**5. Improvement in Heat Tolerance through Exogenous Application of GB**

Exogenous application of GB improves thermotolerance in many plants by enhancing their growth and yield via counteracting metabolic maladjustments caused by HS (Table 2). For example, while appraising the role of exogenous GB application on heat-stressed tomato plants, Li et al. [60] reported enhanced seed germination, expression of heatshock genes, and accumulation of heat-shock protein 70 (HSP70). Likewise, exogenous GB supplementation likely controls many other key metabolic processes in heat-stressed plants. For example, exogenously applied GB protected photosystem II (PSII) in heatstressed plants of *Hordeum vulgare* [61] and *Triticum aestivum* [20] and decreased the relative membrane permeability and leakage of ions such as Ca2+, K+, and NO3 − in heat-stressed barley seedlings [62]. Furthermore, GB supplied to sprouting sugarcane nodal buds under HS markedly reduced H2O2 generation, increased K<sup>+</sup> and Ca2+ contents, and increased the levels of endogenous GB, free proline, and soluble sugars, enhancing the overall growth [63]. Sorwong and Sakhonwasee [23] stated that exogenous GB supplementation alleviated the heat stress-induced reduction in CO2 assimilation rate, stomatal conductance, relative water content, and transpiration rate in marigold. The high-temperature-induced oxidative stress in marigold was mitigated due to reduced levels of H2O2, peroxide, superoxide, and lipid peroxidation [23]. Exogenous application of GB during mid-flowering in heat-stressed tomato in the field increased fruit yield [64]. In apple, the application of GB enhanced photosynthesis under individual HS or drought stress and combined stresses [65]. In a three-year field study, Chowdhury et al. [66] evaluated the role of GB and potassium nitrate in heat-stressed late-sown wheat; these osmolytes improved grain yield under heat stress compared to the control. Hence, it is clear that exogenously applied GB mediates HS. However, future studies should focus on field-based heat stress evaluations of different crops.

**Table 2.** Effect of exogenously applied GB on the regulation of different physio-biochemical attributes in heat-stressed plants.



**Table 2.** *Cont.*

Abbreviations: HSPs: Heat-shock proteins, PSII: Photosystem II, ROS: Reactive oxygen species, *psbA*: PSII protein D1 precursor gene.

#### **6. Genetic Engineering for Enhanced Thermotolerance**

Developing transgenic plants for thermotolerance is a cost-effective and efficient biotechnological approach for achieving optimum agricultural production under the changing climate scenario [17]. Genes involved in encoding GB biosynthetic enzymes in different organisms and plants have been cloned to produce transgenic plants overexpressing one or more of these genes to enhance endogenous GB production, improving HS tolerance [67,68] (Table 3). For example, Zhang et al. [68] compared the thermotolerance ability of two transgenic tomato lines containing the betaine aldehyde dehydrogenase (*BADH*) and choline oxidase (*COD*) genes responsible for GB synthesis. They observed that *codA* transgenic plants had higher GB levels, CO2 assimilation rate, and photosystem II (PSII) photochemical activity and lower accumulation of H2O2, O2 •−, and malondialdehyde (MDA) than wild-type (WT) plants. In addition, the codA transgenic line had higher heat-response gene expression, heat-shock protein 70 (HSP70) accumulation, and expression of a mitochondrial small heat-shock protein (MT-sHSP), heat-shock cognate 70 (HSC70), and heat-shock protein 70 (HSP70) than WT plants under HS. In another study, Yang et al. [69] reported GB accumulation in tobacco by introducing the *BADH* gene in tobacco, which increased tolerance to high-temperature stress and improved photosynthesis. While transferring the *BADH* gene from spinach to tomato, Li et al. [67] reported enhanced accumulation of GB, antioxidant activity, and photosynthetic capacity by improving D1 protein content, which could repair heat stress-induced damage to PSII. Furthermore, transgenic tomato accumulated less MDA and ROS (H2O2 and O2 •−), reducing oxidative stress relative to the WT. Reduced oxidative stress and restored PSII from HS-induced enhanced photoinhibition occurred in transgenic tobacco plants transformed with the *BADH* gene relative to the WT [70]. The role of *BADH* in xerophyllic *Ammopiptanthus nanus* under severe stress was confirmed by transferring the *BADH* gene of this plant into *E. coli* treated with 700 mM NaCl at 55 ◦C; the transgenic bacteria showed tolerance to these combined stresses [71]. The above studies reveal the positive role of GB-related genes in providing stress tolerance in plants. The introgression of GB synthesis-related genes could enhance endogenous GB accumulation to protect plants from heat-induced oxidative stress.

Numerous studies have been conducted on engineering GB biosynthesis [33]. Performance of single-gene-based transgenics under field conditions may not be the same as that reported under controlled or semi-controlled conditions. Thus, the development of transgenic lines by transforming with multiple genes (pyramiding of genes) for enhanced GB biosynthesis under heat stress is plausible.


**Table 3.** Genetic engineering

 for enhanced GB

accumulation

 and improved

thermotolerance

 in different crops.


**Table 3.** *Cont.*
