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Mol Biotechnol (2013) 54:102–123

DOI 10.1007/s12033-012-9538-3

REVIEWS

Bioengineering for Salinity Tolerance in Plants: State of the Art


Pradeep K. Agarwal • Pushp Sheel Shukla •

Kapil Gupta • Bhavanath Jha

Published online: 27 April 2012


Ó Springer Science+Business Media, LLC 2012

Abstract Genetic engineering of plants for abiotic stress SAPK4 Sucrose nonfermenting 1-related protein
tolerance is a challenging task because of its multifarious kinase2 (SnRK2)
nature. Comprehensive studies for developing abiotic stress AtMEK1 MAPK kinase
tolerance are in progress, involving genes from different MYB Myeloblastoma
pathways including osmolyte synthesis, ion homeostasis, NAC No apical meristem, ATAF 1,2 and cup-shaped
antioxidative pathways, and regulatory genes. In the last cotyledon
decade, several attempts have been made to substantiate DRE Drought responsive element
the role of ‘‘single-function’’ gene(s) as well as transcrip- DBF DRE binding factor
tion factor(s) for abiotic stress tolerance. Since, the abiotic DREB Drought responsive element binding protein
stress tolerance is multigenic in nature, therefore, the recent TPS Trehalose-6-phosphate synthase
trend is shifting towards genetic transformation of multiple p5cs D1-Pyroline-5-carboxylate synthase
genes or transcription factors. A large number of crop codA Choline oxidase
plants are being engineered by abiotic stress tolerant genes BADH Betaine aldehyde dehydrogenase
and have shown the stress tolerance mostly at laboratory mt1D Mannitol-1-phosphate dehydrogenase
level. This review presents a mechanistic view of different P5CR P5C reductase
pathways and emphasizes the function of different genes in GutD Glucitol-6-phosphate dehydrogenase
conferring salt tolerance by genetic engineering approach. MIPS L-Myo-Inositol-1-phosphate synthase
It also highlights the details of successes achieved in APX Cytosolic ascorbate peroxidase
developing salt tolerance in plants thus far. DHAR Dehydroascorbate reductase
MDHAR Mono DHAR
Keywords Gene function  Genetic engineering  Salt SOD Superoxide dismutase
tolerance ADC Arginine decarboxylase
ODC Ornithine decarboxylase
Abbreviations SAMDC S-Adenosyl methionine decaroboxylase
CBL Calcineurin B-like protein SPDS Spermidine Synthase
CIPK CBL-interacting protein kinases NHX-1 Vacuolar Na?/H? antiporter
NPK1 Mitogen-activated protein kinase kinase kinase SOS1 Salt overly sensitive
NDPK2 Nucleoside diphosphate kinase 2 TsVP H?-pyrophosphatase
HKT2 High efficiency potassium transport

P. K. Agarwal (&)  P. S. Shukla  K. Gupta  B. Jha


Discipline of Marine Biotechnology and Ecology, Central Salt Introduction
and Marine Chemicals Research Institute (Council of Scientific
and Industrial Research), G.B. Road, Bhavnagar 364021,
Gujarat, India Plants being sessile are highly affected by harsh climatic
e-mail: pagarwal@csmcri.org changes. Although plants gradually evolved a remarkable

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Mol Biotechnol (2013) 54:102–123 103

ability to adapt themselves to such a highly variable Calcineurin B-like proteins (CBL) sense the calcium signal
environmental onslaughts, environmental stresses never- and participate in salt stress signal transduction pathway
theless cause over 50% crop loss [1]. Approximately, 7% and control the influx and efflux of Na?. The signaling
of total land area (1,000 million ha) and 20% of the irri- approach to improve salt tolerance was demonstrated in
gated agriculture land is affected by soil salinity [2] and transgenic tobacco by modulating the expression of a cal-
imposes major constraints to the sustainability of crop cium stress-signaling component from yeast, Ca2?/cal-
yield. modulin dependent protein phosphatase [22]. The
Salinity imparts both ionic and osmotic stresses thus transgenic tobacco plants co-expressing the two catalytic
limiting plant growth and productivity. Plants respond and and regulatory subunits of this protein exhibited substantial
adapt to these conditions by regulating a wide array of NaCl tolerance [22]. The calcineurin B-like (regulatory)
genes. Salinity causes adverse impact on plant growth by Ca2? sensor, SOS3 (Salt overly sensitive 3) has been cloned
disturbing the ionic equilibrium and eliciting sodium tox- from Arabidopsis [23]. This protein triggers the activity of
icity. High Na? concentration is toxic to cell metabolism SOS pathway for plant Na? tolerance, the SOS gene and
and has deleterious effects on the functioning of some of its applications are discussed below along with the
the enzymes [3]. Different plants employ different mech- transporter genes. A CBL protein from maize, ZmCBL4,
anisms to minimize the damage from Na?, e.g., minimize showed salt stress tolerance in Arabidopsis. Expression of
initial influx, maximize efflux, minimize loading into the 35S:ZmCBL4 complemented the salt hypersensitivity in
xylem, and maximize recirculation out of the shoot to Arabidopsis sos3 mutant and enhanced the salt tolerance
phloem, intercellular compartmentalization and even in wild-type Arabidopsis at the germination and seedling
secretion of salt from the leaf surface [4, 5]. stages [24]. Recently, Tripathi et al. [25] showed that
Various genes induced by salt stress could be grouped overexpression of a constitutively active mutant of Ca-
under two categories, namely, ‘‘single-function’’ genes and CIPK6 from chickpea promoted salt tolerance in trans-
regulatory genes. The first category of genes generally genic tobacco. AtNDPK2 activated both AtMPK3 and
facilitates production of protective metabolites, which AtMPK6, and its overexpression showed enhanced salt
include osmolytes, transporters/channel proteins, antioxi- and other abiotic stress tolerance by reducing the ROS
dative enzymes, lipid biosynthesis genes, polyamines, etc. concentration in the plants [26]. SAPK4 is a serine thre-
The second class of genes consists of regulatory proteins onine type of kinase and is known to regulate the stress
like bZIP, DREB, MYC/MYB, and NAC, which control responsive gene expression. The overexpression of SAPK4
the expression of many downstream salt stress tolerant resulted in improved germination, growth and develop-
genes [6, 7]. These genes converge and interact in different ment under salt stress. In response to salt stress, the
pathways related to the abiotic stress and successfully lead SAPK4-overexpressing rice accumulated less Na? and Cl-
to tolerance (Fig. 1). and showed improved photosynthesis [27].
Different genes related to abiotic stress tolerance, have Plants use another common mechanism to translate
been introduced into a variety of plants [1, 8–10]. Many external stimuli into cellular responses through the acti-
reviews have been published recently on oxidative pathway vation of mitogen-activated protein (MAP) kinase cascade
[11], transporters [12–15] and role of regulators in con- [28]. MAPKs are signaling modules that phosphorylate
trolling salt stress tolerance [6, 7, 16–18]. The present specific serine/threonine residues on the target protein
article focuses on new paradigm developed in the recent substrate and regulate a variety of cellular activities. The
past years in developing salt tolerant plants by employing MAPK cascade consists of three functionally interlinked
genes from all the possible pathways. protein kinases: MAPKKK, MAPKK, and MAPK. Based
on sequence alignment, the MAPKKs are placed in four
groups (A–D). The roles of groups A, B, and C have been
Signaling Molecules studied in relation to biotic and abiotic stresses [29].
Overexpression of MAP kinases showed enhanced toler-
Environmental signals are first perceived by signaling ance to salt stress (Table 1). Transgenic rice plants with
molecules and involves protein phosphorylation, dephos- OsMAPK5 and OsMAPK44 genes showed tolerance to salt,
phorylation, phospholipid metabolism, Ca2? sensing, etc. drought and cold stresses [30, 31]. The overexpression of
Their overexpression in many plants showed better stress ZMKK4 from Zea mays in Arabidopsis plants showed
tolerance (Table 1). Stress and other extracellular stimuli enhanced salt tolerance and also higher proline and soluble
change the intracellular Ca2? concentration [19, 20]. Ca2? sugar contents, and higher POD and CAT activities com-
signaling process is considered as one of the earliest events pare to control plants. The transgenic plant showed
in salt signaling, and play an essential role in the ion upregulation of the transcription factor, TF, which even-
homeostasis leading to salt tolerance in plants [15, 21]. tually control the expression of COR47, Rd29a, and P5CS

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Signal sensing

AtHK1, Ca++, SOS3, CDPKs,


SnRK1

Transcription factor

AREB/ABF,bZIP,
CBF/DREB, MYC/MYB,
NAC, WRKY

Detoxification Chaperone

Gene activation & Photosynthesis and


Osmoprotection Stress response related metabolism

Aquaporin and transporters HSps, LEAs, proteinases

Stress tolerance
and resistance

Fig. 1 A generic pathway under salt and drought stress. The signal is perceived by the sensor proteins, which in turn activates the transcription
factors (TFs). The TFs trigger the process of activating the genes of different class resulting to achieve stress tolerance

[32]. Several researchers investigated that on one hand cross-tolerance, it interrelates the signaling responses to
MAP kinase control the stress in plants by overexpressing wounding and salt stress. It showed that mechanical
of antioxidative genes and on other hand it activated the wounding increases salt-stress tolerance by involving the
TFs which further control the downstream genes. Kong signaling peptide systemin and the synthesis of JA (Jas-
et al. [32] has studied that the transgenic plants showed monic Acid) [34]. It is evident from the literature that
upregulation of CBF1, CBF2, CBF3, STZ, DREB2A TFs signaling genes play important role in abiotic stress toler-
and which in turn showed the enhanced expression of the ance by regulating the expression of salt tolerant genes and
COR47, RD29A, P5CS2 genes. The signaling molecules TFs. In spite of the important role of signaling molecule,
are reported to interact for developing cross tolerance, in only few studies had been carried out (Table 1) for engi-
this case when one type of stress renders to plants than it neering salt tolerance in the plants, therefore it is desirable
leads to resistant to another type of stress [33]. LeCDPK1 to have more transgenic lines with this kind of genes in
gene from tomato also is a good example of imparting future.

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Table 1 Overexpression of signaling molecule to develop salt and desiccation tolerance


Gene/source Transgenic plant Performance of transgenic plant References

CBL4/maize Arabidopsis Salt tolerance [24]


CBL5/Arabidopsis Arabidopsis Salt and dehydration tolerance [165]
CIPK6/chickpea Tobacco Salt tolerance [25]
MAPK5/rice Rice Enhanced tolerance to drought, salt and cold stresses [30]
MAPK44/rice Rice Salt tolerance [31]
MEK1/Arabidopsis Arabidopsis Salt and dehydration tolerance [166]
MAPK kinase kinase (MAPKKK/DSM1) Rice Dehydration tolerance [167]
ZmMKK4/maize Arabidopsis Salt tolerance [32]
CIPK03, OsCIPK12, and CIPK15 Rice Salt, dehydration and cold tolerance [168]
AtCPK6/Arabidopsis Arabidopsis Salt and drought tolerance [169, 170]
NPK1 Maize Salt, dehydration, cold and heat tolerance [171]
NDPK2/Arabidopsis Arabidopsis Salt, cold and oxidative stress tolerance [26]
SAPK4/rice Rice Salt stress tolerance [27]
ZmSAPK8/maize Arabidopsis Salt tolerance [172]
GhMPK2/Gossypium hirsutum Tobacco Salt and drought tolerance [173]
OsMSR2/Oryza sativa Arabidopsis Salt and drought tolerance [174]
TaSnRK2.8/Triticum aestivum Arabidopsis Salt and drought tolerance [175]
CBL Calcineurin B-like protein, CIPK CBL-interacting protein kinases, NPK1 Mitogen-activated protein kinase kinase kinase, NDPK2
Nucleoside diphosphate kinase 2, SAPK4 Sucrose nonfermenting 1-related protein kinase2 (SnRK2), AtMEK1 MAPK kinase

Regulatory Genes different regulatory systems during abiotic stress in ABA-


dependent and independent manner has been discussed by
TFs interact with different cis-elements in the promoter Agarwal et al. [16] and Agarwal and Jha [6]. In this review,
regions of various downstream genes and modulate their we have incorporated only the recent developments in the
expression. TFs play a pivotal role in developing stress TFs and their role in only salt and drought tolerance in
tolerance in plants against various environmental stresses plants.
(Fig. 2). TFs corresponds to a large number in plants,
Arabidopsis genome contains 5.9% TFs [35]. TFs are better DREBs
expanded in plants due to significant complexity of plant
metabolism, as compared to other kingdoms [36]. TFs can The ABA-independent stress-responsive genes are regulated
be classified into [50 different families, based on the by dehydration-responsive element binding (DREB) pro-
presence of their DNA-binding domains [35]. In Arabid- teins, which bind to dehydration-responsive element (DRE)
opsis, cis-elements and corresponding binding proteins, cis-elements. DREBs are important plant-specific TFs,
with distinct DNA-binding domains, such as AP2/ERF which induce a set of abiotic stress related genes and impart
(Apetala 2/Ethylene responsive factor), basic leucine zip- stress tolerance. DREB genes have been isolated and char-
per, HD-ZIP (homeodomain leucine zipper), MYC, MYB acterized from a wide variety of plants, and their differential
(myelocytomatosis, myeloblastosis) and different classes regulation and functional analysis is reviewed by Agarwal
of zinc finger domains, have been identified [37]. The et al. [16] and Lata and Prasad [39]. A number of down-
control of specific sets of genes can be accomplished by the stream genes get activated by overexpression of DREB TFs
combinatorial interaction among TFs, between TFs and leading to enhanced abiotic stress tolerance. Microarray
non-DNA-binding proteins, and between TFs and cis-reg- analysis of DREB transgenics showed higher expression of
ulatory elements [38]. Therefore, it is inevitable to study large number of downstream genes belonging to late
the convergence and interaction horizontally or vertically embryogenic abundant (LEA) protein, heat shock, detoxifi-
of TFs for better control of abiotic stresses. cation, seed proteins and enzymes involved in metabolism,
ABA signaling is an important component of abiotic etc. [6]. Recently, it has been reported that DREB2A from
stress transduction pathways. Besides ABA, salicylic acid- Salicornia brachiata showed enhanced salt tolerance in E.
mediated pathway also regulates TFs in abiotic stress. The coli, indicating that SbDREB2A is interacting with tran-
role of ABA in stress signaling and its involvement in scriptional network in the bacterial cells [40].

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Drought, Salt

SIGNAL PERCEPTION

ABA dependent ABA independent

CBF4

MYC/ MYB AREB/


DREB2
ABF
NAC NAC

MYCRS, MYBRS ABRE DRE/CRT


(rd22) (rd29B) NACRS (rd29A)
NACRS
(erd1)

GENE EXPRESSION

PHYSIOLOGICAL AND BIOLOGICAL CHANGES

STRESS TOLERANCE

Fig. 2 A diagrammatic representation of ABA dependent as well as independent regulatory cellular signal transduction pathways between stress
signal perception and gene expression involved in stress responsive gene expression

NAC resulted significantly increased stomata closure under


drought stress. Similarly, Hu et al. [50] reported that
Another plant-specific TF, NAC (NAM-ATAF1,2-CUC2), SNAC2 (Stress-Responsive NAC 2) (identical to OsNAC6)
regulates both the ABA-dependent and independent genes. showed improved tolerance to various stresses in the
These TFs are expressed in different tissues at various transgenic rice. The transgenic lines showed higher seed
developmental stages and are involved in plant growth and germination on 150 mM NaCl compared to control plants
development [41]. The N-terminal region of the NAC whereas there was no difference in the germination rate on
protein contains a highly conserved domain found in NAM MS basal medium. The SNAC2 profiling analysis of
(no apical meristem), ATAF1, 2 and CUC2 (cup-shaped transgenic plants revealed many upregulated genes related
cotyledon) proteins. The domain might form a helix-turn- to stress response and adaptation such as peroxidase,
helix structure that specifically binds to target DNA [42]. ornithine aminotransferase, heavy metal-associated protein,
The C-terminal region of NAC proteins possesses highly sodium/hydrogen exchanger, heat shock protein, GDSL-
divergent sequence. The first NAC gene isolated was NAM like lipase, and phenylalanine ammonia lyase. The up- or
from petunia [43], which played a critical role in deter- downregulated genes compared in the SNAC1 and SNAC2
mining shoot apical meristem and primordia positions [44]. overexpression plants showed sharp difference. This may
Recently, NAC genes are also found to be involved in be because of the difference in the conserved flanking sites
abiotic and biotic stresses [45–47]. Arabidopsis NAC of the target genes between SNAC1 and SNAC2.
genes, namely ANAC019, ANAC055, and RD26/ANAC072, Another rice NAC gene, OsNAC6, was induced both by
showed the upregulation of several stress-related genes and biotic and abiotic stress. The transgenic lines showed
conferred enhanced drought tolerance [48]. Later, Hu et al. upregulation of many biotic and abiotic stress responsive
[49] reported that rice SNAC1 (Stress-ResponsiveNAC 1) genes. The transgenic plants showed higher tolerance to
showed significantly better drought and salinity stress tol- dehydration and salt stress. The OsNAC6 transgenic lines
erance in transgenic rice. The over expression of this gene with stress inducible promoters OSNAC6 and LIP9 (low-

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temperature-induced protein) minimized the negative of protein phosphatases 2C (PP2Cs) genes in microarray
effect on growth compared to control plants and showed analysis and northern blots analysis, whereas the mutant
higher tolerance in the presence of salt [47]. ONAC063 lines showed the higher expression of PP2Cs and reduced
from rice showed enhanced tolerance to salt and osmotic tolerance to salt and drought. This study showed that At-
stress in Arabidopsis plants. Microarray analysis showed MYB44 performs the abiotic stress function by suppressing
upregulation of 29 genes among them oleosin and amylase the negative regulator group of genes. Liao et al. [60] has
expressed more than 100-fold [51]. Two NAC genes studied expression of large number of soybean Myb genes
GmNAC11 and GmNAC20 have been isolated from the under ABA, salt, drought, and/or cold stress. The overex-
soybean. These both genes differentially induced under pression of three Myb genes, viz., GmMYB76, GmMYB92,
stress condition and plant hormones. The overexpression of or GmMYB177 in Arabidopsis showed higher seed germi-
the GmNAC20 showed the tolerance to salinity and freez- nation rate under salt media [60]. Recently, Gao et al. [61]
ing, whereas, GmNAM11 showed tolerance to only salinity. reported that overexpression of apple Myb10 resulted in
Both genes likely regulated the stress tolerance by DREB/ osmotic stress tolerance. The transgenic apple plants
CBF-COR pathway [52]. Liu et al. [53] reported that showed higher flavonoid content, thus manages higher
overexpression of Chrysanthemum DgNAC1 in tobacco antioxidative ability to cope-up osmotic stress. Three dif-
resulted salt tolerance. OsNAC5 [54] when overexpressed ferent Myb TFs were isolated from wheat (Triticum aes-
showed better salt tolerance than RNAi knock-out lines or tivum L)., TaMYB2A, TaMYB2B, and TaMYB2D [62].
wt plants. Different physiological parameters of OsNAC5 TaMYB2A Arabidopsis transgenics showed enhanced tol-
overexpression lines showed that proline and free sugar erance to drought, salt, and freezing stresses and revealed
level get enhanced in transgenic lines. Further, it is also decreased rate of water loss, enhanced cell membrane
observed that MDA and H2O2 content have got reduced in stability, improved photosynthetic potential, and reduced
overexpressed lines compared to knockdown lines [54]. osmotic potential. Another MYB gene TaPIMP1, was also
Similarly, Takahashi et al. [55] also observed higher salt isolated from wheat: TaPIMP1 showed significantly higher
tolerance by overexpression of OsNAC5. The role of NAC transcript level by a fungal pathogen Bipolaris sorokiniana
gene in ABA signaling is reported by Liu et al. [56], the and by drought treatment. TaPIMP1 transgenic tobacco
Arabidopsis transgenic lines with AhNAC2 (Arachis hyp- lines showed tolerance to salinity along with pathogen and
ogaea) showed hyper sensitivity to ABA in root growth, drought. In these transgenic lines, the activities of phen-
seed germination, and stomatal closure compared to wild ylalanine ammonia-lyase (PAL) and superoxide dismutase
type Arabidopsis. The AhNAC2 overexpressed lines (SOD) were significantly increased compared to wild-type
showed higher expression of downstream genes viz. tobacco plants [63].The Solanum lycopersicum abscisic
Rd29A, Rd29b, Rab18, AtMYB2, AtMYC2, ERD1, COR 47, acid-induced myb1 (SlAIM1) TF is reported to induced by
COR15a, KIN1, AREB1, CBF1, AMY1. The upregulation pathogens, plant hormones, salinity and oxidative stress,
of Rd29A, Rd29b can correlate the ABA signaling of Ah- suggesting a function in pathogen and abiotic stress
NAC2 since both the gene possess ABRE element in their responses. The RNAi plant with silenced SlAIM1 showed
promoter region [56]. high susceptibility to fungus Botrytis cinerea, and high
sensitivity to salt and oxidative stress. These responses
Myb correlate with reduced sensitivity to abscisic acid (ABA) in
the SlAIM1 RNAi, but increased sensitivity in the over-
In higher plants, myb (myeloblastosis)-TF family repre- expression plants, suggesting SlAIM1-mediated ABA
sents a large number of genes. In Arabidopsis it is con- responses are required to integrate tomato responses to
sidered as the largest TF family and contains more than 163 biotic and abiotic stresses. The SIAIM1 also shows the
genes [57]. Plant R2R3-MYB TFs play wide functional regulation of ion fluxes, the RNAi plants accumulated more
roles, and involved in imparting stress tolerance against Na? compared to overexpressed lines [64].
various environmental cues in transgenics (Table 2). The
OsMYB3R-2I from rice employed enhanced tolerance to
salt, freezing and dehydration stresses and decreased sen- Osmolytes
sitivity to ABA in transgenic Arabidopsis [58]. In contrast,
Jung et al. [59] reported that the AtMYB44 transgenic were Osmolytes are organic metabolites of low molecular
highly sensitive to ABA and a rapid ABA induced stomatal weight known as compatible solutes and do not deter the
closure was seen during the stress treatment. Transgenic cellular functions. The osmolytes such as glycine betaine,
plants had low rate of water loss and enhanced tolerance to fructans, trehalose, mannitol, sorbitol, ononitol, and pinn-
drought and salt stress compared to wild-type plants. The itol play prominent role as osmoprotectants. Genes for
AtMYB44 overexpressing lines revealed the low expression many osmolytes have been cloned and introduced into

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Table 2 Overexpression of transcription factors for salt and desiccation tolerance


Gene/source Transgenic plant Performance of transgenic plant References

HsDREB1A/Hordeum spontaneum Argentine bahiagrass Salt and dehydration tolerance [176]


PpDBF1/Physcomitrella patens Tobacco Salt, dehydration, and freezing tolerance [177]
DREB1A/Arabidopsis Arachis hypogaea Dehydration tolerance [178]
AIDFa/Triticum aestivum Arabidopsis Salt and dehydration tolerance [179]
DREB1A/Arabidopsis Festuca arundinacea Dehydration tolerance [180]
DgDREB1A/Dendranthema grandiflorum Arabidopsis Dehydration and freezing tolerance [181]
DREB1/Hordeum vulgare Arabidopsis Salt tolerance [182]
DREB2/populus Tobacco Salt tolerance [183]
DREB2A/Pennisetum glaucum Tobacco Salt and dehydration tolerance [184]
DREB2A/Salicornia brachiata E.coli (BL21DE3) Salt and dehydration tolerance [40]
DREB2B/rice Arabidopsis Dehydration and heat tolerance [185]
DREB2A/maize Arabidopsis Dehydration [186]
DREB2/Glycine max Arabidopsis Salt and dehydration tolerance [187]
DREB/cotton Wheat High salt drought and cold tolerance [188]
DREB1D/rice Arabidopsis Salt tolerance [189]
DREB2ACA/Arabidopsis Arabidopsis Drought tolerance [190]
OsDREB1F/rice Arabidopsis/Rice Salt and dehydration tolerance [191]
2 9 35S TaDREB2/wheat Barley and wheat Drought tolerance [192]
2 9 35S TaDREB3/wheat
pRAB17 TaDREB2/wheat
pRAB17 TaDREB3/wheat
LcDREB3a/Leymus chinensis Arabidopsis thaliana Salt and drought tolerance [193]
MtCBF4/Medicago truncatula Medicago truncatula Salt tolerance (enhanced root length in 10 mM NaCl) [194]
VrCBF1/grape Arabidopsis Drought tolerance [195]
VrCBF4/grape
ERF3/Glycine max Tobacco Salt Dehydration tolerance [196]
CpMYB10/Craterostigma plantagineum Arabidopsis Salt and desiccation tolerance [197]
OsMYB3R-2/rice Arabidopsis Salt, drought and freezing tolerance [58]
GmMYB76 or GmMYB177/Glycine max Arabidopsis Salt and freezing tolerance [60]
AtMYB44/Arabidopsis Arabidopsis Drought and salt tolerance [59]
Myb10/apple Arabidopsis Osmotic stress [61]
TaPIMP1(MYB)/wheat Tobacco Salt and drought tolerance [64]
AtMYB52/Arabidopsis Arabidopsis Drought tolerance [198]
TaMYB2A/wheat Arabidopsis Salt and drought tolerance [62]
SNAC2/Rice IRA109 Rice Zhonghua 11 Salt and cold stress tolerance [50]
NTL8(NAC)/Arabidopsis Arabidopsis GA mediated Salt signaling [199]
ONAC063/rice Arabidopsis Salt and Osmotic stress tolerance [51]
SNAC1/rice Rice Salt tolerance [49]
AhNAC2/groundnut Arabidopsis Salt and drought tolerance [56]
DgNAC1/Chrysanthemum Tobacco Salt tolerance [53]
GmNAC11/Glycine max Arabidopsis Salt tolerance [52]
GmNAC20/Glycine max
HvDhn4s:TaNAC69/wheat Wheat In combined salt and drought by producing more biomass [200]
Stress inducible Drought tolerance
HvDhn8s:TaNAC69/wheat
Constitutive
OsNAC5/rice Arabidopsis Salt and drought tolerance [54]
Rice

MYB myeloblastoma; NAC no apical meristem, ATAF1,2 and cup-shaped cotyledon; DBF DRE (drought responsive element) binding factor; DREB
drought responsive element binding protein

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Table 3 Genetic transformation of osmolytes for salt and desiccation tolerance in plants
Gene/source Transgenic plant Performance of transgenic plant References

ScTPS1/yeast Tobacco Dehydration tolerance [67]


ScTPS1/yeast Arabidopsis Salt tolerance [68]
TPS1-TPS2/yeast Alfalfa Salt tolerance [69]
TPS1/yeast Tomato Salt tolerance [70]
OsTPS1/Oryza sativa Rice High salinity and drought tolerance [72]
OtsA and OtsB/E. coli Rice Salt, drought and cold tolerance [71]
mt1D/E. coli Arabidopsis High salt tolerance [73]
mt1D/E. coli Tobacco Salt and dehydration tolerance [74]
mt1D/E. coli Wheat Salt and dehydration tolerance [75]
MIPS/Potresia coarctata Tobacco Growth improvement at 300 mM NaCl [201]
mt1D and gutD/A. tumefaciens Loblolly pine/Pinus taeda Salt tolerance [202]
p5cs/Vigna aconitifolia Tobacco Salt tolerance [78]
p5cs/Vigna aconitifolia Rice Salt tolerance [80]
Antisense ProDH/Arabidopsis Arabidopsis Salt and freezing tolerance [82]
TaP5CR/Triticum aestivum Arabidopsis Salt tolerance [83]
p5cs/Vigna aconitifolia Rice Increased fresh weight at 200 mM NaCl [81]
p5cs/Vigna aconitifolia Wheat Salt tolerance [203]
p5cs/Vigna aconitifolia Tobacco Salt tolerance [79]
p5cs/Arabidopsis Solanum tuberosum Salt tolerance [204]
CodA/A. globiformis Arabidopsis, Rice, Brassica Salt tolerance [205– 208]
betA/E.coli B. oleracea Salt tolerance [89]
AhBADH/ Wheat Salt tolerance [209]
E. Coli betA and betB Tobacco Salt tolerance [85]
ADC/Oat Arabidopsis Drought tolerance [210]
TPS trehalose-6-phosphate synthase, p5cs D1-Pyroline-5-carboxylate synthase, codA Choline oxidase, BADH Betaine aldehyde dehydrogenase,
mt1D Mannitol-1-phosphate dehydrogenase, P5CR P5C reductase, GutD glucitol-6-phosphate dehydrogenase, MIPS L-myo-Inositol-1-phosphate
synthase

plants. Generally, this resulted in higher accumulation of higher expression of RAB16C, HSP70, and ELIP and Os-
osmoprotectants and enhanced salt and drought tolerance TPP1 and OsTPP2 [72].
(Table 3). The E. coli mannitol-1-phosphate (mt1D) gene was
Trehalose is a non-reducing disaccharide and its syn- introduced into Arabidopsis [73], tobacco [74], and wheat
thesis help in achieving dehydration tolerance to living [75] plants. The transgenic Arabidopsis showed enhanced
organism. It protects biomolecules by water replacement, seed germination on high salt (400 mM NaCl) medium
glass formation and chemical stability [65]. The genes for [73]. The wheat transgenic plants with mt1D showed less
trehalose synthesis have been cloned from bacteria, yeast, reduction in biomass compared to wild-type plants under
and eukaryotic plants, and first time overexpressed in the presence of salt and dehydration [75].
tobacco enhancing drought tolerance by incorporation of E. During stress treatment, the low molecular weight
coli otsA gene or ScTPS1, trehalose-6-phosphate synthase metabolite proline gets accumulated in the cells. The bio-
from yeast [66, 67]. Later, ScTPS1 gene was introduced synthesis of proline resulted in improved tolerance to salt
into Arabidopsis [68], alfalfa [69], rice, and tomato [70] and drought stress in a number of crops [76, 77]. The proline
and the transgene imparted enhanced salt tolerance to the precursor, p5cs (D1-Pyroline-5-carboxylate synthase), has
plants. Garg et al. [71] introduced OtsA and OtsB (E. coli been introduced into tobacco [78, 79], rice [80, 81], and
trehalose-6-phosphate synthase) genes in rice and observed Arabidopsis [82, 83] and these transgenic plants showed
increased tolerance to salt, drought, and cold in the trans- better tolerance to salinity stress.
genic plants. The trehalose-6-phosphate synthase gene Glycine betaine (betaine) is a non toxic cellular osmolyte
(OsTPS1) overexpressing rice lines showed improved tol- that raises intracellular osmolarity in response to different
erance to cold, high salinity and drought treatments without stresses in lower organisms as well higher plants and sta-
any morphological changes. These lines also exhibited bilize the biological macromolecules [84, 85]. Glycine

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110 Mol Biotechnol (2013) 54:102–123

betaine is synthesized by two-step process in both pro- hydroxyl ions, and hydrogen peroxide [92–96] and consid-
karyotes and eukaryotes from choline with an intermediate ered as marker for stress activation in the plants [97]. ROS
compound betaine aldehyde. In eukaryotes, choline gets serves as signaling molecules that regulates stress response to
converted in betaine aldehyde with the help of choline maintain the growth and development of plants. In this
monooxygenase [86] in first step, which subsequently pro- review the direct impact of oxidative stress compounds on
duce betaine in second step by betaine aldehyde dehydro- salt stress tolerance by genetic engineering approach is dis-
genase (BADH) [87], whereas in bacteria the choline to cussed. During salt stress available CO2 get reduced in the
betaine aldehyde and finally to betaine is achieved by leaf because of stomata closing, which further causes the
choline dehydrogenase (CDH). However, several crop over-reduction of photosynthetic electron transport chain,
plants are incapable of synthesizing betaine [85]. The E. thereby leading to the generation of ROS [97]. Several
coli betA and betB genes [88] encoding for CDH and BADH enzymes are involved in the detoxification of antioxidative
enzymes, respectively, were introduced in tobacco resulting substances (AOS). Superoxide dismutase (SOD) converts
increased biomass and faster recovery from photoinhibition superoxide to H2O2, which is further scavenged by catalase.
under salt stress [85]. There are examples where only betA Ascorbate peroxidase (APX) also reduces H2O2 and is
gene has been overexpressed in B. oleracea [89] and maize present in different isoforms in different plant organelles
[90], which showed enhanced salt and drought tolerance, [98]. Transgenic plants developed by overexpression of
respectively. In comparison to the CDH and CMO pathways several enzymes such as glutathione peroxidase (GPX),
a direct choline oxidase pathway (COD) do exist which has SOD, APX, and glutathione reductases (GR) showed
single step conversion of choline to glycinebetaine and also improved stress tolerance (Table 4). Appearance of APX
does not require any cofactors for the catalysis [91]. A codA isoenzymes in response to environmental stresses, such as
(Choline oxidase) gene from Arthrobacter globiformis had salinity and drought, has been reported in several plant spe-
been introduced in many plants, resulting in enhanced cies and their overexpression led to salt and drought tolerance
growth under salt stress which showed better germination in tobacco and Arabidopsis [99–101]. A number of SOD
and growth in transgenic at 150 mM of NaCl (Table 3). isoforms are present in different plants as Cu/Zn-SOD iso-
forms is found in the chloroplast and in the cytosol, whereas a
Mn-containing enzyme is located in the mitochondria
Antioxidative Enzymes showed high photosynthetic rate under increased salt stress in
tobacco transgenic plants [102]. A cytosolic Cu/ZnSOD gene
Salinity stress generates reactive oxygen species (ROS) from the mangrove species, Avicennia marina enhanced salt
including singlet oxygen, superoxide anion radicals, tolerance in the indica rice variety [103]. Similarly,

Table 4 Genetic transformation with antioxidative enzyme genes for salt and desiccation tolerance
Gene/source Transgenic plant Response of transgenic plant References

Chl-APX5/Arabidopsis Tobacco Salt and water stress tolerance [99]


APX/pea Tomato Salt tolerance [211]
APXa and APXb/rice Arabidopsis Salt tolerance [100]
APX/tomato Tobacco Salt and osmotic tolerance [98]
GST and GPX/tobacco Tobacco Salt tolerance [107]
GST/Suaeda salsa Arabidopsis Salt tolerance [212]
MnSOD/Arabidosis Arabidopsis Salt and Cold tolerance [104]
MnSOD/pea Rice Drought tolerance [213]
Cu/ZnSOD/Pea, APX and DHAR/human Tobacco Salt and oxidative tolerance [105]
Cu/Zn-SOD/A. marina Rice Salt, drought, and oxidative tolerance [103]
SOD/tomato Pepper Drought tolerance [214]
Cu/Zn-SOD/pea and APX Tobacco Salt and osmotic stress [106]
DHAR1/rice Arabidopsis Salt tolerance [215]
MDAR/Arabidopsis Tobacco Salt, ozone, and PEG [216]
AmMDAR/A. marina Tobacco Salt tolerance [217]
katE/E. coli Tobacco Resistance of chloroplast translation machinery to salt stress [218]
katE/E. coli Rice Salt tolerance [219]
APX cytosolic ascorbate peroxidase, DHAR dehydroascorbate reductase, MDHAR monoDHAR, SOD superoxide dismutase

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Mol Biotechnol (2013) 54:102–123 111

overexpression of MnSOD showed enhanced salt tolerance in division, cell differentiation, growth, development, and cell
Arabidopsis [104]. Many studies have been performed to death [109]. They are also involved in various abiotic and
develop abiotic stress tolerant plants by engineering one gene biotic plant stress responses. The role of polyamines is
but recently interest has changed to incorporate more than amply described in recent reviews [110, 111]. The heter-
one gene for empowering better tolerance. However, on this ologous overexpression of ornithine decarboxylase (ODC),
aspect only a few reports have appeared [105], transforma- arginine decarboxylase (ADC), S-adenosyl methionine
tion of tobacco plants by pyramiding more than one gene for decarboxylase (SAMDC), and spermidine synthase (SPDS)
anti-oxidative enzyme like Cu/ZnSOD (C), APX (A), and in rice, tobacco, and tomato has shown tolerance against
DHAR (D) into chloroplasts and demonstrated that such stress conditions (Table 5). In carrot cell lines the over-
plants were more salt and oxidative stress tolerant compared expression of mouse ODC, which converts ornithine to
to those expressing single or double gene transfer. The putrescine, exhibited high level of salt stress tolerance
overexpression of ‘CAD’ showed approximately 1.6–2.1 [112]. Kumria and Rajam [113] also showed that mouse
times higher dehydroascorbate reductase (DHAR) activity, ODC when introduced into tobacco exhibited enhanced
higher ratios of reduced ascorbate to dehydroascorbate production of constitutively expressed putrescine, confer-
(DHA), and oxidized glutathione (GSSG) to reduced gluta- ring salt tolerance to the plants. A cDNA for ADC from oat,
thione (GSH) compared to ‘CA’ plants. The transgenic expressed in rice under an ABA-inducible promoter,
seedlings with ‘CAD’ showed enhanced tolerance to NaCl, showed higher biomass accumulation in salinity stress as
compared to ‘CA’ plants. The ‘CA’ plants exhibited better compared to the control plants [114]. The overexpression
seed longevity compared to non-transgenic plants [106]. of SAMDC1 in Arabidopsis led to elevated spermidine
In addition to other reported antioxidative genes, GST levels and enhanced tolerance to various abiotic stress
gene from a rice variety ‘Pusa Basmati-1’ showed better conditions. Introduction of human SAMDC into tobacco led
growth under salt and cold stresses by scavenging ROS and to overexpression of putrescine and spermidine resulting in
preventing membrane damage in transgenic tobacco plants enhanced salt and osmotic tolerance [115]. Different
[107, 108]. Antioxidant compounds are proved as good examples confirmed that enhanced expression of poly-
candidates for reducing the cellular toxicity and help plants amines control the salt and drought tolerance in plants.
in maintaining good health in adverse environmental con- However, a gap remains in understanding the mechanism
ditions. The manipulation of antioxidant genes in plants of polyamines involved in abiotic stress tolerance.
seems a good approach for keeping ROS at basal level.
Although, several reports have mentioned that (Table 4)
higher expression of single antioxidant gene control salt- Transporter Genes
induced oxidative stress efficiently, but it is plausible to
incorporate more than one genes for balanced ratio of Plants apply both ionic and osmotic homeostasis to re-
protective enzymes and other metabolites. establish themselves in saline environmental conditions.
Plants employ various strategies for maintaining low Na?
in the cell either by the active exclusion by the plasma
Polyamines membrane Na?/H? antiporter AtSOS1 [116, 117], or by
sequestration of excess sodium into the vacuoles via vac-
The polyamines play essential roles in many fundamental uolar Na?/H? antiporters. Transporter proteins are impor-
cellular processes, gene expression, protein synthesis, cell tant candidates for genetic engineering to develop salt

Table 5 Overexpression of polyamine gene for salt and desiccation tolerance


Gene/source Transgenic plant Performance of transgenic plant References

ADC/oat Rice Salt tolerance [114]


ADC/Datura Rice Drought tolerance [220]
ADC2/Arabidopsis Arabidopsis Drought tolerance [221]
ODC/mouse Tobacco Salt tolerance [114]
SAMDC/Tritodeum Rice Salt tolerance [222]
SAMDC/human Tobacco Salt and osmotic tolerance [115]
SPDS/Cucurbita ficifolia Arabidopsis Salinity, hyperosmosis tolerance [223]
SPDS/apple Pear Salt and drought tolerance [224]
ADC arginine decarboxylase, ODC ornithine decarboxylase, SAMDC S-Adenosyl methionine decaroboxylase, SPDS spermidine synthase

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112 Mol Biotechnol (2013) 54:102–123

Table 6 Overexpression of transporter genes for salt and desiccation tolerance


Gene/source Transgenic plant Performance of References
transgenic plant

NHX1/Arabidopsis cotton, Arabidopsis, tomato, Salt tolerance [44, 119, 163, 225–231]
B. napus Festuca, Fagopyrum,
sugar beet, kiwi fruit, wheat, maize
NHX1/Atriplex gmelini Rice Salt tolerance [232]
NHX1/wild type rice Rice Salt tolerance [233]
NHX1/Gossipium Tobacco Salt tolerance [234]
NHX1/Hordeum Tobacco Salt tolerance [235]
NHX1/Pennisetum B. juncea, rice Salt tolerance [236, 237]
NHX1/Agropyron elongatum Arabidopsis/Festuca Salt tolerance [238]
NHX1/Reed Yeast Salt tolerance [56]
NHX1/Aleuropus littoralis Tobacco Salt tolerance [239]
NHX1/Salicornia brachiata Tobacco Salt tolerance [124]
NHX1/Salsola soda Alfalfa Salt tolerance [240]
NHX1/Malus Apple Salt tolerance [241]
NHX1/Thellungiella halophilla Arabidopsis Salt tolerance [242]
HcNHX1/Halostachys caspica Arabidopsis Salt tolerance [243]
AtNHX1/Arabidopsis Arachis hypogaea Salt tolerance [244]
AmNHX2/Ammopiptanthus mongolicus Arabidopsis Salt and drought tolerance [245]
NHX2/Hordeum Potato Salt tolerance [246]
HAL1/yeast Cucumis melo, tomato Salt tolerance [125, 126]
nhaA/E. coli Rice Salt tolerance [130]
SOD2/yeast Arabidopsis, rice Salt tolerance [129, 247]
SOS1/Arabidopsis Arabidopsis Salt tolerance [117]
SOS1/rice Yeast Salt tolerance [248]
SOS1/Populus euphratica E. coli Salt tolerance [249]
SOS1-RNAi/Thellungiella halophilla Thellungiella halophilla Salt tolerance [131]
SOS1 Yeast [250]
SOS1-RNAi/Solanum lycopersicon Tomato
NHX1,SOS1, SOS3, SOS2 ? SOS3, NHX1 ? Arabidopsis Salt tolerance [132]
SOS3, SOS1 ? SOS2 ? SOS3/Arabidopsis
Avp1(H?-PPases)/Arabidopsis Arabidopsis Salt tolerance [135]
H?-PPase/Rhodospirillum rubrum Tobacco Salt tolerance [251]
TsVP(H?-PPase)/Thellungiella halophila Tobacco, maize, cotton Salt tolerance [252–254]
?
Avp1(H -PPases)/Arabidopsis Alfalfa Salt tolerance [255]
Avp1/Arabidopsis ? NHX1/Saueda salsa Rice Salt tolerance better than [256]
SsNHX1 alone
SsVP1/Saueda salsa Arabidopsis Salt and drought tolerance [257]
?
H -PPase TVP1 and TNHX1/wheat Arabiodopsis Salt and water deprivation [133]
tolerance
Avp1/Arabiodopsis Cotton, Creeping bent grass Salt and drought tolerance [258, 259]
TsVP/Thellungiella halophila Cotton Salt tolerance [260]
ATPase pENA1/Physcomitrella patens Rice Salt tolerance [261]
V-ATPase c subunit gene ThVHAc1/ Saccharomyces cerevisiae Salt and drought tolerance [262]
Tamarix hispida
NHX-1 vacuolar Na?/H? antiporter, SOS1 plasma membrane Na?/H? antiporter, TsVP H?-pyrophosphatase, HKT2 high efficiency potassium
transport

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Mol Biotechnol (2013) 54:102–123 113

tolerant plants. Of late, several efforts have been made to 220 mM NaCl treatment for 3 days, less than 20% of the
develop salt tolerant plants by upregulation of transporter control and transgenic plants overexpressing only AtNHX1
genes as compared to other classes of genes (Table 6). A survived, but over 80% of the transgenic plants over-
gene, homologous to animal plasma membrane Na?/H? expressing SOS1, SOS3, SOS2 ? SOS3, AtNHX1 ? SOS3,
antiporters of the NHE family and the yeast ScNHX1 was or SOS1 ? SOS2 ? SOS3 survived [132]. Brini et al. [133]
first identified from Arabidopsis genome and termed At- reported that overexpression of wheat TVP1 (Tonoplast H?-
NHX1 [118]. Overexpression of Arabidopsis AtNHX1 PPase) and NHX1 in Arabidopsis conferred better growth in
conferred enhanced salt tolerance in Arabidopsis [119]. the presence of 200 mM NaCl and also under a water-
Na?/H? antiporters have been isolated from several gly- deprivation regime, while wild-type plants exhibited chlo-
cophytes and halophytes, i.e., Mesembryanthemum crys- rosis and growth inhibition. The proton pumps present at the
tallinum [120], Atriplex gmelini [121], Saudea salsa [122], cellular membrane work as driving force for nutrient uptake
Beta vulgaris [123], and Salicornia brachiata [124]. The [134]. Three distinct proton pumps are responsible for the
yeast HAL1 gene showed a certain level of salt tolerance generation of the proton electrochemical gradients: (1) the
when expressed in transgenic melon [125] and tomato plasma membrane H-ATPase pump (PM H-ATPase), (2)
[126] plants and retained more K? than the control plants vacuolar type H?ATPase (V-ATPase), and (3) the vacuolar
under salt stress. There are more than 26 reports on H-pumping pyrophosphatase (H-PPase). The PM H-ATP-
acquiring salt tolerance induced by overexpression of ase extrudes H? from the cell and thus generates a proton
NHX. These include five halophytic plants (Table 6). motive force while V-ATPase and H-PPase acidify the
Salt overly sensitive (SOS) pathway was found to be vacuolar lumen and other endomembrane compartments
involved in Na? exclusion. SOS1 is a plasma membrane [12]. Genetic evidence of physiological role of PM
Na?/H? antiporter that excludes Na? by taking H? into the H-ATPase and V-ATPase is very scarce, whereas, a few
cytoplasm. The SOS pathway is regulated by Ca2? studies have been carried out on H-PPase showing its
dependent protein kinase signaling [14]. SOS pathway involvement in salt tolerance. The pioneer work was carried
involves SOS1, SOS2 and SOS3. Ca2? signaling is per- out by Gaxiola et al. [135], overexpression of Avp1 (H?-
ceived by SOS3, a calcium binding protein. SOS3 activates PPase) showed enhanced salt tolerance in transgenic Ara-
SOS2, a protein kinase that activates SOS1 by its phos- bidopsis plants. Later this gene was isolated from Thel-
phorylation. Recently, SOS4 and SOS5 have also been lungiella halophila and engineered in tobacco, maize, and
characterized by Shi et al. [127]. SOS pathway also regu- cotton showed enhanced salt tolerance (Table 6). As evi-
lates vacuolar Na?/H? antiporter exchange activity and dent from the literature (Table 6) a large interest have been
Na? compartmentalization [128]. SOS1 gene from Ara- shown for overexpressing the transporter genes in model as
bidopsis was ectopically expressed first time in Arabidopsis well as crop plants. In future, these transgenic plants should
plant showed reduced Na? accumulation in the presence of be tested in the field to test the efficacy in the adverse
salt [117]. Similar results were obtained when the plasma environment conditions.
membrane Na?/H? antiporters, SOD2 (Sodium 2) from
Schizosaccharomyces pombe and nhaA from Escherichia
coli, were overexpressed in Arabidopsis [129] and rice Glyoxalase Pathway
[130], respectively. The SOS1 from Thellungiella salsugi-
nea suppressed the salt sensitive phenotype when expres- Glyoxalase pathway has emerged as a prospective can-
sed in the yeast cells. This gene showed high salt stress didate for the genetic engineering of salt tolerance. The
tolerance in the Arabidopsis transgenic plants, when the glyoxalase pathway has shown its role in rapidly dividing
SOS1 gene was suppressed by ThSOS1-RNAi in Thel- plant cells [136, 137] and in stress tolerance [138–140].
lungiella salsuginea, the plants showed high salt sensitivity This pathway involves two enzymes, glyoxalase I and
compared to wild-type plants [131]. glyoxalase II, which convert methylglyoxal to lactic acid
Instead of transforming single transporter genes, some in two-step reactions [7]. Methylglyoxal is a potent
researchers have tried manipulation of a combination of two cytotoxic compound, and is the primary substrate for
or more transporters in plants. Yang et al. [132] tested glyoxalase I. Expression of the GlyI gene from Brassica
overexpression of multiple genes to improve salt tolerance juncea showed salt tolerance in transgenic tobacco lines
in Arabidopsis. They produced six different transgenic [139]. Singla-Pareek et al. [141] achieved better salt stress
Arabidopsis plants overexpressing AtNHX1, SOS1, and tolerance in tobacco by overexpression of both GlyI and
SOS3 alone or in different combinations (AtNHX1 ? SOS3, GlyII together. Further, Singla-Pareek et al. [142] trans-
SOS2 ? SOS3, SOS1 ? SOS2 ? SOS3). Surprisingly, the formed only GlyII in rice plants, which showed higher
AtNHX1 alone did not show significant salt tolerance. In tolerance to NaCl.

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114 Mol Biotechnol (2013) 54:102–123

Role of Small RNA in Abiotic Stress Tolerance of Arabidopsis miR395c or miR395e retarded and accel-
erated the seed germination of Arabidopsis under high salt
Recently, a newer approach to understand the mechanism or dehydration stress conditions, respectively. The overex-
of abiotic stress in plant system through miRNA/siRNA pression of miR395c and miR395e, did not show the same
approach is getting attention. The plant response to the cleavage of mRNA targets, APS1, APS3, APS4, and
stress relies on the correct regulation of gene expression, SULTR2;1, in both the overexpressing plants. Wang et al.
which depends on the number of mechanisms. The post- [161] reported suppression of an Arabidopsis bacterial-type
transcription events play a crucial role in regulating gene PEPC gene, Atppc4, by artificial microRNA (amiRNA).
expression at a right time [143]. MicroRNA and short Atppc4-amiRNA transgenic plants showed decreased
interfering RNA are small (approx. 21 nt) non-coding accumulation of Atppc4 transcripts, whereas other three
RNA, involved in regulating gene expression by mRNA plant-type PEPC genes, Atppc1, Atppc2, and Atppc3 were
degradation, translational repression, and chromatin mod- significantly upregulated and these transgenic plants
ification [144, 145]. The small RNA regulate gene showed improved tolerance to salt stress.
expression in stressed plants either by overexpression of These results demonstrated that a given miRNA family
small RNA which resulted in downregulation of negative containing a single nucleotide difference can guide the
regulators, or reduced biogenesis of small RNA which cleavage of various mRNA targets, thereby acting as a
causes downregulation of positive regulators and accumu- positive or negative regulator of seed germination under
lation of beneficial gene products [143]. stress [162]. These studies highlight that microRNA play
Recently, a number of reports have been published an important role in targeting various important genes for
elucidating the role of microRNAs in regulating gene managing stress tolerance in plants. Recently miRNAs is
expression under various environmental conditions [146– getting increased attention and in near future a vast
151]. Some good reviews on role of microRNA in stress knowledge on the mechanism of miRNA for abiotic stress
response elucidating their roles have also been published will be generated.
[143, 152, 153]. Several miRNAs have been isolated dur-
ing salt treatment from Arabidodpsis [144], P. trichocarpa
[154], and rice [155] plants. A detailed mechanism of Conclusion
miR398 has been studied by Sunkar et al. [149], where
mRNA abundance of Cu/Zn SOD1 (CSD1, cytosolic) and Genetic engineering towards developing salt and drought
Cu/Zn SOD2 (CSD2, chloroplastic) was observed inversely tolerant crops is challenging and forefront area of research
correlated by the abundance of miR398 under salt stress. for future crop improvement programs. Conventional
There are very limited studies on the overexpression of breeding has made considerable success in twentieth cen-
miRNA for abiotic stress tolerance. Atmir398 from Ara- tury to improve crop yield and quality but limited work was
bidopsis was overexpressed in tobacco plants, caused carried out for developing abiotic stress tolerant crop plants
reduced seedling and root growth by down regulating with only few successful reports [9]. This is probably due
NtTIR1 expression and causing auxin insensitivity in to the low magnitude of genetically based variation in the
transgenic tobacco plants. These transgenic plants showed plants for salinity tolerance. For achieving the salinity
enhanced resistance to salt stress by suppressing auxin tolerance, the genetic material from the distant wild rela-
signal via degradation of NtTIR1 mRNA [156]. The rice tives or halophytes can be used for transfer the salt tolerant
osa-MIR396c showed a dramatic transcript change under genes into the sensitive plants through conventional
salt and alkali stress conditions in Oryza sativa [157]. The breeding but again the incompatibility of the reproduction
osa-MIR396c, osa-MIR393 transgenic rice, and Arabidop- hinders in achieving the goal. With the advent of plant
sis lines showed that several TFs related to growth, devel- molecular biology and understanding of the stress signal
opment, and stress tolerant genes are targeted by these transduction pathways, it is now possible to generate plants
microRNA and eventually showed reduced salt and alkali with least damaging effect on environmental conditions
stress tolerance compared to that of wild-type plants [157, and concurrently promising an increase in productivity. So
158]. Xia et al. [159] observed that OsmiR393 has same far, many stress-related genes have been isolated and
results in transgenic rice as reported by Gao et al. [158] in characterized in the model plants like tobacco and Ara-
response to salt. The transgenic lines also showed two new bidopsis. With the progress of whole genome sequencing
functions as increased tilling and early flowering. However, of different plant species, it seems now easier to identify
the report of Gao et al. [157] is in contradiction to AtmiR396 unique stress responsive genes. During the last decade,
overexpression in Arabidopsis [160], which conferred the transgenic plants with stress tolerant genes have been
tolerance to drought and other stresses. The overexpression generated using various genes from diverse pathways

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Mol Biotechnol (2013) 54:102–123 115

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