Critical Reviews in Biotechnology

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DEAD box helicases as promising molecular tools

for engineering abiotic stress tolerance in plants

Sridevi Nidumukkala, Lavanya Tayi, Rajani Kant Chittela, Dashavantha

Reddy Vudem & Venkateswara Rao Khareedu

To cite this article: Sridevi Nidumukkala, Lavanya Tayi, Rajani Kant Chittela, Dashavantha Reddy
Vudem & Venkateswara Rao Khareedu (2019): DEAD box helicases as promising molecular
tools for engineering abiotic stress tolerance in plants, Critical Reviews in Biotechnology, DOI:
10.1080/07388551.2019.1566204

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Published online: 03 Feb 2019.

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CRITICAL REVIEWS IN BIOTECHNOLOGY
https://doi.org/10.1080/07388551.2019.1566204

REVIEW ARTICLE

DEAD box helicases as promising molecular tools for engineering abiotic

stress tolerance in plants
Sridevi Nidumukkalaa, Lavanya Tayia, Rajani Kant Chittelab, Dashavantha Reddy Vudema and
Venkateswara Rao Khareedua
a
Centre for Plant Molecular Biology, Osmania University, Hyderabad, India; bMolecular Biology Division, Bhabha Atomic Research
Centre, Mumbai, India

ABSTRACT ARTICLE HISTORY

Diverse abiotic stresses constitute one of the major factors which adversely affect the normal Received 28 December 2017
plant growth and development which results worldwide in decreased agricultural productivity. Revised 11 October 2018
At present, utilization of new molecular tools to achieve improved stress tolerance and increased Accepted 9 November 2018
crop productivity is highly desirable. Abiotic stress in plants induces expression of a wide range
KEYWORDS
of genes like transcription factors, defense related genes and so on, and the products of these Abiotic stress; DNA
genes are important in combating stress conditions. Helicases are one such category of proteins helicases; overexpression;
that play a key role in maintaining the genomic integrity of the cell by participating in nucleic plant DEAD box helicases;
acid mediated processes such as recombination, replication, and repair of DNA as well as the RNA helicases; RNA
unwinding of misfolded RNA structures that are formed during stress conditions. The DEAD box metabolism; stress
helicases are a subgroup of helicases which contain the amino acids Asp-Glu-Ala-Asp (DEAD) and tolerance; transgenic plants
are involved in the above molecular functions that mediate adaptation to stress. Overexpression
of DEAD box helicases is known to provide stress tolerance in various plants and thus their use
in developing stress tolerant plants is gaining importance. The plausible physiological mecha-
nisms of helicases in bestowing abiotic stress tolerance of plants include ROS scavenging,
enhanced photosynthesis, ion homeostasis and regulation of various stress responsive genes. In
this review, the characteristics of plant DEAD box helicases and the stress conditions under
which they express are discussed. We have provided a detailed description on the transgenic
plants overexpressing DEAD box helicases with an emphasis on their stress tolerance abilities.

Introduction characterized by the presence of amino acids in the

Helicases are ubiquitous and highly conserved enzymes
that are involved in nucleic acid mediated transactions.
They have received significant attention in recent years
due to their importance in stress tolerance. These
enzymes constitute the largest class of proteins in
nucleic acid metabolism and they perform a variety of
functions like replication, recombination, repair, tran-
scription, translation, and so forth. [1]. Helicases move
directionally from 50 to 30 or 30 to 50 disrupting the
hydrogen bonds between nucleic acid strands by utiliz-
ing the energy derived from ATP hydrolysis [2]. They
contain nine conserved motifs namely Q motif, motif I,
motif Ia, motif Ib, motif II, motif III, motif IV, motif V, and
motif VI (Figure 1). Helicases are classified into six
superfamilies SF1 to SF6 based on the sequence of con-
served motifs and their organization [3]. Among these
six superfamilies, the SF2 forms the largest helicase
superfamily and the motif II of SF2 proteins is
sequence, Asp-Glu-Ala-Asp (DEAD) and hence these
proteins are named as DEAD box helicases. The Q-motif
and motifs I and II (Walker motifs A and B) are involved
in ATP binding and hydrolysis. Motif III is associated
with the coupling of ATP hydrolysis with helicase activ-
ity. It was suggested that the Motif V might play a role
in substrate binding through the sugar-phosphate
backbone and they interact with NTP [4]. Motif VI is
believed to be associated with binding of the ATP phos-
phates, and loss of this motif’s function was associated
with impaired ATP hydrolysis in some DEAD-box pro-
teins [5]. The remaining motifs (Ia, Ib, IV) are involved in
nucleic acid binding induced ATP hydrolysis [6]. The
helicase domain of DEAD box helicases is surrounded
by auxiliary domains that are needed for essential func-
tion of these enzymes [7]. Based on substrate specifi-
city, helicases are mainly divided into three major
groups. These include: RNA helicases, DNA helicases
and dual helicases that unwind RNA, DNA or both DNA

CONTACT K. V. Rao rao_kv1@rediffmail.com Centre for Plant Molecular Biology, Osmania University, Hyderabad, Telangana 500 007, India
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 S. NIDUMUKKALA ET AL.
Figure 1. Schematic representation of the motifs of DEAD-box helicases. The Q, I, Ia, Ib, II, and III motifs represent domain I, and
IV, V, and VI motifs are in domain II. Capital letters indicate highly conserved amino acid sequences (80%) and “x” indicates less
conserved amino acids (50–79%).
and RNA hybrids. The DNA helicases usually belong to
superfamily1 and possess the sequence (G-X-X-X-X-G-K-
T) in the walker motif A, whereas the RNA helicases
possess the sequence (A-X-X-X-X-G-K-T) in this domain
and belong to superfamily 2 [8]. Major abiotic stresses
such as salinity, drought, cold, and heat are adversely
affecting the yields of various food crops. It is estimated
that more than 50% of the crop losses occur due to
these abiotic stresses worldwide [9]. Plants have devel-
oped various strategies to overcome these stress condi-
tions by modulating the expression of several stress
responsive genes [10]. The DEAD box helicases are a
unique group of genes, known to be expressed under
stress conditions in plants and the products of these
genes play crucial roles in tolerating stress conditions. It
is speculated that the stress tolerance mechanisms of
plant DEAD box helicases might be due to their
involvement in a wide variety of key cellular processes
such as mRNA transport or secondary structure
removal, regulation of gene expression and enhancing
or stabilizing protein synthesis [11–15]. Research con-
ducted so far on plant helicases, demonstrated great
potential for these proteins to protect plants from
many adverse effects caused by several stresses.
Various transgenic plants expressing DEAD box helicase
genes from different sources improved tolerance to dif-
ferent abiotic stress conditions [14,16–27]. Different
DEAD box helicases that exhibit tolerance to various
abiotic stresses are shown in the Figure 2. Earlier
Vashisht and Tuteja [2] suggested the probable action
mechanism of helicases for the stress tolerance of
plants. Nawaz and Kang [28] have recently summarized
the role of chloroplast and mitochondria targeted
DEAD box RNA helicases in RNA metabolism as well as
plant responses to abiotic stress. Baruah et al. [29] have
described the functional role of DEAD box RNA heli-
cases in conferring multiple abiotic stress tolerance. In
the current review, recent advances made on various
plant DEAD box helicases and their stress responsive-
ness are discussed. The expression mode pattern of
various DEAD box helicases under diverse stress condi-
tions have been described. Furthermore, insights
gained from studies on transgenic plants overexpress-
ing DEAD box helicases bestowing perceptible
tolerance against different abiotic stresses are explained
in detail.
The physiological mechanisms of different DEAD box
helicases involved in stress tolerance are described
briefly. The biochemical properties of these stress
responsive DEAD box helicases such as their specific
unwinding activity, polarity (direction of unwinding
50 –30 or 30 –50 ), localization, and so forth are also men-
tioned. The primary objective of this review is to high-
light the effectiveness of helicases in providing
improved abiotic stress tolerance of crop plants
through biotechnological approaches.
Stress responsive RNA helicases
The stress responsive RNA helicases reported from vari-
ous plant species are described in this section. These
RNA helicases show enhanced expression under differ-
ent abiotic stress conditions such as: cold, high salinity,
drought, heat, and so forth. Mutations in some of the
RNA helicases conferred sensitivity to different abiotic
stresses emphasizing their role in stress tolerance. A list
of RNA helicases with the conditions under which they
are expressed or the tolerance that is conferred by
them together with their specific role in RNA metabol-
ism is presented in the Table 1.
RNA helicases show enhanced expression
under various abiotic stress conditions
During exposure to various stresses, plants induce the
expression of a diverse array of genes including heli-
cases to combat the stress conditions. Transcripts of
some of the RNA helicases were found to be upregu-
lated under stress conditions in various plant species.
Among the plant species, the Arabidopsis genome con-
tains the largest number (more than 58 genes) of DEAD
box RNA helicases, and some of these are responsive to
various environmental stresses [43]. The Arabidopsis
thaliana RNA helicase 57(AtRH57) that exhibits ATP
independent RNA helicase activity and localizes to the
nucleus and nucleolus was induced by phytohormone
abscisic acid (ABA), high glucose and salt conditions,
and mutation in the protein inhibited ribosome
 CRITICAL REVIEWS IN BIOTECHNOLOGY 3

Figure 2. Diagram showing stress tolerance functions of different plant DEAD box helicases. DEAD box helicases from different
plant species showing tolerance to a particular type of abiotic stress are grouped in each circle.

Table 1. List of stress responsive RNA helicases and their role in RNA metabolism.
S. No RNA Helicase Plant source Stress condition Role in RNA metabolism References
1 AtRH3 Arabidopsis thaliana Cold and Salt Splicing [30,31]
2 AtRH7 Arabidopsis thaliana Cold Ribosome biosynthesis [32,33]
3 AtRH9 Arabidopsis thaliana Cold ND [16]
4 AtRH25 Arabidopsis thaliana Cold ND [16]
5 AtRH57 Arabidopsis thaliana Salt, ABA, High glucose Ribosome formation [34]
6 AtHELPS Arabidopsis thaliana Cold, low Kþ and Zeatin ND [35]
7 LOS4 Arabidopsis thaliana Cold mRNA transport [11,13]
8 RCF-1 Arabidopsis thaliana Cold Pre-mRNA splicing [36]
9 OsABP Oryza sativa Salt, dehydration, ABA, Blue and m-RNA and t-RNA synthesis, transcription, [37]
Red light nuclear transport
10 OsRH17 Oryza sativa ABA and NAA 16s rRNA maturation [38]
11 GmRH Glycine max Cold, salinity ND [39]
12 HVD1 Hordeum vulgare Cold, Salinity, Drought, ABA ND [40]
13 CbDRH Chorispora bungeana Cold ND [41]
14 TaRH1 Triticum aestivum Cold, Salinity ND [42]
ND: Not Determined.

formation [34]. The Arabidopsis DExD/H box RNA heli-
case (AtHELPS) was upregulated by low potassium, zea-
tin and cold treatments. During low potassium levels,
the mRNA levels of different transport proteins such as
Arabidopsis K þ transporter 1 (AKT1), Calcineurin B like
protein (CBL1 /9), and CBL-interacting protein kinase 23
(CIPK23) were higher in the AtHELPS mutants indicating
the regulation of potassium levels by this protein under
salt stress [35]. Differential expression patterns were
observed for two of the RNA helicases, A. thaliana RNA
helicase 9 (AtRH9) and A. thaliana RNA helicase 25
(AtRH25) when subjected to different abiotic stresses.
The AtRH9 and AtRH25 genes were induced in response
to cold stress while they were down regulated during
drought and salinity stress. Although, it was observed
that the transcript levels of both AtRH9 and AtRH25
were upregulated in response to cold stress, AtRH25
alone was found to confer cold tolerance in transgenic
Arabidopsis [16]. In addition to these RNA helicases,
microarray data of Arabidopsis has revealed many
chloroplast and mitochondria targeted DEAD box heli-
cases that control transcription under many abiotic
stress conditions [44].
Stress responsive RNA helicases were also identified
and studied from other plant species. A rice DEAD-box
RNA helicase protein, Oryza sativa ATP-binding protein
4 S. NIDUMUKKALA ET AL.
(OsABP) was seen to be upregulated in response to salt
stress, dehydration, ABA treatment, blue and red light
exposure, signifying its role in multiple abiotic stress
responses. Participation of this protein in RNA metabol-
ism was demonstrated by in silico analysis, which indi-
cated that OsABP interacts with various proteins
involved in tRNA and mRNA synthesis, transcription,
nuclear transport, and so forth [37]. Another DEAD box
RNA helicase from rice that is nuclear localized, O. sativa
RNA helicase 17 (OsRH17) was upregulated by phyto-
hormones NAA (naphthalene acetic acid) and ABA
treatments. In E. coli cells, expression of the OsRH17
protein was seen to suppress the maturation of 16S
rRNA. However, a similar suppression was not observed
in the transgenic rice overexpressing OsRH17 plausibly
owing to its redundancy under natural conditions [38].
The expression of a putative chloroplast localized ATP
dependent RNA helicase, HVD1 from Hordeum vulgare
was induced by various abiotic stresses such as cold,
salinity, drought, and ABA, with enhanced expression
under salt stress. It was suggested that the role of this
protein in RNA metabolism might be through RNA
binding to RGG motifs present at its C-terminal end of
the protein [40]. Glycine max RNA helicase (GmRH) an
ATP independent, nucleolus localized RNA helicase, is
responsive to cold and salinity stresses [39]. In cryo-
phyte C. bungeana, it was observed that one of the two
splice variants of a cold stress responsive DEAD box
RNA helicase (CbDRH) expresses under cold stress con-
ditions and it interacted with a cold-responsive, gly-
cine-rich RNA binding protein (CbGRP) [41].
Mutational studies on RNA helicases involved
in abiotic stresses tolerance
Studies on mutants of RNA helicases have indicated
their role in tolerating various abiotic stresses but
largely for cold stress tolerance. Cold stress experienced
by plants, i.e., exposure of plants to suboptimal temper-
atures (0–12  C) poses serious threats to crop productiv-
ity. Plants under cold stress show symptoms such as
poor germination, stunted growth of seedlings, chlor-
osis (discoloration of leaves), wilting of leaves, and so
forth, finally leading to their death. At the cellular level,
cold stress induces cell membrane damage, solute leak-
age, metabolite imbalance, and so forth. Cold stress tol-
erance is mediated through cold responsive genes and
transcription factors by their active participation in
repair processes [45].
The Arabidopsis plants expressing low levels of
osmotically responsive gene 4(los4-1) mutants sensitive
to chilling temperatures were identified in a genetic
screen. Under cold stress, the mutant showed
decreased expression of cold responsive genes. The
expression of C-repeat binding factor (CBF) genes,
which act as transcriptional activators of cold respon-
sive genes is also reduced or delayed in los4-1 mutant
plants. The chilling sensitivity of los4-1 mutant plants is
reversed by continuous expression of the CBF3 gene.
Further, the LOS4 gene was found to encode a nucleus
and cytoplasm localized DEAD-box RNA helicase pro-
tein [13]. Later, an Arabidopsis mutant, a cryophyte tol-
erant to chilling and freezing stress but sensitive to
heat and phytohormone was isolated. This mutant
showed enhanced expression of CBF and its down-
stream target genes under cold stress. The mutant
plants were smaller in size and flowered earlier as com-
pared to wild type plants. The mutation was also identi-
fied to be present in the same los4 locus and thus the
mutant is named as los4-2 mutant. The los4-2 mutation
changes Glu-94 to Lys, and the los4-1 mutation changes
Gly-364 to Arg. The mRNA export seems to be affected
differently in these two los4 mutants in response to
temperature stress. Whereas in the los4-2 mutants, the
mRNA export is blocked only at high temperatures but
in the previously identified los4-1 mutant, the export of
mRNA is affected both at high and low tempera-
tures [11].
The knockout mutants of Arabidopsis thaliana RNA
helicase 7(atrh7) also exhibited hyper sensitivity to cold
stress. The mutant lines rh7-5 and rh7-8 with insertions
in exon 4 and 8, respectively, showed various abnormal
morphological features such as short roots, disturbed
vein pattern in leaves, and so forth, that are shown by
mutants of ribosome-related proteins. At low tempera-
ture, the emergence of both radicle and first leaf was
severely delayed in atrh7mutants. The mutants also dis-
played defective pre-rRNA processing as seen by accu-
mulation of 35S and 18S rRNA precursors. The AtRH7
was shown to interact with the RNA processing, cold
shock domain protein A. thaliana Cold Shock Protein 3
(AtCSP3), which is involved in the adaptation to cold
[32]. Subsequently, two more allelic mutants, atrh7-2
and atrh7-3 having T-DNA insertions in exon 8 and
intron 7, respectively, with cold hypersensitivity and
decreased expression of CBF genes were isolated. These
mutants have shown developmental defects and also
accumulated 18S rRNA precursors confirming the role
of AtRH7 in plant development, rRNA biogenesis and
cold tolerance [33].
The phenomenon of splicing also has an important
role in stress tolerance mediated by RNA helicases as
evident from the mutational studies on a couple of RNA
helicases wherein the mutants have displayed improper

splicing. The chloroplast localized A. thaliana RNA heli-
case 3 (AtRH3) was shown to be involved in splicing of
group II introns, maturation of rRNA, and ribosome
assembly. The null mutants of atrh3 were found to be
embryonic lethal, but mutants with weak allele (rh3-4)
developed into pale-green seedlings [30]. In a subse-
quent study, the rh3-4 mutant with T-DNA insertion in
intron 9 or the miRNA mediated knock down mutant
displayed retarded growth and pale-green phenotypes
under salt and cold stresses, while dehydration stress
resulted in only marginal growth defects. Moreover,
intron splicing of ndhA and ndhB genes of chloroplast
was severely affected in mutants under salt and cold
stresses but not under dehydration stress, indicating an
important role for this stress responsive helicase in
intron splicing and plant growth [31]. The cold indu-
cible helicase regulator of CBF gene expression-1(RCF-
1) has a role in pre-mRNA splicing. Under cold stress
conditions, the rcf-1 mutant, where glycine at 808th
position would change to arginine, is hypersensitive to
cold and displayed mis-splicing of various cold respon-
sive genes. This indicated that the role of RCF-1 in cold
tolerance is through proper splicing of cold responsive
genes [36].
Mutations in stress responsive suppressor genes,
strs1 and strs2 (AtRH25), resulted in increased salt,
osmotic and heat stress tolerance. The strs1 and strs2 T-
DNA insertion mutants carry mutations in exon 6 and
promoter regions, respectively. In these strs mutants,
salt, osmotic, and cold stresses induced the expression
of transcriptional activators, while heat stress induced
the expression of the heat shock transcription factor
genes. Taken together, the STRS1 and STRS2 proteins
have attenuated the expression of stress-responsive
transcriptional activators implicating their role in sup-
pressing the effects of multiple abiotic stresses [18]. It
was also shown that these STRS proteins are involved
in epigenetic gene silencing and undergo re-localiza-
tion upon exposure to stress conditions [46,47].
In conclusion, all the above-mentioned studies on
RNA helicases have clearly demonstrated the ability of
plant RNA helicases in mitigating the effects of multiple
abiotic stresses through RNA mediated cellular proc-
esses such as splicing, processing, transport and ribo-
some assembly. But how the stress tolerance is
achieved through these RNA processes is not well
understood. Certain sequences or motifs of helicases
were thought to be essential in interacting with either
RNA or other RNA binding proteins and such interac-
tions seem to be important for stress tolerance function
of helicases [40]. Detailed studies about these protein
interactions are needed for a better understanding of
CRITICAL REVIEWS IN BIOTECHNOLOGY 5
the stress tolerance mechanism exhibited by
RNA helicases.
Transgenic expression of plant DEAD box
helicases confers stress tolerance
The ability of transgenic expression of plant DEAD box
helicases to confer abiotic stress tolerance is well docu-
mented. Transgenic expression of DEAD box helicase
genes from different plant sources and their stress tol-
erance ability are discussed in this section. A list of
stress tolerant transgenic plants developed by overex-
pressing different plant DEAD box helicase genes is
given in the Table 2.
Salinity is one of the major environmental stresses
that adversely affects plant development and yield.
High salinity levels prevent absorption of water and the
movement of nutrients in plants. High levels of reactive
oxygen species (ROS) production and a disturbed ion
homeostasis are usually observed during salt stress.
One of the main adaptive methods employed by plants
to tolerate salt stress is reduction of Naþ ion uptake
and retention of Kþ ions. Hence, plants tend to main-
tain a high Kþ/Naþ ratio under salt stress, which is
essential to retard these stress conditions [53]. The
involvement of DEAD box helicases of plant origin in
salinity tolerance has been demonstrated in great detail
by overexpressing them in various crop plant species.
The DEAD box helicases from the pea plant have been
investigated for stress tolerance in detail. The trans-
genic plants expressing pea family DEAD box helicases
such as PDH45, PDH47, Psp68, and MH1 have been
generated and studied for stress tolerance
[14,17,19–22,26,27,48,49,54,55]. Among these, PDH45
has been extensively studied for salt stress tolerance in
a wide variety of crop plants. The transgenic tobacco,
rice, pea, sugarcane and chili plants expressing PDH45
conferred explicit salinity tolerance [14,19–21,48]. The
PDH45 is a homolog of eukaryotic translation initiation
factor 4 A (eIF4A), which localize to both nucleus and
cytoplasm and it possess an ATP-dependent DNA heli-
case activity in 30 –50 direction as well as RNA helicase
and DNA-dependent ATPase activities. It was also
observed that the activity of topoisomerase I was stimu-
lated upon its interaction with PDH45. The role of
PDH45 in protein synthesis was amply demonstrated
using in vitro protein translation system [12].
The expression analysis of PDH45 in transgenic
tobacco revealed, induction with sodium ions but not
with lithium ions. Other abiotic stresses such as dehy-
dration, wounding, low temperature and ABA treat-
ments also led to the upregulation of PDH45 [14].
6 S. NIDUMUKKALA ET AL.

Table 2. List of stress tolerant transgenic plants expressing DEAD box helicase genes.
Physiological
processes involved in Type of
S. No Gene Plant source Transgenic plant stress tolerance stress tolerance Reference
1 PDH45 Pisum sativum Tobacco Low accumulation of Salt stress [14]
Naþ, ROS and cal-
cium homeostasis
2 PDH45 Pisum sativum Rice Stabilized or Salt stress [19]
enhanced pro-
tein synthesis
3 PDH45 Pisum sativum Rice Induced ion Salt stress [27]
homeostasis
4 PDH45 Pisum sativum Pea Improved cellular Salt stress [20]
level tolerance
5 PDH45 Pisum sativum Sugarcane Enhanced cell mem- Salt stress [21]
brane thermal stabil-
ity and upregulation
of stress
related genes
6 PDH45 Pisum sativum Chilli Enhanced protein Multiple abi- [48]
synthesis by removal otic stresses
of second-
ary structures
7 PDH47 Pisum sativum Rice Regulation of stress Drought stress [26]
responsive genes
8 PSP68 Pisum sativum Tobacco Reduction of oxida- Salt stress [22]
tive stress and
improved photosyn-
thesis machinery
9 PSP68 Pisum sativum Rice Attenuation of ionic Salt stress [49]
adjustment and
ROS scavenging
10 MH1 Medicago sativa Arabidopsis ROS scavenging Multiple abi- [17]
otic stresses
11 OsSUV3 Oryza sativa Rice Improved photosyn- Salinity and [23]
thesis and antioxidant drought stress
machinery
12 OsBIRH1 Oryza sativa Arabidopsis Interaction with dis- Biotic and abi- [25]
ease resistance sig- otic stresses
naling pathways
13 OsBAT1 Oryza sativa Rice ROS detoxification Salt stress [50]
and expression of
genes in stress sig-
nal pathways
14 TOGR1 Oryza sativa Rice Pre rRNA processing Heat stress [51]
at high temperature
15 SlDEAD31 Solanum lycopersicum Tomato Expression of stress Salt stress [24]
responsive genes
16 AvDH1 Apocynum venetum Cotton ROS detoxification Salt stress [52]
17 AtRH25 Arabidopsis thaliana Arabidopsis RNA chaper- Cold stress [16]
one activity

Similar results were obtained when the PDH45 gene
was expressed in the indica rice variety IR64. The trans-
genic rice lines exhibited salinity tolerance besides
improved physiological traits under high salt conditions
and it was hypothesized that the PDH45 is involved in
increasing or stabilizing protein synthesis under stress
conditions [19]. In a different study, it was shown that
expression of PDH45 in IR64 rice lines imparted salt tol-
erance by regulating sodium ion levels, ROS production,
cytosolic calcium ion homeostasis, cation transporters
in rice plants [55]. Further, in the transgenic IR64 rice
plants expressing the PDH45 helicase or O. sativa
Suppressor of Var1-3 (OsSUV3) genes exhibited
decreased levels of apoptosis and some senescence
associated genes were downregulated leading to
delayed leaf senescence [56]. The Bangladeshi rice var-
iety Binnatoa transgenic plants expressing PDH45 also
showed improved salt tolerance by maintaining a
higher Kþ/Naþ ratio in leaf tissue and decreased root
length under normal physiological conditions when
compared to the WT plants. Moreover, the transgenic
plants showed better fertility and higher seed yield
than WT plants [27]. Overexpression of PDH45 gene in
pea plants exhibited salinity tolerance and delayed sen-
escence in the transgenics. Under desiccation stress,
the transgenic plants remained green and displayed
better cell membrane integrity than WT plants. The
transgenic plants also exhibited an improved yield
 CRITICAL REVIEWS IN BIOTECHNOLOGY 7

Table 3. Amino acid sequences of different motifs in various stress tolerant DEAD box helicases.
DEAD box
helicase Q Motif Motif I Motif I a Motif I b Motif II Motif III Motif IV Motif V Motif VI Type of stress tolerance
PDH45 GFEKPSAIQ AQSGTGKT PTRELA TPGRV DESD SRT LYSVNTKR LITTDVWARGLD HRIGRSGR Multiple abiotic stresses
PDH47 GFEKPSAIQ AQSGTGKT PTRELA TPGRV DEAD SAT VIFVNTRR LITTDLLARGID HRIGRSGR Drought stress
P68 EYTRPSSIQ AETGSGKT PTRELA TPGRF DEAD SAT – LVATDVASRGLD HRIGRTGR Salinity stress
MH1 GFEHPSEVQ AKSGMGKT HTRELA TPGRI DECD SAT VIFVKSVS LVATDLVGRGID HRVGRAGR Multiple abiotic stresses
OsBAT1 GFEHPSEVQ AKSGMGKT – TPGRI DECD SAT VIFVKSVS LVATDLVGRGID HRVGRAGR Salinity stress
OsBIRH1 GFPRPSHIQ DQSGSGKT PTAELA TPGRF DEVD – – LVCTDRASRGID RRVGRTAR Oxidative and biotic stresses
SlDEAD31 GWKNPSKIQ AQTGSGKT PTRELA TPGRL DEAD SAT – LICTDVASRGLD HRVGRTAR Salinity stress
AvDH1 GFEKPTPIQ AQTGSGKT PTRELA TPGRL DEAD TAT VIFCNTRR LITTDVWARGID HRIGRTGK Salinity stress
AhRH47 GFEKPSAIQ AQSGTGKT PTRELA TPGRV DEAD SAT VIFCNTRR LITTDLLARGID HRIGRGGR Multiple abiotic stresses
TOGR1 GWKEPTRIQ GQTGSGKT PTRELA TPGRL DEAD SAT – LICTDVASRGLD HRVGRTAR Heat stress

compared to that of control plants, both under non-
stress and stressed conditions due to constitutive
expression of helicase [20]. The PDH45 gene was also
used for the development of stress tolerant sugarcane
cultivars; the transgenic sugarcane expressing the
PDH45 showed better photosynthetic efficiency, higher
cell membrane thermostability, more water content
and gas exchange parameters under moisture stress
[21]. The ability of PDH45 in mitigating multiple abiotic
stresses has been demonstrated in Capsicum annuum;
the transgenic capsicum plants expressing PDH45 dis-
closed similar responses to various abiotic stresses such
as salt stress, oxidative stress, senescence and moisture
stress as other PDH45 transgenic plants [48]. More than
one stress tolerance trait, salinity and herbicide toler-
ance were observed with overexpression of PDH45
along with EPSPS (5-enoylpruvyl shikimate-3-phosphate
synthase) in tobacco [57].
The mechanism of PDH45 mediated salinity stress
tolerance was investigated in transgenic tobacco and
rice using CoroNa green dye based sodium localization
in root and shoot sections [54]. Under salinity stress,
the PDH45 transgenics accumulated lesser sodium in
shoots and higher sodium levels in roots as compared
to the corresponding WT plants. The authors have
explained that PDH45 was probably involved in the
deposition of apoplastic hydrophobic barriers which
inhibited sodium transport to shoot, thus providing sal-
inity stress tolerance in transgenic plants [54].
Transgenic rice expressing PDH45 and transgenic
tobacco expressing a non-DEAD box helicase, Mini
Chromosome Maintenance 6 (MCM6) were studied to
assess the impact of transgenic plants upon the soil
environment, where it was observed that the overex-
pression of PDH45 or MCM6 neither led to any changes
in the microbial population nor the enzyme compos-
ition of the soil was altered, justifying the amenability
of transgenic plants overexpressing helicase genes for
abiotic stress tolerance [58,59].
The pea helicase, PDH47 is also a homolog of eIF4A
that localizes and possess similar activities as that of
PDH45 despite the sequence differences in the motifs II
and III of these two proteins [15,60]. The PDH45 con-
tains DESD and SRT amino acid sequences in the motifs
II and III, respectively, whereas the PDH47 has DEAD
and SAT sequences in these motifs (Table 3).
Phosphorylation of PDH47 at Ser and Thr residues
enhances its activity [15]. The transcripts of PDH47
were observed to be induced under cold and salinity
stresses in both roots and shoots. However, ABA
induced the expression of the PDH47 gene only in roots
but not in shoots indicating that the expression of
PDH47 was mediated through both ABA-dependent
and independent pathways under abiotic stress condi-
tions [15]. Recently, it was reported that the transgenic
rice lines expressing PDH47exhibited explicit tolerance
to drought stress [26].
Another pea helicase, Psp68 transcript was upregu-
lated under salinity stress in pea. It is a homolog of
human p68 helicase and exhibits RNA helicase and
bidirectional (50 –30 and 30 –50 direction) DNA helicase
activities [61]. It was identified as a self-interacting,
cytosol localized helicase. The amino acids in the Q
motif and motif V of Psp68 are slightly different from
that of PDH45 and PDH47, although all of them exhibit
salinity tolerance (Table 3). The functional characteriza-
tion of Psp68 and its role in salinity stress tolerance has
been studied in tobacco. Transgenic tobacco plants
expressing Psp68 gene exhibited enhanced tolerance to
salinity stress by improving photosynthesis and ROS
accumulation. Moreover, the transgenics accumulated
higher Kþ and lower Naþ levels than the WT plants and
this could be responsible for increased salt stress toler-
ance [22]. In a similar study, the transgenic rice lines
expressing Psp68 exhibited enhanced salinity tolerance
by controlling ROS production and accumulating higher
Kþ and Ca2þ and lower Naþ levels than the WT
plants [49].
Medicago sativa helicase 1(MH1) is also a homolog of
PDH45 that is localized only in the nucleus unlike
PDH45 which localizes in both nucleus and cytoplasm.
Mannitol, NaCl, methyl viologen and ABA induced the
8 S. NIDUMUKKALA ET AL.
expression of MH1 helicase and the transgenic expres-
sion of MH1 in Arabidopsis improved seed germination
and plant growth under drought, salt and oxidative
stresses [17]. The O. sativa antigen-B-associated tran-
script 1 (OsBAT1) helicase is a bipolar RNA helicase that
exhibits DNA and RNA-binding and DNA/RNA-depend-
ent ATPase activities. The OsBAT1 expression is induced
under abiotic stresses such as salt stress and ABA [62].
Overexpression of the OsBAT1 in IR64 rice lines pro-
vides salinity stress tolerance by expressing several
stress responsive genes and improving ROS detoxifica-
tion [50]. Both MH1 and OsBAT1 possessed DECD and
SAT sequences in the motif II and III, respectively
(Table 3).
The O. sativa Suppressor of Var1-3 (OsSUV3) helicase,
which is localized in mitochondria, exhibits both DNA
and RNA helicase as well as ATPase activities. It is a
bipolar enzyme which unwinds single stranded nucleic
acid overhangs utilizing ATP or dATP as the energy
source. The IR64 transgenic rice expressing OsSUV3
were found to exhibit high salinity and drought stress
tolerance by improved photosynthesis and antioxidant
mechanisms in transgenics [23]. Higher phytohormone
levels were maintained in OsSUV3 rice transgenics
under salt stress [63]. The OsSUV3 is also shown to pro-
vide metal stress tolerance to cadmium and zinc metals
in transgenic rice [64]. In theOsSUV3overexpressing rice
transgenics, senescence associated genes were downre-
gulated and DNA repair genes were upregulated
together with increased telomere length demonstrating
its role in delaying senescence [56].
Two of the tomato genes, Solanum lycopersicum
DEAD-box RNA helicase 30 and 31 (SlDEAD30) and
(SlDEAD31) were found to be salt responsive. However,
only the SlDEAD31 but not SlDEAD30 transcripts were
upregulated under heat, cold, and dehydration stress
conditions [24]. The transgenics overexpressing
SlDEAD31 exhibited marked salt tolerance and low level
drought tolerance. Moreover, SlDEAD31 transgenics
showed higher survival rate, relative water content
(RWC), chlorophyl content, malondialdehyde (MDA)
production and lower water loss rate as compared to
the WT plants, demonstrating the role of SlDEAD31 in
abiotic stress tolerance [24]. The Arachis hypogaea RNA
helicase 47 (AhRH47) is a multistress responsive heli-
case and high levels of its expression was observed
under salinity and drought stresses. AhRH47 is also
homologous to eIF4A and thought to be involved in ini-
tiation of translation as the protein synthesis in the
AhRH47 transgenic plants is maintained under stress
conditions [65].
The overexpression of Apocynum venetum DEAD box
helicase1 (AvDH1) in transgenic cotton conferred salin-
ity tolerance and showed increased yield potential
when compared to WT plants in the saline field studies.
It was suggested that the low membrane ion leakage
and increased superoxide dismutase activity in trans-
genic plants was responsible for its better performance
[52]. The motif III of the AvDH1 possessed TAT
sequence instead of conserved SAT sequence (Table 3).
A nucleolar RNA helicase of rice, thermotolerant
growth required 1(TOGR1) was shown to be involved in
thermotolerance by associating with preRNA processo-
some and maintaining normal ribosomal RNA levels at
high temperatures. Overexpression of the TOGR1 was
found to increase the growth of rice plants under hot
conditions, thus justifying its role in thermotolerance by
maintaining the rRNA homeostasis [51]. The motif I of
TOGR1 possesses the sequence GQTGSGKT instead of
the conserved sequence AxxGxKT (Table 3). The above
discussed DEAD box helicases have clearly demon-
strated that their transgenic overexpression confers tol-
erance to multiple abiotic stresses.
DEAD box proteins causing negative
regulation of abiotic stress
Overexpression of plant DEAD box proteins often
results in enhanced stress tolerance owing to positive
regulation. However, plants expressing a few proteins
containing DEAD box disclosed sensitivity to different
stresses through their negative regulation. In
Arabidopsis, incidence of mutation in los4 gene ren-
dered the plants tolerant to freezing and cold stresses
but were also sensitive to heat stress [11]. Similarly,
mutations in strs1 and strs2 genes that were down
regulated by multiple abiotic stresses rendered plants
tolerant to salt, osmotic and heat stresses. Further,
these mutants revealed increased expression of tran-
scriptional activators DREB1A/CBF3, DREB2A and that of
the downstream DREB target gene RD29A under salt,
osmotic and cold stresses, while heat shock transcrip-
tion factors HSF4 and HSF7 showed enhanced expres-
sion during heat stress [18]. Through deployment of
recently developed CRISPR/Cas tools for genome edit-
ing, it is feasible to explore the knockout of such nega-
tive genes to develop abiotic stress tolerant plants.
DEAD box helicases and biotic stress
Although the majority of the DEAD box helicases were
demonstrated to provide abiotic stress tolerance, the
Triticum aestivum RNA helicase1 (TaRH1) helicase and O.

sativa BTH-induced RNA helicase 1 (OsBIRH1) were
found to confer tolerance to both abiotic and biotic
stresses. The TaRH1 is an ATP-dependent RNA helicase
involved in the defense signaling pathway. The expres-
sion of TaRH1 was more under cold and salinity stresses
when compared to wounding and PEG treatments. The
results of TaRH1 gene expression upon infection with
Puccinia striiformis f. sp. Tritici (Pst) strains indicated that
it positively regulates resistance to this pathogen.
Further, it was proposed that this defense response,
shown by TaRH1 to Pst infection, in wheat was medi-
ated through either salicylic acid or methyl jasmonic
acid (MeJA) dependent signal transduction path-
ways [42].
A rice helicase, OsBIRH1 that possess both RNA-
dependent ATPase and ATP-dependent RNA helicase
activities was found to confer both biotic and oxidative
stress tolerance [25]. The Arabidopsis transgenics
expressing OsBIRH1 were shown to confer resistance to
pathogens Pseudomonas syringae pathovar tomato
DC3000 and Alternaria brassicicola. The enhanced resist-
ance exhibited by the transgenic plants is mainly due to
the increased expression of pathogenesis related genes
namely PR-1, PR-2, PR-5, and PDF1.2. These transgenics
revealed better seed germination, less growth retard-
ation and increased seedling fresh weight as compared
to WT plants. Moreover, these transgenics disclosed ele-
vated expression levels of oxidative defense genes, viz.,
ascorbate peroxidase 1 (AtApx1), ascorbate peroxidase 2
(AtApx2) and Fe-superoxide dismutase 1 (AtFSD1). It was
therefore suggested that the observed oxidative
response contributes to enhanced abiotic stress toler-
ance [25]. The OsBIRH1 contains a DEVD sequence in
the motif II instead of DEAD box (Table 3).
Localization of plant DEAD box helicases
The major metabolic organelles of the cell, the chloro-
plasts and mitochondria, are also affected under stress
conditions. These organelles perceive and respond to
various biotic and abiotic stresses by modulating the
expression of several stress responsive genes [66–68].
Some of the nucleus encoded RNA helicases are trans-
ferred to these organelles for regulating gene expres-
sion under stress conditions. Hence, cross talk between
nucleus, chloroplast and mitochondria is essential for
plant growth and development under stress [69–71].
The DEAD box helicases, PDH45, PDH47, and LOS4 are
known to be localized in both the nucleus and cytosol.
Localization in more than one place indicates the multi-
functionality of these proteins. The Psp68 was found in
the cytosol as well as in the surroundings of nucleus.
CRITICAL REVIEWS IN BIOTECHNOLOGY 9
The AtRH57, AtRH7, STRS1&2, RCF-1, TOGR1 are local-
ized in nucleus and nucleolus. Under stress conditions,
the nucleus or nucleolus localized DEAD box helicases
might be involved in the early stages of pre-RNA proc-
essing, transcription and repair. The DEAD box helicases
of OsSUV3, AtRH9, and TaRH1 are present in mitochon-
dria, whereas HVD1, OsABP, and AtRH3 are localized in
chloroplasts. The mitochondria and chloroplast local-
ized DEAD box helicases play a role at the post tran-
scriptional level during stress conditions [28].
Role of helicase promoters in stress tolerance
In many studies, transgenic expression of plant heli-
cases for stress tolerance is carried out using constitu-
tive promoters like Cauliflower mosaic virus (CaMV35S),
Porteresia coarctata (Port Ubi 2.3), and so on. Use of
native promoters for the expression of helicases is sug-
gested as their continuous expression may result in lim-
ited growth and low yield [72,73]. In this attempt, a
multi stress inducible promoter from rice, OsXPB2 pro-
moter has been identified. It contains the cis-elements
CACG, GTAACG, CACGTG, CGTCA, and CCGCCGCGCT
that are responsible for various abiotic stresses such as
salt, dehydration, cold, MeJA, and ABA, respectively
[74]. An abiotic stress and hormone responsive pro-
moter of pea helicase, Psp68 was isolated and charac-
terized. This promoter possessed hormone and stress
associated cis regulatory elements such as E-box,
AGAAA, GATA-box, ACGT, GAAAA, and GTCTC [75].
Functionality of these two promoters under various abi-
otic stresses was assessed by Agrobacterium-mediated
transient assay in tobacco [74,75]. A promoter of
another pea helicase gene, PDH45 has been isolated
and in silico analysis of this promoter revealed that this
promoter contained many stress responsive cis regula-
tory elements such as ABRE, CCAAT box, MBS, G-box,
GARE motif, and TGA element [76]. The functional role
of this PDH45 promoter has yet to be determined.
Hence, the use of specific stress inducible promoters for
helicases expression is an ideal choice for developing
stress tolerant plants in the future.
Conclusions and future perspectives
Helicases are proteins that participate in nucleic acid
transactions and are essential for normal functioning of
the cells. This review focuses on the stress responsive
DEAD box helicases and their involvement conferring
abiotic stress tolerance. In the presence of various stress
conditions, DEAD box helicases often exhibit enhanced
expression, and diverse studies have amply
10 S. NIDUMUKKALA ET AL.
demonstrated that overexpression of these helicases
bestows abiotic stress tolerance in plants. The unique
state of the art genome editing technologies using
CRISPR/Cas9 system can be profitably utilized for engin-
eering plants with inbuilt stress tolerance. Plausibly,
adoption of these tools enables generation of knockout
lines of specific DEAD box helicases that are down
regulated under various stress conditions. Thus, DEAD
box helicases serve as promising candidates for engin-
eering different stress tolerant plants.
In future, the following lines of research activities
can be contemplated that probably accelerate the
potential use of DEAD box helicases in modulating the
stress tolerance of different plants:
 A unique set of plant DEAD box helicases have
been found to show great potential for tolerance
to multiple abiotic stresses. Hopefully, homologs of
such helicases can be deployed in economically
important crop plants for developing multiple stress
tolerant plants.
 Co-expression of certain DEAD box helicases in con-
junction with other stress responsive genes might
cause additive effects thus providing tolerance to
multiple stresses.
 Isolation of DEAD box helicases from resilient plants
that thrive under harsh environments and their use
in designing stress tolerant plants would be a bet-
ter option as they likely result in greater levels of
stress tolerance.
 Since both biotic and abiotic stresses are known to
coexist in nature, development of such stress resist-
ant/tolerant plants is always beneficial. However,
DEAD box helicases reported so far have largely
conferred tolerance to only abiotic stresses.
Helicases that bestow tolerance/resistance to both
abiotic and biotic stresses are few. Therefore, more
such proteins need to be identified and functionally
characterized.
 A few RNA helicases with established roles in RNA
metabolism are known; however, their stress toler-
ance ability has not yet been determined. Their per-
formance under stress conditions can be assessed
for their use with evolving stress tolerant plants.
 Structure-function analysis of plant DEAD box heli-
cases might help to understand the molecular
mechanisms of diverse helicases which facilitates
improvement in the stress tolerance of plants.
Acknowledgments
SN wishes to acknowledge the University Grants
Commission, New Delhi, for the award of Dr. D.S. Kothari-
fellowship. LT would like to thank Department of Atomic
Energy (DAE), Government of India, for providing financial
support. We extend our thanks to Prof. T. Papi Reddy of the
Department of Genetics, Osmania University for his critical
reading and improving the manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
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