# Single versus recurrent heat stress: A case study on the impact of a stress at flowering on the sorghum response to a subsequent post-flowering stress

Angélique Berger, Belmys Cakpo, Cyndi Suire, Loic Pagan, Sandrine Roques, Raoul Vial, Gregory Aguilar, Nancy Terrier, Christine Granier

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## To cite this version:

Angélique Berger, Belmys Cakpo, Cyndi Suire, Loic Pagan, Sandrine Roques, et al., Single versus recurrent heat stress: A case study on the impact of a stress at flowering on the sorghum response to a subsequent post-flowering stress. Environmental and Experimental Botany, 2025, 235, pp.106162. 10.1016/j.envexpbot.2025.106162. hal-05158577

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# Single versus recurrent heat stress: A case study on the impact of a stress at flowering on the sorghum response to a subsequent post-flowering stress

Angelique Berger<sup>a,b,*</sup>, Belmys Cakpo<sup>b,c,d</sup>, Cyndi Suire<sup>b</sup>, Loic Pagan<sup>a,b</sup>, Sandrine Roques<sup>a,b</sup>, Raoul Vial<sup>a,b</sup>, Gregory Aguilar<sup>a,b</sup>, Nancy Terrier<sup>b</sup>, Christine Granier<sup>b</sup>

## ARTICLE INFO

### Keywords:

Grain yield

Heat

PSII efficiency

Recurrent stress

Sorghum bicolor

Tillering

## ABSTRACT

The increasing intensity and frequency of heat waves due to climate change pose a growing threat to global food security. Two experiments were conducted under controlled conditions using six temperature scenarios to compare the effects of single and recurrent heat stresses on maximum photosystem II quantum efficiency ($F_v/F_m$), above-ground plant biomass, grain yield and grain yield components in two sorghum genotypes. This study confirmed that the responses of grain number and thousand grain weight are highly dependent on stage development for both single and recurrent heat stresses. Recurrent heat stresses have synergistic effects, leading to around 20% more yield losses than the cumulative responses to the corresponding single stresses. In contrast, $F_v/F_m$ was less reduced by heat stress if the plants had been subjected to a previous stress. The large reduction in thousand grain weight under recurrent heat stresses may result in a strong remobilization of carbohydrates to sinks other than the main stem panicle. The increased tillering induced by single heat stresses and further amplified under recurrent heat stresses could partly explain this effect. The study highlights the complexity of plant responses to heat stress and the challenges of predicting outcomes under recurrent stress conditions.

## 1. Introduction

Climate change has intensified rapidly over the last century, driven by an unprecedented increase in greenhouse gas (GHG) emissions. According to the 2021 report by the Intergovernmental Panel on Climate Change (IPCC, 2021), this increase could lead to a rise in temperature between 1.5 and 3.2 °C by 2100, depending on the different scenarios. Climate change is also characterized by an increase in the frequency, intensity and duration of extreme weather and climatic events, such as heat waves (Lemonsu et al., 2014; Bador et al., 2017). Among abiotic stresses, extreme temperatures are one of the main environmental threats to crop growth and productivity (Xie et al., 2018; Lesk et al., 2016), having a significant impact on both the quantity and quality of harvested products (Jagadish et al., 2021), including cereal crops such as wheat (Trnka et al., 2014; Zampieri et al., 2017), maize (Tigchelaar et al., 2018), rice (Arshad et al., 2017), and sorghum (Impa et al., 2019). Each crop species has its own temperature threshold, beyond which plant developmental and physiological processes are affected by higher

temperatures (Parent and Tardieu., 2012). Sorghum (Sorghum bicolor L. Moench), a monocotyledon of African origin belonging to the Poaceae family, has attracted attention in Northern countries due to its adaptability and resilience to stressful environmental conditions. Sorghum is currently the fifth most widely grown cereal, covering an area of around 42 million hectares, and a global production of around 58.4 million tones (USDA, 2024). Its robustness and C4 photosynthesis pathway enable it to withstand high temperatures and low water supply conditions (Chantereau et al., 2013; Edwards., 2019), which positions it as a potential substitute for crops that are more sensitive to heat or drought. However, despite having a higher temperature optimum than other crops, sorghum suffers the adverse effects of high temperatures, with development rates decreasing above 34 °C (Parent and Tardieu., 2012), and yields being affected above 35 °C (Chadalavada et al., 2021). Numerous studies have been carried out on the effects of single thermal stresses (single period of stress) on sorghum grain yield. They confirm the detrimental effects of high temperature on yield, highlighting the respective effects of the intensity and duration of stress, as well as the

<footnote><sup>a</sup> CIRAD, UMR AGAP Institut, Montpellier, France</footnote>

<footnote><sup>b</sup> UMR AGAP Institut, Univ Montpellier, CIRAD, INRAE, Institut Agro, Montpellier, France</footnote>

<footnote><sup>c</sup> CIRAD, UMR ABSys, Montpellier F-34398, France</footnote>

<footnote><sup>d</sup> ABSys, Univ Montpellier, CIHEAM-IAMM, CIRAD, INRAE, Institut Agro, Montpellier, France</footnote>

<footnote>* Corresponding author at: CIRAD, UMR AGAP Institut, Montpellier, France.
E-mail address: angelique.berger@cirad.fr (A. Berger).</footnote>
stage of the plant at which the stress occurs (Prasad et al., 2015; Chiluwal et al., 2020). Like many species, sorghum is particularly sensitive to high temperatures during its reproductive phase (Prasad et al., 2017, 2021). Heat stress around flowering causes a reduction in grain yield by reducing the number of grains per panicle (Pasternak and Wilson, 1969; Prasad et al., 2008, 2015; Singh et al., 2015, 2016; Sunoj et al., 2017). Physiological processes underlying the reduction in grain number in response to heat stress are related to reduced spikelet fertility or pollen germination (Djanaguiraman et al., 2018, 2020). Heat stresses occurring after flowering also led to a reduction in grain yield but this is due to a reduction in grain size and weight (Prasad et al., 2008, 2015). The physiological processes affected by heat stress during grain filling are mainly related to the photosynthetic efficiency of plants and carbohydrate remobilization. Heat stress alters the ability of leaves to use solar energy via photochemical pathways, which has an effect on the intensity of chlorophyll fluorescence emission (Chaudhry and Sidhu., 2022). Hüve et al. (2011), examined how different intensities and durations of heat shock affect plant photosynthetic function on Phaseolus vulgaris, commonly known as green beans and showed a threshold-like decline in photosynthetic function, with the level of photosynthetic deterioration and cell damage being influenced by the cumulative heat dose. High temperatures also impair photosynthesis in sorghum by affecting photosynthetic pigments and the photosynthetic electron transport chain, ultimately resulting in reduced RuBP regeneration rates and thylakoid membrane damages (Sunoj et al., 2017; Pradhan et al., 2022). In addition, chlorophyll content is significantly affected when heat stress is applied throughout the growth cycle, leading to a decrease in leaf photosynthetic activity, maximum quantum yield of photosystem II and final biomass (Prasad et al., 2008; Jagadish et al., 2021). All these studies gave insights into grain yield reduction due to a single period of high temperatures during the sorghum cycle and mainly during the reproductive phase. However, episodes of high temperature are becoming increasingly frequent and occur unpredictably throughout the plant life cycle (Breshears et al., 2021). It is therefore necessary to examine how successive heat waves, which are more representative of real-life situations, affect plant physiology, growth, and yield.

It might be tempting to think that plant responses to successive periods of stress are independent of each other. However, a period of initial stress could prepare (or “prime”) plants, by inducing “defense” responses at cellular and systemic levels to prevent serious damages from subsequent stress (Bäurle., 2016). These adaptive responses, which depend on the duration and intensity of the stress, can improve the plant’s thermotolerance during a second stress (Ahrens et al., 2021). In wheat, an initial heat stress during the early reproductive stages (stem elongation stage and booting) allows plants to initiate effective tolerance mechanisms to cope with subsequent high temperature conditions (Fan et al., 2018). Recurrent stress in wheat plants, resulted in improved photosynthetic efficiency compared to plants exposed to single heat stress. This improvement was evidenced by a higher concentration of chlorophyll and soluble proteins, and an overall improvement in source productivity, leading to a more substantial allocation of carbohydrates to sinks. Finally, grain weight of plants exposed to recurrent heat stress scenario is less affected than that of plants exposed to a late stress without “priming” stress (Fan et al., 2018). Numerous situations have been reported at different scales in which an initial period of stress positively primed the plant for a subsequent stress and therefore trigger a form of plant ‘memory’ which enables plants to be more tolerant to a second stress (Ahrens et al., 2021; Oberkofler et al., 2021). The length of the time between the two stress periods seems to be a key regulatory factor that dictates whether the plant’s memory is retained or dissolved. During this phase, plants balance the allocation of resources for acclimation against the potential benefits of resetting and reallocating these

resources to growth and/or reproduction (Crisp et al., 2016). In some cases, plants fail to recover from the first stress, leading to an amplification of the negative effects when subsequent stresses occur (Crisp et al., 2016; Ruehr et al., 2019). Recurrent stress responses have been investigated in various crops, and whether similar responses occur in sorghum remains to be further explored. The main objective of this study was to evaluate whether the plant’s response to recurrent heat stresses (two successive periods with high temperature) can be considered as the sum of the plant’s responses to the same stresses (same intensity, same duration and imposed at the same stage of plant development) taken individually. Heat stresses were applied at heat-sensitive stages that were previously well-described in the literature for the effects of a single stress on sorghum yield components: a first heat stress at flowering expecting to reduce grain number and a second one during the post-flowering phase expecting to reduce grain weight (Prasad et al., 2015). We analyzed whether the initial heat stress imposed at flowering had a detrimental, beneficial, or neutral effect on the plant’s subsequent physiological response to a second stress imposed a few days after flowering, and ultimately on grain yield and biomass production.

## 2. Materials and methods

Two independent experiments, Exp. 1 and Exp. 2, were conducted in controlled conditions, in the Abiophen platform (CIRAD Montpellier, France: 43.64994° N, 3.86913° E), including greenhouse and growth-chambers (Aralab Fitoclima 25000HP). The first one was conducted from March to July 2023 and the second one from November 2023 to February 2024.

### 2.1. Plant material and growth conditions in the control treatments in Exp. 1 and 2

Two pure lines of Sorghum bicolor L. Moench, Btx623 and Macia were selected for this study, based on three criteria: (a) similarities in their development avoiding confounding effects in interpretations of heat stress response (similar flowering dates, low tillers production in control conditions, and same panicle shape and length); (b) contrasted responses to heat stress in previous studies (Impa et al., 2019; Prasad et al., 2015; Smith et al., 2023); (c) final stem length compatible with growth-chamber height. Both were grown in Exp. 1 and only Macia in Exp. 2. In each experiment, for each genotype, 120 pots of 7-liter were filled with soil (a mixture of 1/3 Neuhaus n°2, 1/3 Neuhaus n°9, 1/3 CIRAD2 (composition in supplementary data, Table S1) and fertilized with Basacote®Plus16–8–12, 24 g/pot). Experiments were arranged with 15 pots per row, in 8 rows, in 2 greenhouse compartments, one compartment per genotype to avoid cross-pollination for Exp. 1 and only one compartment for Exp. 2. All plants of the control treatments, C, remained in the greenhouse from sowing until grain maturity, with micrometeorological conditions set up to be identical as far as possible between the two greenhouse compartments and between both experiments (Table 1A and B, for Exp. 1 and 2 respectively). In Exp. 1, day/night temperature over plant cycle was 27.9/22.9 °C for Macia and 28.0/23.0 °C for Btx623. Day/night air humidity was 56.6%/64.1 % for Macia and 53.8%/58.8 % for Btx623. Natural light in the greenhouse was supplemented by LED ensuring a 13 h day duration. The average daily PAR was around 350 µmol m⁻² s⁻¹ for Macia and 390 µmol m⁻² s⁻¹ for Btx623 (Table 1A, Fig. S1). In Exp. 2, day/night temperature over plant cycle was 27.2/21.8 °C and day/night air humidity was 46%/50% (Table 1B). Natural light in the greenhouse was supplemented by LED ensuring a 13 h day duration and the average daily PAR was around 494.61 µmol m⁻² s⁻¹ (Table 1B, Fig. S2). Both in Exp. 1 and 2, plants were manually irrigated with water at full soil water retention
<caption>**Table 1**</caption>

Micrometeorological conditions (A) during Exp. 1 in the two greenhouse compartments (one compartment per genotype) and the two growth chambers (one per genotype) and (B) during Exp. 2 in the greenhouse compartment and the two growth chambers (one per thermal condition). Values are means ± SE. The means were calculated over the periods relevant to the scenario comparison (from flowering until the last day of heat stress).

<table><thead><tr><th></th><th>Greenhouse Macia</th><th>Greenhouse Btx623</th><th>Growth chamber Macia</th><th>Growth chamber Btx623</th></tr></thead><tbody><tr><td>A. Experiment 1</td><td></td><td></td><td></td><td></td></tr><tr><td>Temperature day/night (°C)</td><td>27.9/22.9 ± 0.89/0.72</td><td>28.0/23.0 ± 1.44/1.3</td><td>38.7/28.0 ± 0.12/0.31</td><td>38.5/27.9 ± 0.31/0.55</td></tr><tr><td>Relative humidity day/night (%)</td><td>56.5/64.1 ± 6.64/13.54</td><td>53.7/58.8 ± 3.88/7.67</td><td>71.2/69.9 ± 0.98/1.11</td><td>70.0/69.2 ± 0.49/2.74</td></tr><tr><td>PAR (µmol m<sup>-2</sup> s<sup>-1</sup>)</td><td>350 ± 63.7</td><td>390 ± 64.38</td><td>701 ± 15</td><td>703 ± 6</td></tr><tr><td>Type of lights</td><td>Natural + LED</td><td>Natural + LED</td><td>Ceramic</td><td>Ceramic</td></tr><tr><td>Day-length (h)</td><td>13</td><td>13</td><td>13</td><td>13</td></tr><tr><td>B. Experiment 2</td><td></td><td></td><td></td><td></td></tr><tr><td>Temperature day/night (°C)</td><td>27.4/21.8 ± 0.59/0.52</td><td>27.1/21.2 ± 0.66/0.48</td><td>38.5/28.0 ± 1.12/0.32</td><td></td></tr><tr><td>Relative humidity day/night (%)</td><td>47.98/53.32 ± 3.76/6.17</td><td>48.2/55.0 ± 0.89/0.52</td><td>69.9/69.3 ± 0.25/2.4</td><td></td></tr><tr><td>PAR (µmol m<sup>-2</sup> s<sup>-1</sup>)</td><td>494.61 ± 18.67</td><td>726 ± 5.8</td><td>706 ± 8.49</td><td></td></tr><tr><td>Type of lights</td><td>Natural + LED</td><td>Ceramic</td><td>Ceramic</td><td></td></tr><tr><td>Day-length (h)</td><td>13</td><td>13</td><td>13</td><td></td></tr></tbody></table>

twice a week until the emergence of the 7th leaf, then automatic irrigation (drip) was set up at a rate of 21 h⁻¹ for 3 min, twice a day, in the morning and evening.

## 2.2. Heat stress scenarios at and after flowering in Exp. 1 and 2

### 2.2.1. Micrometeorological conditions during heat stress periods in Exp. 1 and 2

Exp. 1 included one control condition described above and five heat stress scenarios (n = 20 plants per treatment). The temperature scenario assigned to each pot was randomized to ensure the objectivity of the results. The five batches of plants for heat stress treatments were subjected to thermal stress at specific stages of their panicle development and for a specific duration (see Fig. 1). During the heat episodes, plants of each genotype were placed in two separate growth chambers, with one chamber per genotype. They were moved from the greenhouse to the growth chamber at the end of the day, when air temperature difference between the growth-chambers and the greenhouse was at its lowest. In the growth-chambers, the temperature was programmed to reach a maximum of 40 °C in the morning, to remain at this maximum for 10 h, then to decrease in the evening to reach a minimum of 28 °C at night. The rise and fall in temperature were progressive over a period of

1h30. Temperature and hygrometry conditions were set up to be identical between the two growth-chambers (Table 1). Air humidity in the growth chamber was increased compared to the greenhouse with the aim to ensure a relatively comparable VPD in both conditions (around 1.7 kPa/day and 1.2 kPa/night). Light in the growth chamber was provided for 13 h by Philips full spectrum ceramic metal halide lamps and the average daily PAR was around 700 µmol m⁻² s⁻¹ in both growth chambers (Table 1A, Fig. S1).

Exp. 2 included the same control condition as Exp. 1 and two heat stress scenarios (n = 20 plants per treatment, Fig. 2). For the two-heat stress scenario, micrometeorological conditions in the growth chamber were set up to be identical between both experiments. Day/night temperature during heat stress periods was 38.5/28.0 °C, day/night air humidity was 69.9%/69.3% with a VPD around 2 kPa/day and 1.4 kPa/night (Table 1B, Fig. S2). The average daily PAR was 706 µmol m⁻² s⁻¹ in the growth chamber with the same light quality than during Exp. 1 (Table 1B, Fig. S2).

### 2.2.2. Duration and position of heat stress scenarios in Exp. 1 and 2

In sorghum, flowering extends over 3–5 days, with a gradient along the panicle from the top to bottom (Chiluwal et al., 2020). In this study, the "flowering" stage was determined on a plant-by-plant basis, by

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Schematic representation of the 6 temperature scenarios during Exp 1. Letters on the right (C, SS0, SS1, SS2, RS1 and RS2) designate the different scenarios and the pink rectangles represent position and duration of the different heat stress periods on a time scale expressed in days after flowering (DAF). The flowering stage of the panicle is illustrated by a picture of both genotypes on the left Macia and Btx623 respectively up and down. C corresponds to the control condition; SS0, the single 2-day heat stress treatment applied at flowering; SS1, the single 4-day heat stress treatment applied 4 days after flowering; SS2, the single 4-day heat stress treatment applied 9 days after flowering. RS1 and RS2 correspond to two recurrent heat stress treatments; RS1 corresponding to heat stresses combining SS0 and SS1, and RS2 combining SS0 and SS2. In the control condition (C), plants remained in the greenhouse at 30 °C during the day and 23 °C at night. During heat stress periods (pink rectangles), plants were transferred to growth-chambers set at 40 °C during the day and 28 °C at night and returned to greenhouse conditions after each heat stress period. (n = 20 per treatment).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Schematic representation of the 5 environmental scenarios during Exp 2. Letters on the right (C, SS1, RS1, CPSS1, CPRS1) designate the 5 different scenarios. The pink rectangles represent position and duration of the different heat stress periods on a time scale expressed in days after flowering (DAF). The flowering stage of the panicle is illustrated by a picture of the Macia genotype. C corresponds to the control condition; SS1, the single 4-day heat stress treatment applied 4 days after flowering; RS1 corresponds to a recurrent heat stress treatment combining SS0 and SS1. The blue rectangles represent position and duration of periods during which plants are moved into the growth-chamber but without heat stress (same temperature as in the greenhouse). CPSS1 refers to a single 4-day transfer in the growth-chamber (same stage and duration as for SS1 but without increase in temperature). CPRS1 refers to a recurrent transfer in the growth chamber, a first 2-day transfer at flowering followed by a 4-day transfer 4 days after flowering (same stage and duration as for RS1 but without increase in temperature). (n = 20 per treatment).</figcaption></figure>

observing the panicle developmental stage daily, and noting when the panicle had fully opened flowers between half and 2/3 of its length.

In Exp. 1, the 5 stress scenarios included 3 single heat stresses designated SS0, SS1 and SS2. Plants in treatment SS0 were subjected to a 2-day heat stress period starting at flowering (Fig. 1). Plants in treatment SS1 and SS2 were subjected to a 4-day heat stress period starting 4 days after flowering for SS1 and 9 days after flowering for SS2. The two other stress scenarios were 2 recurrent heat stresses called RS1 and RS2 (Fig. 1). For both, plants were first exposed to a 2-day heat stress at flowering (equivalent to SS0) and then, plants were subjected to a second 4-day heat stress starting 4 days after flowering (equivalent to SS1) for RS1, and 9 days after flowering (equivalent to SS2) for RS2.

In Exp. 2, the 2 stress scenarios were ‘exact’ repetitions of SS1 and RS1 described in Exp. 1 (n = 20 plants per treatment, Fig. 2).

For both experiments, during their stay in the growth-chamber, plants were manually watered in the morning and evening at full soil water retention capacity to prevent drought stress. After each heat stress event (after a single stress, between the two stresses of a recurrent stress scenario and after the second stress of a recurrent stress scenario), the plants were returned to their initial position in the greenhouse. From 30 days after flowering until the end of the cultivation period, i.e., when the seeds reached maturity, drip irrigation was reduced to once a day, in the morning.

## 2.3. Plant transfer to the growth chamber after flowering without heat stress in Exp. 2

During Exp. 2, two other conditions were added to test an eventual effect of the transfer to the growth chambers conditions on the plants independently of the temperature treatment. These scenarios were labeled CPSS1/CPRS1 with “CP= Control Phytotron” (Fig. 2). Plants of the CPSS1 treatment were transferred 4 days after flowering to a growth-chamber for 4 days, and the temperature of the growth chamber was equivalent to that of the greenhouse (equivalent transfer into a growth chamber than SS1 but without increase in temperature, Table 1B). Plants of the CPRS1 treatment were transferred to a growth-chamber for 2 days at flowering and for 4 days, 4 days after flowering with the temperature of the growth chamber was equivalent to that of the greenhouse (equivalent transfer into the growth chamber than RS1 but without increase in temperature, Table 1B). Relative air humidity was also equivalent between plants in the greenhouse and those in the growth

chamber for these treatments (Table 1B). After periods in the growth-chamber, all the plants returned at their initial position in the greenhouse until the grains of the main panicle reached maturity.

## 2.4. Plant traits measurements

### 2.4.1. The maximum quantum yield of PSII (Exp. 1 and Exp. 2)

The $F_v/F_m$ ratio, representing the maximum quantum yield of photosystem II (PSII), is commonly used as an indicator of the physiological state and functional integrity of photosystem II. The maximum quantum yield of PSII measurements were taken in the morning, on the penultimate leaf (flag leaf Fl-1), in the middle of the leaf using a portable chlorophyll fluorimeter (Hansatech instruments Pocket PEA). For each plant, one measurement was taken on the right and one on the left of the midrib, with 20 plants per treatment for each genotype. The tested leaves were dark-adapted with a leaf clip for half an hour before the measurements.

For the 5 heat stress scenarios in the Exp. 1, measurements were taken before the plants were subjected to stress, during the heat stress period at two and four days after the beginning of the stress period (only two for SS0 which lasted two days), and after the stress, in recovery period, two days after removal of the stress for all treatment to determine whether PSII integrity was restored. For the control scenario, measurements were taken in parallel to all the points studied during the stress scenarios. For the Exp. 2, measurements were taken during the heat stress period at two and four days after the beginning of the stress period for SS1 and RS1 scenario, in parallel for the CPSS1 and CPRS1 scenario in the control growth chamber and for the control in the greenhouse.

### 2.4.2. Plant biomass, yield and related traits (Exp. 1 and Exp. 2)

All plants were harvested at grain maturity, i.e. 50 ± 3 days after flowering in this experiment, for both experiments. Each plant was cut at the panicle base, the tillers were removed, counted and weighed separately from the main stem. The panicle was bagged and placed in a drying oven at 30 °C for 72 hours. The fresh vegetative biomass of the main stem was measured by weighing the main stem with its leaves. The panicles were threshed and the total grain mass was measured. A random grain sub-sample from each plant was placed on the plate of an OptoAgri2 machine, a high-throughput grain-counting device (Opto-machine, France) combined with a high-precision balance, thus
measuring the weight of 1000 grains (thousand-grain weight). The number of grains per panicle was calculated from the total grain weight and the thousand-grain weight.

## 2.5. Data analysis (Exp. 1 and Exp. 2)

Some plants were excluded from the analysis due to human errors (e. g. error of stress duration, broken plants) in applying heat stress treatments or due to abnormal developmental delays (e.g. excessively late flowering). Consequently, measurements were carried out on 15–20 plants per treatments and genotypes.

All data were analyzed using XLSTAT software. The two genotypes were examined separately and statistical significance was set at α= 5 %. Because the sample sizes were small and the data did not follow a normal distribution, non-parametric Kruskal Wallis tests were conducted to assess the effects of different treatments. Following these tests, Steel-Dwass-Critchlow-Fligner pairwise comparisons were performed to group samples according to significant differences.

The expected effects of recurrent stresses were calculated under the assumption that individual stress effects are cumulative. Specifically, for the 1000g-weight assay, let Wc be the average 1000g-weight under control (no-stress) conditions, W1000 the average 1000g-weight after applying one or more stresses, and R the relative weight reduction (%) due to a given stress.

Under this assumption, when two stresses (SS0 and SS1) are applied consecutively, the combined effect is given by:

$$W_{1000} (SS0+SS1) = W_c \times (1 - R_{SS0}) \times (1 - R_{SS1})$$

We compared this calculated combined reduction to the observed reduction after the consecutive application of SS0 and SS1 (RS1):

$$W_{1000} (RS1) = W_c \times (1 - R_{RS1})$$

(See Table 2 for details on each variable and the corresponding experimental conditions.).

# 3. Results

## 3.1. Effects of single and recurrent heat stresses on the maximum quantum yield of PSII ($F_v/F_m$)

The maximum quantum yield of photosystem II ($F_v/F_m$) remained constant (around 0.8) over time in the control condition even if a slight decrease was observed after 9 days or 11 days after flowering in Macia (Fig. 3A, B) and Btx623 (Fig. 3C, D), respectively. For both genotypes, it was significantly reduced around 0.66 during single heat stress periods as shown two days after the beginning of the stress period in SS0, SS1 and SS2 but also four days after the beginning of the heat stress period in SS1 and SS2 (Fig. 3). For both genotypes, $F_v/F_m$ recovered to initial values after a period of single heat stress (Fig. 3A, D), regardless of the duration (2-day stress for SS0 or 4-day stress for both SS1 and SS2) or the phenological stage at which it was imposed (flowering, 4 days or 9 days after flowering for SS0, SS1 and SS2, respectively).

When plants were subjected to the early recurrent heat stress (RS1), $F_v/F_m$ was less reduced during the second heat stress imposed 4 days after flowering than by the first one both for Macia and Btx623 although this difference was not significant for Btx623 (Fig. 3A, C). $F_v/F_m$ values during the post-flowering stress were lower for plants subjected to the SS1 scenario than for those subjected to the initial stress at flowering (RS1). A significant difference was observed after 2 and 4 days of stress in Macia, while in Btx623, the difference was only significant after 4 days (Fig. 3A, C). When plants underwent late recurrent heat stress (RS2), $F_v/F_m$ did not significantly differ between the first and second stress for both genotypes two days after stress imposition (Fig. 3B, D).

No significant differences between plants exposed to recurrent or

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Effects of single and recurrent heat stresses on the time-course of potential quantum efficiency of PSII ($F_v/F_m$) in Macia (A, B) and Btx623 (C, D). Flag leaf −1 $F_v/F_m$ was measured over time before, during, between and/or after heat stress period. Periods of heat stress are represented by vertical pink rectangles. On each plot, $F_v/F_m$ is shown for control plants (grey diamonds) and for the single 2-day stress imposed at flowering (SS0, orange stars). $F_v/F_m$ of plants subjected to early single and recurrent heat stresses (SS1, orange squares and RS1, red triangles) are shown in A and C, for Macia and Btx623, respectively. $F_v/F_m$ of plants subjected to late single and recurrent heat stresses (SS2, pink squares and RS2, purple triangles) are shown in B and D, for Macia and Btx623, respectively. Values are means ± SD with $36 \le n \le 40$ (p < 0.05). Letters represent significant statistical differences between scenarios at each stage from the Steel-Dwass-Critchlow-Fligner test.</figcaption></figure>
single stress (RS2 or SS2) were detected for $F_v/F_m$ in Macia when post-flowering stress was applied from 9 to 13 days after flowering (Fig. 3B), whereas PSII efficiency was better after 4 days in Btx623, when the plants had already undergone a first stress at flowering (Fig. 3D).

## 3.2. Effects of single and recurrent heat stresses on plant biomass and tillering

Vegetative aerial biomass was divided into main stem biomass and tiller biomass. Tillering was characterized by the biomass of tillers per plant (Fig. 4A, D), the number of tillers per plant (Fig. 4B, E) and the percentage of tillering plants in each modality (Fig. 4C, F).

### 3.2.1. Single and recurrent heat stress led to increased plant and tiller biomass compared to control

For both genotypes, single and recurrent heat stress scenarios caused an increase in whole plant fresh vegetative biomass (Fig. 4A, D), although this increase was not significant for Macia in SS0 (Fig. 4A). In contrast, there was no significant difference in the biomass of the main stem (without the tillers) among plants of the same genotype grown in the different scenarios suggesting that differences were only due to tillering (Fig. 4A, D). The increase in tiller biomass when plants were subjected to heat stress was higher for recurrent stress scenarios compared to single stress. In Macia, tiller biomass reached 13 g in the control plants and 15 g for those grown in SS0. Significant increases were detected for tiller biomass in response to single heat stresses SS1 and SS2, with respectively 50 g and 58 g. For recurrent heat stresses, tiller biomass reached 78 and 84 g, for RS1 and RS2 respectively. These

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Effects of single and recurrent heat stresses on final shoot biomass and tiller production in Macia (A, B, C) and Btx623 (D, E, F) genotypes. The total shoot biomass is presented in A and D, detailing the biomass of the main shoot (light grey) and the biomass of the tillers (dark grey), for Macia and Btx623 respectively and for the 6 thermal scenarios including the control, C, the 3 single heat stress treatments SS0, SS1 and SS2 and the 2 recurrent heat stress treatments, RS1 and RS2. The mean number of tillers per plant is shown in B and E for Macia and Btx623 respectively. The percentage of plants with at least one tiller is shown in C and F. Values are means ± SD 16 ≤n≤ 20 for Macia and 15 ≤n≤ 19 for Btx623 (p < 0.05). Upper letters represent the different statistical groups from the Steel-Dwass-Critchlow-Fligner test.</figcaption></figure>
differences were even more pronounced in genotype Btx623. For Btx623, tiller biomass was almost negligible (2.8 g) in control plants, whereas all the heat stress scenarios caused a significant increase in tiller biomass with 40 g, 28 g and 62 g of tillers biomass for stressed plants SS0, SS1 and SS2 respectively, and 111 g and 103 g for recurrent stressed plants, RS1 and RS2 respectively (Fig. 4A, D).

### 3.2.2. Single and recurrent heat stresses led to an increased tillering

Tillering was very low for both genotypes in control conditions with less than one tiller per plant in Macia corresponding to 35 % of tillering plants (Fig. 4C) and only one plant with a single tiller in Btx623, nearly 5 % of tillering plants (Fig. 4F). The average number of tillers per plant increased when plants were subjected to heat stress for both genotypes. In Macia, this difference was not significant when plants were subjected to a two-day heat stress imposed at flowering (SS0) but was significant when they were subjected to 4 days heat stress either at 4 (SS1) or 9 (SS2) days after flowering, with an average of 2 tillers/plant for SS1 and 2.3 tillers/plant for SS2 (Fig. 4B). The percentage of plants with at least one tiller reached 53 %, 75 % and increased to 80 % for plants subjected

to SS0, SS1 and SS2, respectively (Fig. 4C). Tillering was further increased by recurrent heat stress scenarios with 100 % of recurrently stressed plants producing at least one tiller, and an average of 3 tillers/plant for both RS1 and RS2 (Fig. 4B, C).

In Btx623, single stresses, whether at flowering or post-flowering stages, had a significant impact on the number of tillers per plant: 1.84 tiller per plant for SS0, 1.94 tiller per plant for SS1 and 2.84 for SS2 scenarios (Fig. 4E). The percentage of plants with at least one tiller increased to 84–87 % for plants grown in the three single stress scenarios (Fig. 4F). As for Macia, tillering was also further increased by recurrent heat stress scenarios with 100 % of plants producing at least one tiller with an average of 4.5 tillers/plant in the RS1 scenario and 94 % of plants showing at least one tiller with an average of 3.9 tillers/plant in the RS2 scenario (Fig. 4E, F).

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Effects of single and recurrent heat stresses on main yield and yield components in Macia (A, B, C) and Btx623 (D, E, F) genotypes. Yield is represented by total grain weight per panicle (A, D). Yield components are the number of grains per panicle (B, E) and 1000-grain weight (C, F), for the 6 thermal scenarios including the control, C, the 3 single heat stress treatments SS0, SS1 and SS2 and the 2 recurrent heat stress treatments, RS1 and RS2. Data are presented as whisker boxes with the boxes representing data between the 1st and 3rd quartiles (25 and 75 %) The horizontal line within the interquartile range box in the boxplot represents the median of the data distribution ($16 \le n \le 20$ for Macia and $15 \le n \le 19$ for Btx623). The letters above whisker boxes represent the different statistical groups resulting from the Steel-Dwass-Critchlow-Fligner test.</figcaption></figure>
### 3.3. Effects of single and recurrent heat stresses on grain yield and grain yield components of main stem panicle

#### 3.3.1. Single heat stresses decreased grain yield and affected yield components

The 3 single heat stress scenarios caused a decrease in total grain dry weight of the main stem panicle in both genotypes (Fig. 5A, D). SS0, imposed during two days at flowering, caused a reduction in grain weight by 25 % for Macia and 33 % for Btx623 (Fig. 5A, D, Table 2). SS1, imposed for 4 days, at 4 days after flowering, caused a reduction in grain weight by 56 % in Macia and 30 % in Btx623 (Fig. 5A, D, Table 2). SS2, imposed for 4 days, at 9 days after flowering, caused a reduction in grain weight by 50 % in Macia and 47 % in Btx623 (Fig. 5A, D, Table 2).

For both genotypes, the single heat stress imposed at flowering (SS0) significantly reduced grain number compared to the control condition (-34 % for Macia and -36 % for Btx623, Fig. 5B, E, Table 2). In contrast, thousand-grain weight was not significantly affected by SS0 in Btx623 (Fig. 5F, Table 2) but it was significantly increased by this 2-day heat stress scenario in Macia (+12 %, Fig. 5C, Table 2). For later stresses, grain number was not significantly affected by the single heat stress imposed 4 days (SS1) or 9 days after flowering (SS2), in either genotype despite a slight reduction in grain number under these two treatments for Btx623 (Fig. 5B, E, Table 2). The decrease in total grain weight per panicle described before for these two single stress scenarios was mainly due to a significant reduction in thousand-grain weight for both genotypes: -58 % for Macia and -26 % for Btx623, in SS1 and -50 % for Macia and -40 % in Btx623 for SS2 (Fig. 5C, F, Table 2).

#### 3.3.2. Recurrent heat stresses reduced grain yield and yield components

Both recurrent heat stress scenarios caused a significant reduction in total grain weight and the reductions were higher than for the single stresses (Fig. 5A, D). RS1 caused a reduction in total grain weight by 83 % in Macia and 78 % in Btx623 (Fig. 5A, D). For RS2, the reduction in total grain weight was lower than for RS1 for both genotypes but this difference was only significant for Btx623. Total grain weight was reduced by 75 % in Macia and 64 % in Btx623 (RS2, Fig. 5A, D, Table 2).

Grain number was significantly reduced in Macia and Btx623 plants subjected to recurrent stresses. It was reduced by 34 % in Macia and 32 % in Btx623 in the RS1 scenario. RS2 caused a reduction in grain number by 29 % in Macia and 24 % in Btx623 (Fig. 5B, E, Table 2). The reduction in grain number was similar for plants subjected to RS1 or RS2 for both genotypes.

Recurrent heat stresses had a major impact on the thousand-grain weight (Fig. 5C, F, Table 2). The thousand-grain weight of plants subjected to RS1 was reduced by 75 % for Macia and 69 % for Btx623. RS2 caused a thousand-grain weight reduction by 66 % in Macia and 54 % in Btx623 (Fig. 5C, F, Table 2). For both genotypes, thousand-grain weight was significantly more affected in RS1 than in RS2 (Fig. 5C, F, Table 2).

#### 3.3.3. The impact of a recurrent heat stress on grain yield components does not always correspond to the sum of individual responses to each stress

RS1 and RS2 combined the effect of a first heat stress at the flowering stage, SS0, which significantly affected grain number, with the effect of a

second post-flowering heat stress, SS1 or SS2, which significantly affected thousand-grain weight in both genotypes (see above). RS1 and RS2 caused a reduction in grain number that was not significantly different from that caused by SS0 in both genotypes (Fig. 5B, E). In Macia, grain number was reduced by 34 %, 34 % and 29 % for SS0, RS1 and RS2 respectively (Table 2). Consistently, the observed reduction in grain number caused by RS1 and RS2 was almost equal to cumulative effects caused by SS0 and SS1 for calculated RS1 (-33 %) and the cumulative effects of individual reductions caused by SS0 and SS2 for calculated RS2 (-33 %, Table 2). In Btx623, grain number was reduced by 36 %, 32 % and 24 % for SS0, RS1 and RS2 respectively (Table 2). However, the observed reduction in grain number caused by RS1 and RS2 was higher than the cumulative effects caused by SS0 and SS1 for calculated RS1 (-44 %) and the cumulative effects caused by SS0 and SS2 for calculated RS2 (-45 %, Table 2).

In both genotypes, the thousand-grain weight was significantly more affected by the recurrent heat stress scenario than the corresponding single stress imposed after flowering (RS1 vs SS1 and RS2 vs SS2, Table 2). In Macia, thousand-grain weight was reduced by 58 %, 50 %, 75 % and 66 % for SS1, SS2, RS1 and RS2 respectively (Table 2). In Btx623a, thousand-grain weight was reduced by 26 %, 40 %, 69 % and 54 % for SS1, SS2, RS1 and RS2 respectively (Table 2). The observed reduction in thousand-grain weight caused by RS1 and RS2 was higher than the cumulative effects caused by SS0 and SS1 for calculated RS1 (-23 %) and cumulative effects caused by SS0 and SS2 for calculated RS2 (-38 %, Table 2).

### 3.4. Effects on $F_v/F_m$ grain yield, yield components and tillering during single and recurrent heat stresses are mainly due to high temperatures

Effects of single and recurrent heat stresses were studied by transferring plants from a greenhouse at control temperature to growth chambers with higher temperature but also slight differences in air humidity and light conditions (see Material and Methods). Although the objective was to analyze the effect of temperature, it was not possible at this stage of the study to disentangle the effects of temperature from the effects of air humidity or light (quantity and/or quality). Two additional scenarios (CPSS1 and CPRS1) were performed during Exp. 2 to address this issue.

A single or recurrent transfer to the growth chamber with control temperature (CPSS1 and CPRS1) affected slightly and significantly $F_v/F_m$ from 0,8 to 0,75 (Fig. 6). However, higher significant effect on the $F_v/F_m$ (from 0,8 to 0,66) was observed in the growth chamber with high temperature (SS1 and RS1) (Fig. 6).

A single or recurrent transfer to the growth chamber with control temperature (CPSS1 and CPRS1) did not affect grain yield nor its components, grain number and thousand grain weight compared to control plants (Fig. 7A-C). In contrast, single or recurrent transfer to the growth chamber with high temperature caused a significant decrease in yield and both yield components and those differences were consistently similar to those reported before for Exp. 1. A single or recurrent transfer to the growth chamber without changes in air temperature (CPSS1, CPRS1) did not affect the average number of tillers per plant with

**Table 2**

Reduction (-) or increase (+) in grain number and thousand-grain weight (in percent of the control treatment) for SS0, SS1, SS2, RS1 and RS2 during Exp. 1 for both Macia (A) and Btx623 (B) genotypes. Corresponding calculations of the reduction in grain number and thousand-grain weight were calculated as cumulative effects caused by SS0 and SS1 for calculated RS1 and the cumulative effects caused by SS0 and SS2 for calculated RS2 see Material and methods. ($16 \le n \le 20$ for Macia and $15 \le n \le 19$ for Btx623).

<table><thead><tr><th></th><th>SS0</th><th>SS1</th><th>SS2</th><th>RS1</th><th>Calculated RS1</th><th>RS2</th><th>Calculated RS2</th></tr></thead><tbody><tr><td><strong>A-Macia</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Grain number (%)</td><td>-34</td><td>+2</td><td>+1</td><td>-34</td><td>-33</td><td>-29</td><td>-33</td></tr><tr><td>Thousand grain weight (%)</td><td>+12</td><td>-58</td><td>-50</td><td>-75</td><td>-53</td><td>-66</td><td>-45</td></tr><tr><td><strong>B-Btx623</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Grain number (%)</td><td>-36</td><td>-13</td><td>-14</td><td>-32</td><td>-44</td><td>-24</td><td>-45</td></tr><tr><td>Thousand grain weight (%)</td><td>+4</td><td>-26</td><td>-40</td><td>-69</td><td>-23</td><td>-54</td><td>-38</td></tr></tbody></table>
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Effects of single and recurrent heat stresses on *F*<sub>v</sub>*/*F*<sub>m</sub> in Macia plants grown in Exp. 2 compared to the effects of a transfer into the growth-chamber at the same periods without increasing temperature. The 5 scenarios include the control, C, the early single 4-day heat stress treatment, SS1; the early recurrent heat stress treatment, RS1; and 2 additional treatments without heat stress but in which plants are transferred to growth-chambers conditions at the same stage and for a same duration as SS1, CPSS1 or at the same stages and for same durations as RS1, CPRS1. The number after each treatment (x scale) corresponds to the number of days after flowering when the *F*<sub>v</sub>*/*F*<sub>m</sub> was measured (6 or 8). Data are presented as whisker boxes with the boxes representing data between the 1st and 3rd quartiles (25 and 75 %). The horizontal line within the interquartile range box in the boxplot represents the median of the data distribution (26 ≤n≤ 34). The letters above whisker boxes represent the different statistical groups resulting from the Steel-Dwass-Critchlow-Fligner test.</figcaption></figure>

approximately 40 % of plants producing at least one tiller in all these scenarios (C, CPSS1, CPRSS1) (Fig. 8A, B). In contrast, single or recurrent transfer to the growth chamber with high temperature caused a significant increase in tillering that was similar to that observed in Exp. 1 (Fig. 8A, B & Fig. 4B, C).

**4. Discussion**

Complex interactive responses can occur in plants exposed to suc-
cessive environmental stresses (Zandalinas et al., 2024). This has been
reported for stresses of the same nature (Fan et al., 2018), but also for
combined abiotic stresses (Poudyal et al., 2023; Radha et al., 2023).
Here, the effects of single and recurrent heat stress have been reported
on sorghum plant traits measured at the plant and organ level at grain
maturity, but also at the leave photosynthetic (physiological) level
during the heat stress scenario. These experiments were designed to
provide a robust analytical framework while minimizing potential
confounding effects. These results clearly show that an initial heat stress
during flowering influences the plant responses to a subsequent heat
stress during the post-flowering phase.

4.1. *Single and recurrent heat stresses affected grain yield components with a consistent stage dependent effect*

In cereals, the early reproductive stage shows high susceptibility to
heat stress by an increase in flower abortion and pollen sterility, which
ultimately leads to a reduction in grain number, which in turn affects
grain yield (Zenda et al., 2022; Jagadish., 2020). In contrast, heat stress
occurring during the vegetative phase did not affect grain yield (Siebers
et al., 2017). Later heat stresses during reproductive stage delayed grain
growth, resulting in reduced thousand-grain weight (Prasad et al., 2015
in sorghum; Tao et al., 2016 in maize; Farooq et al., 2011 in wheat).
Taking advantage of the indeterminate growth of oilseed rape, with
successive pods at different stages of their development on a same plant
subjected to a unique heat stress period, Magno Massuia de Almeida
et al. (2021) showed that heat stress applied after 20 % of pods had
reached maximum size significantly reduced total seed yield. This
reduction was mainly due to a decrease in yield for pods shorter than
5 cm, caused by fewer seeds and slightly lower seed weight. In contrast,
pods larger than 5 cm were unaffected and their seeds were normally
filled. These results are consistent with the trends observed in this study,
where heat stress imposed at flowering reduced yield by affecting grain
number whereas post-flowering heat stresses also reduced yield but
mainly through reduction of thousand-grain weight. The reduction in
grain number during post-flowering stress in Btx623 (despite

non-significant) may be explained by the sensitivity of post-pollination
processes, such as pollen tube growth, fertilization, endosperm forma-
tion, and embryo development, to heat. These processes, resulting in late
abortion events, suggest that pollination does not always ensure fruit or
seed formation (Driedonks et al., 2016).

The recurrent heat stresses combining both type of stresses (at
flowering and post-flowering) cumulated the two effects: a decrease in
grain number and grain weight and, consistently, had a greater effect on
yield than single stresses, with losses ranging from 64 % to 83 %
depending on the scenario and the genotype whereas it was between
25 % and 55 % for single stresses. However, the response of yield
components to recurrent heat stress, mainly thousand-grain weight but
also grain number in Btx623 was not equal to the sum of the response to
each corresponding stress taken as single. Molecular mechanisms un-
derlying those responses require further studies.

4.2. *Evidences of interactions between a first heat stress imposed at flowering on the response to heat stress during the post-flowering phase at the yield scale*

A positive effect of heat priming on the response to heat stress during
grain filling has been reported in different cereals allowing a better
response of yield or yield components when compared with a single late
heat stress (Wang et al., 2012; Fan et al., 2018, Siebers et al., 2017). This
was the case for grain number in Btx623 as the reduction in grain caused
by the two recurrent stress scenarios was lower than expected from the
succession of responses to corresponding single stresses, possibly due to
lower late abortion events, even if not verified. However, this was not
the case in this study for the response of thousand-grain weight, as the
heat priming stress imposed at flowering exacerbated the negative effect
of heat stress during the post-flowering phase with a higher decrease in
thousand grain weight than expected. The combination of the two suc-
cessive periods had negative effects, resulting in a higher decrease in
thousand-grain weight than anticipated. In wheat, early heat stress
during the vegetative stage (leaf stages 7–9) promotes plant acclimation
as it mitigates the effects of post-flowering heat stress on yield and starch
accumulation in grains (Wang et al., 2012). This temperature acclima-
tion by a priming stress imposed before flowering was related to an
increased remobilization of carbohydrates from the plant to the grains
compared to non-primed plants (Wang et al., 2012). Similarly, for wheat
again, heat priming imposed during stem elongation, booting or anthesis
enhanced plant photosynthesis by increasing green flag leaf area, chlo-
rophyll content, soluble proteins and antioxidant enzyme efficiency,
resulting in better carbohydrate supply to grains during grain filling and
finally reduced grain yield loss compared to non-primed plants (Fan
<figure><img src="image_11.png" /><figcaption>**Fig. 7.** Effects of single and recurrent heat stresses on main stem panicle yield and yield components in Macia plants grown in Exp. 2 compared to the effects of a transfer into the growth-chamber at the same periods without increasing temperature. Yield is represented by total grain weight per panicle (A). Yield components are the number of grains per panicle (B) and 1000-grain weight (C). The 5 scenarios include the control, C, the early single 4-day heat stress treatment, SS1; the early recurrent heat stress treatment, RS1; and 2 additional treatments without heat stress but in which plants are transferred to growth-chambers conditions at the same stage and for a same duration as SS1, CPSS1 or at the same stages and for same durations as RS1, CPRS1. Data are presented as whisker boxes with the boxes representing data between the 1st and 3rd quartiles (25 and 75 %). The horizontal line within the interquartile range box in the boxplot represents the median of the data distribution (13 ≤n≤17). The letters above whisker boxes represent the different groups resulting from the Steel-Dwass-Critchlow-Fligner test.</figcaption></figure>

et al., 2018). Positive, absence or negative heat stress priming effect can be crop dependent, but also depend on the heat stress scenario itself, including intensity, duration and stage of occurrence of stress and can also depend on the duration between two heat stress periods, also called the recovery phase (Bäurle, 2016; Ahrens et al., 2021; Oberkofler et al., 2021). In this study, the more detrimental effect of recurrent heat stress scenario on thousand-grain weight was observed for the recurrent scenario with the shortest recovery phase duration: the length of recovery period between the two stresses for the recurrent scenarios is 2 days for

<figure><img src="image_12.png" /><figcaption>**Fig. 8.** Effects of single and recurrent heat stresses on tillering in Macia plants grown in Exp. 2 compared to the effects of a transfer into the growth-chamber at the same periods without increasing temperature. Tillering is represented by the average number of tillers per plant in A and the percentage of plants with at least one tiller in B. The 5 scenarios include the control, C, the early single 4-day heat stress treatment, SS1; the early recurrent heat stress treatment, RS1; and 2 additional treatments without heat stress but in which plants are transferred to growth-chambers conditions at the same stage and for a same duration as SS1, CPSS1 or at the same stages and for same durations as RS1, CPRS1. Values are means ± SD, 15 ≤n≤ 18 (p < 0.05). Letters represent the different statistical groups from the Steel-Dwass-Critchlow-Fligner test.</figcaption></figure>

RS1 and 7 days for RS2. During this phase, if plants do not recover from the initial period of stress, the negative effects of a second stress may be amplified compared to the first one, as it affects plants that are already in a weakened state (Crisp et al., 2016). The more detrimental effect of recurrent heat stress scenario on thousand-grain weight could be due to a lack of time for recovery between the two stress events.

### 4.3. Evidences of interactions between a first heat stress imposed at flowering on the response to heat stress during the post-flowering phase at the leaf physiological scale

PSII maximal efficiency is reduced in response to various abiotic stresses such as high temperature, drought, nutrient deficiency (Poudyal et al., 2018, 2024). This decrease reflects damage or malfunction in the photosynthetic machinery. Return to optimal $F_v/F_m$ two days after heat stress, regardless of the duration of the stress, the developmental stage at which the stress is applied, or the recurrent nature of the stress indicates that no irreparable damage of photosystem II had occurred in response to heat. The results obtained are consistent with others as heat waves have been shown to have significant immediate effects on photosynthesis or photosynthesis related traits in crops such as soybean and
maize, with a return to values that do not significantly differ from those observed in control conditions at the end of the heat wave (Siebers et al., 2015, 2017). The duration of the priming effect on PSII stability and responsiveness remains an open question and likely depends on several factors, including species, tissue type, developmental stage, and stress intensity. In this recurrent heat stress scenario, $F_v/F_m$ of sorghum leaves was less affected when plants were exposed to heat stress after a priming heat stress, with recovery periods from 2 to 7 days. Similarly, Januškaitienė (2023) showed in their study on *Medicago sativa* L. that the negative effect of a late heat wave on $F_v/F_m$ was lower when plants were previously exposed to an early heat wave and returned to their normal state after 5 days of recovery. In addition, changes in the distribution of energy within photosystem II showed that *Medicago sativa* L. used energy more efficiently during a second heat stress event. Similar results were observed in wheat, where the photochemical efficiency of photosystem II of plants under recurrent stress with a 7-day recovery period tended to be better than that of plants exposed to a single stress event at the same time (Wang et al., 2011). In *Arabidopsis thaliana*, heat stress transcriptional memory can last for several days or even several weeks (Schöffl et al., 1998; Lämke and Bäurle, 2017). Lämke and Bäurle (2017) have shown that for the heat shock proteins (HSP), which are highly specific for heat stress, transcript levels decrease sharply within 24 hours of heat stress induction; however, protein levels remain high for at least 48 hours. Further investigations would be needed to determine whether the memory of heat stress in sorghum persists over longer periods (e.g., several weeks or months) and whether it involves epigenetic or other long-term regulatory mechanisms.

## 4.4. Tillering, by causing a sink competition, could explain the strong impact of recurrent heat stress on thousand-grain weight of the main stem panicle

Impacts of heat stresses on thousand grain weight is the end-result of the integration of many processes occurring during plant cycle combining source limitations and sink competitions whereas heat stress effect on grain number can also be due to direct effects of temperature on short term physiological processes at target stages such as production or viability (Kim et al., 2024). Intriguingly, the thousand-grain weight on main stem panicle was more affected by recurrent heat stress than the calculated cumulative response to the two successive single stresses. The difference between observed and calculated response to recurrent stress was more pronounced for the RS1 than the RS2 scenario in both sorghum genotypes. A first hypothesis could be that the source is differently affected by recurrent heat stress than single stresses. Temperature is a critical factor affecting photosynthetic efficiency (Brestic and Zivcak., 2013), with effects on the carbon source and electron transport chain that cause a decrease in assimilates to sink organs (here mainly grains, tillers and roots). Cereals exposed to high temperatures experience a sharp decrease in Rubisco activity and rapid senescence, which limits photosynthesis (Barnabas et al., 2008; Crafts-Brandner and Law., 2000; Griffin et al., 2004; Sunoj et al., 2017). In this study, photosynthesis was not measured but we observed an acclimation of the maximum efficiency of photosystem II ($F_v/F_m$) during a recurrent stress. This acclimation is stronger during RS1 compared to RS2 whereas the thousand-grain weight is more affected in RS1.

A second hypothesis could be that additional sinks compete with grain filling during recurrent heat stress. Accordingly, it has been described that sorghum has the ability to form tillers in response to abiotic stresses, particularly water stress (Ndlovu et al., 2021; Nguyen and Blum., 2004), also recently in response to single heat stress (Smith et al., 2023). Tillers are additional carbon sinks and their growth subsequently competes with the main stem panicle development. Tillering has been shown to be compensatory mechanism to ensure time-delayed grain production, thereby ensuring offspring through faster emergence of reproductive shoots (Smith et al., 2023). In pearl millet (*Pennisetum glaucum*), another African crop, tiller development plasticity is

also a key adaptive trait under abiotic stress conditions (Djanaguiraman et al., 2024). Under heat stress, when primary tillers fail to flower and produce seeds, secondary tillers develop and compensate yield losses in both the main stem and primary tillers. This compensatory mechanism increases pearl millet resilience to elevated temperatures and contributes to its adaptation to stressful environments (Djanaguiraman et al., 2024). However, tillering could be in strong competition with grain filling of the main stem and explain at least partly why recurrent heat stress with high tiller production causes a stronger reduction in thousand-grain weight than what expected from the cumulative effect of two single stresses.

## 5. Conclusion

This study focuses on understanding the effects of single and recurrent heat stress on sorghum. Exposure to high temperatures during critical stages such as flowering and grain filling reduces PSII maximal efficiency in leaves, reduces final grain yield and induces tillering. Recurrent heat stresses amplify the decrease in grain yield and thousand grain weight compared to the cumulative effect of single stresses. The results highlight the vulnerability of sorghum to repeated thermal stress and the need to explore genetic diversity and crop management strategies to enhance its resilience. A key limitation is that potential strategies for enhancing heat resilience were not addressed. Future research should aim to identify and assess approaches to mitigate the heat stress impacts and maintain sorghum productivity under changing climatic conditions.

## Funding

This work was supported by the AgroEcoSystem division of the French National Research Institute for Agriculture, Food and Environment (INRAE, PARSEMA project) and by the National French Research Agency (ANR, RICOCHETS project grant N° ANR-22-CE20-0041).

## CRediT authorship contribution statement

**Berger Angelique:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. **Suire Cyndi:** Methodology, Investigation. **Cakpo Belmys:** Writing – review & editing, Investigation, Formal analysis. **Terrier Nancy:** Writing – review & editing, Project administration, Investigation, Funding acquisition, Conceptualization. **Aguilar Gregory:** Methodology, Investigation. **Granier Christine:** Writing – review & editing, Project administration, Investigation, Funding acquisition, Conceptualization. **Vial Raoul:** Methodology, Investigation. **Roques Sandrine:** Methodology, Investigation. **Pagan Loic:** Methodology, Investigation.

## Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used ChatGPT and DeepL in order to improve language. After using these tools, the author (s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

We are most grateful to the CIRAD Ecophysiology and plant growing platforms for their technical support. Thanks to Laura Lescroart,
Catherine Giauffret, Valérie Mechin for reviewing.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.envexpbot.2025.106162.

## References

Ahrens, C., Challis, A., Byrne, M., Leigh, A., Nicotra, A.B., Tissue, D., Rymer, P., 2021. Repeated extreme heatwaves result in higher leaf thermal tolerances and greater safety margins. N. Physiol. 232, 1212-1225. https://doi.org/10.1111/nph.17640.

Arshad, M.S., Farooq, M., Asch, F., Krishna, J.S.V., Prasad, P.V.V., Siddique, K.H.M., 2017. Thermal stress impacts reproductive development and grain yield in rice. Plant Physiol. Biochem. 115, 57-72. https://doi.org/10.1016/j.plaphy.2017.03.011.

Bador, M., Terray, L., Boé, J., Somot, S., Alias, A., Gibelin, A.-L., Dubuisson, B., 2017. Future summer mega-heatwave and record-breaking temperatures in a warmer France climate, 07,40-25 Environ. Res. Lett. 12. https://doi.org/10.1088/1748-9326/aa751c.

Barnabás, B., Jäger, K., Fehér, A., 2008. The effect of drought and heat stress on reproductive processes in cereals. Plant, Cell Environ, 31, 11-38. https://doi.org/10.1111/j.1365-3040.2007.01727.

Bäurle, I., 2016. Plant heat adaptation: priming in response to heat stress. F1000Res 5, F1000 Faculty Rev-694. doi: 10.12688/f1000research.7526.1.

Breshears, D.D., Fontaine, J.B., Ruthrof, K.X., Field, J.P., Feng, X., Burger, J.R., Law, D.J., Kala, J., Hardy, G.E.St.J., 2021. Underpracticed plant vulnerabilities to heat waves. N. Phytol. 231, 32-39. https://doi.org/10.1111/nph.17348.

Brestic, M., Zivcak, M., 2013. PSII fluorescence techniques for measurement of drought and high temperature stress signal in crop plants: protocols and applications. In: Rout, G.R., Das, A.B. (Eds.), Molecular Stress Physiology of Plants. Springer, India, pp. 87-131. https://doi.org/10.1007/978-81-322-0807-5-4.

Chadalavada, K., Kumari, B.D.R., Kumar, T.S., 2021. Sorghum mitigates climate variability and change on crop yield and quality. Planta 253, 113. https://doi.org/10.1007/s00425-021-03631-2.

Chantereau, J., Cruz, J.-F., Ratnadass, A., Trouche, G., Fliedel, G., 2013. Le sorgho. Éditions Quae. doi: 10.35690/978-2-7592-2062-5.

Chaudhry, S., Sidhu, G.P.S., 2022. Climate change regulated abiotic stress mechanisms in plants: a comprehensive review. Plant Cell Rep. 41, 1-31. https://doi.org/10.1007/s00299-021-02759-5.

Chiluwal, A., Bheemanahalli, R., Kanaganahalli, V., Boyle, D., Perumal, R., Pokharel, M., Oumarou, H., Jagadish, S.V.K., 2020. Deterioration of ovary plays a key role in heat stress-induced spikelet sterility in sorghum. Plant, Cell Environ. 43, 448-462. https://doi.org/10.1111/pce.13673.

Crafts-Brandner, S.J., Law, R.D., 2000. Effect of heat stress on the inhibition and recovery of the ribulose-1,5-bisphosphate carboxylase/oxygenase activation state. Planta 212, 67-74. https://doi.org/10.1007/s004250000364.

Crisp, P.A., Ganguly, D., Eichten, S.R., Borevitz, J.O., Pogson, B.J., 2016. Reconsidering plant memory: Intersections between stress recovery, RNA turnover, and epigenetics. Sci. Adv. 2, e1501340. https://doi.org/10.1126/sciadv.1501340.

Djanaguiraman, M., Perumal, M., Jagadish, S.V.K., Ciampitti, I.A., Welti, R., Prasad, P.V. V., 2018. Sensitivity of sorghum pollen and pistil to high-temperature stress. Plant, Cell Environ. 41, 1065-1082. https://onlinelibrary.wiley.com/doi/10.1111/pce.13089.

Djanaguiraman, M., Narayanan, S., Erdayani, E., Prasad, P.V.V., 2020. Effects of high temperature stress during anthesis and grain filling periods on photosynthesis, lipids and grain yield in wheat. BMC Plant Biol. 20, 268. https://doi.org/10.1186/s12870-020-02479-0.

Djanaguiraman, M., Priyanka, A.S., Vaishnavi, S.J., Ramasamy Perumal, R., Ignacio, A., Ciampitti, I.A., Prasad, P.V.V., 2024. Impact of drought and high-temperature stresses on growth and development stages, physiological, reproductive, and yield traits on Pearl Millet. In: Perumal, R., Vara Prasad, P., Satyavathi, T., Govindaraj, M., Tenkouano, A. (Eds.), Pearl Mille, t. https://doi.org/10.1002/9780891184034.ch9.

Driedonks, N., Rieu, I., Vrieken, W.H., 2016. Breeding for plant heat tolerance at vegetative and reproductive stages. Plant Reprod. 29, 67-79. https://doi.org/10.1007/s00497-016-0275-9.

Edwards, E.J., 2019. Evolutionary trajectories, accessibility and other metaphors: the case of C₄ and CAM photosynthesis. N. Phytol. 223, 1742-1755. https://doi.org/10.1111/nph.15851.

Fan, Y., Ma, C., Huang, Z., Abid, M., Jiang, S., Dai, T., Zhang, W., Ma, S., Jiang, D., Han, X., 2018. Heat priming during early reproductive stages enhances thermotolerance to post-anthesis heat stress via improving photosynthesis and plant productivity in winter wheat (Triticum aestivum L.). Front. Plant Sci. 9. https://doi.org/10.3389/fpls.2018.00805.

Farooq, M., Bramley, H., Palta, J.A., Siddique, K.H., 2011. Heat stress in wheat during reproductive and grain-filling phases. Crit. Rev. Plant Sci. 30, 491-507. https://doi.org/10.1080/07352689.2011.615687.

Griffin, J.J., Ranney, T.G., Pharr, D.M., 2004. Heat and drought influence photosynthesis, water relations, and soluble carbohydrates of two ecotypes of Redbud (Cercis canadensis). J. Am. Soc. Hortic. Sci. 129, 497-502. https://journals.ashs.org/jashs/view/journals/jashs/129/4/article-p497.xml.

Hüve, K., Bichele, I., Rasulov, B., Niinemets, Ü., 2011. When it is too hot for photosynthesis: heat-induced instability of photosynthesis in relation to respiratory burst, cell permeability changes and H2O2 formation. Plant, Cell Environ. 34, 113-126. https://doi.org/10.1111/j.1365-3040.2010.02229.

Impa, S.M., Perumal, R., Bean, S.R., John Sunjo, V.S., Jagadish, S.V.K., 2019. Water deficit and heat stress induced alterations in grain physico-chemical characteristics and micronutrient composition in field grown grain sorghum. J. Cereal Sci. 86, 124-131. https://doi.org/10.1016/j.jcs.2019.01.013.

IPCC report, 2021 (https://www.ipcc.ch/report/ar6/wgl).

Jagadish, S.V.K., 2020. Heat stress during flowering in cereals effects and adaptation strategies. N. Phytol. 226, 1567-1572. https://doi.org/10.1111/nph.16429.

Jagadish, S.V.K., Way, D.A., Sharkey, T.D., 2021. Plant heat stress: Concepts directing future research. Plant, Cell Environ. 44, 1992-2005. https://doi.org/10.1111/ pce.14050.

Januskaitienė, I., 2023. The second heat wave leads to changes in the efficiency of alfalfa's photosynthetic energy use. Biologija 69, 134-146. https://doi.org/10.6001/ biologija.2023.69.2.2.

Kim, J., Savin, R., Slafer, G.A., 2024. Quantifying pre- and post-anthesis heat waves on grain number and grain weight of contrasting wheat cultivars. Field Crops Res. 307, 109264. https://doi.org/10.1016/j.fcr.2024.109264.

Lämke, J., Bäurle, I., 2017. Epigenetic and chromatin-based mechanisms in environmental stress adaptation and stress memory in plants. Genome Biol. 18, 124. https://doi.org/10.1186/s13059-017-1263-6.

Lemonsu, A., Beaulant, A.L., Somot, S., Masson, V., 2014. Evolution of heat wave occurrence over the Paris basin (France) in the 21st century. Clim. Res. 61, 75-91. https://doi.org/10.3354/cr01235.

Lesk, C., Rowhani, P., Ramankutty, N., 2016. Influence of extreme weather disasters on global crop production. Nature 529, 84-87. https://doi.org/10.1038/nature16467.

Magno Massuca de Almeida, L., Avice, J.-C., Morvan-Bertrand, A., Wagner, M.-H., González-Centeno, M.R., Teissedre, P.-L., Bessoule, J.-J., Le Guedard, M., Kim, T.H., Mollier, A., Brunel-Muguet, S., 2021. High temperature patterns at the onset of seed maturation determine seed yield and quality in oilseed rape (Brassica napus L.) in relation to sulphur nutrition. Environ. Exp. Bot. 185, 104400. https://doi.org/10.1016/j.envexpbot.2021.104400.

Ndlovu, E., van Staden, J., Maphosa, M., 2021. Morpho-physiological effects of moisture, heat and combined stresses on Sorghum bicolor (Moench (L.)) and its acclimation mechanisms. Plant Stress 2, 100018. https://doi.org/10.1016/j.stress.2021.100018.

Nguyen, H.T., Blum, A., 2004. Physiology and Biotechnology Integration for Plant Breeding. CRC Press.

Oberkofler, V., Pratx, L., Bäurle, I., 2021. Epigenetic regulation of abiotic stress memory: maintaining the good things while they last. Curr. Opin. Plant Biol. 61, 102007. https://doi.org/10.1016/j.pbi.2021.102007.

Parent, B., Tardieu, F., 2012. Temperature responses of developmental processes have not been affected by breeding in different ecological areas for 17 crop species. N. Phytol. 194, 760-774. https://doi.org/10.1111/j.1469-8137.2012.04086.

Pasternak, D., Wilson, G.L., 1969. Effects of heat waves on grain sorghum at the stage of head emergence. Aust. J. Exp. Agric. Anim. Husb. 9.

Poudyal, D., Rosenqvist, E., Ottosen, C.-O., 2018. Phenotyping from lab to field - tomato lines screened for heat stress using F~v~/F~m~ maintain high fruit yield during thermal stress in the field. Funct. Plant Biol. 46, 44-55. https://doi.org/10.1071/FP17317.

Poudyal, D., Joshi, B.K., Zhou, R., Ottosen, C.-O., Dahal, K.C., 2023. Evaluating the physiological responses and identifying stress tolerance of Akabare chili landraces to individual and combined drought and heat stresses. AoB PLANTS 15 (6). https://doi.org/10.1093/aobpla/plad083e36239.

Poudyal, D., Joshi, B.K., Dahal, K.C., 2024. Insights into the responses of Akabare chili landraces to drought, heat, and their combined stress during pre-flowering and fruiting stages. Heliyon 10 (16). https://doi.org/10.1016/j.heliyon.2024.e36239.

Pradhan, A., Aher, L., Hegde, V., Jangid, K.K., Rane, J., 2022. Cooler canopy leverages sorghum adaptation to drought and heat stress. Sci. Rep. 12, 4603. https://doi.org/10.1038/s41598-022-08590-6.

Prasad, P.V., Pisipati, S.R., Mutava, R.N., Tuinstra, M.R., 2008. Sensitivity of grain sorghum to high temperature stress during reproductive development. Crop Sci. 48, 1911-1917. https://doi.org/10.2135/cropsci2008.01.0036.

Prasad, P.V.V., Djanaguiraman, M., Perumal, R., Ciampitti, I.A., 2015. Impact of high temperature stress on floret fertility and individual grain weight of grain sorghum: sensitive stages and thresholds for temperature and duration. Front. Plant Sci. 6. https://doi.org/10.3389/fpls.2015.00820.

Prasad, P.V.V., Bheemanahalli, R., Jagadish, S.V.K., 2017. Field crops and the fear of heat stress opportunities, challenges and future directions. Field Crops Res. 200, 114-121. https://doi.org/10.1016/j.fcr.2016.09.024.

Prasad, V.B.R., Govindaraj, M., Djanaguiraman, M., Djalovic, I., Shailani, A., Rawat, N., Singla-Pareek, S.L., Pareek, A., Prasad, P.V.V., 2021. Drought and high temperature stress in Sorghum: physiological, genetic, and molecular insights and breeding approaches. Int. J. Mol. Sci. 22, 9826. https://doi.org/10.3390/ijms22189826.

Radha, B., Sunitha, N.C., Sah, R.P., Azharudheen, T.P., Krishna, M., Umesh, G.K., Thomas, D.K., Anilkumar, S., Upadhyay, C., Kumar, S., Manikanta, A., Ch, L.N., Behera, S., Marandi, B.C., Siddique, K.H.M., 2023. Physiological and molecular implications of multiple abiotic stresses on yield and quality of rice. Front. Plant Sci. 13. https://doi.org/10.3389/fpls.2022.996514.

Ruehr, N.K., Grote, R., Mayr, S., Arneth, A., 2019. Beyond the extreme: recovery of carbon and water relations in woody plants following heat and drought stress. Tree Physiol. 39, 1285-1299. https://doi.org/10.1093/treephys/tpz032.

Schöffl, F., Prändl, R., Reindl, A., 1998. Regulation of the heat-shock response. Plant Physiol. 117, 1135-1141. https://doi.org/10.1104/pp.117.4.1135.

Siebers, M.H., Yendrek, C.R., Drag, D., Locke, A.M., Rios Acosta, L., Leakey, A.D.B., Ainsworth, E.A., Bernacchi, C.J., Ort, D.R., 2015. Heat waves imposed during early pod development in soybean (Glycine max) cause significant yield loss despite a rapid burst, cell permeability changes and H2O2 formation. Plant, Cell Environ. 34, 113-126. https://doi.org/10.1111/j.1365-3040.2010.02229.
recovery from oxidative stress. Glob. Change Biol. 21, 3114–3125. https://doi.org/
10.1111/gcb.12935.

Siebers, M.H., Slattery, R.A., Yendrek, C.R., Locke, A.M., Drag, D., Ainsworth, E.A., Bernacchi, C.J., Ort, D.R., 2017. Simulated heat waves during maize reproductive stages alter reproductive growth but have no lasting effect when applied during vegetative stages. Agric., Ecosyst. Environ. 240, 162–170. https://doi.org/10.1016/
j.agee.2016.11.008.

Singh, V., Nguyen, C.T., Van Oosterom, E.J., Chapman, S.C., Jordan, D.R., Hammer, G.L., 2015. Sorghum genotypes differ in high temperature responses for seed set. Field Crops Res. 171, 32–40. https://doi.org/10.1016/j.fcr.2014.11.003.

Singh, V., Nguyen, C.T., Yang, Z., Chapman, S.C., Van Oosterom, E.J., Hammer, G.L., 2016. Genotypic differences in effects of short episodes of high-temperature stress during reproductive development in Sorghum. Crop Sci. 56, 1561–1572. https://doi.org/10.2135/cropsci2015.09.0545.

Smith, A., Gentile, B.R., Xin, Z., Zhao, D., 2023. The effects of heat stress on male reproduction and tillering in Sorghum bicolor. Food Energy Secur. 12, e510. https://doi.org/10.1002/fes3.510.

Sunoj, V.S.J., Impa, M.S., Chiluwal, A., Perumal, R., Prasad, P.V.V., Jagadish, S.V.K., 2017. Resilience of pollen and post-flowering response in diverse Sorghum genotypes exposed to heat stress under field conditions. Crop Sci. 57, 1658–1669. https://doi.org/10.2135/cropsci2016.08.0706.

Tao, Z., Chen, Y., Li, C., Zou, J., Yan, P., Yuan, S., Wu, X., Sui, P., 2016. The causes and impacts for heat stress in spring maize during grain filling in the North China Plain. J. Integr. Agric. 15, 2677–2687. https://doi.org/10.1016/S2095-3119(16)61409-0.

Tigchelaar, M., Battisti, D.S., Naylor, R.L., Ray, D.K., 2018. Future warming increases probability of globally synchronized maize production shocks. Proc. Natl. Acad. Sci. 115, 6644–6649. https://doi.org/10.1073/pnas.1718031115.

Trnka, M., Rötter, R.P., Ruiz-Ramos, M., Kersebaum, K.C., Olesen, J.E., Žalud, Z., Semenov, M.A., 2014. Adverse weather conditions for European wheat production will become more frequent with climate change. Nat. Clim. Change 4, 637–643. https://doi.org/10.1038/nclimate2242.

USDA report 2024 (https://www.fas.usda.gov/data/production/commodity/0459200?utm_source=chatgpt.com).

Wang, X., Cai, J., Jiang, D., Liu, F., Dai, T., Cao, W., 2011. Pre-anthesis high-temperature acclimation alleviates damage to the flag leaf caused by post-anthesis heat stress in wheat. J. Plant Physiol. 168, 585–593. https://doi.org/10.1016/j.jplph.2010.09.016.

Wang, X., Cai, J., Liu, F., Jin, M., Yu, H., Jiang, D., Wollenweber, B., Dai, T., Cao, W., 2012. Pre-anthesis high temperature acclimation alleviates the negative effects of post-anthesis heat stress on stem stored carbohydrates remobilization and grain starch accumulation in wheat. J. Cereal Sci. 55, 331–336. https://doi.org/10.1016/j.jcs.2012.01.004.

Xie, W., Xiong, W., Pan, J., Ali, T., Cui, Q., Guan, D., Meng, J., Mueller, N.D., Lin, E., Davis, S.J., 2018. Decreases in global beer supply due to extreme drought and heat. Nat. Plants 4, 964–973. https://doi.org/10.1038/s41477-018-0263-1.

Zampieri, M., Ceglar, A., Dentener, F., Toreti, A., 2017. Wheat yield loss attributable to heat waves, drought and water excess at the global, national and subnational scales. Environ. Res. Lett. 12 (6). https://doi.org/10.1088/1748-9326/aa723b.

Zandalinas, S.I., Peláez-Vico, M.A., Sinha, R., Pascual, L.S., Mittler, R., 2024. The impact of multifactorial stress combination on plants, crops, and ecosystems: how should we prepare for what comes next? Plant J. 117, 1800–1814. https://doi.org/10.1111/tpj.16557.

Zenda, T., Wang, N., Dong, A., Zhou, Y., Duan, H., 2022. Reproductive-stage heat stress in cereals: impact, plant responses and strategies for tolerance improvement. Int. J. Mol. Sci. 23, 6929. https://doi.org/10.3390/ijms23136929.