Mechanisms of Plant Genomic Responses to Stresses

#### **Chapter 7**

## Heat Shock Proteins (HSP70) Gene: Plant Transcriptomic Oven in the Hot Desert

*Fatima Batool, Batcho Anicet Agossa, Zainab Y. Sandhu, Muhammad Bilal Sarwar, Sameera Hassan and Bushra Rashid*

#### **Abstract**

Heat stress is considered to induce a wide range of physiological and biochemical changes that cause severe damage to plant cell membrane, disrupt protein synthesis, and affect the efficiency of photosynthetic system by reducing the transpiration due to stomata closure. A brief and mild heat shock is known to induce acquired thermo tolerance in plants that is associated with concomitant production of heat shock proteins' (HSPs) gene family including HSP70. The findings from different studies by use of technologies have thrown light on the importance of HSP70 to heat, other abiotic stresses and environmental challenges in desserts. There is clear evidence that under heat stress, HSP70 gene stabilized the membrane structure, chlorophyll and water breakdown. It was also found that under heat stress, HSP70 decreased the malondialdehyde (MDA) content and increased the production of superoxide dismutase (SOD) and peroxidase (POD) in transgenic plants as compared to nontransgenic plants. Some reactive oxygen species (ROS) such as superoxide, hydrogen peroxide and hydroxyl radical are also synthesized and accumulated when plants are stressed by heat. Hence HSP70 can confidently be used for transforming a number of heat tolerant crop species.

**Keywords:** heat stress, genomic approaches, heat shock protein gene family, plant transformation, plant physiology

#### **1. Introduction**

Heat Shock Proteins (HSPs) are a family of functionally associated proteins which regulates their expression when cells are subjected to high temperatures or other stimuli [1]. As intracellular chaperones, HSP genes are an evolutionarily conserved class of proteins in all living organisms, from bacteria to humans. They are important components that contribute to cellular homeostasis under favorable and harmful growth conditions in prokaryotic and eukaryotic cells [2]. They are responsible for the folding, assembly, translocation and degradation of proteins during normal cell growth and development. Many HSP members perform critically important chaperone roles, such as three-dimensional folding of newly formed proteins and/or proteins weakened by cell stress [3]. For this reason, many chaperones are known to be HSPs due to their existence as aggregates when denatured by heat stress. Under high temperature conditions, their expression levels are increased by transcription of heat shock transcription factors (HSTFs) which are enabled by trimerization of their monomeric forms. This gene regulation system of HSPs is one of the most defined response systems known at molecular level for organisms exposed to extreme temperature conditions [4]. Based on their respective molecular weights, HSPs are referred to small HSP (HSP18, HSP20 and HSP40) and large HSP (HSP60, HSP70, HSP90 and HSP100) [5].

HSP70s are an essential part of the cellular protein folding mechanism and help the cells to defend against stress. Strongly regulated by heat stress, their mechanism interacts with expanded protein peptide segments as well as partly folded proteins to resist aggregation, reshape folding pathways, and regulate activity [6]. When there is no interaction with peptide substrate, HSP70 is normally ATP (Adenosine 5<sup>0</sup> -triphosphate) bound, distinguished by very poor ATPase activity. When newly synthesized proteins emerge from the ribosomes, the HSP70 substrate binding domain identifies and interacts with hydrophobic amino acid residue sequences [7]. Ubiquitously found in all living species, they inhibit aggregation and assist in the reproduction of nonactive proteins in both normal and stress conditions and provide heat-tolerance in plants that are under heat stress. They also inhibit protein folding in mitochondria/ chloroplast during post-translational importation. They are also involved in the import and translocation mechanism of proteins and promote the proteolytic degradation of defective proteins by transferring these proteins to lysosomes or proteasomes [8]. The key role is to manage protein folding and quality control in a crowded cell environment. It also plays a crucial role in signal transduction networks, cell cycle regulation, protein degradation and protein trafficking. In addition, it may also play a role in the morphological evolution and adaptation to stress. They were isolated from the cytosol, the endoplasmic reticulum and the plastid of many plants [9]. Recent studies have shown that HSP70 interacts with the 26S proteasome and plays a crucial function in its assembly and maintenance. As the most abundant proteins in the cells, HSP70 genes have been recorded in many plant species, such as Arabidopsis, soybean, tobacco, rice, maize wheat and Agave [10, 11]. They are also constitutively expressed in plants but their expression is developmentally regulated and caused by various environmental factors such as drought, cold, high temperature and salt. Studies have shown their involvement in cytosols, mitochondria and chloroplasts, play an important role in remodeling machines that contribute to preserving the integrity of the cell proteome by promoting protein remodeling, disaggregation, reactivation and degradation of malformed and inactive proteins [12]. The mechanism for the recovery of proteins from aggregation often requires the assistance of another ATP-dependent chaperone system. The HSP70 family solubilizes the aggregated protein and extracts it in a process that can be repeated with the aid of a specific HSP family of genes [13]. This review focuses on recent discoveries of molecular and cellular mechanisms of HSP70 that govern the tolerance of plants in unfavorable environmental conditions.

#### **2. Biochemical and physiological responses of plants against heat stress**

Heat inducible genes can be categorized in two agencies. The primary organization consists of proteins that maximum probably characteristic in abiotic stress tolerance.

*Heat Shock Proteins (HSP70) Gene: Plant Transcriptomic Oven in the Hot Desert DOI: http://dx.doi.org/10.5772/intechopen.105391*

These encompass molecules along with antifreeze proteins, chaperones osmotin, late embryogenesis abundant (LEA) proteins, mRNA-binding proteins, key enzymes involved in osmolyte biosynthesis, proteins of water channel, transporters like sugars and proline, detoxification enzymes and numerous proteases. The second institution consists of regulatory proteins i.e. protein factors concerned in addition law of signal transduction and stress-responsive gene expression [14, 15]. These encompass diverse transcription factors, protein kinases, protein phosphatases, enzymes worried in phospholipid metabolism, and different signaling molecules inclusive of calmodulinbinding protein. Many transcription factor genes have been stress inducible, suggesting that numerous transcriptional regulatory mechanisms can also function in regulating heat, drought, cold, or excessive salinity stress signal transduction pathways. Those transcription factors could govern expression of stress-inducible genes both cooperatively and independently [16].

#### **3. Methods to study gene identification**

Many techniques have been carried out to evaluate the gene expression for the improvement of plants such as subtractive hybridization of cDNA (Deoxyribonucleic Acid) libraries, homology searching, differential display, genome-wide identification and third generation sequencing [17]. Differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) is a delicate, easy and significant technique to evaluate cDNA [18]. Differential display has benefit as compare to other techniques because big quantity of RNA (Ribonucleic acid) is not needed for analysis. It has been used with great success to identify several differentially expressed genes from plants [19]. The main thing is to do research by using oligonucleotide primers, of which one will be used as anchored primer to the poly-adenylate tail of mRNAs subgroup and the second will be used as arbitrary that will be short in sequence length so that it may combine at different positions as compare to the first primers [20]. The resulted mRNA after using these primer can then be manipulate by using RT-PCR (Reverse transcription polymerase chain reaction) and checked on agarose gel. Multiple primer's pairs can be used to obtain the complementary DNA fragments that depend on strong link with sequence specificity of respective primer. Novel HSP70 genes expressed under stressful conditions have been identified and isolated from tomato, Arabidopsis and wheat by using the DDPCR [21–23]. However, number of genes studied in one attempt of experiment are low as compared to the other advanced techniques like high-throughput expression profiling as qPCR (quantitative polymerase chain reaction) and Northern blotting [24, 25]. Advancements of second-generation sequencing technology offers opportunities for the discovery of millions of novel markers in non-model crop organisms as well as the detection of genes for agronomic traits [26]. Identification of genes within a population gives an understanding that how essential is to control the agronomic traits. The ability to produce sequence data is being supported by increasingly high throughput technologies such as next or second-generation sequencing. It identify the systems that yield vast number (usually millions) of short DNA sequence reads between 25 and 400 bp [27].

The first model plant genome sequenced was *Arabidopsis thaliana* (Arabidopsis Genome, 2000) and first crop genome sequenced was the Rice [28–30]. Current crop genome sequencing programmes are quickly shifting pace with emerging technology that's why second-generation sequencing is adopted to obtain insight into their chosen genome. Although the sequencing and assembly of large and complex crop genomes

remains an important task, a considerable amount of information can be obtained from low-coverage shotgun sequencing of these genomes. Short paired reading data generated using second generation technologies are especially suited for the discovery of genes and gene promoters in crop plants [31, 32]. Many HSP70 genes from different species such as *Agave sisalana* [10]; *Solanum tuberosum* [33, 34]; common bean [35]; *A. thaliana* [36]; Populus [37] have been described at transcription that expresses under heat stress. Four transcriptional regulatory systems have also been reported, two of them are ABA-independent (Abscisic acid) whereas other two are ABA-dependent. Genetic and molecular analyses suggested cross talk between these regulatory systems. Genomic analyses of stress-inducible genes have recently revealed cross talk in stress-responsive gene expression [33, 34, 38, 39].

#### **4. HSP70 gene family leading to improve abiotic stress tolerance in plant**

Plants accumulate specific stress responsive proteins under harsh environmental conditions [40]. Heat-shock proteins (HSPs) and late embryogenesis abundant (LEA) proteins accumulate under salinity, extreme temperature and water stress. These proteins have been shown to be involved in cellular protection during the stress [41, 42]. Enzymes and proteins are not able to function during abiotic stresses. Therefore, it is necessary for cell survival to prevent them from aggregation and maintain their functional conformations [3]. HSP70s are synthesized when environmental changes disturb an organism's whole physiological system to such an extent that results in denaturation of proteins [43, 44]. Under such situation many stress associated proteins especially HSPs have been proven to act as molecular chaperones which play significant role in protein synthesis, maturation, degradation and targeting in an extensive array of ordinary mobile processes. Furthermore, molecular chaperones stabilize the proteins and membranes, in addition to assist in refolding of protein beneath stress conditions [45, 46]. They had been broadly documented in many plant species together with Arabidopsis, soybean, tobacco, rice, maize and wheat. They may be regularly expressed in plants constitutively but their expression is regulated by using various environmental conditions which include heat and salt [47]. Research have verified their presence in cytosol, mitochondria and chloroplast and play vital function in remodeling machines that participate in maintaining the integrity of the mobile proteome via facilitating protein reworking, disaggregation, reactivation or degradation of misfolded and inactive protein [13].

The cDNA coding HSP70 solubilizes and releases the aggregated protein in a kingdom that can be replenished with the assistance of small HSP gene circle of relatives as stated by way of Nillegoda et al., [48]. They're generally cytoprotective, presenting thermo-tolerance that is specifically crucial for plant life [49]. The HSP70 superfamily's genomic evaluation revealed an evolutionary history as phylogenetic tree of all HSP70 participants that cautioned the similarity of HSP70s in 12 subgroups, including the ones expressed formerly to the mammalian HSP110 and GRP170 in the identical sub-cell component. Growth in the expression of HSP70 in one of a kind plant species underneath heat stress situations has been studied appreciably by using proteomics and practical genomics [50]. A widespread osmoprotective effect changed into received in *Escherichia coli* transformed with the cytosolic chaperonin CCP-1a from *Bruguiera sexangula* [51].

HSPs have been studied extensively through plant transformation in response to heat stress [52]. Over expression of HSP101 from Arabidopsis in rice transgenic plants *Heat Shock Proteins (HSP70) Gene: Plant Transcriptomic Oven in the Hot Desert DOI: http://dx.doi.org/10.5772/intechopen.105391*




*Heat Shock Proteins (HSP70) Gene: Plant Transcriptomic Oven in the Hot Desert DOI: http://dx.doi.org/10.5772/intechopen.105391*



*Heat Shock Proteins (HSP70) Gene: Plant Transcriptomic Oven in the Hot Desert DOI: http://dx.doi.org/10.5772/intechopen.105391*

#### **Table 1.**

*Sequence characteristic & subcellular localization of HSP70 gene family in different plants species.*

improved the growth performance after recovery from heat stress [53]. Heikkila et al. [54] demonstrated that exposure of seedlings of corn to ABA, water stress, heat shock, and wounding increases the synthesis of HSP70. Study of HSP70 in Rice, Tomato and Arabidopsis genomes revealed that about 37 genes of HSP70 were identified in rice, 30 in tomato 13 in Arabidopsis and 68 in *A. sisalana* and has been documented to be present within every cellular compartment of plants (**Table 1**).

#### **5. Structure and function of HSP70**

The HSP70s have two huge functional domains, an ATPase domain within the N-terminal part of the protein and a peptide-binding domain within the C-terminal part of the protein [58]. The two domain names are more or less 40 & 25 kDa lengthy, respectively and are separated by means of a hinge location at risk of protease cleavage. Since the HSP70s are determined inside the cytosol, endoplasmic reticulum (ER), mitochondria and plastids, the N-terminal transit peptide of variable sequence is present in the precursor form of these individuals that make part for importation into the organelle [59]. For several HSP70s, a C-terminal subdomain of 5 kDa or much less is needed for plenty co-chaperone interactions (**Figure 1**) [36]. HSP70s exercising their role in several cell techniques by way of binding uncovered hydrophobic residues of non-native proteins during protein folding, stopping protein aggregation, selling the regeneration of combination proteins and keeping proteins in an importable translocation surroundings to subcellular cubicles [9]. HSP70s interact in protein folding by means of chaperone strategies that encompass repeated cycles of peptide binding, ATP hydrolysis and peptide release. Cytosolic Hsp70s are lively in cellular strategies such as protein folding, denatured protein folding, protein aggregation prevention and protein retention in an import-capable eukaryotic surroundings [60]. There are some medical studies of plant cytosolic Hsp70s, but they are acknowledged to behave like different prokaryotic and eukaryotic HSP70s. In vitro experiments have proven that plant cytosolic Hsp70s mixes mysterious precursor polypeptides with nascent. Further, whilst wheat germ extract become removed from cytosolic Hsp70, co-translocation and processing of precursor proteins have become inefficient and the

#### **Figure 1.**

*Structure of HSP70: SBD (substrate binding domain), ATP (adenosine 5*<sup>0</sup> *-triphosphate), ADP (adenosine diphosphate).*

*Heat Shock Proteins (HSP70) Gene: Plant Transcriptomic Oven in the Hot Desert DOI: http://dx.doi.org/10.5772/intechopen.105391*

incorporation of cytosolic Hsp70 restored the translocation and processing of precursor proteins [61]. It is proposed that the cytoplasmic Hsp70s are involved within the ER-translocation precursor protein.

Chloroplast Hsp70 homologs recognized to be energetic in import strategies are within the outer envelope membrane facing the cytoplasm and inter membrane space, the stroma and the thylacoid lumen. Hsp70 is concerned in early protein imports by using associating with chloroplast precursor proteins, based on move-linking and immune precipitation studies [62]. The precise characteristic of Hsp70 stays unclear, however it is proposed that precursor proteins are moved from the cytosolic HSP70s to the chloroplast HSP70s. This is confirmed through studies with Dnak as a model system and shows that HSP70s can join chloroplast precursor protein transit peptides [7]. Mitochondrial precursor protein translocation also occurs post-translationally and precursor proteins are once again preserved in an import-ready state with the useful resource of cytosolic HSP70s. Its homologs are positioned inside the outer mitochondrial membrane facing the cytosol and in the mitochondrial bean matrix [63]. An HSP70 homolog located in the outer membrane changed into suspected to be involved in protein translocation within the outer membrane with the aid of attaching the precursor proteins launched from the cytosolic Hsp70 and integrating them into the outer membrane. However, there may be no proof to guide this process. Matrix Hsp70, which has a strong homology to DnaK, is determined to be closely connected with the inner mitochondrial membrane of bean [36]. It is proposed that the HSP70 matrix may additionally result in internal membrane import by means of pulling precursor proteins into the matrix in an ATP-based method aided by using the GrpE mitochondrial co-chaperone homolog [64].

#### **6. Mechanism of HSP70 in plants**

The ability of plants to tolerate dangerous effects of intense high temperature without irreversible harm is heat stress tolerance [65]. Effect of temperature contributes to a number of bad changes in plants' life: extreme dehydration and dryness, chlorophyll burning and different physiological disorders. The cessation of protein synthesis improves the degradation and accumulation of ammonia poisonous substances [66]. However, the heat tolerance mechanisms in flora have been partly understood that HSP70 gene protect flowers from oxidative damage [67]. Additional mechanisms probably contributing to heat tolerance involve phytohormones, second messenger molecules which includes calcium (Ca++) and an expansion of transcription factors [68]. The various downstream tactics, safety towards oxidative damage and protein aggregation at some stage in heat stress are critical for preserving mobile membrane integrity and photosynthesis. Consequently, over-expression of HSP70s initially regarded to be a promising technique for engineering to evaluate the heat tolerance in vegetation; but, best restricted success has been reported in past many years [69], and no field tests, heat tolerant and transgenic line has been stated. These observations advocate that a single stress tolerance mechanism might not be sufficient and additional mechanisms will be had to generate durable heat-tolerant cultivars. Highly conserved protein, HSP70s are omnipresent proteins first-class acknowledged for their susceptibility to numerous stresses consisting of heat stress [70]. HSP70 assist to place every protein inside the organelles of the cellular and interplay the mitochondrion and chloroplast proteins [11]. They've a link to proteasomal degradation pathway mediated through ubiquitin. In addition unfolded outer membrane proteins in the

**Figure 2.** *Response of HSP70 against.*

intercellular spaces transduce a signal to the inner membrane proteins under hot temperature inside the cytosol. This causes the heat shock transcription factors to be activated [71]. These heat shock transcription factors (HSFs) associated with HSP70 are one of the maximum reported protein families. They commonly hold collectively with heaat shock induced factors (HSEs) within the promoter areas to set off their expression, which transcribes the HSP70 (**Figure 2**).

#### **7. HSP70 confers the tolerance to heat stress in plants**

As the primary pigment of plants, chlorophyll (Chl) plays a crucial role in the mechanism of photosynthesis and its contents. Role of HSP70 in the prevention of heat, stress, chlorophyll and water breakdown was determined in transgenic tobacco and cotton seedlings. As seen in research conducted by Batcho et al., [52] & Wang et al., [72], overall output of chl, chl (a) and chl (b) content of the non-transgenic plants were decreased with the extension of treatment time after treatment with heat stress. However, the total Chl, Chla and Chlb content of HSP70 transgenic plants was higher and the reduction was slower when compared to the controls. Assay of soluble sugar content and comparative electrical conductivity of transgenic plants was improved during heat treatment when compared to control plants. This suggests that the relative electrolyte leakage of the control plants was evidently higher and the damage to the cell membrane was severe. This is consistent with the studies of [36, 73] indicating that HSP70 is involved in response to heat stress in plants.

Overexpression of HSP70 was found to decrease the malondialdehyde (MDA) content and increased production of superoxide dismutase (SOD) and peroxidase (POD) in transgenic plants when compared to control [74]. Some reactive oxygen *Heat Shock Proteins (HSP70) Gene: Plant Transcriptomic Oven in the Hot Desert DOI: http://dx.doi.org/10.5772/intechopen.105391*

species (ROS) such as superoxide, hydrogen peroxide and hydroxyl radicals will be synthesized and accumulated when plants are heat stressed. These ROS are cytotoxic through inactivating enzymes and killing essential cellular components such as cell membranes by oxidative processes' damage. MDA is the final product of peroxidation of the membrane. The higher the peroxidation, larger the amount of MDA produced. Plants have developed several defensive pathways to reduce oxidative damage and mitigate adverse effects. Transgenic tobacco plants demonstrated the higher overall activity of SOD and POD. This suggested that there would be less accumulation of ROS in transgenic and a better state of growth under heat stress. It has been found that overexpression of HSP70 increased the soluble sugar content and decreased the electrical conductivity in transgenic plants. The cell membrane also experiences primary physiological injuries which results the cell electrolyte leakage under heat stress [75].

#### **8. Conclusion and future prospects**

Heat Shock Protein Gene70 (HSP70) is one of the solutions to induce heat stress tolerance in agriculturally important crop plants. These genes identified, isolated from local environment/habitat and local plant species will be helpful to make the genetic transformation of local varieties of desirable plants. The modern genomic approaches will be helpful for the characterization of genes at transcriptional or promoter level to modify the gene and to enhance the gene expression in transgenic crops. Hence the HSP70 will be a suitable target to combat the crops against global warming threat to crops.

#### **Author details**

Fatima Batool<sup>1</sup> \*†, Batcho Anicet Agossa1,2†, Zainab Y. Sandhu<sup>3</sup> , Muhammad Bilal Sarwar<sup>1</sup> , Sameera Hassan<sup>1</sup> and Bushra Rashid<sup>1</sup>

1 University of the Punjab Lahore, Centre of Excellence in Molecular Biology, Pakistan

2 Faculty of Agriculture and Environmental Sciences, Catholic University of the West Africa Cotonou, Benin

3 Montclair State University, Montclair, New Jersey, USA

\*Address all correspondence to: fatima.batool@cemb.edu.pk

† These authors contributed equally.

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## Abiotic Stress in Plants

*Shubham Dey and Ayan Raichaudhuri*

#### **Abstract**

Stress in plants refers to external conditions, which drastically affect the growth, development, or productivity of plants. Stress triggers a wide range of plant responses, such as altered gene expression, cellular metabolism, changes in growth rates, and crop yields. Some abiotic stresses, such as low or high temperature, deficient water, and ultraviolet radiation, make plant growth and development unfavorable, leading to a fall in crop yield worldwide. The following writeup incorporated the abiotic stress factors related to the growth and development of plants, such as temperature, drought, heat, cold, and many more. Abiotic stress factors are the nonliving factors influencing the metabolism, growth, and development of the plant tissues at that particular time when such abiotic stress affects them. As a result of such abiotic stresses, the plants have generated many stress tolerance factors. Various stress-responsive genes are thus being formulated in response to the abiotic stresses, so the plants can survive even in such extreme conditions as well. Henceforth, it can be concluded that the abiotic stress factors imposed on the plants adversely impact their growth and developmental procedures, and at the same time, they also produce some stress tolerance factors to minimize the damage.

**Keywords:** biotic stress, abiotic stress, temperature, salt, stress regulators

#### **1. Introduction**

Abiotic stress can be defined as the adverse impacts created by the abiotic factors on the plant tissues [1]. Abiotic stress is caused by nonliving factors that are in contrast to biotic stress, which is caused by living organisms. The various factors impacting the plant tissues interrupt their normal metabolism. In response to this stress, the plants adapt newer metabolic reactions to resist the stress. The majority of such reactions aid them to regulate and sustain themselves against various environmental factors [1].

In **Figure 1**, stresses influencing the growth and developmental patterns of plants are shown. As stated by the figure, plant stress depends upon the stress factors being living or nonliving and thus the stress gets segmented as biotic (living) stress and abiotic (nonliving) stress in plants [2].

**Figure 1.** *Some of the common plant stresses: both biotic and abiotic stresses have effects on plants.*

#### **2. Stress impacts on plants**

The consequence of the stress factors on the plant tissue is their influence on their growth and development pattern [2]. As a result of the stresses, various types of plant metabolism get triggered, such as the altered expression of the inherited genes, metabolism of the cells of plants, changed patterns of growth types, crop yields, and much more [2]. However, as stated by Zhang et al. [3], there are two types of stresses—biotic and abiotic that are observed among the plant tissues.

#### **2.1 Biotic stress**

Biotic stress is caused by living organisms, such as viruses, bacteria, fungi, nematodes, insects, weeds, and many others [4]. Such stressors deprive the host plants of the growth factors and nutrients within them and eventually the plants die. Thus, biotic stress factors become the major reason for the plants pre-and post-harvest losses.

In **Figure 2**, the influence of the endophytic fungi on desert plants is. The figure shows how fungi grow with the association of the plants and thus take in salts, water, and other nutrients from the plant roots. In such cases, the growing plants become deprived of salt and water and continuous salt stress and water deficiency are encountered [5]. As a result of all such negative impacts, the deterioration of the inherent metabolism of the plant parts occur. Additionally, in the case of the desert plants, already deserts are known for lower availability of water, thus any such further disturbances by the endophytic fungi have adversely impacted the plant growth [5].

#### **2.2 Abiotic stress**

Abiotic stress factors are the nonliving factors influencing the present metabolism, growth, and development of the plant tissues [6]. As stated by Sharma et al. [7], abiotic stress factors impacting plants are excessive hot temperature, extreme cold temperature, salinity, drought, mineral availability or toxicity, and much more. Such abiotic stress factors have, thus, negatively impacted the overall crop yields, and,

#### **Figure 2.**

*Biotic stress on the desert plant: the endophytic fungi are taking the natural product of the soil meant for the plant and creating biotic stress.*

thus, there is a need of generating resistant plant varieties that can sustain against abiotic stress factors [6].

**Figure 3** showcases the various abiotic stress impacting plant parts and their relative outcomes. As stated in the figure, sunlight, cold weather, salinity, and mineral availability or deficiency are the major abiotic stress factors for a plant [8]. As a result of various stress factors, multiple responses can be seen. For example—the amount of ROS (reactive oxygen species) can be increased, plant growth and yield reduced, and simultaneously photosynthetic activity can be reduced. The adverse environmental temperatures deteriorated the plants' growth and developmental patterns [8].

#### **3. Mechanism of abiotic stress**

The abiotic stress is known to impact the internal metabolism of the plant parts, and, thus, the overall productivity of the plants gets reduced [9]. This could be the major adverse impact as the abiotic stress factors are widely known to cause mostly negative impacts only [9]. The several abiotic stress factors and their simultaneous mechanisms are being discussed as follows:

**Figure 3.**

*Abiotic stress factors in plants: major abiotic stress factors for a plant deficiency are sunlight, cold weather, salinity, and mineral availability or deficiency. They are the major abiotic stress factors for plant deficiency.*

#### **3.1 Cold**

Every plant is known to survive at a particular temperature only. The alteration of the required temperature changed the overall sustenance patterns of the individual plants [10]. In case of plants growing in cold temperatures, cold temperature would result in disruption of the plant tissues, as a result, it would lead to deterioration of the life cycle of plants.

**Figure 4** explains the various mechanisms adopted by the plants to mitigate the cold stress factors against the sustenance of plants [10]. As seen from the figure, the incorporation of such stress factors aids the plants to generate signals and further transcriptional control so that the genes of stress signaling could be simultaneously activated. This will ultimately lead to re-establishment of the cellular homeostasis and functional and structural protection of protein and membranes [10]. All such optimistic sequential steps impacted positively internal cellular membranes and, thus, lead to stress tolerance or resistance against abiotic stress factors by plants [10].

#### **3.2 Salt**

The salt concentration is one of the major factors impacting plants' growth and development [11]. Higher amounts of salt lead to the re-release of genes for minimizing stresses against salt concentration and thus optimizing the plants to survive in such hazardous situations as well.

**Figure 5** illustrates the fact that accumulation of solute concentration leads to mineral absorption in an excessive amount and simultaneously it leads to cell wall modification and incorporation of transporters that lead to re-transportation of salts to mesophyll, homeostasis of potassium, and nitrate ions and thus generating optimistic stress responses [12].

#### **Figure 4.**

*Mechanism of abiotic stress factors on plants: genes of abiotic stress signaling could be simultaneously activated. This will ultimately lead to re-establishment of the cellular homeostasis, functional and structural stress tolerance, or resistance protection of proteins and membranes.*

#### **3.3 Toxin**

Toxins are chemicals released by the plant tissues in response to several abiotic stress factors [13]. Toxins are also considered to be the surrounding stress factors adversely impacted by the environment, and, thus, the plants are getting negatively impacted by such abiotic stress factors.

**Figure 6** illustrates the toxins released by the cell wall, cell membranes, cytoplasm, chloroplast, mitochondrion, endoplasmic reticulum, peroxisome, and nucleus [14]. Signal integration of the stress factors occurs now and the stress response genes are activated and released. These responses lead to the sustenance of the overall growth and development of the plant parts [14].

#### **Figure 5.**

*Factors impacting salt stress in plants: excessive amount of mineral absorption leads to cell wall modification and incorporation of transporters that lead to re-translocation of salts to mesophyll, homeostasis of potassium and nitrate ions, and thus generating optimistic stress responses.*

#### **Figure 6.**

*Mitigation strategies against toxins: signal integration of the stress factors occurs and the stress response genes are activated and released. These responses lead to the sustenance of the overall growth and development of the plant parts.*

#### **4. Mitigation strategies adopted by plants for overcoming abiotic stress factors**

The abiotic stress factors are the ones that cannot be sustained and mitigated by the plants externally, henceforth plants are known for developing fresh mechanisms within their inner metabolism to balance the excessive adverse impacts created by the outside environment [7]. Many such mitigation strategies are adopted by plants to overcome such abiotic stress factors [7].

In **Figure 7**, the sequential steps adopted by plants to overcome the abiotic stress impacts are shown. The figure states that with abiotic stresses, the plants tend to develop excessive ROS. Such an excessive production leads to the further incorporation of 3 steps—activation of oxygen antioxidants, up-regulation of osmolytes, and activation of stress-responsive genes [15]. The activation of the stress-responsive genes makes the plants much tolerant and thus they can survive against such hazardous temperatures as well. So identification of targeted genes is necessary as the overall mechanism depends upon such gene regulations only [3]. Henceforth it can be concluded that oxidative stress reduction results in an increase in the stress tolerance factors and long-term sustainability of the plants in such adverse conditions as well [3].

#### **5. Conclusion**

The research on abiotic stress factors on plant growth and development reveals that they are the major factors that influence and lead to the deterioration of the plant

#### **Figure 7.**

*Steps for overcoming abiotic stress by plants: in abiotic stresses, the plants tend to develop excessive ROS in oxidative stress reduction, which results in increase of stress tolerance factors and long-term sustainability of the plants.*

species. Heat, cold, drought, salinity, and toxins are various abiotic stressors impacting adversely the overall development of plants. Various stress-responsive genes are formulated in response to abiotic stresses so that the plants can survive extreme conditions as well. Rapid population growth, economic development, and international economic integration have intensified resource use in every sector of the world. The human population is expected to increase to a total of 9 billion by 2050. So production of more food from the same area of land will be needed and this can happen only by reducing the adverse environmental impacts on plants. This is what has been called sustainable intensification, for feeding, clothing, and providing energy to such a large population. Transgenic approaches have been proven to show as powerful tools to help understand and manipulate the responses of plants to stress. Global research analyses indicate that transcription to proteins and metabolites occurs during abiotic stress. These findings will advance our understanding of major metabolic pathways and provide direction for achieving abiotic stress-tolerant plants. The viable evaluation of transgenes that enhance crop performance under both stress and optimal conditions is a prolonged, tedious, and expensive process. It is being proposed that the current stance on plant stress tolerance can be significantly polished by thorough characterization of individual genes and evaluating their contribution to stress tolerance.

The molecular mechanisms of plants to create stress tolerance against salt, drought, and temperature involve a number of regulatory proteins, such as transcription factors. The study of such mechanisms enabled us to increase our knowledge of enhanced plant survival and increased crop yields in spite of abiotic stresses. Further research is needed for accurate evaluation in the field of genotypes for abiotic stress resistance, a deeper understanding of the transcription factors that regulate major stress-responsive genes, and cross-talks between divergent signaling components. We are to advance our knowledge on traits that are associated with root architecture and plasticity, especially in agronomically superior genotypes under abiotic stress conditions. Crop tolerance to various abiotic stresses is a matter of continued research to increase our knowledge further and to help plants from deterioration and extinction. The stress biotechnology research in the recent future will emphasize on strength and stress-induced expression of the transgenes, combined with the regulatory machinery involving transcription factors as a new genetic manipulation tool for controlling the expression of many different stressresponsive genes.

In conclusion, plant sciences currently achieve good models of how model plants react to environmental factors by transcriptional and metabolic reprogramming. However, especially molecular research efforts in crops have to be strengthened considerably. Plant stress physiology is a very complex matter and needs future biocomputational integration of multiple omics and meta-omics to understand it properly. This needs further effort in developing innovative research tools and fundamental resources for crop plant research, such as reference genomes, proteomes, and metabolomes with comprehensive annotations and structure-function relationships, respectively. Even for the model *Arabidopsis*, these resources are not fully available. Nevertheless, in several cases, *Arabidopsis* and other model plants have already been proved suitable for the translation of fundamental research into agronomically relevant crop traits. This is encouraging but requires further and significant investment into translational research. Besides this, it remains indispensable to investigate abiotic stress resistance mechanisms directly in elite crop plants and in the genetic resources available for breeding.

*Abiotic Stress in Plants DOI: http://dx.doi.org/10.5772/intechopen.105944*

#### **Author details**

Shubham Dey and Ayan Raichaudhuri\* Amity Institute of Biotechnology, Amity University, Kolkata, India

\*Address all correspondence to: araichaudhuri@kol.amity.edu

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## Heterologous Expression of Genes in Plants for Abiotic Stresses

*Shahzad Ali, Nadir Zaman, Waqar Ali, Majid Khan, Muhammad Aasim, Asmat Ali and Muhammad Usman*

#### **Abstract**

Abiotic stresses are considered to be the major factors causing a decrease in crop yield globally, these stresses include high and low temperature, salinity, drought, and light stress etc. To overcome the consistent food demand for the ever-growing population, various genes from micro-organisms and non-plant sources have been expressed in transgenic plants to improve their tolerance against abiotic stresses. Gene expression in transgenic plants through conventional methods are timeconsuming and laborious that's why advanced genetic engineering methods for example *Agrobacterium-*mediated transformation and biolistic methods are more accurate, useful, and less time-consuming. This review provides an insight into various bacterial genes for example *mtID*, *codA*, *betA*, *ADH*, *IPT*, *DRNF1* and *ggpPS*, etc. that have been successfully expressed in transgenic plants against various abiotic stress for stress tolerance enhancement and crop yield improvement which exhibited good encouraging results. Genes from yeast (*Saccharomyces cerevisiae*) have been introduced in transgenic plants against drought and salinity stress. All these genes expressed from non-plant sources in plants can be very helpful to enhance crops for better yield productivity in the future to meet the demands of the consistently rising population of the world.

**Keywords:** abiotic stresses, heterologous expression, bacterial, yeast, fish and insect genes

#### **1. Introduction**

Plant stress is a condition in which plant growing in an unfavorable condition that mainly causes growth problems, deficiencies in crop yields, and even death when the stress-causing factors cross the limit that plants can tolerate [1]. It refers to external environmental conditions that adversely affect the overall growth, progress, or production of plants [2].

There are two types of stresses to which plants are subjected that is abiotic stress and biotic stress. The crop loss worldwide is mainly due to abiotic stress which consists of drought, cold, salinity, high environmental temperature, and radiation, etc. [3]. While biotic stresses are the attack of various pathogens on plants including bacteria, fungi, herbivores, and nematodes) etc. [4]. Due to the sessile nature

of plants they cannot avoid these environmental factors but develop several mechanisms to tackle these abiotic and biotic stresses for their survival and environmental adaptation.

#### **1.1 Abiotic stress mechanism in plants**

Plants usually sense the environmental stress and then stimulate appropriate suitable response takes place, cell surface receives the stimuli and the transformation to the transcriptional system in the nucleus takes place via various pathways that help in transduction, make plants resistant to various environmental stress by the activation of molecular, biochemical, and physiological suitable response [5]. The first line of defense of plants is situated in roots to overcome abiotic stress. If the plant growing in the soil is healthy and there is biological diversity the chances of survival against the abiotic stress of the plant will be high. High salinity affects the growth and development of plants. The disruption of (Na<sup>+</sup> ) and (K<sup>+</sup> ) ratio in the cytoplasm is mainly the primary response shown by the plants against stress. Living microorganisms need to ensure effective growth and generate an effective environmental response, this especially very important in plants because of their immobility and encountering large changes/alterations in temperature, humidity, light, and availability of nutrients in the environment. Massive agricultural losses happen due to environmental stresses [6, 7] and the improvement of crop resistance is a major goal for crop programs.

A genetic locus that keeps productivity maintained even in serious conditions are situated within the germplasm of existing crops, their relative species that are earlier adopted to severe environments. Selective breeding in combination with other loci has improved crops yield in extremely challenging environmental conditions throughout agricultural history. An efficient advanced paradigm is the precise selection of genetic factors of stress adaptation that have been in nature for years and passes on by plants to their higher verities [8]. Abiotic stress causes biosynthetic capacity and nutrient decrease which leads to inhibition in plant growth and has been further elaborated by various researchers in their work by knowing the response to abiotic stress through various signaling pathways involving several genes, mechanism of post-transcriptional modification, and proteins. Those pathways are MAPK, ABF/bZIP, Ca2+-CBL-CIPK, and CBF/DREB which enables much stress responding transcription factors to initiate downstream signals needed for abiotic stress defense [9]. These signaling pathways can predict the effects generated by abiotic stress to control growth and plant adaptation. Recently genes have been identified which control plant growth during stress conditions for example molecular mechanism which controls leaf progress and growth under drought conditions relates both transcriptional signals to the circadian clock. Importantly (ERFs), ERF2 and ERF8 related to ethylene response factors showed to affect leaf in drought and wet conditions [10].

Abscisic acid plays a huge role in helping plants for their environmental adaptation against cold, drought, alteration in temperature, salinity, and wounding [11]**.** During extreme environmental conditions, the level of Abscisic acid goes up through the ABA biosynthesis process. High-level ABA combines with receptor for the initiation of signal transduction which leads to the cellular response to stress [12]. Various mechanisms that help in the protection of plant survival against abiotic stress are very much important, yet they are activated at the cost of plant growth and its productivity which is essential for agriculture. Recent studies in molecular genetics help us to

understand the basis of abiotic stress tolerance [13]. **Figure 1** illustrates the various signaling pathways involved in abiotic stress mechanisms in plants [9].

#### **1.2 Abiotic stresses (factors) that affect plants**

#### *1.2.1 Temperature*

Temperature is a very important abiotic stress factor that affects plant from seed germination to reproduction [14]. Significant temperature changes can lead to permanent disturbance in the plant cycle which even leads to death. It causes plant stress by two means; extremely cold and hot temperature, severe cold conditions below the optimum temperature can cause physical and mechanical changes to the plant and leads to severe cell disruption [15]. In various areas extremely low temperature causes agricultural crop productivity and affects the cultivation process [16]. While due to uncontrolled rise in temperature affects the rate of photosynthesis, water availability to plants, and fruit ripening. Due to climatic changes an appreciable rise in temperature in the coming times will cause rainfall reduction, alteration in wind speed, and snow leads to less growing plant season and eventually will harm crop production and quality [17]. The effects of verglas/frost and high temperature have been evaluated recently on the production of Wheat (*Triticum aestivum L*), fruitless plants and

#### **Figure 1.**

*Illustration of various pathways involved in abiotic stress mechanism in plants [9].*

termination of matured grains was due to frost while extreme temperature caused a decrease in grains number during the filling grain period [18]. These noteworthy effects due to extreme climatic changes in crop production will result in food insecurity and crop production trends in the future [19].

#### *1.2.2 Drought/water stress*

To obtain maximum crops yield globally drought or water stress is a very important factor it affects plants in many ways; during the growth phase, water stress decreases leaf expansion development, photosynthetic process, the height of the plant, and the overall area of leaf. The early symptoms caused by drought stress are leaf rolling and dryness of leaf tip, cell elongation is seriously affected by drought stress water scarcity blocks stomata, and reduces transpiration [20]. It has a huge negative impact on plant growth and the potential quality of yield in the agricultural system. *Miscanthus* has very good potential for the production of biofuel it was observed after an experiment that drought the weight of the plant significantly by about 45% and cell wall composition and biomass were affected by drought stress during the plant growth phase [21]. The availability of water to plants is very necessary however waterlogging in the area surrounding the roots can be very damaging it can cause lack of oxygen and even death of the plant due to its lethality [22]. The transfer of free oxygen exchange between the soil and atmosphere is caused by water stress suffocation [23]. Waterlogging is often caused by floods, heavy rain, and snow in winter, such soils have limited or lack of oxygen due to less gas exchange [24].

#### *1.2.3 Light stress*

For plants, the energy production process through photosynthesis sunlight plays an important role. Plants adapt themselves to change in light which alter considerably at various times. That is why plants can develop certain mechanisms that help in maximum use of existing light during irradiance state while other mechanisms to escape the long-term sunlight exposure [25]. As a result of low light or reduction in solar energy significant decrease happens in metabolic rate which leads to a reduction in crop yields and lower growth rates. An increase in reactive oxygen species (ROS) and photo-damage is caused by prolonged exposure of plants to sunlight [26].

#### *1.2.4 Salinity stress on plants*

Soil salinity is considered as one of the major abiotic stress affecting the performance of crop plants adversely around the globe, it can create a cluster of diverse interactions that harms the nutrition uptake, metabolic process, and plant vulnerability to various biotic stresses as well [27]. Minerals and nutrients present in the soil have valuable importance but the unwanted existence of salts results in extreme ionic and osmotic stress in plants [22]. The cations present in inorganic soils or water includes potassium (K<sup>+</sup> ), magnesium (Mg<sup>+</sup> ), calcium (Ca<sup>+</sup> ), and sodium (Na<sup>+</sup> ) while the important anions are NO3 − , HCO3 − , SO4 2−, Cl− , and CO2 –3 other components include SiO2, Al3+, Sr2+, B, Mo, and Ba2+ [28]. Enzymes inactivation, cell death, and subsequently whole plant can diminish due to high salinity [29]. Salinity stress in plants leads to a huge decrease in dry and fresh weight obtained from stem, roots, and leaves [30]. An excessive amount of salt increases osmotic pressure in plants which reduces the chances of minerals like (K<sup>+</sup> Ca2+) and nutrient uptake for

survival, such primary effects leads to secondary effects as a non-proper expansion of cell, decrease in membrane function and a significant decrease in cytosol metabolic activity [5]. According to FAO world's 6% of the land is affected by salt. **Table 1** shows the distribution of salt-affected land around the world.

#### **2. Heterologous expression of genes in plants for abiotic stresses tolerance**

During the past two decades the use of recombinant DNA technologies, the methods of gene transfer, and tissue culture techniques have improved the transformation and transgenics in many varieties of crop production in agriculture. Transformation techniques provide larger accessibility to the pool of genes as compared to conventional methods because the genes are inserted from bacteria, animals, viruses, yeast, fungi, and even from various synthetic chemicals prepared in the laboratory (Chahal and Gosal 2002). Various methods are used for genetic transformation of crop plants, Biolistic bombardment, and *Agrobacterium-*mediated are the most common methods used for gene transfer in plants [31].

#### **2.1 Genetic engineering through bacterial genes in plants against abiotic stresses**

Cloned genes insertion has produced transgenics against abiotic stresses in plants [32]. Many bacterial genes have been expressed in plants to confer abiotic stresses like salinity, drought, temperature, cold, and light stress. Those bacterial genes include *mtID* which is expressed in several transgenic crops like tomato (*Lycopersicon esculentum* L. *var. Pusa Uphar*) was transformed against salinity, high and cold temperature and drought stress by the insertion of *mtID* gene [33] Wheat (*Triticum aestivum* L.) was transformed by the expression of this gene against salinity and waterlogging stress, as a result, the transgenic plants showed good resistance than WT non-transformed plants [34]. Tobacco plants were transformed by the expression of this gene against various abiotic stresses and the results were very improved in comparison to wild type plants [35]. Finger millet (*Eleusine coracana*) is a major food crop consumed and cultivated around the globe has been genetically transformed by the expression of this bacterial gene against drought and salinity [36]. Peanut (*Arachis hypogaea* L.) has been genetically transformed by the insertion of bacterial *mtID* gene against salinity and drought stress [37]. Moreover Indica rice


#### **Table 1.**

*Various parts of the world affected by salinity stress [5].*

was transformed through the above-mentioned gene to improve its productivity in drought and saline environment [37]. Another Bacterial gene codA which has been isolated from (*Arthrobacter globiformis)* coding for choline oxidase and expressed Arabidopsis thaliana to improve its resistance to salinity, freezing, and high temperature [38]. The same bacterial *codA gene* was expressed in tomato (*Lycopersicon escuelentum*) to enhance its tolerance against high temperature, chilling, and drought stress [39]. Other Bacterial gene *IPT i*solated from *Agrobacterium tumefaciens* and expressed for the enhancement of various crop plants to improve their tolerance against various abiotic stresses like sugarcane (*Saccharum* spp.) cv. enhancement against cold stress [40] the same gene was inserted in rice for its enhancement against drought stress and increase in crop yield as well [41]. ADH *sysr1* gene of *cyanobacteria* was expressed in tobacco plants for salt tolerance improvement and that showed encouraging results in comparison to wild type plants [42]. *IPT* gene was also introduced in canola (*Brassica napus* L.) plants for delayed leaf senescence leading to crop improvement in drought stress [43]. The expression of Bacterial *ggpPS* gene isolated from (*Azotobacter vinelandii*) for glucosyl glycerol biosynthesis confers salt and drought stress tolerance in *Arabidopsis thaliana* [44]. Gene encoding for bacterial chaperons have been inserted in various transgenic plants for the improvement of tolerance to abiotic stresses successfully [45].

Abiotic stress tolerance related genes from micro-organisms are considered to be very valuable for the production of transgenic plants. A cyanobacterium (*Nostoc flagelliforme*) that can tolerate water deficit conditions is proved to be very useful prokaryotic organism for gene isolation. Salt tolerant gene *DRNF1* having P-loop NTPase (nucleosidetriphosphatase) domain, has been expressed in *Arabidopsis thaliana* the results indicated improvement in the growth of shoots and seed germination in saline conditions [46].

#### *2.1.1 The expression of mtID bacterial gene for the improvement of various abiotic stresses in plants*

To improve abiotic stress tolerance in transformed tomato plants a bacterial mannitol-1-phosphate dehydrogenase (*mtID*) gene was inserted through Agrobacterium-mediated method supported by CaMV35S promoter, Rt PCR, and southern blotting analysis was used for the confirmation of transient integration, reverse transcription (RT)-PCR and direct activity of mtID gene was analyzed for the confirmation of transgene expression [33]. Transgenic tomato plants upon exposure to low temperature round about 4°C in a cold chamber survived for almost 2 days as compared to untransformed plants that were not able to survive and their death accrued slowly. During the exposure of transgenic tomato plants to chilling effect showed a significant decrease in electrolyte leakage in the plant membrane, when they are exposed to stress the leakage starts to the surrounding environment, and the damage caused to the cell with hardiness can be identified through the leaked contents conductivity in water by the comparison of injured and non-injured plants [47] while an increase to lipid peroxidation [48], antioxidant enzymes [49] and relative water content [50] as compared to the non-transformed plants. Drought stress was tested through polyethylene glycol in the medium, and salinity by the content of sodium chloride (NaCl) showed a greater response to these stress than non-transformed plants. By the indication of all these observations, it was clear that the introduction of bacterial (mtID) gene in tomato showed a good response to abiotic stress than transformed plants. As **Figure 2** shows the various transformation phases of tomato [33].

*Heterologous Expression of Genes in Plants for Abiotic Stresses DOI: http://dx.doi.org/10.5772/intechopen.105171*

**Figure 2.** *Different tomato (*Lycopersicon esculentum L. *var.* Pusa Uphar*) transformation phases [33].*

The above work has shown that the accumulation of mannitol in several transgenic plants can improve plant tolerance against abiotic stresses. At the cellular level due to *mtID* gene insertion from *E. coli* [51] has been reported for the protection of Wheat (*Triticum aestivum* L.) against the adverse impact of waterlogging and soil salinity. Through the exposure of calli to polyethylene glycol ranging from 8000 mm of NaCl. The stress in the T2 plant was caused by the addition of a large amount of water and 150 mm of NaCl to the nutrition medium, the fresh weight of *mtID* calli was decreased by about 40% in the existence of PEG and 37% during salt stress, no effect was observed in the growth of *+mtID* callus, while in the plants of *−mtID* the content of fresh and dry weight, height of the plant, and leaf areas was decreased by about 70%, 56%, 40%, and 45% in a comparison with 40%, 8%, 18% and 29% comprehensively with that of *+mtID* plants. Salinity stress decreased shoots of fresh and dry weight, changes in the height of plants were observed, and the leaf area was reduced by 77%, 73%, 25%, and 36% in *-mtID* plants as compared to 50%, 30%, 12%, and 20% in plants having *+mtID*. As a result, no effect was seen on the growth of *mtID* callus and transgenic wheat plants showed significant tolerance against salinity and waterlogging stress due to the insertion of this bacterial gene [34].

For expression in higher plants against abiotic stress a bacterial gene that codes for mannitol-l phosphate dehydrogenase, *mtID* was inserted stably in tobacco plants which translated in tobacco through a functional enzyme, led to the accumulation of mannitol, which was identified and detected through NMR and mass spectrometry, the concentration of mannitol increased by d 6 jumol/g (fresh weight) in roots and leaves of the transgenic tobacco while these sugar were not detected in wild or untransformed tobacco plants that were passed through the same treatment. This study could help us in understanding sugar role of alcohol in the enhancement of plant tolerance against abiotic stresses in higher plants comprehensively [35].

In Asia, Nepal, India, and almost 25 countries of Africa, finger millet (*Eleusine coracana*) is cultivated and consumed as a major crop for food, it covers more than 12% of the world's millet cultivating area [52, 53]. It also has better nutritional properties and ingredients than wheat and other major crops [54, 55]. It is very vulnerable to various abiotic stresses like drought and salinity in fields during the early stages of seed germination and the development of seedlings, therefore it is very important to make finger millet plants resistant to drought, salinity, and oxidative stress. Proper radical scavenging capability and cell protection by osmotic modification during several abiotic stresses are major mechanisms in plants, mannitol is an osmolyte [56] that helps in the neutralization of various free hydroxyl radicals produced due to abiotic stresses and decreases stress disruption in various plant species. Through *Agrobacterium-*mediated transformation, the biosynthetic pathway gene from bacteria mannitol-1-phosphate dehydrogenase (*mtID*) was expressed in finger millet plants to understand the performance of transgenic plant upon their exposure to drought and salinity stress simultaneously. The results obtained through these experiments showed that transgenic finger millet had better performance in saline and drought stresses as compared to wild type plants [36].

Peanut (*Arachis hypogaea L*.) is an important crop grain cultivated in tropical and sub-tropical zones in about 21–24Mha production areas, it is generally grown in rain-fed areas where drought is a major crop decreasing factor occurring in semiarid lands that cover 70% of the peanut cultivation areas [57] one of the many strategies is to genetically transform peanut plants that can resist in drought conditions [34]. Plant breeding through conventional methods is time-consuming with lesser success and far more laborious, while genetic engineering techniques show great potential to develop peanuts plants that can tolerate drought conditions. Plants have developed several mechanisms against drought stress for their survival [58]. To overcome drought stress in peanuts bacterial *mtID* genes were expressed through CaMV35S promotor using *Agrobacterium tumefaciens*-mediated transformation. The transformed plants showed significant resistance to water deficit conditions as a result of mannitol accumulation during these experiments, **Figure 3** illustrates the whole process [37].

The accumulation of mannitol an osmolyte plays an important role in abiotic stress. So, through the insertion of *mtID* gene from *E. coli* for the improvement of *Indica* Basmati rice against salinity and drought stress, by agrobacterium-mediated transformation, many putative transformed plants were generated. Transgene existence was confirmed in early transformed plants by PCR through *mtID* and hygromycin phosphotransferase, the transgenic lines showed better performance against salinity and drought stress as compared to wild type plants [59].

#### *2.1.2 RNA chaperones genes of bacteria confer abiotic stresses in transgenic plants*

With a consistent increase in the world's population, constant supply of food demands, and a decrease in water shortage alongside cultivating land, it is necessary

#### **Figure 3.**

*Schematic illustration of the T-DNA section of pCAMBIA 1380 binary plasmid used for transformation of deembryonated cotyledons with Agrobacterium tumefaciens strain LBA 4404. The location of the primers used in PCR assays is shown by arrows on the top of the mtID gene. LB, left T-DNA border sequence; RB, right border sequence; 35S, CaMV35S promoter; and mtID, mannitol-1-phosphate dehydrogenase [37].*

#### *Heterologous Expression of Genes in Plants for Abiotic Stresses DOI: http://dx.doi.org/10.5772/intechopen.105171*

to transform crops like rice that can grow in salt-affected areas [60]. High saline condition seriously affects the growth of rice like leaf expansion ability, root, and shoot formation [61]. The decrease in leaf expansion occurs in rice due to the low rate of osmotic turgor pressure under saline and cold conditions [62]. Results obtained from various research studies have shown that chloroplast and mitochondria in rice plants are seriously affected by salt and chilling stress [63, 64]. During abiotic stress conditions, bacterial RNA chaperones play a major role in stable messenger RNA expression, in salinity stress these bacterial genes develop transgenic rice plants that can tolerate even cold stress apart from salinity [60].

Drought is the major factor that causes crop yield reduction globally leading to socioeconomic complications. During an estimation, it was observed that a 40% loss in Maize crop is caused by drought stress alone in North America annually [65]. Maize crops are vulnerable to drought stress through-out their growing stages, effects of stresses that initiate during the flower development phases either before the start of floral events or post pollination results in a significant reduction of crop yields at the end of the season [66, 67]. In 2013 the first drought-resistant maize crop was genetically transformed by the expression of bacterial genes that codes for chaperonin showed significant improvement in resistance to water deficit stress [45].

The expression of bacterial CSPs (cold shock proteins) exhibited improvement against cold stress in transgenic *Arabidopsis thaliana* seedlings cultivated at very low temperatures on standard agar media in Petri dishes as illustrated in **Figure 4**. The tests were conducted of the transformed Arabidopsis seedlings having *CspA* and *CspB* to check the improvement for cold stress using non-transformed seedling as controls. The seedlings were exposed to low temperatures for 6 weeks and the results suggested improve tolerance against cold stress in comparison to non-transformed wild type plants [45].

#### *2.1.3 The expression of* ADH (alcohol dehydrogenase gene) *isolated from*  cyanobacteria Synechocystis *sp. improves salt tolerance in tobacco plants*

A gene PCC 6906 (*sysr1*) from *synechocystis* that shows a good response to salt was engineered and stably inserted in higher developed tobacco plants. The tolerance response of the gene *sysr1* (An ADH superfamily member) was investigated through quantitative real-time PCR, gas chromatography-mass spectrometry, and bioassays. The tobacco plants having *ADH* showed considerably improved tolerance to salt stress, besides that the activity of many

stress-responsive genes was up-regulated and enhanced due to the expression of (*sysr1*). The results suggested that the expression of *ADH* genes could significantly improve transgenic tobacco plants against salt stress through genetic engineering techniques in the future. **Figure 5** shows the identification of *sysr1* gene plants in three transgenic lines (1, 4 and 7) [42].

#### *2.1.4 Bacterial* codA *gene enhances tolerance against various abiotic stresses in plants*

*Arabidopsis thaliana* was genetically transformed by a gene isolated from *Arthrobacter globiformis* that codes for choline oxidase, an enzyme used for the synthesis of glycine betaine from choline, which remarkably improved cold or freezing stress in plants. Moreover, the photosynthesis machinery was more resistant to freezing/cold stress than non-transformed plants. These results also indicated the accumulation of glycine betaine in transformed plants, enhanced their ability to extremely cold temperatures [68].

In the control of RNA CaMV35S promotor, *A. thaliana* was genetically transformed by *codA* gene of bacterial *Arthrobacter globiform* coding for choline oxidase. Subsequently, the accumulation of increased glycinebetaine occurred in the seeds of transformed plants. The transformation of *codA* gene significantly boosted

#### **Figure 5.**

*Identification of transgenic plants with sysr1 gene. (a) Results obtained from the analysis of quantitative real-time PCR in three transformed lines (lines 1, 4 and 7) used for the assays of salt tolerance. (b) the ADH activity of leaf of transgenic tobacco plants with the control [42].*

#### *Heterologous Expression of Genes in Plants for Abiotic Stresses DOI: http://dx.doi.org/10.5772/intechopen.105171*

the ability of plants to high-temperature stress in the period of seed germination and the growth phase of young seedlings. The level of improvement of the resistance to high temperature was evaluated with the extent of the expression of choline oxidase and the accumulation of glycinebetaine in transgenic plants [69].

Tomato (*Lycopersicon escuelentum*) was genetically transformed by the introduction of *codA* gene from *Arthrobacter globiform* bacterium for choline oxidase that had been allowed to target both mitochondria and cytosol. The accumulation of glycinbetaine was detected in the seeds of transformed plants by about 1 μmol g−1 dry weight while no accumulation of glycinbetaine was seen in wild type/non-transformed plants. The transformed codA seeds germinated at fast speed during high temperatures. After heat stress, the content of small mitochondrial heat shock proteins, 70 heat shock proteins, and cognate 70 were much increased in transformed seeds during the heat stress phase than non-transformed seeds. Cognat 70 (HSP70) accumulation was more obvious in codA transgenic seeds than non-transgenic seeds. The results suggested that the transformation of tomato seeds with codA gene showed improved tolerance to a higher temperature in tomato plants [70].

Genetically transformed tomato (*Lycopersicon escuelentum*) plants which can synthesize glycinbetain was produced by the introduction of the bacterial *codA* gene. The expression of the gene was examined through RT-PCR analysis and in combination with RNA blotting hybridization. During the seed germination phase, the transformed plants exhibited greater tolerance to salt stress following the growth of young seedlings as well. The insertion of *codA* gene resulted in high-stress resistance ability in leaves and overall plants. Results from the experiments revealed that the developed leaves of *codA gene* transformed plants showed more water content, chlorophyll content, and enhanced proline levels in comparison with non-transformed plants during salinity and water stress [39].

They are vulnerable to chilling stress because of lower glycinbetaine synthesis ability. The cold temperature lower than 10°C causes severe injuries to tomato plants leading to lesser yield production. Bacterial *codA* gene has been introduced into the genome of the tomato by targeting chloroplast. The transformed plants expressed this gene and synthesize choline, by the accumulation of glycinbetain in leaves and the formation of shoots up to 0.3 and 0.2 μm/g fresh weight. The chloroplast of transgenic plants contained 86% of glycinbatain, in different developmental stages during the seed sprouting and fruit production process, the glycinbetain containing plants were more resistant to chilling stress than their wild types, 10–30% increase was seen in fruit production on average during abiotic stress, thus the introduction of GB biosynthesis pathways is an important strategy against chilling stress in tomato plants [71].

*A. thaliana* was genetically transformed through the bacterial *codA* gene which encodes for choline oxidase. The photosynthetic activity examined for chlorophyll fluorescence of transgenic plants were more resistant to light stress than non-transformed wild type plants. This improvement in resistance to light stress was eventually because of the high speed of the recovery process of the photosystem II complex from the photo-inactive stage. It showed that in vivo production of glycinbetain and no changes in the lipids membrane or H2O2 level, it ensured the protection of photosystem II complex in transgenic plants from the possible damage due to light stress [38].

#### *2.1.5 The expression of* IPT *gene against various abiotic stresses in plants*

To increase the cold stress tolerance, *IPT* gene was introduced in sugarcane (*Saccharum* spp.) cv. RB855536, in the control of a promoter (AtCOR15a), through biolistic, non-biological transformation method. The leaves extracted from genetically transformed plants showed good resistance and decrease leaf senescence upon their exposure to low temperature as compared to wild-type control plants. Improved enhancement against cold stress was seen due to the expression of this gene in non-acclimatized plants when the transgenic plants were exposed to extremely cold temperatures. The chlorophyll content of leaf was 31% more than non-transformed plants. A decrease in malondialdehyde level and the leakage of electrolyte showed lesser damage caused by chilling stress in transgenic plants. So, stress-inducible promoter *COR15a* used in the insertion of the *IPT* gene in transgenic plants shown no adverse effect while improving them against cold stress [40].

To delay the process of leaf senescence would allow capturing sunlight for longer periods, which leads to photosynthetic improvement and its contribution to plant growth and enhanced seed yield. Moreover, delayed senescence would allow the slow degeneration of source tissues so that the metabolites, proteins, nutrients could be slowly and gradually released to the sink tissues. Increase in plant potential biomass, maintenance of photosynthetic process, the higher influx of nitrate, increase in the life of flowers after harvesting, improved drought resistance, and greater seeds yield are the benefits of delayed leaf senescence [72, 73]. Cytokinin; a plant hormone that plays an important role in the process of cell division, cell growth, and differentiation, and it influences various developmental and phycological characteristics in plants ranging from seed germination, the flowering period of the plant, apical dominance, developmental process of flowering, fruits and leaf senescence [74, 75]. In various plants the role of a plant hormone cytokinin in delaying leaf senescence has been reported by [73, 76–78]. A gene *IPT* isolated from *Agrobacterium tumefaciens* has been inserted in several plants to enhance cytokinin level as this gene synthesize the rate-limiting step in cytokinin. Canola plants were transformed by *IPT* gene in combination with *AtMYB32* promoter, the insertion of *IPT* gene in transformants caused delayed leaf senescence cultivated under control condition and various field experiments at two separate geographical areas for one season. As a result, the transformed Canola (*Brassica napus* L.) plants maintained high chlorophyll content for a longer period and an increase in seed yield under drought and irrigated conditions was observed as compared to wild type non-transformed plants. In comparison to control plants, all of the seed quality parameters and oleic acid content in transformed plants were exactly similar, as a result of the experiments, it was concluded that the introduction of bacterial *IPT* gene can significantly improve crop yield and seed quality under irrigated and drought stress conditions in various plants [43].

In rain-fed areas drought is a major hindrance to rice crop productivity [79, 80]. To fulfill the constant demand of rice by 2030 a remarkable increase by almost 35% in yield is necessary [81] that is why the development of transgenic rice to drought stress and improved productivity is an important challenge, various studies have indicated that the expression of bacterial *IPT* gene using different promotors could help in delaying leaf senescence to improve crop productivity under drought stress conditions [82]. In tobacco plants *IPT* gene was introduced under the control of senescencerelated receptor [83] and a promotor to induce stress exhibited enhancement in photosynthetic capacity leading to improvement in drought tolerance in tobacco plants [73]. Moreover, transgenic rice plants resistant to drought stress were produced by the insertion of *IPT* gene under the control of PSARK a stress-inducible promoter. The plants were tested against drought stress tolerance at two yield sensitive developmental phases; pre and post-anthesis. During both treatments, the transformed rice

plants showed remarkable resistance to drought stress and an increase in yield grain as compared to non-transformed wild type control plants [41].

#### *2.1.6 The expression of bacterial* ggpPS *gene isolated from* Azotobacter vinelandii *for glucosyl glycerol biosynthesis confers salt and drought stress tolerance in transgenic plant*

Various organisms generally accumulate compatible solutes to show response against salt and drought stress, which includes heterotrophic and cyanobacteria which shows resistance to salty environment and produces glucosyl glycerol as their major compound for protection. To know the potential of glucosyl glycerol to enhance salt resistance in higher plants, a gene *ggpPS* that codes combinedly for GG-phosphate synthase/phosphatase was isolated through PCR from the chromosomal DNA of the cells treated with lysozyme from a heterotrophic (*proteobacterium A. vinelandii*) and introduced into model plant *A. thaliana*. The high accumulation of glucosyl glycerol was observed due to the expression of this gene. In various growth experiments, three separate Arabidopsis lines were tested that showed varied glucosyl glycerol levels. Plants having a low level of glucosyl glycerol within leaves showed no changes in growth development in the control condition, rather an improvement to salt tolerance. While plants having a very low or higher glucosyl glycerol content exhibited growth delay and no enhancement of salt resistance was observed, the results suggested that the suitable solute synthesis has a positive impact on the stress tolerance of plants as long as the accumulation extent does not interfere adversely with the metabolic process of plants [44].

#### *2.1.7 The expression of bacterial* betA *gene confers abiotic stress tolerance in transgenic plants*

Drought stress exists in most of the areas where sugarcane is grown and cultivated, which has no support of irrigation system and has lower rainfall. To know psychological and biochemical mechanisms better, underlying plants response to water deficit stress, have been overcome by the development of drought-resistant plants through biotechnological techniques. To tackle water stress plants use various strategies like variations in gene expression and the accumulation of compatible solutes for survival and growth. A bacterial gene *betA* that codes for *CDH* choline dehydrogenase has been effectively expressed in sugarcane to produce drought resistant plants. The function of *CDH* is the conversion of choline in betaine aldehyde that is then transformed into glycinebetaine GB, the expression of *betA* gene improves the level of glycinbetaine that act as an osmoprotectant and help in the acclimatization of sugarcane in water deficit stress, the drought-resistant sugarcane was first developed by Ajinomoto Company in Tokyo [84].

Transgenic cotton (*Gossypium hirsutum* L.) was genetically transformed by the expression of a bacterial *betA* gene from *E. coli* for the enhancement of glycinbetaine, its accumulation was identified at three stages. Five lines expressing this gene showed significant improvement to drought stress than wild type non-transformed plants from seedlings to flowering plants. The five transgenic lines showed better relative water content, a decrease in the leakage of ions, and less malondialdehyde content in comparison to wild-type plants. The glyycinbetaine content was positively related with water deficit tolerance in water stress, the results indicated that the expression of the *betA* gene not only provide protection to cell membrane against drought stress but also act in the osmotic adjustment in transgenic cotton plants, more importantly,

line 4 among five lines showed a significant increase in cotton seed yield after exposure to drought stress which will help a great deal in cotton production in future [85].

The similar *betA* gene of *E. coli* was expressed through *Agrobacterium-*mediated transformation in maize to improve its tolerance against cold or chilling stress, five transgenic lines were tested in which four lines exhibited a higher level of glycinebetaine than WT plants. At lower temperatures 10 and 15°C three transformed lines showed an increase in germination stages, as identified through the progress of germination and presented lower inhibition in the speed of shoot growth in seedlings than nontransformed lines. Upon exposure to chilling stress the tolerance of transgenic plants was significantly improved in cell membrane injury, the level of damage caused by cold stress, survival rate, and photosynthetic capacity in transgenic lines than WT plants [86].

Tobacco plants were also genetically transformed by the expression of this gene from *E. coli* and improvement in glycinbetaine was observed leading to improvement in the resistance of transgenic plants to chilling and salinity stress than wild-type plants [87, 88].

#### **3. Gene expression from yeast (***Saccharomyces cerevisiae***) in plants against abiotic stresses tolerance**

Just like the above bacterial genes expression in plants to improve their tolerance against abiotic stresses, yeast genes have also been introduced in transgenic plants to enhance their tolerance, *TPSI* gene of *Saccharomyces cerevisiae* has been expressed in transgenic tobacco against salt and drought stress and the results were very much better than WT plants [89] *HAL1* gene was expressed in tomato against salt tolerance and the results showed better improvement in comparison to wild type plants [90] similarly *HAL1* and *HAL3* genes were introduced in *A. thaliana* for its enhancement against saline stress and the transgenic plants exhibited much better tolerance than wild type non transformed lines [90], the procedures of the expression of these genes have been discussed below.

#### **3.1 Insertion of a yeast gene** *TPSI* **in transgenic tobacco plants against drought and salt stress**

A gene trehalose-6-phosphate synthase from yeast was introduced in tobacco plants by the control of Cauliflower mosaic virus (CaMV35S) regulation sequence. *Agrobacterium-*mediated transformation method was used for the introduction of a gene into the genomic DNA of tobacco (*Nicotiana tabacum* L) plants. The accumulation of trehalose was found in transgenic plants through ion-exchange chromatography in combination with ampometry detection procedure. The disaccharide that was non-reducing accumulated almost 0.17 per gram of fresh weight in leaf extracts of the transformants. The plants with trehalose accumulation had various changes in phenotypes like dwarfness, pointed or lancet leaves pattern, and decrease in sucrose level. Moreover, the expression of *TPS1* gene in tobacco plants showed significant tolerance to drought and salt stress as illustrated in **Figure 6** [91].

#### **3.2 The role of yeast** *HAL1***, and** *HAL3* **genes against salt tolerance in plants**

To overcome salinity stress in *Arabidopsis thaliana*, the yeast genes *HAL1* and *HAL3* were introduced under the control of 35S promoter via the

#### **Figure 6.**

*Drought tolerance in transgenic tobacco plants by the overexpression of the TPSI gene from yeast. The left 2 rows consist of non-transformed control plants while the right two rows contain the transgenic homozygous plants. No water has been given to all plants for almost 15 days. The results obtained are similar by exposure to drought stress with 400 mM NaCl. The visible better changes can be seen in transgenic plants with TPSI gene [91].*

*Agrobacterium-*mediated method. Almost 33 plants showing resistance to kanamycin were obtained from 70,000 plus seeds. Southern blotting analysis showed that *HAL1* and *HAL3* genes were introduced into all the genomes of the transgenic plants. The copy number of the yeast gene in all plants was in the range of 1–3 by the confirmation of southern blotting analysis, there was no difference in the phenotype of the transgenic plants compared to wild ones. Most of the transformants were self-pollinated, the progenies of transformants and non-transform *A. thaliana* plants were observed through different experiments for gene expression to know the salt resistance. The measurement of (K+ ) and (Na<sup>+</sup> ) showed that the transgenic plants accumulated fewer (Na+ ) as compared to the control lines. In light of several tests, it was observed that the introduction of yeast *HAL1* gene exhibited more resistance to saline soil in comparison to non-transform plants [92].

In past, remarkable advancements have been made in the identification and isolation of various genes which could be used in the process of abiotic stress protection in plants. It is hard to believe that a single gene insertion would make a dramatic improvement to salt stress directly producing a fresh salt-resistant transgenic plant that could be enough for breeding purpose point of view. Yeast *HAL1* gene was introduced in tomato (*Lycopersicon esculentum*) through a well-modified plasmid containing the elements of enhancer and salt resistance was evaluated in transgenic plants from progenies. The result showed that transgenic lines having one copy of the *HAL1* gene had higher salt tolerance than non-transformed plants [90].

For the production of transformed watermelon plants, and adjusted *agrobacterium* mediated protocols were maintained. The efficient transformation rate was 2.8–5.3% in the cultivars. Yeast *HAL1* gene under the control of 35S cauliflower mosaic virus having a double sequenced enhancer was cloned in pBiN19 plasmid. RNa4 from Alfalfa mosaic virus was used alongside 35S. The vector was introduced in the LBA4404 strain of *agrobacterium tumefaciens* for the inoculation of watermelon cotyledon explants. PCR and Southern hybridization analysis were used for the assessment of the *HAL1* gene in new transformants. Improved elongation of leaves and new roots emergence was seen in plantlets in culture media having NaCl. It was

observed that the *HAL1* gene as a molecular tool for genetic engineering could be very useful to protect crop plants in the future [93].

#### **3.3** *HAL1* **gene mode of action in (***Saccharomyces cerevisiae***)**

Yeast (*S. cerevisiae*) *HAL1* gene was initially found in the screening process for various genes that could be expressed in various plasmid copies that improve saline resistance in yeast (*S. cerevisiae*). It codes for a soluble protein in the cytoplasm, even though there is no significant information available about this gene, still it is a major affective ions regulator during the homeostatic process, slight expression of its promotor generally have an impact on the potassium levels inside the cells [94]. However, a significant expression by strong promotor had an impact on (K<sup>+</sup> ) and (Na<sup>+</sup> ) homeostasis [95]. The expression of the *HAL1* gene decreases the loss of (K+ ) from cells affected by salt stress a phenomenon initiated through an unknown K<sup>+</sup> efflux system. The cells with *HAL1* contain a high level of potassium in cells, and a low level of sodium within the cells, and an increased K<sup>+</sup> /Na+ ratio as compared to control cells the last one indicating the enhancement in salt tolerance [96]. Currently, it is not known how a protein product from the cytoplasm of the *HAL1* gene can control the transportation of sodium and potassium efflux. Besides the lack of information available about this process *HAL1* gene possess a high capability to improve salt tolerance of various plants, and it was selected in the first trials for expression of genes in transgenic plants [89].

#### **4. Anti-freeze proteins**

During the study on fishes in the waters of temperate oceans proteins that act as antifreeze elements were found, in winter the temperature of these waters reaches (−1.9°C) but fishes under these waters still survive. NaCl is the most common electrolyte in blood serum of most species, but to inhibit freezing environment it only helps in 40–50% of the examined freezing point depression [97] the other substances due to which freezing point depression occurred were marked as proteins and glycoproteins [98–100] the molecular masses of antifreeze-glycoproteins ranges from 2.6 to 34 kD. They consist of tripeptide repeats (A l a-A l a-T h r) along with the moiety of disaccharide (-Naga-Gal) having the residue of threonyl [101].

#### **4.1 AFP gene mechanism of action**

Many researchers have studied the ant-freeze protein from winter flounder because of their small size and are very effective for structural mechanism requirements, there are some changes in the size and AFP amino acid composition which depends on the isolation technique from the serum of the fish [102]. Through southern blot and restriction maps of genomic clones analysis, the pattern of antifreeze protein multigene family was observed in winter flounder [103]. Most of them are equal in number to 40 AFP genes in this fish are present in 7–8 kbp DNA elements which act like tandem repeats, in every repeat, there is 1 kbp long AFP gene having same transcription shape and orientation, they also have some restriction site polymorphism ability even though the repeats are homologous. When winter flounder genomic DNA goes through the digestion phase mainly by Restriction endonuclease which normally does not cut inside the repeats, many of the AFP genes goes to 40 kbp long fragments that represent five or more repeats in tandem as clusters. After the digestion of genomic DNA, these genes reside in the fragments of extremely high mol. Weight indicating the groups of clusters in the genome [104]. By the combination of protein and DNA sequencing methods, the precursor of amino acid in the second AFP protein B gene has been observed in winter flounder. The precursor containing 82 amino acid residues is only different in three main sites to AFP, A gene that acts in the process of substitution, various other changes, all are grouped inside the DNA that codes for the mature portion of protein. In the process of post-transcriptional modification, the c-terminal glycine residue removal takes place [105].

#### **4.2 The introduction of fish antifreeze AFP gene in transgenic plants**

The quality of fruits and vegetables can be compromised by adverse effects due to the formation of ice crystals inside the frozen tissues. At lower concentrations, some proteins from the blood of fishes have shown the ability to help in the inhibition of ice crystals formation. To know whether the expression of certain genes improves freezing properties of the plant tissues, the transgenic tomato and tobacco have been produced by the expression of anti-freeze gene *AFA3* were introduced at higher steady mRNA levels in the leaves of transgenic plants but no crystals inhibition was observed in tissues extracts. As a result of these experiments, ice crystal inhibition was seen in transformed tomato and tobacco plant tissues [106]. The freezing rate and temperature of the storage site are the two factors that influence ice formation in frozen fruits and vegetables, when plants tissues gradually freeze the large and randomly distributed extracellular gaps are filled by ice crystals in comparison to small intracellular and extracellular gaps which freezes rapidly during storage temperature changes lead to large shaped ice crystals and reorganization of ice in food [107].

AFP genes isolated from fish and insects are more useful in the inhibition of frost or crystal formation in several crop plants. AFPs isolated from insects and then their expression in plants against freezing stress are much better than those of fish because of their survival ability in freezing temperatures. AFPs can decrease water freezing level (thermal hysteresis) has generated the phenomenon that the damage could be avoided by those plants which are much more sensitive to frost at the end of autumn and the start of spring due to the expression of higher activity genes coding antifreeze proteins allowing them to be unfrozen in extremely cold and freezing temperatures. During the last two decades, the effectiveness of this idea has been conducted in several different research studies that produce transgenic plants by the expression of various AFPs. Earlier the anti-freezing proteins isolated from fish were used in these studies but later on, as AFPs of insects with high levels of anti-freezing activity were discovered and now being used for plant transformation studies as a choice. A chemically synthesized antifreeze gene from winter flounder fish was introduced through the *Agrobacterium-*mediated transformation method in potato *solanum Tuberosum L. cv.* which decreased the electrolyte leakage from the leaves at freezing temperature [108].

Spring wheat which is vulnerable to the damage caused by frost can also be transformed to show tolerance to frost by the expression of winter flounder gene AFPs in the cytoplasm and apoplast of the plant where ice formation leads to damage at the cellular level. The transformed wheat lines which were targeted by apoplast antifreeze proteins showed the highest anti-freezing activity and exhibited remarkable protection against frost at very lower temperatures [109].

Various marine species survive in extremely cold seawater below the freezing point temperature of their non-protected blood serum by producing anti-freezing proteins and glycoproteins [110, 111]. These proteins and glycoproteins have subsequently been considered for the neutralization of ice nucleator agents [112] to protect the cell from ice crystallization potential damage by hypothermic temperatures [113]. The introduction of these proteins in transgenic plants has been a very important tool for increasing their cold stress tolerance against freezing temperatures. In early work, an AFP gene that codes for alanine-rich, α-helical Type I AFP from winter flounder fish was introduced into tobacco plants through the *Agrobacterium-*mediated transformation method. The transformed plants produced antifreeze proteins mRNA and upon exposure to cold showed the accumulation of AFP to a detectable extent. The observation from the results was that fish antifreeze gene could be very useful in protecting plants from cold and freezing stress [114].

An anti-freezing gene (IIA7 cDNA) was isolated from a fish winter flounder *Pseudopleuronectes americanus* which can survive below the freezing temperature point under cold sea waters, which encodes for 91 amino acid and then proceeded to a mature protein of 53 amino acids. Only mature antifreeze proteins are encoded by this gene, a start methionine was also cloned alongside a plasmid that allowed improved expression from a double cauliflower mosaic virus CaMV 35S promotor. A binary vector pMON200 and intermediate vector pBI121 was used for the subcloning of anti-freezing protein. Various Kanamycin resistant seedlings were tested against the frost tolerance more than 30% of plants survived as compared to the control wild type plants these results confirmed that these genes can help in the resistance to frost in tobacco plants [115].

#### **4.3 Transformation of plants with insects AFPs**

The first transgenic plants were produced by the expression of insect AFPs [116], a chemically synthesized gene based on the anti-freezing proteins from an insect *Choristoneura fumiferana*, was introduced into (*Nicotiana Tubaccum*) tobacco plants through Cauliflower mosaic virus 35S promoter. The transformation success was determined by the levels of properly shaped transcripts through real-time PCR, in crude leaf homogenates the recrystallization inhibition activity and the apoplast plant extracts, and the most importantly the degree of water freezing point 0.37% in the apoplast liquid [117].

Transgenic *A. thaliana* was produced by the gene isolated from an insect (*Dendroides canadensis*), AFPs were introduced by *agrobacterium* mediated transformation. The AFP genes simultaneously with and without peptide signals sequence were expressed in transgenic plants. The thermal hysteresis activity showed the existence of active AFPs in proteins isolated from plants that expressed both proteins and were found in fluids of leaf apoplast of plants expressing AFPs alongside signal peptide. The transformed lines did not show any enhancement to survive in freezing temperatures in comparison to wild type plants, however, when cooled under four different stages the transformed lines containing active AFPs apoplast fluid froze at significantly low temperatures in comparison to wild type, especially when there was no intrinsic nucleation [118].

To illustrate the activity of AFPs from beetle (*Microdera punctipennis*) from the deserts of Xinjiang *China,* for freezing stress resistance in plants the *MpAFP149* gene, alongside the signal peptide sequence used for the secretion of *MpAFP149* into the

#### *Heterologous Expression of Genes in Plants for Abiotic Stresses DOI: http://dx.doi.org/10.5772/intechopen.105171*

apoplast gaps in the control of cauliflower virus 35S was expressed in tobacco plants through *Agrobacterium tumefaciens* transformation method. The transformants were determined by reverse transcription-polymerase chain reaction analysis of leaf fragments, and those plants having higher transcripts contents were identified for further experiments and analysis. The introduced AFPs were restricted to cell walls of transformants by the use of immune-gold label procedure, and the existence of AFPs in apoplast liquid was indicated by western blot. The inhibition of crystal formation and thermal hysteresis tests to observe the expressed AFPs active state were not done. However, it could be expected that a slight extent of activity existed, the resistance to freez stress of transformed plants near to non-transformed plants was identified through ion-exchange chromatography technique, ion leakage, and malondialdehyde (membrane lipid peroxidase product) measurement release leading to the plant exposure to −1**°**C for varied periods for 72 h. Upon exposure to −1°C for 2 and 3 days, non-transformed plants were observed to be more adversely affected than the transformed, as by the assessment of more wilted leaves in them. The transgenic plants seemed fully recovered after 1 day at 25–28°C while the non-transformed plants appeared stressed by the indication of wilted leaves, which was obvious even after 5 days of recovery time. After 1 day at −1°C lower ion leakage and malondialdehyde level was observed in transformed and wild type plants but the level increased significantly after 2–3 days at the same temperature. Therefore the AFPs genes protected transgenic tobacco plants from frost stress [119].

The AFPs synthesized from Spruce budworm (*C. fumiferana*) an insect in the *Choristoneura* genus and introduced into *A. thaliana* by plant codon and a peptide with PR-signal of tobacco, the expression vector in plants had a synthesize gene of AFP with double 35S promotor. The transgenic lines showing the high content of anti-freezing protein transcript were selected based on RT-PCR of total RNA from *Arabidopsis* leaves. After 3 weeks growth progress was determined at 23°C under the condition of extended photoperiod, wild and transformed plants were moved to 4°C for 48 h (at long and short photoperiods simultaneously) and further exposed to a very low temperature of −20°C for 30 min. The plants were then maintained at 4°C at night and transferred back to the facility of growth chamber having 23°C temperature. Through visual inspection, the death of most wild-type plants in comparison to the survival of most transformed plants was observed, although the exact numbers were not determined. The transformed lines having a high level of *AFP* transcript showed better survival ability in comparison with wild type plants that exhibited very poor survival capabilities. The rise in electrolyte leakage and malondialdehyde content was observed in all plants upon their exposure to cold treatment, but the levels were much higher in wild type than transgenic plants. The results showed that the expression of AFPs gene from Spruce budworm (*C. fumiferana*) in transgenic *A. thaliana* plants increased their tolerance to freezing temperatures and helped in the removal of injuries [120].

The lists of genes that have been expressed in plants for abiotic stresses tolerance improvement are shown in **Tables 2** and **3**.

Several genes have been expressed in transgenic plants from bacteria for abiotic stresses tolerance that exhibited good results in many transgenic plants for example tomato, tobacco, finger millet, peanut*,* potato, *A. thaliana*, wheat etc. are shown in the following **Table 2**.

Other genes from insects, fish, and yeast have been introduced in transgenic plants that exhibited better tolerance against various abiotic stresses are shown in **Table 3**.


#### **Table 2.**

*Bacterial genes expressed in plants for abiotic stresses tolerance.*


#### **Table 3.**

*List of heterologous expression of genes in transgenic plants for abiotic stresses tolerance from yeast, fish and insects.*

#### **5. Conclusion**

In this study, the use of various genes isolated from non-plant sources have been expressed in plants for improving their tolerance against abiotic stresses that adversely affect plant growth, and crop yield productivity are reviewed comprehensively. Gene expression in transgenic plants through conventional methods are time consuming and laborious that is why advanced genetic engineering methods for example *Agrobacterium-*mediated transformation and biolistic methods are more accurate, useful, and less time consuming. This review of the chapter provides an extensive insight into various bacterial genes for example *mtID*, *codA*, *betA*, *ADH*, *IPT*, *DRNF1* and *ggpPS*, etc. that have been successfully expressed

*Heterologous Expression of Genes in Plants for Abiotic Stresses DOI: http://dx.doi.org/10.5772/intechopen.105171*

in transgenic plants against various abiotic stresses for stress tolerance enhancement and crop yield improvement which exhibited good encouraging results. Genes from yeast (*Saccharomyces cerevisiae*) have been introduced in transgenic plants against drought and salinity stress, other genes isolated from fish for example *AFA3* and *AFA5* which codes for anti-freezing proteins improve transgenic plants against frost stress. Genes from insects have also been inserted in plants to improve their resistance. According to the available literature, several genes isolated from bacteria, yeast, fish, and insets have been expressed in transgenic plants for their enhancement against high and low temperatures, drought, light, and salinity stress. Various research studies have been conducted to improve transgenic plants for the fulfillment of the constant demands of the ever-increasing population. Further work can be done in the future to enhance crop and transgenic plants through new sophisticated technologies. The above mentioned genes can be tested on various other crops to improve their resistance, better yield productivity, longevity in shelf life and enhanced resistance against abiotic and biotic stresses.

#### **Acknowledgements**

First of all, I am extremely thankful to ALMIGHTY ALLAH, the ever-magnificent, greatest, merciful, gracious, and pervasive, who provided me the audacity and knowledge to commence and complete this task.

I am also extremely thankful to our holy Prophet Mohammad (SAW), who is a light for humanity and who began his preaching from learning.

I convey my bottomless sense of gratitude to my supervisor Dr. Nadir Zaman associate professor, Department of Biotechnology, University of Malakand, for his welcoming support, and guidance from the beginning till the completion of the present work, who has always been kind in all phases.

I would like to express my profound sense of admiration to all teachers especially Dr. Fazal Hadi chairman department of Biotechnology for his able guidance and support throughout the B.S program, Dr. Syed Muhammad Jamal, Dr. Aftab Ali Shah, Dr. Waqar Ali, Dr. Ayaz Ali Khan, Dr. Muhammad Aasim, Dr. Tariq khan, Dr. Alam Zeb ex-chairman Department of Biotechnology, and Mr. Taqweem Ul Haq for sharing expertise and providing a friendly learning environment.

I am cordially thankful to my family for the generous support they provided me throughout my entire life and particularly through the process of pursuing the BS (Hons.) degree. Because of their unconditional love and prayers, I have the chance to complete this thesis.

I also want to express my profound gratitude to all my friends for their company and continue support.

#### **Abbreviations**



### **Author details**

Shahzad Ali1 \*, Nadir Zaman<sup>2</sup> , Waqar Ali1 , Majid Khan1 , Muhammad Aasim3 , Asmat Ali3 and Muhammad Usman3

1 Institute of Biotechnology and Genetic Engineering (IBGE), The University of Agriculture Peshawar, Pakistan

2 Department of Biotechnology, University of Malakand, Pakistan

3 University of Malakand, Pakistan

\*Address all correspondence to: alis60597@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 10**

## Reactive Oxygen Species, Oxidative Damage and Their Production, Detection in Common Bean (*Phaseolus vulgaris* L.) under Water Stress Conditions

*Asmat Ara, Mahroofa Jan, Parvaze A. Sofi, Munezeh Rashid, Ajaz Ahmad Lone, Zahoor Ahmad Dar, Mohd. Ashraf Rather and Musharib Gull*

#### **Abstract**

Reactive oxygen species (ROS) being small and highly reactive oxygen containing molecules play significant role in intracellular signaling and regulation. Various environmental stresses lead to excessive production of ROS causing progressive oxidative damage and ultimately cell death. This increased ROS production is, however, tightly controlled by a versatile and cooperative antioxidant system that modulates intracellular ROS concentration and controls the cell's redox status. Furthermore, ROS enhancement under stress serves as an alarm signal, triggering acclimatory/defense responses via specific signal transduction pathways involving H2O2 as a secondary messenger. Nevertheless, if water stress is prolonged over to a certain extent, ROS production will overwhelm the scavenging action of the anti-oxidant system resulting in extensive cellular damage and death. DAB (3,3′-diaminobenzidine) test serves as an effective assessment of oxidative damage under stress. It clearly differentiates the lines on the basis of darker staining of leaves under water stress. The lines showing greater per cent reduction in yield parameters show greater staining in DAB assay underlining the reliability of using this assay as a reliable supplement to phenotyping protocols for characterizing large germplasm sets.

**Keywords:** ROS, cell death, oxidative stress, DAB

#### **1. Introduction**

Abiotic stresses such as drought and high temperature invariably cause unfavorable changes in water status of plant cells as well as evolution of reactive oxygen species in cellular compartments resulting in acceleration of leaf senescence through lipid peroxidation and other oxidative damage [1]. Omae et al. [2] discovered a link between genotypic differences in bean leaf water status and crop productivity under drought conditions.

This implies that there are differences in leaf water status among bean cultivars, which could be related to drought tolerance mechanisms. Among the reactive oxygen species hydrogen peroxide (H2O2) is a non-radical reactive oxygen species (ROS) produced in a two- electron reduction of molecular oxygen. H2O2 being a strong oxidant, it can initiate localized oxidative damage in leaf cells leading to disruption of metabolic function and loss of cellular integrity, actions that result in senescence promotion.

In plants, reactive oxygen species (ROS) are formed by the leakage of electrons from the electron transport activities of mitochondria, chloroplasts and plasma membranes or as a byproduct of various biotic and abiotic stresses due to disruption of cellular homeostasis [3–5]. A cell is said to be under oxidative stress when the level of ROS exceeds the defense mechanism. Increased ROS production during various stresses endangers cells, causing lipid peroxidation, protein oxidation, enzyme inhibition, nucleic acid damage, activation of the programmed cell death pathway, and ultimately cell death [6–8]. The overproduction of H2O2 has been observed in plants exposed to a number of stress conditions and is considered as one of the factors causing oxidative stress [9].

#### **2. ROS, sites of production and their effects**

Reactive oxygen species are a group of free radicals, reactive molecules and ions that are derived from o2. ROS are known for playing role as both deleterious and beneficial species depending on their concentration in plants. They are produced at several locations within the cell in both stressed and unstressed cells (**Figure 1**).

Production and removal of ROS needs to be controlled to avoid oxidative stress. When this level exceeds the defense mechanisms, a cell is said to be in a state of "oxidative stress". Increased level of ROS can cause damage to biomolecules like lipids, proteins and DNA (**Figure 2**). These reactions can alter intrinsic membrane properties like fluidity, loss of enzyme activity, ion transport, protein cross-linking, DNA damage, inhibition of protein synthesis ultimately resulting in cell death.

Under water stress, ROS production is enhanced in various ways. Inhibition of carbon dioxide assimilation coupled with changes in photosystem activities and photosynthetic

#### **Figure 1.**

*Sites of production of reactive oxygen species (ROS) in plants.*

*Reactive Oxygen Species, Oxidative Damage and Their Production, Detection in Common Bean… DOI: http://dx.doi.org/10.5772/intechopen.106164*

#### **Figure 2.**

*Reactive oxygen species (ROS) induces oxidative damage to lipids, proteins and DNA.*

transport capacity results in increased production of ROS [10]. Excess light energy dissipation in the PSII core and antenna generates ROS, which are potentially dangerous under water stress conditions [11]. The photorespiratory pathway is also increased, especially when RUBP oxygenation is optimum due to CO2 fixation limitation.

DAB (3,3′-diaminobenzidine) assay has been suggested as an effective qualitative assessment of plant response to biotic and abiotic stress and measures the intensity of oxidative burst under stress. Since the oxidative burst is an early response to stress, in terms of production of reactive oxygen species (ROS) including hydrogen peroxide through either NADPH oxidases or peroxidises (Bindschedler et al., 2006) that may exist singly or in combination in different plant species have been proposed for the generation of ROS. The qualitative evolution can be differentially tracked in different parts of plant under stress to assess the most vulnerable part under stress. It is done by staining with 3,3′-diaminobenzidine (DAB) which is oxidized by hydrogen peroxide and generates a dark brown precipitate.

#### **3. Experimental method**

The present study was conducted during 2016-2018 at the Division of Genetics & Plant Breeding, Faculty of Agriculture Wadura, SKUAST-K, Sopore. In the current study, fifty genotypes of common bean were evaluated under controlled conditions.

The genotypes used were chosen based on their yield screening trial performance and represented a wide range of market classes in terms of use category, growth habits, and seed characteristics. The material included 47 breeding lines as well as three released varieties, SR-1, SFB-1, and Arka Anoop. The experiment was designed in a completely randomized design.

The DAB assay was performed in accordance with Daudi & O'Brien [12]. In this protocol, hydrogen peroxide (one of several reactive oxygen species) is detected in situ by staining with 3,3′-diaminobenzidine (DAB). In the presence of some haemecontaining proteins, such as peroxidases, DAB is oxidized by hydrogen peroxide to produce a dark brown precipitate. This precipitate is used as a stain in plant cells to detect the presence and distribution of hydrogen peroxide. DAB staining solution was prepared by adding 50 mg DAB and 45 ml sterile H2O for a final 1 mg ml−1 DAB solution in a 50 ml falcon tube. The tube was covered with aluminum foil as DAB is light-sensitive. About 25 μl Tween 20 (0.05% v/v) and 2.5 ml 200 mM Na2HPO4 to the DAB solution to produce 10 mM Na2HPO4 DAB staining solution. Similar, fully opened leaves were selected from each treatment and incubated for one hour in falcon tubes with 2 ml of the DAB staining solution with the volume being adjusted to ensure that leaves were immersed. The leaves from irrigated treatment were incubated with 2 ml of 10 mM Na2HPO4. All the falcon tubes from both drought and irrigated treatments were shaken for 4-5 h at 80-100 rpm. Following the incubation, the aluminum foil was replaced and the DAB staining solution replaced with bleaching solution (ethanol: acetic acid: glycerol in ratio of 3:1:1). For 15 minutes, the falcon tubes were immersed in a boiling water bath (90-95°C). The chlorophyll will be bleached out, but the brown precipitate formed by the DAB reacting with the hydrogen peroxide will remain. The time should be adjusted (5 minutes) depending on how the leaves look (they should be completely devoid of chlorophyll). After 15 minutes of boiling,

#### **Figure 3.**

*DAB staining of common bean (*Phaseolus vulgaris *L.) genotypes under water stress conditions. Largely stained genotypes (3b) show higher production of hydrogen peroxide under stress conditions causing oxidative damage to cell structure.*

*Reactive Oxygen Species, Oxidative Damage and Their Production, Detection in Common Bean… DOI: http://dx.doi.org/10.5772/intechopen.106164*

the bleaching solution was replaced with fresh bleaching solution and left to stand for 30 minutes. DAB staining was visualized directly on leaves.

#### **4. Results and discussion**

The DAB staining of common bean genotypes under irrigated and water stress conditions clearly differentiates the lines on the basis of darker staining of leaves under drought (**Figure 3**). The lines showing greater per cent reductions in yield parameters show greater staining in DAB assay underlining the reliability of using this assay as a reliable supplement to phenotyping protocols for characterizing large germplasm sets. However, DAB is only a qualitative test for evolution of reactive oxygen species such as H2O2 and the genotypes showing greater staining under drought can be further analyzed for the amount of H2O2 through various analytical methods.

The DAB staining of common bean genotypes clearly differentiates the genotypes on the basis of darker staining of leaves under water stress. The lines showing greater per cent reductions in yield parameters show greater staining in DAB assay underlining the reliability of using this assay as a reliable supplement to phenotyping protocols for characterizing large germplasm sets (**Table 1**). Reactive oxygen species play an important role as signaling molecules that initiate stress responses in plants. Environmental stresses are known to induce the production of H2O2 and other toxic oxygen species in cellular compartments, resulting in the acceleration of leaf senescence via lipid peroxidation and other oxidative damage, according to Upadhyaya et al. [1]. Because H2O2 is a strong oxidant, it can cause localized oxidative damage in leaf cells, disrupting metabolic function and causing cellular integrity loss, both of which promote senescence. Overproduction of H2O2 has been observed in plants subjected to a variety of stress conditions and is thought to be one of the causes of oxidative stress [9]. According to Foyer and Noctor [13], among the various forms of ROS, the central role in plant signaling, regulating plant development, and adaptation to abiotic and biotic stresses is played by hydroxyl radicals. ROS also act as signaling molecules to regulate development and stress responses [14]. Increased availability of H2O2 is commonly observed feature of plant stress response signature. The physiological context involves a continuous supply of environmental stimuli that can trigger intracellular H2O2 accumulation or modulate the response to such accumulation.

The detection of cellular levels of H2O2 was done by DAB staining method and our results shows a clear difference in the degree of staining achieved in the stressed plant. Under water stress, there was significant variation in staining in different genotypes indicating differential oxidative damage on account of production of H2O2. The lines which showed fair amount of tolerance to water stress in terms of higher yield and lower reduction had almost negligible staining while as the genotypes which showed lower yield showed higher reduction, distinctly darker staining. Less tolerant cultivars accumulated more H2O2 than more tolerant ones, and vulnerable variety showed noticeably greater increases in lipid peroxidation. Similar findings were reported by a number of prior studies (Chai et al., 2005; Zlatev et al., 2006).

Plants accumulate reactive oxygen species during drought stress (Verslues et al., 2006). ROS can cause cell death by destroying DNA, proteins, and carbohydrates through partially reduced or activated oxygen derivatives [3]. DAB staining investigations can efficiently differentiate ROS levels in transgenic lines of rice, with reduced staining in transgenic lines compared to control plants after drought-stress treatment, according to Jiang et al. (2016). DAB assay results were consistent with those



### **Table 1.**

*Mean performance under different water regimes and percent reductions for yield and yield parameters under water stress in common bean (Phaseolus vulgaris L.).*

#### *Reactive Oxygen Species, Oxidative Damage and Their Production, Detection in Common Bean… DOI: http://dx.doi.org/10.5772/intechopen.106164*


#### **Table 2.**

*Comparative performance of tolerant susceptible cultivars for various physio- biochemical parameters under stress in common bean.*

obtained using membrane stability and other biochemical parameters in tolerant and sensitive wheat cultivars, according to Chakraborty and Pradhan (2012). However, only a qualitative assessment of DAB was performed in this work. Ghahfarokhi et al. (2016) performed an experiment to examine the effects of drought stress caused by withholding irrigation at the vegetative stage (4-5 leaves) and reproductive stage on crop production, physiological, and biochemical features in hybrids of maize (*Zea mays* L.) (anthesis). Results indicated that these traits were significantly impacted by drought stress (**Table 2**). Under water stress, both the yield and its constituent parts significantly reduced. The main causes of the yield reduction were a decrease in the quantity of grain ear−1 and the weight of 1000 grains. In comparison to other hybrids, short maturity hybrids had a larger yield reduction. These results suggested that water stress lead to the production of reactive oxygen species (ROS), which caused an increased membrane permeability and oxidative stress in the maize plants. The reliability of the DAB test can be further validated by conducting a quantitative assessment of hydrogen peroxide evolution in common bean under water stress.

#### **5. Conclusion**

ROS are produced by electron transport activities of mitochondria, chloroplast, plasma membrane or as a byproduct of various metabolic pathways localized in different cellular compartments. Study of formation and fate of ROS using advanced and analytical techniques help in developing broader view of the role of ROS in plants. DAB assay is employed to delineate genotypic response in terms of qualitative differentiation of oxidative damage as indicated by differential staining under DAB treatment. All the genotypes revealed almost similar staining in irrigated conditions. While as, under drought conditions, genotypes which showed better resilience to water stress in terms of higher yield and drought had significantly lesser staining as compared to susceptible ones. Therefore, DAB staining can be used as complementary method for differentiating genotypes to water stress.

*Reactive Oxygen Species, Oxidative Damage and Their Production, Detection in Common Bean… DOI: http://dx.doi.org/10.5772/intechopen.106164*

#### **Author details**

Asmat Ara1 \*, Mahroofa Jan2 , Parvaze A. Sofi1 , Munezeh Rashid1 , Ajaz Ahmad Lone1 , Zahoor Ahmad Dar1 , Mohd. Ashraf Rather1 and Musharib Gull1

1 Division of Genetics and Plant Breeding, Wadura, Sopore, India

2 Department of Botany, University of Kashmir, Srinagar, India

\*Address all correspondence to: mirasmat35@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### Section 3
