Rice is a staple crop that provides food for more than half of the world’s population and 60% of the human population in China (He et al., 2023; Yuan, 2014). Climate change, especially increases in temperature driven by rapid economic development and the greenhouse effect, poses major challenges for human society. Heat waves occurred in many countries in the summer of 2022 (Lu et al., 2023), and they have been particularly common in central–eastern China since the 1960s (Ma Y. Y. et al., 2023; Zhang et al., 2023). Rice yield has declined by 10% for each 1°C increase in the nighttime minimum temperature in the dry season (Peng et al., 2004). Under the heat stress, rice yield could decrease by up to 10% in 2030 in South Asia (Lobell et al., 2008) and by 15%–26% by 2080 in developing countries; yields could decrease by 30%–40% in India, and grain losses could reach devastating levels, such as more than 50% in Senegal and Sudan (Cline, 2008). The global human population is rapidly increasing (Rahman and Zhang, 2023). Therefore, warming will have a significant effect on global food security (Driedonks et al., 2016). In light of continuous global warming and the frequent occurrence of high temperatures over short periods, ensuring the sustainable production of rice will require studies of its heat tolerance.

Rice prefers warm temperatures and short periods of sunshine. However, it is sensitive to high temperatures in the booting stage, flowering stage, and grain-filling stage (Jagadish et al., 2007). It is particularly sensitive in the flowering stage given that heat stress over 35°C can result in substantial decreases in pollen fertility and vigor, inhibit anther dehiscence, and lead to poor pollen germination on the stigma (Jagadish et al., 2010; Wang Y. et al., 2019), which affects the grain filling rate and grain yield. Subsequently, it was found that heat stress results in poor rice quality, expressed as reduced palatability, undesirable grain appearance, and increased grain chalkiness at the grain-filling stage (Nevame et al., 2018; Kaneko et al., 2016). The effect of heat stress on rice was directly related to the duration of heat stress, and the higher and longer the heat stress, the more serious the effect on rice (Guo et al., 2010). Therefore, many studies have examined the high-temperature tolerance of rice to mitigate heat damage and losses.

Proteomics studies of rice under heat stress at the flowering stage (Kim et al., 2015; Mu et al., 2017), filling stage (Liao et al., 2014), and milk stage (Timabud et al., 2016) have shown that the differentially expressed proteins (DEPs) vary among stages. These are mainly involved in biosynthesis, energy and metabolism, redox homeostasis, photosynthesis, and signaling, and temporary protective mechanisms play a role in enhancing heat tolerance. Jagadish et al. (2010) first identified 13 DEPs by two-dimensional gel electrophoresis. One cold protein and one heat shock protein (HSP) were found to be significantly upregulated in the heat-tolerant variety N22. Comparison of rice anther protein expression patterns in high-temperature environments and natural environments revealed 139 DEPs, including HSPs, energy metabolism proteins, DnaK family proteins, and chaperonins (Kim et al., 2015). Mu et al. (2017) found that heat stress only induced the degradation of ribosomal proteins in heat-sensitive varieties, and the abundance of HSPs, β-extension proteins, and lipid transfer proteins was increased in heat-tolerant varieties; these changes in protein abundances might be related to differences in high-temperature tolerance. Due to differences in local heat conditions, researchers have studied rice plants under different types of heat stress (stepped temperature gradient and maximum temperature). Compared with the previous studies, the temperature setting was different in the present study, at 38°C for 6 h (from 9:00 a.m. to 3:00 p.m.) lasting 3 days, which was a valid test condition derived from repeated trials locally in Nanning, Guangxi (Liang et al., 2016). In addition, anthers were chosen as the subject, which is distinguished from tissues such as spikes, glume, and leaves in previous studies. Furthermore, tandem mass tags (TMT) (Thompson et al., 2003) has not been frequently used to study the DEPs of rice anthers under heat stress.

In previous studies, we identified various heat-tolerant resources from different rice varieties at the flowering stage from 2012 to 2018 (Wang, Q. et al., 2019) and localized the heat-tolerant QTL qHTT8 on chromosome 8 in the F2:3 population of the heat-tolerant rice variety Huang Hua Zhan (HZ) and the heat-susceptible rice variety Yangdao6 (YD) in 2021 (Chen et al., 2021). However, the physiological mechanism and the mechanism of protein regulation in the anthers of HZ and YD in response to heat stress remain unclear. We examined the physiological responses, gene expression responses, and proteomic differences in the anthers of HZ and YD under heat stress. The main aim of this study was to clarify the physiological mechanisms underlying the heat tolerance of rice to facilitate the screening of heat-tolerant varieties.

The results of the KEGG pathway and functionally annotated statistics revealed DEPs involved in heat stress tolerance ( Table 1 ). Six representative DEPs with functions in metabolism, genetic information, and environmental information processing were validated, including Q01IS7 (LOC_Os04g33740) and A2Y2C4 (LOC_Os05g15770), which are related to carbohydrate metabolism; A2YDE3 (LOC_Os06g30970), which is involved in folding, as well as sorting and degradation; A2YGL2 (LOC_Os06g48200), which is involved in signal transduction; B8BN36 (LOC_Os12g42810), which plays a role in translation; and Q01IX6 (LOC_Os04g39980), which plays a role in amino acid metabolism. The consistency of the expression patterns inferred via RT-qPCR with the proteomic data indicated that our data were robust ( Figure 11 ).

The successful identification of heat-resistant quantitative-trait loci (QTL) revealed significant differences in the response to heat stress between HH and YD (Chen et al., 2021). Physiological, gene expression, and proteomic analyses of these two varieties were conducted to elucidate the regulatory mechanisms underlying the heat stress tolerance of rice, which ensures the robustness of our results. Plants do not remain passive when exposed to heat stress; they can adapt to heat stress and rapidly perceive stimuli, generate and transduce signals, and activate antioxidant enzyme systems, which accelerate the removal of ROS and maintain ROS homeostasis (Hasanuzzaman et al., 2013; Zafar et al., 2020). Thus, improving the high-temperature tolerance and effects of the antioxidant system of rice depends on the heat-tolerance capacity of each variety (Visakh et al., 2024). Under heat stress, increases in the activity of the antioxidant enzymes SOD, CAT, POD, and APX were greater in the plants of heat-tolerant varieties at the flowering stage than in heat-sensitive varieties, which is consistent with the results of previous studies (El-Remaly, 2023; Yang et al., 2022b). The content of soluble sugars and proline was higher in HH than in YD, which promoted osmoregulation and the structural stability of cell membranes after exposure to high-temperature stress. In addition, it has been shown that MDA was able to inhibit the activity of cytoprotective enzymes and reduce the content of antioxidants, which aggravated the peroxidation of membrane lipids (Xu et al., 2021), and the results of this study showed that HZ produced significantly less MDA than YD under heat stress. This indicates that anther cell membrane disruption and electrolyte permeation were more severe in YD than in HZ under heat stress, which led to a reduction in its physiological functions and thus the lack of resistance to heat stress. This is also one of the reasons why HZ is more resistant to high temperature than YD. In terms of the same variety, the antioxidant enzyme activity of anthers was increased under high stress compared to control temperature, and the results were similar to those of Zhang et al. (2014), but different from some studies (Tang et al., 2008; Zhao et al., 2018); it was caused by the difference in sampling time. The activity of antioxidant enzyme increased, and reached the peak on the third day. With the prolongation of high temperature treatment, the antioxidant enzyme activity were hurt and decreased (Zhang et al., 2014).

When plants are subjected to environmental stress, the content of ABA increases sharply, and there is a significant positive correlation between the accumulation of ABA and the enhancement of stress tolerance (Dar et al., 2017). This is also the reason why the ABA content of HH was higher under heat stress than under normal temperature. Excessive ABA inhibits the expression of sucrose-converting enzymes and monosaccharide transporter protein genes, which leads to the accumulation of a large amount of sugar in anthers, including sucrose. Given that sucrose cannot be transported to pollen, this leads to a reduction in rice pollen fertility and the fruiting rate (Ji et al., 2011). In our study, the spikelet fertility of YD was 14.3%, which was lower than 54.5% of HZ under high-temperature stress (Chen et al., 2021), which was probably caused by the accumulation of a higher amount of ABA in the anthers in the HZ. A physiological and biochemical response is often regulated not only by a single hormone but by multiple hormones and their balance with each other. GA is also key under stressful environments. High-temperature stress increased ABA content but decreased the content of active cytokinin (CK), gibberellin A₁ (GA₁), and indole-3-acetic acid (IAA). These factors reduced rice grain weight (Wu et al., 2016). Due to enhanced biosynthesis, reduced degradation, or release of bound forms, ABA concentrations increase under high stress (Chen et al., 2022). It induced a significant increase in ROS and ABA content in developing the anther of rice at the spike stage under high-temperature stress, and ABA regulated PCD and microspore apoptosis in anther chorioallantois cells by “triggering” ROS production, which leads to the formation of high-temperature pollen abortion (Zhao et al., 2023). In the present study, the content of endogenous ABA and GA3 was increased under heat stress. The difference of GA₃ may be caused by the different types of GA measured and varieties. The ratio of GA₃/ABA content is more convincing; the ratio of endogenous GA3/ABA in HZ was 10.65 under heat stress, which was significantly higher than that in YD (3.84), and responses in hormone levels ultimately enhance reproductive performance.

A number of rice heat tolerance genes, including transcription factors that are induced by exposure to heat, that are expressed in the floral organs were identified. In 2021, our team screened two candidate genes LOC_Os08g07010 and LOC_Os08g07440 by spikelets and QTL (Chen et al., 2021). In the present study, anthers were used for RT-qPCR validation under the same high-temperature treatment, and the results were consistent with the results of previous studies that LOC_Os08g07010 and LOC_Os08g07440 were highly induced in HZ compared with those in YD under heat stress. Both candidate genes were expressed in spikelets, glumes, and anthers (Zhao et al., 2019; https://rice.uga.edu/). However, according to the NCBI and UniProt databases, the proteins encoded by the genes in the BSA region did not intersect with the differential proteins in this study. The candidate genes in the BSA interval may be involved in the regulation of resistance to high temperature, but there is no significant difference in the protein level, or the protein expression is so low that it could not be detected. Furthermore, we homologated the protein sequences encoded by the genes in the BSA interval and found the proteins expressed in this study, of which seven had a similarity greater than 80% ( Supplementary Table S1 ). Based on gene function annotation, we focused on three genes. LOC_Os08g06430 encodes mitochondrial NADH ubiquinone oxidoreductase, which is involved in rice environmental stresses such as stress tolerance under compartmentalized stress (Chatterjee et al., 2024) and salt tolerance (He et al., 2024). LOC_Os08g07760 (OsBAK1) encodes a kinase for the BR signaling receptor BRI1, which has important roles in rice growth and development and stress tolerance (Chen et al., 2014; Song et al., 2022). LOC_Os08g06550 encodes acyl-coenzyme A-binding proteins, a class of lipid transport proteins, which have a high binding capacity for acyl-coenzyme A and phospholipids and play an important role in plant growth and development and stress response process (Xu et al., 2019).

TMT proteomics has been widely applied because of its higher quantitative accuracy, fewer missing values, and higher efficiency compared with other methods of proteomic analyses (Creskey et al., 2023). Previous proteomics studies of the response to heat stress have identified proteins involved in heat shock, energy metabolism, DnaK family proteins and chaperonins (Jagadish et al., 2007), DOF family, HSPs, ROS-related proteins (Zhang et al., 2019), HSPs, β-expansins, and lipid transfer proteins (Mu et al., 2017). In this study, we found that the identified heat-resistant proteins also contained energy metabolism and HSP, which was consistent with previous studies. Through GO annotation, we found that the classification functions annotated by DEPs of both HH and YD were basically the same at the CC, MF, and BP levels in the present study. Concurrently, it showed the pathways enriched in HH and YD partially overlaps and some difference by KEGG pathway. Overlapping functions included carbohydrate metabolism, folding, sorting and degradation, transcription, and biosynthesis of secondary metabolites. We identified new upregulated proteins involved in HSPs, UBC core domain-containing protein, thioredoxin-like proteins (Trxs), thiamine thiazole synthase (THI1), beta-fructofuranosidase (BFru), and xyloglucan endotransglycosylase/hydrolases (XTH) in HH. In YD, We founded that amino acid metabolism, carbohydrate metabolism, and COP9 signalosome (CSN) were downregulated. We queried the literature and found that the differential protein accessions A2WKD2, Q01IS7, A2YDE3, A2YM28, B8B881, A2XEW6, and B8BD19 in this study corresponded to the coding genes LOC_Os01g04340, LOC_Os04g33740, LOC_Os06g30970, LOC_ Os07g34570, LOC_Os10g34520, LOC_Os10g34520, LOC_Os03g16020, and LOC_Os09g31486, which were also recognized as differential genes in high temperature stress transcriptome analysis (Vitoriano and Calixto, 2021; Wilkins et al., 2016; Wang et al., 2020).

HSPs were verified to play a role in the response to heat stress by preventing the irreversible aggregation of denatured proteins (Sarkar et al., 2009; Wang et al., 2004; Yang et al., 2022a). HSPs are one of the major types of molecular chaperone proteins identified to date. They are usually induced by exposure to high temperatures, contribute to the correct folding of specific proteins, and assist in trans-membrane translocation during plant stress responses. According to the former study, it showed that high molecular mass Hsps include Hsp70, Hsp90, and Hsp100, and small heat shock proteins (sHsps) include Hsp20 (Yang et al., 2022a). This study identified that sHsps domain-containing protein were upregulated in the heat-tolerant variety HZ. This suggested that sHsps played a role in the regulation of heat resistance which was congruent with that of the present study (Jagadish et al., 2007; Zhang et al., 2019; Mu et al., 2017). Concurrenly, We found that the expression of UBC core domain-containing proteins was upregulated in HZ, similar to sHsps, under heat stress ( Table 1 ). Endoplasmic reticulum-associated degradation (ERAD) is one of the pathways maintaining the homeostasis of the endoplasmic reticulum (ER), and UBC has a major effect on the stability of certain ERAD substrates under certain types of stress (Cui et al., 2012; Ma, W. et al., 2023). In addition, UBC enzymes play indispensable roles in the biological processes of plants, such as plant growth and stress responses (Jue et al., 2015). Although HSPs have been thought to be solely responsible for the response to heat stress (Umarani et al., 2020), other proteins, including ubiquitin, cytosolic Cu/Zn-SOD, and Mn-POD, are also expressed following exposure to heat stress indicating that they are also involved in the response to heat stress. In algae, the content of the ubiquitin protein complex increases under heat shock (Li et al., 2014). The present study identified COP9 signaling associated with ubiquitination. The initial discovery of the COP9 signalosome (CSN) complex was made via a genetic analysis of light control in Arabidopsis seedling development. CSN itself is a target of kinase activity and regulates the activity of kinases via the entire ubiquitin degradation pathway, which affects the biological functions associated with target proteins (Orit Harari-Steinberg, 2004). It was found that CSN plays an important regulatory role in plants in response to both heat and cold stress (Qin et al., 2020). This study’s conclusion was the same. In addition to its interaction with E3s, CSN can regulate protein hydrolysis by binding to protein kinases and deubiquitinating enzymes (Schwechheimer, 2004). In this study, the activity of CSN was reduced under high temperatures in YD, and it may be one of the reasons why its heat resistance is relatively poor.

Thioredoxin-like proteins (Trxs) are mainly involved in the splicing and transcription of mRNAs according to the Uniprot database. In previous studies, Trxs have been shown to play an important role in chloroplast metabolic pathways (Kang et al., 2019). Trxs play key roles in maintaining the function of the b6f complex, which drives ATP synthesis, maintains the energy balance, and maintains redox homeostasis in chloroplasts (Chen et al., 2024). Trx is involved in the light-induced activation of key enzymes in the Calvin Cycle to improve photosynthetic efficiency (Da Fonseca-Pereira et al., 2021). The Trx/Prx/Srx system is involved in plant signaling under stress, especially under abiotic stress (Sevilla et al., 2015), and these signals are important cues that affect plant yield and growth. Trx and Trx-like proteins can regulate chloroplast function by controlling the redox state of various photosynthesis-associated proteins in Arabidopsis (Yokochi et al., 2021). In the present study, TRX was also upregulated in HH suggesting that it is mainly from photosynthesis and chloroplasts that heat tolerance is improved. Similarly, thiamine thiazole synthase (THI1) is also expressed as an upregulated protein. THI1 is involved in abscisic acid (ABA) signal transduction, stomatal closure in guard cells, and in the response to drought stress in Arabidopsis thaliana (Li et al., 2016). Stomata also affect photosynthesis to some extent, thereby increasing resistance to abiotic adversity. Thiamine plays an important role as an enzyme cofactor in glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle. It can regulate cell tolerance to DNA damage, and it is activated after plants are attacked by pathogens. The abundance of THI1 protein has been shown to increase under heat stress in previous studies (Xu and Huang, 2018; Zhang et al., 2013), which suggests that it might be involved in DNA protection or repair. In our study, THI1 regulates the metabolism of cofactors and vitamins to provide more energies to resist heat stress; the expression of THI1 was also upregulated in the heat-tolerant variety HZ under heat stress ( Table 1 ).

Heat stress can interfere with carbohydrate metabolism in rice, disrupt the energy balance, and inhibit the antioxidant capacity and heat stress protein accumulation, which lead to decreases in yield and quality. The expression of enzymes involved in carbohydrate interconversion, including putative beta xylosidase, aldose 1-epimerase, and pectinesterase, was downregulated in YD compared with that in HZ under heat stress, which indicates that energy deficiency leads to reductions in the heat tolerance of rice. Similarly, the expression of beta-fructofuranosidase, insoluble isoenzyme 2 (BFru), which is involved in carbohydrate metabolism, was also downregulated. BFru mediates the degradation of sucrose into glucose and fructose, which is the first step in starch biosynthesis (Zhang et al., 2024); the glucose and fructose products then enter the glucuronate pathway, which ultimately produces energy and carbon dioxide. This is attributed to the expression of proteins that mediate decreases in energy metabolism under high-temperature stress. This indicates that high temperatures interfere with energy metabolism and severely inhibit the growth and development of organisms.

DEPs involved in environmental information processing were identified. For example, xyloglucan endotransglycosylase/hydrolases (XTH) can disrupt and reconnect xyloglucan chains and modify the structure of cellulose–xyloglucan complexes to rebuild the cell wall. XTH plays a critical role in the aluminum (Al) tolerance of tea trees (Camellia sinensis) (Wu et al., 2021). XTH plays a role in determining the structure and composition of plant cell walls. Therefore, elucidating changes in XTHs during the response of plants to abiotic stress is important for studying plant cell wall signaling-mediated stress regulation mechanisms in plants (Chen et al., 2024). The TaXTH gene regulates the drought response in wheat. The expression of TaXTH12.5a enhances the drought tolerance of Arabidopsis thaliana (Han et al., 2023). In this study, XTH is related to signal transduction in heat-tolerant rice varieties; in subsequent studies, we will investigate its signaling role in regulating heat-tolerant functions.

In summary, we found that the proteins associated with heat tolerance mainly included HSPs proteins, carbohydrate metabolism, maintain endoplasmic reticulum stability, photosynthetic system, cell wall signaling, and are ubiquitination-related by TMT. Through the KEGG pathway analysis, these DEPs were found to be mainly involved in the pathways of phenylpropanoid biosynthesis, ubiquitin-mediated proteolysis, carbohydrate metabolism, thiamine metabolism, and protein processing in the endoplasmic reticulum in this study.

We analyzed the physiological characteristics, expression of the heat tolerance genes, the candidate genes LOC_Os08g07010 and LOC_Os08g07440 that our team located in 2021 under heat stress, and protein expression patterns in heat-tolerant and susceptible varieties of rice via TMT proteomics. We found that the antioxidant capacity was stronger, the content of osmoregulatory substances and the GA/ABA ratio were higher, and DEPs were mainly involved in the pathways of phenylpropanoid biosynthesis, ubiquitin-mediated proteolysis, carbohydrate metabolism, thiamine metabolism, and protein processing in the endoplasmic reticulum. Folding, sorting, and degradation were upregulated to a greater degree in HZ than in YD, all of which likely contribute to the greater heat tolerance of HZ compared with that of YD. Additional studies are needed to clarify the associations of these proteins with heat stress regulation through signaling.