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

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ability to adapt themselves to such a highly variable
environmental onslaughts, environmental stresses never-
theless cause over 50% crop loss [1]. Approximately, 7%
of total land area (1,000 million ha) and 20% of the irri-
gated agriculture land is affected by soil salinity [2] and
imposes major constraints to the sustainability of crop
yield.
Salinity imparts both ionic and osmotic stresses thus
limiting plant growth and productivity. Plants respond and
adapt to these conditions by regulating a wide array of
genes. Salinity causes adverse impact on plant growth by
disturbing the ionic equilibrium and eliciting sodium tox-
icity. High Na? concentration is toxic to cell metabolism
and has deleterious effects on the functioning of some of
the enzymes [3]. Different plants employ different mech-
anisms to minimize the damage from Na?, e.g., minimize
initial influx, maximize efflux, minimize loading into the
xylem, and maximize recirculation out of the shoot to
phloem, intercellular compartmentalization and even
secretion of salt from the leaf surface [4, 5].
Various genes induced by salt stress could be grouped
under two categories, namely, ‘‘single-function’’ genes and
regulatory genes. The first category of genes generally
facilitates production of protective metabolites, which
include osmolytes, transporters/channel proteins, antioxi-
dative enzymes, lipid biosynthesis genes, polyamines, etc.
The second class of genes consists of regulatory proteins
like bZIP, DREB, MYC/MYB, and NAC, which control
the expression of many downstream salt stress tolerant
genes [6, 7]. These genes converge and interact in different
pathways related to the abiotic stress and successfully lead
to tolerance (Fig. 1).
Different genes related to abiotic stress tolerance, have
been introduced into a variety of plants [1, 8–10]. Many
reviews have been published recently on oxidative pathway
[11], transporters [12–15] and role of regulators in con-
trolling salt stress tolerance [6, 7, 16–18]. The present
article focuses on new paradigm developed in the recent
past years in developing salt tolerant plants by employing
genes from all the possible pathways.
Signaling Molecules
Environmental signals are first perceived by signaling
molecules and involves protein phosphorylation, dephos-
phorylation, phospholipid metabolism, Ca2? sensing, etc.
Their overexpression in many plants showed better stress
tolerance (Table 1). Stress and other extracellular stimuli
change the intracellular Ca2? concentration [19, 20]. Ca2?
signaling process is considered as one of the earliest events
in salt signaling, and play an essential role in the ion
homeostasis leading to salt tolerance in plants [15, 21].
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Calcineurin B-like proteins (CBL) sense the calcium signal
and participate in salt stress signal transduction pathway
and control the influx and efflux of Na?. The signaling
approach to improve salt tolerance was demonstrated in
transgenic tobacco by modulating the expression of a cal-
cium stress-signaling component from yeast, Ca2?/cal-
modulin dependent protein phosphatase [22]. The
transgenic tobacco plants co-expressing the two catalytic
and regulatory subunits of this protein exhibited substantial
NaCl tolerance [22]. The calcineurin B-like (regulatory)
Ca2? sensor, SOS3 (Salt overly sensitive 3) has been cloned
from Arabidopsis [23]. This protein triggers the activity of
SOS pathway for plant Na? tolerance, the SOS gene and
its applications are discussed below along with the
transporter genes. A CBL protein from maize, ZmCBL4,
showed salt stress tolerance in Arabidopsis. Expression of
35S:ZmCBL4 complemented the salt hypersensitivity in
Arabidopsis sos3 mutant and enhanced the salt tolerance
in wild-type Arabidopsis at the germination and seedling
stages [24]. Recently, Tripathi et al. [25] showed that
overexpression of a constitutively active mutant of Ca-
CIPK6 from chickpea promoted salt tolerance in trans-
genic tobacco. AtNDPK2 activated both AtMPK3 and
AtMPK6, and its overexpression showed enhanced salt
and other abiotic stress tolerance by reducing the ROS
concentration in the plants [26]. SAPK4 is a serine thre-
onine type of kinase and is known to regulate the stress
responsive gene expression. The overexpression of SAPK4
resulted in improved germination, growth and develop-
ment under salt stress. In response to salt stress, the
SAPK4-overexpressing rice accumulated less Na? and Cl-
and showed improved photosynthesis [27].
Plants use another common mechanism to translate
external stimuli into cellular responses through the acti-
vation of mitogen-activated protein (MAP) kinase cascade
[28]. MAPKs are signaling modules that phosphorylate
specific serine/threonine residues on the target protein
substrate and regulate a variety of cellular activities. The
MAPK cascade consists of three functionally interlinked
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
studied in relation to biotic and abiotic stresses [29].
Overexpression of MAP kinases showed enhanced toler-
ance to salt stress (Table 1). Transgenic rice plants with
OsMAPK5 and OsMAPK44 genes showed tolerance to salt,
drought and cold stresses [30, 31]. The overexpression of
ZMKK4 from Zea mays in Arabidopsis plants showed
enhanced salt tolerance and also higher proline and soluble
sugar contents, and higher POD and CAT activities com-
pare to control plants. The transgenic plant showed
upregulation of the transcription factor, TF, which even-
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
MAP kinase control the stress in plants by overexpressing
of antioxidative genes and on other hand it activated the
TFs which further control the downstream genes. Kong
et al. [32] has studied that the transgenic plants showed
upregulation of CBF1, CBF2, CBF3, STZ, DREB2A TFs
and which in turn showed the enhanced expression of the
COR47, RD29A, P5CS2 genes. The signaling molecules
are reported to interact for developing cross tolerance, in
this case when one type of stress renders to plants than it
leads to resistant to another type of stress [33]. LeCDPK1
gene from tomato also is a good example of imparting
cross-tolerance, it interrelates the signaling responses to
wounding and salt stress. It showed that mechanical
wounding increases salt-stress tolerance by involving the
signaling peptide systemin and the synthesis of JA (Jas-
monic Acid) [34]. It is evident from the literature that
signaling genes play important role in abiotic stress toler-
ance by regulating the expression of salt tolerant genes and
TFs. In spite of the important role of signaling molecule,
only few studies had been carried out (Table 1) for engi-
neering salt tolerance in the plants, therefore it is desirable
to have more transgenic lines with this kind of genes in
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-

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

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

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temperature-induced protein) minimized the negative
effect on growth compared to control plants and showed
higher tolerance in the presence of salt [47]. ONAC063
from rice showed enhanced tolerance to salt and osmotic
stress in Arabidopsis plants. Microarray analysis showed
upregulation of 29 genes among them oleosin and amylase
expressed more than 100-fold [51]. Two NAC genes
GmNAC11 and GmNAC20 have been isolated from the
soybean. These both genes differentially induced under
stress condition and plant hormones. The overexpression of
the GmNAC20 showed the tolerance to salinity and freez-
ing, whereas, GmNAM11 showed tolerance to only salinity.
Both genes likely regulated the stress tolerance by DREB/
CBF-COR pathway [52]. Liu et al. [53] reported that
overexpression of Chrysanthemum DgNAC1 in tobacco
resulted salt tolerance. OsNAC5 [54] when overexpressed
showed better salt tolerance than RNAi knock-out lines or
wt plants. Different physiological parameters of OsNAC5
overexpression lines showed that proline and free sugar
level get enhanced in transgenic lines. Further, it is also
observed that MDA and H2O2 content have got reduced in
overexpressed lines compared to knockdown lines [54].
Similarly, Takahashi et al. [55] also observed higher salt
tolerance by overexpression of OsNAC5. The role of NAC
gene in ABA signaling is reported by Liu et al. [56], the
Arabidopsis transgenic lines with AhNAC2 (Arachis hyp-
ogaea) showed hyper sensitivity to ABA in root growth,
seed germination, and stomatal closure compared to wild
type Arabidopsis. The AhNAC2 overexpressed lines
showed higher expression of downstream genes viz.
Rd29A, Rd29b, Rab18, AtMYB2, AtMYC2, ERD1, COR 47,
COR15a, KIN1, AREB1, CBF1, AMY1. The upregulation
of Rd29A, Rd29b can correlate the ABA signaling of Ah-
NAC2 since both the gene possess ABRE element in their
promoter region [56].
Myb
In higher plants, myb (myeloblastosis)-TF family repre-
sents a large number of genes. In Arabidopsis it is con-
sidered as the largest TF family and contains more than 163
genes [57]. Plant R2R3-MYB TFs play wide functional
roles, and involved in imparting stress tolerance against
various environmental cues in transgenics (Table 2). The
OsMYB3R-2I from rice employed enhanced tolerance to
salt, freezing and dehydration stresses and decreased sen-
sitivity to ABA in transgenic Arabidopsis [58]. In contrast,
Jung et al. [59] reported that the AtMYB44 transgenic were
highly sensitive to ABA and a rapid ABA induced stomatal
closure was seen during the stress treatment. Transgenic
plants had low rate of water loss and enhanced tolerance to
drought and salt stress compared to wild-type plants. The
AtMYB44 overexpressing lines revealed the low expression
107
of protein phosphatases 2C (PP2Cs) genes in microarray
analysis and northern blots analysis, whereas the mutant
lines showed the higher expression of PP2Cs and reduced
tolerance to salt and drought. This study showed that At-
MYB44 performs the abiotic stress function by suppressing
the negative regulator group of genes. Liao et al. [60] has
studied expression of large number of soybean Myb genes
under ABA, salt, drought, and/or cold stress. The overex-
pression of three Myb genes, viz., GmMYB76, GmMYB92,
or GmMYB177 in Arabidopsis showed higher seed germi-
nation rate under salt media [60]. Recently, Gao et al. [61]
reported that overexpression of apple Myb10 resulted in
osmotic stress tolerance. The transgenic apple plants
showed higher flavonoid content, thus manages higher
antioxidative ability to cope-up osmotic stress. Three dif-
ferent Myb TFs were isolated from wheat (Triticum aes-
tivum L)., TaMYB2A, TaMYB2B, and TaMYB2D [62].
TaMYB2A Arabidopsis transgenics showed enhanced tol-
erance to drought, salt, and freezing stresses and revealed
decreased rate of water loss, enhanced cell membrane
stability, improved photosynthetic potential, and reduced
osmotic potential. Another MYB gene TaPIMP1, was also
isolated from wheat: TaPIMP1 showed significantly higher
transcript level by a fungal pathogen Bipolaris sorokiniana
and by drought treatment. TaPIMP1 transgenic tobacco
lines showed tolerance to salinity along with pathogen and
drought. In these transgenic lines, the activities of phen-
ylalanine ammonia-lyase (PAL) and superoxide dismutase
(SOD) were significantly increased compared to wild-type
tobacco plants [63].The Solanum lycopersicum abscisic
acid-induced myb1 (SlAIM1) TF is reported to induced by
pathogens, plant hormones, salinity and oxidative stress,
suggesting a function in pathogen and abiotic stress
responses. The RNAi plant with silenced SlAIM1 showed
high susceptibility to fungus Botrytis cinerea, and high
sensitivity to salt and oxidative stress. These responses
correlate with reduced sensitivity to abscisic acid (ABA) in
the SlAIM1 RNAi, but increased sensitivity in the over-
expression plants, suggesting SlAIM1-mediated ABA
responses are required to integrate tomato responses to
biotic and abiotic stresses. The SIAIM1 also shows the
regulation of ion fluxes, the RNAi plants accumulated more
Na? compared to overexpressed lines [64].
Osmolytes
Osmolytes are organic metabolites of low molecular
weight known as compatible solutes and do not deter the
cellular functions. The osmolytes such as glycine betaine,
fructans, trehalose, mannitol, sorbitol, ononitol, and pinn-
itol play prominent role as osmoprotectants. Genes for
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
osmoprotectants and enhanced salt and drought tolerance
(Table 3).
Trehalose is a non-reducing disaccharide and its syn-
thesis help in achieving dehydration tolerance to living
organism. It protects biomolecules by water replacement,
glass formation and chemical stability [65]. The genes for
trehalose synthesis have been cloned from bacteria, yeast,
and eukaryotic plants, and first time overexpressed in
tobacco enhancing drought tolerance by incorporation of E.
coli otsA gene or ScTPS1, trehalose-6-phosphate synthase
from yeast [66, 67]. Later, ScTPS1 gene was introduced
into Arabidopsis [68], alfalfa [69], rice, and tomato [70]
and the transgene imparted enhanced salt tolerance to the
plants. Garg et al. [71] introduced OtsA and OtsB (E. coli
trehalose-6-phosphate synthase) genes in rice and observed
increased tolerance to salt, drought, and cold in the trans-
genic plants. The trehalose-6-phosphate synthase gene
(OsTPS1) overexpressing rice lines showed improved tol-
erance to cold, high salinity and drought treatments without
any morphological changes. These lines also exhibited
higher expression of RAB16C, HSP70, and ELIP and Os-
TPP1 and OsTPP2 [72].
The E. coli mannitol-1-phosphate (mt1D) gene was
introduced into Arabidopsis [73], tobacco [74], and wheat
[75] plants. The transgenic Arabidopsis showed enhanced
seed germination on high salt (400 mM NaCl) medium
[73]. The wheat transgenic plants with mt1D showed less
reduction in biomass compared to wild-type plants under
the presence of salt and dehydration [75].
During stress treatment, the low molecular weight
metabolite proline gets accumulated in the cells. The bio-
synthesis of proline resulted in improved tolerance to salt
and drought stress in a number of crops [76, 77]. The proline
precursor, p5cs (D1-Pyroline-5-carboxylate synthase), has
been introduced into tobacco [78, 79], rice [80, 81], and
Arabidopsis [82, 83] and these transgenic plants showed
better tolerance to salinity stress.
Glycine betaine (betaine) is a non toxic cellular osmolyte
that raises intracellular osmolarity in response to different
stresses in lower organisms as well higher plants and sta-
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-
karyotes and eukaryotes from choline with an intermediate
compound betaine aldehyde. In eukaryotes, choline gets
converted in betaine aldehyde with the help of choline
monooxygenase [86] in first step, which subsequently pro-
duce betaine in second step by betaine aldehyde dehydro-
genase (BADH) [87], whereas in bacteria the choline to
betaine aldehyde and finally to betaine is achieved by
choline dehydrogenase (CDH). However, several crop
plants are incapable of synthesizing betaine [85]. The E.
coli betA and betB genes [88] encoding for CDH and BADH
enzymes, respectively, were introduced in tobacco resulting
increased biomass and faster recovery from photoinhibition
under salt stress [85]. There are examples where only betA
gene has been overexpressed in B. oleracea [89] and maize
[90], which showed enhanced salt and drought tolerance,
respectively. In comparison to the CDH and CMO pathways
a direct choline oxidase pathway (COD) do exist which has
single step conversion of choline to glycinebetaine and also
does not require any cofactors for the catalysis [91]. A codA
(Choline oxidase) gene from Arthrobacter globiformis had
been introduced in many plants, resulting in enhanced
growth under salt stress which showed better germination
and growth in transgenic at 150 mM of NaCl (Table 3).
Antioxidative Enzymes
Salinity stress generates reactive oxygen species (ROS)
including singlet oxygen, superoxide anion radicals,
hydroxyl ions, and hydrogen peroxide [92–96] and consid-
ered as marker for stress activation in the plants [97]. ROS
serves as signaling molecules that regulates stress response to
maintain the growth and development of plants. In this
review the direct impact of oxidative stress compounds on
salt stress tolerance by genetic engineering approach is dis-
cussed. During salt stress available CO2 get reduced in the
leaf because of stomata closing, which further causes the
over-reduction of photosynthetic electron transport chain,
thereby leading to the generation of ROS [97]. Several
enzymes are involved in the detoxification of antioxidative
substances (AOS). Superoxide dismutase (SOD) converts
superoxide to H2O2, which is further scavenged by catalase.
Ascorbate peroxidase (APX) also reduces H2O2 and is
present in different isoforms in different plant organelles
[98]. Transgenic plants developed by overexpression of
several enzymes such as glutathione peroxidase (GPX),
SOD, APX, and glutathione reductases (GR) showed
improved stress tolerance (Table 4). Appearance of APX
isoenzymes in response to environmental stresses, such as
salinity and drought, has been reported in several plant spe-
cies and their overexpression led to salt and drought tolerance
in tobacco and Arabidopsis [99–101]. A number of SOD
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
showed high photosynthetic rate under increased salt stress in
tobacco transgenic plants [102]. A cytosolic Cu/ZnSOD gene
from the mangrove species, Avicennia marina enhanced salt
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
overexpression of MnSOD showed enhanced salt tolerance in
Arabidopsis [104]. Many studies have been performed to
develop abiotic stress tolerant plants by engineering one gene
but recently interest has changed to incorporate more than
one gene for empowering better tolerance. However, on this
aspect only a few reports have appeared [105], transforma-
tion of tobacco plants by pyramiding more than one gene for
anti-oxidative enzyme like Cu/ZnSOD (C), APX (A), and
DHAR (D) into chloroplasts and demonstrated that such
plants were more salt and oxidative stress tolerant compared
to those expressing single or double gene transfer. The
overexpression of ‘CAD’ showed approximately 1.6–2.1
times higher dehydroascorbate reductase (DHAR) activity,
higher ratios of reduced ascorbate to dehydroascorbate
(DHA), and oxidized glutathione (GSSG) to reduced gluta-
thione (GSH) compared to ‘CA’ plants. The transgenic
seedlings with ‘CAD’ showed enhanced tolerance to NaCl,
compared to ‘CA’ plants. The ‘CA’ plants exhibited better
seed longevity compared to non-transgenic plants [106].
In addition to other reported antioxidative genes, GST
gene from a rice variety ‘Pusa Basmati-1’ showed better
growth under salt and cold stresses by scavenging ROS and
preventing membrane damage in transgenic tobacco plants
[107, 108]. Antioxidant compounds are proved as good
candidates for reducing the cellular toxicity and help plants
in maintaining good health in adverse environmental con-
ditions. The manipulation of antioxidant genes in plants
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-
induced oxidative stress efficiently, but it is plausible to
incorporate more than one genes for balanced ratio of
protective enzymes and other metabolites.
Polyamines
The polyamines play essential roles in many fundamental
cellular processes, gene expression, protein synthesis, cell
Table 5 Overexpression of polyamine gene for salt and desiccation tolerance
Gene/source Transgenic plant
ADC/oat Rice
ADC/Datura Rice
ADC2/Arabidopsis Arabidopsis
ODC/mouse Tobacco
SAMDC/Tritodeum Rice
SAMDC/human Tobacco
SPDS/Cucurbita ficifolia Arabidopsis
SPDS/apple Pear
ADC arginine decarboxylase, ODC ornithine decarboxylase, SAMDC S-Adenosyl methionine decaroboxylase, SPDS spermidine synthase
111
division, cell differentiation, growth, development, and cell
death [109]. They are also involved in various abiotic and
biotic plant stress responses. The role of polyamines is
amply described in recent reviews [110, 111]. The heter-
ologous overexpression of ornithine decarboxylase (ODC),
arginine decarboxylase (ADC), S-adenosyl methionine
decarboxylase (SAMDC), and spermidine synthase (SPDS)
in rice, tobacco, and tomato has shown tolerance against
stress conditions (Table 5). In carrot cell lines the over-
expression of mouse ODC, which converts ornithine to
putrescine, exhibited high level of salt stress tolerance
[112]. Kumria and Rajam [113] also showed that mouse
ODC when introduced into tobacco exhibited enhanced
production of constitutively expressed putrescine, confer-
ring salt tolerance to the plants. A cDNA for ADC from oat,
expressed in rice under an ABA-inducible promoter,
showed higher biomass accumulation in salinity stress as
compared to the control plants [114]. The overexpression
of SAMDC1 in Arabidopsis led to elevated spermidine
levels and enhanced tolerance to various abiotic stress
conditions. Introduction of human SAMDC into tobacco led
to overexpression of putrescine and spermidine resulting in
enhanced salt and osmotic tolerance [115]. Different
examples confirmed that enhanced expression of poly-
amines control the salt and drought tolerance in plants.
However, a gap remains in understanding the mechanism
of polyamines involved in abiotic stress tolerance.
Transporter Genes
Plants apply both ionic and osmotic homeostasis to re-
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
membrane Na?/H? antiporter AtSOS1 [116, 117], or by
sequestration of excess sodium into the vacuoles via vac-
uolar Na?/H? antiporters. Transporter proteins are impor-
tant candidates for genetic engineering to develop salt
Performance of transgenic plant References
Salt tolerance [114]
Drought tolerance [220]
Drought tolerance [221]
Salt tolerance [114]
Salt tolerance [222]
Salt and osmotic tolerance [115]
Salinity, hyperosmosis tolerance [223]
Salt and drought tolerance [224]
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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
tolerant plants. Of late, several efforts have been made to
develop salt tolerant plants by upregulation of transporter
genes as compared to other classes of genes (Table 6). A
gene, homologous to animal plasma membrane Na?/H?
antiporters of the NHE family and the yeast ScNHX1 was
first identified from Arabidopsis genome and termed At-
NHX1 [118]. Overexpression of Arabidopsis AtNHX1
conferred enhanced salt tolerance in Arabidopsis [119].
Na?/H? antiporters have been isolated from several gly-
cophytes and halophytes, i.e., Mesembryanthemum crys-
tallinum [120], Atriplex gmelini [121], Saudea salsa [122],
Beta vulgaris [123], and Salicornia brachiata [124]. The
yeast HAL1 gene showed a certain level of salt tolerance
when expressed in transgenic melon [125] and tomato
[126] plants and retained more K? than the control plants
under salt stress. There are more than 26 reports on
acquiring salt tolerance induced by overexpression of
NHX. These include five halophytic plants (Table 6).
Salt overly sensitive (SOS) pathway was found to be
involved in Na? exclusion. SOS1 is a plasma membrane
Na?/H? antiporter that excludes Na? by taking H? into the
cytoplasm. The SOS pathway is regulated by Ca2?
dependent protein kinase signaling [14]. SOS pathway
involves SOS1, SOS2 and SOS3. Ca2? signaling is per-
ceived by SOS3, a calcium binding protein. SOS3 activates
SOS2, a protein kinase that activates SOS1 by its phos-
phorylation. Recently, SOS4 and SOS5 have also been
characterized by Shi et al. [127]. SOS pathway also regu-
lates vacuolar Na?/H? antiporter exchange activity and
Na? compartmentalization [128]. SOS1 gene from Ara-
bidopsis was ectopically expressed first time in Arabidopsis
plant showed reduced Na? accumulation in the presence of
salt [117]. Similar results were obtained when the plasma
membrane Na?/H? antiporters, SOD2 (Sodium 2) from
Schizosaccharomyces pombe and nhaA from Escherichia
coli, were overexpressed in Arabidopsis [129] and rice
[130], respectively. The SOS1 from Thellungiella salsugi-
nea suppressed the salt sensitive phenotype when expres-
sed in the yeast cells. This gene showed high salt stress
tolerance in the Arabidopsis transgenic plants, when the
SOS1 gene was suppressed by ThSOS1-RNAi in Thel-
lungiella salsuginea, the plants showed high salt sensitivity
compared to wild-type plants [131].
Instead of transforming single transporter genes, some
researchers have tried manipulation of a combination of two
or more transporters in plants. Yang et al. [132] tested
overexpression of multiple genes to improve salt tolerance
in Arabidopsis. They produced six different transgenic
Arabidopsis plants overexpressing AtNHX1, SOS1, and
SOS3 alone or in different combinations (AtNHX1 ? SOS3,
SOS2 ? SOS3, SOS1 ? SOS2 ? SOS3). Surprisingly, the
AtNHX1 alone did not show significant salt tolerance. In
113
220 mM NaCl treatment for 3 days, less than 20% of the
control and transgenic plants overexpressing only AtNHX1
survived, but over 80% of the transgenic plants over-
expressing SOS1, SOS3, SOS2 ? SOS3, AtNHX1 ? SOS3,
or SOS1 ? SOS2 ? SOS3 survived [132]. Brini et al. [133]
reported that overexpression of wheat TVP1 (Tonoplast H?-
PPase) and NHX1 in Arabidopsis conferred better growth in
the presence of 200 mM NaCl and also under a water-
deprivation regime, while wild-type plants exhibited chlo-
rosis and growth inhibition. The proton pumps present at the
cellular membrane work as driving force for nutrient uptake
[134]. Three distinct proton pumps are responsible for the
generation of the proton electrochemical gradients: (1) the
plasma membrane H-ATPase pump (PM H-ATPase), (2)
vacuolar type H?ATPase (V-ATPase), and (3) the vacuolar
H-pumping pyrophosphatase (H-PPase). The PM H-ATP-
ase extrudes H? from the cell and thus generates a proton
motive force while V-ATPase and H-PPase acidify the
vacuolar lumen and other endomembrane compartments
[12]. Genetic evidence of physiological role of PM
H-ATPase and V-ATPase is very scarce, whereas, a few
studies have been carried out on H-PPase showing its
involvement in salt tolerance. The pioneer work was carried
out by Gaxiola et al. [135], overexpression of Avp1 (H?-
PPase) showed enhanced salt tolerance in transgenic Ara-
bidopsis plants. Later this gene was isolated from Thel-
lungiella halophila and engineered in tobacco, maize, and
cotton showed enhanced salt tolerance (Table 6). As evi-
dent from the literature (Table 6) a large interest have been
shown for overexpressing the transporter genes in model as
well as crop plants. In future, these transgenic plants should
be tested in the field to test the efficacy in the adverse
environment conditions.
Glyoxalase Pathway
Glyoxalase pathway has emerged as a prospective can-
didate for the genetic engineering of salt tolerance. The
glyoxalase pathway has shown its role in rapidly dividing
plant cells [136, 137] and in stress tolerance [138–140].
This pathway involves two enzymes, glyoxalase I and
glyoxalase II, which convert methylglyoxal to lactic acid
in two-step reactions [7]. Methylglyoxal is a potent
cytotoxic compound, and is the primary substrate for
glyoxalase I. Expression of the GlyI gene from Brassica
juncea showed salt tolerance in transgenic tobacco lines
[139]. Singla-Pareek et al. [141] achieved better salt stress
tolerance in tobacco by overexpression of both GlyI and
GlyII together. Further, Singla-Pareek et al. [142] trans-
formed only GlyII in rice plants, which showed higher
tolerance to NaCl.
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114
Role of Small RNA in Abiotic Stress Tolerance
Recently, a newer approach to understand the mechanism
of abiotic stress in plant system through miRNA/siRNA
approach is getting attention. The plant response to the
stress relies on the correct regulation of gene expression,
which depends on the number of mechanisms. The post-
transcription events play a crucial role in regulating gene
expression at a right time [143]. MicroRNA and short
interfering RNA are small (approx. 21 nt) non-coding
RNA, involved in regulating gene expression by mRNA
degradation, translational repression, and chromatin mod-
ification [144, 145]. The small RNA regulate gene
expression in stressed plants either by overexpression of
small RNA which resulted in downregulation of negative
regulators, or reduced biogenesis of small RNA which
causes downregulation of positive regulators and accumu-
lation of beneficial gene products [143].
Recently, a number of reports have been published
elucidating the role of microRNAs in regulating gene
expression under various environmental conditions [146–
151]. Some good reviews on role of microRNA in stress
response elucidating their roles have also been published
[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
miR398 has been studied by Sunkar et al. [149], where
mRNA abundance of Cu/Zn SOD1 (CSD1, cytosolic) and
Cu/Zn SOD2 (CSD2, chloroplastic) was observed inversely
correlated by the abundance of miR398 under salt stress.
There are very limited studies on the overexpression of
miRNA for abiotic stress tolerance. Atmir398 from Ara-
bidopsis was overexpressed in tobacco plants, caused
reduced seedling and root growth by down regulating
NtTIR1 expression and causing auxin insensitivity in
transgenic tobacco plants. These transgenic plants showed
enhanced resistance to salt stress by suppressing auxin
signal via degradation of NtTIR1 mRNA [156]. The rice
osa-MIR396c showed a dramatic transcript change under
salt and alkali stress conditions in Oryza sativa [157]. The
osa-MIR396c, osa-MIR393 transgenic rice, and Arabidop-
sis lines showed that several TFs related to growth, devel-
opment, and stress tolerant genes are targeted by these
microRNA and eventually showed reduced salt and alkali
stress tolerance compared to that of wild-type plants [157,
158]. Xia et al. [159] observed that OsmiR393 has same
results in transgenic rice as reported by Gao et al. [158] in
response to salt. The transgenic lines also showed two new
functions as increased tilling and early flowering. However,
the report of Gao et al. [157] is in contradiction to AtmiR396
overexpression in Arabidopsis [160], which conferred the
tolerance to drought and other stresses. The overexpression
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Mol Biotechnol (2013) 54:102–123
of Arabidopsis miR395c or miR395e retarded and accel-
erated the seed germination of Arabidopsis under high salt
or dehydration stress conditions, respectively. The overex-
pression of miR395c and miR395e, did not show the same
cleavage of mRNA targets, APS1, APS3, APS4, and
SULTR2;1, in both the overexpressing plants. Wang et al.
[161] reported suppression of an Arabidopsis bacterial-type
PEPC gene, Atppc4, by artificial microRNA (amiRNA).
Atppc4-amiRNA transgenic plants showed decreased
accumulation of Atppc4 transcripts, whereas other three
plant-type PEPC genes, Atppc1, Atppc2, and Atppc3 were
significantly upregulated and these transgenic plants
showed improved tolerance to salt stress.
These results demonstrated that a given miRNA family
containing a single nucleotide difference can guide the
cleavage of various mRNA targets, thereby acting as a
positive or negative regulator of seed germination under
stress [162]. These studies highlight that microRNA play
an important role in targeting various important genes for
managing stress tolerance in plants. Recently miRNAs is
getting increased attention and in near future a vast
knowledge on the mechanism of miRNA for abiotic stress
will be generated.
Conclusion
Genetic engineering towards developing salt and drought
tolerant crops is challenging and forefront area of research
for future crop improvement programs. Conventional
breeding has made considerable success in twentieth cen-
tury to improve crop yield and quality but limited work was
carried out for developing abiotic stress tolerant crop plants
with only few successful reports [9]. This is probably due
to the low magnitude of genetically based variation in the
plants for salinity tolerance. For achieving the salinity
tolerance, the genetic material from the distant wild rela-
tives or halophytes can be used for transfer the salt tolerant
genes into the sensitive plants through conventional
breeding but again the incompatibility of the reproduction
hinders in achieving the goal. With the advent of plant
molecular biology and understanding of the stress signal
transduction pathways, it is now possible to generate plants
with least damaging effect on environmental conditions
and concurrently promising an increase in productivity. So
far, many stress-related genes have been isolated and
characterized in the model plants like tobacco and Ara-
bidopsis. With the progress of whole genome sequencing
of different plant species, it seems now easier to identify
unique stress responsive genes. During the last decade,
transgenic plants with stress tolerant genes have been
generated using various genes from diverse pathways
Mol Biotechnol (2013) 54:102–123
(Tables 1–6). However, abiotic stress tolerant plant varie-
ties are yet to be released for the benefit of the farmers. The
sincere effort in this area is needed, in most cases trans-
genic plant performance are being evaluated under con-
trolled growth environments. Therefore, a gap remains
between the success in the laboratory and the application of
these techniques to develop the crops in the field. The
performance of the transgenic plants for salt tolerance
should be carried out in the laboratory by mimicking dif-
ferent stress together as in the field. Therefore, this area is a
serious concern and much attention needs to be focussed
towards the plants response to a combination of stresses.
Attempts made by Xue et al. [163] and Waterer et al. [164]
for evaluating the transgenic wheat and potato plants,
respectively, in the field to asses abiotic stress tolerance are
paragons. The AtNHX1 transgenic wheat lines produced
higher grain yields and heavier and larger grains in the field
of saline soils [163]. The potato transgenic lines showed
the higher yield in drought condition [164].
The basic genetic studies have shown that the stress
tolerant traits are multigenic in nature. Therefore, it is
significant to transfer multiple genes for better control of
stress tolerance traits. However, as evident from the tables,
there are very few studies, in which more than one gene has
been introduced [106, 132] into the same plant to confer
better stress tolerance.
A meticulous fine-tuning of the expression of the known
candidate genes for stress tolerance in specific temporal
and spatial patterns is also one of the essential parameters
for avoiding negative effects in plant growth and devel-
opment. The usage of specific TFs controlled by stress
inducible promoters could be a good choice for developing
diverse stress tolerance without compromising plant
energy. The genetic engineering of stress tolerance can
develop future crops to survive better in adverse environ-
mental conditions and lead to agricultural benefits.
Acknowledgments The financial support received from CSIR and
DST New Delhi is gratefully acknowledged. Kapil Gupta acknowl-
edges the award of CSIR-SRF.
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