15
19 Helicases in Improving Abiotic Stress Tolerance in Crop Plants Narendra Tuteja, Sarvajeet Singh Gill, and Renu Tuteja Abiotic stress conditions adversely affect plant growth and limit agricultural pro- duction worldwide. Minimizing these losses is a major area of concern for all countries. Salinity, drought, and cold are among the major environmental stresses that greatly inuence the growth, development, survival, and yield of plants. Several genes including the genes for helicases are known to express under the inuence of various abiotic stresses. The helicases are ubiquitous enzymes that catalyze the unwinding of energetically stable duplex DNA (DNA helicases) or duplex RNA secondary structures (RNA helicases). Most helicases are members of DEAD-box protein superfamily that play essential roles in basic cellular processes regulating plant growth and development, such as DNA replication, repair, recombination, transcription, ribosome biogenesis, and translation initiation. It seems, therefore, that DEAD-box helicase might also be playing an important role in stabilizing growth in plants under stress conditions by regulating some stress-induced pathways. There are now few reports on the upregulation of DEAD-box helicases in response to abiotic stresses. The exact mechanism of helicase-mediated tolerance of stress has not yet been understood. There could be two possible sites of action for the helicases: (i) at the level of transcription or translation to enhance or stabilize protein synthesis or (ii) in an association with DNA multisubunit protein complexes to alter gene expression. Here, we have described all the known plant helicases, which play a role in stress responses. The exploitation of abiotic stress-responsive helicase genes of new path- ways of RNA and DNA unwinding will be helpful for engineering stress-tolerant crop plants. 19.1 Introduction Stress is fundamentally a mechanical concept, dened by engineers and physical scientists as a force per unit area applied to an object. It is difcult to dene stress so accurately in a biological sense. The most useful denition of biological stress is an adverse force or inuence that tends to inhibit normal system from functioning [1]. Improving Crop Resistance to Abiotic Stress, First Edition. Edited by Narendra Tuteja, Sarvajeet Singh Gill, Antonio F. Tiburcio, and Renu Tuteja Ó 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA. j 435

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19Helicases in Improving Abiotic Stress Tolerance in Crop PlantsNarendra Tuteja, Sarvajeet Singh Gill, and Renu Tuteja

Abiotic stress conditions adversely affect plant growth and limit agricultural pro-duction worldwide. Minimizing these losses is a major area of concern for allcountries. Salinity, drought, and cold are among the major environmental stressesthat greatly influence the growth, development, survival, and yield of plants. Severalgenes including the genes for helicases are known to express under the influence ofvarious abiotic stresses. The helicases are ubiquitous enzymes that catalyze theunwinding of energetically stable duplex DNA (DNA helicases) or duplex RNAsecondary structures (RNA helicases). Most helicases are members of DEAD-boxprotein superfamily that play essential roles in basic cellular processes regulatingplant growth and development, such as DNA replication, repair, recombination,transcription, ribosome biogenesis, and translation initiation. It seems, therefore,that DEAD-box helicasemight also be playing an important role in stabilizing growthin plants under stress conditions by regulating some stress-induced pathways. Thereare now few reports on the upregulation ofDEAD-box helicases in response to abioticstresses. The exact mechanism of helicase-mediated tolerance of stress has not yetbeen understood. There could be two possible sites of action for the helicases: (i) atthe level of transcription or translation to enhance or stabilize protein synthesis or (ii)in an associationwithDNAmultisubunit protein complexes to alter gene expression.Here, we have described all the known plant helicases, which play a role in stressresponses. The exploitation of abiotic stress-responsive helicase genes of new path-ways of RNA andDNAunwindingwill be helpful for engineering stress-tolerant cropplants.

19.1Introduction

Stress is fundamentally a mechanical concept, defined by engineers and physicalscientists as a force per unit area applied to an object. It is difficult to define stress soaccurately in a biological sense. The most useful definition of biological stress is an�adverse force or influence that tends to inhibit normal system from functioning� [1].

Improving Crop Resistance to Abiotic Stress, First Edition.Edited by Narendra Tuteja, Sarvajeet Singh Gill, Antonio F. Tiburcio, and Renu Tuteja� 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

j435

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World population is increasing continuously and in 2050 it may reach more than 9billion (http://www.unfpa.org/swp/200/); on the other hand, the crops� productivityis decreasing because of many negative factors including stresses (Figure 19.1a).Also, the demand for oilseeds, wheat, rice, pulses, and so on is going to muchincrease in near future (Table 19.1). Because of these factors, in future there may bedanger to food security; therefore, it is important to develop stress-tolerant crops.Plants being immobile in nature have to bear a wide range of environmental stresses.They can respond to stress in several ways and have evolvedmechanisms by which toincrease their tolerance of these stresses through both physical adaptation andinteractivemolecular and cellular changes that begin after onset of stress [2]. Stressescan be broadly classified into two classes, namely, abiotic and biotic (Figure 19.1b).Low temperature, drought, and high salinity are common abiotic stress conditions.

Decline in agricultural yieldIncrease in population(a)

Actuall

Potential

Bill

ion

6

9

0

3

Present 2050

AbioticAbiotic1. Heat1. Heat2. Cold2. Cold

Drought

Biotic (imposed by other Biotic (imposed by other organisms)organisms)1. Pathogens (B,F,V)1. Pathogens (B,F,V)

Herbivores2. Herbivores2.

(b)

Drought3. Drought4. Salinity5. Wind6. Waterlogging7. Ozone

Heavy metals

2. Herbivores2. Herbivores3. Weeds4. Insects6. Nematodes7. Mycoplasma

8.9. Wounding10. Nutrient loss11. Excess light12. Anoxia/Hypoxia

It is necessary to obtain stress-tolerant crops to cope with the upcoming

13. Genotoxic problem of food security

Figure 19.1 (a) Population of the world isincreasing, while the crop production isdecreasing due to the negative impactof different stresses. (b) Different typesof abiotic and biotic stresses and their

negative effect leading to decliningagricultural production. To cope upwith the impending danger to foodsecurity, it is important to developstress-tolerant crops.

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Biotic stresses include diseases caused by various pathogens. Overall, the environ-mental stresses adversely affect plant growth and productivity.

Abiotic stress in plants induces changes in the expression of many importantgenes, which affect the plant growth and development. Plants are continuouslyexposed to a plethora of different signals, which prevent them fromreaching their fullgenetic potential. All these stress conditions adversely affect plant growth and limitagricultural productionworldwide. To increase their tolerance, plants have developedmechanisms that involve both physical adaptation and interactive molecular andcellular changes. The responses to abiotic stresses are multigenic and the molecularmechanisms underlying these are not very well understood. The extracellular stresssignal is first perceived by the membrane receptors and it then activates a large andcomplex signaling cascade intracellularly, including the generation of secondarysignal molecules. The signal cascade results in the expression of multiple stress-responsive genes, the products of which can provide the stress tolerance directly orindirectly. Overall, the stress response could be a coordinated action of many genes,which may crosstalk with each other. Because the abiotic stresses affect the cellulargene expression machinery, it is possible that molecules involved in nucleic acidmetabolism including helicase are likely to be the target. DNA helicases are motorproteins that catalyze the unwinding of duplex DNA in an ATP-dependent mannerand thereby play an important role in most of the basic genetic processes includingreplication, repair, recombination, transcription, and translation [3–6] (Figure 19.2a).Usually, they need single-stranded (ss) DNA or ss/dsDNA junction as loading zoneand translocate on DNA either in the 30–50 or in the 50–30 direction [3, 4].

All the helicases are also associated with intrinsic DNA-dependent ATPase activity,which provides energy for the helicase action [7, 8]. RNA helicases catalyze the ATP-dependent unwinding of local RNA secondary structures and play a broader role inremodeling RNA structures [9–12]. Many helicases share a core region (�200–700amino acids) of highly conserved nine sequencemotifs (designatedQ, I, Ia, Ib, II, III,IV, V, and VI) and belong to the rapidly growing DEAD-box or DEAH-box proteinfamily, which is conserved from bacteria to humans [6, 9–12] (Figure 19.2b). Theseconserved motifs are involved in different activities such as ATP-binding andhydrolysis, Mg2þ binding, DNA or RNA binding, unwinding, and so on(Figure 19.2b). Multiple DNA helicases are present in single cell in each systembecause of different structural requirement of the substrate at various stages of DNA

Table 19.1 Target productivity requirement to meet the demand in 2020.

Food items 2009 Production (mt) Demand 2020 Increase required

Oilseeds 30 8.5 243%Pulses 35 �15 140%Wheat 110 80 38%Rice 130 �100 30%Total cereals 285 220 30%

Source: FAO, 2009.

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transaction [3, 4, 13]. For example, at least 14 different DNA helicases have beenreported from Escherichia coli, 6 from bacteriophages, 12 from viruses, 15 from yeast,11 from calf thymus, and 34 from humans [5]. However, the exact biological roles ofonly few DNA helicases have been defined [3, 4, 6]. Still very little is known aboutDNA helicases from plant system. Till to date only 11 biochemically active DNAhelicases have been reported in the purified form from plants [5, 14]. In plants, theexact role of plant DEAD-box helicases has not been defined properly. Recently, therehave been some indications of a new role of helicases in stress-regulated processes.

19.2Stress-Regulated Helicases

Many important crops and fruits that originated from the tropics or subtropics, suchas rice, maize, tomato, banana, and orange, are injured or killed by exposure to low,nonfreezing temperature in the range of 0–12 �C [15, 16]. Low temperature is animportant environmental factor that greatly influences the growth, development,survival, and distribution of plants [17]. Plant response to chilling aremultigenic andthe molecular mechanism of chilling sensitivity or resistance is not well under-stood [18]. Low temperature induces the expression of a diverse array of genes [19].The product of these genes helps plants to adapt to subsequent freezing stress.

R iRibosome assembly R i

• Stress tolerance (New)• rRNA processing ••

Role of Helicases and Conserved Motifs

Replication • RNA synthesis •

RNA helicases DNA helicases(a)

••

epa r• Recombination • Transcription• Translation

• Splicing• Replication• Translation initiation • Editing

• Nuclear mRNA export • mRNA stabilization and degradation

• Stress tolerance (New)

B esaPTAA esaPTA 2 niamoD1 niamoD(b)

NWalker I Walker II

AxxGxGKT PT RELA T PGR DEAD/H SAT RGxD HRIGRxxRGFccPoSIQ FVNT

I II III VIIa Ib IV VQ

C

ATP binding/hydrolysis

Nucleic acidbinding

Nucleic acid unwinding

RNA bindingMg2+

binding

ibosomeR assembly

rRNA processing Stress tolerance (New)

Figure 19.2 (a) Role of RNA and DNAhelicases in different processes of nucleic acidmetabolism. A new function in stress toleranceis alsomentioned. (b)Helicase-conservedmotifstructure of DEAD and DEAH helicase family ofproteins. Helicases are characterized by thepresence of an Asp-Glu-Ala-Asp (DEAD) or an

Asp-Glu-Ala-His (DEAH)motif (shown asmotifII here). Both families of helicases containamino-terminal (N) and carboxy-terminaldomains (domain 1 and domain 2, respectively)and nine characteristic motifs (Q, I, Ia, Ib, andII–VI) within these. The known or proposedfunctions of each motif are shown.

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DEAD/H-box helicase genes were reported to induce under chilling and freezingstress [20, 21]. Although multiple DNA helicases have also been isolated fromplants [5, 14], the molecular and biochemical characterization of the plant stress-induced DNA helicase(s) has not been achieved. Though the involvement ofRNA helicase genes in response to stress has been reported from nonplant sys-tems [22–24], the role of DNA helicases in stress has not been well studied. Analysisof genes whose expression is induced under stress condition is important tounderstand the mechanism of abiotic stress tolerance and possibly to use it forbreeding stress-tolerant plants. However, the functions of DNA/RNA helicases arepoorly understood in plants [25, 26]. Among all the sequenced genomes includingthose of humans, fly, worm, and yeast, Arabidopsis has the largest number of DEAD/H-box helicase genes [26–28]. There are 94 helicases reported from Arabidopsis(TAIR) that are regulated with stress. The Affymetrix 22K ATH1 oligonucleotideexpression data were obtained from the Genevestigator Response Viewer (https://www.genevestigator.com) [29] available as an external link in TAIR database.

19.3Expression Profiling of Arabidopsis Helicase Genes under Abiotic Stress

The log2 fold expression values for 113 genes in various stresses such as anoxia, cold(3 independent replicates), drought, genotoxic, heat (2 independent replicates),hypoxia, osmotic, oxidative, salt, and wounding were imported into the Genesissoftware. The hierarchical clustering of 113 different transcriptomes revealedexpression patterns for helicase genes under 10 different stress conditions.A dendrogram was constructed after integrating together the similar expression ofgenes into rows to form a cluster. The heatmap resulting from the clustering analysisshowed high expression of large set of helicases under anoxia, cold, and heat stresses.The expression analysis revealed overexpression of SDE3, RH55, chromatin remo-deling 31, three genes for helicase domain-containing proteins, RH18 and RH11, indrought stress; RecQl3, helicase-related, CHR31 and MCM8 in genotoxic stress;MEE29, RH42, helicase domain-containing protein, SNF2, RH55, and MER3 inhypoxia. In osmotic stress, MEE29, RH55, CHR31, and RH45 showed increasedexpression, while in oxidative stress SDE3, helicase domain-containing protein,RH28, RNA helicase DRH1, and RH37 were overexpressed. The genes that showedhigh expression in salt stress wereMEE29, SNF2 domain-containing protein, RH55,CHR31, CHR9, EDA16, RH30, RH40, andRNAhelicaseDRH1. Inwounding stress,SNF2, CHR42, MER3, and PIF1 showed increased expression levels [28].

Since RNA molecules are more prone to forming stable nonfunctional secondarystructures, their proper functioning requires RNA chaperones [28]. DEAD/H-boxRNA helicases are the best candidates for RNA chaperones because these proteinscan use energy derived from ATP hydrolysis to actively disrupt misfolded RNAstructures so that correct folding can occur [28].

Kujat and Owttrim [30] reported that in photosynthetic organisms light-drivenshift in redox potential acted as a sensor that initiates alteration in gene expression at

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the level of both transcription and translation. This study provides evidence that theexpression of a cyanobacterial RNA helicase gene crhR is controlled at the level oftranscription and RNA stability by a complex series of mechanisms that are redoxregulated. RNA helicase would not be directly involved in photosynthetic reactionper se to coordinate regulation of crhR expression implying that modulation of RNAsecondary structure is required to elicit electron flow. CrhR-induced RNAunwindingactivity could remove secondary structures that inhibit efficient translation ofmRNAs whose products are required under these conditions. Whether crhR hasspecific RNA targets such as redox-induced mRNA, enhancing translation orassembly of ribonucleoprotein complex, or RNA turnover, remains to beexplored [30].

19.3.1Arabidopsis FL25A4 Helicase

Using biotinylated CAP trappermethod, full-length cDNA libraries fromArabidopsisplants grown under different conditions such as drought treated, cold treated, orunstressed were constructed. By cDNA microarray analysis of 1300 Arabidopsisgenes, Seki et al. [31] reported a DEAD-box helicase gene (accession numberAB050574) as a cold stress-inducible gene suggesting a new role of helicases instress signaling [25], but it has not been characterized further.

19.3.2Arabidopsis LOS4 Helicase (AtRH38)

During a genetic screening for Arabidopsis mutants with deregulated expression ofthe RD29A-LUC reporter gene, a mutant named los4-1 was isolated, which showed areduced RD29A-LUC expression in response to cold, but not ABA or high salt.Northern blot analysis indicated that the mutation also decreases expression ofendogenous RD29A and other COR/RD genes under cold stress. The CBF geneshows reduced or delayed cold induction in los4-1mutant plants.Unexpectedly, los4-1mutant plants are very sensitive to chilling temperature in dark. The constitutiveexpression of the CBF-3 gene reverses the chilling sensitivity of los4-1mutant plants.LOS4 gene was isolated by map-based cloning and found to encode for a DEAD-boxRNA helicase protein (AtRH38) that is localized both in cytoplasm and in nucle-us [21]. A novel Arabidopsis mutant (cryophyte) was isolated as having an enhancedcold induction of CBF2 and its downstream genes. Compared to wild type, mutantplantsflower earlier and are smaller in size. The gene in thismutantwas found to be aDEAD-box RNA helicase identical to LOS4 (low expression of osmotically responsivegene). Cryophyte was given the name los4-2. It has an RNA-dependent ATPaseactivity, and los4-2 mutants are defective in mRNA export (Table 19.2). Consistentwith its role inmRNAexport, the LOS4protein appears highly enriched at the nuclearrim. The los4-2 and los4-1mutation affect cold response but in an opposite way. Thelos4-1 plants appear to be sensitive to chilling stress, while los4-2 show chillingresistance compared to thewild type. The los4-2mutant disrupts RNAexport at warm

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Table 19.2 Stress upregulated helicases from plants.

S. No. Organism Type of stress Gene Possible role References

1. A. thaliana Low temperature (4 �C) FL25A4 Suggested a new role for helicases in stress signaling [31]2. A. thaliana Low temperature (22–4 �C) los-4-1, los-4-2 Involved in mRNA export [21, 32]3. Hordeum vulgare Salt and cold stress HVD1 Regulates transcript(s) concernedwith salt tolerance, or

important metabolism such as photosynthesis, inchloroplast

[34]

4. Pisum sativum Salt stress PDH45 Possible role in translation or regulating DNA/RNAmetabolism under stress conditions

[35, 36]

5. P. sativum Salt stress and cold stress PDH47 Efficient translation under stress condition orregulating the DNA/RNA metabolism

[37]

6. A. thaliana Salt, osmotic, and heat STRS1 andSTRS2

Mutations in either gene cause increased tolerance tosalt, osmotic, and heat stresses, suggesting that thehelicases suppress responses to abiotic stress

[38]

7. Apocynum venetum Salt and cold stress AvDH1 ATP-dependent DNA helicase and ATP-independentRNA unwinding activities

[39]

8. M. sativa Mannitol, NaCl,methyl viologen, andabscisic acid

MH1 The ectopic expression ofMH1 inArabidopsis improvedseed germination and plant growth under drought, salt,and oxidative stress

[40]

9. A. thaliana Cold AtRH9 andAtRH25

AtRH25, but not AtRH9, enhanced freezing tolerancein Arabidopsis plants

[41]

10. Glycine max(soybean)

Low temperature andhigh salinity

GmRH GmRH plays an important role in RNA processingduring low-temperature and high-salinity stresses inplants

[43]

11. P. sativum High salinity and cold MCM6 MCM6 single subunit from pea functions as DNAhelicase and its overexpression in tobacco plant pro-motes salinity stress tolerance without affecting yield

[44, 45]

19.3Expression

ProfilingofA

rabidopsisHelicase

Genes

underAbiotic

Stressj441

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andhigh temperature but not at low temperature,while los4-1 impairsmRNAexport atwarm and low temperature. So, los4-1 appears to be a heat-sensitive allele that mayeven enhance mRNA export at low temperature, whereas los4-1 appears to be aconstitutive allele that affects mRNA export in both low and warm temperatures.Analysis of mRNA export-defective mutant cryophyte/los4-2 has provided an uncom-mon opportunity to understand the contribution of mRNA export to higher plantdevelopment and stress response [32]. Overall, LOS4 helicase confers freezingtolerance by regulating mRNA export from the nucleus to the cytoplasm under coldstress conditions [21, 32, 33]. LOS4 helicase is also involved in many physiologicalprocesses suchasgermination (ABAhypersensitivity of los4-2) andplant development(los4mutant flowers earlier), in addition to its role in low-temperature responses [32].

19.3.3Sorghum HVD1 Helicase

In sorghum [34], a salt-responsive transcript HVD1 (Hordeum vulgare DEAD-boxprotein), encoding a putative ATP-dependent DEAD-box RNA helicase, was reported(Table 19.2). The transcript accumulation was induced under salt stress, cold stress,and ABA treatment. In addition to the conserved helicase domain, the encodedprotein contained five repeats of RGG known as RNA recognition motif, at itshydrophilic C terminus. The transcript also dramatically increased during recoveryfrom salt stress. The protein was found to localize in chloroplast by immunogoldlabeling. Thus, it was anticipated that HVD1 protein regulates the function oftranscript(s) concerned with salt tolerance or important metabolism such as pho-tosynthesis, in chloroplast. cDNA is essential for functional analysis of plant genes.

19.3.4Pea DNA Helicase 45

It exhibits striking homology to eukaryotic translation initiation factor 4A (eIF4A)and contains ATP-dependent DNA and RNA helicase activity and DNA-dependentATPase activity [35]. It is also reported that the pea DNA helicase 45 (PDH45) mRNAis upregulated in pea seedling in response to high salt (200mM of NaCl), and whenthis gene was transferred to tobacco it provided the salinity stress tolerance [36]. Thisresponse was specific to Naþ ion stress because treatment with Liþ did not inducethe transcript. This studywas the first direct evidence of the possible role of a helicasein promoting the salinity stress tolerance in plants. The PDH45 transcript was alsoupregulated in response to other abiotic stresses (dehydration, wounding, and lowtemperature), which suggested that the transcript increase could be due to waterstress resulting from salinity- and mannitol-induced desiccation [36]. The inductionof PDH45 transcript was observed to be induced by the phytohormone, ABA, whichsuggested that the stress effect may take place through ABA-mediated pathways. Theexact mechanism of PDH45-mediated tolerance to salinity stress is not understood.This protein may act at translational level or may associate with DNA multisubunitprotein complex to alter gene expression [36].

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19.3.5Pea DNA Helicase 47 (PDH47)

The pea DNA helicase 47 (PDH47) also belongs to DEAD-box protein family andshows 93% homology to tobacco eIF4A. The purified recombinant protein (47 kDa)was reported to contain ATP-dependent DNA helicase and DNA-dependent ATPaseactivities. These activities are upregulated after phosphorylation of PDH47 at Ser andThr residues with protein kinase C. Using Western blot analysis and in vivoimmunostaining followed by confocalmicroscopy, PDH47 is localized in the nucleusand cytosol. The level of transcript of PDH47 is more in shoot than in root. Thetranscript was induced in both shoot and root under cold (4 �C) and salinity (300mMof NaCl) stress, but there was no change in response to drought stress. It is a uniquebipolar helicase that contains both the 30–50 and 50–30 directional helicase activities.The anti-PDH47 antibodies immunodeplete the activities of PDH47 and inhibitin vitro translation of protein. Furthermore, the PDH47 protein showed induction ofprotein synthesis [37].

19.3.6Arabidopsis STRS1 and STRS2

Two DEAD-box RNA helicases from Arabidopsis were reported to be downregulatedbymultiple abiotic stresses. However, themutations in their coded genes resulted inincreased tolerance to salt, osmotic, and heat stresses (Table 19.2). This suggestedthat these helicases suppress responses to abiotic stress. The genes were,therefore, named stress response suppressor 1 (STRS1; At1g31970) and STRS2(At5g08620) [38]. Thesemutants showed greater tolerance than wild type to multipleabiotic stresses and also showed more highly induced expression of genes encodingstress-responsive transcription factors and their downstream target genes. The ABAis observed to reduce the expression of the STRS genes, but the STRSs were reportedto be regulated by both ABA-dependent and -independent stress signaling networks.Overall, this study indicated the importance of RNA metabolism in the control ofstress-responsive gene expression.

19.3.7Dogbane AvDH1 Helicase

The AvDH1 helicase is a salt-responsive gene isolated from the halophytedogbane (Apocynum venetum). It also contained the nine conserved helicasemotifs of the DEAD-box protein family. The purified recombinant proteincontains ATP-dependent DNA and RNA helicase activities and DNA- or RNA-dependent ATPase activities. The AvDH1 gene was reported to be present as asingle copy in the dogbane genome. This gene was found to be upregulated inresponse to NaCl and not in drought and abscisic acid. The AvDH1 transcript wasalso induced by cold stress, but its accumulation was first increased thendecreased with time [39].

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19.3.8Alfalfa MH1 Helicase

The cDNA of this helicase was cloned fromMedicago sativa (alfalfa) and was found tobe homologous to PDH45, and was named M. sativa helicase 1 (MH1). The MH1gene was found to be expressed in roots, stems, and leaves, and was upregulated inresponse to mannitol (drought), NaCl, or H2O2 treatments (Table 19.2). Theexpression of MH1 in Arabidopsis thaliana conferred tolerance to drought andsalinity to the transgenic plants. The enhanced stress tolerance in MH1-expressingArabidopsis was observed to be correlated with an increase in superoxide dismutase(SOD) and ascorbate peroxidase (APX) activities and proline content. The findingssuggested that MH1may function in abiotic stress by elevating the capacities forreactive oxygen species (ROS) scavenging and osmotic adjustment [40].

19.3.9Arabidopsis AtRH9 and AtRH25

Two Arabidopsis helicases, AtRH9 (At3g22310) and AtRH25 (At5g08620), wereselected by Kim et al. [41] to study the basis of the observation that these twoDEAD-box RNA helicases were among the genes highly upregulated in the tran-scriptome ofArabidopsis plants subjected to cold stress [42]. Both these helicases werefound to be upregulated in response to cold stress, whereas their transcript levelswere downregulated by salt or drought stress (Table 19.2). Phenotypic analysis of theoverexpression of AtRH9 or AtRH25 transgenic plants showed the retarded seedgermination of Arabidopsis plants under salt stress conditions. AtRH25, but notAtRH9, was also reported to enhance freezing tolerance in Arabidopsis plants [41].

19.3.10Soybean GmRH

A novel RNA helicase GmRH has been isolated from soybean and characterized byChung et al. [43]. This helicase was shown to contain a bipartite lysine-rich nuclearlocalization signal (NLS) to ward the N-terminal variable region of GmRH. Thesoybean genome was reported to contain two copies of GmRH gene. The gene wasreported to be upregulated in response to low-temperature or high-salinity stress, butnot in response to abscisic acid or drought stress. The GmRH recombinant proteincontained dsRNA unwinding activity independent of ATP in vitro. The authorsproposed that GmRH might play an important role in RNA processing duringlow-temperature and high-salinity stresses in plants (Table 19.2).

19.3.11Pea MCM6 Single-Subunit DNA Helicase

The eukaryotic prereplicative complex (Pre-RC), including heterohexameric mini-chromosomemaintenance (MCM2–7) proteins, ensures that the DNA in genome is

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replicated only once per cell division cycle. The MCM complex provides DNAunwinding function during the DNA replication. However, the unwinding functionin the single subunit ofMCMand its role in abiotic stress tolerance were not reportedso far in any systems. Recently, we have shown the first direct evidence of theidentification of a DNA unwinding activity in a single-subunit (MCM6) of the MCMcomplex of pea [44]. The pea MCM6 single subunit is also reported to form ahomohexamer that actually functions as a DNA helicase (Table 19.2). The DNAhelicase activity is in 30–50 direction and the activity is found to be stimulated byreplication fork-like structure of the substrate [44]. Since MCM proteins play anessential role in cell division and most likely are affected during stress conditions,their overexpression in plants may help in stress tolerance. Furthermore, we havetested its role in salinity stress tolerance. Recently, we have reported that (a) theMCM6 transcript is upregulated in pea plant in response to high-salinity and coldstress and not with ABA, drought, and heat stress (Table 19.2); (b) MCM6 over-expression driven by a constitutive cauliflowermosaic virus-35S promoter in tobaccoplants confers salinity tolerance. The T1 transgenic plants were able to grow tomaturity and set normal viable seeds under continuous salinity stress, without yieldpenalty. It was observed that in salt-grown T1 transgenic plants, the Naþ ions ismostly accumulated in mature leaves and not in seeds of T1 transgenic linescompared to the wild-type (WT) plants. T1 transgenic plants exhibited better growthstatus under salinity stress conditions in comparison to WT plants. Furthermore,the T1 transgenic plants maintained significantly higher levels of leaf chlorophyllcontent, net photosynthetic rate, and therefore higher dry matter accumulation andyield with 200mM of NaCl compared to WT plants. Tolerance index data showedbetter salt tolerance potential of T1 transgenic plants in comparison to WT. Thesefindings provide first direct evidence that overexpression of single-subunit MCM6confers salinity stress tolerance without yield loss [45]. The possible mechanism ofsalinity tolerance is discussed. These findings suggest that DNA replicationmachin-ery can be exploited for promoting stress tolerance in crop plants.

19.4Possible Mechanisms of Helicase Action During Stress

The exact mechanism of helicase-mediated tolerance of stress has not yet beenunderstood. There could be twopossible sites of action for the helicases: (i) at the levelof transcription or translation to enhance or stabilize protein synthesis or (ii) in anassociation with DNAmultisubunit protein complexes to alter gene expression. It isevident that mRNA and protein synthesis are very sensitive to stress, so factorsinvolved in transcription and translation are potential targets of salt toxicity in plants.In bacteria, the toxic effect of Naþ is mainly in translation rather than in RNAsynthesis. Themechanisms of translation initiation are conserved among eukaryotesand the regulation of translation occurs at the step of initiation. The RNA helicaseactivity ofDEAD-box proteins could facilitate transcription by altering the structure ofnascent RNA, a process that can stimulate reinitiation and/or elongation.

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The initiation step of translation is impaired after cold shock and the reactivation oftranslation machinery might represent a bottleneck during cold adaptation. RNAhelicases not only remove RNA secondary structure but also protect mRNA fromdegradation in particular under condition where transcription and translation areuncoupled as a result of inefficient translation initiation [46].

There are also temperature-dependent changes in the cellular ribosome profile.Upon cold shock, the number of polysomes existing at optimal temperature decreasesin favor of increasing amounts ofmonosomes, 70 S particles, and free ribosomal 30 Sand 50 S subunits. This effect has been suggested to result from a cold-induced blockin translation initiation. As a consequence, additional mRNA structuring may occurthat would further complicate protein biosynthesis at low temperature [47]. There is atemperature-dependent alteration of mRNA structures affecting ribosomal proteinsynthesis. ThemRNA secondary structures at the 50 untranslated region (50UTR) canmask the ribosomal binding site, and at 30UTR canmask the stop codon, which finally

5′ UTR inhibitory structure

5′m7G(cap)

AAAn mRNA AUG 3′

(No translation)

(cap)

eIF4BATPeIF4A

A

(Stress- inducedhelicase)

B

AUG AAAn

Rib b it

helicase)

A

A B

40S

60S

AUGAAAn

40S

60S

AUG

osome su unitsB Unwinding

Translation start

Figure 19.3 Hypothetical model for thepossible mechanism of stress tolerance by ahelicase. The eIF4A is a prototypic memberof the DEAD-box RNA helicase family. Stressesmay enhance formation of the inhibitorysecondary structure at the 50UTR of mRNAsof many essential genes. The eIF4A isresponsible for removal of the secondary

structure of the mRNA. eIF4A, along witheIF4B, binds to 50UTR and unwinds theinhibitory secondary structure in an ATP-dependent manner. This facilitates thebinding of ribosome. After this, ribosomescans for the start codon (AUG) and proteinsynthesis begins normally, which wasinhibited due to negative impact of the stress.

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leads to impairment of protein translation initiation. The stress-inducedhelicasesmayresolve these inhibitory structures during stress adaptation.

The possiblemechanism of helicase action during stress is depicted in Figure 19.3.In response to stress, the extra secondary structures could be formed in the50-untranslated region in mRNA of many essential genes, which could be inhibitoryfor translation. These inhibitory secondary structures need to resolve in order tohave active translation, as otherwise theseRNAswill act as nonfunctional RNAswhereprotein synthesis cannot proceed. The stress-induced RNA helicase(s) recognizedthese nonfunctional RNAs and unwound to resolve the secondary structures, whichpermit the translation initiation to proceed (Figure 19.3). Overall, these stress-inducedhelicases help in recovering the functions of the genes for stress adaptation, whichwere stopped previously because of the negative impact of the stress.

The involvement of DEAD-box helicases in various metabolic processes in plantcells might have general implications. In plants, the role of these helicases in stressresponses is just beginning to be understood. The overexpression of stress-inducedDEAD-box helicase(s) can provide an example of the exploitation of DNA/RNAmetabolism pathways for engineering stress-tolerant crop plants. Overall, DEAD-box helicases are conserved and have emerged as newmolecules to understand stresssignaling inplants.Afewstudiesof stress-inducedDNAandRNAhelicasessuggestedthat salinity stress affects the stability of nucleic acid base pairing. Therefore, theexploitationof salinity stress-responsivegenesofnewpathways, includingDNA/RNAmetabolism, will be useful in elucidating the less-known stress signaling networksand will also be helpful for engineering salinity-tolerant crop plants.

Acknowledgment

This work was partially supported by the grants from the Department of Biotech-nology and the Department of Science and Technology, Government of India.

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