Targeting Glycinebetaine for Abiotic Stress Tolerance in Crop Plants: Physiological Mechanism, Molecular Interaction and Signaling

: In the era of climate change, abiotic stresses (e.g., salinity, drought, extreme temperature, flooding, metal/metalloid(s), UV radiation, ozone, etc.) are considered as one of the most complex environmental constraints that restricts crop production worldwide. Introduction of stress-tolerant crop cultivars is the most auspicious way of surviving this constraint, and to produce these types of tolerant crops. Several bioengineering mechanisms involved in stress signaling are being adopted in this regard. One example of this kind of manipulation is the osmotic adjustment. The quarternary ammonium compound glycinebetaine (GB), also originally referred to as betaine is a methylated glycine derivative. Among the betaines, GB is the most abundant one in plants, which is mostly produced in response to dehydration caused by different abiotic stresses like drought, salinity, and extreme temperature. Glycinebetaine helps in decreased accumulation and detoxification of ROS, thereby restoring photosynthesis and reducing oxidative stress. It takes part in stabilizing membranes and macromolecules. It is also involved in the stabilization and protection of photosynthetic components, such as ribulose-1, 5-bisphosphate carboxylase/oxygenase, photosystem II and quarternary enzyme and protein complex structures under environmental stresses. Glycinebetaine was found to perform in chaperone-induced protein disaggregation. In addition, GB can confer stress tolerance in very low concentrations, and it acts in activating defense responsive genes with stress protection. Recently, field application of GB has also shown protective effects against environmental adversities increasing crop yield and quality. In this review, we will focus on the role of GB in conferring abiotic stress tolerance and the possible ways to engineer GB biosynthesis in plants.


Introduction
From the prehistoric era, natural calamities came in the form of abiotic stress and hampered the growth and productivity of plants. As a sessile organism, plants frequently face a glut of abiotic stresses such as salinity, drought, flooding and submergence, low and high temperature, UV radiation, ozone, nutrient deficiency and metal toxicity [1]. With the present global climatic change, these abiotic stress factors are taking place more frequently than earlier times leading to the vulnerability of crop productivity, and creating transduction mechanisms towards cellular responses for conferring abiotic stress tolerance are the focal points of this review.

Glycinebetaine Biosynthesis and Metabolism in Plants
Glycinebetaine is a naturally occurring quaternary ammonium compound (N, N, N-trimethylglycine), which is dipolar yet electrically neutral at physiological pH [27]. The biosynthesis of GB is usually characterized by the double step oxidation of choline using the toxic intermediate, betaine aldehyde. The enzymes participating in this process are usually localized in the stroma of the chloroplast. These are the ferredoxin-dependent soluble Rieske-type protein, choline monooxygenase (CMO) and the soluble NAD + -dependent betaine aldehyde dehydrogenase 1 (BADH1) [28]. Choline formed from the L-serineethanolamine pathway is reduced to the toxic betaine aldehyde via the action of CMO. Betaine aldehyde dehydrogenase 1 uses NAD + as the proton acceptor and oxidizes betaine aldehyde into GB (Fig. 1) [29]. Ladyman et al. [30] identified GB as an inert end product in barley plants, which accumulate in the young shoots exposed to stress but does not get degraded even after the stress cues are removed. However, radiotracers showed that the distribution of GB among the plant tissues changed. Although GB produced in mature leaves during stress, but is translocated to new expanding leaves through the phloem under well-watered conditions [30].

Figure 1:
The biosynthesis of GB involves two-step catalysis of choline formed from the serineethanolamine pathway. Choline is acted upon by choline monooxygenase and reduced to betaine aldehyde along with simultaneous oxidation of ferredoxin (Fdox). The betaine aldehyde formed is again oxidized to GB via reduction of NAD + to NADH by betaine aldehyde dehydrogenase

Glycinebetaine Transport and Trafficking in Plants
Glycinebetaine is a crucial osmolyte, and its transport and optimum distribution throughout the cell is essential for maintaining the cellular osmotic pressure and generating abiotic stress tolerance [31]. Considerable structural specificity has been observed in case of GB transport in Escherichia coli, where the kinetics of GB entry could be associated with the Michaelis-Menten relationship reproducing an affinity (Km) of 35 μM and a maximum velocity (Vmax) of 42 nmol min -1 mg -1 protein [32]. It was also observed that the GB transport is critically regulated by an active and specific system dependent on the osmotic strength of the growth medium [32]. Lai et al. [33] also identified a high-affinity and high-specificity GB transporter in the halophilic archaeon, Methanohalophilus portucalensis, which allowed GB uptake at an observed Vmax of 8 nmol min -1 mg -1 proteins. The Gram-negative bacterium, Azospirillum brasilense, plays an important role in nitrogen fixation in association with the roots of graminaceous plants. Addition of GB in the medium during salt stress enabled trafficking of the osmolyte within the bacterial cells due to the presence of a periplasmic GB-binding protein. This stimulated the nitrogen fixation activity even under salt stress [34]. The mechanism of GB transport in plants is not yet elaborately understood. However, some reports indicate the presence of a strictly operating transporter, which is responsive to sub-optimal conditions and extracellular ionic imbalances [35].
Two full-length GB transporter genes (AmT1 and AmT2) were cloned from the mangrove species, Avicennia marina [35]. These genes exhibited high homology with the proline (Pro) transporters of Arabidopsis and tomato. When E. coli cells were complemented with GB and Pro transporters, it resulted in efficient uptake of GB and Pro. Interestingly, mutations of Thr290-Thr291-Ser292 in the GB transporters to Arg290-Gly291-Arg292 resulted in a constitutively active form, independent of ionic concentrations. These results indicate that incorporation of the positive amino acids yields a positive conformational change in the quarternary structure of the transporters [35]. In another study, Schwacke et al. [36] identified Pro transporter 1 (ProT1) in tomato pollen, which transported Pro and γ-aminobutyric acid (GABA) with low affinity and GB with high affinity. This membrane protein was observed to be the member of the amino acid permeases (AAPs), which ensure proton-coupled uptake of diverse molecules ranging from amino acids to compatible solutes [36]. Fig. 2 proposes a general mechanism of GB trafficking within plant cells.

Figure 2:
Glycinebetaine transport into the cell is induced by ionic imbalance due to osmotic stress. This protein belongs to the class amino acid permeases. The GB transporter co-transports H + ions and is also permeable to Pro and γ-amino butyric acid to a lower extent (dotted arrow)

Variation of Glycinebetaine Content in Plants Under Different Stress
Glycinebetaine, the most important organic osmolytes, show variation in their accumulation in diverse plant species due to the imposition of different environmental stresses such as salinity, drought, high temperatures, heavy metals, chilling, UV radiation, etc. [8,37,38]. It is involved in the protection of many plant species against a range of oxidative stress under stressful conditions, such as, salinity [39][40][41][42], drought [43][44][45][46], metal/metalloid stress [47][48][49], high temperature [45,50] and chilling [23,51,52]. A lot of research findings confirmed the positive relationship between accumulation of GB and plant stress tolerance. Glycinebetaine accumulation also greatly varied depending on the stress type and plant species (Tab. 1). Raza et al. [53] checked the GB-induced modulation of antioxidant enzyme activities and ion accumulation in two wheat cultivars differing in salt tolerance. They observed that the salt-tolerant cv. S-24 accumulated significantly higher GB in the leaves than that in cv. MH-97 (salt-sensitive MH-97 under saline conditions. Two Tunisian populations of the Mediterranean shrub, Atriplex halimus L., [plants originating from a salt-affected coastal site (Monastir) or a non-saline semi-arid area (Sbikha)] were exposed to a nutrient solution containing 160 mM NaCl or drought-inducing 15% polyethylene glycol (PEG). Both drought and salt stress condition drastically increased GB content of the two plants, but higher GB content was observed under salt stress condition and in Montasir genotypes [44]. Under salt stress condition, the increase of GB content was 712 and 326% higher than the control in Montasir and Sbikha genotypes, respectively, whereas it was 559 and 216% higher in Montasir and Sbikha genotypes, respectively under drought condition. Kholová et al. [54] observed the effect of long-term soil salinity [S0 (0, 0, 0 mM), S1 (50, 25, 25 mM), S2 (100, 50, 50 mM) and S3 (150, 75, 75 mM) NaCl, Na2SO4 and CaCl2, respectively] on two maize (Z. mays L.) genotypes, PEHM 3 (comparatively tolerant) and Navjot (susceptible) at vegetative and anthesis stages during summer/rainy season. Compared with control, salinity stress increased GB content by 9, 39, 20% in Navjot genotypes and 20, 70, 83% in PEHM 3 genotypes under S1, S2, and S3 stress condition, respectively. Two eggplant (Solanum melongena L.) cultivars, Dilnasheen and Bemisal, showed a similar result under salt stress (100 mM NaCl). Glycinebetaine accumulation in the leaves of both eggplant cultivars was considerably enhanced due to NaCl stress; GB accumulation in cv. Dilnasheen was higher than that in cv. Bemisal under salt stress [55]. Khan et al. [56] screened out four wheat (Triticum aestivum L.) cultivars 711, PBW343, 3765 and WH542 for checking variations in GB concentration under 100 mmol L −1 NaCl stress. Salt-treated wheat plants showed greater accretion of GB than control plants. Maximum accumulation of GB (45.4%) was observed in wheat cultivar 711, whereas minimum accumulation was found in cultivar WH542 (14.3%) in contrast to control. The other two cultivars, PBW343 and 3765 accumulated 33.3% and 22.2% GB, respectively, under salt stress, compared to their respective control. Khan et al. [40] checked the effect of salt stress in mungbean (Vigna radiata L.) under saline condition (100 mM NaCl) and found that GB content increased by 31.9% in salt-treated plants in comparison with control plants. Tsutsumi et al. [41] treated Atriplex gmelini plants with three levels of NaCl (0, 50 or 250 mM). They observed that all salinity levels increased GB content in both bladder hairs and leaf laminae, but high salinity (250 mM NaCl) conditions showed a significant increase of GB content in bladder hairs than leaf laminae. Wei et al. [42] used a transgenic line and wild-type tomato (S. lycopersicum cv. 'Moneymaker') plants in a salinity experiment. They showed GB content increased in both wild-type and transgenic lines under 200 mM NaCl; the GB content in the transgenic lines was significantly higher than that in wild-type plants.
Drought stress also increased the endogenous level of GB. Ma et al. [43] checked the performance of two T. aestivum cultivars, HF9703 (drought-tolerant) and SN215953 (drought-sensitive) under drought stress condition and found that GB content of wheat plant increased with the increase of drought stress in HF9703 cultivar; however, in SN215953, it first increased and then declined. Wang et al. [45] used a transgenic T. aestivum line T6 and its wild-type Shi4185 for checking the GB-induced tolerance of the photosynthetic apparatus to drought (30% PEG-6000) and heat (40°C) stress. Stress-induced GB levels increased significantly in the leaves of the two lines, especially in T6, showing that the accumulation of GB sensitized the wheat plants to these stresses. Two maize cultivars, Agaiti-2002 (drought tolerant) and EV-1098 (relatively drought sensitive), were also exposed to water deficit conditions. As a result, endogenous GB content considerably increased in both leaf and seed. The cultivars did not differ significantly in leaf GB contents under both water stress and non-stress conditions; however, in case of seed, slightly higher GB contents were observed in cv. Agaiti-2002 than those of EV-1098 [60]. Basu et al. [61] subjected three rice cultivars to PEG-induced dehydration stress and have shown that GB level was maximally triggered after stress imposition in Pokkali, the salt-tolerant cultivar, while it was induced to a lesser extent in both the salt-sensitive cultivars, IR-29 and Pusa Basmati. Later, Abbas et al. [37] tested thirteen genotypes of sugarcane (Saccharum officinarum L.) under drought stress (5%, 7.5%, 10% and 12.5% PEG) and found that GB content increased significantly with the increase of drought severity.
Duman et al. [47] exposed an aquatic plant (Lemna gibba L.) to different levels of cadmium (Cd) and found that in contrast to control, GB content increased by 26, 189 and 111% under 0.5, 1.0 and 3.0 mM CdCl 2 stress, respectively. Glycinebetaine content also increased in perennial ryegrass under Cd stress condition [48]. In contrast to control, Cd stress (0.5 mM CdCl2) increased GB content by 164% in shoots and 56% in roots of perennial ryegrass. Farooq et al. [49] experimented with a cotton genotype MNH 886 under Cd stress (1.0 and 5.0 μM CdCl2). They observed GB concentrations significantly increased in both leaves and roots with increasing Cd levels in the growth medium in contrast to control. The up-regulation of BADH1 transcript has been noted in the rice varieties, IR-64 and Nonabokra, during 1.5 mM CdCl2 stress that highlights a direct correlation between Cd-tolerant capacity of each variety and BADH1 gene expression level and hence GB level [62].
Sugarcane (Saccharum sp. cv. HSF-240) plant showed higher GB content under heat stress condition (42 o C) in contrast to control plants [57]. Sorwong and Sakhonwasee [58] checked the effects of GB under heat stress conditions. They exposed three marigold cultivars, namely, 'Narai Yellow', 'Bali Gold', and 'Columbus Orange' to heat stress (39°C/29°C day/night temperature). Heat stress conditions caused an increase of endogenous GB content. Zhang et al. [59] used wild-type T. aestivum cv. Shi 4185 and three transgenic lines (T1, T4, and T6) expressing the BADH gene isolated from A. hortensis L. under cold stress. Following two days of cold treatment (2/0°C day/night temperature), the GB content increased significantly in all the plants. The increase of GB content was higher in the transgenic lines than the wild-type. Nayyar et al. [63] recorded that chilling stress (12-14/3-5°C day/night temperatures) in chickpea (Cicer arietinum L.) increased the GB content more than two-folds on the 4th day of stress in comparison with the 1st day of stress, but it started to quickly decline subsequently. Xing and Rajashekar [51] experimented with A. thaliana (Columbia) under cold stress (4/2°C (day/night, 4 weeks and GB (10 mM) was applied exogenously to the plants as a foliar spray for increasing stress tolerance. The endogenous level of GB in the leaves augmented noticeably throughout the cold acclimation treatment. During the first day of cold acclimation, the endogenous GB level was increased by three-folds and following three days of cold acclimation, the endogenous GB content reached at the maximum. Later, fluctuation in GB content was observed during the whole stress period. Rasheed et al. [52] cultivated sugarcane (HSF-240) bud under chilling stress for 2 d. They maintained stress treatments by imposing temperature in a growth chamber. Under chilling stress, the GB accumulation of plant notably increased. A similar increase of GB content was observed in cotton cv. Lumianyan19 after 3 d of cold stress [5 o C; 23].

Glycinebetaine-Induced Improvement in the Photosynthetic Machinery
The chloroplast is the site of GB production. The final steps of GB biosynthesis are localized in the stroma. Synthesis of this zwitter ionic osmolyte leads to improved photosynthetic efficiency during abiotic stresses [64]. Glycinebetaine protects the soluble stromal and luminal enzymes, lipids and proteins involved in photosynthesis [2; Fig. 3]. Abiotic stresses induce the generation of excess electrons from photosynthesis. Glycinebetaine reportedly acts as an additional or alternative biochemical sink for quenching the excess electrons and prevents the photosynthetic apparatus from over-reduction. Thus, GB reverses photosynthetic inhibition [65]. Glycinebetaine accumulation under sub-optimal conditions enhances the photosynthetic CO2 fixation, stomatal conductance and water-use efficiency [66]. In addition, GB inhibits the stress-induced reduction in the energy trapping efficiency by protecting the oxygen-evolving center of photosystem II (PSII) and maintaining the PSII activity. Moreover, GB protects the photosynthetic apparatus from chronic photoinhibition by preventing over-reduction of QA [31]. Ohnishi and Murata [67] reported the abilities of GB to repair stress-induced photo damages of PSII. The inhibition of the PSII reaction core protein, D1, and its degradation was also reversed by GB [67]. Glycinebetaine also counteracts abiotic stresses by inducing RuBisCO activase and promoting CO2 fixation [10].

Figure 3:
Glycinebetaine acts as an alternate sink for absorbing the excess electrons leaked from the electron transport chains (ETCs) of mitochondria and chloroplast. GB inhibits chlorosis and protects lipids and proteins. It also exhibits chaperonin activity and preserves enzyme structures during oxidative stress. GB recharges the antioxidant machinery by activating the enzymes involved in the ascorbateglutathione (AsA-GSH) cycle and glyoxylase I (GlyI). The latter enables efficient detoxification of the toxic methylglyoxal (MG). GB also induces the expression of downstream stress-inducible genes encoding late embryogenesis abundant (LEA) proteins

Glycinebetaine-Induced Antioxidant Defense
Being an osmolyte, GB acts as a potential scavenger of the toxic ROS produced during abiotic stress [68]. The ROS consists of hydrogen peroxide (H2O2), superoxide (O2 •− ) and hydroxyl radicals (OH • ), which are produced when electrons from the mitochondrial and chloroplastic electron transport systems leak and interact with O2 in the dearth of conventional electron acceptors. The ROS degrades the membrane systems by inducing uncontrolled lipid peroxidation and triggers distortions in protein and nucleic acid structures [69]. Almost all kinds of abiotic stresses result in a burst in the production of ROS. Effective ROS scavenging by non-enzymatic antioxidants like Pro, polyamines (PAs), ascorbate (AsA), glutathione (GSH), etc. and enzymatic antioxidants like superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione reductase (GR), etc. is crucial for maintaining the physiological processes under adverse situations [68]. The cellular accumulation of GB recharges the antioxidant machinery and preserves the optimum osmotic pressure during desiccation [43]. Interestingly, GB itself does not exhibit antioxidant activity but induces the ROS-defense machinery [70]. Exogenous application of GB in tobacco cell cultures induced the activities of the enzymes like APX, dehydroascorbate reductase (DHAR) and GR participating in the AsA-GSH cycle [71]. However, such GB-mediated induction was visible only under salt stress and not under control conditions, indicating towards the fact that GB regulates ROS detoxification by sensing its level within the cell [71]. Increased GB accumulation reduces cellular ROS production and stabilizes the rate of CO2 assimilation in plants grown under unfavorable conditions [72].
The glycolytic metabolism produces methylglyoxal (MG) as a toxic by-product, which severely inhibits cell growth in plants. The MG content steeply increases during abiotic stresses and is required to be neutralized in order to maintain the cellular physiology [73]. Banu et al. [74] reported the exogenous application of GB to decrease the MG and H2O2 content in tobacco cells cultured in a saline medium. The cells exhibited increased transcript accumulation of GPX. Hoque et al. [75] reported that application of GB reduced protein carbonylation in tobacco cell cultures in a saline medium. The treated cells also exhibited increased GSH redox state and activities of GPX, glutathione S-transferase (GST) and glyoxalase I (Gly I) during salinization. This effectively indicated that GB activates the integrated systems involved in antioxidant defense and MG detoxification [75; Fig. 3].

Glycinebetaine-Induced Abiotic Stress Tolerance in Plants
Glycinebetaine is a compatible solute capable of performing multiple functions as a non-enzymatic antioxidant. From our previous discussion, it is clear that GB crucially maintains normal cellular physiology under harsh environmental conditions. Such properties of this osmolyte have enabled its diverse participation in generating abiotic stress tolerance across plant species.

Drought
Water deficit or physical drought stress is one of the most prevalent types of edaphic stress, which severely limits the growth and yield of susceptible plants [64,76]. Some plant species have developed evolutionary strategies to tackle such stressful situations by triggering the accumulation of protective compatible solutes and antioxidative enzymes [77,78]. Several plant studies showed that GB protects the plants against drought stress (Tab. 2). Ibrahim et al. [79] compared the level of osmolyte accumulation in two cotton genotypes, Zhongmian 23 (stress tolerant) and Zhongmian 41 (sensitive). It was observed that the former exhibited its tolerance due to enhanced accumulation of GB, soluble sugar and K + under both drought (4% soil moisture) and salt stress (200 mM NaCl). Zhongmian 23 also displayed higher activities of SOD, POD, CAT, APX, and Na + /K + -ATPase [79]. In another recent study, it was observed that exposure to drought stress increased GB, Pro and soluble sugar content in the flag leaves of wheat plants [80]. Further, proteomic investigations using two-dimensional difference gel electrophoresis (2D-DIGE) identified 66 and 105 differentially accumulated protein (DAP) spots in the flag leaves and grains during drought stress. These proteins were mainly associated with oxidative stress responses, photosynthesis, starch, and energy metabolism [80].
Drought priming of wheat seeds promoted drought tolerance in the progenies of the treated seeds [81]. Successive application of drought priming in three generations resulted in improved photosynthetic rate and antioxidant capacity in the offspring plants. The physiological basis of such drought tolerance was attributed to higher BADH and Δ 1 -pyrroline-5-carboxylate synthase (P5CS) activities, leading to enhanced accumulation of GB and Pro in the offspring seedlings of the treated plants. Thus, GB acts as a crucial osmolyte, which promotes parental priming-induced drought tolerance [81]. Hydropriming and osmopriming of bread wheat seeds with 1.5% CaCl2 promoted trans-generational drought memory and improved salt tolerance due to increased accumulation of leaf GB and Pro. These osmolytes reduced malondialdehyde (MDA) and Na + contents and improved leaf area and water relation parameters in the offspring plants exposed to stress [82]. Analysis of the upstream promoter of phosphoethanolamine N-methyltransferase (PEAMT) gene (involved in choline and hence GB biosynthesis) led to the identification of stress-inducible cis-acting elements like abscisic acid (ABA)-responsive elements (ABREs) involved in salt and drought stress responses [91]. The promoter assay using β-glucuronidase (GUS) fusion displayed significant GUS expression under drought, salt, and oxidative stresses. This indicates the inducible status of this gene in accelerating GB accumulation under abiotic stress [91]. In another study, Kumar et al. [83] showed the involvement of GB in conferring drought and salt tolerance in ornamental plant Nerium oleander. The stressed plants also accumulated soluble carbohydrates and exhibited mechanisms, which inhibited the transport of toxic ions from the roots to the aerial biomass [83].

Salt
Salinity is regarded as the physiological drought stress, where Na + ions present in the soil at a toxic level infiltrate the cell membrane and disrupt endogenous osmotic pressure. A large array of enzymes and proteins are inhibited in the presence of excess Na + ions [92]. Being a potential osmoprotectant, GB mitigates salt-induced injuries by maintaining the cellular turgor, protecting biomacromolecules and inducing antioxidant levels (Tab. 2).
Foliar treatment of zinc (Zn) and copper (Cu) mitigated the injurious effects of salt by enhancing the contents of GB, Pro, total free amino acids and sugars in Z. mays seedlings [85]. Ghorbani et al. [86] recently reported that inoculation of tomato plants with the symbiotic fungus, Piriformospora indica, improved the growth parameters under salt stress. Symbiotic association triggered the accumulation of GB and Pro, which efficiently protected the chlorophyll (chl) pigments, and hence, the plants could maintain enhanced photosynthetic efficiency under sub-optimal edaphic conditions. This finding opens a new avenue, where GB is associated with symbiosis to regulate abiotic stress tolerance in plants. Glycinebetaine, Pro, total phenolics and soluble carbohydrate content sharply increased in the rootstock and leaves of pistachio plants on exposure to salt stress [87]. The accumulation of the osmolytes also facilitated exclusion of Na + and Clin the rootstocks of the assayed cultivars grown under saline conditions [87].
Kageyama et al. [93] recently identified a novel methyltransferase, TpGSDMT in the marine diatom, Thalassiosira pseudonana that catalyzed a three-step synthesis of GB. Due to the induction of this gene during salt stress, it was proposed to be involved in salt tolerance in T. pseudonana cells [93]. In an interesting study, Carillo [94] reported the accumulation of GABA and not GB in durum wheat plants exposed to salt stress. It has been proposed that the stress associated metabolomic plasticity favors the GABA shunt instead of GB production in salt-stressed durum wheat seedlings [94]. Sabzalian et al. [88] observed that the GB content did not markedly increase in the halophytic plant species Distichlis spicata on exposure to salt stress. This might be due to the fact that the species being a halophyte maintains high endogenous levels of GB, which efficiently maintains cellular osmotic pressure under stress. However, the GB content steeply increased under salt stress in another halophytic species, Suaeda aegyptiaca indicating towards the stress-induced metabolic plasticity existing across plant species [88]. Leon et al. [89] also reported the effectiveness of GB and arsenobetaine in ameliorating salt-induced damages in the halophile, Spiribacter salinus. The radiolabeled GB was used to study its transport, and it was observed that the GB pool was intricately associated with the presence of salt in the growth medium. Recently, the salt-induced aminoaldehyde dehydrogenase (AMADH) similar to the conventional BADH gene was characterized in the halophyte Lycium ruthenium Murr. [90]. Induction of AMADH1 upon salinization led to significant GB accumulation in the seedlings as well as in fruits. On the contrary, virus-mediated AMADH1 silencing interfered with GB accumulation and reduced the salt tolerating capacity of the plants [90]. This indicates the promising roles of GB in conferring salt tolerance traits across plants.

Toxic Metals/Metalloids
Over increasing population of the world has accelerated industrialization and urbanization resulting in accumulation and release of excessive amounts of different toxic metal/metalloids to the environment [95]. Excess concentrations of toxic metal/metalloids impose severe threats on both flora and fauna. Most of the toxic metal/metalloids are not bio-degradable and can bio-accumulate in living organisms; consequently, they can contaminate the entire food chain [96]. They affect the surroundings by destroying soil, water, and air properties, which reduce the quality and quantity of crop and eventually destroy human health [95]. Toxic metal/metalloids in the plant-growing media facilitate their entry to the plant cell, which causes oxidative damage due to overproduction of ROS. Every organism has well established antioxidant defense systems to defend its cells against ROS at a certain level. Moreover, plant cell increases different osmolyte synthesis to reduce ROS-induced oxidative damage [95]. Glycinebetaine is one of the most abundant osmolytes produced in higher plants under stressful environments and involved in the protection of plants against different toxic metal/metalloids stress [12,97,98;Tab. 3]. However, the natural manufacture of GB in cellular level is not sufficient to defend plants from severe stress. Under these circumstances, exogenous application of GB gradually becomes a familiar strategy to overcome abiotic stresses in plants including toxic metal/metalloids stress. However, its response differs with plant genotypes and stress type and intensity [12]. In most of the cases, exogenous application of GB decreased the accumulation of toxic metal/metalloids in the plant [49,98,99]. However, Duman et al. [47] showed the reverse result. They suggested that the addition of GB in the growing media under stress decreased the accumulation of Cd in the plant. In general, the increase or decrease of metal accumulation depends on the dose of both stress and GB. As GB scavenges a range of ROS, promotes osmotic adjustment, stabilizes proteins and protects membrane integrity in plants under adverse conditions, it plays a vital role to confer tolerance in plants against different metal/metalloids toxicity [100]. Islam et al. [101] exposed suspension-cultured cells of Nicotiana tabacum L. to 100 mM CdCl2 with or without 1 mM and 10 mM GB. They recorded that exogenous GB at 10 mM notably decreased the amount of MDA under Cd stress, but the application of 1 mM GB did not result in a noteworthy decrease of MDA content. Both the doses of GB decreased the Cd accumulation of plant. A similar reduction of MDA due to supplementation of GB was recorded in Cd-affected mung bean seedlings [102]. Duman et al. [47] treated Lemna gibba plant with different doses of Cd (0.5, 1, and 3 mM CdSO4.7H2O) and GB (0.5, 1, 2, and 5 mM). Exposure to 0.5 mM GB reduced the Pro content, but an increase was observed for 2 and 5 mM GB. Significant MDA content reduction was observed under 0.5 mM GB treatment in Cd affected plants. Later Bharwana et al. [99] checked the protective role of GB in cotton (MNH 886) plant against two levels of lead (Pb) stress [50 μM and 100 μM Pb(NO3)2 ]. They observed that supplementation of 1 mM GB in Pb-affected plant reduce the adverse effects of Pb stress in cotton plants. Excessive electrolyte leakage (EL) and MDA production due to Pb stress notably decreased in both leaf and root of the cotton plant because of GB treatment. Moreover, transpiration rate (TR) and water use efficiency (WUE) significantly increased in the Pb-affected plant due to GB supplementation, which resulted in improved plant stress tolerance. Rasheed et al. [103] conducted an experiment with two wheat cultivars, viz., Millat-2011 and Punjab-2011. They exposed two-week old wheat plants to Cd stress. After one week of Cd treatment, they sprayed GB on the leaves of Cdaffected wheat plants. Two weeks later, they observed that spraying of GB showed tolerance to Cd toxicity. They recorded that MDA content sharply decreased in the Cd-treated plants after GB application. In another experiment, 53 day-old perennial ryegrass (Lolium perenne) seedlings were exposed to seven different doses of Cd (0 and 0.5 mM CdCl2) and GB (0, 20, and 50 mM) separately or in combination in hydroponic culture for seven days [48]. Cadmium stress considerably decreased the normalized relative transpiration (NRT) and increased EL and MDA content. Exogenous GB ameliorated the adverse effects of Cd stress on perennial ryegrass by increasing NRT and decreasing EL and MDA content in Cd-stressed plants. Furthermore, perennial ryegrass with 20 mM GB application suppressed the Cd accumulation in both shoots and roots. Their findings concluded that exogenous application of GB with 20 mM is the best strategy to ameliorate the detrimental impacts of Cd stress on perennial ryegrass. Application of GB contributes to decreased Cr concentrations in wheat plants and plays different biochemical function for detoxification of heavy metals [98]. Farooq et al. [49] treated four-weeks-old cotton genotype (MNH 886) with different Cd levels (0 μM, 1.0 μM, and 5.0 μM CdCl2) and GB (0 and 1 mM GB) for six weeks. Cadmium treatments significantly decreased TR and WUE but increased the MDA content and EL in both leaves and roots as compared to control in a dosedependent manner. On the other hand, GB application in Cd-stressed plants considerably increased the TR and WUE [except (1.0 μM CdCl2 + 1 mM GB) treatments] but decreased the levels of MDA and EL in both leaves and roots in contrast to respective only-Cd treatments. Radicle bearing seeds of Pennisetum typhoideum were treated with Ni, GB and aspirin separately or in combination, where Ni increased the level of MDA, 4-hydroxy-2-nonenal and lipoxygenase (LOX), whereas decreased the membrane stability index (MSI). Supplementation of GB with Ni reversed the above-mentioned parameters [104]. Exogenous GB increased MSI by 122% and decreased the level of MDA, 4-hydroxy-2-nonenal and LOX by 37, 17 and 23% in P. typhoideum plants.

Extreme Temperature
Due to the global climate change, extreme temperature (both high and low) is becoming the most important concern for the plant scientists, worldwide [106]. Every biochemical process of the plant is governed by a crucial temperature range. Where the rise or fall of temperature is beyond the critical threshold for a considerable period, it causes irreversible damage to plant growth and development [106]. In many research, both endogenous and exogenous GB confirmed its positive interaction with improving plant stress tolerance (Tab. 3).
Heat stress adversely affects plant physiology and biochemistry, while GB plays a protective role under stressful conditions. For confirmation, Wahid and Shabbir [50] pretreated the seeds of Hordeum vulgare L. cv. Haider-93 with different doses (10, 20, 30, 40 and 50 mM) of GB for 24 h. The seedlings were next exposed to heat stress (40 ± 1/32 ± 1°C day/night temperature). They observed that 20 mM GB increased water potential and reduced relative membrane permeability and leakage of important ions including Ca +2 , K +, and NO3under heat stress condition. Similarly, GB improved the accumulation of soluble sugars and protected the developing tissues from heat stress effect in sugarcane (Saccharum sp. cv. HSF-240; [57]). Addition of GB to the germination medium or imbibition of seeds in a solution of GB enhanced the tolerance of wild-type tomato (S. lycopersicum L. cv. 'Moneymaker') seeds to high temperature. The seeds treated with exogenous GB also expressed heat-shock genes at elevated levels and accumulated more HSP70 than controls [107]. Sorwong and Sakhonwasee [58] checked the effects of GB under heat stress conditions. They exposed three marigold cultivars, namely, 'Narai Yellow', 'Bali Gold', and 'Columbus Orange' to heat stress (39°C/29°C day/night temperature). Heat stress conditions caused photoinhibition and low levels of CO2 assimilation rate, stomatal conductance, and transpiration rate in all marigold cultivars in comparison with their respective control plants. Foliar applications of GB at 0.5 and 1 mM alleviated the above-mentioned negative impacts, improved water status and lowered the production of MDA as well as increased stress tolerance.
Nayyar et al. [63] grew C. arietinum plants under chilling stress (12-14/3-5°C day/night temperatures) in combination with exogenous GB (1 mM). They observed that GB reduced the chilling-induced damages of the plant. Importantly, GB reduced EL by 63% and increased cellular respiration by 69%. Xing and Rajashekar [51] experimented with A. thaliana (Columbia) under cold stress (4/2°C day/night temperature) up to four weeks. They recorded that exogenous GB (10 mM) increased freezing stress tolerance in A. thaliana (Columbia) plant. Sugarcane (HSF-240) bud, pretreated with 10 mM GB for 8 h, showed improvement in chilling stress tolerance [52]. Pretreatment by GB improved the K + and Ca +2 nutrition, increased free Pro and GB content and levels of soluble sugars, which finally developed chilling stress (15 o C for 2 d) tolerance in sugarcane. Recently, Cheng et al. [23] demonstrated that exogenous GB treatment of seeds increases the chilling tolerance of cotton at the seedling stage. Moreover, GB decreases the membrane damage and improves the antioxidant defense system in the cotton plant.

Waterlogging and UV Radiation
Waterlogging/flooding stress inflicts serious hazard to plant survival by upsetting the nutritional balance and obstructing diverse physiological and biochemical activities of the plant, which results in a disturbance in growth and development, and ultimately yield loss [1]. Use of different exogenous protectant to overcome waterlogging/flooding is common in plant science, but the potential use of GB in alleviating the effects of waterlogging/flooding stress in plants is very rare. Rasheed et al. [105] investigated the effect of GB on the growth and physiology of tomato plants under waterlogged conditions (Tab. 3). They imposed waterlogging at 15 day-old tomato seedlings (cv. Roma) for 25 days. This was achieved by blocking the drainage holes and supplying water to form a three cm film of stagnant water at the surface of each pot. Glycinebetaine (50 mM) was applied 10-day after waterlogging imposition and continued up to 50 d old plant. After 15-day, they observed different data. Waterlogging enhanced MDA content and deteriorated membrane integrity. It also increased the concentration of Na and reduced the concentrations of K and Ca (root only) in the different parts of the plant. On the other hand, GB application in waterlogged plant decreased MDA content; improved membrane integrity and nutritional status of the plant, which resulted in waterlogging stress tolerance.
Due to depletion of stratospheric ozone layer, ultraviolet radiation (UV) can easily enter to the ground surface of the earth and hamper the living organism. Ultraviolet radiation is capable of negatively influencing plant growth and developmental processes [26,108]. Different research work is going on to increase tolerance to UV-induced stress. Moussa and Jaleel [109] studied the effect of GB on Fenugreek plant (cv. Giza 2) under UV-induced stress. They exposed dry seeds to different doses of irradiation of 0.0, 25, 50, 100, and 150 Gray (1 Gray = 100 rad), using a gamma source, 60Co, with the strength of 500 Ci and dose rate of 0.54 Gy·min -1 . Then, they soaked the irradiated and non-irradiated seeds in 50 mM of GB for 24 h. Finally, plants were grown for 30 days from the seeds to check the effect of both radiation and GB. They observed that gamma radiation treatment alone decreased the accumulation of reducing, non-reducing and total soluble sugars of the plant, but GB-soaked gamma irradiated plants showed an increase in the above sugar contents. Hence, GB protection was more pronounced against gamma radiation stress at lower doses.

Interaction of GB with Phytohormones and Other Signaling Networks
Phytohormones are the chief signaling molecules in plants, which critically regulate abiotic stress responses across plant species. Stress hormones like ABA, ethylene, salicylic acid (SA) and other traditional plant growth regulators (PGRs) are essentially involved in the signalosome regulation in plants [110]. Other molecules like the gasotransmitter nitric oxide (NO), charged PAs and even plant nutrients equilibrate the cellular environment during responses against sub-optimal conditions [111]. Glycinebetaine induces the antioxidant machinery in order to detoxify ROS molecules and stabilize the cellular osmoticum during abiotic stresses. This process demands intricate interactions between the molecular factors involved in signaling.

Abscisic Acid
Abscisic acid is regarded as the universal stress phytohormone involved in myriad physiological regulations under abiotic stresses. Plant tissues accumulate high ABA contents in response to stress [112]. This allows rapid stomatal closure and protects the plants from unwanted desiccation and water loss [113]. Application of exogenous ABA stimulated GB accumulation in barley plants exposed to salt, drought and low-temperature stress [114]. According to the report of Ishitani et al. [115], it can be suggested that such ABA-induced GB accumulation was due to increased BADH transcription in the leaves and roots of barley plants. In an interesting study, it was observed that exogenous application of either ABA or GB increased freezing tolerance in Arabidopsis. However, treatment with only ABA stimulated GB production, indicating the fact that ABA possibly acts upstream of the GB biosynthetic pathway [51].
The inter-connection between ABA and GB synthesis has been reported [116]. Goggin et al. [117] showed that treatment of plants with fluridone (1-methyl-3-phenyl-5-[3-trifluoromethyl] phenyl]-4[1H]pyridinone) inhibited ABA biosynthesis by blocking the carotenoid pathway. Interestingly, application of fluridone suppressed ABA levels and as well as BADH transcription in Sorghum bicolor [116]. Thus, reduction in endogenous ABA content also suppressed the endogenous GB level. Similar fluridonemediated inhibition of ABA and GB was also reported by Zhang et al. [118] in corn plants. In a study involving the temporal regulation between ABA and GB, it was observed that drought stress initially increased the endogenous ABA content followed by BADH transcription and then GB content within the cells of the Z. mays seedlings [118].
Accumulation of ABA upon desiccation stress effectively induces stomatal closure, which could lead to reduced photosynthetic activities. However, in previous sections, we have discussed regarding the roles of GB in promoting the photosynthetic efficiency during abiotic stresses. Since ABA positively regulates the production of GB, the functions of ABA and GB might seem to be contradictory. However, Franks and Farquhar [119] reported that exogenous ABA application reduced stomatal conductance without affecting the photosynthetic capacity in Tradescantia virginiana. This inference probably highlights the deeply correlated functioning of ABA and GB in response to abiotic stresses.

Salicylic Acid
Endogenous SA accumulation is also associated with abiotic stress tolerant phenotype in plants. Salicylic acid triggers the production of enzymatic and non-enzymatic antioxidants, which efficiently scavenge ROS and confer protection against oxidative stress [120]. Exogenous application of SA has also been useful in generating tolerance against heavy metal toxicity, salt, drought and extremes of temperatures [121]. Aldesuquy et al. [122] showed that co-application of SA and GB further increased the yield in drought-stressed wheat plants compared to the amount of grain production in the stressed plants treated individually with SA or GB.

Ethylene
The gaseous hormone ethylene is constitutively required for optimal plant growth and development [2]. Ethylene also promotes tolerance to moderately intense forms of freezing, etc. by promoting stem elongation, expansion of leaf area, stem radial growth and leaf thickness [123]. Ethylene and GB biosynthesis are intricately linked with each other due to the involvement of the common mediator molecule, choline. Ethylene biosynthesis is facilitated by the transfer of a methyl group from S-adenosyl methionine (SAM) to choline. Thus, ethylene production might occur at the expense of GB synthesis or vice versa. Plants subjected to severe abiotic stress accumulate endogenous GB for ensuring growth and survival [28]. This possibly occurs at the expense of ethylene production due to channelization of choline towards GB synthesis. High ethylene accumulating species like tobacco and tomato did not accumulate GB under cold or other forms of stress. It was also observed that the species with low ethylene accumulation exhibited increased GB synthesis in response to abiotic stresses [123].

Traditional PGRs
Traditional PGRs consist of auxins, gibberellic acids (GAs) and cytokinins (CKs), which are involved in growth, development, stem elongation, polar transport of solutes, phototropism and gravitropism, cell division and reproductive initiation [124,125]. The antagonistic relationship between ABA and GA is known. Increase in the endogenous ABA content suppresses GA synthesis during abiotic stresses [112]. Since ABA accumulation triggers GB synthesis, it can be inferred that GB and GA are negatively associated with each other. In line with this proposition, Bao et al. [126] observed lower GA1 levels in the transgenic L. perenne plants overexpressing the CMO and BADH genes from spinach chloroplasts. Cytokinin and GB accumulation are also negatively correlated. Kathuria et al. [127] observed that transgenic rice plants overexpressing the codA gene exhibited increased expression of CK dehydrogenase 1 (CKX1), which encodes a CK degrading enzyme. The plants accumulated high amounts of GB and low concentration of CKs. Auxin also manifested negative regulation of GB synthesis. The exogenous application of the synthetic auxin, dicamba reduced CMO expression and hence GB accumulation in Kochia scoparia plants exposed to salt stress [128].

Gasotransmitters
Hydrogen sulfide (H2S) and nitric oxide (NO) are gaseous molecules involved in myriad signaling processes in plants. These gasotransmitters are also involved in promoting plant tolerance against multiple abiotic stresses [78,129]. They act as crucial concentration-dependent redox-related signaling moieties.
Exogenous application of sodium hydrosulfide (NaHS) in S. oleracea seedlings increased the cellular production of H2S. As a result, the content of GB and trehalose steeply increased. The transcription of BADH and CMO genes were also up-regulated upon NaHS treatment. The H2S-induced accumulation of GB programmed the antioxidant machinery and promoted drought tolerance in the treated seedlings [84]. These reports pointed towards unknown signaling interactions between H2S and GB, which have not been exhaustively investigated. Recently, Zanganeh et al. [130] also showed that seed priming with SA and NaHS triggered the accumulation of GB and NO in Z. mays seedlings grown under Pb stress. The treated plants also exhibited lowered expression of the ethylene biosynthetic gene, 1-aminocyclopropane-1carboxylate synthase 6 (ACS6). This was due to channelization of choline towards GB synthesis instead of ethylene production. The reduction of PA biosynthesis gene, SAM decarboxylase (SAMDC) and accumulation of NO under Pb stress indicate that arginine metabolism is probably channelized towards NO production instead of PA synthesis [130]. Thus, H2S, NO, and GB altogether positively interact to confer Pb tolerance in maize plants. The positive regulatory association between NO and GB was observed in maize seedlings exposed to oxidative stress [131]. Seedlings treated with nitric oxide synthase (NOS) inhibitor exhibited reduced BADH expression and hence GB accumulation. Due to reduced GB accumulation, the activity of the antioxidant enzymes was also suppressed. The CAT expression was reduced leading to elevated H2O2 accumulation in the NOS-inhibitor-treated seedlings [131].

Polyamines
Polyamines and GB are compatible solutes, which together regulate the cellular osmoticum during abiotic stresses [132,133]. Polyamines like putrescine, spermidine and spermine perform similar functions as GB and hence are correlated with the latter. Seedlings primed with spermidine and spermine, prior to salt treatment, showed BADH1 expression to be higher in the shoots of salt-tolerant rice cultivar, Nonabokra than the sensitive rice cultivar, IR-64, as compared to non-primed seedlings under stress. On the contrary, in case of roots, priming with spermidine and spermine increased BADH1 transcript accumulation in Nonabokra, while it decreased gene expression in IR-64 roots exposed to 75 mM NaCl [134]. Polyamine priming of salt stressed-seedlings likewise enhanced BADH1 expression in the shoots and roots of another salt-sensitive, aromatic rice cultivar, Gobindobhog [135]. All these results clearly show the cross-talk between PAs and GB-biosynthesis machinery in rice under salt stress. Overexpression of aldehyde dehydrogenase 10A8 (ALDH10A8) and ALDH10A9 (encoding functional BADH) in Arabidopsis increased GB content under salt stress. The transgenic plants also exhibited an increase in the levels of free PAs like agmatine, cadaverine, and tyramine along with a reduction in the putrescine and spermidine content upon salinization [136]. Rippa et al. [137] reported the ability of BADHs to oxidize 3aminopropanal (an intermediate compound in PA catabolism). Oxidation of spermine by polyamine oxidase (PAO) produced 3-aminopropanal, which was utilized as a substrate by ALDH10A8 and ALDH10A9 [136]. This shows that BADH isoforms are directly involved in the catabolism of spermine. Choudhary et al. [138] observed that exogenous application of epibrassinolide (an active brassinosteroid) and spermidine remarkably increased the titers of cellular antioxidants like GB, Pro, AsA, GSH and total phenolics in Raphanus sativus seedlings exposed to Cr stress. As a result, excess ROS was efficiently scavenged, and stress tolerance was conferred. This evidently illustrates the tripartite regulation of PAs, GB, and brassinosteroid in generating stress-induced responses. Synthetic PA (diamino hydroxyl ethyl phospho mineral and diaminohexanoic mineral amino ethanoic acid) supplementation of the perennial medicinal herb, Stevia rebaudiana, increased the endogenous GB and Pro contents on exposure to cold stress [139].

A model for GB-Induced Cross-Tolerance Under Abiotic Stress
In previous discussions, we revealed the diverse roles of GB in generating abiotic stress tolerance in plants. Since any kind of abiotic stress generates oxidative damages, GB, being an osmolyte, protects the system from such injuries. It can be proposed that increasing the endogenous accumulation of GB against one type of stress can also promote cross-tolerance against other abiotic stresses. In this section, we have proposed an outline of interactions between GB, phytohormones, photosynthetic efficiency and amelioration of ROS-induced injuries (Fig. 4) in order to suggest an interactive model on GB-induced cross-tolerance against abiotic stresses. Under abiotic stress, SAM is more channelized towards GB and PA biosynthesis, which results in reduced ethylene content in GB accumulating plants Plants exposed to any abiotic stress experience an increase in the PSII excitation pressure, resulting in imbalances in the electron transport chain (ETC) and the stromal redox equilibrium [2]. This initiates a burst in ROS production leading to extreme oxidative stress. In order to tackle uncontrolled damages, GB accumulation is stimulated via the channelization of SAM (common precursor of ethylene, PA and GB) towards the GB biosynthetic pathway [130]. Excess generated electrons are utilized in the process of choline oxidation to betaine aldehyde catalyzed by CMO. Thus, GB synthesis reduces PSII excitation pressure, restores the redox balance within the chloroplast and confers protection to the photosynthetic machinery [123]. The stress phytohormones like ABA and SA, which are synthesized in the majority of the abiotic stresses co-operatively stimulate the accumulation of GB within the cell [112,121]. The redox imbalance and ROS accumulation also signals the up-regulation of ABA biosynthetic genes and promotes ABA production, which again empowers the generation of GB and associated compatible solutes, dedicated towards physiological protection [2]. Under stress situations, the plant induces a systemic response, which channelizes the metabolic equivalents towards amelioration and protection rather than growth and development. In line with this theory, stress-triggered GB accumulation is accompanied by a reduction in the levels of traditional PGRs like auxins, GAs, and CKs [127]. Glycinebetaine also cooperates with gasotransmitters like NO and H2S via less studied interactive pathways to promote stress tolerance [130]. The model presents a compiled outcome of different observations across various plant species under diverse sub-optimal conditions. Thus, it can be stated that GB acts as a crucial compatible solute conferring protection against the basic photosynthetic damages and ROS-induced injuries.

Exogenous Use of GB: From Lab to the Field
Many plant species naturally accumulate GB as major organic osmolytes, responsive to abiotic stresses and play adaptive roles in osmotic adjustment and protecting subcellular structures. However, these plants also show better performance when grown under normal conditions. Not all plants accumulate GB in a considerable amount to alleviate the adverse effect of stresses because of the inability of synthesizing or accumulating GB. Thus, extensive research has been undertaken to induce GB-mediated abiotic stress tolerance via genetic improvement as well as generating transgenic plants. However, all of these approaches have limitations and were unable to synthesize adequate amounts of GB. Therefore, the third and short-cut method for inducing GB-mediated tolerance, which has gained popularity is the exogenous GB application through seed treatment/priming, application through irrigation water and foliar spray [39]. Hence, this section of review article will highlight examples of the successful exogenous application of GB to improve growth and yield of plants under normal as well as stress conditions.

Exogenous GB Application and Plant Growth Promotion
Exogenous applications of GB to plants, as seed priming or pretreatment (before stress), co-treatment (during stress), and/or post-stress period, have been shown to increase the internal GB levels, thereby enhancing plant growth both under stressed or non-stressed conditions [39;Tab. 4]. However, the response differed greatly among plant species. The first thing to be considered is that not all species are responsive to the exogenous application and secondly, the effective dose varied from species to species, and sometimes over-dose may show toxic effects resulting in growth inhibition. Therefore, optimum concentrations and appropriate plant developmental stages must be carefully identified for the exogenous application. Abdullah et al. [157] Exogenously applied GB was tested in Brassica under normal and saline conditions, where different growth parameters, viz., plant height, shoot and root fresh and dry weight were positively influenced by GB under normal growing condition. Moreover, GB efficiently reduced the deleterious effect of salinity [13]. Foliar application of GB at 25 and 50 mM on onion growth and physio-biochemical attributes was investigated under normal and 4.80 dS m -1 salt stress, where GB showed significant increase in growth indices in terms of shoot length, leaf area per plant, shoot fresh weight (FW) and dry weight (DW) under non-stressed and stressed condition as well [14]. Foliar GB application resulted in significant increases in growth attributes of lettuce grown under 100 mM NaCl, compared with non GB treated plants, where 25 mM GB showed the best performance for mitigating salinity damage and growth improvement. Yet, 25 mM foliar-applied GB increased FW and DW of shoot by 54.9% and 80.5%, respectively, whereas it increased FW and DW of root by 40.0% and 64.3%, respectively under stress conditions [150]. Application of low levels of GB (15 mM) was slightly effective in increasing root fresh and dry biomasses of NIAB-IRRI-9 rice under non saline condition, but efficiently improved the growth parameters under 60 mM and 120 mM salt in both NIAB-IRRI-9 and Super Basmati variety of rice [141]. Glycinebetaine applied as foliar spray increased shoot DW and root FW in Pisum sativum L. cv. Pea 09, while decreased root FW and root length in P. sativum L. cv. Meteor Fsd under non-saline conditions [151]. Exogenous application of GB markedly mitigated the inhibitory effects of Cd on T. aestivum plant growth. In this context, foliar application of GB had higher shoot length and dry mass under control and Cd stress [103].
In Vigna unguiculata, 50 mM NaCl significantly reduced growth parameters as compared with the control (non-saline). However, under the saline condition, foliar application of GB showed significant increments in all growth parameters. As for dry weight, 10 mM GB without salinity increased significantly the dry matter compared to the control seedlings. There was no significant difference in dry weight with 5 mM GB in control and NaCl-treated seedlings. Moreover, GB treatments without salinity and under 50 mM NaCl increased leaf area as compared with the control [153]. Glycinebetaine treatments were efficient for improving the growth under water stress, as well as under well-irrigated conditions. Among seed treatment and foliar spray of GB, the foliar spray was more effective, and foliar application with 100 ppm GB (0.85 mM) was the best, contributing the highest seedling FW and DW in aromatic fine-grain rice cv. Super-Basmati rice [158]. Aziz et al. [147] tested the G. hirsutum to normal and drought stress condition in combination with GB. Their results indicated that only germination rate and plant height were positively altered by GB application. However, under drought condition, GB significantly effected the number of monopodial and sympodial branches, a number of main stem nodes, plant height, and the ratio of height to the node. A similar result was also postulated in cotton under dehydration stress [146].

Exogenous GB application and yield improvement
In most of the crop plants, the reproductive stage is the most vulnerable period to abiotic stresses, which reduce the final yield. Hence, protection against abiotic stress at the reproductive stage is considered as the key to higher yields from crops in stress-prone areas globally. On the other hand, scientists are now targeting the yield gap in order to get higher yield using some biostimulants [159]. Therefore, exogenous GB application as a stimulant in crop plants appears to be an effective strategy for lessening the yield gap as well as improving abiotic stress tolerance. Exogenous application of GB to plants, as seed priming or pre treatment, co-treatment, and/or post-stress period, have been shown to increase the internal GB levels, thereby enhancing plant growth and final crop yield [158;Tab. 5]. However, the response differed greatly among plant species. The first thing to be considered is that not all species are responsive to the exogenous application and secondly, the effective dose varied from species to species, and sometimes over-dose may show toxic effects resulting in growth or yield reduction [39]. Therefore, optimum concentrations and appropriate plant developmental stages must be carefully identified for the exogenous application. Glycinebetaine up-regulates the yield under normal or abiotic stress conditions due to its osmoprotective role on photosynthetic machinery and maintaining the ion homeostasis [160]. Moreover, it improves assimilation of CO2 under drought stress [163] and biosynthesis and transport of hormones, for example, CKs, which have a positive role in the transportation of photoassimilates [165].
A field experiment was conducted to investigate the influence of exogenous application of 100 mM GB on the performance of wheat at maximum tillering and anthesis stage. Foliar-applied GB improved grain spike -1 and thousand grain weight under control and stressed ones [161]. The response also suggested that the optimum level of GB is required for yield improvement under irrigated condition and drought, which varied among susceptible and tolerant genotypes [161,166,167]. Pre-soaking of seeds with GB was not significant in alleviating the adverse effects of water deficit on the yield of sunflower, but a positive effect was obtained by foliar spray under both water limited and well-irrigated conditions. Severety of drought in P. sativum is due to drought at the vegetative and flowering stage, which is mainly owing to a reduction in pod number plant -1 [168]. However, control (unstressed) plants produced the maximum pod number plant -1 followed by drought-stressed one treated with GB at the vegetative or reproductive stage. When the drought-stressed plants were subjected to recovery, although the growth increased, pod development remained paused and did not alleviate the detrimental effect of drought stress. On the other hand, re-watering at the flowering stage resumed reproductive activity, but the new pods failed to reach maturity, which in turn reduced the pod number per plant. In this connection, exogenously supplied GB and Pro increased the pod number per plant either in well-watered plants or under all treatments of drought [152]. In this regard, Rezaei et al. [162] also reported maximum seed yield of soybean under control and salt stress due to foliar application of GB, which was associated with an increase in pod number. If the drought stress was imposed at the vegetative stage, foliar application of GB was more effective and enhanced the hundred achene weight of sunflower [164].
Exogenous GB treatment can significantly increase the seed yield, as well as the oil, carbohydrate, and protein contents in B. napus seeds in either unstressed plants or plants irrigated with 75% or 50% field capacity (FC). In this regard, 20 mM GB was effective in alleviating the detrimental effect of mild and severe drought stress, but more effective under severe drought than the mild stress [148]. Exogenous GB (100 mM) applications under irrigated and water stressed conditions improved grain yield and number of grains per spike by modulating the photosynthetic ability through maintaining turgor potential of leaf [160].
Foliar application of GB enhanced biomass production and significantly increased grain yield of maize and sorghum. It also resulted in more grains of maize, sorghum, and wheat, with interactions between water and GB treatments [169]. Exogenous GB recuperated the rice plants from water stress as indicated by greater plant height as well as an increase in the yield from GB pretreated plants compared to no-GB (untreated) ones [142]. Glycinebetaine pretreatment considerably increased hundred grain weight as well as total grain yield of wheat in contrast to GB untreated plant [160]. A similar result was also found in maize [170] and dicotyledonous species such as bean [154]. Moreover, researchers observed that pretreatment with GB maintained the healthy condition of the mature plants particularly at the reproductive stage, which facilitated more yield in crops.
Although a number of examples exist for successful GB application to improve plant growth and yield under normal and abiotic stress, the field application is yet to be practiced widely. To gain the maximum output along with ensuring economic feasibility of GB application, further investigations are required to determine the most effective dose and frequency of applications, along with the most responsive growth stage(s) of specific crop plants, which may vary from crop to crop. Moreover, a better understanding of the mode of action of exogenous GB may aid its effective utilization for the farming community.

Glycinebetaine-Induced Regulation of the Molecular Machinery
Glycinebetaine is postulated to be involved in the protection of the transcriptional and translational machinery via the induction of genes encoding osmoprotective proteins [10]. In vitro studies have revealed that GB lowers the melting temperature of double-stranded DNA. This enables GB to qualify as a candidate to activate transcription under high salt concentrations within the cell [171]. Cold responsive genes encoding a late embryogenesis abundant group of proteins, viz., wheat cold-regulated 410 (WCOR410), wheat cold specific 120 (WCS120) and WCS413 were found to be up-regulated in GBtreated wheat seedlings exposed to low-temperature stress [172]. Tomato plants exposed to chilling stress and exogenously treated with GB exhibited high transcription of cat1 gene encoding CAT [173]. Transgenic tomato plants overexpressing the GB biosynthetic gene choline oxidase A (codA) from soil bacterium Arthrobacter sp., resulted in the induction and repression of 30 and 29 genes, respectively in the flower buds due to GB accumulation [174]. These reports concomitantly propose the ability of GB to directly or indirectly regulate transcription and hence stress-induced gene regulation. Bourot et al. [175] also showed that GB behaves like a chaperon in vivo. Thus, GB might confer protection to the proteins and the translational machinery itself to prevent unwanted misfolding and aggregation under osmotic stress (Fig. 3).

Glycinebetaine Versus Proline: A Comparison on Their Mimicry
Proline is an amino acid, which acts as an osmolyte and compatible solute. Like GB, Pro also accumulates under almost all kind of abiotic stress [176]. The biosynthesis of GB and Pro can be related via arginine and the PA synthesizing pathway. Though principally formed from glutamate (Glu), Pro can also be produced from ornithine (Orn). The Orn aminotransferase (OAT) catalyzes the formation of Δ 1pyrroline-5-carboxylate (P5C) from Orn. P5C is then acted upon by P5C reductase (P5CR) to form Pro [8]. Orn decarboxylase (ODC) again catalyzes Orn to form putrescine. Contrary, the PA and GB biosynthetic pathways are connected to each other through the common precursor, SAM [130]. Hence, it seems that channelization of Orn towards Pro and siphoning of SAM towards GB synthesis might enable the simultaneous accumulation of both the osmolytes at the expense of PAs. The experiments to undermine the greater efficacy between Pro and GB are not available in the literature. Like GB, exogenous application of Pro or generating transgenics that overexpress Pro biosynthetic genes have also led to recharging of the entire antioxidant machinery along with up-regulation of several abiotic stressinducible genes encoding late embryogenesis abundant proteins, heat shock proteins, etc. [176]. Hasanuzzaman et al. [39] reported that exogenous treatment of rice seedlings with Pro triggered GB accumulation under salt stress. In another study, Sobahan et al. [177] showed the co-operative action of GB and Pro in salt stress amelioration of sensitive rice seedlings. It was found that exogenous application of GB and Pro promoted stomatal closure and induced the production of vacuoles, which could store the excess Na + ions [177]. A recent review by Murmu et al. [178] contains many more instances on the positive correlation between GB and Pro under abiotic stresses.
Zhang et al. [179] observed a heritable change in the methylation pattern of Pro metabolizing genes, P5C synthase (P5CS). The existence of a demethylated allele of P5CS was proposed even after removal of the stress cues indicating towards the development of stress memory [176]. Drought-and osmopriming-induced trans-generational drought tolerance (stress memory) mediated by GB has also been reported in wheat seedlings [81,82]. Thus, abiding by the current understanding, it would be better to relate GB and Pro as two synchronized arms of osmotic-stress response, which simultaneously cooperate under sub-optimal conditions. However, over-accumulation of Pro has been observed to be detrimental for mitochondrial and chloroplastic ultrastructures. Treatment with 40-50 mM or higher concentration of Pro also promotes unwanted necrosis [180]. Extremely high concentrations of Pro inhibit P5CS by feedback regulation, resulting in retarded organogenesis and growth in Arabidopsis and Distichlis spicata [181]. At such concentration, Pro lowers the DNA melting point, destabilizes the double helix and makes DNA hyper-susceptible to S1 nuclease [181]. However, no such adverse effects of GB have been observed in plants. Hence from this point of view, it can be hypothesized that GB could be a safer osmolyte compared to Pro, though such a proposition will require rigorous experimentations using high concentrations of GB.

Engineering Plants for Enhanced GB Biosynthesis
Crop loss due to abiotic stress-induced injuries is a severe agro-economic problem. The biotechnological advancements including maneuvering with molecular targets and generation of transgenic plants have paved the way to better understand GB-mediated abiotic stress tolerance.

Molecular Approaches
The field of molecular biology has not been exhaustively investigated in case of GB production. Some recent data have validated the molecular basis of GB accumulation under stress. Proteomic investigations identified BADH as a salt stress marker in T. monococcum seedlings [182]. The proteome of the salt-stressed seedlings was analyzed by two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF-TOF mass spectrometry. The proteins involved in regulatory, stress protection, protein dynamics, and diverse metabolic pathways were identified. Further analysis verified BADH, leucine aminopeptidase 2, Cu/Zn SOD and 2-Cys peroxiredoxin BAS1 as the molecular markers of the salt stress response [182]. Meng et al. [183] also dissected the proteome of the monocot perennial halophyte, Puccinellia tenuiflora exposed to chilling stress. Along with altered protein synthesis and turnover in the cytoplasm and chloroplast, the investigators found an accumulation of osmolytes like GB, Pro, and soluble sugars to be responsible for promoting such adaptation [183]. The importance of BADH in abiotic stress tolerance was illustrated by a knockdown experiment using RNA interference (RNAi) technique. The BADH1-RNAi repression lines in rice exhibited reduced yield and tolerance to salt, drought, and cold. This was accounted for due to enhanced MDA and H2O2 accumulation within the tissue. Interestingly, the repression lines did not display any change in the endogenous GB content [184]. Hence, the decrease in stress tolerance was possibly due to the reduced dehydrogenation of the aldehydes accumulating from diverse metabolic processes.

Transgenic Approaches
Due to the inability of the major cereals (barley, wheat, maize) to normally accumulate GB, transgenic approaches to overexpress GB biosynthetic genes have been undertaken to enhance plant tolerance against abiotic stresses [185]. The principal staple food crop, rice has one CMO and two BADH homologues. The removal of the translational initiation codon, loss of functional domains and generation of premature stop codons due to frameshift mutations altogether result in the formation of non-functional BADH2 homologues. This gene is involved in the production of the aroma compound, 2-acetyl-1pyrroline in aromatic cultivars of rice [186]. The functional BADH1 homologue, on the contrary, is involved in the production of GB under osmotic stresses [187]. Engineering GB biosynthesis in GB nonor low-accumulators by genetic engineering has been tested in diverse plant species ranging from Arabidopsis, tobacco, Brassica, tomato, maize, rice, potato, and wheat. This resulted in increased tolerance against multiple stresses [185].
Transgenic rice plants overexpressing choline oxidase (cox) gene from Arthrobacter pascens under stress-inducible and constitutive promoters resulted in increased GB accumulation and tolerance towards salt [188]. Similar observations were also put forward for transgenic tomato plants with chloroplast targeted GB accumulation. These exhibited better tolerance towards chilling stress compared to the sets, where GB was produced in the cytosol and also in greater amounts [189]. These observations might be accounted for due to the effective GB-mediated protection of the photosynthetic apparatus, which is the prime machinery to be affected under oxidative stress [190].
Overexpression of the codA gene from Arthrobacter globiformis resulted in high GB accumulation in the chloroplasts and the cytoplasm of tomato seedlings. This ensured higher photosynthetic rates, antioxidant activities and efficient scavenging of ROS in the transgenics compared to the wild-type plants exposed to salt stress [42]. The transgenic plants also exhibited better regulation of the ion channels like K + transporter, Na + /H + antiporter and H + -ATPase under stress, which led to a higher K + /Na + ratio compared to the stressed seedlings [42]. The relation between ion channel regulation and GB accumulation has also been observed by Ahire et al. [191] where overexpression of vacuolar proton pyrophosphatase (VPPase) from Sorghum bicolor in Bacopa monnieri conferred salt tolerance via enhanced production of GB. The shoots of the transgenic plants also accumulated low MDA and exhibited lesser membrane damage due to GB-mediated recharging of the antioxidant machinery and effective scavenging of toxic ROS [191]. Overexpression of the S. oleracea chloroplastidic BADH in sweet potato cv. Sushu-2 increased GB production within the chloroplast [192]. This resulted in improved tolerance to salinity and oxidative stress in the transgenic sweet potato plants due to better photosynthetic efficiency, membrane integrity, and ROS scavenging compared to the stressed plants [193].
Overexpression of genes encoding stress-associated proteins also induced GB synthesis within transgenic plant tissues [185]. Transformation of chilli pepper with tobacco osmotin gene resulted in enhanced salt tolerance due to accelerated GB and Pro synthesis. The transgenics exhibited higher activities of SOD, GR, APX and elevated relative water content (RWC) under stress compared to the control plants [194]. The G-protein, RabAc4, involved in membrane trafficking has been reported to cooperate with GB to promote chilling tolerance in tomato seedlings [189]. Tab. 6 presents a concise list of transgenic approaches applied to enhance GB accumulation and promote abiotic stress tolerance.

Conclusion and Outlook
Glycinebetaine, the most important organic osmolyte, plays numerous functions in plant growth and development. Under different environmental stresses, many higher plants naturally accumulate GB as a protectant. Besides acting as an osmoprotectant, GB stabilizes cytosolic pH, proteins, enzymes, and membranes, scavenges toxic ROS, and maintains the balance of cell redox for protecting subcellular structures in stressed plants. Moreover, GB maintains the ionic balance of plants under different stress conditions. Extensive work in recent past confirmed how endogenous and exogenous GB confers tolerance to plants at the time of diverse abiotic stresses. New findings recommend the involvement of differentially expressing endogenous genes in GB arbitrated stress tolerance in higher plants by ROS scavenging, maintaining dealings with oxidative stress, gene expression and the increasing accumulation of GB under the condition of abiotic stress. Because of its broad range of function, the genes of the GB biosynthesis pathway are being used to develop transgenic plants. Such plants are very efficient to accumulate GB and show improved tolerance to a variety of abiotic stresses, including secondary oxidative bursts. Even though scientists are performing different research work with GB, the signaling behavior mediated by GB is still unclear and needs investigation.