**4. Discussion**

### *4.1. Seed Germination*

Seed germination is an important stage in the life cycle of higher plants and is one of the periods with the greatest sensitivity to the external environment. Under the condition of sufficient water, seeds will break dormancy and start germination after sensing the appropriate ambient temperature. High temperatures above 35 ◦C will inhibit the germination of rice seeds. The germination performance of 304 lettuce materials cultured at 21 ◦C, 28 ◦C and 35 ◦C for 40 h were compared. The results showed that the seeds of all 304 materials germinated at 21 ◦C, with an average germination rate of 87.72%. The average germination rate decreased to 42.84% at 28 ◦C and 1.01% at 35 ◦C [56]. The germination percentage of *B. subalternans* weed seeds reached above 77% for a wide alternating temperature (15/20 ◦C to 30/35 ◦C night/day). The highest germination and uniformity occurred at 25/30 ◦C night/day. Only 11% of the seeds germinated at a temperature of 35/40 ◦C night/day [57].

According to our seed germination test, when the external ambient temperature was 35 ◦C or below, the germination of rice seeds was not greatly affected, and the germination rate of all the treated seeds could be maintained at more than 90%. In a previous study, a drying temperature of 35◦C was safe for rice seeds with high initial moisture content, whereas higher drying temperatures (41 ◦C and 47 ◦C) remarkably reduced rice seed vigor. The metabolism of ROS, antioxidant enzymes, GA, ABA and α-amylase may be closely involved in the regulation of drying temperature on the seed vigor of rice seeds with high initial moisture content [58]. However, when the temperature reached 38 ◦C, the germination of most rice seeds was significantly inhibited, and there was almost no germination. Under heat stress, plants accumulate some soluble substances to adapt to the stress, and betaine is one of the quaternary amines that is effective at alleviating heat stress [23]. Because rice cannot accumulate betaine naturally, we investigated the effect of betaine on rice seed germination under heat stress by exogenous seed immersion. We found the best effect at a concentration of 10 mM, which demonstrated that the betaine immersion efficiently increased the rice seed germination under heat stress. We further investigated the regulatory mechanism of the betaine promotion of seed germination under heat stress by transcriptome analysis. Compared with the control treatment, the 10 mM betaine-soaked seeds significantly induced the differential expressions of 528 genes under heat stress, of which 332 genes were downregulated, and 196 genes were upregulated. The betaine regulated the seed germination mainly by downregulating related genes. According to the GO function analysis of the differential genes, the 10 mM betaine soaking treatment under heat stress mainly affected the cellular and metabolic processes in the biological processes; the cells, membranes, membrane parts and organelles in the cell components; binding in the molecular function; and the expressions of genes related to catalytic activity to promote seed germination.

#### *4.2. Physiological Changes of Rice under Heat Stress*

Heat stress will cause plants to produce a heat stress response, which disturbs the structural and metabolic integrity of cells to damage cell homeostasis and then causes irreversible damage to plants [59]. At the physiological level, heat stress leads to the excessive production of ROS in mitochondria and chloroplasts through disordered electron transmission, which induces lipid peroxidation to cause serious damage to the cell membrane and eventually causes cell death [60]. Therefore, enhancing the ability to eliminate excess ROS caused by heat stress is an effective defense measure to alleviate heat stress.

Soaking seeds in 10 mM of betaine considerably reduced the MDA content in rice seeds and the level of membrane lipid peroxidation under 38 ◦C heat stress. The activities of SOD, POD and CAT in the antioxidant enzyme system were significantly increased, which enhanced the ability to remove excess ROS. According to the results of this study s KEGG enrichment analysis, soaking rice seeds in betaine reduced the expression of *MEKK1* in the H2O2 signaling pathway and prevented the buildup of reactive oxygen species. On the one hand, a total of 10 mM of betaine can enhance the ability of scavenging

reactive oxygen species by increasing the activity of antioxidant enzymes under heat stress. A previous study had confirmed that 10 mM exogenous betaine could effectively improve the germination potential and germination rate of *medicago sativa* seeds under drought stress, which were significantly increased by 46.8% and 30.5% compared with the control [61]. Some researchers also found that 10 mM exogenous betaine did not significantly promote the germination rate of *elymus nutans* seeds under 100 mM NaCl stress but could significantly increase the shoot length of seeds [62]. Previous research also confirmed that betaine, either applied exogenously or accumulated in vivo in *codA*transgenic seeds, enhanced the expression of heat-shock genes and improved tolerance of high temperatures in tomato seeds during germination [22]. On the other hand, it inhibits the accumulation of reactive oxygen species by downregulating the expression of *MEKK1*. In addition, heat stress leads to a decline in the osmotic adjustment ability of crops.

Soluble protein is an important small molecular substance that participates in osmotic adjustment when crops are stressed and that participates in the relevant physiological repair after rehydration [63]. After soaking seeds with 10 mM of betaine, the content of soluble protein in the rice seeds substantially increased, and the osmotic adjustment ability was enhanced. Physiologically speaking, the 10 mM betaine treatment increased the activity of antioxidant enzymes and decreased the level of MDA, improving the removal of excess reactive oxygen species, thereby reducing the degree of membrane lipid peroxidation, and increased the content of soluble protein, enhancing osmotic regulation and promoting rice seed germination under heat stress.

#### *4.3. Synthesis and Metabolism of IAA, GAs and ABA under Heat Stress*

The germination of rice seeds is regulated by plant hormones. ABA is a typical plant defense hormone that can improve the stress resistance of plants by inducing resistance and that can also regulate seed dormancy and grain maturation [64]. GAs positively promote seed germination, and they regulate seed germination with ABA in an antagonistic manner. IAA was recently found to be the second hormone that positively regulates seed dormancy after ABA, and it also responds to the induction of heat stress. The hormone level in cells is regulated by the balance between biosynthesis and catabolism. According to the KEGG enrichment analysis, betaine soaking induced the differential expressions of some genes in carotenoid and diterpene biosynthesis and plant hormone signal transduction pathways under heat stress, which were related to ABA synthesis and metabolism, GA metabolism and IAA metabolism. *NCED* is a key rate-limiting enzyme in the ABA synthesis pathway. *NCED* catalyzes the cleavage of C40 9–cis–epoxy carotenoids, 90 CIS neoflavin and 9–cis– violet, which are considered to be the main regulatory steps of ABA biosynthesis [50]. Under heat stress, the ABA levels in seeds soaked with 10 mM betaine were significantly lower than those in the control. According to the transcriptome results, the betaine treatment significantly reduced the expression level of the *NCED* gene and inhibited the synthesis of ABA. ABA is metabolically inactivated by hydroxylation or coupling. The hydroxylation of 8 –methyl is the main step in the catabolic pathway of ABA, which is catalyzed by *CYP707A*, a member of cytochrome *P450* monooxygenase [51]. The betaine treatment not only downregulated the expression level of the *NCED* gene, but also inhibited the expression of *CYP707A*. The inhibition of *NCED* gene expression reduces the ABA level and then promotes seed germination. At the same time, a decrease in the *CYP707A* gene expression level means that the ABA metabolism has slowed down, which ensures that rice seeds still maintain a certain ABA level under heat stress to trigger the defense mechanism that requires ABA stimulation to reduce heat-stress damage. Betaine substantially increased the GA levels under heat stress. The *OsGA2ox3* gene belongs to a small polygenic family encoding GAs 2–oxidase, which is a major catabolic enzyme in plants and can convert active GAs into inactive GAs [65]. The betaine treatment substantially decreased the expression level of the *OsGA2ox3* gene and inhibited the inactivation of GAs. We did not find the expressions of differential genes in the synthesis pathway of GAs, which indicates that

betaine mainly increases the level of GAs by inhibiting the inactivation of GAs, rather than by promoting their biosynthesis.

In response to the induction of heat stress, the IAA levels increased, while the betaine treatment substantially reduced them. In general, the IAA levels in plant cells are regulated in three different ways: dynamic balance, polar transport and auxin response [66]. In this experiment, there were no differential gene expressions in the biosynthesis and polar transport pathways of IAA, but we found that *GH3* family genes were differentially expressed in the IAA signal transduction pathway. *GH3–4* is very sensitive to changes in the IAA level. After the increase in the IAA level, the expression level of *GH3–4* is one of the most substantial [53]. *TLD1* encodes an IAA amination synthase. In the *TLD1–D* mutant, the expression of *TLD1* was significantly increased, which promoted the chelation of IAA with amino acids and reduced the content of free IAA [54]. The decrease in the IAA level in the betaine treatment under heat stress may be related to the high expression of *TLD1*, which suggests that betaine may regulate the level of IAA in plant cells through the auxin response. In terms of the regulation of seed germination by plant hormones, betaine reduces the level of ABA and increases the level of GAs by inhibiting the synthesis of ABA and the inactivation of GAs under heat stress, promotes the inactivation of IAA to reduce the level of IAA, and finally, promotes the germination of rice seeds.
