*4.4. Expression and Crosstalk of IAA, GA and ABA Signals*

Plant hormones transmit endogenous and environmental signals through specific signal pathways to trigger output responses [67]. According to the KEGG enrichment analysis, the betaine induced the expressions of differential genes in the IAA, GA and ABA signal transduction pathways under heat stress. The expressions of these differential genes affect hormone signal transduction. In the IAA signal transduction pathway, the *GH3* expression was significantly upregulated in the betaine treatment, while the *SAUR* expression was significantly downregulated. The decrease in the IAA level is related to the upregulation of *TLD1* expression in the *GH3* gene family. *SAUR*, as an auxin early-response gene, participates in the regulation of cell elongation and growth. The overexpression of *SAUR* can promote the elongation of *Arabidopsis* cells [55]. *SAUR* promotes cell elongation and is related to *PP2C* in the ABA signal transduction pathway. *SAUR* inhibits the activity of *PP2C* and leads to the elevated activation of AHA2 phosphorylation, thereby derepressing PM H+–ATPases (e.g., AHA2) to acidify the cell wall and ultimately promote elongation growth [52]. In this study, we found that the expression level of *SAUR* was downregulated in response to decreased growth hormone levels; however, at the same time, the expression level of *PP2C* was also downregulated by decreased ABA levels. The promotion of seed germination by betaine under heat stress indirectly indicates the promotion of elongated cell growth, which may be mainly caused by the downregulation of the *PP2C* expression levels.

Under heat stress, the expression level of *DELLA* in the GA signal transduction pathway was downregulated in the betaine treatment. DELLA protein is an inhibitor of GA signal transduction. In the presence of GAs, the GAs bind to the *GA–INSENSITIVE DWARF 1* (*GID1*) receptor and cause conformational changes that trigger *DELLA* degradation or that lead to its inactivation, ultimately allowing the output of the GA signaling pathway [49]. Notably, ABA can inhibit the expression of GA signaling by increasing the stability of *DELLA*, thereby inhibiting the growth of *Arabidopsis* roots [68]. In addition, the GA stimulation of *Arabidopsis* root elongation requires the involvement of growth hormone. GA-induced root elongation was inhibited when the stem tip, which is the main source of growth hormone, was removed; however, this inhibition was reversed when growth hormone was reapplied. The participation of auxin in GA-induced root elongation is completed by promoting the degradation and inactivation of DELLA protein, which is a prerequisite for GAs to stimulate root elongation [69]. This suggests that betaine can promote the degradation of DELLA protein under heat stress and enhance the output of the GA signal through three aspects: first, by increasing the level of GAs, promoting the combination of GAs and *GID1* to degrade or inactivate DELLA protein; second, by

inhibiting the promoting effect of ABA on the stability of DELLA protein; third, through the DELLA protein degradation pathway participated in by IAA. According to our results, the crosstalk of plant hormones also occurs in the seed germination stage under stress, and it can coordinate the balance between signals through exogenous substances to adapt to changes in the external environment. This provides a feasible solution to alleviate the plantgrowth crisis caused by global warming. The regulatory pathways of 10 mM exogenous betaine enhance the protrusion vigor of rice seeds under heat stress, as shown in Figure 9. In the future, we will explore the molecular regulatory mechanisms of betaine with respect to rice seed heat tolerance.

**Figure 9.** Regulatory pathways of 10 mM exogenous betaine enhance the protrusion vigor of rice seeds under heat stress.

#### **5. Conclusions**

In conclusion, according to our study, under 38 ◦C heat stress, the 10 mM betaine soaking treatment could alleviate the heat stress and promote the germination of rice seeds. In terms of physiology, the 10 mM betaine seed-soaking treatment promoted rice seed germination by increasing the SOD, POD and CAT antioxidant enzyme activities, decreasing the MDA content, increasing the soluble protein content to reduce ROS accumulation and mitigate membrane lipid peroxidation, and enhancing the osmoregulatory capacity to alleviate heat stress. By comparing the transcriptomes of the seed coleoptile and mesocotyl elongation stage, we obtained many DEGs involved in seed germination. According to the differential expression analysis, the betaine downregulated key genes in the H2O2 signaling pathway to reduce the accumulation of reactive oxygen species to mitigate the oxidative damage caused by heat stress. In addition, the betaine treatment affected the expressions of key genes related to the synthesis, metabolism and signal transduction of three endogenous hormones (GAs, IAA and ABA), increased the level of GAs, decreased the levels of IAA and ABA, and enhanced the output of the GA signal in rice seeds through the signaling crosstalk among the three hormones, which ultimately promoted the germination of rice seeds. The qRT-PCR results and the detection of the endogenous hormone levels further validated the expression patterns of these key genes. Our study reveals the molecular mechanism of betaine in regulating rice seed germination under heat stress, which provides an effective and feasible way to ensure the normal production of rice under the global warming trend.

**Author Contributions:** G.C. and Y.W. conceived and supervised the work; X.M. and J.Q. conducted the experiments, analyzed the data and prepared the figures; P.L. and H.Z. assisted in the data analysis; G.C. and Y.W. drafted the manuscript, together with X.M., J.Q., P.L. and H.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the National Natural Science Foundation of China (31971923; 31301650); the National Key R&D Program of China (2017YFD0301501); the Hunan Provincial Natural Science Foundation of China (2020JJ4360); the Key Scientific Research Project of Hunan Provincial Education Department of China (19A220); and the Innovation and Entrepreneurship Training Program for college students of Hunan Agricultural University (XCX2021038).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All of the data is contained within the article.

**Acknowledgments:** The authors thank Kai Wang and Zilu Liu of Hunan Agricultural University for all their help during the experiment.

**Conflicts of Interest:** The authors declare no conflict of interest.
