Priming effect of exogenous ABA on heat stress tolerance in rice seedlings is associated with the upregulation of antioxidative defense capability and heat shock-related genes

Heat stress is a major restrictive factor that suppresses rice production. In this study, we investigated the potential priming effect of exogenous abscisic acid (ABA) on heat tolerance in rice seedlings. Seedlings were pretreated with 10 µM ABA by root-drenched for 24 h and then subjected to heat stress conditions of 40 °C day/35 °C night. ABA pretreatment significantly decreased leaf withering by 2.5–28.5% and chlorophyll loss by 12.8–35.1% induced by heat stress in rice seedlings. ABA pretreatment also mitigated cell injury, as shown by lower malondialdehyde content, relative electrolytic conductivity, and expression of cell death-related genes OsKOD1, OsCP1, and OsNAC4, while expression of OsBI1, a cell death-suppressor gene, was upregulated by ABA pretreatment. Moreover, ABA pretreatment improved antioxidant defense capacity, as shown by an obvious upregulation of ROS-scavenging genes and a decrease in ROS content (O2− and H2O2), and downregulation of the OsRbohs genes. Application of fluridone, an ABA biosynthesis inhibitor, increased membrane injury and the accumulation of ROS under heat stress. Exogenous antioxidants (proanthocyanidins) significantly alleviated leaf withering by decreasing ROS overaccumulation and membrane injury induced by heat stress. In addition, ABA pretreatment significantly superinduced the expression of ABA-responsive genes SalT and OsWsi18, the ABA biosynthesis genes OsNCED3 and OsNCED4, and the heat shock-related genes OsHSP23.7, OsHSP17.7, OsHSF7, and OsHsfA2a. Taken together, these results suggest that exogenous ABA has a potential priming effect for enhancing heat stress tolerance of rice seedlings mainly by improving antioxidant defense capacity and heat shock-related genes.


Introduction
Global warming has become a severe ecological and environmental problem due to the development of industry and population growth. Heat stress is caused by an extremely high temperature or by a lasting threshold of high-temperature weather, which has resulted from global warming (Quint et al. 2016;Xu et al. 2018). Heat stress results in a serious threat to crop production worldwide, and the yields of wheat, rice, and maize were decreased by 6.0%, 3.2%, and 7.4% Communicated by Hong-Xia Zhang.
* Xiaolong Liu lxl032202@163.com 1 due to 1 °C increase in the mean temperature, respectively (Zhao et al. 2017a;Janni et al. 2020). Rice (Oryza sativa L.) is a staple food crop for most of the world's population, while the main temperature during the growing season of rice occurs during the annual long hot summer season (Shi et al. 2015). Rice production during this time suffers from heat stress which directly inhibits the survival and transplanting of late rice, as well as the heading, flowering, and grain-filling of early season rice (Yang et al. 2020). Furthermore, heat stress results in significantly decrease in grain yield, total milled rice yield, head rice yield and total milling revenue with the increasing average growing temperature (Lyman et al. 2013). Thus, the demand for an expanding population under the current situation of global warming remains a huge challenge for food production.
The effect of heat stress on rice production occurs during the entire growth stage. The germination of rice seeds was suppressed when the growth temperatures was ≥ 35 °C, as shown by a significant decrease in the germination rate and growth in buds (Yang et al. 2021). The optimal temperature of rice seedlings ranges from 22 to 28 °C, while the growth of rice seedlings is severely inhibited under growth temperature of ≥ 35 °C (Soda et al. 2018). Heat stress results in withering, browning, abnormalities, and water loss of rice leaves, suppresses the growth of seedlings and roots and causes complete wilting of rice plants (Liu et al. 2016;Kilasi et al. 2018;. Heat stress suppresses photosynthetic efficiency by decreasing chlorophyll content, disturbing the combination of chlorophyll-protein complexes, and damaging the photosystem structure Fan et al. 2017). In addition, heat stress causes excess accumulation of reactive oxygen species (ROS), which results in severe damage to the membrane and cell death (Zhao et al. 2017b). Rice plants are more sensitive to heat stress at the reproductive and grain-filling stages. Heat stress results in the degradation of flowering florets, abortion of pollen, and devitalization of pollen viability of rice at the booting stage Zhang et al. 2018), and suppresses the grain-filling rate in rice at the grain-filling stage Wang et al. 2019). Thus, it is indeed necessary to explore an effective pathway for the improvement of heat tolerance in rice.
Plants generate and accumulate ROS such as superoxide anions (O 2 − ) and hydrogen peroxide (H 2 O 2 ) under various environmental stress conditions, such as drought, salinity, alkalinity, and extreme temperature (Choudhury et al. 2017). These ROS molecules play an important role in the regulation of development and adaptation to the environment (Mittler 2017). However, excess accumulation of ROS causes oxidative stress in plants, which results in severe damage to the plant cellular membrane, RNA, DNA, and proteins (Sewelam et al. 2016). We previously reported that overaccumulation of ROS in rice roots is a main limiting factor in cell damage and plant growth inhibition in rice seedlings under alkaline stress conditions . ROS accumulation is an important harmful pathway in the physiological effects of heat stress on plants . Heat stress causes a remarkable upregulation of respiratory burst oxidase homolog (RBOH) genes and an accumulation of ROS in rice seedlings, and excessive ROS levels disturb the balance of ROS production and scavenging, which further results in membrane lipid peroxidation, cell damage, changes in a series of antioxidant enzymes, and even plant death (Liu et al. 2019). Previous studies have shown that excessive accumulation of ROS induced by heat stress causes the decline of germination rate, pollen viability, spikelet fertility, and grain chalkiness in rice (Suriyasak et al. 2017;Zhao et al. 2017b;Yang et al. 2021), indicating that ROS level is involved in the regulation of yield formation of rice under heat stress condition and enhancing the antioxidative defense capacity promotes anther development and yield formation under heat stress conditions (Dwivedi et al. 2019). Therefore, studies on the improvement of rice tolerance to various stress factors by reducing oxidative stress induced by overaccumulation of ROS are still required for further insight and will provide a potentially useful pathway in rice field production in the future (Kerchev et al. 2015).
The phytohormone abscisic acid (ABA) plays an important role in the regulation of plant growth and adaptive to various stress factors (Dar et al. 2017;Lang et al. 2021). One important pathway of ABA action in stress tolerance is the priming effect on plants (Vishwakarma et al. 2017;Wei et al. 2017;Liu et al. 2019), which confer the potential enhanced ability to mount defense responses to impending stress factors (Aranega-Bou et al. 2014). Priming is progress that plants were pretreated with a range of chemical compounds before being treated in various external stresses and the state of chemically treated plants are referred to as "primed" (Beckers and Conrath 2007;Aranega-Bou et al. 2014). The priming effect of ABA has been demonstrated as shown by rice seeds or seedlings pretreated with ABA improved plants survival, growth, and grain yield under saline-alkaline stress conditions (Gurmani et al. 2011;Wei et al. 2015Wei et al. , 2017. Further analysis on the underlying mechanism of ABA priming showed that ABA priming potentiated to improve the downstream antioxidant defense capacity and stress tolerance-related gene expression for an increased adaptive response to alkaline stress (Liu et al. 2019).
ABA also functions in crops' response to heat stress and plays a vital role in crop production under climate warming conditions (Suzuki et al. 2016;Li et al. 2021). Application of exogenous ABA improves plant heat tolerance as shown by keeping water balance, regulating stomatal conductance, and the regulation of gene expression Li et al. 2014). Additionally, ABA also functions in the regulation of rice in the reproductive and grain-filling stages (Islam 1 3 et al. 2018;Li et al. 2021). The ROS signal pathway plays an important role in plant response to environmental stress and excess accumulation induced by various stress factors results in membrane injury, root damage, and even the death of plants Qiu et al. 2019). ABA confers to regulate ROS levels in plants' response to environmental stress factors Liu et al. 2019), as well as under high-temperature conditions (Hu et al. 2010). And the ABA-deficient mutants exhibited more sensitivity to heat stress (Larkindale et al. 2005). These studies demonstrate a strong correlation between ABA application and regulation of ROS levels in plants' response to stress conditions (Suzuki et al. 2016;Liu et al. 2019). Current studies on the relationship between ABA and heat stress in rice were mainly focused on the ABA-dependent signal pathway and spraying of exogenous ABA . Research of ABA priming effect on stress tolerance in rice was mainly validated in the response to salt or alkaline stress (Wei et al. 2015(Wei et al. , 2017Liu et al. 2019).
This study aimed to gain insights into the effect and mechanism of ABA priming on heat tolerance in rice by focusing on the effect of ABA priming on ROS-formation or ROS-scavenging pathway. We hypothesized that the priming effect of exogenous ABA on heat stress tolerance in rice seedlings is associated with improvement of antioxidative defense capacity and expression of heat stress-tolerant genes.

Plant material and growth conditions
Rice cultivar Huanghuazhan, an elite cultivar suitable to be spread in eastern China, was used in this study. It was bred by crossing 'Huangxinzhan' with 'Fenghuazhan' and was resistant to heat stress (China Rice Data Center). Rice seeds were surface-sterilized with 75% (v/v) alcohol for 5 min and rinsed with deionized water five times. After that, seeds were immersed in water for 2 days and then were sprinkled onto a petri dish with wet filter paper for pre germination in an incubator under dark conditions at 28 °C for 24 h. Eighteen uniformly germinated seeds were transplanted onto a multiwell plate floating on a 320-mL cup containing deionized water for 7 days and then grown in half-strength Kimura B nutrient solution (Miyake and Takahashi 1983) for another 7 days. All rice seedlings were grown in a controlled growth chamber under the following conditions: 28 °C day/22 °C night, 12 h photoperiod, and 350 mmol photons m − 2 s − 1 light intensity.

ABA pretreatment and heat stress treatment
ABA (Sigma, Inc., St, Louis, MO, USA) was dissolved in a small amount of absolute ethanol and then diluted with deionized water to the desired concentrations (Wei et al. 2015;Liu et al. 2019). Rice seedlings at the approximately three-leaf stage were pretreated with 10 µM ABA or deionized water by root-drench for 24 h, respectively. Then these two sets of rice seedlings were transferred to the control or heat stress conditions after being rinsed with deionized water, respectively. Thus, four treatments were set: root-drench with deionized water in unstressed condition (CK); root-drench with 10 µM ABA in unstressed condition (ABA); root-drench with deionized water in heat stress (HS), root-drench with 10 µM ABA in heat stress condition (ABA + HS). We used growth temperature of 40 °C to simulate heat stress. The temperature of unstressed condition was set as 28 °C day/22 °C night, and the temperature of heat stress was set as 40 °C day/35 °C night. This growth temperature was set according to the description of rice response to heat stress in South China by Shi et al. (2015) and Huang et al. (2017).

Treatment of rice seedlings with exogenous Fluridone and Proanthocyanidins (PC)
In this study, exogenous fluridone and PC was used to examine the mechanism of the ABA priming effect. Fluridone is an ABA biosynthesis inhibitor and inhibited rice seedlings' growth under alkaline stress (Wei et al. 2015). Proanthocyanidin is an antioxidant that effectively scavenged superoxide anion radicals and hydroxyl radicals, and alleviated alkalinity-induced suppression of rice seedling growth by inhibiting ROS overaccumulation (Rue et al. 2017;Zhang et al. 2017). Two-week-old rice seedlings were pretreated in the solution with deionized water, 10 µM fluridone, and 1% PC, by root-drench for 24 h, respectively; and then transferred to control or heat stress conditions aforementioned. The treatments were set as follows: CK, Fluridone, PC, HS, Fluridone + HS, PC + HS, respectively.

Measurement of seedling growth
Photograph of the growth condition of rice seedlings was taken at the indicated treatment hours. The withered leaf rate was investigated at 48, 72, and 96 h of heat stress, respectively. The withered leaf rate was recorded as 1 if the whole leaf was dry and brown, while it was recorded as 0.5 if half of the leaf was dry and brown, respectively (Liu et al. 2020). The withered leaf rate of each treatment was represented by the proportion of withered leaves of whole leaves in one cup.

Measurement of chlorophyll content
The chlorophyll content was measured according to the theory as described by Wellburn and Lichtenthaler (1984), with some modifications as described by Liu et al. (2019). Leaf samples (0.1 g) were extracted using a 10 mL mixture of ethanol (5 mL) and acetone (5 mL) under dark conditions. The absorbance of the supernatant was determined at 645 and 663 nm using a spectrophotometer (UV-2700, Shimadzu, Kyoto, Japan) until the whole leaves whitened. The total chlorophyll content unit fresh weight was calculated using the following formula: (20.29×A 645 + 8.05×A 663 ) V/ (1000×W).

Measurement of malondialdehyde (MDA) content and relative electrolytic conductivity (REC)
The MDA content was determined by the thiobarbituric acid reaction as described by Heath and Packer (1968). Leaf samples were homogenized in 1 mL of 50 mM phosphate buffer (pH 7.8) after being smashed at the refrigerated condition with liquid nitrogen and centrifuged at 12,000×g for 15 min. Subsequently, 400 µL of supernatant was mixed with 1 mL of 0.5% thiobarbituric acid, and the mixture was boiled for 20 min. The absorbance of the resulting supernatant after cooled and centrifuged was measured at 532, 600, and 450 nm using a spectrophotometer (UV-2700, Shimadzu, Kyoto, Japan). The MDA concentration was calculated using the following formula: 6.45× (A 532 − A 600 ) − 0.56 × A 450 . Finally, the MDA content in the leaf was calculated according to the fresh weight of the leaf of each treatment.
The relative electrolytic conductivity (REC) was an important index for evaluating the membrane injury (Tantau and Dörffling 1991;Wei et al. 2015). Relative electrolytic conductivity was represented by the electrolytic conductivity of the effusion with leaf in it before and after boiling (Wei et al. 2015). Rice leaves (2 g fresh weight) were randomly selected from each treatment group, washed with deionized water to remove surface-adhered electrolytes. Leaf samples were submerged in 15 mL of deionized water in 50 mL conical tubes and kept at room temperature for 1 h. The electrical conduction of the effusion was then measured with a DDS-12 conductivity meter (Lida Inc., Shanghai, China) and recorded as R1. The tissue samples were killed by heating tubes in a boiling bath for 40 min, cooled to room temperature, and the electrical conduction of the effusion was measured again which was recorded as R2. The REC was evaluated using the formula REC (%) = R1/R2 × 100%.

Measurement of ROS levels
The O 2 − contents were measured as described by Elstner and Heupel (1976) by monitoring nitrite formation from hydroxylamine in the presence of O 2 − , with some modifications as described by Jiang and Zhang (2001). For the determination of O 2 − contents, the fresh leaves (0.1 g) were loaded in a 2 mL tube and frozen in liquid nitrogen, then homogenized with 1 mL of 50 mM potassium phosphate buffer (pH 7.8) and centrifuged at 10,000×g for 10 min at low temperatute of 4 °C, and then collected the supernatant. The incubation mixture contained 0.9 mL of 50 mM phosphate buffer (pH 7.8), 0.4 mL of 10 mM hydroxylamine hydrochloride, and 1 ml of the supernatant were mixed for incubation at room temperature for 20 min. Then, 0.3 mL of 17 mM sulfanilamide and 0.3 mL of 7 mM α-naphthylamine were added to the incubation mixture. After reaction at room temperature for 20 min, ethyl ether with the same volume was added and centrifuged at 8000×g for 5 min. The absorbance values in the aqueous solution were read at 530 nm to calculate the contents of O 2 − from the chemical reaction of O 2 − and hydroxylamine. The H 2 O 2 contents were measured as described by monitoring the A 415 of the titanium-peroxide complex (Brennan and Frenkel 1977). For the determination of H 2 O 2 contents, the fresh leaves (0.1 g) were loaded in a 2 mL tube and frozen in liquid nitrogen, then homogenized with 1 mL of acetone and centrifuged at 8000×g for 10 min at low temperature of 4 °C, and collected the supernatant. The incubation mixture contained 1 mL of the supernatant, 0.1 mL of titanium sulfate, and 0.2 mL of stronger ammonia water and then was centrifuged at 4000×g for 10 min at room temperature. The precipitate was solubilized in 1 mL of 2 mol/L H 2 SO 4 and then reacted at room temperature for 5 min. The absorbance values in the aqueous solution were read at 415 nm to calculate the contents of H 2 O 2 . The analytical reagent used to measure the H 2 O 2 and O 2 − contents were acquired from the determination kit, according to the manufacturer's instructions (Comin Biotechnology Co., Ltd. Suzhou, China) Liu et al. 2019).
The housekeeping gene β-actin (GenBank ID: X15865.1) was used as an internal standard. PCR was conducted in a 20 µL reaction mixture containing 1.6 µL of cDNA template (50 ng), 0.4 µL of 10 mM specific forward primer, 0.4 µL of 10 mM specific reverse primer, 10 µL of 2× SYBR® Premix Ex Taq™ (TaKaRa, Bio Inc.), and 7.6 µL of double-distilled H 2 O in a PCR machine (qTOWER2.2. Analytic Jena. GER). The procedure was performed as follows: 1 cycle for 30 s at 95 °C, 40 cycles for 5 s at 95 °C, and 20 s at 60 °C, and 1 cycle for 60 s at 95 °C, 30 s at 55 °C, and 30 s at 95 °C for melting curve analysis. The level of relative expression was computed using the 2 −△△CT method (Livak and Schmittgen 2001).

Experimental design and statistical analyses
All of the experiments were conducted in a controlled growth chamber with five biological replicates, each consisting of 3 cups of rice seedlings, with eighteen seedlings each cup. Statistical analyses were performed using the statistical software SPSS 21.0 (IBM Corp., Armonk, NY). Based on a one-way analysis of variance (ANOVA), Duncan's multiple range test (DMRT) was used to compare differences in the means among treatments. The significance level was P < 0.05.

ABA pretreatment rescued rice seedlings from wilting and death under heat stress
There was no significant difference in leaf withering with or without ABA pretreatment at 24 and 48 h of heat stress (Fig. 1A, B). While pretreatment with exogenous ABA significantly rescued rice seedlings from wilting and death as shown by the lower withered leaf rates of seedlings pretreated with ABA at 72 and 96 h of heat stress (Fig. 1C-E), the withered leaf rate of rice seedlings was decreased by 28.5% and 15.8% by ABA pretreated under heat stress condition (Fig. 1F). And this mitigative effect was sustained to 108 h of heat stress (Fig. S1). Almost the whole leaves were withered and there was no significant difference with or without ABA application, after 120 h of heat stress (Fig.  S1). Rice seedlings pretreatment with ABA significantly increased chlorophyll content by 11.3%, 25.9%, and 13.8%, compared with without ABA pretreatment under heat stress conditions (Fig. 1G).

ABA pretreatment mitigated membrane injury induced by heat stress
Exogenous ABA pretreatment significantly mitigated cell injury as shown by a lower accumulation of MDA and REC (Figs. 2A, B and S2). Compared to HS treatment, MDA content was decreased by 22.4%, 22.1%, and 10.8%, and REC was decreased by 14.1%, 13.2%, and 7.9%, at 48, 72, and 96 h of heat stress, respectively ( Fig. 2A, B). In addition, a cell death suppressor, OsBI1, was significantly downregulated and the cell death-related genes, OsKOD1, OsCP1, OsNAC4, were significantly upregulated by ABA pretreated under heat stress conditions (Fig. 2C-F). The relative expression level was increased by 37.1%, 47.2%, and 50.2% with ABA pretreatment at 48, 72, and 96 h of heat stress condition, respectively (Fig. 2C).

ABA pretreatment decreased ROS accumulation and improved ROS-savaging capacity under heat stress
Pretreatment with exogenous ABA significantly inhibited ROS accumulation as shown by lower O 2 − and H 2 O 2 content in rice leaves under heat stress conditions (Fig. 3). Compared to HS treatment, the content of O 2 − was decreased by 5.9%, 19.6%, and 22.2% (Fig. 3A), and content of H 2 O 2 was decreased by 8.3%, 16.5%, and 16.1% with ABA pretreatment at 48, 72 and 96 h, respectively (Fig. 3B).
ABA pretreatment also suppressed the transcriptional expression of OsRboh genes. As shown in Fig. 4, an obvious upregulation was shown in the OsRbohs family genes by heat stress, and the relative expression level of OsRboh1, OsRboh4, OsRboh5, OsRboh6, and OsRboh7 was reached to a higher level. Among these OsRbohs family genes, OsR-boh2, OsRboh3, OsRboh5, and OsRboh7 were significantly suppressed by ABA pretreatment (Fig. 4).
We further analyzed the relative expression levels of 20 ROS-scavenging genes, as shown in Fig. 5, almost all ROSscavenging genes were upregulated by heat stress. Furthermore, ABA pretreatment significantly super-upregulated the expression level of 16 ROS-scavenging genes except for R5, R7, R13, and R20 (Fig. 5).

Exogenous ABA biosynthesis inhibitor (Fluridone) suppressed rice seedlings' growth under heat stress
As shown in Fig. 6A, the growth condition of rice seedlings with the application of fluridone (treatment of Fluridone + HS), an ABA biosynthesis inhibitor, was similar to the HS treatment. The withered leaf rate and chlorophyll content were not statistically significant between Fluridone + HS and HS treatment (Fig. 6B, C), as well as the accumulation of O 2 − and H 2 O 2 (Fig. 6F, G). Accumulation of MDA and MI with the application of fluridone was significantly higher than that of HS treatment at 48, 72 h of heat stress condition, respectively (Fig. 6D, E).

Application of exogenous antioxidant (Proanthocyanidins, PC) rescued rice seedlings from leaf withering induced by heat stress
In this study, application of exogenous PC significantly rescued rice seedlings from leaf withering as shown by a decrease of 47.8%, 44.7%, and 33.5% of leaf withered rate at 48, 72, and 96 h, compared to HS treatment (Fig. 6A,  B). Content of chlorophyll was increased by 13.6%, 31.3%, and 34.8% with the application of PC under heat stress conditions, compared to HS treatment (Fig. 6C). In addition, membrane injury was significantly mitigated by PC as shown by a lower accumulation of MDA content and MI in rice seedlings of PC + HS treatment (Fig. 6D, E). Consistently, the accumulation of O 2 − and H 2 O 2 was decreased by 25.3-41.1% and 39.8-45.6% with the application of PC, compared to HS treatment (Fig. 6F, G). Values are means ± SD, n = 5. Different letters on the column represent significant differences (P < 0.05) between different treatments based on Duncan's test Two-week-old rice seedlings were root-drenched with or without 10 µM ABA for 24 h, and then subjected to unstress or heat stress conditions. Malondialdehyde (MDA) content (A) and relative electrolytic conductivity (REC) (B) of rice seedlings were measured at the indicated treatment hours. Values are means ± SD, n = 5. Expression levels of cell death-related genes, OsBI1 (C), OsKOD1 (D), OsCP1 (E), and OsNAC4 (F) were measured at the indicated treatment hours. A quantitative real-time polymerase chain reaction was performed using OsACT1 as an internal standard. The expression levels of unpretreated control (CK) were set as the unit to calculate the expression levels, shown as fold changes relative to the CK. Values are means ± SD, n = 3. Different letters on the column represent significant differences (P < 0.05) between different treatments based on Duncan's test

ABA pretreatment upregulated stress tolerance-related genes under heat stress
ABA signaling pathway was exactly activated by heat stress and ABA pretreatment as shown by an upregulation of two ABA-responsive genes, Salt and OsWsi18 (Fig. 7A,  B). While the expression levels of Salt and OsWsi18 were significantly superinduced by 34.2-47.8% and 25.9-26.9% with ABA pretreatment, compared to HS treatment (Fig. 7A,  B). The expression levels of two ABA biosynthesis genes, OsNCED3 and OsNCED4, were increased by 40.8-71.3% and 32.5-54.0% by ABA pretreatment, compared to HS treatment (Fig. 7C, D).
To gain further insights into the mechanism of ABA pretreatment for heat stress, two heat shock protein (HSP) genes, OsHSP23.7 and OsHSP17.7, and two heat shock transcription factors (HSF), OsHSF7 and OsHsfA2a, were analyzed in this study. All these stress tolerance-related genes were significantly upregulated by ABA, HS, and ABA + HS treatment, while the relative expression levels of these four genes were significantly super-upregulated by ABA pretreatment under heat stress conditions (Fig. 7E-H). The gene relative expression levels of OsHSP23.7, OsHSP17.7, OsHSF7 and OsHsfA2a was increased by 28.4-36.9%, 32.9-49.7%, 31.0-42.6% and 33.3-50.5% with ABA pretreatment under heat stress condition, respectively (Fig. 7E-H). Two-week-old rice seedlings were root-drenched with or without 10 µM ABA for 24 h, and then subjected to unstress or heat stress conditions. Expression levels of ROS generation-related genes, OsRboh1 OsRboh8 (H), and OsRboh9 (I) were measured at the indicated treatment hours. A quantitative real-time poly-merase chain reaction was performed using OsACT1 as an internal standard. The expression levels of unpretreated control (CK) were set as the unit to calculate the expression levels, shown as fold changes relative to the CK. Values are means ± SD, n = 3. Different letters on the column represent significant differences (P < 0.05) between different treatments based on Duncan's test

Discussion
Heat stress is characterized by an extreme or lasting hightemperature climate for a long time, which has become an enormous meteorological disaster for crop production . Heat stress results in severe inhibition in crop growth and yield formation as shown by increasing leaves withering and death (Wei et al. 2012;Kilasi et al. 2018;Liu et al. 2018), damaging cell membrane and photosynthetic structure (Essemine et al. 2017;Soda et al. 2018), impairing pollen swelling (Das et al. 2014;Wang et al. 2019), reducing spikelets Zhang et al. 2016Zhang et al. , 2018 and ABA priming increased ROS-scavenging capacity under heat stress. Two-week-old rice seedlings were root-drenched with or without 10 µM ABA for 24 h, and then subjected to unstress or heat stress conditions. Expression levels of 20 ROS-scavenging related genes (R1-R20) were measured at the indicated treatment hours. A quantitative real-time polymerase chain reaction was per-formed using OsACT1 as an internal standard. The expression levels of unpretreated control (CK) were set as the unit to calculate the expression levels, shown as fold changes relative to the CK. Values are means ± SD, n = 3. Different letters on the column represent significant differences (P < 0.05) between different treatments based on Duncan's test the grain filling (Chen et al. 2017;Suriyasak et al. 2017). Recently, it was shown that the application of exogenous phytohormones alleviated heat-induced damage in plants and enhanced plant heat tolerance . ABA plays an important role in crops' response to environmental stress. Previous studies have reported the priming effect of exogenous ABA on tolerance to alkaline stress in rice seedlings (Wei et al. 2015(Wei et al. , 2017Liu et al. 2019). And we previously showed rice seeds soaked with exogenous ABA significantly improved seed growth under lasting heat stress conditions (Yang et al. 2021). In the present study, rice seedlings pretreated with exogenous ABA significantly mitigated the heat-induced leaf withering (Fig. 1), membrane injury (Fig. 2), and overaccumulation of ROS (Figs. 3 and 4), and improved ROS-scavenging capability (Fig. 5). In addition, there was some evidence that showed that application of the ABA biosynthesis inhibitor, fluridone, compromised tolerance to heat stress in rice seedlings (Fig. 6), while application of the antioxidant, PC, improved tolerance of the seedlings to heat stress (Fig. 6). Pretreatment with ABA also upregulated gene expression levels related to ABA signal and heat shock and transcription factor (Fig. 7). These data collectively suggest that pretreatment with exogenous ABA enhanced heat tolerance in rice seedlings mainly by improving ROS-scavenging capability and upregulating heat shockrelated genes (Fig. 8).
ABA is an important "stress phytohormone" in plants, which has been evidenced by the action in various stress conditions such as drought, salt, alkali, cold, and high temperature (Dar et al. 2017;Vishwakarma et al. 2017). Exogenous ABA plays a vital in the improvement of stress tolerance by multiple methods, such as foliage spray, adding into solution, or seed soaking (Gurmani et al. 2011;). An important mechanism of ABA for enhancing stress tolerance in plants is the priming effect, which helps plants to acquire a potential capacity to enhance defense response to subsequent stress factors (Aranega-Bou et al. 2014;Wei et al. 2017). This priming effect has recently been validated in rice response to salt or alkali stress as shown by seed presoaking or root drenching with exogenous ABA Fig. 6 Effect of exogenous ABA biosynthesis inhibitor (Fluridone) and antioxidant (Proanthocyanidins, PC) on rice seedlings' growth under heat stress conditions. Two-week-old rice seedlings were rootdrenched with or without 10 µM fluridone or 1% proanthocyanidins for 24 h, and then subjected to unstress or heat stress conditions. Photographs of seedling growth (A) were taken at 48 h, 72 h, and 96 h, respectively. Withered leaf rate (B), chlorophyll content (C), REC (D), MDA content (E), O 2 − content (F), and H 2 O 2 content (G) of rice seedlings were counted at 48, 72, and 96 h, respectively. Values are means ± SD, n = 5. Different letters on the column represent significant differences (P < 0.05) between different treatments based on Duncan's test significantly improved the survival rate, plant growth, and grain yield of rice (Gurmani et al. 2011;Wei et al. 2015Wei et al. , 2017. Application of exogenous ABA plays an active effect in plants' response to heat stress (Islam et al. 2018). However, few studies have reported on the priming effect of ABA in the heat stress responses. We previously reported that ABA primes rice seeds for enhanced heat stress tolerance as shown by ABA presoaking improved ROS-scavenging capacity, inhibiting ROS overaccumulation and mitigating membrane injury (Yang et al. 2021). Results of the present study showed that the ABA-responsive genes, Salt and OsWsi18 (Fig. 7A, B), and ABA-biosynthesis genes, OsNCED3 and OsNECD4 (Fig. 7C, D), were significantly upregulated by heat stress, and application of the ABA inhibitor suppressed rice seedlings growth (Fig. 6), which indicated that the ABA signal indeed participated in the Fig. 7 ABA priming upregulated transcriptional expression of stress tolerance-related genes under heat stress. Two-week-old rice seedlings were rootdrenched with or without 10 µM ABA for 24 h, and then subjected to unstress or heat stress conditions. Expression levels of ABA-responsive genes, Salt (A) and OsWsi18 (B), ABA biosynthesis genes OsNCED3 (C) and OsNCED4 (D), and stress tolerance-related genes OsHSP23.7 (E), OsHSP17.7 (F), OsHSF7 (G), and OsHs-fA2a (H) were measured at 72 h. A quantitative real-time polymerase chain reaction was performed using OsACT1 as an internal standard. The expression levels of unpretreated control (CK) were set as the unit to calculate the expression levels, shown as fold changes relative to the CK. Values are means ± SD, n = 3. Different letters on the column represent significant differences (P < 0.05) between different treatments based on Duncan's test 1 3 response to heat stress in rice seedlings. However, rice seedlings were withered or eventually dead induced by heat stress for more than 4 days (Fig. 1), indicating that these activation levels of ABA-induced by heat stress may not be sufficient to effectively cope with the heat stress factor. Nevertheless, ABA pretreatment upregulated the expression of ABA-responsive and ABA-biosynthesis genes to a higher degree (Fig. 7), as well as a great increase of ROS-scavenging genes (Fig. 5), heat shock-related genes (Fig. 7), and a remarkable decrease of ROS accumulation (Fig. 3) and cell death in rice seedlings under heat stress condition (Fig. 2). These results suggest that exogenous ABA enhances tolerance to heat stress in rice seeds or seedlings by the priming effect which potentiates multiple downstream pathways response to heat stress.
ROS plays a vital role in the regulation of plants' response to various stress factors (Choudhury et al. 2017;Mittler 2017). ROS serves as the signaling messenger in a series of physiological processes required for the growth regulation and stress response at low levels (Sewelam et al. 2016;Mittler 2017). However, environmental stress induces overaccumulation of ROS in cells, which results in oxidative stress and even cell death in plants (Choudhury et al. 2017;Zhang et al. 2017). In rice, excessive accumulation of ROS has been identified as a key causal factor in the inhibition of seed germination and seedlings growth under various stress conditions due to the result of oxidative stress, especially for the severe cellular damage to roots (Guan et al. 2017;Zhang et al. 2017). Heat stress caused multiple physiological effects to rice including membrane and photosynthesis damage, disturbance of ROS accumulation, and carbohydrate . We previously showed that increasing intracellular ROS levels was the primary for the inhabitation of seed germination and bud growth under lasting heat stress conditions (Yang et al. 2021). In the present study, heat stress caused a remarkable increase of ROS in rice seedlings as shown by a gradually rising accumulation of O 2 − and H 2 O 2 in leaves at the indicated time (Fig. 3A, B), as well as the upregulation of a series of OsRboh genes (Fig. 4). Meanwhile, rice seedlings presented a significant membrane injury as shown by the increase of MDA and REC ( Fig. 2A, B), as well as several cell death-related genes, OsKOD1, OsCP1, and OsNAC4, (Fig. 2D-F) under heat stress condition. In addition, several ROS-scavenging genes were significantly upregulated by heat stress (Fig. 5). These results indicated that the ROS signal pathway was activated in the response to heat stress in rice seedlings; however, the ROS levels were too high in turn led to severe injury to the cell membrane, and finally resulted in withering and even death of rice seedlings (Fig. 1). Application of exogenous antioxidant, PC, significantly rescued rice seedlings from death by decreasing the ROS content and membrane injury (Fig. 6), indicating that overaccumulation of ROS is an important mechanism for inhibiting rice seedlings induced by heat stress. Nevertheless, rice seedlings pretreatment with ABA significantly improved antioxidative defense capacity as shown by a series of ROS-scavenging genes (Fig. 5), and decreased the ROS accumulation (Fig. 3A, B) and membrane injury (Fig. 2), which achieved a similar effect with the PC. On the contrary, the application of fluridone was ineffective to decrease the ROS accumulation and membrane injury (Fig. 6). These data demonstrate that ABA primes for enhanced heat tolerance in rice seedlings mainly by improving the ROS-scavenging capacity (Fig. 8), which is accordant to our previous study in alkaline stress (Liu et al. 2019).
The ROS levels in plants are codetermined by the ROS formation which is mainly regulated by the RBOH genes, and the scavenging pathway that is constituted by antioxidant enzymes (Choudhury et al. 2017). The ROS formation may be induced by various stress factors, as well as ABA, while ROS levels would affect the ABA biosynthesis and catabolism (Ishibashi et al. 2015;Suriyasak et al. 2017). Thus, the "cross-effect" of ROS and ABA levels play a vital role in plants' response to environmental stress conditions (Ye et al. 2011;Liu et al. 2019;Zhao et al. 2021). In this study, almost all these OsRboh genes were upregulated Fig. 8 A schematic mechanism of ABA in response to heat stress in rice seedlings. Heat stress-induced overaccumulation of ROS in leaves caused severe membrane injury in rice seedlings and even the plant death. Exogenous ABA application super-induced the ABA signal in rice, to improve the antioxidant defense capability to inhibit ROS overaccumulation and upregulate the expression of heat shockrelated genes for enhanced tolerance to heat stress in rice during the heat stress process, indicating that heat stress resulted in the accumulation of ROS by inducing the transcriptional expression of OsRboh genes in rice seedlings. Among these OsRboh genes, OsRboh1, OsRboh4, OsR-boh6, OsRboh8, and OsRboh9 were induced by ABA priming and heat stress, which indicated that ABA-induced the expression of OsRboh genes to increasing ROS levels in the regulation of plant growth and response to stress factors . Nevertheless, OsRboh2, OsRboh3, OsRboh5, and OsRboh7 was suppressed by ABA pretreatment under heat stress (Fig. 4), which may demonstrate another potential mechanism in the "cross-effect" of ROS and ABA in plants response to environmental stress, that was ABA priming may inhibit the expression of OsRboh2, OsRboh3, OsRboh5, and OsRboh7 to decrease ROS formation under heat stress condition (Fig. 8). In further studies, it would be interesting to gain further insights into the correlation between ROS formation and ABA levels by using the mutants or transgenic plants in ROS or ABA pathways.
Reprograming the gene expression is an important pathway for plants to cope with multiply environmental stress conditions. Recently, several genes have been identified for enhancing heat tolerance in plants (Hoang et al. 2019;Su et al. 2019). Heat shock proteins and heat shock transcription factors are known as the vital defense mechanism for plants or animals to resist heat stress conditions and numerous studies have demonstrated that overexpression of the HSP or HSF genes contributed to improving stress tolerance in rice (Cheng et al. 2015;Liu et al. 2015). ABA has a potential regulation effect in the genetic network in plants' response to the stresses (Liu et al. 2019). In this study, the ABA-responsive genes and ABA biosynthesis genes were superinduced by ABA pretreatment under heat stress ( Fig. 7A-D), indicating the ABA signal pathway was indeed activated by ABA priming. In addition, two HSP genes, OsHSP23.7 and OsHSP17.7; and two HSF genes, OsHSF7 and OsHsfA2a were significantly upregulated by ABA-priming under heat stress ( Fig. 7E-H). These data represented another important mechanism of the ABA-priming effect, indicating that ABA was involved in the multiple gene transcriptional regulatory network in plants under heat stress conditions. As shown in Fig. 8, heat stress-induced the expression of OsRboh genes in rice seedlings, which caused overaccumulation of ROS in leaves and further resulted in severe membrane injury as shown by higher cell and plant death of rice seedlings. Exogenous ABA application super-induced the ABA signal, to inhibit expression of OsRboh2, OsR-boh3, OsRboh5, and OsRboh7 and upregulate the antioxidant defense capability to decrease ROS accumulation in leaves for enhanced tolerance to heat stress, which achieved the same effect with antioxidant (PC). In addition, ABA application upregulated the expression of HSP and HSFrelated genes for activating the activity of heat shock protein.
In summary, in this study, ABA priming super-increased the ABA signal in rice seedlings under heat stress, to greatly upregulate ROS-scavenging capability and expression of heat shock-related genes, for increasing adaptive response to heat stress.