Open access peer-reviewed chapter
Abstract
In last decades, plants were increasingly subjected to multiple environmental abiotic stress factors as never before due to their stationary nature. Excess urbanization following the intense industrial applications introduced combinations of abiotic stresses as heat, drought, salinity, heavy metals etc. to plants in various intensities. Technological advancements brought novel biotechnological tools to the abiotic stress tolerance area as an alternative to time and money consuming traditional crop breeding activities as well as they brought vast majority of the problem themselves. Discoveries of single gene (as osmoprotectant, detoxyfying enzyme, transporter protein genes etc.) and multi gene (biomolecule synthesis, heat shock protein, regulatory transcription factor and signal transduction genes etc.) targets through functional genomic approaches identified abiotic stress responsive genes through EST based cDNA micro and macro arrays. In nowadays, genetic engineering and genome editing tools are present to transfer genes among different species and modify these target genes in site specific, even single nuclotide specific manner. This present chapter will evaluate genomic engineering approaches and applications targeting these abiotic stress tolerance responsive mechanisms as well as future prospects of genome editing applications in this field.
Keywords
- GMO
- abiotic stress
- genetic engineering
- genome editing
1. Introduction
Before the first examples of the crop domestication by late hunter-gatherer man approximately 10.000 years ago, plants evolved most of their traits following the most basic rule of evolution: success in reproduction and producing next generations. To achieve this goal, plants evolved various adaptation mechanisms against harsh environmental conditions described as abiotic stress factors as well as biotic stress factors like diseases and herbivores [1, 2]. The early domesticated agronomical traits desired by the primal farmers were mostly controlled by the limited number of genes. Traits as panicle size (rice, pearl millet), fruit size (tomato, eggplant), seed size (rice, sorghum, maize, common bean), seed dispersal (cereals in general) are easily controlled by small number of genes [3, 4]. Traits as bitter taste which is one of the adaptation mechanism of plants against herbivores can be controlled by single or multiple genes due to the complexity of the biochemical pathway of the compound. In the absence of genetic knowledge, first breeders domesticated almond as its bitterness is controlled by single gene, while the similar bitter taste in oak acorns is controlled my multiple genes and it was never domestication target [5]. Human intervention to natural selection of plants helped plants to achieve their reproduction goal at the cost of losing most of the gene pool diversity including abiotic stress tolerance and disease resistance genes in favor of human desired traits. Until Gregory Mendel’s breakthrough findings, plant breeding was based on easily made crosses, observations and mass selections without knowing sexual recombination of alleles recombined in meiosis to produce new traits for hundreds of years. Even after the knowledge of Mendelian segregation of traits, crossing of distantly related plant species for desired traits as disease resistance became problematic due to the abnormal chromosome pairing and infertile hybrids not to mention horizontal gene transfer between different kingdoms as bacteria and viruses. Following the studies on molecular structure and function of genes during 1940s and the discovery of the structure of DNA by Watson and Crick in 1953, technical developments as PCR and use of restriction enzymes led to the first transfer of bacterial antibiotic resistance gene to plant cells in 1983. Foreign bacterial gene introduced to petunia cell cultures and plantlets derived from transformed cells retained antibiotic resistance [6]. Following these developments, tomato was the first genetically engineered plant which was intended to be used in commercial practices in 1982. Commercial use of genetically engineered crops started in 1996 in US, since four genetically engineered plants were allowed by USDA for field testing in 1985. Technical and technological research kept improving on this field. Certain trends in engineered plants divided the use of this technology in three categories. Targeted traits of the first generation genetically engineered plants were mostly oriented according to farmer’s benefit as abiotic stress tolerance or herbicide resistance. Second generation of genetically engineered plants emphasized more on commercial benefits as shelf life or nutritional value. The third generation of genetically engineered plants represent the idea of functional foods which were enhanced with pharmaceutical product that are not present in the plant itself [7].
Use of genetic engineering techniques in abiotic stress tolerance of plants requires knowledge on physiology and biochemical processes to identify key gene targets. Functional genomic approaches utilize transcriptome analysis of plants under abiotic environmental stress factor by using techniques as quantitative, real-time PCR, microarray and high-throughput RNAseq. Gene expression profiles of specific tissues under development stage of choice can be searched between previously submitted Expressed Sequence Tags (ESTs) in various cDNA libraries. Abiotic stress responsive genes in particular abiotic stress tolerance mechanisms can be evaluated before progressing further in experimental design [8]. Molecular regulation type of the targeted trait is also important in genetic engineering approach. Abiotic stress tolerance is mostly regulated by multiple genes depending on stress and targeted tolerance mechanism. One of the early effects of the abiotic stresses on plants is disruption of osmotic balance in cells. Therefore, biosynthesis and accumulation of osmoprotectant molecules as proline, glycine betaine, polyamines, mannitol, trehalose can be targeted as they are regulated by single individual genes. Likewise, reactive oxygene species scavenging genes (superoxide dismutase, glutathione reductase, ascorbate peroxidase), late embryogenesis abundant (LEA) proteins, ion transport genes are some single gene targets used in genetic engineering approach for abiotic stress tolerance which will be further detailed in this chapter. Targeting a tolerance mechanism which is regulated by multiple genes is more complicated. Still, there are many successful genetic engineering applications targeting heat shock proteins (HSPs), transcription factors (TFs), signal transduction genes for abiotic stress tolerance [9, 10]. After all of these decision making process which requires intense precision, promoter type is one of the key factors since all transferred genes requires expression regulation. Calliflower Mosaic Virus 35S (CaMV35S), maize ubiquitine-1 (Ubi-1) and rice actin-1 (act-1) are some of the mostly used strongly expressed constitutive promoters which were used in abiotic stress tolerant genetically engineered plant studies. Despite their advantages to plants as strong expression in all developmental stages in all tissues without any stimulation from the environment, they are not economical for plants in contexts of energy and biochemical source consumption. Signal transduction genes as SAPK4, OsITPK2, OsCPKI2, transcription factor genes as JERF3, ZFP182, antioxidant genes as katE, CAT1, GST, OsMIOX, osmotic homeostasis genes as codA, ion homeostasis genes as AgNHX1, nhaA, OshAK5 are some examples which are regulated by CaMV35S promoter in abiotic stress tolerant genetically engineered plants while Ubi-1 promoter is utilized for DSM1, OsCPK21, OsCPK4 signal transduction genes, OsWRKY45-1, OsWRKY45-2, ZmCBF3, OsbZIP46 transcription factor genes, Sod1 antioxidant gene, otsA, otsB, adc osmotic homeostasis genes and OsKAT1 ion homeostasis genes [11]. Constitutive and avoidable use of biochemical sources in stress free periods or environments leads these genetically engineered plants to reduced growth and loss of yield in some cases. Therefore, regulation of abiotic stress induced or tissue/developmental stage specific promoters are more viable options. General aspects of desired stress induced promoters are described as: (i) having basal expression level under stress free conditions, (ii) strongly promoting expression of resistance genes, (iii) having dose dependent and dose sensitive stress induction, (iv) being stress specific and (v) having reversible induction which rapidly reduce to basal level as removal of stress factor. Arabidopsis rd29A promoter is the most frequently used stress inducible promoter in genetically engineered plant studies. Some stress inducible promoters may lack some desired features as stress induced rab16A, LIP9, OsNAC6 which have higher promoting regulation under basal conditions or some may need further characterization and knowledge as HVA1s, Dhn8s and Dhn4s from barley and DGP1 from tobacco [12].
Most of the abiotic stress tolerant genetically engineered plant trials are limited to laboratory experiments and do not have commercial uses due to the regulations or inadequate field performance. Commercial genetically engineered plants need to tolerate or resist to multiple biotic and abiotic stresses in different combination, duration and concentrations. Tolerance or resistance should not be limited only to developmental stages but also reproductive stage which plants are more vulnerable to abiotic stresses.
As an alternative to gene transfer approach, many studies utilize genome editing techniques as they allow researchers to modify the genome even in a few nucleotide level as well as they can be used to alter or replace allels, silence or insert new gene(s) to targeted sites in genome. Genome editing by using site specific endonucleases is not a new concept in developing crop plants by means of biotic and abiotic stress tolerance. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is the most recent site specific genome editing technique which dominated the alternatives as transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs) due to the accuracy, cost and time efficiency and by the means of application advantages as allowing multiple site editing at the same time. Even this new approach evolved in short time due to the occurring limitations during application and new alternatives to Cas9 as Cpf1 are seriously in consideration recently. Also transgene free genome editing applications are introduced lately as the law regulations and public acceptance on this topic are crippling for researchers [13, 14].
Consumers and farmers are interested to the traits as taste, productivity, yield, shape of commercial genetically engineered and genome edited varieties more than just survival under abiotic stresses. Therefore, most of the abiotic stress tolerant plant strategies mentioned further in this chapter are laboratory applications. Table 1 represents successful commercial transformation events of abiotic stress tolerant maize, soybean and sugarcane developed by Monsanto, Verdeca and Persero Companies.
| MON87427 x MON87460 x MON89034 x TC1507 x MON87411 x 59122 / Monsanto | insect resistance, drought and herbicide (glyphosate and spesifically glufosinate ammonium) tolerance | cp4 epsps, cspB, cry2Ab2, cry1A.105, cry1F, pat, cry34Ab1, cry35Ab1, cry3Bb1, dvsnf | Food Feed Biofuel | |
| MON87460 (Genuity® DroughtGard™) / Monsanto | cold / heat drought tolerance | cspB, nptII | Food Feed Biofuel | |
| MON87460 x MON88017 / Monsanto | insect resistant (coleopteran and lepidoptera), drought and herbicide (glyphosate) tolerant | cspB, cp4 epsps, cry1A.105, cry2Ab2, cry3Bb1, nptII | Food Feed | |
| MON87460 x MON89034 x MON88017 / Monsanto | cold / heat, drought, herbicide (glyphosate) tolerance and insect resistance (lepidoptera and coleopteran) | cspB, cry1A.105, cry2Ab2, cp4epsps and cry3Bb1 | Food Feed | |
| MON87460 x MON89034 x NK603 / Monsanto | cold / heat, drought, herbiside (glyphosate) tolerance and insect (lepidoptera) resistance | cspB, cry1A.105, cp4 epsps, cry2Ab2, nptII | Food Feed | |
| MON87460 x NK603 / Monsanto | drought and herbicide (glyphosate) tolerance | cspB, cp4 epsps nptII | Food Feed | |
| MON89034 x MON87460 / Monsanto | drought tolerance and insect (lepidoptera) resistance | Cry2Ab2, cry1A.105 and cspB | Food Feed | |
| HB4 / Verdeca | drought and hyper salinity tolerance | Hahb-4 | Food Feed | |
| HB4 x GTS 40-3-2 / INDEAR | drought, salinity, herbicide (glyphosate) tolerance | Hahb-4 and cp4 epsps | Food Feed | |
| NXI-1T / PT Perkebunan Nusantara XI (Persero) | drought tolerance | EcBetA, nptII, aph4 (hpt) | Food | |
| NXI-4T / PT Perkebunan Nusantara XI (Persero) | drought tolerance | RmBetA | Food Feed | |
| NXI-6T / PT Perkebunan Nusantara XI (Persero) | drought tolerance | RmBetA | Food |
Table 1.
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2. Genetic engineering applications in plant abiotic stress tolerance
One of the key points in breeding better crops under various stress conditions is understanding the cellular, biochemical and molecular changes that occur in response to stress [17]. Understanding the mechanisms underlying plants responses to abiotic stress is an important step for genetic engineering that focuses on improving or enhancing tolerance to these stresses (i.e. salinity, cold, dehydration). Identifying key genes that positively affect tolerance within these mechanisms and introduction and overexpression of them allow improvement of genetically modified plants tolerant to abiotic stress. Additionally, understanding the mechanisms by which plants perceive and transmit stress signals is another important point for genetic engineering of crops [18, 19]. Growing number of molecular and biochemical studies on this subject provide an understanding of the signal transduction network involved in the response to abiotic stress and the pathways associated with this network. The development of plants by conventional breeding methods has certain limitations as transfer of a gene requires repeated cycles of crossing and selection. Moreover, the classical breeding process is limited to species with sexual reproduction. Another disadvantage of classical breeding is that genes with undesirable characteristics are transferred together with target genes in this process. With the advent of modern biotechnology tools, it has become possible to modify genetic structure of plants using genes from other living organisms, and recombinant DNA technology has provided an effective alternative to traditional approach. In addition, it has become possible to exchange genetic material between sexually incompatible species. Genes encoding proteins known to play a role in resistance and tolerance to biotic (virus, bacteria, nematode, fungus, herbivores) and abiotic stresses (drought, salinity, temperature, cold) are widely used in the obtaining genetically modified plants. Plants have developed a signal transduction pathway that regulates various stress-response genes such as kinases, molecular chaperones, osmoprotectants, transcription factors, and thus gives an idea of how tolerance to environmental factors is achieved. Since it is known that this signal transduction and associated physiological, biochemical and molecular pathways are governed by more than one gene, it is extremely difficult for only one gene to achieve complete abiotic stress tolerance. Among the molecules known to have protective roles against abiotic stress are proline, which acts as an osmoprotectant, metal chelator, antioxdative defense molecule and signal molecule [20]; trehalose, which acts as an osmoprotectant and is involved in ROS scavenge during abiotic stress [21]; heat shock proteins (HSPs) that serve as molecular chaperones responsible for the folding, assembly, translocation and degradation of proteins [22]; Late embryogenesis abundant (LEA) proteins with antioxidant functions involved in protein protection, membrane protection, and ion binding [23]; aquaporins involved in the transport of water and neutral molecules [24]; calcineurin B-like proteins that act as calcium sensors and play a role in signal transmission. Transcription factors (NAC, WRKY, MYB, bZIP, DREB/CBF), kinases and phosphatases serve a function in stress perception and signal transduction and in the regulation of stress-inducible genes. In addition to the identification, isolation and characterization of genes involved in all this abiotic stress response, introduction of these genes to plants is an effective molecular approach to understand the roles of genes and products in plant tolerance, behavior and phenotypes.
2.1 Late embryogenesis abundant proteins
Late embryogenesis abundant proteins, which were first discovered in the late developmental stages of plant seeds, are among the proteins most commonly used by plants in their response to abiotic stresses such as drought, high salt, extreme temperature and oxidative stress. Most of the LEA proteins and their mRNAs accumulate in high amounts in the embryo tissues in the final stages of seed development when drought initiates [23]. In addition, LEA proteins also accumulate in plant tissues exposed to dehydration, osmotic and low temperature stress. There is strong evidence that LEA proteins or their genes classified into 6 groups according to their specific domains are correlated with stress resistance [25, 26]. Many studies have reported that stress tolerance occurs as a result of the introduction of LEA genes to various plants such as Arabidopsis, tobacco, rice, barley, and corn by genetic engineering techniques (Table 2). Amara [30] reported that the Rab28 LEA gene was overexpressed in maize plants in which they transferred the Rab28 LEA gene with a constitutive maize promoter by particle bombardment method. With the expression of the Rab28 transcripts, the Rab28 protein accumulated in transgenic plants and the transgenic plants continued their growth in the dehydration condition in medium containing polyethyleneglycol (PEG) compared to wild-type controls. These results showed that LEA Rab28 (belonging to the 5th subgroup of the LEA protein family) protein is one of the important candidates that can be used to increase stress tolerance in maize plants. In another transformation study performed with the LEA genes, the barley (HVA1) gene encoding the late embryogenesis abundant protein was transferred to mulberry, which is an important industrial plant used in silk production, by
| LEA | ROB5 from Bromegrass, DHN4 from | CaMV35S Cor78 | Drought Heat Cold | Agrobacterium | [27] | |
| LEA | Hva1 from | rd29A | Salinity Drought Cold | [28] | ||
| LEA | OsLEA3-2 from | CaMV35S | Salinity Drought | Agrobacterium EHA105 | [29] | |
| LEA | LEA Rab28 gene from | UBQ | Water stress | Biolistic system | [30] | |
| LEA | JcLEA from | CaMV35S | Salinity | [31] | ||
| LEA | ZmLEA3 from | CaMV35S | Osmotic, Oxidative stress | [32] | ||
| LEA | WCI16 from | CaMV35S | Freezing | [33] | ||
| LAE | ZmLEA5C from | CaMV35S | Osmatic stress Cold | [34] | ||
| LAE | SiLAE from | Super promoter | Osmotic stress Salinity | [35] | ||
| LEA | AdLEA from | CaMV35S | Salinity Oxidative stress | [36] |
Table 2.
Summary of transgenic plants over-expressing late embryogenesis abundant (LEA) genes for abiotic stress tolerance.
2.2 Aquaporins (AQPs)
Aquaporins (AQPs) are a family of membrane water channel proteins that control the osmatic movement of water and other molecules such as carbon dioxide, glycerol, urea, and ammonia [24]. This system, which has high osmotic water permeability, operates with lower activation energy [37]. Since environmental conditions such as high temperatures and winds encountered by plants living on land cause an increase in the rate of transpiration. These plants have developed various strategies such as controlling transpiration with hormones, cellular signaling mechanisms, ion channels, and transporters to protect hemeostasis [38, 39]. However, the current situation worsens when osmotic pressure increases, water is absent and root hydraulic conductivity (Lpr) decreases [18]. In order to overcome this negative situation, the isolation of water channel genes and their introduction to the desired plant by using genetic transformation emerges as one of the most important solutions. Thus, this situation can be reorganized by transgenic expression of water channels that ensure the continuity of processes such as transcellular water transport, stomatal closure, osmoregulation, vacuolar differentiation and plant cell expansion (Table 3). Under saline conditions, plant growth is suppressed due to osmotic stress, and in addition, this suppression is further increased due to reduced photosynthesis and gas exchange due to salt accumulation in the leaves. Aquaporins are thought to be extremely effective in plant growth thanks to their effects on leaf gas exchange as well as root water intake. While overexpression of plasma membrane intrinsic proteins (PIP) causes an increase in leaf gas exchange under normal conditions, it increases assimilation and development under salt stress [43]. Moreover, it has been reported that shoot length and fresh weight significantly increased in PIP introduced plants under salt stress [43]. They indicated that transgenic plants developed by transferring the GmPIP1;6 genes to soybean plant (
| Aquaporin (plasma membrane intrinsic proteins) | TdPIP1 and TdPIP2 from | CaMV35S | Osmotic stress Salinity | [40] | ||
| Aquaporin (plasma membrane intrinsic protein) | GhPIP2;7 from | GhPIP2;7 | Drought | [41] | ||
| Aquaporin (plasma membrane intrinsic protein) | OsPIP1 from | UBQ | Salinity | [42] | ||
| Aquaporin (plasma membrane intrinsic protein) | GmPIP1;6 from | CaMV35S | Salinity | [43] | ||
| Aquaporin (plasma membrane intrinsic protein) | BvCOLD1 from | CaMV35S | Cold | [44] |
Table 3.
Summary of transgenic plants over-expressing aquaporin genes for abiotic stress tolerance.
2.3 Osmoprotectants
Trehalose is a non-reducing disaccharide sugar found in bacteria, fungi, invertebrates, including plants. The accumulation of trehalose, which is an important component of plant growth, affecting sugar metabolism and causes an osmoprotectant effect under abiotic stresses [46]. Trehalose is considered as an osmoprotectant as it counteracts against the effects of dehydration of plants caused by drought, salinity or low temperature [47]. Studies have shown that enzymes, proteins and lipid membranes can be stabilized by trehalose and that biological structures can be protected from damage by trehalose during abiotic stress [18]. Trehalose is an osmolite that both regulates osmosis and protects macromolecules. In higher plants, overexpression of trehalose-6-phosphate synthases (TPS) and/or trehalose-6 phosphate phosphatases genes originated from microorganisms has led to tolerance to various abiotic stresses. In addition, several genes involved in trehalose metabolism have been used together to improve the stress tolerance of some plants. The results show that the increased production of trehalose with overexpression of the bifonional fusion gene (TPSP) of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase genes increases the soluble sugar content and increases the tolerance to drought and salt stress in rice [48]. As a result of the expression of the TPSP fusion gene, tomatoes that accumulate high amounts of trehalose in their leaves exhibited a tolerance to drought and salt stress, and showed higher photosynthetic rates under salt stress conditions than wild-type plants [49]. Proline, another osmoprotectant, is an amino acid that plays a role in plant functions exposed to various abiotic stresses. Proline accumulates in response to many environmental stresses such as salinity, drought, heavy metals, and there is a positive correlation between plant stress and proline accumulation. Proline protects proteins and other molecules in the cell against denaturation by stabilizing them. In addition to being an excellent osmolyte, it also functions as a metal chelator, antioxidant defense molecule and signal molecule [20]. In addition, it plays an important role in the protection of subcellular structures, adjustment of cell turgor and osmatic balance, prevention of electrolyte leakage by stabilizing membranes, and balancing the concentration of reactive oxygen species (ROS). Proline synthesis in plants is carried out by two important pathways; glutamate pathway and orinithine pathway. However, among these, the glutamate pathway is the primary pathway of proline accumulation during osmatic stress [50]. This reaction is catalyzed in most plants by the enzyme Δ’-pyrroline-5 carboxylate synthetase (P5CS) encoded by two genes and the enzyme Δ’-pyrroline-5-carboxylate reductase (P5CR) encoded by a gene. In addition to the effects of proline on the physiology and biochemical processes of various plants, a high amount of endogenous proline has been shown to act as a regulator and/or signaling molecule that can alter the transcription levels of stress-related genes such as ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) [51, 52]. A P5CS gene that can be transferred in addition to the P5CS genes present in plants by genetic transformation helps plants to adapt to osmatic conditions by regulating proline accumulation, and also supports legume plants for optimum nodule formation and maintenance of nodule function [53]. Ghanti [54] reported that when they transferred the
Plants synthesize and accumulate another osmoprotectant, glycine betaine (GB), in response to environmental stresses. Glycine betaine, which also plays a role in osmoregulation, protects the activity of macromolecules and the integrity of the membranes against stresses and scavenge the ROS. Glysine betaine synthesis reactions are catalyzed in two steps by the enzymes choline monooxygenase (CMO) and NADC-linked betaine aldehyde dehydrogenase. Many studies have been carried out showing that the abiotic stress tolerance of transgenic plants obtained as a result of isolating these genes in the biosynthetic pathway and transferring them to other plants has developed. Several osmoprotectant target for gene transfer approach are listed in Table 4.
| Pyrroline-5-carboxylate synthetase | P5CS from | CaMV35S | Salinity | [53] | ||
| Pyrroline-5-carboxylate synthetase | P5CS from | CaMV35S | Salinity | [56] | ||
| Pyrroline-5-carboxylate synthetase | P5CS from | CaMV35S | Salinity | [57] | ||
| Pyrroline-5-carboxylate synthetase | VaP5CSF129A from | CaMV35S | Drought | - | [54] | |
| Pyrroline-5-carboxylate synthetase | MtP5CS3 from | CaMV35S | Salinity | - | [58] | |
| Pyrroline-5-carboxylate synthetase | P5CSF129A from | CaMV35S | Salinity | [59] | ||
| Trehalose-6-phosphate synthase | OsTSP1 from | CaMV35S | Salinity Drought Cold | [60] | ||
| Trehalose-6-phosphate synthase/ phosphatase | Recombinant fusion TPSP from | UBQ | Drought Salinity | - | [51] | |
| Trehalose-6-phosphate synthase | TPS1 from yeast | StDS2 | Drought | [61] | ||
| Trehalose-6-phosphate synthase/ phosphatase | Recombinant fusion TPSP from | CaMV35S | Drought Salinity | [52] | ||
| Glycine betaine | codA (Choline oxidase) from | SWPA2 | Cold Salinity | [62] | ||
| Glycine betaine | BADH from | CaMV35S | Salinity Cold | [63] | ||
| Glycine betaine | betaine aldehyde dehydrogenase gene (AhBADH) from | CaMV35S | Salinity | [64] | ||
| Glycine betaine | betA gene from | UBQ | Salinity | [65] | ||
| Glycine betaine | betA gene from | UBQ | Drought | [66] | ||
| Glycine betaine | Mpgsmt and Mpsdmt from | CaMV35S | Drought Salinity | [67] | ||
| Glycine betaine | codA from | - | Heat | [68] | ||
| Glycine betain | BADH betaine aldehyde dehydrogenase from | CaMV35S | Heat | [69] | ||
| Glycine betaine | codA from | CaMV35S | Salinity | Solanum | [70] |
Table 4.
Summary of transgenic plants over-expressing osmoprotectant-related genes for abiotic stress tolerance.
2.4 Calcineurin B-like proteins (CBLs), calcium-dependent protein kinases (CDPKs)
Plants perceive the stimuli from the external environment through receptors in their membranes. After transmitting this signal, they respond to this stimulus. Ca2+ acts as one of the secondary messengers in the ABA signaling pathway, which plays an active role in this process. Calcium acts as a central link in numerous signaling pathways, and thus external signals such as hormone, light, biotic and abiotic stresses cause changes in Ca2+ concentration in the cell. The fluctuation in the cytosolic calcium concentration (calcium signatures) initiates various biochemical and physiological processes in the cell. Cellular calcium sensor molecules such as calcineurin B-like proteins (CBLs), calcium-dependent protein kinases (CDPKs) and calcium-dependent protein kinases (CIPKs), calmodulin (CaM) detect Ca2+ signals and enable Ca2+ ions to be transmitted to downstream pathways. Especially, calcineurin family of B-like proteins (CBLs) interacts and activates calcium-dependent protein kinases (CIPKs). Many studies have shown that this regulatory pathway plays a role in plants in response to environmental stresses (Table 5). It has been reported that overexpression of GmCBL1 in Arabidopsis increases tolerance to both high salt and drought stress in transgenic plants. It has also been shown that hypocotyl elongation is promoted under light conditions by overexpression of GmCBL1. Calcineurin B-like proteins (CBLs) can regulate stress tolerance by activating stress-related genes such as DREB1A, DREB2A, RD29A and KIN1, while also controlling hypocotyl development by changing the expression of genes related to gibberellin biosynthesis [68]. BdCIPK31, a CIPK gene belonging to
| CIPK | OsCIPK23m from | UBQ | Drought | [71] | ||
| CBL-CIPK | BnCBL1-BnCIPK6 from | CaMV35S | Salinity | [72] | ||
| CBL protein | GmCBL1 from | CaMV35S | Salinity Drought | [68] | ||
| CBL protein | PeCBL10 from | CaMV35S | Salinity Drought Cold | [73] | ||
| CBL protein | PtCBL10A PtCBL10B from | CaMV35S | Salinity | [74] | ||
| CBL protein | NsylCBL10 from | CaMV35S | Salinity | [70] | ||
| CBL protein | SpCBL6 from | CaMV35S | Cold | [75] | ||
| CIPK | BdCIPK31 from | CaMV35S | Drought Salinity | [69] |
Table 5.
Summary of transgenic plants over-expressing calcineurin B-like (CBL) and CBL-interacting protein kinase (CIPK) genes for abiotic stress tolerance.
2.5 Heat shock protein
Heat shock proteins (HSPs) were originally identified as proteins that respond to high temperature conditions and involve in eukaryotes and prokaryotes. However, many studies have revealed the relationship of these proteins with various abiotic stresses. Heat shock proteins are powerful chaperones produced in response to many physiological and environmental stresses. In general, heat shock proteins function in the cytoplasm, while they are also located in organelles such as the nucleus, mitochondria, chloroplasts, endoplasmic reticulum [22]. These proteins that are evolutionarily conserved in prokaryotes and eukaryotes are also abundant in plants. HSPs divided into 5 different groups including HSP100, HSP90, HSP70, HSP60 and sHSP (small heat shock protein) according to their molecular weights. Among these groups, small heat shock proteins are also induced by other stresses such as drought, salinity and cold stress, and also play an active role in processes such as seed germination, embryogenesis and fruit development [76]. Transgenic
| Heat Shock | GHSP26 from | CaMV35S | Drought | [81] | ||
| Heat Shock | OsHSP6 from | CaMV35S | Oxidative stress Heat | [77] | ||
| Heat shock | TaHSP26 from | CaMV35S TaHSP26 | Heat | [76] | ||
| Heat Shock | ZmHSP16.9 from | CaMV35S | Heat | [82] | ||
| Heat Shock | GmHsp90s from | CaMV35S | Osmotic stress Salinity | [83] | ||
| Heat Shock | EaHSP70 from | Port Ubi 2.3 | Salinity Water deficiency | [80] | ||
| Heat Shock | HSP70 from | UBQ | Salinity | [84] | ||
| Heat Shock | sHSP18.6 from | CaMV35S | Heat Drought Salt Cold | [78] |
Table 6.
Summary of transgenic plants over-expressing heat shock genes for abiotic stress tolerance.
2.6 Transcription factors
The characterization and identification of genes involved in the stress response of plants is a prerequisite for producing genetically modified plants that are tolerant of various stresses. The fact that these identified genes are regulatory genes responsible for regulation become prominent as an effective approach in terms of ensuring the efficient control of many other genes involved in stress management. The most important regulatory gene candidates that can be used in the development of stress-tolerant plants are transcription factors (TFs) that also regulate the expression of stress genes. Transcription factors belonging to many different families such as AP2/EREBP, NAC, WRKY, MYB, bZIP are involved in various abiotic and biotic stress processes.
NAC proteins stand out as one of the largest family of transcription factors in plants. In particular, they perform vital tasks in the developmental processes of plants and their response to environmental stresses. They play a role in the regulation of many processes such as cell division, flower development, lateral root development, senescence, phytohormone homeostasis, secondary cell wall formation, abiotic and biotic stress responses. There are many studies that develop tolerance to abiotic stress as a result of transformation of plants with NAC transcription factors. Huang [86] transferred the TaNAC29 gene, which they isolated from wheat (
Another important transcription factor family that is widely distributed in plants is MYB TFs. Many MYB transcription factors function in many physiological and biochemical processes, including cell development, cell cycle, hormone cycle, primary and secondary metabolism and signal transduction, biotic and abiotic stresses [89]. There are many studies reporting that transgenic plants enhance tolerance by overexpression of MYB transcription factors (Table 7). GmMYBJ1 introduced transgenic Arabidopsis plants have been shown to have an increased tolerance to drought and salinity compared to wild-type plants. At the same time, these plants showed higher plant height and less water loss rate when exposed to dehydration and cold stresses. Moreover, less MDA accumulation was detected in transgenic plants under stress with the overexpression of MYB [115]. TaMYB3R1 (
| AP2-ERFBP | GmERF3 from | Salinity Drought | Agrobacterium-mediated transformation | [90] | |
| DREB1 | OsDREB1D from | Cold Salinity | [90] | ||
| AP2-ERFBP | SbDREB2 from | Drought | [91] | ||
| DREB1A | AtDREB1A from | Drought | Particle-bombardment transformation | [92] | |
| AP2-ERFBP | LcDREB2 from | Salinity | [93] | ||
| AP2-ERFBP | OsERF4a from | Drought | [94] | ||
| AP2ERFB | EaDREB2 from | Drought Salinity | [95] | ||
| DREB1A | AtDREB1A from | Drought | [96] | ||
| AP2-ERFBP | LcERF054 from | Salinity | [97] | ||
| AP2-ERFBP | TaPIE1 from | Freezing | Biolistic bombardment | [98] | |
| AP2-ERFBP | VrDREB2A from | DroughtSalinity | [99] | ||
| AP2-ERF | OsEREBP1 from | Drought | [100] | ||
| AP2-ERFBP | SsDREB from | Salt Drought | [101] | ||
| bZIP | PtrABF from | Drought | [102] | ||
| bZIP | GmbZIP1 from | DroughtSalinity Cold | [103] | ||
| bZIP | OsbZIP39 from | ER stress | [104] | ||
| bZIP | MsbZIP from | Drought Salinity | [105] | ||
| bZIP | ZmbZIP72 from | Drought Salinity | [106] | ||
| bZIP | LrbZIP from | Salinity | [107] | ||
| bZIP | OsbZIP71 from | Drought Salinity | [108] | ||
| bZIP | TabZIP60 from | DroughtSalinity Freezing | [109] | ||
| bZIP | CaBZ1 from | Drought | [110] | ||
| bZIP | AtTGA4 from | Drought | [111] | ||
| MYB | AtMYB15 from | Drought Salinity | Agrobacterium-mediated floral dip | [112] | |
| MYB | TaPIMP1 from | Drought Salinity | Agrobacterium-mediated leaf disc method | [113] | |
| MYB | LcMYB1 from | Salinity | [114] | ||
| MYB | GmMYBJ1 from | Drought Cold | [115] | ||
| MYB | TaMYB3R1 from | Drought Salinity | [116] | ||
| MYB | LeAN2 from | Heat | [117] | ||
| MYB | SbMYB2 and SbMYB7 from | Salinity | [118] | ||
| NAC | ONAC063 from | Salinity Osmotic stress | [119] | ||
| NAC | GmNAC20 GmNAC11 from | Salt Freezing | Agrobacterium-mediated vacuum infiltration | [120] | |
| NAC | ZmSNAC1 from | Cold Salinity Drought | [121] | ||
| NAC | TaNAC2a from | Drought | Agrobacterium-mediated transformation | [122] | |
| NAC | AhNAC3 from | Drought | [123] | ||
| NAC | SNAC1 from | Drought Salinity | Bombardment with a biolistic gun | [124] | |
| NAC | OsNAP from | Cold Salinity Drought | [125] | ||
| NAC | TaNAC67 from | Cold Salinity Drought | [87] | ||
| NAC | TaNAC29 from | Drought Salinity | [86] | ||
| NAC | MLNAC5 from | Drought Cold | [126] | ||
| NAC | TaNAC47 from | Salt Cold Drought | [127] | ||
| WRKY | OsWRKY45 from | Drought Salinity | [128] | ||
| WRKY | VvWRKY11 from | Drought | [129] | ||
| WRKY | AtWRKY28 from | Salinity | [130] | ||
| WRKY | ZmWRKY33 from | Salinity | [131] | ||
| WRKY | TaWRKY79 from | Drought | [132] | ||
| WRKY | BdWRKY36 from | Drought | [133] | ||
| WRKY | GhWRKY34 from | Salinity | [134] | ||
| WRKY | MtWRKY76 from | Drought Salinity | Agrobacterium-mediated transformation | [135] | |
| HomeoBox 4 | HaHB4 from | Drought | [136] |
Table 7.
Summary of transgenic plants over-expressing transcription factor genes for abiotic stress tolerance.
The basic leucine zipper (bZIP) is a class of transcription factors with a conserved bZIP domain consisting of a region for DNA binding and nuclear localization at the N-terminus and a lysine-rich region for dimerization at the C-terminus. Similar to other TFs, it not only plays a role in developmental processes but also plays a regulatory role in the response to various abiotic stresses. In this context,
Large regulatory WRKY transcription factors protein family contain two conserved WRKY domains, consisting of approximately 60 amino acids containing the amino site sequence WRKYGQK at the N-terminus and zinc-finger motif at the C-terminus [138]. WRKYs manage many developmental and physiological processes including leaf senescence, regulation of biosynthetic pathways, hormone signaling, embryogenesis, trichome development. Various WRKY transcription factors are also known to be involved in abiotic stress responses. Overexpression of transcription factors, such as ZmWRKY33 (
The Apetala2/Ethylene responsive factor family is characterized by the highly conserved APETALA2 (AP2)/Ethylene Responsive Element Binding Factor (EREB) domain containing 40–70 amino acid sequences involved in DNA binding. AP2/ERFs are divided into four main groups; Apetala2 (AP2), related to abscisic acid intensive 3/Viviparous (RAV), dehydration-responsive element binding protein (DREB), ethylene responsive factor (ERF). These transcription factors play an important role in the regulation of both plant growth and the response to various stresses [139]. The AP2 family of Ethylene Responsive Element Binding Factors supports the survival of plants under stress by activating jasmonate and abscisic acid signaling pathways. The amount of α linolenate, some jasmonate derivatives and abscisic acid increased in rice plants, in which the EREB1 transcription factor gene from AP2/ERF transcription factor family was transferred. The role of this gene family in the response to both biotic and abiotic stress is very important for the regulation of multiple stress tolerance for genetic engineering [100]. Dehydration responsive element-binding proteins (DREB) cooperated with the genes involved in polyamine biosynthesis to enable plants to tolerate salt stress [93]. Thanks to the transfer of another subgroup of ERF transcription factor LcERF054 (
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3. Genome editing applications for abiotic stress tolerance in plants on emphasis to recent CRISPR applications
Since the bacterial defense mechanism against biotic agents as viruses based on the detection and elimination of invader nucleic acids suggested as novel tool for site specific genome editing tool by Jinek [140] in 2012, CRISPR/Cas9 is used in developing crop plants for abiotic stress tolerance as well as many other plant biotechnology applications. All of the abiotic stress tolerance mechanisms mentioned earlier in this chapter which were targets for gene transfer applications can be targeted also by CRISPR/Cas9 system as it allows both induction (CRISPRactivation) or repression (CRISPRinterference) of genes. Therefore, it can be used for activation of tolerance genes (T genes) as well as suppressing sensitivity (S genes) genes [141].
Targeting hormonal regulation for abiotic stress tolerance is one of the viable options. Abscisic acid (ABA) regulates physiological processes as seed dormancy, stomatal closure, plant development, as well as plant responses to environmental stimuli and multiple stresses. 9-cis-epoxycarotenoid dioxygenase (NCED), which is responsible from regulation of ABA in rice, targeted for multiple abiotic stress tolerance including salinity, drought and subsequent H2O2 stress.
Transcription factors (TFs) regulate the expression patterns of the genes on promoter regions. Therefore, they are important targets for both genetic engineering and genome editing applications in abiotic stress tolerance of plants. CRISPRi system designed for 696–amino acid B-type response regulator transcription factor encoding
Antioxidant scavenging is also very important in abiotic stress tolerance. Hence, elevation of antioxidant enzyme capacities in plants is a legitimate approach to improve tolerance. There are several successful transgenic application as described before.
Ion homeostasis is another key factor in abiotic stress tolerance in plants especially against salinity. In Cucurbitaceae family, salt sensitive cucumber and salt tolerant pumpkin varieties were compared in context of K+ uptake during salt stress and salt tolerant pumpkins were found superior to cucumber plants for this trait. CRISPRi knocking out of NADPH oxidase (respiratory burst oxidase homolog D; RBOHD), which was previously tested by its inhibitor diphenylene iodonium, presented decrease in salinity tolerance. On the contrary, ectopic expression of pumpkin RBOHD in Arabidopsis enhanced the salinity tolerance [150].
Along with CRISPRa and CRISPRi application to enhance tolerance to abiotic stresses, it can also be used to identify roles of particular genes in abiotic stress tolerance. Nonexpressor of pathogenesis-related gene 1 (NPR1) is well known gene in plant pathogen response. To evaluate its involvement in drought stress CRISPRi derived tomato
Despite all of these present efforts and obviously more to come in near future, there are some bottlenecks on this topic beyond the genome editing technique itself. The topic of law regulations and public opinion on genetically engineered plants still have many uncertainties. In these days, when many countries regulated or starting to regulated their laws for genetically engineered plants, CRISPR/Cas9 derived products started another debate due to the lack of standardized detection methods and difficulty to distinguish these changes from naturally occurring mutations. Court of Justice of the European Union (ECJ) ruled their opinion on genome edited products in 25 July 2018 as they are to be subjected under the same stringent regulations previously determined for genetically engineered products in 2001 directives. On the other hand, United States Department of Agriculture (USDA) announced their opinion as regulation is not needed for genome edited mutations since they can already occur naturally. Likewise, some countries as Brazil, Argentina and Australia shared the same opinion. In 2016, France inquired to ECJ about reviewing 2001 directive in favor of genome editing techniques as many researchers share their opinion as this method should be evaluated similar to use of radiation in mutation breeding allowing random mutations on genome. But the deliberate and intentional nature of the genome editing mutations brings the opposition. Still, Calyxt, an agricultural biotechnology company in Minnesota, announced the first US sale of high-oleic-acid oil product generated from gene edited soybeans in February, 2019. Following this development, Intrexon Company in Maryland announced start of commercial non-browning gene edited lettuce trials [13, 14].
Woo [155] suggested a novel approach to overcome this dilemma as DNA-free genome editing. In contrary to existing CRISPR/Cas9 system which is based on delivery of RNA guided nucleases into plants either by
In conclusion, following the footsteps of the data generated by the genetic engineering application for abiotic stress tolerance in crop plants, the most recent developments in genome editing techniques as transgene free approach combined with superior endonucleases provides promising results on this topic from many perspectives as law regulations, public acceptance, technical issues.
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