**2.2 Glycine betaine and environmental stress**

Many plants are able to accumulate naturally GB and diverse osmoprotectants to balance the disruption of plant cell homeostasis caused by environmental stress such as drought, chilling, salinity or high temperature [8, 35, 36]. Many studies have been reported on the positive effect of endogenous GB in plants under abiotic stresses. The role of glycine betaine in osmotic adjustment was related in *Amaranthus tricolor* [37] and *Hordeum maritimum* [38] under salinity. The role of GB against oxidative stress via scavenging the reactive oxygen species and increasing the antioxidant activities was reported in many studies [39–41]. For these reasons, the use of glycine betaine in non accumulator and accumulator plant species become more popular in plant physiology. Indeed, several reports have related the positive effect of GB in transgenic plants (**Table 1**).

#### **2.3 Glycine betaine engineering**

The idea of introducing GB pathway and its high accumulation in plant under environmental stresses has long been a target for metabolism engineering stress tolerance. The feasibility of this process was based on comparative physiology and genetic evidence from a maize mutant [15, 54]. Metabolic engineering of the biosynthesis of GB from choline by using various genes such as cod*A* or BADH gene gained more attention to improve stress tolerance in crop and woody plants that are incapable of synthesizing GB under abiotic stresses [8, 18, 55]. Moreover, genetic engineering is also use to increase GB accumulation in various plant species which

*Insights into Metabolic Engineering of the Biosynthesis of Glycine Betaine and Melatonin… DOI: http://dx.doi.org/10.5772/intechopen.97770*


#### **Table 1.**

*Reported roles of GB in transgenic plant under abiotic stresses.*

produce a low concentration of GB that might not be sufficient for osmoregulation to counteract with abiotic stress [56].

The genes (codA or cDNA BADH) and enzymes involve in GB biosynthesis have been identified and cloned. GB has been successfully synthesized in various targeted organisms and provided stress tolerance via genetic engineering (**Table 2**).

#### *2.3.1 Genetic engineering of GB via codA gene*

As shown in **Table 2**, many species that can accumulate or not GB have been targeted via genetic engineering to synthesize or over accumulate GB under both stressed and non-stressed conditions. The choline oxidase (codA) from *A. globiformis* has been widely used in various transgenic plant species to synthesize GB, and *codA* has the ability to convert choline in one reaction [56].

The catalytic activity of choline oxidase (EC: 1.1.3.17) in *A. globiformis* results in this following equation: (Choline + H2O + 2 O2 = glycine betaine + H+ + 2 H2O2) [63].


#### **Table 2.**

*Overview of GB genetic engineering in various plant species.*

The codA gene is of particular interest with respect to the engineering of desirable productive traits in crop plants and stress tolerance. In transgenic tomato and brown mustard the codA was targeted to the chloroplast and cytosol which allowed GB accumulation for an increase of stress tolerance [19, 59]. Further, transgenic *indica* rice showed a significant increase of water-stress tolerance and transcriptome changes via codA gene expression [51]. One of the advantages of using choline oxidase pathway as a tool for engineering GB synthesis in plant is that the addition of a single gene codA is enough for the conversion of choline to GB [8]. The codA transgenic plant has showed their abilities to counteract with environmental stresses such as salinity, high temperature, high light, cold stress and freezing in different plant growth stages [64].

*Insights into Metabolic Engineering of the Biosynthesis of Glycine Betaine and Melatonin… DOI: http://dx.doi.org/10.5772/intechopen.97770*

#### *2.3.2 Genetic engineering of GB via BADH gene*

The other pathway that provided successful results in genetic engineering of GB biosynthesis in various transgenic plant species is the BADH pathway (**Table 2**). BADH is one of the most prominent genes involved in the biosynthetic pathway of GB, and its utilization in various plant species has led to an increased tolerance to a variety of environmental stresses [65]. Indeed, the second step of GB biosynthesis is performed by betaine aldehyde dehydrogenase (BADH) that can be encoded by *betB* or *betA* gene from *E. coli*. BADH is an NDA-dependent dehydrogenase that has been characterized and cloned from plants species belong to the Amaranthaceae and Gramineae families [15]. The BADH pathway has been targeted in the chloroplasts in *N. tabacum* [13] and in peroxisomes in Gramineae [60]. Many studies showed positive results in stress tolerance in transgenic plants with genes *betA*, *betB* or both from *Escherichia coli* encoding Oxygen-dependent choline dehydrogenase (CHDH) and BADH [8]. The catalytic activities of CHDH (EC: 1.1.99.1) encode by *betA* from *E. coli* can be resume by this following Eq. (A + choline = AH2 + betaine aldehyde), A (hydrogen acceptor) and AH2 (hydrogen donor) [66]. Meanwhile the catalytic activities of the NAD/NADP-dependent betaine aldehyde dehydrogenase (EC: 1.2.1.8) are done by this equation: (betaine aldehyde + H2O + NAD+ = glycine betaine +2 H<sup>+</sup> + NADH) [66, 67]. The equation for the catalytic activities is similar for chloroplastic betaine aldehyde dehydrogenase in sugar beet or spinach compared to those of *E. coli*.

### **3. Metabolism engineering of melatonin**

Melatonin (**Figure 3**) as an ancient pleiotropic bio-molecule which can be traced back to the origin of life, is present in both animal and plant organisms [24, 68]. In plant, melatonin has been found in diverse family and at different stage of growth: Asteraceae, papaveracea, apiaceae, linaceae, fabaceae, poaceae, rosaceae, lamiaceae, solanaceae, musaceae or vitacea etc. [69].

Melatonin (N-acetyl-5-methoxytryptamine), a multifunctional plant hormone, was discovered in plants in 1995 [70]. Moreover, the presence of melatonin in plant was confirmed in *Chenopodium rubrum* via chromatography/tandem mass spectrometry and radio-immuno-assays [71]. Melatonin has multi-functional actions

**Figure 3.** *N-acetyl-5-methoxytryptamine.*

that improve cellular and organ health in various plant species and it is a powerful antioxidant in both animals and plants [72].

Melatonin functions as a metabolite with numerous roles in plant, including plant stress responses such as chilling, oxidative stress, drought, salt stress and nutrients deficiency, moreover melatonin can regulates plant growth and development, such as root organogenesis, flowering, and senescence [9, 73, 74]. Plenty of studies have focused on the function and regulation of melatonin in transgenic plants because of its crucial role in plant regulation.

#### **3.1 Melatonin biosynthesis pathways in plant**

The **Figure 4** shows a schematic representation of the biosynthesis of MT, in which the tryptophan is synthesized via shikimic acid pathway that is also responsible for the synthesis of vitamins and aromatic amino acids such as phenylalanine and tyrosine. In plants, tryptophan is converted to Tryptamine via a reaction catalyzed by tryptophan decarboxylase (TDC) [75], and the production of serotonin from Tryptamine is activated by tryptamine 5-hydroxylase [76]. The formation of melatonin is preceded by two reactions from serotonin; the first reaction catalyzed by ASMT transform serotonin to 5-methoxytryptamine, and the last step is catalyzed by N-acetyltransferase [77].

As far as we know, there are 6 genes which are involved in plant melatonin biosynthesis: TDC, TPH, T5H, SNAT, ASMT, and COMT [68], and the keys enzymes they encoded are the; L-tryptophan decarboxylase, tryptophan hydroxylase, serotonin-N-acetyltransferase, N-acetylserotonin methyltransferase and hydroxyindole-O-methyltransferase [24].

**Figure 4.** *A schematic representation of melatonin biosynthesis in brief.*

*Insights into Metabolic Engineering of the Biosynthesis of Glycine Betaine and Melatonin… DOI: http://dx.doi.org/10.5772/intechopen.97770*
