**2. Materials and Methods**

#### *2.1. Test Materials*

The tested material was XZX45, which is a rice variety that is mainly planted in rice-growing areas in the middle and lower reaches of the Yangtze River in China. The high-quality variety Zhouyou 903 was used as the female parent to prepare an inter-variety hybrid combination with Zhefu 504, which had a short growth period and strong resistance. The offspring was named XZX45 after purification. The entire growth period of XZX45 is 106 days, which is characterized by strong lodging resistance and excellent rice quality. In China's provincial regional test of varieties, the average yield was 7573.5 kg ha<sup>−</sup>1. It is also mainly used as the national grain reserve. All test seeds were harvested in 2021.

#### *2.2. Experimental Design*

We carried out seed germination in a germination box (length, width and height of 12 cm, 12 cm and 5 cm, respectively). According to the previous research results of this experiment, the betaine seed-soaking solution was set at 0 mM, 0.1 mM, 1 mM and 10 mM. We selected seeds with full grains, soaked them and disinfected them with 5% sodium hypochlorite for 30 min, and we then rinsed them 5 times with sterile distilled water. After soaking the seeds with betaine solutions of 0 mM, 0.1 mM, 1 mM and 10 mM concentrations for 24 h, we placed them in germination boxes with sterilized germination paper, placing 100 seeds in each germination box. We added 7 mL of sterile distilled water to each germination box for seed germination, and we repeated each treatment 20 times. We conducted the germination test in a light incubator. We set the temperature for the heat stress treatment at 26 ◦C, 32 ◦C, 35 ◦C and 38 ◦C in 16 h light/8 h dark, and the humidity

was 70%. The germination of seeds is subject to the condition that the bud length is equal to half of the seed length, and the root length is equal to the seed length. We recorded the germination every 24 h after sowing, and we counted the final germination rate on the seventh day of germination. We referred to the method of Yu et al. (2022) to determine the germination potential and rate [32]. The germination potential and germination rate were investigated on days 3 and 7 after initiation, respectively. The germination potential was calculated as germination potential (%) = (number of seeds germinated on day 3/total number of experimental seeds) × 100. The germination rate was calculated as germination rate (%) = (number of seeds germinated on day 7/total number of experimental seeds) × 100. After 24 h of the high-temperature treatments of the seeds, we measured and sampled the antioxidant enzyme activity, MDA content, soluble protein content, endogenous hormone content, transcriptome determination and related gene expressions. We quickly put the samples into a 2 mL centrifuge tube with RNase inactivation. After quick freezing with liquid nitrogen, we placed them in a refrigerator at −80 ◦C for storage.

#### *2.3. Determination of Physiological Indexes*

We determined the activity of superoxide dismutase (SOD) according to the method of Dhindsa et al. [33]. We referred to the method of Kar et al. [34] to determine the activity of peroxidase (POD). We used the UV absorption method [35] to determine the activity of catalase (CAT). We determined the content of malondialdehyde (MDA) by using the method of Li et al. [36]. We determined the content of soluble protein by the Coomassie brilliant blue G–250 method [37]. We repeated each measurement 5 times.

#### *2.4. Determination of Endogenous Hormones*

We accurately weighed 50 mg of the sample that we had ground and crushed in liquid nitrogen. We conducted the extraction and quantification of the endogenous IAA, ABA and GAs according to the operation guidelines of Wuhan Metwell Biotechnology Co., Ltd., China. We performed data collection using ultra-performance liquid chromatography (UPLC) (the Shim–pack UFLCSHIMADZU CBM30A system, Kyoto, Japan) and tandem mass spectrometry (MS) (Applied Biosystems, Waltham, MA, USA). Wuhan Metwell Biotechnology Co., Ltd., Wuhan, China (http://www.metware.cn/) (accessed on 17 November 2021) analyzed the data. We performed three biological replicates for each treatment.
