**3. Results**

#### *3.1. Betaine Soaking Treatment Can Promote the Germination of Rice Seeds under Heat Stress*

Germination potential and germination rate are two of the main indicators of seed quality. When germination rates are identical, seeds with high germination potential demonstrate greater vigor. Germination potential determines the regularity of seedling emergence [39]. Therefore, when the germination potential and germination rate of rice seeds are high, it indicates that the seeds emerge quickly and evenly. Meanwhile, the seedlings are robust. If rice seeds have low germination potential and a high germination rate, it indicates uneven seedling emergence and weaker seedlings. After soaking seeds in betaine at different concentrations, we conducted the germination test for rice seeds at different temperatures (Figure 1A,B). At 25 ◦C, soaking seeds with betaine at various concentrations had little effect on the germination rate and potential. At 32 ◦C, soaking seeds with betaine at all concentrations improved the germination rate and potential, which were 5.82–6.19% and 5.82–7.26% higher than the control (0 mM betaine), respectively. At 35 ◦C, soaking seeds with 0.1 mM, 1 mM and 10 mM of betaine had no significant effect on the germination potential and rate. At 38 ◦C, soaking seeds with 10 mM betaine significantly improved the germination potential and rate, which were 57.96 times and 43.77 times higher than the control (0 mM betaine), respectively.

**Figure 1.** Effects of soaking seeds with different concentrations of betaine at 25 ◦C, 32 ◦C, 35 ◦C and 38 ◦C on germination characteristics of rice seeds. The difference in the average value of each letter indicates that there is a significant difference in the parameters (*p* < 0.05): (**A**) germination potential; (**B**) germination rate.

#### *3.2. Betaine Increased the Antioxidant Enzyme Activity and Soluble Protein Content of Rice Seeds under Heat Stress*

The seed germination activities of the 0 mM and 10 mM betaine soaking treatments were significantly different under heat stress for 24 h. By measuring the antioxidant enzyme activity, MDA content and soluble protein content, we found that the activities of SOD, POD and CAT in the HT + BT treatment were significantly higher than those in the HT treatment (Figure 2A–C) and were increased by 243.06%, 66.10% and 63.37%, respectively. The MDA content in the HT+BT treatment was significantly reduced by 70.69% compared with the HT treatment (Figure 2D), while its soluble protein content was significantly increased by 45.88%, compared with the HT treatment (Figure 2E).

**Figure 2.** Effects of soaking seeds with 10 mM betaine on antioxidant enzyme system and soluble protein content of rice seeds under heat stress. Each parameter \* indicates significant statistical difference, \* (*p* < 0.05): (**A**) SOD activity; (**B**) POD activity; (**C**) CAT activity; (**D**) MDA content; (**E**) soluble protein content.

#### *3.3. Hormone Content during Seed Germination*

Seed germination is regulated by endogenous plant hormones, so we measured the ABA, IAA and GA contents of the seed protrusion of both the HT- and HT + BT-treated seed endosperm breakthroughs. Under heat stress, the contents of ABA and IAA in the seeds were significantly higher than those of the betaine treatment, of which the ABA was 1.7 times that of the betaine treatment (Figure 3A), and the levels of indole-3-carboxylic acid (ICA), indole-3-carboxaldehyde (ICAld), methyl indole-3-acetic acid ester (ME–IAA) and IAA were 2.49 times, 2.04 times, 3.28 times and 2.21 times that of the betaine treatment, respectively (Figure 3E,H). The contents of various GAs were significantly inhibited under heat stress. Except for gibberellic acid 15 (GA15), the contents of gibberellic acid 19 (GA19) and gibberellic acid 24 (GA24) in the betaine treatment were significantly higher than those in the heat-stress treatment and were 2.13 times and 1.47 times higher than those in the HT treatment, respectively (Figure 3B–D).

**Figure 3.** Comparison of hormone levels in HT- and HT + BT-treated seeds: (**A**) comparison of ABA content; (**B**) comparison of GA15 content; (**C**) comparison of GA19 content; (**D**) comparison of GA24 content; (**E**) comparison of ICA content; (**F**) comparison of ICAld content; (**G**) comparison of ME–IAA content; (**H**) comparison of IAA content. Each parameter \* indicates significant statistical difference, \* (*p* < 0.05), \*\* (*p* < 0.01).

*3.4. Transcriptome Analysis of the Effect of Betaine Treatment on Rice Seed Germination under Heat Stress*

During seed germination, we performed a transcriptomic analysis of the seed coleoptile and mesocotyl elongation stage after 24 h of immersion under heat stress at 38 ◦C. We performed three biological replicates of each treatment (Table 2). We present the sequencing data outputs and quality for the six samples in the table. Each sample produced an average of over 44 Mil. total raw reads. After quality control to remove low-quality reads, splice contamination and reads with high unknown base N content, we obtained an average of more than 6 Gb of total clean bases per sample, and all six samples had a Q30 of more than 85% with homogeneous base content.


**Table 2.** Output and quality of sample sequencing data.
