**1. Introduction**

Rice (*Oryza sativa* L.) is one of the most important food crops, feeding more than half of the world s population [1]. Rice is a thermophilic crop, and temperature is one of the most important factors affecting its growth and development. However, greenhouse gas emissions are warming the planet s temperature because of a variety of human and natural forces. Researchers estimate that, by the end of the 21st century, the global average temperature will increase by 2–4 ◦C, and for every 1 ◦C increase in the minimum temperature in the growing season, the rice yield will be reduced by 10% [2,3]. According to estimates, the effects of global warming will cause a 41% reduction in the rice yield by the end of the 21st century [4].

**Citation:** Mo, X.; Qian, J.; Liu, P.; Zeng, H.; Chen, G.; Wang, Y. Exogenous Betaine Enhances the Protrusion Vigor of Rice Seeds under Heat Stress by Regulating Plant Hormone Signal Transduction and Its Interaction Network. *Antioxidants* **2022**, *11*, 1792. https://doi.org/ 10.3390/antiox11091792

Academic Editor: Nafees A. Khan

Received: 15 August 2022 Accepted: 7 September 2022 Published: 11 September 2022

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The reduction in the rice yield caused by heat stress mainly affects the important period of rice grain formation. For example, heat stress at the booting stage of rice leads to the degeneration of spikelets, pollen abortion, a decline in pollen vitality and then to an increase in dry grains [5,6]. Heat stress during the tassel and filling stages can prevent pollination, increase the number of blighted grains in the spike and reduce the fruit set rate, which results in the reduced weight of a thousand seeds and, hence, a lower rice yield [7,8]. Therefore, in the future development of rice, high temperature will be one of the main factors limiting stable and high rice yields.

Temperature is one of the main factors affecting seed germination. The optimum temperature for the germination of rice seeds is 25 ◦C. When the temperature is 32 ◦C, the germination is fast and neat; however, when the germination period encounters heat stress of more than 35 ◦C, the germination of rice seeds is inhibited, which involves the accumulation of reactive oxygen species (ROS) and changes in plant hormone levels [9]. Heat stress decouples the enzymes and metabolic pathways of plants. Heat stress further induces and triggers an oxidative stress reaction by destroying the stability of the cell membrane through membrane lipid peroxidation and protein denaturation, which result in the accumulation of a large number of reactive oxygen species, such as singlet oxygen, superoxide radical, hydrogen peroxide (H2O2) and hydroxyl radical [10]. Efficient enzymatic and nonenzymatic antioxidant defense systems play an important role in scavenging and detoxifying ROS to reduce membrane lipid peroxidation and maintain ROS homeostasis and redox signaling [11]. Heat stress leads to a reduction in the activity of important members of the antioxidant enzyme system, such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), which can further lead to an imbalance in the ROS system, accelerating the accumulation of ROS and ultimately inhibiting seed germination [12,13].

In addition to the excessive accumulation of reactive oxygen species, the imbalance of endogenous hormone levels caused by heat stress is also an important reason for inhibiting seed germination. Abscisic acid (ABA) inhibits seed germination, and gibberellins (GAs) promote it. Abscisic acid and gibberellins act antagonistically with each other in the plant to regulate seed germination [14]. The inhibition of seed germination by heat stress is called the thermal inhibition of seed germination. Thermal inhibition inhibits the expression of GA biosynthesis genes and ABA inactivation genes; however, it promotes the expression of ABA synthesis genes [15]. The mitigation of heat stress by ABA is accomplished through synergistic effects with nitric oxide (NO) to increase the antioxidant enzyme activity, reduce the levels of H2O2 and thiobarbituric acid reactive substances (TBARS), and increase carbohydrates, adenosine triphosphate and heat-shock proteins in plants [16,17]. Auxin (IAA) is the second hormone after ABA that positively regulates seed dormancy [18]. In response to heat stress induction, the expression levels of the growth hormone synthesis genes *OsIAA13* and *OsIAA20* were increased, and the growth hormone response gene *SAUR* was also substantially induced to be expressed [19]. The elongation and growth of *Arabidopsis* hypocotyl under heat stress is related to the increase in the auxin content [20].

Betaine is a quaternary amine compound that is commonly found in bacteria, fungi, higher plants and animals [21]. It plays an extremely important role in plant resistance to heat stress. The heat tolerance of tomato lines transformed with the *codA* gene of the betaine synthesis gene was significantly higher than that of tomato lines without betaine [22]. During the germination of *Arabidopsis* seeds, transgenic plants with the *codA* gene increased the level of betaine, thereby enhancing the high-temperature tolerance of the seeds and promoting seed germination [23]. Endogenous betaine can enhance the high-temperature tolerance of plants. After spraying betaine on leaves, the photoinhibition caused by the heat stress of *Tagetes erecta* was reduced, and the CO2 assimilation rate, stomatal conductance and transpiration rate improved. Betaine also reduced the levels of hydrogen peroxide, superoxide, lipid peroxidation and cell death [24]. Spraying betaine on tobacco leaves significantly increased the activities of SOD and POD, increased the contents of chlorophyll and proline, and decreased the contents of malondialdehyde (MDA), alleviating heat stress and promoting the accumulation of tobacco biomass under the combined stress of high

temperature and drought [25]. Spraying leaves with 10mM betaine improved the average total number of florets obtained in summer maize Jingnongke 728 under the different treatments of early sowing date, normal sowing date and late sowing date, increasing by 5.09%, 4.70% and 2.27%, respectively. The yield of summer maize Jingnongke 728 after spraying betaine increased by 3.05–12.81% [26]. Rice is a typical non-betaine-accumulating species [27].

At present, researchers conduct investigations into betaine to alleviate the stress damage suffered by rice by introducing the betaine enzyme system into rice or by exogenous application through transgenic technology. Transforming the *BADH* gene encoding betaine aldehyde dehydrogenase from barley into rice promoted the accumulation of betaine in rice and significantly improved the tolerance of rice to salt, cold and heat stress [28]. The exogenous application of betaine can also effectively improve the adaptability of rice. Spraying 50 mM or 100 mM concentrations of betaine on the leaves effectively enhanced the osmotic adjustment ability of rice and improved its adaptability to osmotic stresses, such as salt, drought and heat stress [29–31]. These reports mostly investigate the effect of betaine on rice-plant morphogenesis; however, we lack research on the seed germination stage before seedling morphogenesis, and we do not yet understand the molecular mechanism of betaine to alleviate stress damage. In order to study the mechanism of exogenous betaine during rice seed germination, we took XZX45 as the test material, soaked seeds with different concentrations of betaine and carried out seed germination tests at different temperatures. We further measured the antioxidant enzyme activity, MDA content, soluble protein content and endogenous hormone content under heat stress. After heat stress at 38 ◦C for 24 h, we constructed the cDNA libraries for transcriptome sequencing. We identified differentially expressed genes (DEGs) related to plant endogenous hormone synthesis and metabolism, plant hormone signal transduction and reactive oxygen species accumulation, and we verified the credibility of these differentially expressed genes by qRT-PCR. Finally, we clarified the physiological characteristics of soaking seeds in betaine solutions to alleviate the seed coleoptile and mesocotyl elongation stage under heat stress, and we revealed the molecular mechanism of the betaine promotion of seed germination by regulating plant hormone synthesis and metabolism, as well as signal transduction.
