**1. Introduction**

Temperature can adversely affect the normal functioning of plant metabolism [1,2]. In the last few decades, climate change-induced rising temperatures have beome a major challenge for modern crop production, especially in southern Mediterranean regions [3]. Thus, efforts to achieve maximum crop yields to ensure food security for an ever-increasing human population remain challenging. Global food security could be jeopardized if mitigation efforts are not implemented aggressively [4]. Any fluctuation in an environmental cue such as temperature can directly affect the production of temperature-sensitive crops, and hence food security, causing considerable losses to growers and other stakeholders. According to the Inter-Governmental Panel on Climate Change, global temperatures increased by 0.74 ◦C within a century (1906–2005), which was ascribed to ongoing anthropogenic activities and their resultant greenhouse gases [5]. The terrestrial surface temperature is expected to increase by a further 1–6 ◦C by 2050, with arable areas projected to see the greatest increases [6]. There is little doubt that the ongoing climate change will affect all societies inhabiting the globe [7,8], decreasing crop production, and threatening food security. Therefore, strategies are needed to ameliorate its effects on modern agriculture.

Heat stress is a condition of unfavorable temperature causing irreversible damage to plants [9]. High-temperature stress adversely affects plant physio-biochemical and molecular characteristics, resulting in poor plant growth and development [10]. At the physiological level, heat stress negatively influences photosynthesis by adversely affecting

**Citation:** Zulfiqar, F.; Ashraf, M.; Siddique, K.H.M. Role of Glycine Betaine in the Thermotolerance of Plants. *Agronomy* **2022**, *12*, 276. https://doi.org/10.3390/ agronomy12020276

Academic Editors: Channapatna S. Prakash, Ali Raza, Xiling Zou and Daojie Wang

Received: 18 November 2021 Accepted: 20 January 2022 Published: 21 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the oxygen evolving complex, photosystem II, RuBisCo, and energy-(ATP) producing processes [11,12]. Furthermore, heat stress-induced disturbance of the electron transport chain leads to excessive reactive oxygen species (ROS) production in different cell organelles, such as mitochondria and chloroplasts, causing severe damage to DNA and cell membranes by inducing lipid peroxidation and ultimately causing cell death 13]. Increasing the capacity of heat stress-induced excessive ROS scavenging is considered an efficient defense strategy for ameliorating heat stress in plants [13]. A plant's thermotolerance ability is attributed to enhanced plasma membrane thermostability and reduced toxic ROS levels [14]. Plants have naturally adapted various defense mechanisms to counteract harsh environmental conditions such as heat stress. These defense mechanisms include an antioxidant machinery, osmolyte accumulation, maintenance of membrane integrity, and increased biosynthesis of heat-shock proteins (HSPs) by upregulating their associated genes' expression [15,16]. These defense mechanisms are involved in cellular defense against heat stress. Osmolyte accumulation has a significant role in mediating stress tolerance in plants (reviewed in Zulfiqar et al. [17]). Various studies have reported enhanced GB accumulation in plants under heat stress, revealing the positive role of this osmolyte in heat stress tolerance [18–22]. Sorwong and Sakhonwasee [23] stated that exogenous GB application alleviated the heat stress-induced reduction in leaf gas exchange traits.

There are no critical reviews on the role of GB in heat stress tolerance. Here, we summarize the fundamental impact of GB in inducing heat stress tolerance in economically important crops.

#### **2. GB Structure and Biosynthesis in Plants**

Glycinebetaine is an *N*,*N*,*N*-trimethylglycine quaternary ammonium compound that is naturally produced in numerous living organisms, including plants [24]. At physiological pH, GB is electrically a neutral molecule, but dipolar in nature. There are two pathways related to GB biosynthesis in plants [25]. The initiating metabolites for these pathways are choline and glycine [26]. GB biosynthesis in plants occurs via N methylation of glycine or the oxidation of choline [27]. GB biosynthesis, particularly in higher plants, is a two-step pathway beginning with choline, which is catalyzed by a ferredoxin-dependent Riesketype protein, namely, choline monooxygenase (CMO), and by a soluble NAD+-dependent enzyme [28,29]. Betaine aldehyde is oxidized by NAD+-dependent betaine aldehyde dehydrogenase (*BADH*) to produce GB. Both *BADH* and CMO generally exist in chloroplast stroma [24,30] (Figure 1).

**Figure 1.** Biosynthesis of GB in plant cells (Figure adapted from Sakamoto and Murata [24]; Ashraf and Foolad [31]).

#### **3. Glycine Betaine-Accumulating and -Non-Accumulating Plants**

Glycine betaine is a vital compatible osmolyte that plays multifarious roles in plant growth and metabolism. However, plant species differ in naturally accumulating GB. It is now evident that GB synthesis occurs in both chloroplasts and cytosol [32,33], but chloroplastic GB has been positively related to stress tolerance, whereas cytosolic GB has not shown such a relationship [32]. Thus, the presence of high amounts of GB in a plant may not necessarily account for its enhanced stress tolerance. Metabolic restriction of GB synthesis in plants has been ascribed to the availability of the substrate (choline) [33]. Since choline occurs in the cytosol [34], its transport to chloroplasts through appropriate transporters is essential for GB synthesis [34,35]. GB-non-accumulating plants have either limited amounts of intrinsic choline or poor activity of choline transporters in the chloroplast envelope [35]. Thus, genetic engineers need this information to generate transgenic lines with high GB-accumulating ability.

Plants accumulate various amounts of GB; naturally accumulating plants under normal and stress conditions are categorized as GB accumulators, while non-accumulating plants do not increase GB level under stress [36]. Table 1 lists GB accumulators and non-accumulators. Natural GB accumulators accrue a certain amount of GB solely under stressful cues [18,21]. For example, Alhaithloul et al. [22] studied the responses of *Catharanthus roseus* and *Mentha piperita* under HS and drought stress, individually and in combination. They reported that the level of GB increased by 46% and 58% for *C. roseous* and *M. piperita*, respectively, in response to HS, and more so under combined heat and drought stress. This evolutionary adaptation enables plants to survive under a varied range of climatic conditions. Screening plants for their ability to accumulate osmolytes, particularly GB, offers a way to target such plants for acquiring GB biosynthesis-related genes to develop GB-producing plants.

**Table 1.** Glycine betaine accumulating crops.

