Emerging Technologies and Management of Crop Stress Tolerance || Genetic Engineering of Crop Plants...

CHAPTER

5Genetic Engineering of Crop Plantsfor Abiotic Stress Tolerance

Surbhi Goel and Bhawna Madan

5.1 IntroductionGrowth and survival of plants are greatly affected by unfavorable environmental factors such as

high salinity, water drought, low and high temperatures, flooding, and deprivation of nutrients

(Kasuga et al., 1999; Sreenivasulu et al., 2007; Bhatnagar-Mathur et al., 2008). These abiotic stress

factors affect plant growth, reduce productivity, and deteriorate seed quality, thus reducing yield

potential (Levitt, 1980). It has been estimated that abiotic stresses lead to more than 50% of yield

reduction (Rodrıguez et al., 2005; Acquaah, 2007). Plant species that are more genotypically

advanced than others show better survival under abiotic stress conditions, but to sustain the growth

of agriculturally important plants lacking this advantage, measures need to be taken to increase

stress resistance. For this, selection of stress-resistant plants and conventional breeding programs

has been used; however, no significant success was achieved (Richards, 1996; Singla-Pareek et al.,

2001). Agricultural plants require maintenance of their growth and development to ensure high pro-

ductivity and yield along with high quality standards. To feed the increasing population, it is

required to explore new lands where agriculture is impractical due to water-drought condition, low

and high temperatures, heavy metal poisoning, and high saline concentration. This can be achieved

by engineering plants with improved abiotic stress tolerance (Reguera et al., 2012).

Genetic engineering approaches are one of the important ways to produce plants with increasing

abiotic stress tolerance. Abiotic stress leads to up-regulation of expression of a number of genes in

plants, which enables them to survive in stress conditions. These abiotic stress-inducible genes are

classified into two groups: (1) genes whose products are directly involved against stress such as

osmoprotectants, antifreeze proteins, chaperons, detoxification enzymes, and (2) genes that regulate

expression of stress-related genes and signal transduction in stress response such as transcription fac-

tors, protein kinases, etc. (Kasuga et al., 1999). The stress-inducible genes and their regulatory genes

can be altered, transferred, and expressed in different species via an Agrobacterium-mediated trans-

formation system or expressed ectopically, thus inducing biochemical, molecular, and physiological

alteration leading to increase in growth, development, and yield of plants in stress conditions

(Bhatnagar-Mathur et al., 2008). Overexpression of stress-inducible genes is controlled under strong

constitutive promoters like CaMV 35S, which improves stress tolerance, but this causes expression

in an uncontrolled manner, which costs cells their energy reserve resulting in growth retardation

(Kasuga et al., 2004). Recently, the use of stress-inducible promoters for expression of stress-

inducible genes ensured a time-specific and optimum level expression of genes (Katiyar et al., 1999).

99P. Ahmad (Ed): Emerging Technologies and Management of Crop Stress Tolerance, Volume 1.

DOI: http://dx.doi.org/10.1016/B978-0-12-800876-8.00005-9

© 2014 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/B978-0-12-800876-8.00005-9

In this chapter, the various approaches used to improve abiotic stress tolerance by genetic engineer-

ing of transcription factors, ion transport channels, overexpression of osmoprotectants and stress-

signaling genes, and maintenance of osmotic balance have been discussed.

5.2 Overexpression of genes for transcriptional regulationTranscriptional factors (TFs) are proteins that interact with the cis-elements of promoter regions

and up-regulate the expression of stress-signaling genes, imparting enhanced tolerance towards

multiple stresses (Bhatnagar-Mathur et al., 2008; Hussain et al., 2011). The various TFs involved in

stress signaling, e.g., zinc-finger domains, dehydration response elements, basic leucine zippers,

MYB, and NAC transcriptional factors, are described below (Figure 5.1). As the overexpression of

the TFs in transgenic plants has shown increased stress tolerance (Mukhopadhyay et al., 2004;

Fujita et al., 2005; Ito et al., 2006; Jung et al., 2008), it has, therefore, emerged as a powerful tool

for development of drought-tolerant crops.

5.2.1 Zinc-finger domainsCys-2/His-2-type zinc fingers, also called the classical or TFIIIA-type fingers, are DNA binding

motifs found in eukaryotic transcription factors. They contain about 30 amino acids, having a con-

sensus sequence of Cx2-4Cx9-12Cx2Cx4Cx2Hx5HxC, where x represents any amino acid, and two

ABA dependent

MYBbZIPDREB

Enhanced abiotic stress resistance

Expression of stress inducible genes

Activation of transcription factors

Stress perception

Abiotic stress

ZFDNAC

ABA independent

FIGURE 5.1

A schematic representation of activation of transcriptional factors leading to abiotic stress tolerance.

100 CHAPTER 5 Genetic Engineering of Crop Plants for Abiotic Stress Tolerance

pairs of conserved Cys and His bound tetrahedrally to a zinc ion (Pabo et al., 2001). In multiple-

fingered proteins, the adjacent fingers are separated by a long spacer that is highly variable in

length and sequence from one protein to another. The EPF family is a subfamily of TFIIIA-type

zinc-finger proteins of plants, which are characterized by a highly conserved sequence QALGGH

in their zinc-figure motifs (Frugier et al., 2000). This family includes soybean SCOF-1 for cold

tolerance (Kim et al., 2001), Arabidopsis STZ/ZAT10 for salt tolerance (Lippuner et al., 1996), and

Arabidopsis RHL41/ZAT12 for light acclimatization response (Lida et al., 2000). In Petunia

hybrida both ZPT2-2 and ZPT2-3 (EPF family zinc-finger motifs) were induced by stress, desicca-

tion, cold, wounding, etc. (Van Der Krol et al., 1999; Sugano et al., 2003). The constitutive overex-

pression of ZPT2-3 in transgenic Petunia plants results in increased tolerance to dehydration

(Sugano et al., 2003). In Arabidopsis, four ZPT2-related proteins (AZF1, AZF2, AZF3, and STZ)

were characterized. RNA gel-blot analysis showed that expression of AZF2 and STZ was strongly

induced by dehydration, high-salt and cold stresses, and abscisic acid treatment. The transgenic

Arabidopsis plants overexpressing AZF2 and STZ show increased stress tolerance following growth

retardation (Sakamoto et al., 2004). In rice, the gene OSISAP1 encoding a zinc-finger protein is

induced after different types of stresses such as cold, desiccation, salt, submergence, heavy metals as

well as injury. The overexpression of this gene OSISAP1 in transgenic tobacco conferred tolerance to

cold, dehydration, and salt stress at the seed-germination/seedling stage (Mukhopadhyay et al., 2004).

5.2.2 Dehydration-responsive elementDehydration-responsive element/C-repeat (DRE/CRT), a 9 bp conserved DNA sequence,

TACCGACAT, a cis-acting element, plays an important role in both dehydration and low tempera-

ture tolerance. In Arabidopsis, DRE-related motifs have been reported in the promoter regions of

cold- and drought-inducible genes such as kin1, cor6.6, and rd17 (Wang et al., 1995; Iwasaki et al.,

1997). In addition, two DRE binding proteins, DREB1 and DREB2, have been isolated using the

yeast one-hybrid screening technique (Liu et al., 1998). DREB1 is up-regulated in response to cold

stress whereas DREB2 is up-regulated in response to drought and salinity (Liu et al., 1998). Both

the DREB1A and DREB2A homologues belong to the family of EREBP/AP2 transcription factors.

The DREB binding proteins have also been reported in other plants such as Triticum aestivum,

Zea mays, and Oryza sativa, and are found to be induced by low temperature, salt, and drought

(Kizis et al., 2001; Shen et al., 2003). The overexpression of DREB1A under a strong constitutive

promoter in transgenic Arabidopsis plants induced strong expression of the stress genes in unstressed

conditions, which in turn imparts freezing and dehydration tolerance along with dwarf phenotype.

However, no growth alterations were observed when the DREB1A was overexpressed under the con-

trol of a stress-inducible promoter (Jaglo-Ottosen, 1998; Liu et al., 1998; Kasuga et al., 1999). The

transgenic plants overexpressing DREB2A proteins caused growth retardation but did not show stress

resistance (Liu et al., 1998). Similarly, in Oryzae sativa and Zea mays, the overexpression of

OsDREB1 and ZmDREB1A, respectively, has resulted in increased tolerance to drought, salt, and

freezing stress (Qin et al., 2004; Ito et al., 2006). This indicates that both OsDREB1 and ZmDREB1A

show functional similarity to Arabiopsis DREB1. The transgenic rice and Arabidopsis plants overex-

pressing DREB1 also accumulated osmoprotectants such as proline and various other sugars (Gilmour

et al., 2000; Ito et al., 2006).

1015.2 Overexpression of genes for transcriptional regulation

5.2.3 NAC transcription factorNAC are a class of transcription factors expressed in plants in response to stress and have shown to

play an important role in development. The NAC protein is characterized by the presence of a highly

conserved region, the NAC domain (Kikuchi et al., 2000; Nakashima et al., 2012). In Petunia, the

first NAC gene NAM was reported (Souer et al., 1996) and the first cDNA of the NAC protein for

the “RESPONSIVE TO DEHYDRATION 26” (RD26) gene was described in Arabidopsis

(Yamaguchi-Shinozaki et al., 1992). In Arabidopsis and rice, 106 and 149 NAC genes have been pre-

dicted, respectively (Gong et al., 2004; Xiong et al., 2005). Overexpressing SNAC1 (stress-responsive

NAC1) in rice enhances drought resistance significantly at the reproductive stage without any

changes in phenotype and yield. The transgenic rice showed increased sensitivity to abscisic acid and

reduced water loss by stomata closing. Also, a number of stress-related genes were found to be

up-regulated in SNAC1 overexpressing rice plants suggesting its promising role in increasing drought

and salt tolerance (Hu et al., 2006). Rice SNAC1 gene was expressed in Chinese wheat variety

Yangmai 12 under maize ubiquitin promoter for improving salinity and drought tolerance. SNAC1

expressing plant showed increased tolerance with high water and chlorophyll content in leaves and

increased fresh and dry weight of roots and high yield. Also, expression of SNAC1 increased the

expression of other abiotic stress-signaling genes such as wheat 1-phosphatidylinositol-3-phosphate-

5-kinase, sucrose phosphate synthase, and type 2C protein phosphatases resulting in enhanced toler-

ance (Saad et al., 2013).

Transgenic rice plants overexpressing rice NAC gene ONAC045 showed enhanced drought and

salt tolerance and up-regulation of other stress-responsive genes (Zheng et al., 2009). In Arabidopsis,

a stress-responsive NAC gene, ATAF1, is induced by drought, salinity, infection, etc. The transgenic

Arabidopsis plants overexpressing ATAF1 increased the sensitivity of plants to salt, drought, and oxi-

dative stress (Wu et al., 2009). The transgenic rice plants overexpressing rice NAC gene ONAC045

showed enhanced drought and salt tolerance and up-regulation of other stress-responsive genes

(Zheng et al., 2009). Gao et al. (2010) identified and isolated a NAC gene OsNAC52 from Oryza

sativa L cDNA library using rapid amplification of cDNA ends (RACE) and studied its effect on

drought tolerance in Arabidopsis. Overexpression of OsNAC52 in Arabidopsis increased drought tol-

erance by activation of expression of a number of downstream genes. Also, increased drought, salt,

and freezing tolerance was observed in transgenic Arabidopsis lines overexpressing TaNAC2, a NAC

transcription factor from wheat, which activated the expression of abiotic stress-responsive genes

(Mao et al., 2012). The transgenic Arabidopsis overexpressing Populus euphratica stress-responsive

gene PeNAC1 showed enhanced tolerance to salt stress, with lower Na1/K1 ratios in roots and leaves

and reduced expression of AtHKT1 (Wang et al., 2013).

5.2.4 Basic leucin zipper transcription factorsABA, a plant hormone, show increased expression under stress conditions such as drought, and

induced responses essential for growth and yield. Under stress conditions, ABA is responsible for up-

regulation of a number of genes containing the cis-element called ABA-responsive element (ABRE)

with (C/T)ACGTGGC consensus sequence in the promoter region (Bray, 1994; Giraudat et al., 1994;

Busk and Pages, 1998). Many of these ABRE elements contain G-box (CACGTG) sequence and


another group of ABREs known as coupling element (CE) has CGCGTG core sequence (Giuliano

et al., 1988; Busk and Pages, 1998).

The ABA-responsive element binding factors (ARBE or ABF) belong to the class of basic leu-

cine zipper transcription factors that binds to the ABA-responsive element (ABRE) motif in the

promoter region of ABA-inducible genes. Four ARBEs (ABF1�ABF4) were isolated first by using

yeast one-hybrid screening of Arabidopsis cDNA expression library, were shown to be induced by

ABA and stress, and are capable of binding to ABREs and transactivation of ABRE-containing

genes (Uno et al., 2000; Choi et al., 2000). In Arabidopsis, the constitutive overexpression (two- to

10-folds) of ABF3 and ABF4 leads to ABA hypersensitivity, reduced transpiration and enhanced

drought tolerance (Kang et al., 2002), suggesting their role in ABA-mediated stress response.

Similarly, constitutive expression of ABF3 (Arabidopsis gene) in rice resulted in expression of

ABA-related genes such as LEA, Hsp 70, and protein phosphatase 2C leading to stress tolerance to

drought (Oh et al., 2005).

Fujita et al. (2005) showed that under normal conditions, the expression of the AREB1 gene is not

sufficient to induce the expression of downstream genes. To overcome this, an activated form of

AREB1 (AREB1ΔQT) was created. Plants overexpressing AREB1ΔQT showed ABA hypersen-

sitivity and enhanced drought tolerance, and late embryogenesis abundant class genes and ABA and

drought stress-inducible regulatory genes with two or more ABRE motifs in the promoter regions

were greatly up-regulated.

Overexpression of OsbZIP23, a member of bZIP transcription factor in rice (Oryza sativa), resulted

in improved drought tolerance, high-salinity stresses, and sensitivity to ABA (Xiang et al., 2008). On

the other hand, a null mutant of this factor showed decreased sensitivity to ABA. Also, it was shown

that expressions of hundreds of genes were altered in transgenic rice overexpressing OsbZIP23, indi-

cating that OsbZIP23 is a transcriptional regulator of stress-related genes through an ABA-dependent

manner (Xiang et al., 2008; Nijhawan et al., 2008).

5.2.5 MYB transcription factorsMYB is one of the largest family of transcription factors in the plant kingdom involved in the

expression of stress-induced genes. Based on the presence of repeats in DNA binding domain,

MYB proteins are classified into three subfamilies: MYBR2R3, MYBR1R2R3, and MYB related

(Stracke et al., 2001; Chen et al., 2006). In plants, the most common family present is MYB with

R2R3 repeat (Stracke et al., 2001). In rice and Arabidopsis, about 126 and 109 R2R3 MYB proteins

were found, respectively (Chen et al., 2006). MYB genes are known to play roles in a number of

developmental and stress-responsive processes (Dubos et al., 2010).

The first plant MYB gene was isolated from Zea mays C1 involved in anthocyanin biosynthesis

by encoding c-myb-like transcription factor (Paz-ares et al., 1987). In Arabidopsis, two transcription

factors, AtMYC2 and AtMYB2, were found to be induced by drought and salt stress and function in

an ABA-dependent manner (Abe et al., 2003). In Arabidopsis, AtMYB44, an R2R3 MYB tran-

scription factor gene, showed activation under dehydration, low temperature, and salinity. When

overexpressed, transgenic Arabidopsis showed reduction in water loss during dehydration and

enhanced salt stress (Jung et al., 2008).

Overexpression of Osmyb4 cDNA in Arabidopsis thaliana showed significant increase in freeze

tolerance and has also affected the expression of genes involved in cold-induced pathways

1035.2 Overexpression of genes for transcriptional regulation

suggesting its major role in cold tolerance (Vannini et al., 2006). Similarly, the constitutive expres-

sion of Chrysanthemum CmMYB2, an R2R3-MYB transcription factor gene in Arabidopsis thaliana,

leads to increased expression of stress-tolerant genes resulting in increase drought and salinity

tolerance (Shan et al., 2012). Also, overexpression of OsMYB2 in rice imparts enhanced tolerance

to salt, cold, and dehydration stress. The enhanced accumulation of soluble sugar and proline and

less accumulation of H2O2 and malondialdehyde was observed in transgenic rice plants overexpres-

sing OsMYB2. Microarray analysis also revealed the increased expression of a number of genes

involved in stress tolerance suggesting the role of transcription factor in tolerance to dehydration,

salt, and cold stress (Yang et al., 2012).

In wheat (Triticum aestivum), the function of MYB transcription factor TaMYB73 was studied

by He et al. (2011). TaMYB73 is an MYB protein of R2R3 type with transactivation activity

known to be induced by NaCl, dehydration, and stress-responsive cis-elements. Overexpression of

TaMYB73 in Arabidopsis showed increased salinity tolerance to NaCl, LiCl, and KCl. Also, the

transgenic plants showed superior germination ability compared to wild type (He et al., 2012). In

another report, ectopic overexpression of TaMYB33 in Arabidopsis showed enhanced drought toler-

ance and NaCl stress, but did not show tolerance towards LiCl and KCl stress (Qin et al., 2012).

5.3 Overexpression of genes for osmoprotectantsOsmoregulation involves up-regulation of osmolytes such as amino acids (proline) and quaternary

amines (glycine betaine, polyamines, dimethylsulfonioproprionate, and polyol/sugars), which main-

tains cell turgor, regulates redox potentials and the hydroxy radical scavenger, protects macromole-

cules against denaturation, and reduces acidity in the cell (McNeil et al., 2001; Vinocur and

Altman, 2005). Many crops lack the ability to synthesize osmoprotectants; therefore, the crops are

engineered to overexpress osmoprotectants, resulting in enhanced stress tolerance (Bhatnagar-

Mathur et al., 2008).

5.3.1 Glycine betaineBetaine, an osmoprotectant, is a quaternary ammonium compound with a methyl group on every nitro-

gen atom. Of these, glycine betaines (GB, N, N, N-trimethyglycine) are widely distributed in higher

plants and microorganisms, and their increased levels are found under various environmental stresses

(Rhodes and Hanson, 1993; Chen and Murata, 2008). The halotolerant plants accumulate GB in chloro-

plasts and plastids in response to abiotic stress (Kishitani et al., 1994; Allard et al., 1998). In vitro stud-

ies have shown that GB, known as a “compatible solute,” effectively stabilizes the complex enzymes

and protein structures and helps in maintaining the ordered structure of membranes at high salt concen-

tration and temperature (Papageorgiou and Murata, 1995). The synthesis of glycine betaine takes place

either by dehydrogenation of choline or N-methylation of glycine (Chen and Murata, 2002). In higher

plants, choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) mediate GB

synthesis in choloroplast with choline as precursor. In microorganisms, GB is synthesized by choline

dehydrogenase (CDH) and BADH (Rathinasabapathi et al., 1997; Takabe et al., 1998). In some

bacteria such as Arthrobacter globiformis and Arthrobacter pascens, a single enzyme choline oxidase

is involved in GB synthesis (Ikuta et al., 1977). The transgenic Arabidopsis and rice plants


overexpressing the Arthrobacter globiformis COD gene for glycine betaine synthesis showed a high

level of salt and cold tolerance (Hayashi et al., 1997; Sakamoto et al., 1998; Huang et al., 2000;

Mohanty et al., 2002). The Oryza sativa contains two BADH genes, but lacks a functional CMO gene,

which is unable to accumulate GB. A CMO gene from spinach (Spinacia oleracea) is transferred in

rice plants by the Agrobacterium-mediated transformation method. This overexpression leads to the

accumulation of GB at the level of 0.29�0.43 μmol/g21 dry weight and showed increased salt and

temperature stress at the seedling stage (Shirasawa et al., 2006). The transgenic tobacco (Nicotiana

tabacum) plant after transformation with E. coli genes for GB biosynthesis accumulated glycine betaine

and showed increased tolerance to salt stress (Holmstrom et al., 1994, 2000). Also, betaine can be syn-

thesized directly from glycine by two N-methyltransferase enzymes. The transgenic Arabidopsis plant

expressing N-methyltransferase (ApGSMT and ApDMT) showed high accumulation of betaine and

increased seed yield under stress conditions (Waditee et al., 2005). Accumulation of GB was found to

be low in transgenic plants even after transformation with GB synthesis genes (CMO, COD), which

may be due to insufficient supply of choline. A key enzyme in choline synthesis, phosphoethanolamine

N-methyltransferase, was overexpressed in tobacco and transgenic plants showed increased levels of

phosphocholine (five-fold) and free choline (50-fold), which in turn led to increased production of GB

via an engineered GB pathway (McNeil et al., 2001).

5.3.2 ProlineAmino acid proline is accumulated in plants under salt stress. In Arabidopsis, salt stress and water

deficiency resulted in increased expression of Δ1-pyrroline-5-carboxylate synthase (P5CS), a key

enzyme involved in proline biosynthesis proving its role as an osmoprotectant. Proline synthesis

takes place in cytosol from glutamate via glutamate semialdehyde (GSA), which undergoes cycliza-

tion to Δ1-pyrroline-5-carboxylate (Hare et al., 1999).

Overexpression of the mothbean (Vigna aconitifolia) Δ1-pyrroline-5-carboxylate synthase (p5cs)

gene in tobacco plant resulted in 10- to 18-fold increased proline production than control plants, and

showed enhanced biomass in stress conditions (Kishor et al., 1995). The transgenic tobacco plants

overexpressing proline were reported to show enhanced freezing tolerance (Konstantinova et al.,

2002), reduced levels of reactive oxygen species (ROS), and increased salt tolerance (Hong et al.,

2000). Also, the high level of proline was achieved by down-regulating its catabolism. Proline

dehydrogenase catalyzes the first step of proline degradation. In Arabidopsis, transformation with an

antisense proline dehydrogenase cDNA resulted in enhanced proline accumulation and stress toler-

ance (up to 600 mM NaCl) and freezing (up to 27�C) (Nanjo et al., 1999).

The transgenic Arabidopsis plant overexpressing LcSAIN2 (Leymus chinensis salt-induced 2)

gene from sheepgrass (Leymus chinensis) showed increased salt tolerance. These plants also

showed accumulation of osmolytes, especially proline, and improved expression of other stress-

inducible genes such as RD29B, RAB18, and transcription factors MYB2 and RD26, which are sug-

gested to be the factors responsible for increased salt tolerance (Li et al., 2013).

5.3.3 PolyaminesPolyamines (PAs), putrescine (Put), spermidine (Spd), and spermine (Spm) are low-molecular-weight

organic polycations, found ubiquitously in a wide range of organisms (Fariduddin et al., 2012).

1055.3 Overexpression of genes for osmoprotectants

These play crucial roles in a wide range of plant growth and developmental processes such as cell

division, root formation, embryogenesis, organogenesis, floral initiation, leaf senescence, fruit devel-

opment, and fruit ripening (Bagni and Tassoni, 2001; Kusano et al., 2008). The overexpression of PA

biosynthetic genes, such as arginine decarboxylase (Masgrau et al., 1997; Roy and Wu, 2001; Capell

et al., 2004), ornithine decarboxylase (Kumria and Rajam, 2002), S-adenosylmethionine decarboxyl-

ase (Torrigiani et al., 2005), and spermidine synthase (Franceschetti et al., 2004; Kasukabe et al.,

2004, 2006) in rice, tobacco, Arabidopsis, and sweet potato plants leads to increased tolerance to

environmental stress. The polyamines have stress protective properties and increase tolerance to

stresses such as salinity (Shevyakova et al., 2006), drought (Yamaguchi et al., 2007), chilling

(Nayyar, 2005), oxidative stress (Ye et al., 1997), metal toxicity (Shevyakova et al., 2011), and para-

quat (PQ) (Benavides et al., 2000). Put is produced either by decarboxylation of L-ornithine to

putrescine catalyzed by ornithine decarboxylase (ODC, EC 4.1.1.17) or by arginine decarboxylase

(ADC, EC 4.1.1.19). Spd is synthesized by spermidine synthase (SPDS, EC 2.5.16) by addition of

the aminopropyl group to ODC. The transgenic rice overexpressing ADC gene under the control of

an ABA-inducible promoter resulted in the accumulation of PAs and improved biomass against salt

stress than the wild-type plants and have shown more tolerance to high salinity (Roy and Wu,

2001). The transformed tobacco (Nicotiana tabacum) plants with human ODC gene showed

that human ODC accumulated to high levels in both the cytosol and apoplast in transgenic tobacco

leaves and putrescine levels increased by up to 8.5-fold (P, 0.05) compared to wild-type plants

(Nolke et al., 2008). Also, the transgenic rice plants containing Datura stramonium ODC produced

much higher levels of putrescine under stress, promoting spermidine and spermine synthesis

and ultimately protecting the plants from drought. The transgenic tomato plants expressing

Saccharomyces cerevisiae S-adenosyl-l-methionine decarboxylase (SAMDC) produced 1.7- to

2.4-fold higher levels of spermidine and spermine than wild-type plants under high temperature

stress. Also, enhanced antioxidant enzyme activity and the protection of membrane lipid peroxida-

tion were also observed, which in turn improved the efficiency of CO2 assimilation and protected

the plants from high temperature stress (Cheng et al., 2009).

5.3.4 LEA proteinsLate-embryogenesis-abundant (LEA) proteins are low molecular weight proteins synthesized during

maturation of embryo and seed desiccation. Their transcription is induced in response to low temper-

ature, osmotic stress and drought (Ingram and Bartels, 1996). Based on their amino acid sequence,

mRNA homology, and expression pattern, LEA proteins are classified into six different groups

(Wise, 2003).

The role of group 1, 2, and 3 LEA proteins in abiotic stress is well known, but the role of group

4 proteins LEA4-1 from Brassica napus was studied by expressing it in both E. coli and

Arabidopsis. Overexpression of BnLEA4-1 cDNA conferred salt, temperature, and drought tolerance

displaying its role in abiotic stress (Dalal et al., 2009). Also, overexpression of B. napus group 3

LEA proteins in Chinese cabbage (Brassica campestris ssp. pekinensis) and lettuce (Lactuca sativa

L.) resulted in enhanced growth under salt and drought conditions (Park et al., 2005a,b). Barley

HVA1, a group 3 LEA protein, was expressed in rice (Chandra Babu et al., 2004; Xu et al., 1994),

wheat (Sivamani et al., 2000), and mulberry (Lal et al., 2008), which resulted in maintenance of


plant growth, delayed development of damage symptoms, and relatively high leaf water content

under drought condition, as the production of HVA1 protein maintains the cell membrane stability

under stress conditions. Similarly, the expression of barley HVA1 gene in a drought-intolerant

grass, creeping bentgrass (Agrostis stolonifera var. palustris), resulted in high water content in

leaves and delayed wilting in water-deficient conditions (Fu et al., 2007). A novel LEA gene

(DQ663481) from Tamarix androssowii transformed in tobacco resulted in increased height and

reduction in wilted leaves under drought conditions (Wang et al., 2006). Two wheat genes, PMA80

(encoded for LEA group I protein) and PMA1959 (encoded for LEA group 2 protein), were shown

to increase in drought tolerance in transgenic rice (Cheng et al., 2002).

A cDNA clone of the LEA gene, OsLEA3-1, was overexpressed in drought-sensitive japonica

rice Zhonghua 11 rice under the control of three different promoters: the drought-inducible pro-

moter of OsLEA3-1 (OsLEA3-H), the CaMV 35S promoter (OsLEA3-S), and the rice Actin1 pro-

moter (OsLEA3-A). Transgenic rice with OsLEA3-H and OsLEA3-S constructs showed higher

grain yield under drought and salt stress (Xiao et al., 2007). Transgenic tobacco plants overexpres-

sing Rab16A, an LEA gene from indica rice Pokkali, displayed increased salt stress tolerance with

increased production of osmolytes, delayed onset of damage symptoms, and maintenance of min-

eral balance (RoyChoudhury et al., 2007). Two dehydration-responsive genes BhLEA1 and BhLEA2

from Boea hygrometrica when ectopically expressed in tobacco plant conferred dehydration toler-

ance with relatively high leaf water content and photosystem II activity after a prolonged drought

period (Liu et al., 2009). These experiments clearly display the efficiency of LEA proteins in con-

ferring water and salt stress tolerance in plants.

Dehydrins are an extensively studied group II LEA protein family and are known to provide

drought stress tolerance in plants by ion sequestration (Bray, 1993), maintenance and stabilization

of protein and membrane structure (Baker et al., 1988; Koag et al., 2003), chaperons (Close, 1996),

and transporting specific molecules to the nucleus (Goday et al., 1994). The transgenic tobacco

plants overexpressing citrus (Citrus unshiu Marcov.) dehydrin showed less electrolyte leakage,

early germination, and seedling growth than wild type (Hara et al., 2003). To study the stress toler-

ance effect of dehydrins, chimeric double constructs of RAB18 and COR47 or LTI29 and LTI30

were prepared under the CaMV 35S promoter and its overexpression in Arabidopsis resulted in

accumulation of dehydrin up to levels similar to cold-acclimated wild-type plants and increased

survival rate (Puhakainen et al., 2004). Wheat (Triticum durum) plants containing DHN-5 (dehy-

drin), induced in response to salt and abscisic acid (ABA) when ectopically expressed in

Arabidopsis thaliana, showed improved growth under high salt concentrations and drought condi-

tions through osmotic adjustments (Brini et al., 2007).

The transgenic maize plant overexpressing the maize Rab28 LEA gene under the constitutive

maize ubiquitin promoter resulted in increased desiccation tolerance. The transgenic seedlings showed

better growth than wild type with increased water content, leaf and root area, lowering chlorophyll

loss, and malondialdehyde production under dehydration stress conditions (Amara et al., 2013).

5.3.5 PolyolsPolyols or sugar alcohols are reduced forms of aldose or ketose monosaccharide (Brimacombe and

Webber, 1972; Williamson et al., 2002). They are present in algae, certain halophytic plants, and

1075.3 Overexpression of genes for osmoprotectants

insects exposed to freezing (Yancey et al., 1982). The most frequently found polyols in plants are

mannitol, sorbitol, galactitol, and cyclitol myo-inositol. The primary pathway for synthesis of man-

nitol is the reduction of fructose 6-phosphate to mannitol 1-phosphate and subsequent

dephosphorylation to mannitol. In Aspergillus niger, mannitol has proved to be involved in

conidial stress protection, particularly oxidative and high temperature stresses (Solomon et al.,

2007). The transgenic tobacco containing mannitol 1-phosphate dehydrogenase (mtlD) from

Escherichia coli resulted in mannitol production and a salinity-tolerant phenotype (Tarczynski

et al., 1993; Thomas et al., 1995). The cyclitols such as myo-inositol have shown better stress

protection. The myo-inositol is synthesized by myo-inositol 1-phosphate synthase and is induced

by salinity (Ishitani et al., 1996). In Mesembryanthemum crystallinum, myo-inositol is converted

to the osmoprotectants, D-ononitol, and D-pinitol (Vernon and Bohnert, 1992; Adams et al.,

1992; Nelson et al., 1998). The methylation of myo-inositol forms O-methyl inositol (D-ononitol)

under abiotic stress using myo-inositol methyltransferase (IMT). The transgenic Nicotiana

tabacum plants expressing myo-inositol O-methyltransferase (IMT1) accumulated the methyl-

ated inositol O-ononitol 35 pmol/g21 fresh weight. These transgenic tobacco plants have shown

less inhibition on photosynthetic CO2 fixation during salt stress and drought, and the plants

recovered faster than wild type (Sheveleva et al., 1997). The transgenic Arabidopsis plants over-

expressing Glycine max IMT have displayed improved tolerance to dehydration stress treatment

and high salinity stress treatment (Ahn et al., 2011). The transgenic Arabidopsis thaliana plants

expressing halophyte Mesembryanthemum crystallinum myo-inositol-O-methyltransferase (Imt1)

in response to low temperature stress have shown elevated cold tolerance. These transgenics

were confirmed by accumulation of malondialdehyde and higher levels of proline and soluble

sugar contents (Zhu et al., 2012). The transgenic Arabidopsis plant overexpressing SaINO1

gene, which encodes for myo-inositol 1-phosphate synthase (MIPS) from the grass halophyte

Spartina alterniflora, showed improved growth, development, and germination under 150 mM

NaCl. This transgenic plant protects its photosystem II by increased retention of chlorophyll

and carorenoids, indicating the effect of SaINO1 overexpression in increasing salt stress toler-

ance (Joshi et al., 2013).

5.3.6 EctoineEctoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a common compatible

osmolyte in halophilic bacteria (Regev et al., 1990; Wohlfarth et al., 1990; Farwick et al., 1995;

Bernard et al., 1993; Del Moral et al., 1994; Malin and Lapidot, 1996). It is synthesized from

L-aspartate β-semialdehyde and requires three enzymes: L-2,4-diaminobutyric acid aminotransferase

(gene: ectB), L-2,4-diaminobutyric acid acetyl transferase (gene: ectA), and L-ectoine synthase (gene:

ectC). The transgenic Nicotiana tabacum overexpressing ectB, ectA, and ectC showed increased toler-

ance to hyperosmotic shock (900 mOsm) (Nakayama et al., 2000). The uptake and translocation of

nitrate-N in roots is impaired by salinity and the impairment can be alleviated by accumulation of

ectoine in the roots of ectoine-transformed tobacco plants (Moghaieb et al., 2006). The genetically

engineered tomato plants expressing the three Halomonas elongata genes (ectA, ectB, and ectC) have

shown increased photosynthetic rates through enhancement of cell membrane stability in oxidative

conditions under salt stress (Moghaieb et al., 2011).


5.4 Engineering of ion transportPlants have developed a number of mechanisms to survive under high salt concentration, which

involve synthesis of compatible solutes, variations in membrane structure and photosynthetic activ-

ity, sequestration of ions into vacuoles, selective uptake, and removal of ions (Parida and Das,

2005). Compartmentalization of ions (Na1) into vacuoles enables plants to carry out their normal

metabolic function under saline conditions (Figure 5.2) (Adams et al., 1992; Reddy et al., 1992). By

regulating the expression of H1 pumps, the vacuolar type H1-ATPase and the vacuolar pyropho-

sphatase (V-PPase) plants maintain high concentrations of K1 in the cytosol and exclude Na1

(Zhu et al., 1993; Dietz et al., 2001). By overexpressing these pumps, salt tolerance can be induced

in plants thereby increasing their ability to grow under saline conditions.

The tobacco seedling overexpressing apple vacuolar H1-ATPase subunit A (MdVHA-A) showed

enhance drought tolerance with extended lateral root growth and osmotic adjustments (Dong et al., 2013).

The overexpression of Na1/H1 antiporter gene AtNHX1, which encodes a tonoplast antiporter

AtNHX1 in plant salt tolerance, resulted in salt tolerance to transgenic Arabidopsis plants

Na+Na+

Na+

Na+

Na+ Na+ - H+ antiporter

Na+

Na+

Na+

Na+Na+

Na+

Na+

Vacuole

V-ATPase

Cytosol

H+

H+

H+ - PPase

H+

H+

H+

Na+

H+

H+

ADP + Pi

ATP

PPi

Na+

NHX

Osmotic or saltstress

FIGURE 5.2

Compartmentalization of Na1 ions in vacuole: Under highly saline conditions, Na1 concentration in the plant is

increased to toxic level. To overcome this toxicity, cells expel this Na1 to the outside via plasma membrane

Na1/H1 antiporter, but the transported Na1 ions may create problem for neighboring cells. Therefore, the Na1

ions are sequestered into vacuoles via Na1/H1 antiporter. The H1 concentration is maintained higher inside the

vacuole by H1-ATPase and V-ATPase. (Gaxiola et al., 2002; Bayat et al., 2011).

1095.4 Engineering of ion transport

(Apse et al., 1999). Arabidopsis vacuolar Na1/H1 antiporter gene was overexpressed in tomato

plants and in Brassica napus with the assumption that these transgenic plants, with increased ability

to sequester sodium in their vacuole, would show ability to grow in high salinity. At a salt concen-

tration of 200 mM, transgenic plants showed growth with fruit and flower and seed production

(Zhang and Blumwald, 2001; Zhang et al., 2001). These results strongly suggest that modification

of a single Na1 transporter on the tonoplast can significantly improve salinity tolerance.

Overexpression of the yeast HAL1 gene in tomato showed increased fruit production and

enhanced growth following salt treatment than wild-type plants. Increased water and K1 content

and decreased intracellular Na1 in transgenic plants were found in the presence of salts (Gisbert

et al., 2000; Rus et al., 2001). Overexpression of the Schizosaccharomyces pombe SOD2 (Sodium2)

gene in Arabidopsis resulted in improved seed germination and salt tolerance. Na1 and K1 content

analysis showed that transgenic lines accumulated less Na1 and more K1 in the symplast than

wild-type plants. Also, the photosynthetic rate and fresh weight of transgenic plants were found to

be higher after salt treatment than wild-type plants (Gao et al., 2003).

Rajgopal et al., (2007) studied that vacuolar Na1/H1 antiporter (PgNHX1) from Pennisetum

glaucum, a glycophyte with a natural ability to withstand high salinity, drought, and heat stress,

confers salt tolerance when overexpressed in Brassica juncea. Up-regulation of PgNHX1 transcript

was found after NaCl and ABA treatment, and transgenic B. juncea plants showed normal flower-

ing and produced normal seeds when grown up to 70 mM NaCl. Similarly, this vacuolar Na1/H1

antiporter was also overexpressed in rice and resulted in a high level of salt tolerance. The trans-

genic rice plants showed increased root system, set flowering, and seeds in the presence of 150 mM

NaCl (Verma et al., 2007). The overexpression of H1-pyrophosphatase AVP1 in Arabidopsis

showed increased salt and drought tolerance, mainly by increased accumulation of ions into

vacuoles (Gaxiola et al., 2001). This Arabidopsis AVP1 (vacuolar H1-PPase) gene was then

expressed in tomato and cotton, and co-expressed in rice along with Suaeda salsa SsNHX1 (vacuo-

lar membrane Na1/H1 antiporter) where enhanced salt and drought tolerance was observed in trans-

genic plants with larger root systems (Park et al., 2005c; Zhao et al., 2006; Zhang et al., 2011).

Tomato, an important crop, is sensitive even to moderate salinity. To generate salt tolerant

tomato plants, AVP1, a vacuolar H1-pyrophosphatase gene from Arabidopsis thaliana, and

PgNHX1, a vacuolar Na1/H1 antiporter gene from Pennisetum glaucum, were co-expressed and

transformants were self-pollinated. In vitro and in vivo salt tolerances of progeny were studied.

Enhanced salt tolerance was observed when AVP1 and PgNHX1 were co-expressed as they showed

growth in the presence of 200 mM NaCl in comparison to a single gene transgenic plant. About 1.5

times higher Na1 sequestration in leaf tissue was found in transgenic plants, thus reducing its toxic

effect (Bhaskaran and Savithramma, 2011). Also, transgenic Arabidopsis plant overexpressing

HvNHX2, a vacuolar Na1/H1 antiporter from barley, showed normal growth in the presence of

200 mM NaCl and longer roots in early seedling stage (Bayat et al., 2011).

5.5 Overexpression of genes for stress signalingA cascade of signal perception and transduction genes is activated under abiotic stress by a variety of

signaling pathways: inositol 1,4,5-trisphosphate [Ins (1,4,5) P3] pathway, protein phosphorylation


involving mitogen-activated protein kinases (MAPKs) (Kyriakis and Avruch, 1996; Gustin et al.,

1998; Viswanathan and Zhu, 2004). High salinity leads to increased cytosolic Ca21, which initiates the

stress signal transduction pathways for stress tolerance. In the inositol 1,4,5-trisphosphate [Ins (1,4,5)

P3] pathway, signals activate phosphatidylinositol-specific phospholipase C (PI-PLC), which in turn

catalyzes the hydrolysis of phosphoinositide (4,5) bisphosphate [PtdIns (4,5)P2] to form diacylglycerol

(DAG) and Ins (1,4,5) P3 (IP3) (Meijer and Teun Munnik, 2003). IP3 is converted to IP6, which pro-

vokes Ca21 release from intracellular stores (Alexandre et al., 1990; Lemtir-Chlieh et al., 2003). DAG

is rapidly phosphorylated by the diacylglycerol kinase (DGK) to phosphatidic acid (PA) (Munnik

et al., 1998) and this molecule is believed to function as a second messenger in plants (Testerink and

Munnik, 2005). The enhanced PA levels are observed when plant cells received different kinds of

stresses such as osmotic (Meijer et al., 2001; Munnik and Meijer, 2001), water deficit (Frank et al.,

2000; Munnik et al., 2000), and cold stresses (Ruelland et al., 2002). Also, the transgenic maize plants

overexpressing phosphatidylinositol-specific phospholipase C (PI-PLC) improved the drought toler-

ance under drought stress conditions. These transgenic plants in drought stress conditions had higher

relative water content, better osmotic adjustment, increased photosynthesis rates, lower percentage of

ion leakage, less lipid membrane peroxidation, and higher grain yield than the wild type (Wang et al.,

2008). The yeast calcineurin (CaN) is a Ca21- and calmodulin-dependent protein phosphatase (PP2B),

which modulates the K1 and Na1 uptake system in order to have higher affinity for K1 and restrict

uptake of Na1. The calcineurin also maintains cytosolic Ca21 homeostasis. The functional expression

of yeast CaN in transgenic tobacco cells resulted in substantial salt tolerance of transgenic plants,

which suggests that essential components of Ca21 and calmodulin-dependent CaN salt stress signal

pathway is present in plants and functions in conjunction with yeast CaN to facilitate salt adaptation

(Pardo et al., 1998). In Arabidopsis thaliana, overexpression of cysteine-rich, receptor-like protein

kinase CRK45 leads to enhanced tolerance to drought. Also, these plants show enhanced expression of

stress-responsive genes in response to salt stress (Zhang et al., 2013).

In yeast, animals, and plants, MAPK pathways induce the production of osmolytes and antioxi-

dants (Hasegawa et al., 2000). These MAPK pathways are activated by receptors/sensors such as

protein tyrosine kinases, G-protein-coupled receptors, and two-component histidine kinases. H2O2

is reported to activate a specific Arabidopsis mitogen-activated protein kinase, ANP1, which initi-

ates a phosphorylation cascade involving two stress MAPKs, AtMPK3 and AtMPK6, which in turn

induce stress-responsive genes and enhanced tolerance to multiple stresses (Sharma et al., 1996;

Schweizer et al., 1997; Alvarez et al., 1998; Chamnongpol et al., 1998). The transgenic tobacco

plants, expressing a constitutively active tobacco ANP1 orthologue, NPK1, display enhanced toler-

ance to multiple environmental stress conditions (Kovtun et al., 1999).

5.6 Quenching of reactive oxygen speciesReactive oxygen species (ROS) include superoxide anion (O2

•2), hydrogen peroxide (H2O2),

hydroxylic free radical (OH•), singlet oxygen (1O2), methyl radical (CH3•), and lipid peroxidation

free radicals (LOO•, ROO•). O2•2 induces a series of electron transfer processes in plants, such as

the spontaneous or enzyme-assisted superoxide dismutase (SOD) dismutation to H2O2. H2O2 reacts

with Fe31 (or Cu21) to generate Fe21 (or Cu1), which will then react with H2O2 to generate

1115.6 Quenching of reactive oxygen species

hydroxyl radical (OH•) (which is highly active and affects the cell membrane). Abiotic stresses

result in generation of ROS, which are detoxified either directly by non-enzymatic antioxidants

(reduced glutathione (GSH), ascorbate (ASH), tocopherols, carotenoids, etc.) or by antioxidative

enzymes (superoxide dismutase, ascorbate peroxidase (APx), glutathione peroxidase (GPx), and

glutathione-S-transferase (GST), etc). The transgenic tobacco plants overexpressing glyoxalase

pathway enzymes resist an increase in the level of methyl glyoxal (MG) that increased to over 70%

in wild-type plants under salinity stress. These plants showed increased activity of glutathione-

related antioxidative enzymes (Yadav et al., 2005). These transgenic potato plants (Solanum tubero-

sum) overexpressing the strawberry GalUR gene under the control of the CaMV 35S promoter with

increased ascorbic acid levels. These transgenic lines retained higher chlorophyll compared to con-

trol plants and, therefore, better tolerance to abiotic stresses (Upadhyaya et al., 2009).

The manganese stabilizing protein (MSP, 33 kDa) represents a key component of the oxygen

evolving complex (OEC) of photosynthetic machinery of plants. Fischer et al. (2008) demonstrated

lack of MSP isoforms in mutant potato lines showing delayed senescence, reduced rooting, and

enhanced tuberization. The transgenic potato plants with reduced expression of MSP accumulated

increased levels of proline and low-molecular-weight metabolites such as ascorbate and tocopherol,

and have a significant increase in stress tolerance as compared to wild-type plants (Gururani et al.,

2013). The ectopic expression of plant helicase, M. sativa helicase 1 (MH1) in Arabidopsis, resulted

in improved seed germination and plant growth under drought, salt, and oxidative stresses. In these

transgenic plants, the capacity for osmotic adjustment, superoxide dismutase (SOD), ascorbate eroxi-

dase (APX) activities, and proline content were also elevated (Luo et al., 2013).

Selenium results in decreased levels of ROS subjected to various stresses such as in grain sor-

ghum (Sorghum bicolor) exposed to high temperature (O2•2 and H2O2) (Na2SeO4) (Djanaguiraman

et al., 2010), wheat (Triticum aestivum) seedlings under UV-B radiation stress (O2•2) (Na2SeO3)

(Yao et al., 2010, 2011) and cold stress (O2•2) (Na2SeO3) (Chu et al., 2010), rapeseed seedlings

under salt and drought stress (H2O2) (Na2SeO4) (Hasanuzzaman et al., 2011; Hasanuzzaman and

Fujita, 2011), and Trifolium repens under water stress (H2O2) (Na2SeO4) (Wang, 2011).

5.7 Conclusion and future perspectivesAbiotic stress factors severely affect growth, development, and productivity of crops. Many plants in

the course of their evolution develop physiological, metabolic, and biochemical mechanisms to with-

stand these stresses, which includes activation of specific stress signaling pathways followed by expres-

sion of stress signaling genes, accumulation of compatible salts, and stress proteins. With a better

understanding of underlying mechanisms responsible for stress tolerance, genetic engineering of crop

plants, overexpressing stress signaling pathway genes, and stress tolerant genes have been achieved.

Genetic approaches have been tremendously successful in obtaining transgenic plants with increased

abiotic stress tolerance, which uses the constitutive or stress-induced overexpression of stress-inducible

genes and transcription factors, engineering of pathways, which leads to accumulation of compatible

solutes, sequestering of salts, and quenching of ROS. Extensive studies of gene expression under differ-

ent abiotic stress conditions will help to choose the suitable regulatory sequence, promoter transcription

factor, and gene for generating transgenic stress-tolerant plants. Also, the co-expression of various

stress-tolerant genes can lead to genetically superior crop plants with increased abiotic stress resistance.


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