Reported roles of GB in transgenic plant under abiotic stresses.
Metabolic engineering in plant can be describe as a tool using molecular biological technologies which promotes enzymatic reactions that can enhance the biosynthesis of existing compounds such as glycine betaine (GB) in plant species that are able to accumulate GB, or produce news compounds like GB in non-accumulators plants. Moreover we can include to these definition, the mediation in the degradation of diverse compounds in plant organism. For decades, one of the most popular ideas in metabolic engineering literature is the idea that the improvement of gly betaine or melatonin accumulation in plant under environmental stress can be the main window to ameliorate stress tolerance in diverse plant species. A challenging problem in this domain is the integration of different molecular technologies like transgenesis, enzyme kinetics, promoter analysis, biochemistry and genetics, protein sorting, cloning or comparative physiology to reach that objective. A large number of approaches have been developed over the last few decades in metabolic engineering to overcome this problem. Therefore, we examine some previous work and propose some understanding about the use of metabolic engineering in plant stress tolerance. Moreover, this chapter will focus on melatonin (Hormone) and gly betaine (Osmolyte) biosynthesis pathways in engineering stress resistance.
- metabolic engineering
- abiotic stress
- stress tolerance
The global climate change influence negatively plant growth and development via the increase of the intensity of various abiotic stresses such as drought, chilling, salinity, waterlogging or flooding. Environmental stresses are one of the most threatening factors that can cause massive losses in agricultural crop production, ranging from 50–70% . Plant biotechnology and engineering are promising platform for exploring the unlimited potential of many various plants species . In recent years, plant metabolism engineering provides successful pathways to increase the production of metabolites that can significantly counterattack the damages caused by diverse abiotic stresses . To improve stress tolerance in plant, various metabolic engineering technologies were used to introduce or increase the synthesis of diverse osmolytes, secondary metabolites or hormones. The adaptation of various plant species to stressful environments can be managed through: (i) the identification of diverse mechanisms developed by plants to counterbalance abiotic stresses (ii) and the improvement of these processes in plants by metabolic engineering [4, 5]. Plant by-products including hormone (melatonin, MT) and osmoprotectant (glycine betaine, GB) that play a prominent roles in plant stress tolerance have been targeting in various plant species to counterattack environmental stresses. The clarification of the biosynthetic pathway of various plant compounds has provided the possibility to metabolically engineer new capabilities in plants as well as successfully engineer whole pathways into microbial systems . Under environmental stresses plant is able to accumulate different molecules such as melatonin or glycine betaine to provide stress tolerance by counteracting with oxidative stress caused by drought, chilling, salinity or heavy metal stresses [7, 8, 9]. The protective properties of GB and MT in plant under abiotic stresses had made these substances targets for plant engineering resistance.
The natural biosynthesis of glycine betaine takes place in marine algae and various higher plant species belong to diverse families, counting the Gramineae, Malvaceae, Asteraceae, or Amaranthaceae [10, 11, 12, 13, 14]. Glycine betaine accumulation in non-accumulators and accumulators plant species under environmental stresses has long been a target for engineering stress resistance [15, 16]. The biosynthesis of glycine betaine passes by choline → betaine aldehyde → glycine betaine pathways. Most of the enzymes involving in these pathways such as choline monooxygenase (CMO) or betaine aldehyde dehydrogenase (BADH) have been identified, and genes for some of them have been cloned [4, 13].
Indeed, GB as a non-toxic molecule is biosynthesized through two phases of choline oxidation: the first step (Choline → betaine aldehyde) is catalyzed by CMO, and the second step (Betaine aldehyde → glycine betaine) is activated by BADH [13, 17]. The expression of CMO or BADH in tobacco has been done via the cDNA from two natural glycine betaine accumulators; spinach and sugar beet plants. The 35S promoter from plant virus, cauliflower mosaic virus which is a fundamental element of transgenic constructs in the majority of genetically modified plant species was used in transgenic tobacco to control the expression of cDNA for BADH pathway . Also, a crucial tool in metabolism engineering of glycine betaine pathway is the use of a single gene codA from
Melatonin a plant hormone identified in a wide variety of animals and plants, has been extensively studied in plants for its properties to counteract with various environmental and biotic stresses [21, 22]. Transcriptome analyses indicated that melatonin primarily affects the pathways of plant hormone signal transduction and biosynthesis of secondary metabolites . In plant the biosynthesis of melatonin is initiated with tryptophan which is converted in serotonin, and between the tryptophan and melatonin, the enzymes hydroxyindole-
2. Glycine betaine and metabolism engineering
Glycinebetaine is a quaternary ammonium compound that appears commonly in a large diversity of plants, animals and microorganisms, the first betaine discovered was trimethylglycine (Figure 1) named also N, N,N-trimethylglycine [8, 12]. The glycine betaine as a osmolytes is a crucial non-toxic molecule that is accumulated in various plant species under environmental stresses .
2.1 Glycine betaine biosynthesis
GB synthesis begins with an essential molecule named choline, synthesized through three sequential adenosyl-methionine dependent methylations of phospho-ethanolamine catalyzed by the cytosolic enzyme phosphoethanolamine methyltransferase (phosphoethanolamine N-methyltransferase) . In plant, the biosynthesis of GB is two steps of oxidation initiated with choline and then betaine aldehyde (Figure 2). In plant such as
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
|Transgenic species||GB Acc./GB N-Acc.||Type of abiotic stress||Role in stress tolerance||References|
|GB N-Acc.||Salinity||Protection of the photosynthetic apparatus|||
|GB Acc.||Chilling stress||Protect photosynthesis, Homeostasis|||
|GB N-Acc.||Low-Temperature||Enhanced Photosynthesis|||
|GB N-Acc.||Salinity, Chilling stress||Improve photosynthesis and phenotype|||
|GB Acc.||Drought||Osmotic adjustment, enhance yield|||
|GB N-Acc.||Salinity||Phenotypic traits|||
|GB Acc.||Heat and drought stress||Promoted photosynthesis, antioxidant and water status|||
|GB N-Acc.||Salinity||Protect photosynthesis and reproductive organs|||
|GB N-Acc.||High temperature||Enhanced the expression of heat-shock genes|||
|GB N-Acc.||Water stress||Enhance Survival rate and agronomic traits|||
|GB N-Acc.||Chilling stress||Promoted ROS scavenge|||
|GB N-Acc.||High salinity and high temperature||Promote photosynthesis|||
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
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).
|Transgenic species||GB Acc./GB N-Acc.||Genes targeted||Protein Encoded||Organism sources/Promoter||Roles in plant||References|
|GB N-Acc.||codA||Choline oxidase||Water stress tolerance|||
|GB Acc.||GB1(novel gene)||GB1 protein|
- Rice actin and
- 35S promoter
|Enhanced endogenous GB synthesis|||
|GB N-Acc.||cDNA sequence||BADH||Betaine aldehyde resistance|||
|GB N-Acc.||codA||Choline oxidase||Modulation of phosphate homeostasis under stress|||
|GB N-Acc.||codA||Choline oxidase||Reproductive organs regulation|||
|GB Acc.||codA||Choline oxidase||Photo inhibition tolerance|||
|GB N-Acc.||BADH cDNA||BADH||GB synthesis in non accumulator plant|||
|GB N-Acc.||BADH cDNA||BADH||Salt tolerance|||
|GB Acc.||codA||Choline oxidase||Enhance of GB biosynthesis|||
|GB Acc.||codA||Choline oxidase||GB accumulation|||
|GB Acc.||BADH gene||BADH||Stress tolerance|||
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
The catalytic activity of choline oxidase (EC: 184.108.40.206) in
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 . Indeed, the second step of GB biosynthesis is performed by betaine aldehyde dehydrogenase (BADH) that can be encoded by
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. .
Melatonin (N-acetyl-5-methoxytryptamine), a multifunctional plant hormone, was discovered in plants in 1995 . Moreover, the presence of melatonin in plant was confirmed in
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) , and the production of serotonin from Tryptamine is activated by tryptamine 5-hydroxylase . 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 .
As far as we know, there are 6 genes which are involved in plant melatonin biosynthesis: TDC, TPH, T5H, SNAT, ASMT, and COMT , and the keys enzymes they encoded are the; L-tryptophan decarboxylase, tryptophan hydroxylase, serotonin-N-acetyltransferase, N-acetylserotonin methyltransferase and hydroxyindole-O-methyltransferase .
3.2 Melatonin involve in abiotic stress tolerance
Melatonin is well know as a hormone which can significantly increase the plant survival rates, photosynthetic efficiency and antioxidant activities in plant under environmental stress [74, 78]. For these reasons, many studies were focused on the effects of exogenous melatonin on various plant species under abiotic stress. Indeed, exogenous melatonin could stimulate the biosynthesis of cold tolerance agents and contribute to increase the plant growth and development under cold stress . As show Table 3, the alleviation of environmental stresses by melatonin has been investigated in many plant species: under drought (Zea mays) , under heavy metal (Caryaca thayensis) , under chilling stress (
|Plant species||Transgenic/exogenous||Stress||Role in stress||References|
|Exogenous MT||Salt stress||Boost antioxidant system|||
|Transgenic||Drought||Enhanced melatonin content|||
|Exogenous MT||Salt stress||Enhanced the rate of germination|||
|Transgenic||Drought||Enhanced melatonin content|||
|Transgenic||Heavy metal stress (Cadmium)||Enhanced stress tolerance|||
|Transgenic||Herbicide||oxidative stress resistance|||
|Transgenic||UV-B radiation||Reduced DNA damages|||
|Exogenous MT||high temperature and light||Promoted germination|||
|Transgenic||Salt stress||ROS scavenge|||
|Transgenic||Salt stress||Increase in autophagy and rebalance homeostasis|||
In Transgenic Arabidopsis the over expression of N-acetyltransferase gene increased salt tolerance via the increase in autophagy, and the reestablishment of redox and ion homeostasis . Furthermore, increase of over-expressing N-acetyltransferase gene enhances the endogenous content in transgenic rice that provoked pleiotropic phenotypes, including enhanced seedling growth, delayed flowering, and low grain yield .
3.3 Melatonin in plant metabolism engineering
Previous studies using genetic engineering (transgenic plant) in various plants species with low or high MT accumulation has been achieved to determined the role of MT in plant growth regulation, stress tolerance or MT function in plant (Table 4). Indeed it was reported the implication of MT in seed germination, root development, fruit ripening, senescence, yield, circadian rhythm and plant homeostasis . Ectopic over-expression (transgenesis) of human serotonin N-acetyltransferase increased endogenous melatonin that allowed transgenic rice seedlings to face chilling stress . The increase of endogenous melatonin in various transgenic plant organisms compared to the wild type has been reported in
|Transgenic species||Genes targeted||Protein encoded||Organism source/ transformer/vector||Functions||References|
|N-acetylserotonin methyltransferase||Ameliorated Plant Growth|||
|arylalkylamine N-acetyltransferase /|
|Ovine/||Improved growth and salt tolerance|||
|N-acetylserotonin deacetylase||Rice/||Regulation of melatonin in plant|||
|Caffeic acid 3-O-methyltransferase||Wheat/||Promoted drought tolerance|||
|Serotonin N-acetyltransferase||Sheep/||Homeostasic regulation of melatonin|||
|serotonin N-acetyltransferase||Alfalfa/||Salt tolerance|||
|N-acetylserotonin-O-methyltransferase||Apple/35S promoter||Drought tolerance|||
|hydroxyindole O-methyltransferase||Ovine/||biosynthetic and physiological functional networks of melatonin|||
|arylalkylamine N-acetyltransferase/hydroxyindole-O-methyltransferase||Inhibited UV-B-induced DNA damage|||
|caffeic acid O-methyl-transferase||Tomato/||Salt tolerance|||
Most of the studies in MT transgenesis are based on the ability of
The elucidations of these reactions and techniques provided a huge benefit to increase the use of those compounds in metabolic engineering. There are others areas to explore and clarify to shed light the use of melatonin or glycine betaine metabolic engineering.