12 May 2025 / Revised: 9 June 2025 / Accepted: 10 June 2025 / Published: 13 June 2025
(This article belongs to the Special Issue Qualitative and Quantitative Plant Screening Measurements for Yield and Quality Enhancement)
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Abstract

:
High temperature has an adverse effect on apple production worldwide. Photosynthesis is a process especially vulnerable to heat stress, which can reduce photosynthetic efficiency, plant growth, development, and ultimately yield. Although the effects of heat stress on apples have been partially examined, the photochemical reactions and heat tolerance of specific rootstocks have still not been sufficiently investigated. Identification of rootstocks with better photosynthetic performance and adaptation to heat stress enables the selection of rootstocks, which could contribute to stable yields and good fruit quality even at elevated temperatures. In this study, chlorophyll a fluorescence (ChlF) induction kinetics was used to investigate the heat tolerance between two apple rootstocks (M.9 and G.210). In addition, we employed lipid peroxidation measurements, hydrogen peroxide quantification, proline content, and total phenolic and flavonoid assessments. Analysis of chlorophyll fluorescence parameters and OJIP curves (different steps of the polyphasic fluorescence transient; O–J–I–P phases) revealed significant differences in their responses, with higher values of the PIABS parameter indicating better PS II stability and overall photosynthetic efficiency in M.9 rootstock. The higher contents of chlorophyll, carotenoids, proline, and significant increase in the accumulation of phenolics, and flavonoids in this rootstock also contributed to its better adaptation to heat stress. Oxidative stress was more pronounced in G.210 through higher H2O2 and MDA levels, which could point to its lower capacity to adjust to heat stress conditions. This research can provide a scientific basis for further breeding programs and growing plans due to climate change and the occurrence of extremely high temperatures.

1. Introduction

Temperature has a significant role in the growth, reproduction, and development of fruit trees because key physiological processes like germination, flowering, and fruiting occur within specific temperature ranges [1]. Due to global warming with more frequent extreme temperatures, heat stress has become one of the serious problems affecting apple trees, which can cause reduced overall productivity of apple orchards. It occurs when apple trees are exposed to high temperatures beyond their optimal range, disrupting key physiological and biochemical processes necessary for survival and reproduction which consequently decrease yield and fruit quality [2]. Kang et al. [3] found that frequent occurrence of high temperatures during the growing season causes a decrease in the production of high-quality apples because of weak fruit coloration and a general decline in their quality. High solar radiation combined with elevated temperatures causes physiological disorders in the fruit skin, which further reduces its quality and market value [4,5]. Hasan et al. [6] conducted long-term experiments under controlled conditions in greenhouses in the field with the aim of assessing possible future changes in apple production in the context of climate change. They found that increased temperatures negatively affect the quality of apple fruit, which is manifested in a decrease in their weight, firmness, acidity, and color, as well as in a shortening of the period for a suitable harvest. Such climatic conditions indicate the importance of studying in more detail how the apple tree responds to heat stress, especially in view of the increasing global warming.
Plants experience permanent damage from heat stress, particularly when they become exposed to temperatures beyond the critical point [7]. Photosynthesis is an essential process by which plants transform solar energy into chemical energy and store it in organic molecules like carbohydrates, enabling their growth, development, and biomass accumulation [8]. This indirectly increases the yield and improves the fruit quality.
Photosystem II (PS II) represents most heat-sensitive components of the photosynthetic machinery, and its stability and efficiency have an important role in maintaining optimal photosynthetic performance. Heat stress reduces the plant’s ability to absorb and use light energy effectively, causing the accumulation of excess energy. If absorbed energy overcomes the capacity for its utilization, it often disrupts or inactivates PS II’s function [9]. It reduces the efficiency of electron transport, impairs ATP and NADPH synthesis, limits carbon fixation, reduces ribulose-1,5-bisphosphate carboxylase–oxygenase (Rubisco) in the dark reaction, and inactivates the oxygen-evolving complex (OEC) [10,11,12]. Furthermore, exposure of plants to heat stress has a great influence on the composition and stability of chlorophyll pigments, which is mostly reflected in the reduction of their amount [13]. Lower levels of chlorophyll content affect reduced light absorption and thus reduced photosynthetic efficiency [14,15,16]. Photosynthesis represents the most important vital process in plants, and it is often used as an indicator of plant vitality and stress tolerance. By monitoring photosynthetic efficiency, it is possible to detect plant stress and gain insight into how plants respond and adapt to specific unfavorable environments. The measurement of chlorophyll fluorescence and analysis of chlorophyll fluorescence parameters have been widely used in studying plant tolerance to various environmental stresses in other plant species [17,18,19,20]. This non-destructive technique quickly detects the damage in PS II by providing insights into the efficiency of PSII and overall energy fluxes within the photosynthetic apparatus.
One of the strategies by which fruit growers tend to minimize the influence of heat stress is using heat-tolerant rootstocks. It is known that grafting on specific rootstock has a significant influence on health, yield, and overall fruit productivity [21,22]. Rootstocks also play a significant role in the adaptation of fruit trees to biotic stresses by improving disease resistance [23], as well as to various abiotic stresses [24,25,26], thereby enhancing their overall stress tolerance and reducing the negative consequences of stress. A previous study observed variations in photosynthesis at the canopy level in some rootstocks and found that more heat-resistant rootstocks retain better photochemical efficiency [27]. Depending on the rootstock, this particular characteristic differs, which points out the significance of selecting heat-tolerant rootstocks for sustainable apple production. Despite these observations, the response of some apple rootstocks, particularly the PS II response, under heat stress conditions has not been well investigated and understood. Since fruit trees are perennial, and rootstocks possess different levels of tolerance to specific environmental conditions, choosing the right rootstock represents a key role in determining the overall success and sustainability of apple production for a long period. In modern intensive apple orchards, growers usually set up high-density planting systems, using dwarfing rootstock to optimize productivity and efficiency [28]. M.9 rootstock is one of the most popular apple rootstocks, ideal for high-density planting because it has a compact tree, early fruiting, high yield, and high fruit quality [29]. Although it has some disadvantages, such as low anchoring and sensibility to pests and diseases like woolly apple aphids and fire blight [30,31], apple growers still choose this rootstock because of its advantages in terms of orchard profitability. Geneva rootstocks, including G.210, are generating a lot of interest lately in apple production. It is a semi-dwarf rootstock with a strong root structure that enhances nutrient uptake and adaptation to different soil types and is appropriate for high-density planting systems. Furthermore, they have good tolerance to woolly apple aphids and fire blight [32,33], which makes these rootstocks very suitable for apple production in areas where these problems are common. Studies on the tolerance of these two rootstocks to abiotic stresses are limited, especially to high temperatures, although some research and findings exist regarding their response to drought. Liu et al. [34] compared the drought tolerance of the new semi-dwarf rootstock Chistock 1 with the commonly used rootstocks M.9, M.26, and M. prunifolia where M.9 showed the weakest drought tolerance among the tested rootstocks. In another study between the rootstocks G.41, G.890, M.9, and B.9, G.890 showed the highest sensitivity to water limitation [35].
Therefore, in this experiment, we specifically chose G.210 rootstock to investigate its tolerance to heat stress in comparison to the widely used M.9 rootstock. We aimed to understand more about the sensitivity of PS II and the overall photosynthetic efficiency of these two rootstocks under heat stress conditions by comparing them using the chlorophyll fluorescence technique. With the obtained results, we will gain further insights into their resistance to high temperatures based on their photosynthetic sensitivity and antioxidative responses to heat stress. By evaluating these markers, we might identify the rootstocks’ capacity to withstand heat and offer practical solutions for enhancing apple heat tolerance in the context of future climate change.

2. Materials and Methods

2.1. Experimental Setup and Growth Condition

The experiment was conducted to assess the photosynthetic efficiency and heat stress tolerance in two apple rootstocks (M.9 and G.210) under controlled conditions during short-term heat stress. The rootstocks were obtained from a commercial nursery (Domaine de Castang, Bergerac, France) and planted in 4 L pots using a substrate with the following composition: 65% white peat, 35% black peat, 150 L of clay/m3, and 1500 g of NPK fertilizer/m3. Before the experiment, the plants were acclimatized in a greenhouse for three months, where they were regularly irrigated with an automatic drip system. Before the start of the experiment, the plants were divided into two groups: control treatment and heat stress treatment. Five plants of each rootstock were used for each treatment. For further research, the plants were placed in a controlled plant growth chamber (Fitoclima 10.000 HP, Aralab, Rio de Mouro, Portugal) for 1 day. They were subjected to a night temperature conditions (25 °C, relative humidity 70%) for 8 h and a day temperature conditions when plants were exposed to heat. Temperature was adjusted to 42 °C for 16 h, with a light intensity of 600–800 µmol m−2 s−1, simulating the conditions during a warm day in a natural orchard environment. The relative humidity during the heat treatment was maintained at 40%. Control plants were put in a climate chamber under optimal growth conditions (25 °C, 70% relative humidity, and light intensity of 600–800 µmol m−2 s−1). At the end of both treatments, chlorophyll fluorescence was measured immediately. The same leaves were then collected and used for further analyses of their chlorophyll, proline, total phenolics, flavonoids, hydrogen peroxide (H2O2), and malondialdehyde (MDA) contents.

2.2. Chlorophyll Fluorescence Measurement

Chlorophyll fluorescence was measured using a plant efficiency analyzer Handy PEA (Hansatech Instruments Ltd., Norfolk, UK). Measurements were performed on three fully developed leaves on each tree, making a total of 15 replicates per rootstock and treatment. They were conducted on the eastern side of the canopy between 8:00 and 10:00 a.m. to avoid photoinhibition caused by high solar radiation in the afternoon.
To achieve complete reduction of plastoquinone and ensure the opening of all reaction centers, the leaves were dark-adapted for 30 minutes prior to measurement using special plastic clips. A pulse of red saturating light induced an increase in fluorescence from the minimal level (F₀), when all reaction centers were open, to the maximum level (Fₘ), corresponding to the closure of all reaction centers. Parameters were calculated from the measured values and analyzed using the OJIP test to assess the efficiency of photosystem II (PS II) [36]. The analysis of PSII under heat stress in the investigated rootstocks was based on 19 chlorophyll fluorescence parameters, which are presented in Table 1. In this study, we compared chlorophyll fluorescence transients of two apple rootstocks by normalizing the data between F0 and Fm (O-P steps). The normalized OJIP transients, which represent the relative variable fluorescence kinetics, were plotted on a logarithmic time scale and expressed as VOP WOP = (Ft − F0)/(Fm − F0). Furthermore, to gain insight into the structural and functional integrity of the photosynthetic apparatus, two differential curves (L and K band) were calculated to analyze heat-induced changes in the OJIP transients. The L band (~150 μs) was calculated as VOK = [(Ft − F0)/(FK − F0)], while the K band (~300 μs) was calculated as VOJ = [(Ft − F0)/(FJ − F0)] [37].

2.3. Measurement of Photosynthetic Pigment Concentration

The same leaves used for chlorophyll a fluorescence measurements were also used for pigment determination. A pooled leaf sample (0.1 g) of each rootstock and treatment was ground with the addition of magnesium carbonate. Pigment extraction was performed using 5 mL of acetone. The absorbance of the obtained extracts was measured on a spectrophotometer Specord 200, Analytik, Jena, Germany at wavelengths of 470, 645, and 662 nm. The concentrations of chlorophyll a and b and carotenoids were calculated using extinction coefficients according to [38] and expressed as milligrams per gram of dry weight (mg/g DW).

2.4. Lipid Peroxidation and Hydrogen Peroxide Measurements

A total of 0.1 g of leaf tissue taken from both treatment and rootstock was mixed with 1 mL of 0.1% trichloroacetic acid and centrifuged at 14,000× g for 15 min to prepare the supernatants for lipid peroxidation and hydrogen peroxide analyses. For the estimation of lipid peroxidation, we used the method described by Verma and Dubey [39], which involves determining the content of MDA, a lipid peroxidation product commonly used as a stress indicator. Supernatants were measured at 532 and 600 nm using a spectrophotometer Specord 200, Analytik, Jena, Germany. MDA content was calculated using an extinction coefficient of 155 mM−1 cm−1 and expressed as nanomole per gram of dry weight (nmol/g DW). H2O2 concentration in the leaves of investigated rootstocks was determined using the previously described method by Velikova et al. [40]. Absorbance was read at 390 nm using an Epoch microplate spectrophotometer (Bio-Tek, Bad Friedrichshall, Germany), and H2O2 content was expressed as micromoles per gram of dry weight (µmol/g DW).

2.5. Proline, Total Phenolic and Flavonoid Measurements

To prepare supernatants for proline and total phenolic analyses, 0.1 g of ground leaf tissue was homogenized with 1 mL of 80% ethanol. Free proline content was determined according to the procedure of Woodrow et al. [41]. Absorbance was read at 520 nm on Epoch microplate spectrophotometer (Bio-Tek, Bad Friedrichshall, Germany), and proline content was calculated using the standard curve and expressed as micromoles per gram of dry weight (μmol/g DW). We used the modified Folin–Ciocalteu method to determine the total phenolic content [42]. Obtained absorbance was measured at 765 nm using an Epoch microplate spectrophotometer (Bio-Tek, Bad Friedrichshall, Germany), and phenolic content was expressed as milligrams of gallic acid equivalents (GAE) per g using a gallic acid calibration curve. The total flavonoid content was assayed using the modified aluminum chloride (AlCl3) colorimetric method [43]. The absorbance was determined at 510 nm against a reaction blank (MeOH) instead of the sample. The total flavonoid content was expressed as the milligrams of catechin hydrate equivalent (mg CE/g of DW) based on the catechin hydrate calibration curve.

2.6. Statistical Analyses

The data in our study were represented as the mean ± standard error (SE) of 15 biological replicates for chlorophyll fluorescence parameter, while for MDA, H2O2, proline, pigments, phenolics, and flavonoids used 5 biological replicates. To reveal differences between treatments and investigated rootstocks, factorial analyses of variance (ANOVA) followed by the least significant difference (LSD) test (p < 0.05) were performed. All analyses were conducted using JASP 0.18.1. [44].

3. Results

3.1. Chlorophyll Fluorescence Parameters

The values of selected JIP parameters were calculated and presented in Table 2. The obtained results indicate that most of the examined parameters were affected by heat stress.
The initial fluorescence intensity (F0) parameter increased significantly under heat stress, with a greater increase observed in the leaves of the G.210 rootstock compared to M.9. Maximal fluorescence intensity (Fm) values were significantly lower in heat-stressed plants than in control plants for both rootstocks; however, the LSD test showed no significant differences between them. Under heat stress, both rootstocks displayed increased relative variable fluorescence intensity at the J-step (VJ) and relative variable fluorescence intensity at the I step (VI), with G.210 showing higher values than M.9. Significant differences in the area above chlorophyll fluorescence curve between F0 and Fm (AREA) were observed between rootstocks and temperature treatments. Heat stress caused a significant reduction in this parameter, with G.210 showing a more pronounced decline. Similarly, high temperature led to a decrease in the parameter normalized total complementary area above the OJIP transient (Sm) in both rootstocks, but the decreasing trend was more pronounced in G.210 than in M.9. The obtained results revealed a significant increase in the parameter approximated initial slope of the fluorescence transient (M0), under heat stress of both rootstocks, but the values of this parameter increased more for G.210 than for M.9. ANOVA revealed significant differences in absorption per RC (ABS/RC) and trapped energy per RC (TR0/RC) between rootstocks and temperature treatments. Under control conditions, both M.9 and G.210 exhibited similar values with no significant differences. However, heat stress led to a significant increase in both parameters for both rootstocks, with G.210 exhibiting a significantly higher value. The electron transport flux per RC (ET0/RC) remained consistent across both rootstocks and temperature treatments, showing no significant difference between control and heat-stressed plants. Both rootstocks exhibited minimal changes under heat stress with no significant differences between them. The values of dissipated energy flux per RC (DI0/RC) increased in the case of both rootstocks under heat stress with significantly higher values in the G.210 rootstock. A significant decrease in the parameter electron flux reducing end electron acceptors at the PSI acceptor side, per RC (RE0/RC), was observed in both investigated rootstocks under heat stress, and reduction was more pronounced in G.210. Under heat stress, both rootstocks experienced a decline in the density of RCs (RC/CS0). M.9 exhibited a higher RC/CS₀ value compared to G.210 under both control and heat-stressed conditions. A significant increase in the parameter’s quantum yield for electron transport (ET0/ABS) and quantum yield for reduction of end electron acceptors at the PSI acceptor side (RE0/ABS) was observed only in the leaves of G.210 when exposed to heat stress, while no changes were observed in M.9. Although both rootstocks exhibited a reduction in the parameters probability that trapped exciton moves an electron into the electron transport chain beyond QA− (ET0/TR0) and efficiency with which an electron from PQH2 is transferred to final PSI acceptors (RE0/ET0) under heat stress, M.9 maintained higher values of these parameters compared to G.210. The results of the ANOVA and LSD tests showed that the values of the parameter performance index on an absorption basis (PIABS) were significantly influenced by rootstock and temperature treatments. Under controlled conditions, M.9 rootstock exhibited higher PIABS values compared to G.210. When plants were exposed to heat stress, both rootstocks experienced a significant decline in PIABS values, with the decline being more pronounced in G.210 compared to M.9. For the parameter maximum quantum yield of primary photochemistry (Fv/Fm), the results showed significant differences only between treatments, where the values of this parameter decreased in both rootstocks below 0.75. No differences were recorded between rootstocks in the control treatment or the heat stress treatment.

3.2. OJIP Transients

The effects of heat stress on the OJIP curves of two different rootstocks are shown in Figure 1. Under control conditions, both rootstocks exhibited similar fluorescence curves, with only minor differences in their OJIP steps. Heat stress treatment caused significant changes in fluorescence transients, leading to an increase in fluorescence intensity, particularly at the J and I step, which was more pronounced in G.210. Figure 2. illustrates the appearance of an additional step (between the O-K step), labeled as the L step, at 300 ms of the fluorescence transient. The L band of both rootstocks, when exposed to heat stress, presented a positive transient value, with G.210 exhibiting a higher L band amplitude compared to M.9. Similarly, the appearance of an additional K band (between the O-J step) at 150 ms showed an increase in both rootstocks, with a more pronounced K band in G.210 (Figure 3).

3.3. Photosynthetic Pigments Contents

M.9 consistently exhibited higher levels of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoid contents under both control and heat stress conditions. Both rootstocks showed an increase in Chl a, Chl b, and carotenoid content under heat stress, with M.9 maintaining higher values (Figure 4).

3.4. MDA, H2O2, Proline, Phenoicsl and Flavonoid Contents

The malondialdehyde (MDA) contents showed a significant increase under heat stress only in the G.210 rootstock, while M.9 maintained stable levels under both control and heat stress conditions (Figure 5A). A similar observation was noted regarding H2O2 content, with a significant increase observed only in G.210, whereas M.9 did not show any significant changes under heat stress (Figure 5B).
Under heat stress, a significant increase in proline content was observed only in G.210 (Figure 5C). In M.9, the highest proline levels were recorded in both the control and heat stress treatments; however, there was no significant difference between these two treatments (Figure 5C). Under high temperatures, the phenolic content increased significantly only in M.9, while it remained unchanged in G.210 (Figure 5D). Similarly, a significant increase in flavonoid content was observed in M.9, whereas G.210 showed a decrease in flavonoids under heat stress (Figure 5E).

4. Discussion

The results of our study demonstrate that heat stress significantly affected the photosynthetic performance and related parameters of M.9 and G.210 rootstocks, with obvious variations and different responses between them.

4.1. Influence of Heat Stress on OJIP Parameters

The parameter Fv/Fm represents the maximum quantum yield of PSII, and the decrease of Fv/Fm is an important indicator of photoinhibition [45] often used for stress tolerance evaluation in many plant species and cultivars. In our study, the values of this parameter under heat stress were below 0.75 in both rootstocks, indicating that photoinhibition occurred in both, but without significant differences between them. Although both rootstocks achieved similar PSII maximum efficiency, other fluorescence parameters revealed significant differences in their overall photosynthetic efficiency under heat stress. This observation is consistent with other reports that Fv/Fm alone does not fully explain declines and sensitivity in photosynthetic efficiency [46,47]. Similar findings were observed in our study. Parameter PIABS, which serves as an indicator of overall photosynthetic performance, was significantly reduced in both rootstocks at high temperature with obvious differences between rootstocks. Some authors consider PIABS as a more sensitive parameter than Fv/Fm, since it integrates energy absorption, trapping, and electron transport and thus explains the functionality of both PS II and partially PSI [45,48]. Therefore, to comprehensively evaluate overall photosynthetic efficiency, we used additional parameters to gain a more detailed understanding of the photochemical reactions in the investigated rootstocks and their response to heat stress. According to Aro et al. [49], a possible sign of photoinhibition is an increase in F0 parameter. In our study, heat stress led to a significant increase in F₀ in both rootstocks, with a greater increase observed in G.210. This suggests a higher proportion of damaged or inactivated reaction centers in this rootstock, leading to reduced energy transfer efficiency and a decrease in the quantum efficiency of PS II [50]. Heat stress reduced Fₘ the maximum fluorescence when all PSII reaction centers are closed, probably because of inhibited electron transfer from the reaction center to the quinone pool [51]. According to previous similar studies, increases in F₀ and decreases in Fm values were observed under high temperatures, implying that F₀ could be used as an indicator of heat tolerance [52,53]. The rise of parameters VJ and VI suggests a reduction in electron transport on the acceptor side of the PSII [54]. VJ (J step) and VI (I step) values increased more in G.210 under heat stress conditions, suggesting that G.210 experiences lower efficiency in electron transport beyond QA, leading to a higher accumulation of reduced QA− within PSII. This also indicates a possible slowdown in plastoquinone reduction due to limitations in downstream electron acceptors [55]. This finding is further supported by the observation of the Sm, M0, and AREA parameters, which provide information regarding the efficiency and limitations of electron transport in photosystem II (PSII). The higher Sm values observed in the M.9 rootstock indicate that it requires more energy to close all reaction centers compared to G.210, which could be due to a more efficient electron transport system, helping it cope with stress [36]. Similar findings for this parameter were observed in the study by Gautam et al. [56], where heat-tolerant wheat cultivars exhibited higher Sm values. The parameter AREA, which represents the pool size of reduced plastoquinone on the reducing side of PSII [57], decreased more in G.210, suggesting greater limitations in downstream electron transport that could reduce the overall photosynthetic efficiency. Similarly, Faseela et al. [51] observed that the reduction of this parameter in stressed plants was due to the blockage of electron transfer from the RC to the quinone pool. Higher AREA values in M.9 indicate a better electron transport system with more efficient energy flow under stress conditions. Gautam et al. [56] also reported that more tolerant wheat plants exposed to heat exhibited a higher value of this parameter, reflecting an increase in the pool size of the reduced PQ pool. An increased M₀ value is often associated with inhibited re-oxidation of QA− to QA by QB, suggesting a disruption in electron transport [58]. The higher M₀ observed in the G.210 rootstock compared to M.9 suggests altered QA− re-oxidation, potentially indicating greater changes in PSII activity, as also observed by Shanker et al. [59]. Since higher M₀ is usually linked to photodamage, the lower M₀ in M.9 could imply better regulation of QA− re-oxidation, supporting a more stable photosynthetic process under heat stress.
Parameters that represent specific fluxes per RC and reflect the absorption, conversion, and dissipation of light energy (such as ABS/RC, TR0/RC, and DI0/RC) were significantly higher under heat stress than in the control for both rootstocks. However, G.210 exhibited significantly higher values for these parameters compared to M.9. Obtained results suggest that the higher average absorption (ABS/RC) and trapping (TR0/RC) per active RC in G.210 are due to the inactivation of some RCs, while the increase in DI0/RC values can be attributed to the higher dissipation of inactive RCs [60]. Inactivation of RCs in plants is often associated with photoinhibition, where photosynthetic performance declines when the amount of light absorbed exceeds the capacity of the photosystems to utilize it [61]. This aligns with the more pronounced decline in PIABS observed in G.210, indicating that its photosynthetic system is less efficient in utilizing absorbed energy under stress. Furthermore, the inactivation of RCs was supported by decreasing the parameter RC/CS0 under heat stress conditions, which represents the total amount of active reaction centers per cross-section. In comparison to G.210, higher RC/CS0 values in heat-stressed leaves of M.9 indicate a greater proportion of active RCs of PSII, as was also seen in apple cultivars more tolerant to high temperatures [37]. Previously, it was noticed that the parameter which describes electron transport flux per RC (ET0/RC) decreases in parallel with increases in ABS/RC, TR0/RC, and DI0/RC [62]. However, in our study, despite the increased light energy absorption and significant changes in PSII activity, ET0/RC remained unchanged between the investigated rootstocks and stress treatments. This was probably due to more inactive centers and inefficient electron transfer from QA to QB, as observed in the study by Shanker et al. [59] on heat-stressed pearl millet plants. Our results are consistent with the findings of Van Heerden et al. [63] in P. sibirica under drought stress, where, despite changes in other photosynthetic parameters (ABS/RC and TR0/RC), the plants were able to sustain stable ET0/RC values through compensatory mechanisms. In our previous study, on plum leaves exposed to high temperature, we also did not find significant changes in this parameter [64].
Schansker et al. [65] stated that a decrease in the RE0/ET0 parameter suggests slow electron transport on the acceptor side of PSI. Thus, significantly lower values of this parameter observed in G.210 indicate reduced electron flow from PQH2 to PSI electron acceptors compared to M.9. This is further supported by a greater decrease in the RE0/RC parameter in G.210, highlighting a weakened electron flow beyond PSI. The parameter ET0/TR0, which describes the probability that a trapped exciton is transferred as an electron into the electron transport chain beyond QA, was also lower in heat-stressed leaves of G.210. Similarly, Rastogi et al. [66] reported lower ET0/TR0 values in more salt-sensitive sorghum genotypes. In contrast, M.9 maintained higher RE0/RC and ET0/TR0 values, suggesting that it retains greater functionality in electron transport to PSI under high temperature, which may contribute to its overall greater tolerance. According to Hornyák et al. [67], higher values of parameter RE0/ABS in heat-sensitive buckwheat imply the occurrence of cyclic phosphorylation in which only ATP is produced without NADPH synthesis, suggesting that this parameter could have a great influence on photosynthetic activity in this cultivar. Similarly, our results showed that under heat stress, the RE0/ABS values of the more heat-sensitive rootstock G.210 were significantly higher than those of M.9, indicating that in M.9 rootstock NADPH was synthesized in sufficient amounts during the reduction phase of the Calvin cycle and that electron transport functioned as a noncyclic.

4.2. Influence of Heat Stress on OJIP Transient

The OJIP transient analysis further supports these differences, and the effect of heat stress on the investigated apple rootstocks was evident when comparing the shapes of the corresponding fluorescence transient curves. It was previously observed that under abiotic stress conditions, a K step appears between the O and J steps, and it has been widely recognized as a sensitive indicator of stress in plants, as its appearance is associated with damage to the OEC of PSII [68]. Both apple rootstocks exhibited a positive K band around 300 µs, indicating inhibition or damage to the OEC, which was confirmed in previous studies where leaves were exposed to high temperatures [69,70]. However, the K band of G.210 was higher than that of M.9, suggesting that the OEC of M.9 was less damaged and has a better tolerance to heat stress than G.210. Heat stress damaged the photosynthetic machinery by reducing energetic cooperativity among PSII units, and this assumption is supported by the appearance of a positive L band in heat-stressed leaves of both rootstocks. Lower connectivity between PSII units leads to poor excitation energy utilization and a decrease in active RCs [45,71]. A higher positive L band in G.210 implies greater dissociation of the antenna pigment complex, with increased distance between PSII antennae and, consequently, less efficient energy exchange in this rootstock compared to M.9 [72]. The obtained results showed that the OJIP transient could be a useful method for detecting and quantifying heat stress. These findings align with the observed decline in the heat-sensitive parameter PIABS, which integrates different components of the photosynthetic apparatus, including energy absorption, energy trapping, and electron transport efficiency within PSII [63]. Therefore, a decrease in this parameter indicates a reduction in the overall functionality of the photosynthetic system. The more pronounced decline in G.210 confirms that its photosynthetic apparatus was more severely disrupted by heat stress compared to M.9, which may possess a more effective protective mechanism or greater thermal stability.

4.3. Influence of Heat Stress on Pigments

In addition to photosynthetic parameters, we also evaluated several biochemical markers to gain a more comprehensive understanding of the physiological response to heat stress in investigated rootstocks. We measured the content of pigments to assess plant vitality, hydrogen peroxide, and lipid peroxidation levels to evaluate oxidative stress, and the content of proline, phenolics, and flavonoids due to their role as non-enzymatic antioxidants that contribute to scavenging of ROS and thus reduce potential stress in plants.
Many previous studies have demonstrated that heat stress reduces chlorophyll production and increases its degradation, negatively impacting leaf chlorophyll content and photosynthesis [13], ultimately affecting yield [73]. This suggests that chlorophyll loss can be used as a screening indicator for heat tolerance [74,75]. Although a reduction in chlorophyll content is a typical symptom under heat stress, our study found that high temperature led to a significant increase in chlorophyll content in both rootstocks. We hypothesize that this increase is associated with the plants’ acclimatization as an adaptive response to heat. Moloi et al. [76] reported that, despite an increasing trend in chlorophyll content in heat-stressed soybean plants, light energy was not efficiently converted into chemical energy, highlighting that increased pigment accumulation does not necessarily improve photosynthesis, and elevated carotenoid levels cannot fully prevent the decline in photochemical reactions. A similar reaction of photosynthetic pigments in heat-stressed plum and tomato leaves has been previously observed [64,77]. Despite both rootstocks experiencing an increase in chlorophyll content, there were obvious differences between them. The significantly higher chlorophyll content observed in the M.9 rootstock reflects its greater pigment stability and enhanced light absorption, which are associated with improved photosynthetic performance [57]. Carotenoids are protective pigments that accumulate in plants under stress conditions, including heat, and have a protective role to scavenge ROS in chlorophyll [78]. Therefore, the higher level of carotenoids observed in the M9 rootstock could also be one of the reasons why it showed a better tolerance to heat. Our findings agree with previous studies related to carotenoid behavior and plant acclimation to adverse environmental conditions [79,80].

4.4. Influence of Heat Stress on MDA, H2O2, Proline, Phenolic and Flavonoid Contents

Along with a significant reduction in photosynthetic activity, further results from our study demonstrated that G.210 rootstock was exposed to higher stress, as revealed by increased ROS generation and membrane damage under heat stress. Significantly higher levels of H2O2 and MDA content in G.210 imply its higher oxidative damage compared to M.9. Despite the observed photoinhibition, unchanged MDA and H2O2 levels in M.9 suggest that, because of acute and short-term stress, there may not have been sufficient accumulation of ROS to cause lipid peroxidation. The stable MDA and H2O2 levels could also be due to the effective mitigation of oxidative stress by the antioxidant defense system. It is known that structural adaptations or the production of specific protective molecules like proline, as an osmoprotectant, protect plants against various abiotic stresses and that phenolics and flavonoids, as efficient antioxidants, help plants survive in unfavorable environments by scavenging oxygen radicals [81,82]. The higher accumulation of proline, along with significant increases in phenolics, and flavonoids in M.9 plants exposed to heat stress likely contributed to better photosynthetic efficiency, more efficient osmotic adjustment, membrane stability, and better ROS scavenging ability, which together contributed to greater heat tolerance of this rootstock.

5. Conclusions

Our study showed that short-term heat stress had a significant impact on the photosynthetic efficiency of both rootstocks, with clear differences in their sensitivity to heat stress. Although both rootstocks showed signs of photoinhibition, detailed analysis of chlorophyll fluorescence and other parameters indicated greater stability and efficiency of PS II and lower levels of oxidative stress in M.9 compared to G.210 rootstock, indicating its greater tolerance. In addition to contributing to a better understanding of the physiological and biochemical mechanisms underlying heat tolerance, this study also has significant practical value, as it can serve as a guideline for the selection of rootstocks for apple growing in regions experiencing rising temperatures. However, for final recommendations, further research under field conditions is necessary to confirm the observed difference in real agroecological conditions.

Author Contributions

Conceptualization, I.M.; methodology, I.M. and M.V.V.; formal analysis, V.T. and M.V.V.; writing—original draft preparation, I.M.; writing—review and editing, I.M.; supervision, Z.Z.; project administration, D.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Agriculture in Croatia through the project entitled “Support Program for Research on the Impact of Various Technologies and Agro-technical Measures on the Maintenance of High-Category Mother Trees for the Period 2022–2024”; 320-01/21-01/34.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 15 01442 g001
Figure 1. The chlorophyll a fluorescence transients recorded in apple rootstocks (M.9 and G.210) under control and heat stress, plotted on a logarithmic time scale. O—(O step), minimal fluorescence intensity when all PS II reaction centers (RC) are open; J—(J step), the intensity at 2 ms; I—(I step), the intensity at 30 ms; P—(P step), the maximal intensity when all PS II reaction centers (RC) are closed.
Figure 1. The chlorophyll a fluorescence transients recorded in apple rootstocks (M.9 and G.210) under control and heat stress, plotted on a logarithmic time scale. O—(O step), minimal fluorescence intensity when all PS II reaction centers (RC) are open; J—(J step), the intensity at 2 ms; I—(I step), the intensity at 30 ms; P—(P step), the maximal intensity when all PS II reaction centers (RC) are closed.
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Figure 2. Visualization of the L band in two apple rootstocks (M.9 and G.210), OJIP transients normalized between O and K steps, expressed as difference [ΔVOK = VOK(heat stress) − VOK(control)].
Figure 2. Visualization of the L band in two apple rootstocks (M.9 and G.210), OJIP transients normalized between O and K steps, expressed as difference [ΔVOK = VOK(heat stress) − VOK(control)].
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Figure 3. Visualization of the K band in two apple rootstocks (M.9 and G.210), OJIP transients normalized between O and J steps, expressed as difference [ΔVOJ = VOJ(heat stress) − VOJ(control)].
Figure 3. Visualization of the K band in two apple rootstocks (M.9 and G.210), OJIP transients normalized between O and J steps, expressed as difference [ΔVOJ = VOJ(heat stress) − VOJ(control)].
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Figure 4. Effect of heat stress on chlorophyll a (A), chlorophyll b (B), total chlorophyll (a + b) (C), and carotenoid contents (D) in the leaves of two apple rootstocks. Different letters represent significant differences between rootstocks and treatments (p ˂ 0.05).
Figure 4. Effect of heat stress on chlorophyll a (A), chlorophyll b (B), total chlorophyll (a + b) (C), and carotenoid contents (D) in the leaves of two apple rootstocks. Different letters represent significant differences between rootstocks and treatments (p ˂ 0.05).
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Figure 5. Effect of heat stress on MDA (A), H2O2 (B), proline (C), phenolics (D), and flavonoid contents (E) in the leaves of two apple rootstocks. Different letters represent significant differences between rootstocks and treatments (p ˂ 0.05).
Figure 5. Effect of heat stress on MDA (A), H2O2 (B), proline (C), phenolics (D), and flavonoid contents (E) in the leaves of two apple rootstocks. Different letters represent significant differences between rootstocks and treatments (p ˂ 0.05).
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Table 1. Data obtained from measurements of chlorophyll a fluorescence polyphasic rise.
Table 1. Data obtained from measurements of chlorophyll a fluorescence polyphasic rise.
ParametersDefinitions
F0Initial fluorescence intensity, when all PSII RCs are open
FmMaximal fluorescence intensity, when all PSII RCs are closed
VJ = (FJ − F0)/(Fm − F0)Relative variable fluorescence intensity at the J step
VI = (FI − F0)/(Fm − F0)Relative variable fluorescence intensity at the I step
AREAThe area above chlorophyll fluorescence curve between F0 and Fm (total complementary area between fluorescence induction curve)
Sm = (Area)/(FM − F0)Normalized total complementary area above the OJIP transient (reflecting multiple turnover QA reduction events)
M0 = 4(F300μs – F0)/(Fm − F0)Approximated initial slope of the fluorescence transient
TR0/ABS = φP0 FV/Fm = [1 − (F0/Fm)]Maximum quantum yield of primary photochemistry
ET0/ABS = φE0 = [1 − (F0/Fm)] × (1 − VJ)Quantum yield for electron transport
ET0/TR0 = ψE0 = 1 − VJProbability that trapped exciton moves an electron into the electron transport chain beyond QA−
RE0/ET0 = δ0 = (1 − VI)(1 − VJ)Efficiency with which an electron from PQH2 is transferred to final PSI acceptors
RE0/ABS = φR0 = φP0 × ψE0 × δR0Quantum yield for reduction of end electron acceptors at the PSI acceptor side
ABS/RC = M0 (1/VJ) × (1/φP0)Absorption flux (of antenna Chls) per RC
TR0/RC = M0 (1/VJ)Trapped energy flux per RC
ET0/RC = M0 (1/VJ) × ψ0Electron transport flux per RC
DI0/RC = ABS/RC − TR0/RCDissipated energy flux per RC
RE0/RC = M0 (1/VJ)ψE0δR0Electron flux reducing end electron acceptors at the PSI acceptor side, per RC
RC/CS0 = φP0 (VJ/M0) F0Density of RCs (QA reducing PSII reaction centers)
PIABS = (RC/ABS)(TR0/DI0)[ET0/(TR0 − ET0)]Performance index on absorption basis
Table 2. JIP test results, the effect of heat stress on selected chlorophyll fluorescence parameters in two apple rootstocks (M.9 and G.210). Different letters represent significant differences between rootstocks and treatments (p ˂ 0.05).
Table 2. JIP test results, the effect of heat stress on selected chlorophyll fluorescence parameters in two apple rootstocks (M.9 and G.210). Different letters represent significant differences between rootstocks and treatments (p ˂ 0.05).
ParameterRootstockControl TreatmentHeat Stress Treatment
F0M.9376.73 ± 4.24 c425.86. ± 15.90 b
G.210360.60 ± 5.63 c472.93 ± 15.76 a
FmM.92175.86 ± 29.75 a1723.46 ± 60.98 b
G.2102120.46 ± 24.90 a1804.33 ± 35.54 b
VJM.90.42 ± 0.01 d0.54 ± 0.02 b
G.2100.48 ± 0.01 c0.65 ± 0.03 a
VIM.90.78 ± 0.01 c0.80 ± 0.01 b
G.2100.81 ± 0.00 b0.84 ± 0.01 a
AREAM.942,690.33 ± 1089.01 a22,157.33 ± 1449.52 c
G.21036,765.67 ± 780.95 b15,755.33 ± 1021.33 d
SmM.923.84 ± 0.77 a17.19 ± 0.71 c
G.21020.89 ± 0.39 b11.76 ± 0.57 d
M0M.90.64 ± 0.03 c0.99 ± 0.08 b
G.2100.77 ± 0.02 c1.59 ± 0.09 a
Fv/FmM.90.83 ± 0.00 a0.74 ± 0.02 b
G.2100.83 ± 0.00 a0.75 ± 0.01 b
ET0/ABSM.91.43 ± 0.02 b1.64 ± 0.07 b
G.2101.61 ± 0.03 b2.36 ± 0.18 a
ET0/TR0M.90.58 ± 0.01 a0.46 ± 0.02 c
G.2100.52 ± 0.01 b0.34 ± 0.03 d
RE0/ET0M.90.13 ± 0.01 a0.08 ± 0.01 b
G.2100.10 ± 0.00 b0.05 ± 0.01 c
RE0/ABSM.93.93 ± 0.16 b3.97 ± 0.10 b
G.2104.32 ± 0.12 b5.18 ± 0.35 a
ABS/RCM.91.84 ± 0.05 c2.46 ± 0.15 b
G.2101.93 ± 0.03 c3.28 ± 0.08 a
TR0/RCM.91.52 ± 0.04 c1.84 ± 0.09 b
G.2101.60 ± 0.03 c2.41 ± 0.05 a
ET0/RCM.90.87 ± 0.02 a0.83 ± 0.05 a
G.2100.82 ± 0.02 a0.89 ± 0.07 a
DI0/RCM.90.31 ± 0.01 c0.61 ± 0.09 b
G.2100.33 ± 0.01 c0.87 ± 0.05 a
RE0/RCM.90.11 ± 0.00 a0.07 ± 0.01 b
G.2100.08 ± 0.00 b0.04 ± 0.01 c
RC/CS0M.9207.13 ± 7.12 a176.10 ± 6.80 b
G.210187.14 ± 4.10 b144.82 ± 5.24 c
PIABSM.93.65 ± 0.17 a1.12 ± 0.12 c
G.2102.74 ± 0.10 b0.56 ± 0.12 d
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Mihaljević, I.; Viljevac Vuletić, M.; Tomaš, V.; Vuković, D.; Zdunić, Z. Characterization of Heat Tolerance in Two Apple Rootstocks Using Chlorophyll Fluorescence as a Screening Method. Agronomy 2025, 15, 1442. https://doi.org/10.3390/agronomy15061442

AMA Style

Mihaljević I, Viljevac Vuletić M, Tomaš V, Vuković D, Zdunić Z. Characterization of Heat Tolerance in Two Apple Rootstocks Using Chlorophyll Fluorescence as a Screening Method. Agronomy. 2025; 15(6):1442. https://doi.org/10.3390/agronomy15061442

Chicago/Turabian Style

Mihaljević, Ines, Marija Viljevac Vuletić, Vesna Tomaš, Dominik Vuković, and Zvonimir Zdunić. 2025. "Characterization of Heat Tolerance in Two Apple Rootstocks Using Chlorophyll Fluorescence as a Screening Method" Agronomy 15, no. 6: 1442. https://doi.org/10.3390/agronomy15061442

APA Style

Mihaljević, I., Viljevac Vuletić, M., Tomaš, V., Vuković, D., & Zdunić, Z. (2025). Characterization of Heat Tolerance in Two Apple Rootstocks Using Chlorophyll Fluorescence as a Screening Method. Agronomy, 15(6), 1442. https://doi.org/10.3390/agronomy15061442

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