24-Epicastasterone and KH2PO4 protect grain production of wheat crops from terminal heat impacts by modulating leaf physiology

ABSTRACT Heat stress during the grain filling is one of most critical factors impacting grain yield in winter wheat. In a two-year field experiment, we studied the effects of 24-Epicastasterone and KH2PO4 on heat-stressed wheat applied during different times. A completely randomized block design was used with three reagents, that is, 24-Epicastasterone, KH2PO4 and H2O and three spraying times, that is, pre-heat, under-heat and post-heat. Two cultivars, that is, Huaimai 33 and Annong 0711 were subjected to a 7 d post-flowering heat stress using plastic tents. Our study suggests that greater grain weight in heat-stressed plants was achieved through sustaining leaf greenness and higher assimilates supplies to grains. When heat-stressed plants sprayed either with 24-Epicastasterone or KH2PO4 could sustain a relatively higher leaf photosynthetic capacity by up-regulating antioxidant enzymes with reduced damage on lipid membranes and leaf chlorophyll. We also found that KH2PO4 was relatively more effective in improving leaf physiology and grain yield of heat-stressed plants than 24-Epicastasterone. Further analysis showed that pre-heat or post-heat was more pronounced in protecting plants from heat injury than spraying the plants under heat stress. Thus, spraying KH2PO4 either prior to or post stress incidence is recommended to protect wheat crops from terminal heat stress.


Introduction
Wheat (Triticum aestivum L.) is one of most important food crops, which significantly contributes to global food security (Shewry and Hey 2015). Approximately 60% increase in global wheat production is needed to feed 9.7 billion people by this middle of the twenty-first century (United Nations 2019). However, this challenge is further complicated by abiotic stresses such as drought (Nadeem et al. 2019a), heat (Farooq et al. 2011), cold (Hassan et al. 2021), and salinity (Nadeem et al. 2019b;Soliman et al. 2022), which significantly affects wheat grain yields. For example, in Huang-Huai-Hai region, one of the major grain production areas in China, (Jiang et al. 2015), annual wheat yields are significantly affected by post-flowering heat (Li et al. 2022). Terminal heat stress is widely reported in major wheat growing regions including in China (Jing et al. 2010), Australia (Flohr et al. 2017), Mediterranean regions (Elia et al. 2018), and the U.S. Great Plains (Lollato et al. 2020). Post-anthesis heat stress accelerates leaf senescence and reduces chlorophyll index (SPAD) (Bergkamp et al. 2018), inhibiting carbon assimilation (Camejo et al. 2005). For example, a brief episode of heat (i.e. 10°C above the optimum temperature) during grain filling phase of wheat crop reduces maximum grain filling rate, grain size and grain yield by 35.3%, 11.4% and 10.1%, respectively (Feng et al. 2018). Modeling studies suggest more frequent and severe heat stress during grain-filling stage of wheat in the near future (Ababaei and Chenu 2020).
Exogenously applied plant hormones and mineral nutrients remarkably improve abiotic stress tolerance and offer an economically feasible option to minimize the impact of terminal heat stress on crops (Eisvand et al. 2018;Shahid et al. 2019). For example, brassinosteroids (BRs) has attracted wide attention as plant growth regulator (Mussig 2005;Haubrick and Assmann 2006;Krishna et al. 2017). BRs can regulate many physiological processes, such as cell elongation and division, and ATPase activity in plants (Haubrick and Assmann 2006;Krishna et al. 2017;Fang et al. 2019;Peres et al. 2019). BRs have been found effective in improving growth of naked oat under high temperature by modulating its root growth and antioxidant enzyme activity, and reducing malodialdehyde (MDA) content (Wang and Guo 2017). Exogenously sprayed BRs can enhance superoxide dismutase (SOD) enzyme activity, and increase proline and soluble sugar content in heat-stressed rice plants (Gao et al. 2019). During differentiation stage of young rice panicles, BRs cause promoted carbohydrate transport to young panicles, inhibited decomposition of cytokinin, and reduced peroxidation damage, thus protecting spikelet from heat injury (Chen et al. 2019).
Phosphorus (P) and potassium (K) are important nutrients that significantly affect crop growth and grain yield formation in wheat. P serves various basic biological functions as a structural element in nucleic acids and phospholipids, energy metabolism, the activation of metabolic intermediates, signal transduction cascades, and regulation of enzymes (Poirier and Bucher 2002). Similarly, K regulates the important physiological processes such as protein biosynthesis, transport of assimilation products and osmotic regulation (Lv et al. 2017). Monopotassium dihydrogen phosphate (KH 2 PO 4 ) is a common high-quality and efficient foliar fertilizer. KH 2 PO 4 is widely used as a foliage spraying reagent in wheat (Lv et al. 2017;Yang et al. 2019), which can promote the grain filling rate and grain yield in wheat by extending flag leaf greenness (Dang et al. 2003). Further, compared with hormones, KH 2 PO 4 is an easily accessible and economical nutrient commercial use in cropping systems.
24-epicastasterone and KH 2 PO 4 have been tested on wheat crop during different developmental stages (Thussagunpanit et al. 2015;Ahammed et al. 2020;Rafiullah et al. 2020). However, application of these reagents for heat stressed wheat crops is rarely reported. Particularly, the regulatory mechanisms through which they protect developing wheat grains from heat injury is yet to determine. Within this context, the objectives are to (i) compare the efficacy of two reagents, that is, 24-epicastasterone and KH 2 PO 4 in protecting wheat crops from terminal heat injury, and (ii) compare the effects at different timings, that is, in pre-heat, under-heat or post-heat event, in order to identify the most suitable spraying time.

Field experiment
A two-year field study was conducted at the experimental field of Anhui Agricultural University (31 ○ 52′ N, 117 ○ 14′ E), Hefei, Anhui Province, China. The soil at the experimental site contained 10.6 g kg −1 readily available organic matter for, 1.13 g kg −1 total nitrogen, 81.5 mg kg −1 available nitrogen, 33.1 mg kg −1 available phosphorus and 76.2 mg kg −1 available potassium.
Two wheat varieties, that is, Huaimai 33 and Annong 0711, were used in the study. These are commercially cultivated varieties in the south of Huang-Huai-Hai Plain, and were selected based on their similar phenology including time to anthesis and to physiological maturity. Seeds were manually sown on 5 November 2017 and on 25 October 2018 in 6 m 2 (2 m × 3 m) plots at a sowing density of 2,250,000 ha −1 , with a row spacing of 20 cm. Wheat grains were harvested on May 25 in 2018 and on May 26 in 2019. The NPK compound fertilizer (N-P 2 O 5 -K 2 O = 15 − 15 − 15%) and urea (46.4% N) were used, at the total rates of 225.0 kg N ha −1 , 112.5 kg P 2 O 5 ha −1 and 112.5 kg K 2 O ha −1 . Prior to sowing, 112.5 kg ha −1 N, 112.5 kg P 2 O 5 ha −1 and 112.5 kg K 2 O ha −1 were applied as base fertilizer. The residual N was applied at jointing stage.
Heat stress treatment was applied from 12th to 18th day after anthesis (DAA) from 10:00 to 16:00 each day (only day time temperatures were increased). Insulation tent of galvanized steel frame structure and covered with the transparent polyethylene film (20 cm of the ground for ventilation) were used for heat stress treatment according to Yang et al. (2016) with minor modifications. The transparent polyethylene film covering steel frame of the tunnel was 0.08 mm thick, which allowed transmission of over 90% of the solar radiation. The thermohygrometers  were mounted inside and outside of the tunnel, and the atmospheric temperature was recorded for every 10 min. When the temperature inside the plastic tunnel exceeded atmospheric temperature by 8°C, the plastic tunnel was manually rolled up for heat dissipation in achieving the desired temperature ( Figure 1). Each of the treatments contained three plots as replicates.
A completely randomized block design was used with three reagents, that is, 24-Epicastasterone, KH 2 PO 4 and H 2 O, and three application times, that is, pre-heat, under-heat and post-heat. The experiment was repeated three times under the same conditions. Two cultivars are independently used. Treatments include CK1 (normal growth conditions outside the tunnel); HT, under artificially simulated high temperature conditions; HT+24-epicastasterone, HT+KH 2 PO 4 and HT+H 2 O. 24epicastasterone and KH 2 PO 4 were purchased from Beijing Solarbio Science & Technology Co., Ltd. 24-epicastasterone and KH 2 PO 4 were sprayed at 9th-10th DAA (prior to the heat stress treatment), at 14th-15th DAA (during heat stress treatment), and at 20th-21th DAA (post-heat stress treatment). All the plants were sprayed for two consecutive days at 17:30 PM each day for each treatment. The regents were sprayed by vapor-pressure type sprayers (13 cm wide×31 cm high, capacity 2 L) at 673.8 L ha −1 according to the method suggested by Lv et al. (2017) and Peirce et al. (2019). The spray concentration was 0.1 mg L −1 for 24-epicastasterone and 0.2% for KH 2 PO 4 . All of the solutions contained 0.01% (V/V) Tween-20. The same volume of deionized water containing the same concentration of Tween-20 was applied to the control plants (HT+H 2 O).

Data acquisition
Crop phenology was recorded using the Zadoks (Zadoks et al. 1974) and Li (Li et al. 2019). 50% flowering of each plot plants were considered as flowering time. Two hundred individual spikes were selected and tagged at an identical flowering time in each plot. Data on SPAD, photosynthetic rate, antioxidant enzyme activities and MDA for the flag leaf were collected at the 30th DAA from the selected plants.

Leaf chlorophyll content and photosynthetic rate
The SPAD value of flag leaves were measured using a hand-held SPAD chlorophyll meter (Model 502, Minolta Co. Ltd., Osaka, Japan). The tip, middle, and the bottom part of a flag leaf were measured, respectively, and averaged to represent a flag SPAD index.
Gas exchange traits such as net photosynthesis rate (Pn), stomatal conductance (gs), intercellular CO 2 concentration (Ci) and transpiration rate (E) of the flag leaves were measured in the sunny day (9:00am-12:00am) with CIRAS-3 (American PP SYSTEMS) photosynthetic instrument. Three plants from each plot were measured. The chamber was equipped with a red/blue LED light source. Photosynthetic measurements were performed under light-saturated conditions (1000 µmol photons m −1 s −1 of PPFD) at 25°C, and an ambient CO 2 concentration controlled by a surge flask.

Antioxidant enzyme activities and MDA
The peroxidase (POD) activity and malondialdehyde (MDA) content of the flag leaves were assayed by following the methods reported by Zheng et al. (2009). The activities of superoxide dismutase (SOD) and catalase (CAT) of the flag leaf were measured according to Almeselmani et al. (2006). The above measurements were cut into pieces from six leaves, and four biological replicates were carried out.

Grain yield and thousand grain weight
According to the description of Li et al. (2018), wheat plants from a 1.2 m 2 area in each plot were harvested at maturity and threshed for fresh grain weighing. Some of grain samples were further used for calculating grain moisture content by oven-dried at 75°C until a constant weight was achieved. Grain yield and 1000-grain weight were then corrected at a 13.0% moisture basis and all values were the averaged from three independent replicates.

Statistical analysis
Analysis of variance (ANOVA) was performed using SPSS statistical software (SPSS ver. 24.0, SPSS, Chicago, IL, United States). The ANOVA and multiple comparison were conducted on SPAD, SOD, POD, CAT and MDA for the flag leaf, 1000-grain weight and grain yield. The means were analyzed using the least significant difference (LSD) method at P= 0.05. All figures were created by Origin Pro 2021 (Origin Lab Corp., Northampton, MA, USA).

Flag leaf SPAD
In 2017-2018 and 2018-2019 growth seasons, the SPAD value was significantly influenced by reagent (R) and spaying time (T) ( Table 1). Compared to HT+H 2 O treatment, the heat-stressed plants treated with 24epicastasterone or KH 2 PO 4 had a significantly higher flag leaf SPAD value. Averaged across all treatments, 24-epicastasterone and KH 2 PO 4 increased flag leaf SPAD value of heat-stressed plants by 27.5% and 30.6%, respectively, in 2017-2018, and by 29.6% and 52.5%, respectively, in 2018-2019 ( Figure 2). Across the reagents, the effect of exogenous spraying KH 2 PO 4 is better than 24-epicastasterone, except for Huaimai 33 in 2017-2018 ( Figure 2, Table S1). KH 2 PO 4 was relatively more effective in sustaining leaf greenness in wheat cultivar of Huaimai 33 and Annong 0711 compared with 24-Epicastasterone under all treatments. Across the reagent application times, all three spraying times improved the SPAD value, 'preheat' and 'post-heat' were effective in increasing leaf SPAD value of wheat cultivars followed by 'underheat' in 2017-2018, however, pre-heat better in 2018-2019 (Table S2).

Flag leaf gas exchange parameters
In 2017-2018 and 2018-2019 growth seasons, the Pn, gs, Ci and E were significantly influenced by reagent (R), spaying time (T) and the interaction between reagent and spaying time (R × T) ( Table 1). Spraying 24-epicastasterone and KH 2 PO 4 significantly increased flag leaf Pn, gs and E but reduced Ci of the heat-stressed wheat plants during both years of study (Table 2). Compared with HT+H 2 O treated plants, averaged across all treatments, 24-epicastasterone and KH 2 PO 4 increased flag leaf Pn of heatstressed plants by 26.2% and 33.3%, respectively, in 2017-2018, and by 169.4% and 211.7%, respectively, in 2018-2019. Averaged across all treatments, KH 2 PO 4 was relatively more effective in increasing leaf Pn than 24-epicastasterone during both years (Table S1). Across the reagent application times, all three spraying times improved the Pn, 'pre-heat' and 'post-heat' were effective in increasing leaf Pn of wheat cultivars followed by 'under-heat', except for Huaimai 33 in 2018-2019 (Table S2). A similar positive effect was recorded on the leaf of gs and E of two wheat cultivars in this study in 2017-2019.

Flag leaf MDA content
In 2017-2018 and 2018-2019 growth seasons, the MDA content was significantly influenced by reagent (R) and spaying time (T) (  Figure 4). Across the reagents, the effect of exogenous spraying KH 2 PO 4 is better than 24-epicastasterone in reduced flag leaf MDA content in wheat cultivar of Huaimai 33 and Annong 0711 compared with 24-Epicastasterone under all treatments ( Figure 4, Table S1). Across the reagent application times, all three spraying times reduced the MDA content, 'pre-heat' and 'post-heat' were effective of wheat cultivars, followed by 'under-heat' (Table S2).

Grain yield and yield components
In 2017-2018 and 2018-2019 growth seasons, the 1000-grain weight and grain yield were significantly influenced by reagent (R), spaying time (T) and the interaction between reagent and spaying time (R × T) (Table 1). HT treatment significantly reduced 1000-grain weight and grain yield of Huaimai 33 by 17.4% and 22.1%, respectively, in 2018; and HT treatment also significantly reduced 1000-grain weight and grain yield of Annong 0711 by 18.3% and 32.2%, respectively, in 2018. Compared to HT+H 2 O, 1000-grain weight and grain yield for the heated plants sprayed with exogenous reagents was significantly increased in 2017-2019. Averaged across all treatments, 24epicastasterone and KH 2 PO 4 increased 1000-grain weight and grain yield of heat-stressed plants by 5.9%, 7.6% and 10.2%, 13.0%, respectively, in 2017-2018, and by 6.8%, 9.7% and 10.6%, 14.7%, respectively, in 2018-2019 (Table 3). In 'pre-heat', 'under-heat' and 'post-heat', the 1000-grain weight for the plants with HT+24-epicastasterone and HT+KH 2 PO 4 average increased by 11.2%, 8.1% and 9.3%, and the grain yield increased by 11.4%, 8.5% and 9.8% across all treatments. Exogenous spraying KH 2 PO 4 under high temperature treatment (HT+KH 2 PO 4 ) was shown to be more effective for improving 1000-grain weight and grain yield than HT+24-epicastasterone, and the results Figure 2. Changes in content of SPAD of Huaimai 33 and Annong 0711 at high temperature plus 24-epicastasterone (HT+24epicastasterone), and high temperature plus KH 2 PO 4 (HT+KH 2 PO 4 ) in 2017-2019. 'pre-heat' denoted the spraying of growth regulator prior to the occurrence of high temperature; 'under-heat' denoted the spraying of growth regulator during the occurrence of high temperature; 'post-heat' denoted the spraying of growth regulator post the occurrence of high temperature. The data are means ± SE (n = 3). Lowercase letters refer to significant differences between the treatments (P < 0.05). Vertical lines indicate error bars. 3.63 ± 0.02b p're-heat' denoted the spraying of growth regulator prior to the occurrence of high temperature; 'under-heat' 'denoted the spraying of growth regulator during the occurrence of high temperature. 'post-heat' denoted the spraying of growth regulator post the occurrence of high temperature. The data are means ± SE (n = 3). Lowercase letters refer to significant differences between the treatments (P < 0.05). Vertical lines indicate error bars. Figure 3. Changes in superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity in Huaimai 33 and Annong 0711 at high temperature plus 24-epicastasterone (HT+24-epicastasterone), and high temperature plus KH 2 PO 4 (HT+KH 2 PO 4 ) in 2017-2019. 'pre-heat' denoted the spraying of growth regulator prior to the occurrence of high temperature; 'under-heat' denoted the spraying of growth regulator during the occurrence of high temperature; 'post-heat' denoted the spraying of growth regulator post the occurrence of high temperature. The data are means ± SE (n = 3). Lowercase letters refer to significant differences between the treatments (P < 0.05). Vertical lines indicate error bars.
indicated that spraying two reagents in 'pre-heat' or 'post-heat' was relatively more effective in improving 1000-grain weight and grain yield compared to spraying in 'under-heat' (Table S1,  Table S2).

Discussion
Post-flowering heat significantly reduced grain size and final grain yield of the tested wheat cultivars of Huaimai 33 and Annong 0711 in this study. High temperature impairs assimilate supplies to developing wheat grains by accelerating leaf senescence. We found that both 24-epicastasterone and KH 2 PO 4 could protect grain-filling process of wheat cultivars from heat injury by improving leaf physiological functioning. Although, KH 2 PO 4 was a relatively more effective in improving growth and grain yield formation of heat-stressed wheat plants than 24-epicastasterone in this study. Positive effect of foliar application of KH 2 PO 4 on leaf transpiration, senescence, and recovery from heatinduced injury has already been reported in wheat (Lv et al. 2017). Heat stress significantly decreased the green flag leaf area compared to CK, leading to premature of wheat plants, SOD, POD and CAT activity decreased rapidly . Meanwhile, Heat stress induced early maturity by reducing grain filling duration compared to control in almost all the varieties except the shorter duration variety (Bergkamp et al. 2018). Our study showed that KH 2 PO 4 modulated antioxidant enzyme activities (SOD, POD, and CAT), which could possibly protect leaf membranes due to heat injury (as evident from MDA contents) and thus delayed senescence process ). This sustained photosynthesis and assimilates supply to developing grains under additional nutrient (P and K), so that grains could achieve their potential size (Lv et al. 2017). Cellular K concentration also significantly regulate leaf zeatin and abscisic acid (ABA) and ethylene, which are the key source-sink regulators in wheat (Lv et al. 2017). Sustained flag leaf Pn under K application may promote carbohydrate transfer from source to sink of the small-sink cultivar, promoting the increase of grain filling rate. Similar, positive effects of 24-epibrassinolide heat-stressed barley has been reported (Janeczko et al. 2010), in protecting the carbon assimilation process from oxidative damage by high temperature (Tanveer et al. 2018).
In this study, we found that pre-or post-heat KH 2 PO 4 and 24-epicastasterone applications were relatively more effective in protecting wheat cultivars from heat injury than their application during heat stress period. This low efficacy of regents during heat period suggests relatively poor ability of wheat plants to uptake and utilize plant growth regulators/nutrients when stressed. Pre-heat stressed applied plant growth regulators could promote plant growth and physiological functioning, thereby improving their ability to resist heat stress (Jing et al. 2020). When the plants are stressed, if further post-stress injury is arrested, some physiological indexes could return to normal functioning (Yang et al. 2011). This is how post-heat 24-epicastasterone and KH 2 PO 4 application may have improved this resilience.
Heat stress tolerance in wheat is a complex trait controlled by genotype and environment interactions. Achieving high yield of wheat under heat stress depends on the selection of varieties, and cultivation methods. Apart from choosing elite varieties, there are many cultivation ways including sowing time, irrigation and tillage system that can be used to mitigate the effects of heat (Wang et al. 2007;Tack et al. 2011;Dhyani et al. 2013). In China, chemical reagents were often used to prevent pests, abiotic stress, premature senescence, and promoting growth (Yang et al. 2016;Lv et al. 2017;Luo et al. 2018). During grain filling phase of wheat crops, additional supply of growth regulators/nutrients could provide a fast and simple solution to sustain grain development under 'CK1' denote the control (normal growth conditions outside the greenhouse), and 'HT' denote artificially simulated high temperature. 'pre-heat' denoted the spraying of growth regulator prior to the occurrence of high temperature; 'under-heat' denoted the spraying of growth regulator during the occurrence of high temperature; 'post-heat' denoted the spraying of growth regulator post the occurrence of high temperature. The data are means ± SE (n = 3). Lowercase letters refer to significant differences between the treatments (P < 0.05). Vertical lines indicate error bars.
high temperature. However, it is important to select and optimize their application protocols for ensuring sustainable economic gains. Our study suggested that KH 2 PO 4 is a cost-effective plant growth regulator compared with ABA, SA and 24-epicastasterone.

Conclusions
Terminal heat stress significantly reduces grain yield formation of wheat crops. We found that spraying either 24-epicastasterone or KH 2 PO 4 can help to alleviate high temperature impacts on grain-filling and final grain yield. These reagents could protect leaf chlorophyll, photosynthesis from heat injury by modulating protective enzyme and reducing lipid membrane peroxidation. Our study suggests that KH 2 PO 4 is relatively more effective compared to 24-Epicastasterone under high temperature due to better effect and the low cost. To maximize the efficacy, these reagents should be sprayed on wheat crops either prior to or after that heat stress event.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This study was funded by National Key Research and Development Program of China (2017YFD0300204-3)