Brassinosteroids Mediate Endogenous Phytohormone Metabolism to Alleviate High Temperature Injury at Panicle Initiation Stage in Rice

Chen Yanhua, Wang Yaliang, Chen Huizhe, Xiang Jing, Zhang Yikai, Wang Zhigang, Zhu Defeng, Zhang Yuping

Research Paper

Brassinosteroids Mediate Endogenous Phytohormone Metabolism to Alleviate High Temperature Injury at Panicle Initiation Stage in Rice

Chen Yanhua#, Wang Yaliang#, Chen Huizhe, Xiang Jing, Zhang Yikai, Wang Zhigang, Zhu Defeng, Zhang Yuping

(National Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China; These authors contributed equally to this work)

High temperatures cause physiological and biochemical changes and significantly affect young panicle development of rice (L.). Brassinosteroids play important roles in enhancing crop stress resistance. In this study, we subjected rice cultivars Huanghuazhan (heat-resistant) and IR36 (heat-sensitive) to high temperature (HT, 40 ºC) or normal temperature (NT, 33 ºC) for 7 d at the panicle initiation stage, in conjunction with application of 24-epibrassinolide [EBR, a synthetic brassinolide (BR)] or brassinazole (BRZ, a BR biosynthesis inhibitor) at the beginning of the treatments. HT exacerbated spikelet degeneration and inhibited young panicle growth, which were partially prevented by EBR application, while BRZ application aggravated the reduction in spikelet number. HT decreased the contents of BR, active cytokinins (aCTK), active gibberellins (aGA) and indole-3-acetic acid (IAA), but increased the content of abscisic acid (ABA) in young panicles. The activities of key enzymes involved in sucrose hydrolysis, glycolysis and the tricarboxylic acid cycle in young panicles were decreased with the change of endogenous hormone levels under HT. In addition, the contents of H2O2and malondialdehyde (MDA) were increased and the activities of antioxidant enzymes were decreased in young panicles. Exogenous application of EBR induced the expression of phytohormone biosynthesis-related genes and down-regulated the expression of phytohormone catabolism-related genes to increase the contents of endogenous BR, aCTK, aGA and ABA, thus promoting the decomposition and utilization of sucrose in young panicles, enhancing the activities of superoxide dismutase, catalase and peroxidase, and reducing the accumulation of H2O2and MDA in young panicles, whereas application of BRZ had the opposite physiological effects. These results showed that brassinosteroids mediate endogenous phytohormone metabolism to alleviate HT injury at the panicle initiation stage in rice.

rice; high temperature; panicle initiation stage; phytohormone metabolism; physiological and biochemical indices

Rice (L.) provides dietary calories for over half of the world’s population. However, each increase in global mean temperature of 1 ºC is predicted to reduce global rice yields by 3.2% on average with current conditions and cultivation practices (Zhao et al, 2017). The panicle initiation and anthesis stages are the most sensitive stages of rice to high temperature (Arshad et al, 2017; Xu et al, 2021). The maximum temperature suitable for rice panicle initiation is 33.1 ºC ± 1.7 ºC (Sánchez et al, 2014), and above which inhibits the development of young panicles. High temperature reduces spikelet differentiation and aggravates spikelet degeneration, thus exacerbating the reduction in spikelet number per panicle (Wang Y L et al, 2020). In addition, high temperature lowers spikelet fertility rate and grain weight (Wu et al, 2016; Chen et al, 2021).

Phytohormones are a kind of organic compounds produced by plant cells and induced by specific environmental signals, which regulate plant physiological responses at a low concentration. In the process of plant growth and development (cell division and elongation, tissue and organ differentiation, flowering and fruiting, maturation and senescence, and dormancy and germination), any physiological responses is not the single action of a phytohormone, but the result of the interaction of multiple phytohormones (Wang, 2000; Weiss and Ori, 2007). The high temperature response of phytohormones is closely related to high temperature resistance in rice, and the levels of endogenous phytohormones in rice change significantlyunder high temperature stress to coordinate the response of yield traits to high temperature stress (Wu et al, 2019).

Endogenous hormones jointly regulate young panicle development and spikelet formation, which directly or indirectly affect rice panicle length and yield (Wu et al, 2016). Young panicle development depends on the oxidative metabolism of sugars produced by photo- synthesis to supply energy. A low cellular content for non-structural carbohydrates at the meiotic stage causes inferior spikelets to degenerate (Skazhennik et al, 2015). Li et al (2020) found that higher energy supply enhances plant tolerance to high temperature. Sucrose synthesized in leaves provides the necessary energy for young panicle development through glycolysis and the tricarboxylic acid (TCA) cycle, but high temperaturerepresses the activities of sucrose-metabolizing enzymes (Kato et al, 2019; Wang W T et al, 2021). However, all of these physiological effects are regulated by endogenous hormones. Cytokinin (CTK), auxin and gibberellin (GA) act as central regulators that modulate the development of young panicles and spikelet formation in rice (Patel and Mohapatra, 1992; Ashikari et al, 2005; Zhang et al, 2021). Wu et al (2016) reported that the active CTKs, GA1and indole-3-acetic acid (IAA) concentrations in rice panicles are decreased by high temperature stress, but are increased by bound CTKs, resulting in the inhibition of panicle development. Abscisic acid (ABA) can improve the stress resistance of plants, which is called stress hormone. High temperature induces the expression of ABA biosynthesis-related genes and represses ABA catabolism-related genes to increase ABA content (Toh et al, 2008). High levels of endogenous ABA are often associated with heat stress tolerance, which can be mimicked using exogenous ABA treatment to alleviate the adverse effects of heat stress (Shinohara and Leskovar, 2014). In rice, high temperature also raises ABA content (Wu et al, 2016). Brassinosteroids are known as the sixth phytohormone, and brassinolide (BR) is one of the most active brassinosteroids, which plays an important role in promoting crop growth, increasing yield and enhancing crop stress resistance (Altmann, 1998; Wang, 2000). However, high temperature promotes BR decomposition to reduce endogenous brassinosteroid content (Chen et al, 2021).

Brassinosteroids play an important role in various stress responses in rice, such as heat stress (Cao and Zhao, 2008; Chen et al, 2019; Pantoja-Benavides et al, 2021), low temperature stress (Wang S Q et al, 2020), drought stress (Zhang et al, 2019), salt stress (Anuradha and Rao, 2003) and alkali stress (Sharma et al, 2019). Application of BR significantly increases the contents of chlorophyll and protein, and enhances the activities of peroxidase (POD) and superoxide dismutase (SOD), and reduces the content of malondialdehyde (MDA) in the leaves of heat-sensitive rice Xieqingzao B under high temperature (Cao and Zhao, 2008). Our previous research showed that high temperature blocks sucrose transport from leaves to young panicles, and inhibits the activities of crucial enzymes involved in sucrose hydrolysis, glycolysis and the TCA cycle to reduce sugar utilization, whereas exogenous 24-epibrassinolide (EBR) application promotes sucrose transport to young panicles and improves sucrose utilization under high temperature (Chen et al, 2021). Zhao et al (2016) reported that brassinosteroid of certain concentration can increase root CTK and GA contents through regulation of growth-related enzymes to improve plant development. BR enhances the stress resistance of rice by improving many physiological metabolisms. Although many studies have explored the relationship between phytohormones and rice yield as well as yield components, little is known about the relationship between phytohormone contents and various physio- logical metabolisms. Furthermore, it is unclear whether BR has an effect on the biosynthesis and metabolism of other phytohormones, and whether BR alleviates the damage of high temperature to the development of young panicles in rice by regulating the contents of other endogenous hormones. Combined with the current research progress, we employed a heat-resistant rice cultivar Huanghuazhan (HHZ) and a heat-sensitive rice cultivar IR36 to investigate the effects of changes in phytohormone contents on young panicle development, as well as the relationship between BR and other phytohormone biosynthesis and metabolism under high temperature conditions. We also characterized how the physiologic reactions (sugar utilization and antioxidant enzyme activity) are regulated by phytohormones to influence young panicle development in rice.

Fig. 1.Effects of exogenous 24-epibrassinolide (EBR) or brassinazole (BRZ) treatments on the number of differentiated spikelets per panicle (A), the number of spikelets per panicle (B) and proportion of degenerated spikelets (C) under normal temperature (NT, 33 ºC) and high temperature (HT, 40 ºC) conditions.

Values are Mean ± SD (= 3). Different lowercase letters above the bars indicate significant differences between the treatments (< 0.05).

RESULTS

Spikelet formation and yield

Table 1. Effects of exogenous 24-epibrassinolide (EBR) or brassinazole (BRZ) treatments on rice yield and its components under normal temperature (NT, 33 ºC) and high temperature (HT, 40 ºC) conditions.

Values are Mean ± SD (3). Different lowercase letters in the same column indicate significant differences between the treatments (0.05).

Compared with control plants sprayed with distilled water under normal temperature (NT, 33 ºC), plants with distilled water under high temperature (HT, 40 ºC) formed fewer numbers of spikelets per panicle by increasing the number of degenerated spikelets per panicle in the both IR36 (heat-sensitive) and HHZ (heat-resistant) cultivars, with a rise in the proportion of degenerated spikelets under HT of 62% for IR36 and 17% for HHZ (Fig. 1-B and -C). Compared with HT, plants with EBR treatment under HT (HT + EBR) showed a reduction in the proportion of degenerated spikelets in the both cultivars, especially in IR36. We observed the opposite effect when plants were treated with the BR biosynthesis inhibitor brassinazole (BRZ) under HT (HT + BRZ) (Fig. 1-C). However, there were no significant differences among the temperature and EBR/BRZ treatments with respect to the number of differentiated spikelets per panicle (Fig. 1-A).

HT treatment significantly reduced rice yield compared with NT, especially in IR36 (Table 1). HT slightly influenced the number of effective panicles per hill, and the yield loss under HT was caused by reducing seed-setting rate, grain weight and the numberof spikelets per panicle (Fig. 1 and Table 1). Comparedwith HT, HT + EBR alleviated the yield loss, increased the number of spikelets per panicle, seed-setting rate and grain weight in both IR36 and HHZ, whereas the BRZ treatment aggravated the yield loss in both IR36 and HHZ. This result indicated that young panicle development, rice yield and yield components were associated with BR under HT and that yield loss was more pronounced in the heat-sensitive rice cultivar compared with the heat-resistant rice cultivar.

Phytohormone content in panicles

Table 2. Effects of exogenous 24-epibrassinolide (EBR) or brassinazole (BRZ) treatments on phytohormone concentrations in rice panicles under normal temperature (NT, 33 ºC) and high temperature (HT, 40 ºC) conditions. ng/g

BR, Brassinolide; Z, Zeatin; ZR, Zeatin riboside; iPA, N6-(Δ2-isopentenyl) adenosine riboside; aCTK, Active cytokinins (Z + ZR + iPA); GA1, Gibberellin A1; GA3, Gibberellin A3; GA4, Gibberellin A4; GA7, Gibberellin A7; aGA, Active gibberellins (GA1+ GA3+ GA4+ GA7); IAA, Indole-3-acetic acid; ABA, Abscisic acid.

Values are Mean ± SD (3). Different lowercase letters in the same column indicate significant differences between the treatments (0.05).

Compared with NT, HT reduced the contents of BR, active cytokinins [aCTK, zeatin (Z) + zeatin riboside (ZR) + isopentenyl adenine riboside (iPA)] and active gibberellins (aGA, GA1+ GA3+ GA4+ GA7), but increased the contents of ABA in young panicles in the both cultivars, especially in IR36 (Table 2). The BR contents under HT were reduced by 35% for IR36 and 8% for HHZ, and the aCTK contents under HT were reduced by 22% for IR36 and 13% for HHZ. HT also reduced the IAA content in IR36. However, for HHZ, there was no significant differences in IAA contents between NT and HT. Exogenous EBR increased the BR contents in young panicles in both IR36 and HHZ, whereas exogenous BRZ significantly reduced the BR contents under both NT and HT. Interestingly, the HT + EBR treatment increased the contents of aCTK and aGA in the both cultivars under NT and HT, and increased the ABA contents in IR36 under both NT and HT, but it slightly influenced the IAA contents in the two cultivars. Exogenous BRZ reduced the aCTK and aGA contents under both NT and HT. These results showed that HT inhibited young panicle development and reduced rice yield, which was associated with the changes of endogenous phytohormone contents, and greater effect were observed in IR36 than in HHZ. Therefore, exogenous EBR/BRZ influenced the metabolism of endogenous BR, aCTK, aGA and ABA.

Brassinosteroid biosynthesis and catabolism

The transcript levels of BR biosynthesis-related genesandwere increased under HT compared with those under NT in HHZ, as well as theandexpression in IR36, but HTreducedexpression in IR36 (Fig. 2-A to -C). Notably, HT significantly induced the expression of the BR catabolism genes,andin young panicles in both IR36 and HHZ (Fig. 2-D to -F). Similarly, exogenous EBR induced the expression of,andin IR36 under both NT and HT conditions, as well asandexpression in HHZ under HT. Conversely, exogenous BRZ repressed the,andexpression in the two cultivars under both NT and HT (Fig. 2-A to -C). The BR catabolism genes,andwere more highly expressed under NT + EBR compared with NT, but were expressed at lower levels under HT + EBR compared with HT, in both IR36 and HHZ (Fig. 2-D to -F). Together with our analysis of BR contents, these results showed that the lower levels of endogenous BR under HT were the result of BR catabolism brought upon by HT, especially in the heat-sensitive rice cultivar, while exogenous EBR induced BR biosynthesis and blocked BR catabolism under HT.

Cytokinin biosynthesis and catabolism

As the contents of aCTK decreased upon HT treatment, we analyzed the transcript levels of CTK biosynthesis- related genes (and) and CTK oxidase genes (and). Compared with NT, HT repressed the expression levels ofandin IR36, but rose them in HHZ (Fig. 3-A and -B). Theandtranscript levels were increased in both IR36 and HHZ in response to HT compared with NT (Fig. 3-C and -D). Exogenous EBR or BRZ also affected the transcript levels of CTK-related genes. The HT + EBR treated plants showed an up-regulation ofandexpression in both IR36 and HHZ compared with the HT treated plants, while exogenous EBR repressed the expression ofand(Fig. 3-A to -D). Exogenous BRZ largely showed the opposite effects, with lowertranscript levels in IR36 and HHZ under both NT and HT, although theexpression was up- regulated in IR36 under NT (Fig. 3-A and -B).andshowed lower expression levels under HT + BRZ than under HT (Fig. 3-C and -D). These results suggested that CTK biosynthesis and catabolism were influenced by brassinosteroids, and greater influence was observed in IR36 than in HHZ under the HT and exogenous EBR treatments.

Fig. 2.Effects of exogenous 24-epibrassinolide (EBR) or brassinazole (BRZ) treatments on brassinolide biosynthesis (A‒C) and catabolism (D‒F) genes under normal temperature (NT, 33 ºC) and high temperature (HT, 40 ºC) conditions.

Transcript levels under NT conditions were set to 1.0. The samples were collected from the young panicles of rice on the 7th day of treatment.The qRT-PCR experiments had three biological replicates withas an internal reference gene. Values are Mean ± SD (= 3). Different lowercase letters above the bars indicate significant differences between the treatments (< 0.05).

Gibberellin biosynthesis and catabolism

encodes the first catalytic step in GA biosynthesis, andis a representative gene at the third step of GA biosynthesis. Except the expression ofin HHZ was reduced, the transcript levels ofandwere slightly influenced under HT compared with those under NT in both IR36 and HHZ (Fig. 3-E and -F). GA2ox enzymes are involved in the deactivation of GA, and the expression levels of encoding genesandwere up-regulated under HT in both IR36 and HHZ (Fig. 3-G and -H). However, exogenous EBR induced theandexpression in the two cultivars under both NT and HT compared with their corresponding controls (Fig. 3-E and -F), and repressedandtranscript levels under HT (Fig. 3-G and -H). However,theandexpression was up- regulated under NT + BRZ compared with NT in both IR36 and HHZ. These results suggested that brassinosteroids participated in the regulation of GA biosynthesis and catabolism in young panicles of rice.

Abscisic acid biosynthesis and catabolism

For ABA biosynthesis, theandtranscript levels rose in IR36 and HHZ upon HT treatment compared with NT (Fig. 3-I and -J). For ABA catabolism, the expression level ofwas increased under HT in IR36, while theexpression was down-regulated. In addition, theandtranscript levels showed no appreciable response to HT in HHZ (Fig. 3-K and -L). Exogenous EBR increased theandtranscript levels and lowered theexpression in both IR36 and HHZ. The expression ofwas induced by exogenous EBR in IR36 under both NT and HT. By contrast, exogenous BRZ repressed the expression levels ofandin the two cultivars under both NT and HT, whereas exogenous BRZ did not follow an appreciable pattern for theortranscript levels (Fig. 3-I to -L). These results showed that brassinosteroids induced the expression of ABA biosynthesis-related genes and repressed the expression of ABA catabolism genes, thus increasing the ABA contents.

Fig. 3.Effects of exogenous 24-epibrassinolide (EBR) or brassinazole (BRZ) treatments on transcript levels of cytokinin biosynthesis and catabolism (A‒D), gibberellin biosynthesis and catabolism (E‒H), and abscisic acid biosynthesis and catabolism (I‒L) genes under normal temperature (NT, 33 ºC) and high temperature (HT, 40 ºC) conditions.

Transcript levels under NT conditions were set to 1.0. The samples were collected from the young panicles of rice on the 7th day of treatment.The qRT-PCR experiments had three biological replicates withas an internal reference gene. Values are Mean ± SD (= 3). Different lowercase letters above the bars indicate significant differences between the treatments (< 0.05).

Sucrose metabolism and utilization in young panicles

Carbohydrates are energy sources for young panicle development, and sucrose catabolism and utilization affect the energy supply. We determined the contents of nonstructural carbohydrate (NSC) and sucrose in young panicles of rice. Compared with NT, HT significantly reduced the NSC contents in the two cultivars (44% and 41% declined in IR36 and HHZ, respectively) (Fig. 4-A). However, the sucrose contents exhibited the opposite pattern, with a higher level upon HT treatment compared with NT, and the increase was more pronounced in IR36 (38% and 24% increased in IR36 and HHZ, respectively) (Fig. 4-B). By contrast, the glucose and fructose contents declined under HT compared with NT (Fig. 4-C and -D). To explain the increased sucrose contents in young panicles of rice, we determined the activities of key enzymes related to sucrose hydrolysis, sucrose synthase and soluble acid invertase. Compared with NT, HT led to a decrease in the activities of sucrose synthase I and soluble acid invertase (Fig. 4-E and -F). However, the HT + EBR treatment resulted in an increase in the activities of sucrose synthase I and soluble acid invertase compared with HT, which was accompanied by higher glucose and fructose contents. These results indicated that exogenous EBR can increase the activities of sucrose synthase I and soluble acid invertase, promote the decomposition of sucrose in young panicles and increase the content of NSC, whereas exogenous BRZ treatment had the opposite effect, which suggested that enhancement of endogenous brassinosteroids may alleviate the inhibition of sucrose hydrolysis induced by HT.

Fig. 4.Effects of exogenous 24-epibrassinolide (EBR) or brassinazole (BRZ) treatments on contents of nonstructural carbohydrate, sucrose, glucose, and fructose as well as activities of sucrose synthase I and soluble acid invertase in young panicles of rice under normal temperature (NT, 33 ºC) and high temperature (HT, 40 ºC) conditions.

A‒F,Nonstructural carbohydrate content (A), sucrose content (B), glucose content (C), fructose content (D), sucrose synthase I activity(E) and soluble acid invertase activity (F) under different treatments. Values are Mean ± SD (= 3). Different lowercase letters above the bars indicate significant differences between the treatments (< 0.05).

Glucose produced by sucrose hydrolysis is mainly metabolized and utilized through the glycolysis and the TCA cycle pathways, and provides energy for young panicle development. Hexokinase and pyruvate kinase are key regulatory enzymes in glycolysis. Compared with NT, HT treatment caused a drop in hexokinase and pyruvate kinase activities in IR36, leading to lower pyruvic acid content, while exogenous EBR increased the activities of both hexokinase and pyruvate kinase, thus increasing the pyruvic acid content. Conversely, exogenous application of BRZ was associated with lower enzymatic activity (Fig. 5). However, the pyruvate kinase activity and the pyruvic acid content increased upon the HT treatment in HHZ, with pyruvate kinase activity also increasing in response to exogenous EBR application.

The activities of key enzymes in the TCA cycle (citrate synthase, isocitrate dehydrogenase, α-ketoglutaratedehydrogenase, succinate dehydrogenase and malate dehydrogenase) for all the treatments were shown in Fig. 6. In IR36, we observed a decrease in the activities of key enzymes in the TCA cycle upon the HT treatment compared with NT, while EBR caused an increase in the activities of all the enzymes under both NT and HT. The exogenous BRZ treatment had the opposite effect on enzymatic activity. We obtained similar results in HHZ, with the exception of isocitrate dehydrogenase (Fig. 6-B). Compared with NT, the α-ketoglutarate contents were lower in IR36 under HT, NT + BRZ and HT + BRZ, especially in HT + BRZ (Fig. 6-C). The HT treatment had a smaller effect than either the EBR or BRZ treatment in HHZ, and exogenous EBR led to a rise in the α-ketoglutarate content, whereas we observed the opposite in response to exogenous BRZ under both the NT and HT conditions. Taken together with the analysis of sucrose hydrolysis and sugar utilization in young panicles of rice, the results suggested that HT blocked sucrose hydrolysis and glucose metabolism in young panicles, and exogenous EBR alleviated this damage. We concluded that exogenous EBR increased the contents of endogenous BR, aCTK, aGA and ABA in young panicles of rice under HT, thus promoting sucrose metabolism and supporting young panicle development. In addition, the HT damage was less severe in HHZ than in IR36.

Fig. 5.Effects of exogenous 24-epibrassinolide (EBR) or brassinazole (BRZ) treatments on hexokinase and pyruvate kinase activities and pyruvic acid contents in young panicles of rice under normal temperature (NT, 33 ºC) and high temperature (HT, 40 ºC) conditions.

A‒C,Hexokinase activity (A), pyruvate kinase activity (B) and pyruvic acid content (C) under different treatments. Values are Mean ± SD (= 3). Different lowercase letters above the bars indicate significant differences between the treatments (< 0.05).

H2O2 and MDA contents and antioxidant enzyme activities

Compared with NT, HT increased the contents of H2O2and MDA in young panicles of rice (15% and 14% increase in IR36 and 20% and 2% increase in HHZ, respectively) (Table 3). We observed an increase for both compounds in response to the exogenous BRZ treatment in both IR36 and HHZ, especially under HT. Compared with the controls, the EBR treatment led to a decrease in H2O2and MDA contents in the two cultivars under both NT and HT (Table 3). We also tested the activities of selected components of the enzymatic antioxidant system. SOD, catalase (CAT) and POD activity levels were decreased upon HT in IR36 compared with their respective levels under NT. Notably, the HT treatment enhanced the activities of SOD and POD in HHZ (Table 3). Exogenous application of EBR led to an increase in SOD, CAT and POD activities in IR36 under the both NT and HT conditions, while exogenous BRZ application lowered the activity levels. These results indicated that changes in phytohormone levels were accompanied by changes in the contents of H2O2and MDA and the activities of antioxidant enzymes of young panicles in rice under HT, and the activities of antioxidant enzymes in HHZ were higher than those in IR36.

Table 3. Effects of exogenous 24-epibrassinolide (EBR) and brassinazole (BRZ) treatments on antioxidant ability in rice panicles under normal temperature (NT, 33 ºC) and high temperature (HT, 40 ºC) conditions.

H2O2, Hydrogen peroxide; MDA, Malondialdehyde; SOD, Superoxide dismutase; CAT, Catalase; POD, Peroxidase.

Values are Mean ± SD (3). Different lowercase letters in the same column indicate significant differences between the treatments (0.05).

DISCUSSION

Effects of phytohormone levels on panicle development and yield under HT

The reproductive growth stage is the most sensitive to stress in rice (Fageria, 2007). Panicle initiation stage is a key period for the formation of panicles in rice. Wu et al (2016) reported that high temperature exposure during the early reproductive stage of rice reduces yield due to lower spikelet fertility, fewer spikelet number per panicle and lighter grains. Exposure to HT during the panicle initiation stage inhibits the development of young panicles in rice (Wang Y L et al, 2020). In this study, the HT treatment increased the proportion of degenerated spikelets in the two cultivars and showed a reduction in the number of spikelets per panicle, to be a greater degree in IR36 than in HHZ (Fig. 1). In terms of yield, the HT treatment reduced the yield by reducing the seed-setting rate and grain weight, especially the number of spikelets per panicle compared with NT, and the yield loss was more pronounced for IR36 than for HHZ (Table 2). These findings were consistent with our previous research results (Chen et al, 2019, 2021).

Phytohormones are key regulators of stress resistance in rice, especially temperature stress and water stress (Yang et al, 2001; Tang et al, 2008; Tang et al, 2018). The changes of phytohormones are closely related to the formation of rice yield (Wu et al, 2016; Zhang et al,2019). Our results confirmed that HT treatment decreases the contents of aCTK, GA1and IAA, but increases the contents of ABA and bound CTK (Wu et al, 2016). In this study, HT decreased the contents of BR, aCTK and aGA and increased the contents of ABA in young panicles of rice. HT induced a significant change in the contents of endogenous phytohormones in rice, which inhibited the development of young panicles and increased the proportion of degenerated spikelets. A small decrease or stability of endogenous phytohormone content is required to avoid large rice yield loss under the HT stress (Wu et al, 2016).

Responses of phytohormone metabolism to exogenous brassinosteroids

Brassinosteroids play essential roles in enhancing plantstress tolerance (Divi and Krishna, 2009). Transcriptome analysis demonstrated that the expression of BR metabolism-related genes is affected by high temperatures (Wang et al, 2019), which was aligned well with the reported heat sensitivity of loss-of-function mutations in BR biosynthesis genes (Setsungnern et al, 2020). Our previous research showed that inhibition of spikelet formation under HT is associated with BR catabolism, and exogenous EBR increases the endogenous BR content and alleviates the decrease in spikelet number (Chen et al, 2021). Exogenous application of EBR induced the expression of the BR biosynthetic genes,and, and repressed the expressionof the BR catabolism genes,andunder HT, whereas their transcript levels increased in response to exogenous EBR under NT (Fig. 2). Generally, exogenously applied EBR increased endogenous brassinosteroid content and promoted young panicle development. These results are similar to findings by Anwar et al (2018), who reported that exogenous BR application increases the biosynthesis of endogenous brassinosteroid. However, the effect of exogenous EBR on the contents of other endogenous phytohormones is unclear. In the present study, exogenous EBR increased the contents of aCTK, aGA and ABA in young panicles of rice. Exogenous BRZ application resulted in opposite effects (Table 2).

CTKs regulate panicle size and mediate heat tolerance in rice, and their accumulations in inflorescence meristems can increase the number of reproductive organs, resulting in higher grain yield (Ashikari et al, 2005; Wu et al, 2017). Werner and Schmülling(2009) reported that CTK directly affects the initiation and development of reproductive organs. Z, ZR and iPAs are the main endogenous CTKs with biological activities in plants (Miyawaki et al, 2004). The content of endogenous CTK is determined by biosynthesis and metabolism. Cytokinin oxidase (CKX) is the only enzyme that can specifically degrade the unsaturated bond of cytokinin isoprene side chain (Houba-Hérin et al, 1999). Liu et al(2018) reported that spraying 0.15 mg/L BR at the seedling stage increases the CTK concentration of rice leaves at the late growth period, and alleviates the stress of alkali soil on sugar beet growth. Our previous study showed that exogenous EBR decreases the expression of cytokinin oxidase genesandunder HT, leading to a higher ZR content in young panicles of rice (Chen et al, 2019). In this study, HT repressed the expression ofandin IR36 while induced the expression ofand(Fig. 3-A to -D), which significantly reduced the content of aCTK in young panicles of rice (Table 2). Exogenously applied EBR promoted the expression ofand, and repressed the expression ofand, especially under HT, thus raising the levels of Z + ZR and iPA in young panicles of rice. These results are similar to findings by Yuldashev et al(2012), who reported that exogenous EBR treatment inhibits gene expression and enzyme activity of cytokinin oxidase, leading to the increase of CTK levels in the roots and shoots of wheat seedlings. Exogenous BR also increases CTK content in the leaves at the late growth stage of sugar beet under saline alkali stress (Wang X et al, 2021). These results showed that there is an interaction between BR and CTK on plant growth regulation (Hu et al, 2000).

Brassinosteroids and GAs are the two most important growth-promoting phytohormones (Tong et al, 2014). Previous research showed that there is a complex relationship between brassinosteroids and GAs (Weiss and Ori, 2007; Gao et al, 2018). Early research showed that brassinosteroids and GAs act antagonistically (Bouquin et al, 2001). Exogenous BR application is suggested to repress the levels of aGA by down- regulating(encoding a GA biosynthetic enzyme) expression and up-regulating(encoding a GA catabolism enzyme) expression, in rice roots (de Vleesschauwer et al, 2012). However, many studies have shown that there are synergistic interactions between BR and GA. Matusmoto et al(2016) reported that application of Yucaizol (a specific inhibitor of BR biosynthesis) significantly retards stem elongation, while Trinexapac-ethyl (a commercially available inhibitor of GA biosynthesis) does not, but when application of Yucaizol combined with Trinexapac-ethyl to rice plants, the mixture of these two inhibitors retards stem elongation of rice at a lower dose, indicating there is a synergistic interaction between GA and BR biosynthesis. In this study, exogenous EBR promoted the expressionofandand inhibited the expression ofand, thus raising the levels of aGA in young panicles of rice (Fig. 3-E to -H). Tong et al (2014) reported that BR promotes the accumulationof aGA by regulating the expression of GA metabolism- related genes to stimulate cell elongation in rice. GA promotion of cell elongation requires BR signaling, whereas BR or active brassinazole-resistant 1 (control BR-responsive gene expression) suppresses the GA- deficient dwarf phenotype (Bai et al, 2012). OsmiR159d- OsGAMYBL2 (an early BR-responsive module) is a common component functioning in both BR and GA pathways, which connects BR signaling and GA biosynthesis, and thus coordinating the regulation of BR and GA in plant growth and development (Gao et al, 2018). Collectively, these results demonstrated that brassinosteroids enhance rice stress tolerance at least in part by regulating GA level.

ABA is a key phytohormone involved in stress responses, and the level of ABA increased by various abiotic stresses, thus enhancing stress tolerance in plants (Zhu, 2002). Endogenous ABA content first increases and then decreases with prolonging the high temperature stress. Generally, OsABA8ox family genes improve plant stress resistance by negatively regulating endogenous ABA content (Cai et al, 2015). However, long-term heat stress accelerates the decomposition of ABA by up-regulating the expression of OsABA8ox family genes, thus reducing the content of endogenous ABA in rice (Liu et al, 2019). BR and ABA antagonistically regulate many aspects of plant growth and development (Nemhauser et al, 2006; Zhang et al, 2009; Ha et al, 2018; Wang et al, 2018). However, other studies have found that ABA interacts synergistically with BR in plants (Li et al, 2021; Lv et al, 2022). BR increases the ABA content ofcells subjected to short-term heat stress (Bajguz and Hayat, 2009). Zhang et al (2011) reported that BR treatment up-regulates the expression of ABA biosynthetic geneand increases the ABA content in maize leaves, resulting in greater drought tolerance. In this study, exogenously EBR increased ABA content by simultaneously promoting the expression of the biosynthetic genesandand by inhibiting the expression of the catabolism geneunder both HT and NT (Fig. 3-I to -L). These results suggested that increasing plant ABA content may be a mechanism by which BR enhances stress tolerance (Yuan et al, 2010; Zhang et al, 2011).

Effects of phytohormone levels on physiological and biochemical indexes of young panicles under HT

Glycolysis and the TCA cycle are the main pathways of carbon metabolism (sugar utilization) during young panicle development. A higher energy charge would be expected to reduce the proportion of spikelet degeneration (Zhang et al, 2020). Phytohormones are closely related to carbon and nitrogen metabolisms (Kang and Turano, 2003; Yu et al, 2004; Lu et al, 2015). HT treatment decreases the contents of endogenous phytohormones in young panicles of rice and thus blocking sucrose hydrolysis and sugar utilization by reducing the activities of sucrose synthase and soluble acid invertase (Fig. 4-E and -F), and inhibiting glycolysis and the TCA cycle in young panicles (Figs. 5 and 6). We established that exogenous EBR increased the contents of endogenous BR, aCTK, aGA and ABA, thereby increasing the activities of key enzymes involved in sucrose hydrolysis, glycolysis and the TCA cycle. These results are similar to findings by Kang et al (2011), who reported that endogenous brassinosteroids increase the activities of major glycolytic enzymes in cucumber () roots exposed to hypoxia. In conclusion, increasing the contents of endogenous BR, aCTK, aGA and ABA can promote the metabolism and utilization of sucrose, and then provide energy for young panicle development, thus improving rice yield.

At high temperature, the activities of SOD and POD decrease and eventually lead to MDA accumulation (Tang et al, 2018). The inhibition of the TCA cycle and electron transport disorder may induce the greater accumulations of H2O2and MDA in young panicles of rice (Bolouri-Moghaddam et al, 2010; Jiang et al, 2020), which promotes cell death in spikelets and results in a higher proportion of degenerated spikelets (Ali et al, 2019). Wu et al (2016) reported that high temperature stress at the beginning of panicle initiation can reduce the contents of aCTK, GA1and IAA, but increase ABA contents in young panicles of rice. These changes in phytohormone levels induced by HT may modulate the differentiation of branches and spikelets. As a stress hormone, ABA priming enhances stress tolerance by up-regulating the antioxidant defense system and stress tolerance-related genes (Liu et al, 2019; Yan et al, 2021; Yang et al, 2021). Transcriptome analyses confirmed that about 60% of low-concentration ABA early response genes can be regulated by BR in the same directions (Li et al, 2021). EBR application increases the concentration of ABA in tomato, thus increasing the activities of antioxidant enzymes under drought stress (Yuan et al, 2010). In this study, exogenous application of EBR increased the ABA content, and lowered H2O2and MDA contents by promoting the activities of SOD, CAT and POD under HT (Table 3). By contrast, the exogenous application of BRZ resulted in the opposite effects, which agrees with the results by Zhang et al (2011), in which BR enhances oxidative stress tolerance at least in part via BR-mediated induction of ABA biosynthesis in maize. In addition, the exogenous ABA or ABA + BR treatment can alleviate heat damage on grape quality by increasing the accumulation of osmotic adjustment substances and endogenous hormone contents (Lv et al, 2022). These findings suggested that BR is involved in regulating the metabolism of endogenous hormones, thereby at least partially enhancing the antioxidant capacity of young panicles in rice.

Inhibited young panicle development and reduced rice yield under HT are associated with the decreased endogenous BR, aCTK and aGA contents, especially the decreased BR and aGA contents. Exogenous EBR induces BR, CTK, GA and ABA biosynthesis and inhibits their catabolism, thereby increasing endogenous BR, aCTK, aGA and ABA contents under HT. Increase in the endogenous hormone contents can enhance the activities of sucrose synthase and soluble acid invertase, and increase the activities of the crucial enzymes involved in glycolysis and the TCA cycle in young panicles of rice. In addition, higher endogenous hormone contents under HT promote the activities of SOD, CAT and POD, and then reduce the contents of H2O2and MDA in young panicles of rice. Blocking BR biosynthesis with the inhibitor BRZ is associated with the opposite physiological effects. In conclusion, raised the contents of endogenous BR, aCTK, aGA and ABA, and then maintained high energy supply and antioxidant capacity in young panicles of rice under HT, can alleviate the damage by HT to young panicle development (Fig. 7).

Fig. 7.Descriptive model of brassinosteroids mediate endogenous phytohormone metabolism to alleviate high temperature injury at panicle initiation stage in rice.

High temperature decreases endogenous BR, aCTK, aGA and IAA contents, inhibits the activities of crucial enzymes involved in sucrose hydrolysis, glycolysis and the TCA cycle, and reduces the nonstructural carbohydrate content in young panicles of rice. In addition, high temperature decreases the activities of antioxidant enzymes and increases H2O2and MDA contents, and ultimately inhibits young panicle development and reduces rice yield. Thus, a relationship between BR-regulated endogenous hormone metabolism and young panicle development is inferred. The blue ‘→’ indicates induction, and red ‘˧’ indicates inhibition.

BR, Brassinolide; EBR, 24-epibrassinolide; aCTK, Active cytokinins; aGA, Active gibberellins; IAA, Indole-3-acetic acid; ABA, Abscisic acid; NSC, Nonstructural carbohydrate; TCA, Tricarboxylic acid cycle; MDA, Malondialdehyde; SOD, Superoxide dismutase; POD, Peroxidase; CAT, Catalase.

METHODS

Rice materials and growth conditions

Pot experiments were conducted between May and October in 2021 at the China National Rice Research Institute (119º55′48′′ E, 30º2′24′′ N), Hangzhou, China. The heat-resistant inbredrice cultivar HHZ and the heat-sensitive inbredrice cultivar IR36 were used. The rice seedlings were raised with greenhouse substrate, and the seedling raising soil was the special seedling matrix, which had all the nutrients required for rice seedling development at the seedling stage without adding fertilizer and pesticide. The size of seed tray was 58.0 cm × 28.0 cm × 2.8 cm, with 120 g seeds per seed tray. During the period from sowing to emergence, the seedling matrix was kept moist, the day temperature in the greenhouse was controlled at 25 ºC‒ 30 ºC, and the night temperature at no less than 15 ºC. During the period from emergence to the first leaf, the seedling matrix surface was kept dry and white, and the temperature in the greenhouse was controlled at 22 ºC‒ 25 ºC. During the second leaf to the transplanting period, the number and time of water spraying were increased, and the temperature in the greenhouse was controlled to be close to the outdoor temperature. At 20 d after seeding, seedlings were transferred into plastic pots (20.0 cm length × 18.0 cm width × 25.0 cm height) with two seedlings per hill and two hills per pot. Each pot contained 10.0 kg of air-dried local paddy soil (pH 5.9, with 28.2 g/kg of organic matter, 1.5 g/kg of total nitrogen, 128.2 mg/kg of alkali-hydrolysable nitrogen, 0.9 g/kg of total phosphorus, 44.8 mg/kg of available phosphorus, 25.3 of g/kg total potassium and 130.0 mg/kg of available potassium). Before transplanting, 3.5 g of compound fertilizer (the ratio of N, P and K as 15%: 15%:15%) were added to each pot as base fertilizer, and after 7 d of transplanting, 0.6 g of urea were added to each pot as tillering fertilizer. Before high temperature treatment, 2.0 g of compound fertilizer were added as panicle fertilizer, and primary tillers were tagged.

Temperature treatments

The methods for cultivation, temperature treatment and exogenous chemical application were as previously described (Chen et al, 2021). Rice plants were grown under natural conditions until the panicle initiation stage(the panicle length was approximately 0.2–0.5 cm) before being subjected to HT (the maximum temperature was 40 ºC, and the mean temperature was 36 ºC) and NT (control, the maximum temperature was 33 ºC, and the mean temperature was 29 ºC) treatments for 7 d and then returned to natural conditions until maturity. The temperature was set in automatic temperature- and humidity-controlled climatic chambers as indicated in Table S1, and the humidity of the climate chambers was set to 70%‒80%. When natural sunlight was insufficient, supplemental light was provided by artificial light at about 625 µmol/(m2·s) with a wavelength of 400‒700 nm.

Exogenous chemical application

At the beginning of the temperature treatments, plants were sprayed with 0.15 mg/L (0.31 µmol/L) EBR (a synthetic brassinolide), or 0.15 mg/L (0.46 µmol/L) BRZ (a BR biosynthesis inhibitor) with the same volume of distilled water (as control) (Chen et al, 2019). NT, NT + EBR and NT + BRZ represent the distilled water treatment, exogenous EBR treatment and exogenous BRZ treatment under NT, respectively; and HT, HT + EBR and HT + BRZ represent the distilled water treatment, exogenous EBR treatment and exogenous BRZ treatment under HT, respectively. For different treatments, each pot was sprayed with 150 mL of exogenous chemical (distilled water, EBR and BRZ). Before and after the treatment of potted plants, all treated rice plants were grown in outdoor natural conditions. During rice growth, a 1.5‒2.5-cm layer of water was maintained. The managements were conducted according to local recommendations of high yield production of rice control of diseases, pests and weeds. Each treatment consisted of three replicates (30 pots per replicate).

Determination of spikelet differentiation and degeneration

Three primary tillers were sampled from three pots per replicate at the heading stage to quantify spikelet differentiation and degeneration. The number of degenerated spikelets was calculated by counting the vestiges present or small protrusions remaining on the panicle. The sum of surviving and degenerated spikelets was counted as the number of differentiated spikelets. The proportion of degenerated spikelets was calculated as The number of degenerated spikelets per panicle / The number of differentiated spikelets per panicle × 100%.

Determination of yield traits

At maturity, rice plants were harvested from three pots for each replicate to determine the following yield traits: the number of spikelets per panicle, seed-setting rate, grain weight and grain yield per plant.

Determination of phytohormone content

Young panicles were collected from plants in each treatment using scissors and tweezers on the 7th day of treatment. Samples were quickly rinsed with ice-cold distilled water and then frozen in liquid nitrogen for 20 min and stored at -80 ºC for analysis of phytohormone, H2O2and MDA contents, and products of carbon and nitrogen metabolism, and to determine enzymatic activities and transcript levels. The frozen samples of young panicles (0.1 g) were ground in liquid nitrogen, extracted in 80% cold methanol, and mixed with 1 mmol/L of butylated hydroxytoluene to prevent oxidation. Samples were then centrifuged at 10 000 ×for 20 min at 4 ºC, and the supernatant was filtered through a C18 Sep-Pak cartridge (Waters, Milford, MA, USA) and dried under nitrogen stream. The residues were resuspended in 0.01 mol/L phosphate buffer (pH 7.5). The contents of Z + ZR, iPA, GA1+ GA3, GA4+ GA7, IAA and ABA were determined by the enzyme-linked immunosorbent assay (ELISA) using the Z + ZR, iPA, GA1+ GA3, GA4+ GA7, IAA and ABA ELISA kits provided by China Agricultural University (Beijing, China).

Endogenous BR was extracted and purified from samples of young panicles according to the method of Ding et al (2013) with minor modifications. Liquid nitrogen was added to 0.1 g frozen sample. The sample was homogenized in an ice bath, placed in a 10.0-mL centrifuge tube, and then extracted in acetonitrile (5.0 mL/g) overnight at -20 ºC. The mixture was then centrifuged at 10 000 r/min for 10 min at 4 ºC. The supernatant was collected, and the remaining residue was extracted again with acetonitrile. The supernatants were combined and then concentrated under a gentle nitrogen stream for later use. Extraction, dehydration, and double-layer solid phase extraction were performed according to the method of Chen et al (2009). The extract was collected and evaporated to dryness under a gentle stream of nitrogen. The measurement and analysis of endogenous BR were conducted in a multi-stage reaction mode by a high-performance liquid chromatography- electrospray ionization-tandem mass spectrometry according to Ding et al (2013). The data acquisition and analysis were performed using the Xcalibur Data System (Thermo Fisher Scientific, NY, USA). The BR content was expressed as ng/g fresh weight.

Determination of NSC, sucrose, glucose and fructose contents

NSC content was calculated as the sum of soluble sugar content and starch content according to the method of DuBois et al (1956). Starch content was measured with the anthrone method (McCready et al, 1950). Sucrose, glucose and fructose contents were determined according to Zhang (1977) with slight modifications as described by Wang Y L et al (2020).

Determination of soluble acid invertase and sucrose synthase I activities

The activities of soluble acid invertase (EC 3.2.1.26) and sucrose synthetase I (EC 2.4.1.13) were determined using the Soluble Acid Invertase Determination Kit and Sucrose Synthase Determination Kit, respectively, provided by the Suzhou Grace Biotechnology Company, Ltd, Suzhou, China. Frozen young panicles (0.1 g) were homogenized using a chilled mortar and pestle with 1 mL phosphate buffer. The mixture was then centrifuged at 12 000 ×for 10 min at 4 ºC. The supernatant was used to determine the activities of soluble acid invertase and sucrose synthetase I with commercial kits.

Determination of hexokinase and pyruvate kinase activities

The activities of hexokinase (EC 2.7.1.1) and pyruvate kinase (EC 2.7.1.40) were determined using the test kits from the Grace Biotechnology Company Ltd, Suzhou, China. Following the instructions of the Hexokinase Determination Kit and Pyruvate Kinase Determination Kit, 0.1 g sample was homogenized on ice with 1 mL buffer (supplied in the kit) and then centrifuged at 12 000 ×for 10 min at 4 ºC. The supernatant was used to determine the hexokinase and pyruvate kinase activities.

Determination of key enzymatic activities of TAC cycle

Before determination of enzymatic activities, mitochondria were extracted according to the method of Reid et al (1977). The activities of pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 2.3.3.1), isocitrate dehydrogenase (EC 1.1.1.42), α-ketoglutarate dehydrogenase (EC 1.2.4.2), succinate dehydro- genase (EC 1.3.5.1) and malate dehydrogenase (EC 1.1.1.37) were determined using the corresponding test kits from the Grace Biotechnology Company Ltd, Suzhou, China.

Determination of pyruvic acid and α-ketoglutarate contents

Pyruvic acid and α-ketoglutarate contents were determined using the test kits from the Grace Biotechnology Company Ltd, Suzhou, China. Pyruvic acid reacts with 2,4-dinitrophenylhydrazine to form 2,4-dinitrophenylhydrazone, which appears brownish red in alkaline solution, allowing the determination of pyruvic acid content by reading absorbance value at 520 nm. The α-ketoglutarate content was determined as for glutamic acid content. Alanine aminotransferase was used to convert α-ketoglutarate to glutamic acid enzymatically, and the glutamic acid content was detected by glutamate dehydrogenase.

Determination of antioxidant capacity parameters

H2O2content was determined using the method of Rao et al (2000). MDA content was determined by the thiobarbituric acid method as described by Heath and Packer (1968). The activity of SOD (EC 1.15.1.1) was determined using the method of Giannopolitis and Ries (1977). The activity of CAT (EC 1.11.1.6) was determined according to the method of Aebi (1984). The activity of POD (EC 1.11.1.7) was determined based on conversion of guaiacol to tetraguaiacol (Maehly and Chance, 1954), which was monitored at 470 nm.

RNA extraction and qRT-PCR analysis

Samples of young panicles (the panicle at pistil and stamen formation-pollen mother cell formation with the panicle length of approximately 1.0‒2.5 cm) were used to determine the relative transcript abundance. Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, USA). First-strand cDNA was synthesized from 1 μg of total RNA with ReverTra Ace quantitative PCR RT Master Mix with gDNA Remover Kit (TOYOBO, Japan). qRT-PCR analyses were performed according to the method of Czechowski et al (2004) with three biological replicates, andwas used as an internal reference gene. The primer sequences used for these analyses are listed in Table S2.

Statistical analyses

Data were managed in Excel 2016. Duncan’s new multiple range method was conducted to compare differences between the NT, NT + EBR, NT + BRZ, HT, HT + EBR, and HT + BRZ treatments (=3) using SAS 9.4 (SAS Corp, Cary, NC, USA). Figures were constructed using the OriginPro 9.0 software (OriginLab, Wellesley Hills, MA, USA).

ACKNOWLEDGEMENTS

This study was funded by the Natural Science Foundation of Zhejiang Province, China (Grant No. LQ20C130009), the Science and Technology Project of Zhejiang Province, China (Grant No. 2022C02034), and the Special Fund for China Agricultural Research System (Grant No. CARS-01-22).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

Table S1. Temperature conditions in climate chambers.

Table S2. Primers used for qRT-PCR analysis.

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(Managing Editor: Wu Yawen)

17 January 2022;

18 May2022

Copyright © 2023, China National Rice Research Institute. Hosting by Elsevier B V

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Peer review under responsibility of China National Rice Research Institute

http://dx.doi.org/10.1016/j.rsci.2022.05.005

Zhang Yuping (cnrrizyp@163.com)