**3. Discussion**

Extremely high temperatures are challenging for rice production, as heat stress usually reduces yields, along with the increasing global mean temperature and shortage of varieties with thermotolerance. For example, rice variety 9311 with its excellent agronomic trait performance has

been widely planted in China. However, this variety set fewer seeds when subjected to heat stress at anthesis in this study, with a seed setting rate of only 31.2% [20]. Thermotolerant varieties are becoming a necessity for rice production.

Rice germplasm SDWG005, belonging to *Oryza sativa* L. spp. *xian*, is more tolerant to high temperature at the flowering stage than the well-known thermotolerant germplasm N22. In addition, SDWG005 was more thermotolerant at the seedling stage than 9311, as indicated by physiological and growth factors related to thermotolerance, such as photosynthesis and shoot and root fresh and dry weights. Therefore, SDWG005 has promise for breeding thermotolerant rice varieties, and N22 has been successfully used as a source of thermotolerance and drought tolerance in rice breeding [22,23].

It has been well documented that anthesis is the most temperature-sensitive stage in rice [10]. Many physiological processes during this stage are negatively influenced by heat stress, such as anther dehiscence, pollination, pollen germination on the stigma, and pollen tube growth to reach the ovule [24], resulting in reduced fertility. Thus, understanding the thermotolerance of anthers at anthesis is essential to elucidate thermotolerance in rice, which is directly associated with production under high temperature. Our morphological analysis showed that the anthers of SDWG005 and 9311 differ in their response to heat stress. Heat stress not only led to severe wilting of the anther sac wall in 9311 but also caused anther dehiscence without mature pollen release (Figure 2). However, SDWG005 anthers only slightly differed in the control and heat treatments (Figure 2). This finding suggests that heat stress damages rice anthers, particularly in the temperature-sensitive genotype 9311, because pollen development is the most sensitive process to heat stress. In rice, spikelet sterility occurs when temperatures exceed 35 ◦C for just 1 h [15]. Therefore, differences in the structure and response of SDWG005 and 9311 anthers under heat stress may explain their differences in the timing of seed set.

Transcriptional profiling analysis of stressed and unstressed plants would help to identify genes involved in acclimation and protection against heat stress. Since the relative seed setting rates of SDWG005 and 9311 differed, theoretical transcriptome analyses need to be performed for rice anthers. Although the current study focused on the dynamic gene expression profiles of anthers of the thermotolerant SDWG005 cultivar under heat treatment, we also analyzed the expression of some of these DEGs in 9311 under the same heat treatments using qRT-PCR. We found that these genes showed different expression patterns in SDWG005 and 9311 (Figure S3). This suggests that genotypes with different thermotolerances may respond differently at the molecular level. It was reported previously that induction levels of some heat-responsive genes in anthers correlated well with heat tolerance in rice [16]. Since both of the two studies focused on the genotypes with different genetic background, it was essential to conduct analysis on near-isogenic lines with different heat tolerances to illustrate the key factors responsible for thermotolerance of SDWG005 in the future.

In this study, 3559 genes were modulated at the transcription level under high-temperature stress; these genes were involved in various processes, including transcription regulation, nucleic acid synthesis and metabolism, protein synthesis and modification, hormone signal transduction, reactive oxygen species (ROS) elimination, and photosynthesis. All of these enriched functional categories have been associated with heat responses or thermotolerance in many species, including rice, wheat, maize, tomato, barley, and brassica [11,12,25–28], suggesting a relatively conserved mechanism in response to this kind of stress. For example, heat shock proteins (HSPs) are the most commonly detected molecules and act as an intermediate in protein folding or determination of the protein conformation during stress [29,30]. Moreover, the expression of HSPs and various other heat-responsive genes is controlled by heat shock transcription factors (HSFs) [31]. In the current study, most of the genes annotated as HSP (e.g., LOC\_Os04g01740, LOC\_Os03g14180, LOC\_Os01g42190 and LOC\_Os05g35400) and heat shock factors (e.g., LOC\_Os05g49310, LOC\_Os10g03730, LOC\_Os02g34260) were also significantly induced by heat stress.

Since other studies have focused on heat-responsive gene profiles in rice reproductive tissues, including anthers [17] and pollinated pistil [16], common or specific responsive genes were identified by comparing our results to those of previous studies. We detected 48 heat-responsive genes in rice

anthers, as done by Li et al. [17] and in our study, and 168 common DEGs in rice anthers (this study) and pollinated pistils under heat treatment [16] (Table S3). However, only 20 DEGs overlapped in the three studies mentioned above, 13 of which were HSPs, as expected (Table 1). These 20 genes are probably a core gene set that responds to heat stress in the floral organs of rice. Moreover, the few overlapping DEGs suggested that the response of rice to heat treatments varies greatly and could be dependent on the stress duration, tissue, or genotype.

Recent years have witnessed a breakthrough in the analysis of the molecular mechanisms of thermotolerance in plants. Increases or decreases in some genes could enhance the thermotolerance of rice or other plant species, which could be used to improve thermotolerance using modern breeding methods (reviewed by Grover et al. [32]). As mentioned above, LOC\_Os03g47860 (*OsACT*) was significantly upregulated in thermotolerant SDWG005 at all time points examined under high-temperature stress but not in 9311 (Figure 8a). The *OsACT* gene may specifically express at the reproductive stage according to in silico data and real-time PCR data. Actually, in order to illustrate whether these DEGs were anther specific or not, we also analyzed expressions of the ten genes mentioned above in heat treated leaves in SDWG005 and 9311 at seedling stage. It was very interesting that only four of the ten genes were identified to be responsive to heat stress at seedling stage both in SDWG005 and 9311, however, the other six genes could not be detected either under the control or high temperature condition in both SDWG005 and 9311 at the seedling stage. It was worth mentioned that three of the four genes were heat shock proteins genes, which indicated that this kind of genes may be important to heat response and tolerance in the whole life of rice. While the other six genes could not be detected in leaves at seedling stage, suggesting that there may be unique mechanisms related to heat tolerance at certain development stages.

On the other hand, agmatine coumarin acyltransferase (ACT) is a member of the BAHD acyltransferase family that is unique to plants and participates in the acylation of phenolamides [33]. Phenolamines are not only involved in pollen development but also in resistance to abiotic stresses such as extreme temperature, drought, high salinity, and UV [34–36]. Phenolamine, as a substrate of peroxidase, also participates in the scavenging of hydrogen peroxide and strengthening of the extraplastid cell wall, so it functions as an antioxidant and free radical scavenger, which can improve the ability of plants to resist abiotic stress [37]. The most important is that spermidine, a precursor in phenolamine synthesis, can enhance thermotolerance in rice seeds by modulating endogenous starch and polyamine metabolism [38]. Therefore, to characterize the possible role of the *OsACT* in thermotolerance of rice anther at anthesis, this gene was cloned and sequenced from SDWG005, 9311 and N22. Sequences of *OsACT* were compared between SDWG005, 9311, N22, and other 11 rice core collections with different thermotolerance. The results showed that there were five haplotypes in WK-box in promoter region of *OsACT* (Figure 8b), which was located in the −872 bp to −880 bp upstream region. It was interesting that the core sequence in 9 out of the 10 thermo-sensitive lines was TCTACT, except for 9311, nevertheless, a considerable variation in WK-box was observed in these thermotolerant lines (Figure 8b). WK-box has been reported to be WRKY12 binding site in tobacco [21]. Variation of WK-box in these accessions indicated that *OsACT* may play an important role in anther thermotolerance during rice anthesis. Functional analysis of *OsACT* is still essential to investigate its importance in thermotolerance of rice.

Overall, a dynamic heat-responsive transcriptome analysis of the anthers of the thermotolerant rice cultivar SDWG005 provided the basis for illustrating the molecular mechanism of its thermotolerance. On the other hand, the DEGs identified in this study pave the way for further research regarding the use of heat-tolerant genes in rice breeding, aimed at improving the performance of rice varieties under challenging environments.


