2.6.2. Nucleic Acid Synthesis and Modification

About 229 genes involved in nucleic acid synthesis and modification were identified as DEGs in this study. Of these genes, 139 were annotated as putative proteins containing pentatricopeptide repeats, which were mainly upregulated in the intermediate (WD\_6) and long-term heat treatments (WD\_12) (Table S2). It is notable that 73 genes were also induced specifically by these two heat treatments that participated in RNA biosynthesis and metabolism, including reverse transcriptase, RNA helicase, RNA polymerase, RNA recognition motif, RNA methylase, and tRNA synthetase genes. 17 DEGs encoding DNA polymerases, DNA mismatch repair proteins, DNA ligases, DNA-directed RNA polymerases, and exonucleases were also detected. In addition to the above genes, the expression of other genes essential for DNA and RNA synthesis and repair, such as ABC transporter and ATPase genes, were altered by the heat treatments in our study.

#### 2.6.3. Protein Synthesis and Posttranslational Modification

The expression of about 220 genes that participate in translation, including ribosome biogenesis, translation initiation and elongation, was altered by heat stress. For instance, 35 genes related to ribosomal proteins and ribosome biogenesis were upregulated specifically by WD\_6 and WD\_12, but not by the short-term heat treatments, WD\_1 and WD\_2. Furthermore, 28 ubiquitin metabolism-related genes were inducible under heat treatment, such as U-box domain-containing protein, ubiquitin carboxyl-terminal hydrolase, ubiquitin-conjugating enzyme, and ubiquitin-protein ligase genes. Amino acid permeases, amino acid transporters, and proton-dependent oligopeptide transporter (POT) family members involved in the transport of amino acids into cells were also identified as DEGs. It is telling that not only was protein synthesis affected by heat stress, but regulation of protein degradation also occurred under heat stress.

Among the DEGs observed in this study, 144 and 44 were annotated as protein kinases and phosphatases, respectively, according to the Nr database. Interestingly, 113 of the 144 protein kinases were tyrosine kinases. Moreover, these heat-responsive phosphatases mainly included protein phosphatase 2C proteins. In addition to phosphorylation, some genes participating in acylation and methylation were also heat-responsive in SDWG005 anthers, including histone deacetylase, acyltransferase, acyl carrier protein and methyltransferase genes.

As expected, the expression of 23 heat shock protein genes was similar to that of the HSFs, being upregulated by at least one of the heat treatments; more than half were small heat shock proteins, which were distinctly induced by 1 h of heat stress. In addition to the heat shock proteins, other chaperone proteins were heat-responsive in our study, including *ClpB1*, *DnaJ* domain-containing genes, and peptidyl-prolyl cis-trans isomerase.

#### 2.6.4. Physiological Processes Involved in the Heat Stress Response in Rice Anthers

Heat stress damages cell membranes in plants, and causes ion leakage from cells. Fatty acids are important components of the cell membrane. Hence, the relative electrical conductivity and concentration of fatty acids are commonly used as indicators of thermotolerance. In this study, we noted that six genes involved in fatty acid metabolism, including fatty acid desaturase and hydroxylase genes, were responsive to heat stress. In addition, 39 genes associated with ion transfer were regulated, such as heavy-metal-associated domain-containing, sodium/hydrogen exchanger, iron transporter, potassium channel, K<sup>+</sup> potassium transporter, magnesium transporter, and sodium/hydrogen exchanger genes.

Phytohormones play a central role not only in plant development but also in biotic and abiotic stress responses. 26 hormone synthesis and signal transduction-related genes were identified as heat stress responsive, encoding proteins responsible for auxin synthesis signal transduction (inositol-3-phosphate synthase, auxin response factor, auxin-responsive protein, AUX/IAA family gene), gibberellin-regulated proteins, and proteins related to ethylene insensitivity (Table S2).

Our previous physiological study suggested that the excellent tolerance of SDWG005 to heat stress at the seedling stage be attributed to its strong ability to degrade reactive oxygen species (ROS) under heat treatment. Consistent with this notion, about 126 genes responsible for the clearance of ROS or involved in antioxidant defenses showed altered expression levels under heat stress, such as glutathione S-transferase, oxidoreductases, P450, peroxidase, thioredoxin, cysteine protease, and dehydrogenase genes.

As one of the most heat-sensitive physiological processes in the plant kingdom, the expression of photosynthesis-related genes was inhibited by heat stress in this study, especially by the prolonged heat treatments. For example, 19 chlorophyll A-B-binding proteins were induced by 1 h of heat stress but repressed by 6 and 12 h of heat stress. We observed a similar expression pattern for other 22 genes involved in photosynthesis, such as photosynthetic reaction center protein, oxygen-evolving enhancer protein, and photosystem I and II reaction center subunit genes.

Carbohydrates play important roles in anther and pollen development during heat stress. In this study, 67 glycosyl hydrolase genes, 24 UDP-glucosyl transferase genes and 18 glycosyltransferase genes were regulated by heat stress. Additionally, our study showed that 42 genes participating in sugar or glucose metabolism and other types of carbohydrate metabolism were regulated by heat stress, such as genes annotated as sugar transporters, trehalose-phosphatases, glucose/sorbosone dehydrogenases, alpha/beta hydrolases, and galactosyl transferases. It is worth mentioning that most of these genes were down-regulated.
