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Isolation and characterization of a pigeonpea cyclophilin(CcCYP) gene, and its over-expression in Arabidopsisconfers multiple abiotic stress tolerancepce_2151 1324..1338

KAMBAKAM SEKHAR, BHYRI PRIYANKA, VUDEM DASHAVANTHA REDDY &KHAREEDU VENKATESWARA RAO

Centre for Plant Molecular Biology, Osmania University, Hyderabad 500007, A.P., India

ABSTRACT

A full-length cDNA clone of pigeonpea (Cajanus cajan L.)encoding cyclophilin (CcCYP) has been isolated from thecDNA library of plants subjected to drought stress. Aminoacid sequence of CcCYP disclosed similarity with that ofsingle-domain cytosolic cyclophilins of various organisms.Expression profile of CcCYP in pigeonpea plants is stronglyinduced by different abiotic stresses, indicating its stress-responsive nature. Compared to the control plants, thetransgenic Arabidopsis lines expressing CcCYP exhibitedhigh-level tolerance against major abiotic stresses, viz.,drought, salinity and extreme temperatures as evidenced byincreased plant survival, biomass, chlorophyll content andprofuse root growth. The CcCYP transgenics, compared tothe controls, revealed enhanced peptidyl-propyl cis-transisomerase (PPIase) activity under stressed conditions,owing to transcriptional activation of stress-related genesbesides intrinsic chaperonic activity of the cyclophilin. Thetransgenic plants subjected to salt stress exhibited higherNa+ ion accumulation in roots as compared to shoots, whilea reverse trend was observed in the salt-stressed controlplants, implicating the involvement of CcCYP in themaintenance of ion homeostasis. Expression pattern ofCcCYP:GFP fusion protein confirmed the localization ofCcCYP predominantly in the nucleus as revealed by intensegreen fluorescence. The overall results amply demonstratethe implicit role of CcCYP in conferring multiple abioticstress tolerance at whole-plant level.

Key-words: Cajanus cajan cyclophilin; ion homeostasis;major abiotic stress tolerance; nuclear localization; PPIase;stress-inducible CcCYP gene; subtracted cDNA library.

INTRODUCTION

Survival, growth and yield potential of diverse crop plantsare adversely impacted by rapid changes in environmentalconditions caused by global warming.Abiotic stresses act asprimary cause of crop yield losses worldwide, and pose a

major threat to the sustainable food production as theyreduce the potential yields of various crop plants by ~50–70% (Bray 1997; Agarwal et al. 2006). Plants often respondand adapt to the rapid climate changes through modulationof various physiological and molecular mechanisms. Stressis perceived and transmitted through signal transductionwhich affects regulatory elements of stress-inducible genesinvolved in the synthesis and/or alteration of differentclasses of proteins, viz., transcription factors, enzymes,molecular chaperones, ion channels, transporters, etc.,resulting in stress tolerance (Knight & Knight 2001; Chenet al. 2002). Molecular genetic and genomic tools have facili-tated the identification of both functional and regulatorygenes, while transformation methods have enabled geneticengineering of plants for production of abiotic stress-tolerant crops (Umezawa et al. 2006).A clear understandingof the functions of stress-inducible genes also helps inunraveling the underlying mechanisms of stress tolerance.Functional genomic approaches, such as subtractive hybrid-ization, differential screening, differential display, micro-array analyses, reverse genetics, etc., have been employedto identify and define the functionality of various stress-inducible genes (Chen & Zhu 2004; Sreenivasulu, Sopory &Kavi Kishore 2007; Priyanka et al. 2010a).

Cyclophilins (CYPs) are ubiquitous proteins and wereoriginally identified to act as intracellular targets for theimmuno-suppressant drug cyclosporine A (CsA) (Hands-chumacher et al. 1984; Wang & Heitman 2005). CYPsbelong to the immunophilin family with a peptidyl-propylcis-trans isomerase (PPIase) activity, and catalyse the cis-trans conversion of X-Pro peptide bonds (Schreiber 1991).CYPs have been found to occur in diverse organismsranging from bacteria, fungi, plants to animals and humans(Wang & Heitman 2005). Thus far, large families of CYPgenes have been identified in various organisms. Humangenome was found to contain 16 CYP genes (Galat 2003),while Arabidopsis thaliana revealed 29 CYP genes(Romano, Horton & Gray 2004a). In yeast, CYP1 andCYP2 CYPs were shown to play an essential role in therecovery of cells when subjected to heat shock treatment(Sykes, Gething & Sambrook 1993). Moreover, CYPs werealso found to play a key role during cell division (Faure,

Correspondence: K. V. Rao. Fax: +91 40 27096170; e-mail: [email protected]

Plant, Cell and Environment (2010) 33, 1324–1338 doi: 10.1111/j.1365-3040.2010.02151.x

© 2010 Blackwell Publishing Ltd1324

Gingerich & Howell 1998), in transcription regulation(Rycyzyn & Clevenger 2002), as well as pre-mRNA splicing(Horowitz et al. 2002).

Plant CYPs consist of a wide variety of isoforms withvaried function and cellular location, and are deemed toplay crucial role(s) during growth and development,protein maturation and trafficking, besides processing ofthe nucleic acids (Ferreira et al. 1996; Schiene-Fischer & Yu2001). In Arabidopsis, AtCYP59 CYP was found to regu-late pre-mRNA processing (Gullerova, Barta & Lorkovic2006). Expression of AtCYP20-3 CYP, which codes for achloroplastic isoform, was found restricted to the photo-synthetic tissues and was strongly induced by light (Chou& Gasser 1997; Romano et al. 2004a). AtCYP38 was foundto play a unique role in the assembly and maintenance ofphotosystem II (PSII) in Arabidopsis (Fu et al. 2007). Ara-bidopsis plants defective in the CYP20-3 gene, causedby T-DNA insertion, showed hypersensitivity to oxidativestress created by high light, rose Bengal (a water-solublexanthene dye), high salt levels and osmotic shock(Dominguez-Solis et al. 2008). Plant CYPs were inducedand expressed in response to various stresses, viz., drought,salt, low temperature, heat shock (Marivet, Frendo &Burkard 1992; Luan, Lane & Schreiber 1994; Marivet et al.1994; Mera-Zepeda et al. 1998), wounding, chemical elici-tors, as well as pathogens (Scholze et al. 1999; Kong, Lee &Hwang 2001). In Solanum tuberosum, elevated levels ofCYP transcripts were detected under cold, salt anddrought stresses (Godoy et al. 2000). The ThCYP1 gene ofThellungiella halophila was induced by salt, abscisic acid(ABA), H2O2 and heat shock, and upon its over-expressionin fission yeast and tobacco cells resulted in increased tol-erance to salt stress (Chen et al. 2007). Molecular chaper-ones and catalytic isomerases, viz., protein disulphideisomerases and PPIases, present in diverse organisms, wereshown to assist in the folding and assembly of newlysynthesized proteins (Freskgard et al. 1992; Gething &Sambrook 1992). Participation of different CYPs hasbeen implicated in diverse cellular mechanisms such assignalling (Oh et al. 2006), transfer of reducing power(Wood et al. 2003) and preservation of protein structure(Marivet et al. 1994).

Pigeonpea (Cajanus cajan L.) is a major grain legumecrop of the semi-arid tropics, and is endowed with a sub-stantial ability to withstand drought stress conditions. Itgrows well in hot, humid climates, and has an excellent deeproot system with profuse laterals that facilitate extraction ofmoisture during drought periods (Nene & Sheila 1990).Identification of genes involved in environmental stressresponse offers scope for genetic engineering of crop plantsfor enhanced tolerance against abiotic stresses (Priyankaet al. 2010a). In this investigation, subtractive hybridizationtechnique has been adopted to isolate different drought-responsive genes from pigeonpea plants. A gene coding forCYP – induced by various abiotic stresses – has been iso-lated and characterized. Over-expression of Cajanus cajanCYP (CcCYP) gene in Arabidopsis plants afforded markedtolerance against major abiotic stresses.

MATERIALS AND METHODS

Plant materials and treatments

A highly drought-tolerant pigeonpea accession ICP 8744,obtained from International Crops Research Institute forthe Semi-arid Tropics (ICRISAT), Hyderabad, India, wasused as the source material for isolation of drought-responsive genes. Seeds were surface-sterilized with 0.1%(w/v) mercuric chloride for 5 min, and washed thoroughlywith sterile water. The sterilized seeds were thoroughlywashed and germinated in Petri plates containing sterilewet blotting paper. Later, the germinated seedlings weretransferred to pots containing Hoagland solution (Hoag-land & Arnon 1938), and were maintained in the green-house. Seeds were also germinated in soil for water stresstreatment. For subtractive cDNA library construction,water stress was given to 4-week-old seedlings by withhold-ing water for 4 d. To monitor the stress-inducible nature ofthe isolated gene, pigeonpea seedlings were subjected toPEG-6000 (10, 15 and 20% w/v); NaCl (0.4, 0.6, 0.8 and1.0 m) for 6 h; cold (4 °C) for 6 and 12 h; and heat (37 and42 °C) for 2 and 4 h, respectively. Relative water content(RWC) of the control and stressed plants was measured asdescribed (Scholander et al. 1964).

Construction of subtractive cDNA library andisolation of CcCYP from pigeonpea

Total RNA was isolated from the 4-week-old control andwater-stressed pigeonpea plants by guanidinium thiocynate(GTC) method (Sambrook & Russell 2001). mRNA wasisolated from the total RNA through biotin-labelled oligo(dT) probe using mRNA isolation kit (Promega, Madison,WI, USA). cDNA library was constructed through subtrac-tive hybridization using one part of poly (A)+ RNA fromstressed (tester) plants and five parts of 5′-biotinylated first-strand cDNA from unstressed (driver) plants.The poly (A)+

RNA–cDNA hybrids and the excessive cDNA wereimmobilized onto streptavidin-coated magnetic beads. Theunbound subtracted poly (A)+ RNA was used to synthesizethe first-strand followed by the second-strand cDNA(Mishra et al. 2007). The cDNA fragments were ligated to alambda-ZAP vector, in vitro packaged and allowed to infectXL1 blue MRF Escherichia coli cells as per the manufactur-er’s instructions, using a Uni-ZAP XR cDNA library con-struction kit (Stratagene, Lajolla, CA, USA). Cloned cDNAfragments were sequenced independently with T7 and T3

promoters using automated DNA sequencer,and nucleotideand amino acid sequences were analysed employing BLAST(NCBI) and ExPASy tools. Based on sequence analysis ofthe cDNA clone, it was designated as Cajanus cajan CYPgene (CcCYP).Multiple sequence alignment was performedemploying CLUSTALW using Bioedit software.

Northern blot analysis

Northern blot was carried out with 10–20 mg of total RNAisolated from pigeonpea and Arabidopsis plants. Northern

Pigeonpea cyclophilin confers abiotic stress tolerance 1325

© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1324–1338

blot analysis was performed according to Sambrook &Russell (2001).The a-32P-dCTP-labelled CcCYP cDNA wasused as a probe, and hybridization was detected by autora-diography. Ethidium bromide-stained r-RNA bands wereused to assess the quality and quantity of RNA.

Construction of plant expression vector andArabidopsis transformation

Full-length CcCYP (GU 238312) coding sequence wasamplified with Pfu DNA polymerase using 5′-GCCTCGAGATGCCTAACCCTAAGGTTTT-3′ (forward, XhoIsite underlined), 5′-GCTCTAGACTAAGAGGGTTGACCGCAG-3′ (reverse, XbaI site underlined) primers. TheCcCYP coding region was cloned into XhoI and XbaI sitesof pRT100 plasmid (Topfer et al. 1987) in the sense orien-tation, and the expression unit (35S:CcCYP: PolyA) wasexcised with HindIII and cloned into the HindIII site of thepBI121 vector (Clontech, Mountain View, CA, USA) con-taining gusA and nptII (kanamycin) expression units.The pBI121 and CcCYP constructs were then mobilizedinto Agrobacterium tumefaciens strain (EHA105) by tri-parental mating. Agrobacterium-mediated transformationwas performed via the vacuum infiltration method of A.thaliana (ecotype Columbia) (Bechtold & Pelletier 1998).Seeds were harvested from transformed plants, and platedon kanamycin (50 mg mL-1) selection medium to identifythe putative transgenic plants. Kanamycin-resistant T1

transgenic plants were screened for the presence of T-DNAby GUS staining of seedlings, and also confirmed by PCRanalysis using gene-specific primers of gusA (5′-GGAAAAGTGTACGTATCACCGTTTG-3′ and 5′-TATCAGCTCTTTAATCGCCTGTAAG-3′) and CcCYP (asdescribed above). PCR products were analysed on 0.8%(w/v) agarose gel containing ethidium bromide. Later, PCRproducts were blotted onto Hybond-N+ charged nylonmembrane, and were hybridized with the gusA codingsequence labelled with a-32P-dCTP (Sambrook & Russell2001). Two transgenic lines of CC2 and CC4 (T3 generation)along with vector containing line (control) were selected forfurther stress tolerance studies.

Functional analysis of transgenics for abioticstress tolerance

Seeds of the the control and transgenic Arabidopsis weresurface-sterilized and grown on MS salt medium (Murashige& Skoog 1962) or in soil (mixture of 1 vermiculite:1 perlite:1soilrite), and were kept at 4 °C in dark for 3 d for stratifica-tion. Later, they were transferred to Conviron growthchamber (model TC16, Winnipeg, Manitoba, Canada) andwere allowed to grow at 20 � 1 °C under long-day condi-tions (16 h light/8 h dark cycles) with fluorescent light(7000 lux at 20 cm). To test for drought and salt tolerance,2-week-old seedlings were grown on MS medium supple-mented with mannitol (0.3 m) or NaCl (0.1 m) for 1 week.To test the cold sensitivity, 2-week-old seedlings were trans-ferred to incubator set at 4 °C for 7 d. For heat treatment,2-week-old seedlings were exposed to 37 °C for 90 min

(pre-treatment) followed by 42 °C for 2 h.After stress treat-ments, the seedlings were allowed to recover on MS mediumunder normal conditions (20 � 1 °C, 16 h light/8 h darkcycles, 7000 lux at 20 cm) in the growth chamber, and sur-vival rate, root length and biomass were recorded after 15 dof recovery. Hypocotyl elongation assay was performed bysubjecting the germinated seedlings to 37 °C for 90 min,followed by 2 h of recovery under normal conditions, andwere exposed to 42 °C for 2 h. Data were recorded on hypo-cotyl elongation after 3 d of recovery (Queitsch et al. 2000).All the experiments were replicated thrice using 20 seedlingsper treatment.

Two-week-old seedlings of the control and transgenicswere grown on MS medium added with mannitol (0.3 m)/NaCl (0.1 m), or subjected to cold (4 °C), for 15 d, andseedling survival rate, total biomass and root length wererecorded without any recovery period.

Peptidyl prolyl cis-trans isomerase(PPIase) assay

Three-week-old transgenic and control plants treated with0.05¥ MS salts containing 300 mm mannitol/100 mm NaClfor 3 d were used for extraction of total proteins asdescribed (Lippuner et al. 1994). Protein concentration ofsamples was determined as per the method of Bradford(1976). PPIase activity was measured in a coupled assaywith chymotrypsin as described by Breiman et al. (1992)with certain modifications. Test peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilidine (Sigma, St Louis, MO, USA)at 60 mm final concentration was added to a solution of theassay buffer [50 mm HEPES (pH 8.0), 0.015% Triton X-100] and plant extract (300 mg) in a final volume of1 mL. The reaction was initiated by adding chymotrypsin(50 mg mL-1), and change in the absorbance at 390 nm wasmonitored for 300 s. CYP-associated PPIase activity wasdetermined by the extent of inhibition of reaction in thepresence of CsA (60 mm). CsA inhibitor was added to theassay mix for 30 min before the start of the reaction, andincubated at 4 °C. For calculating the PPIase activity, differ-ences between the catalysed and uncatalysed first-orderrate constants, derived from the kinetics of absorbancechange at 390 nm, were multiplied with the amount of sub-strate in each reaction (Breiman et al. 1992). The PPIaseactivity recorded in the control plants is deemed as theinnate activity (IPA). Transgene-specific PPIase activity(TPA) is derived by subtracting the activity of theunstressed control plants (IPA) from that of the activity ofthe unstressed transgenics.Transgene-induced PPIase activ-ity (TIPA) is calculated by subtracting the activity of thestressed control (IPA) and transgene-specific activity (TPA)from the total PPIase activity of the stressed transgenics.

Measurement of chlorophyll content understress treatments

Leaf discs from 3-week-old transgenic and control plantswere floated in a 20 mL solution of NaCl (100 mm)/

1326 K. Sekhar et al.


mannitol (300 mm) or water (experimental control) for 72 hat room temperature (28 � 2 °C). For heat and coldstresses, leaf discs were floated in 20 mL of water and keptat 42/4 °C for 72 h.The treated leaf discs were then used formeasuring chlorophyll spectrophotometrically after extrac-tion in dimethylsulphoxide (DMSO) for 2 h.

Measurement of Na+ ion content

For measurement of sodium ion content, 3-week-olduntreated control and transgenic plants, as well as plantstreated with 0.05¥ MS salts containing100 mm NaCl, for 5 dwere used. Later, leaves and roots from untreated andtreated plants were harvested separately, and dry weights ofsamples were recorded after thorough drying at 80 °C for2 d. The samples were digested with HNO3, and Na+ ionconcentration was assayed by atomic emission spectrom-etry (model GBCAAS932).

Subcellular localization of CcCYP protein

A cDNA fragment containing the pigeonpea CcCYP ORFwas fused with the 5′ end of the green fluorescent protein(gfp) coding region, and the fused product was subclonedinto the pBI121 expression vector under the control ofCaMV 35S promoter. The plasmid vector containingCaMV35S-gfp-nos was used as the control.Two microgramsof the plasmid construct was used to coat tungsten particlesfor transformation of onion epidermal cells. The epidermiswas peeled off and carefully placed onto MS mediumcontaining 2% agar. Epidermal peels were bombardedwith plasmid-coated tungsten particles using a gene gun(Genepro, Hyderabad, India 2000He) with 1100 psi under avacuum of 28 in. Hg and target distance of 6 cm.After bom-bardment, the epidermal peels were incubated at 25 °C for24 h in the dark, and were then visualized using a laserscanning confocal microscope (TCS ST; Leica microsystem,Heidelberg, Germany).

Quantitative Real-time PCR (qRT-PCR)

qRT-PCR was performed for A. thaliana salt overly sensi-tive (AtSOS1) gene using oligonucleotide primers of5′-CCAATGAAACTGCGTGGTG-3′ and 5′-GCACTTTCCTGCCAAAGG-3′. First-strand cDNA was synthe-sized from RNA samples of the control and transgenic Ara-bidopsis seedlings subjected to NaCl (0.1 m) stress for 7 d,as well as from unstressed plants. The resultant cDNAswere used as templates for qRT-PCR analysis. DNase treat-ment was given for removing contaminating genomic DNAfrom RNA samples. RT-PCR analysis was carried out usingEurogentec SYBR Green qPCR Master mix with Real-Plex4 (Eppendorf, Hamburg, Germany) at 94 °C (1 min),58 °C (1 min) and 72 °C (1 min) for 30 cycles. Later, theproducts were analysed through a melt curve analysis tocheck the specificity of PCR amplification. Each reactionwas performed twice, and the relative expression ratio wascalculated using 2-DDCt method employing actin gene as

reference. Oligonucleotide primers of 5′-GGCGATGAAGCTCAATCCAAACG-3′- and 5′-GGTCACGACCAGCAAGATCAAGACG-3′ were used for amplification of actingene.

Statistical analysis

Mean values, standard error and t-test were computed withthe help of pre-loaded software in Excel, programmed forstatistical calculations (http://www.Physics. csbsju.edu/stats/t-test.html).

RESULTS

Isolation and characterization of CcCYP gene

A cDNA clone (GU 238312) coding for a CYP wasobtained from the cDNA library of pigeonpea plants sub-jected to water stress (50–60% RWC) by subtractivehybridization. The clone contained 519 bp coding sequencethat codes for a polypeptide of 172 amino acids (aa) and hasbeen designated as CcCYP gene. Amino acid sequenceanalysis of CcCYP protein divulged the presence of a singleconserved CYP PPIase domain including R, F and H resi-dues required for PPIase activity. The CcCYP showed highidentity of >73% with Glycine max (GmCYP1), 67% withLycopersicon esculentum (LeCYP1), >65% with A. thaliana(AtCYP18-3/ROC1), >65% with T. halophila (ThCYP1),>57% with Oryza sativa (OsCYP) and >59% with that ofHomo sapiens (HsCYPA) (Fig. 1).

Expression profiles of CcCYP in response toabiotic stress in pigeonpea

To examine the stress-inducible nature of CcCYP, Northernblot analysis was performed using the total RNA isolatedfrom pigeonpea plants treated with different concentrationsof polyethylene glycol (PEG-10, 15 and 20%) and NaCl(0.4, 0.6, 0.8 and 1.0 m) for 6 h along with untreated plants.Increased levels of CcCYP transcripts were detected inPEG- and NaCl-treated plants as compared to untreatedplants (Fig. 2a). Northern analysis of plants subjected tohigher temperatures at 37 and 42 °C, and cold stress at 4 °C,revealed increased transcript levels of CcCYP when com-pared with the control plants grown at 28 � 2 °C (Fig. 2b,c).

Development of ArabidopsisCcCYP transgenics

To investigate the role of CcCYP against abiotic stress, thecoding sequence of the gene was cloned downstream toCaMV 35S promoter in pBI121 vector containing nptII asa selectable marker along with gusA gene (Fig. 3a). Agro-bacterium strain (EHA105) carrying pBI121–nptII–gusA(control) or pBI121 containing CcCYP, and nptII and gusAexpression units were employed for transformation ofA. thaliana. Transformed seedlings were selected on MSmedium supplemented with kanamycin (50 mg mL-1). PCRanalysis of the genomic DNA of control,T1 andT2 transgenic



plants, employing gusA gene-specific primers, revealed a>800 bp amplified fragment, while no such band wasobserved in the DNA of wild-type plants (SupportingInformation Fig. S1). When the genomic DNA of T1 andT2 transgenic plants were subjected to PCR with CcCYPgene-specific primers, they disclosed a >500 bp amplifiedfragment; however, no such band was observed in thevector (pBI121–nptII–gusA)-transformed plants (Support-ing Information Fig. S1). Southern analysis of PCR productsobtained with gusA primers,when probed with gusA codingsequence, showed a hybridizable band of ~800 bp in thecontrol and transgenic plants (Supporting InformationFig. S1). Furthermore, transformed lines of CcCYP- andvector-containing plants showed the expression of gusA asevidenced by intense blue colour (Supporting InformationFig. S1).Northern analysis of four independentT3 transgeniclines showed varied levels of transgene expression (Fig. 3b).Transgenics CC2 and CC4 with distinctly higher levels ofCcCYP transcripts were chosen for subsequent stress toler-ance studies.

Functional validation of CcCYP transgenic linesfor multiple abiotic stress tolerance

To evaluate the stress tolerance nature of CcCYP transgen-ics, 2-week-old seedlings were subjected to 300 mm mannitol(drought stress) and 100 mm NaCl (salt stress) for 7 d alongwith vector-containing seedlings.Both transgenic lines,whensubjected to drought stress, showed higher survival rates of~95 and ~97% as compared to the control (~60%) plants

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Figure 2. Northern blot analysis of Cajanus cajan cyclophilin(CcCYP) in pigeonpea under different abiotic stress conditions.(a) Four-week-old pigeonpea plants subjected to differentconcentrations of PEG and NaCl; (b) heat stress (37 and 42 °C);and (c) cold stress (4 °C). Duration of exposure to stress isindicated in (h). About 20 mg of total RNA was used forNorthern blot analysis. The blot was hybridized with the cDNAfragment of CcCYP. Ethidium bromide-stained rRNA is shownfor equal amount of RNA loading. C represents untreatedpigeonpea plants.

Figure 1. Comparison of the deduced amino acid sequences of Cajanus cajan cyclophilin (CcCYP) with CYPs from other species.Multiple sequence alignment of CcCYP with GmCYP1 (Glycine max – acc. no. AAL51087), LeCYP1 (Lycopersicon esculentum – acc. no.M55019), AtCYP18-3/ROC1 (Arabidopsis thaliana – acc. no. L14844), ThCYP1 (Thellungiella halophila – acc. no. AY392408), OsCYP1(Oryza sativa – acc. no. NP_001063993) and HsCYPA (Homo sapiens – acc. no. BC005982). Identical and conserved amino acids arerepresented in dark and grey colours, respectively.



(Figs 4a & 5a). These transgenics, compared to the controlplants, disclosed substantial increases in the total biomass of~60 and ~68% (Fig. 5b). Similarly, the transgenic lines, sub-jected to salt stress, showed higher survival rates of ~75 and~88%, and total biomass of ~119 and ~216% than that of thecontrol plants under similar conditions (Figs 4b & 5a,b).Likewise, as compared to the control plants, the CcCYPtransgenics also displayed marked increases in root growth

of ~68 and ~97% under 300 mm mannitol, and ~76 and~114% in 100 mm NaCl stress (Fig. 5c).

The CcCYP transgenics (CC2 and CC4), upon exposure tohigh (42 °C) temperature, disclosed increased survival ratesof ~73 and ~82% in comparison with ~35% survivalobserved in the control plants (Figs 4c & 5a), and alsorevealed increased total biomass of ~230 and ~250%(Fig. 5b). In addition, notable increases of ~44 and ~84%

Figure 3. Structure of the T-DNA region ofpBI121 containing Cajanus cajan cyclophilin(CcCYP), npt-II and gusA expression unitsand expression pattern of CcCYP intransgenic Arabidopsis plants. (a) Restrictionmap of the CcCYP expression cassette usedfor Arabidopsis transformation. The CcCYPgene is driven by the cauliflower mosaic virus35S promoter. Nos. (nos terminator); RB(right) and LB (left) borders of T-DNA.(b) Northern blot analysis of CcCYPexpression in control (vector transformed)and transgenics (CcCYP) of Arabidopsisplants. Each lane was loaded with 10 mg oftotal RNA. C represents vector-transformedArabidopsis, and CC1–CC4 represent fourindependent CcCYP transgenic lines ofArabidopsis. Ethidium bromide-stainedrRNA is shown for the amount of RNAloaded in each well.

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Figure 4. Effect of Cajanus cajancyclophilin (CcCYP) protein in transgenicArabidopsis plants subjected to drought,salt, heat and cold stresses. (a)Two-week-old seedlings of control andtransgenics subjected to 300 mm mannitolfor 7 d; (b) 100 mm NaCl for 7 d; (c) 37 °Cfor 90 min (pre-treatment) followed by42 °C for 2 h; and (d) cold (4 °C) stressfor 7 d. Photographs of seedlings weretaken 10 d after recovery. C representscontrol; CC2 and CC4 represent twoindependent CcCYP transgenic lines.

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were observed in the elongation of hypocotyls of bothtransgenics (Supporting Information Fig. S4). The trans-genic plants, when subjected to cold (4 °C) stress, showeddistinct increases in the total biomass of ~89 and ~110%compared to the control plants (Figs 4d & 5b). Moreover,the transgenics also showed increased root growth underheat (~50 and ~80%) and cold (~70 and ~90%) treatments(Fig. 5c).

Two-week-old seedlings of transgenic lines, when sub-jected to 300 mm mannitol (drought stress)/100 mm NaCl(salt stress) for 15 d, showed higher survival rates of ~75 to~82%, and ~86 to ~91%, respectively, as compared to thecontrol (~48%) plants. In addition, substantial increases inthe total biomass of ~110 to ~122%,and ~109 to ~117% wereobserved in the transgenic lines under drought and salt stresswhen compared to the controls (Supporting Information

Figs S2 & S3). Likewise, as compared to the control plants,the CcCYP transgenic lines revealed significant increases inroot growth of ~68 and ~97% under 300 mm mannitol, and~105 and ~120% in 100 mm NaCl treatment (SupportingInformation Figs S2 & S3). The transgenic plants, subjectedto cold (4 °C) stress for 15 d,showed survival rates of ~98 and~100% compared to ~88% in the control plants. In addition,significant increases were observed in the total biomass ofthe transgenic lines (~80 and ~88%) compared to that of thecontrol plants (Supporting Information Figs S2 & S3). Simi-larly, the transgenics showed increased root growth of ~38and ~65% compared to the control plants under cold stress(Supporting Information Fig. S2).

Leaf discs of CC2 and CC4 transgenic lines, subjected tomannitol (300 mm) stress, revealed higher mean chlorophyllcontent of ~43 and ~53%, respectively, as compared to the

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****** **

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Figure 5. Effect of mannitol, NaCl, heat and cold stresses on control and Cajanus cajan cyclophilin (CcCYP) transgenics of Arabidopsis.Two-week-old seedlings of control and CcCYP transgenics were grown on 300 mm mannitol, 100 mm NaCl and cold stress (4 °C) for 7 d;for heat stress, seedlings were subjected to 37 °C for 90 min (pre-treatment) followed by 42 °C for 2 h. Seedlings were allowed to recoveron MS plates. Data on (a) survival rate, (b) total biomass and (c) root length were recorded after 15 d of recovery. In each treatment, 20seedlings of control and two transgenic lines were used. Percent surviving seedlings is considered as survival rate. Chlorophyll content(d) was determined from the leaf discs of control and CcCYP transgenics after 72 h of incubation in 0, 300 mm mannitol and 100 mmNaCl solutions independently at room temperature (28 � 2 °C); for heat and cold stress, leaf discs were incubated in water at 42 and 4 °C,respectively. Bar represents mean, and I represents SE from three independent experiments. ***, ** and * indicate significant differencesin comparison with the control at P < 0.001, P < 0.01 and P < 0.1, respectively. WS represents without stress; CC2 and CC4 represent twoindependent CcCYP transgenic lines; FW represents fresh weight.



control plants. Likewise, the transgenic plants treated withNaCl (100 mm) disclosed substantially higher chlorophyllcontent (~56 and ~59%) when compared to the controlplants. Furthermore, the transgenic plants subjected to heat(42 °C) and cold (4 °C) treatments divulged increased (>50and >40%) total chlorophyll contents in comparison withthe control plants (Fig. 5d).

Furthermore, the transgenic plants expressing CcCYPcould successfully complete their reproductive cycle,while the control plants (except under cold stress)turned chlorotic and failed to reach the reproductivephase under drought, salt and heat stress conditions(Fig. 6).

PPIase-specific activity in CcCYP transgenicand control plants under mannitol andsalt stress

CcCYP-expressing transgenic lines, subjected to 0.3 mmannitol stress for 72 h, showed increased PPIase activityof ~0.27 and ~0.30 nmol s-1 mg-1 protein compared to~0.15 nmol s-1 mg-1 protein observed in the control plantsunder similar stress conditions. Likewise, the CcCYP trans-genics grown under 0.1 m NaCl stress for 72 h also exhibitedenhanced PPIase activity (~0.30 and ~0.31 nmol s-1 mg-1

protein) compared to the control plants (Fig. 7a). Underboth stress conditions, the transgenic lines, compared tothe control, showed additional PPIase activity of 0.095–0.126 nmol s-1 mg-1 protein (Table 1). However, no such

additional activity was noticed in the control and transgen-ics under unstressed conditions. In the presence of CsA, theunstressed transgenic lines exhibited ~47 and ~50% inhibi-tion of PPIase activity, while the control plants showed~42% inhibition. Whereas, under stressed conditions, thetransgenic lines and control plants revealed >70 and ~63%inhibition of PPIase activity, respectively (Fig. 7b).

Estimation of Na+ ion levels in roots andshoots of A. thaliana plants

The roots of both transgenic lines, grown under salt stress(100 mm NaCl), accumulated higher levels of Na+ ions(3.6 � 0.09 and 3.9 � 0.09 mg g-1 dry weight) than that ofthe control (2.5 � 0.19 mg g-1) plants (Fig. 8). Conversely,the control plants, compared to the transgenics (2.8 � 0.17and 2.6 � 0.26 mg g-1), accumulated higher levels of Na+

ions (3.8 � 0.12 mg g-1) in shoots when grown under similarstress conditions. However, under unstressed conditions, theroots and shoots of the transgenics and control plants accu-mulated low levels of Na+ ions exhibiting minor differencesbetween them (Fig. 8).

Subcellular localization of CcCYP in the onionepidermal cells

To examine the subcellular localization of CcCYP protein,the CcCYP:gfp fusion and gfp (control) constructs wereindependently bombarded into the onion epidermal cells.

Figure 6. Evaluation of Cajanus cajancyclophilin (CcCYP) transgenics againstdifferent abiotic stress conditions.Two-week-old seedlings of control andCcCYP transgenics were subjected to300 mm mannitol (drought), 100 mm NaCl(salt) for 7 d; heat treatments were givenat 37 °C for 11/2 h (pre-treatment) followedby 42 °C for 2 h. Treated seedlings wereallowed to recover for 7 d at normal(20 � 1 °C) temperature. Later, seedlingsfrom the plates were transferred to potsand allowed to grow for 3 weeks undernormal conditions, and werephotographed. C represents control; andCC2 and CC4 represent two independentCcCYP transgenic lines.

C CC2 CC4

Without stress

Drought stress(0.3 M mannitol)

Salt stress(0.1 M NaCl)

Heat stress(42 0C)



Epidermal cells containing pBI121–gfp plasmid showedfluorescence throughout the cell owing to the expression ofGFP in the cytoplasm and nucleus (Fig. 9c). However, theCcCYP:GFP fusion protein was found to fluoresce pre-dominantly in the nucleus, while it was weak in the cytosol(Fig. 9d).

Quantitative RT-PCR analysis of AtSOS1in the control and transgenic plantsexpressing CcCYP

The transgenic Arabidopsis lines expressing CcCYP andcontrol plants, subjected to 100 mm NaCl stress as well asunstressed conditions, were analysed for the expressionlevels of AtSOS1 gene by using quantitative RT-PCR.

PP

Iase

-spe

cifi

c ac

tivi

ty(n

mol

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* ** *

(a)

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0.1

0.15

0.2

0.25

0.3

0.35

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% I

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itio

n of

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y C

sA

(b)

0

20

40

60

80

100

WS Mannitol (0.3 M)

NaCl (0.1 M)

Control

CC2

CC4

Figure 7. Estimation of peptidyl-propyl cis-trans isomerase(PPIase) activity and its inhibition in control and Cajanus cajancyclophilin (CcCYP) transgenic Arabidopsis lines. (a)PPIase-specific activity in control and transgenics. (b). Inhibitionof PPIase-specific activity in the presence of cyclosporine A(CsA) inhibitor. Three-week-old unstressed and stressed (300 mmmannitol/100 mm NaCl) transgenic and control plants were usedfor extraction of total proteins. The PPIase activity was measuredin a coupled assay using chymotrypsin (50 mg mL-1), and changein the absorbance at 390 nm was monitored for 300 s. Forinhibition of PPIase activity, 60 mm CsA was added to thereaction. Bar represents mean, and I represents SE from threeindependent experiments. ** indicates significant differences incomparison with the control at P < 0.01, respectively. CC2 andCC4 represent two independent CcCYP transgenic lines.

Tab

le1.

Pep

tidy

l-pr

opyl

cis-

tran

sis

omer

ase

(PP

Iase

)ac

tivi

ty(n

mol

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anus

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(dro

ught

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l(0.

1m

)(s

alt

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IPA

IPA

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IPA

0.09

3�

0.00

2–

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152

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0.16

4�

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0.11

8�

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0.09

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114

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YP

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Under unstressed conditions, the shoots of CcCYP trans-genic plants showed increase (2.67) in the relative expres-sion of AtSOS1 gene compared to that of the controlplants (2.00). Similarly, under NaCl stress, the shoots ofthe transgenic plants revealed increased (4.62) AtSOS1expression as compared to the control plants (2.30). Theroots of the unstressed transgenic plants exhibitedincreased (7.46) relative expression of AtSOS1 comparedto the control plants (2.25). Likewise, the roots of thetransgenic plants under salt stress disclosed enhanced(12.99) AtSOS1 transcripts compared to the control plants(6.19). The roots of the transgenic and control plants, com-pared to the shoots, exhibited higher levels of AtSOS1transcripts both under stressed and unstressed conditions(Fig. 10).

DISCUSSION

Major abiotic stresses such as drought, salinity and extremetemperatures have been found to cause extensive damage

to the productivity of crop plants worldwide. For evolve-ment of crops with inbuilt tolerance to multiple stresses, it isimperative to prospect for exotic abiotic stress tolerancegenes from resilient species acclimated to diverse environ-mental conditions. Pigeonpea is an important pulse crop ofthe arid and semi-arid regions, and is bestowed with theinnate ability to withstand various environmental stresses.CYPs, occurring in diverse organisms, are ubiquitous pro-teins and are remarkably conserved during their evolution(Romano et al. 2005). CYPs belonging to various familiesare known to possess enzymatic peptidyl-prolyl isomeraseactivity essential for protein folding in vivo, thereby sug-gesting their cardinal importance in different metabolicprocesses.

In our earlier study, various stress-specific clones havebeen identified from the pigeonpea cDNA library anddeposited in the GenBank (Priyanka et al. 2010b). Aminoacid sequence of one of the clones, representing the stress-inducible CcCYP gene, disclosed significant similarity withthat of previously characterized single-domain cytosolicCYPs (Fig. 1) of G. max (AAL51087), L. esculentum(M55019), A. thaliana (L14844), T. halophila (AY392408),O. sativa (NP_001063993) and H. sapiens (BC005982),implying that the CcCYP belongs to the single-domaincytosolic type. Northern blot analysis of stressed pigeonpeaplants, using CcCYP as a probe, revealed intense hybridiza-tion signals owing to enhanced transcript levels underdrought, salt, heat and cold stresses compared to weaksignals in the unstressed plants, thus indicating the stress-responsive nature of CcCYP gene (Fig. 2a–c). DifferentCYP genes of maize, bean, Solanum commersonii and S.tuberosum were found to show increased transcript levelsduring drought, salinity and extreme temperatures (Marivetet al. 1992; Mera-Zepeda et al. 1998; Godoy et al. 2000).Similarly, the ThCYP1 CYP gene from T. halophila washighly induced by different abiotic stresses (Chen et al.2007). The expression of chloroplast-localized fava beanCYP (pCYP B) was regulated by light and was also inducedby heat stress (Luan et al. 1994). These findings amplysuggest that, most probably, plant CYPs have originatedand diverged into various forms from a common ancestralgene source during the course of evolution, so as to copewith the adverse effects of various environmental condi-tions encountered by the plants.

Na+

con

tent

(m

g g–1

DW

)

***

* ***

* *

*

0

1

2

3

4

5

0 mM

NaCl100 mM

NaCl0 mM NaCl

100 mM

NaCl

Shoots Roots

Control

CC2

CC4

Figure 8. Estimation of Na+ ion content in control and Cajanuscajan cyclophilin (CcCYP) transgenic plants of Arabidopsis. Na+

ion levels in roots and shoots of control and transgenic plantswere estimated after subjecting them to 100 mm NaCl treatmentfor 5 d. Bar represents mean, and I represents SE from threeindependent experiments. *** and ** indicate significantdifferences in comparison with the control at P < 0.001 andP < 0.01, respectively. CC2 and CC4 represent two independentCcCYP transgenic lines; DW represents dry weight.

Figure 9. Subcellular localization of thetransiently expressed Cajanus cajancyclophilin (CcCYP):GFP fusion proteinin onion epidermal cell as observed underconfocal laser scanning microscope. Onionepidermal cells corresponding to GFPalone and CcCYP:GFP protein underbright (a,b) and fluorescence (c,d) field.

= 20 mm. N represents nucleus.

N

(a) (b)

(c) (d)

N



For functional validation of the CcCYP gene in a heter-ologous host, A. thaliana plants were transformed withCcCYP to evaluate its role in abiotic stress tolerance.Trans-genic Arabidopsis lines expressing CcCYP were able towithstand abiotic stresses imposed by drought (mannitol),salinity (NaCl), heat (42 °C) and cold (4 °C) treatments, andcould produce healthy seedlings with profuse root system incontrast to the stunted and weak control plants (Fig. 4;Supporting Information Fig. S2). The transgenic Arabidop-sis plants expressing CcCYP showed increased (~2-fold)PPIase activity as compared to the control plants underboth drought and salt stress conditions (Fig. 7a), therebyestablishing the role of CcCYP in mitigating the effects ofabiotic stress by minimizing partial folding of proteins or bypromoting the dissociation of protein aggregates (Boston,Viitanen & Vierling 1996; Miernyk 1999; Asadulghani et al.2004). The PPIases were shown to assist chaperones byaccelerating the slow rate-limiting isomerization steps(Dobson 2004). Earlier, it was reported that PPIases play akey role in the stress responses of plants and other organ-isms (Sykes et al. 1993; Andreeva, Heads & Green 1999;Kullertz et al. 1999; Dwivedi, Breiman & Herman 2003; Ohet al. 2006).

Under stress conditions, the transgenics exhibited addi-tional PPIase activity besides innate and transgene-encoded activity (Table 1), suggesting the plausible role ofCcCYP in the transcriptional activation of certain Arabi-dopsis gene(s) coding for PPIase(s) associated with stresstolerance. Drought-tolerant cultivars of sorghum exhibitedhigher levels of induced PPIase activity as compared tosusceptible genotypes when subjected to drought stress(Sharma & Singh 2003). Furthermore, enhanced levels ofPPIase activity under stress conditions might regulate theexpression of other stress tolerance genes involved in signaltransduction (Harrar, Bellini & Faure 2001). The higherPPIase activity noticed in CcCYP transgenics than that of

control plants, under stress conditions, signifies that the C.cajan CYP functions as an efficient chaperone. CYPs ofPhaseolus vulgaris, fava bean and T. halophila were foundto show innate ability to act as efficient chaperones (Luanet al. 1994; Marivet et al. 1994; Chen et al. 2007). Chaper-ones, in general, were found to play active role(s) in theproper folding, assembly and transport of newly synthe-sized proteins besides protecting them from the proteolyticdegradation, as well as protein aggregation, under severestress conditions (Freskgard et al. 1992; Gething & Sam-brook 1992;Wang & Heitman 2005). PPIases have also beenimplicated in the chromatin remodelling and cell cycle pro-gression, as well as in the regulation of protein kinases bycontrolling the activity or stability of key regulatory pro-teins (Shaw 2007; Nigam et al. 2008).

Under different stress conditions, when compared tocontrol plants, CcCYP transgenic plants displayed increasedsurvival rate (Fig. 5a; Supporting Information Fig. S3a),enhanced plant biomass (Fig. 5b; Supporting InformationFig. S3b) and profuse root growth (Fig. 5c; SupportingInformation Fig. S3c) besides higher chlorophyll content(Fig. 5d). The striking differences observed between trans-genic and control plants testify the profound effect ofCcCYP in bestowing multiple abiotic stress tolerance atthe whole-plant level. Furthermore, the over-expression ofCcCYP in transgenic plants facilitated the development ofnormal flowers and seed set under different abiotic stressconditions. Conversely, the control plants became stuntedand could not reach the flowering stage (Fig. 6). Arabidopsiscyp20-3 (At5g62030) T-DNA insertional mutant plants,when grown under drought, salt and high-intensity light,became hypersensitive to stress conditions (Dominguez-Solis et al. 2008). Rice OsCYP2 gene introduced into thecyp2

- yeast mutant showed complementation and also con-tributed to the growth of wild-type yeast under salinity, hightemperature and osmotic and oxidative stresses (Kumariet al. 2009). Over-expression of ThCYP1 CYP gene in yeastand tobacco cells was found to confer increased toleranceagainst salt stress (Chen et al. 2007).An overview of accruedresults clearly suggests that the pigeonpea CYP (CcCYP)gene plays a crucial role in conferring tolerance to multipleabiotic stresses.

Adaptation of plants to salt stress conditions requiresthe operation of cellular ion homeostasis involvingnet intracellular Na+ and Cl- uptake, and their subse-quent vacuolar compartmentalization (Blumwald 2000;Hasegawa et al. 2000). To gain an insight into the possiblerole of pigeonpea CYP in ion homeostasis, Na+ ion contentwas estimated in transgenic and control plants subjected tosalt stress. Transgenic plants harbouring CcCYP, under saltstress, disclosed higher Na+ ion concentration in the rootscompared to shoots, while a reverse trend was observed inthe salt-stressed control plants (Fig. 8), implying that theCcCYP is involved in the ion homeostasis. A similar trendwas noticed when PcSrp gene from Porteresia coarctataand HAL1 gene from yeast were expressed in fingermillet and tomato plants, respectively (Gisbert et al. 2000;Mahalakshmi et al. 2006). The ability to restrict Na+

Rel

ativ

e ex

pre

ssio

n

tooRtoohS

* *

* *

* *

02468

101214

WS 0.1 MNaCl

WS 0.1 MNaCl

Control CC2 CC4

Figure 10. Relative expression of AtSOS1 gene transcripts inshoot and root of Cajanus cajan cyclophilin (CcCYP) transgenicsand control plants of Arabidopsis under salt and unstressedconditions. Bar represents mean, and I represents SE from twoindependent experiments. * indicates significant differences incomparison with the control at P < 0.1. CC2 and CC4 representtwo independent CcCYP transgenic lines; WS represents withoutstress.



transport from roots to aerial shoots seems to play a majorrole in determining the salt tolerance of higher plants(Lazof & Bernstein 1999). Over-expression of SOS gene inArabidopsis conveyed salinity tolerance by reducing theaccumulation of Na+ ions in the aerial parts (Shi et al.2003). The role of CcCYP in salinity tolerance plausiblyinvolves limiting of Na+ ion loading into the transpirationalstream, thereby reducing Na+ accumulation in the aerialparts through maintenance of ion homeostasis.

Some of the single-domain CYPs of cytosolic type areknown to enter into the nucleus and play regulatory roles.When CcCYP:gfp fusion construct was bombarded intoonion epidermal cells, the fusion protein was found pre-dominantly in the nucleus (Fig. 9d), implicating that CcCYPinteracts with other nuclear proteins to regulate the expres-sion of various stress-responsive genes. In Arabidopsis,different CYP isoforms, occurring in cytosol, nucleus,mitochondria and chloroplasts, were found involved invarious cellular processes, viz., signal transduction, tran-scriptional activation, m-RNA processing and proteinfolding (Romano et al. 2004a,b, 2005; Fu et al. 2007;Dominguez-Solis et al. 2008).The tomato LeCYP1 cytosolicCYP – found essential for expression of subset LeIAAgenes – was localized in the nucleus for interaction withother nuclear proteins, leading to regulated expression ofearly auxin-responsive genes (Oh et al. 2006). Likewise, theT. halophila cytosolic CYP (ThCYP1), when introducedinto onion epidermal cells, was found localized in thenucleus, suggesting its involvement in the stress-responsivesignalling mechanism (Chen et al. 2007). Similarly, thehuman CYP A cytosolic CYP, upon expression in yeast, wasfound primarily in the nucleus and interacted with YY1-zinc finger transcription factor, resulting in altered tran-scriptional activity (Yang, Inouye & Seto 1995). In addition,the cytosolic CYP CYP E of Dictyostilium was foundmainly in the nucleus and could interact with the transcrip-tion co-regulator SnwA (Skruzny et al. 2001). The accruedresults amply indicate that CcCYP has the intrinsic abilityto enter into the nucleus to interact with other nuclearproteins and to participate in the signal transductionpathway. However, the nature of interaction(s) of CcCYPwith various other stress proteins needs further in-depthinvestigations.

In the present study, CcCYP transgenic Arabidopsisplants subjected to NaCl stress showed higher Na+ ion accu-mulation in the roots compared to the shoots, while areverse trend was observed in the salt-stressed controlplants, thus suggesting the plausible role of ion homeostasisin the enhanced salinity tolerance of transgenic plants. Fur-thermore, the presence of higher AtSOS1 transcript levelsin the roots of CcCYP transgenic plants, compared to theshoots, both under stressed and unstressed conditions(Fig. 10), indicate the probable involvement of CcCYP inthe regulation of AtSOS1 in Arabidopsis. AtSOS1 wasshown to enhance the loading of Na+ ions and their pre-ferred accumulation in the roots of Arabidopsis under saltstress conditions (Zhu 2003). Earlier studies revealedthat AtSOS1 is critical for controlling long-distance Na+

transport from root to shoot in A. thaliana (Shi et al. 2002).Enhanced levels of AtSOS1 transcripts observed in theroots, as compared to the shoots, of salt-stressed CcCYPtransgenics suggest the involvement of CcCYP in modulat-ing the expression of SOS1 in conjunction with otherstress-related genes, thereby contributing to increased salttolerance. However, further investigations are needed toclarify the association of CcCYP with various other genesinvolved in salinity tolerance.

In this investigation, a potent CcCYP gene – induced bydifferent abiotic stress conditions – has been isolated fromthe subtracted cDNA library constructed from the water-stressed pigeonpea plants. Expression of CcCYP in Ara-bidopsis imparted high-level tolerance against majorabiotic stresses, viz., drought, salinity and high and lowtemperatures, as evidenced by increased plant survival,higher biomass, profuse root growth, higher chlorophyllcontent and increased PPIase activity in stressed trans-genics compared to control plants. Localization studiesusing CcCYP:gfp fusion gene construct confirmed thatCcCYP protein could enter the nucleus as revealed byintense fluorescence, indicating its plausible interactionwith nuclear proteins. Based on overall observations, wepropose a general mechanism of action of CcCYP com-prising transcriptional activation of stress-related genes,chaperonic activity, maintenance of ion homeostasisbesides its involvement in signal transduction, culminatingin multiple abiotic stress tolerance. As such, the multifunc-tional CcCYP holds promise as a prime candidate genefor enhancing abiotic stress tolerance in diverse cropplants.

ACKNOWLEDGMENTS

This project is supported by grants from the AndhraPradesh-Netherlands Biotechnology Programme(APNLBP), Hyderabad, India. We thank Prof T. PapiReddy of the Department of Genetics, Osmania University,for his helpful suggestions and for evaluation of the manu-script. We are grateful to Dr Imran Siddiqi of Centre forCellular and Molecular Biology, Hyderabad, India for pro-viding the confocal microscope facility, and to Dr C.Maheswari of Central Research Institute for Dryland Agri-culture (CRIDA), Hyderabad, India for the help renderedin measuring the RWC of samples.

REFERENCES

Agarwal P.K., Agarwal P., Reddy M.K. & Sopory S.K. (2006) Roleof DREB transcription factors in abiotic and biotic stress toler-ance in plants. Plant Cell Reports 25, 1263–1274.

Andreeva L., Heads R. & Green C.J. (1999) Cyclophilins and theirpossible role in the stress response. International Journal ofExperimental Pathology 80, 305–315.

Asadulghani, Nitta K, Kaneko Y., Kojima K., Fukuzawa H., KosakaH. & Nakamoto H. (2004) Comparative analysis of the hspAmutant and wild type Synechocystis sp. strain PCC 6803 undersalt stress: evaluation of the role of hapA in salt stress manage-ment. Archives of Microbiology 182, 487–497.



Bechtold V. & Pelletier G. (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants byvacuum infiltration. Methods in Molecular Biology 82, 259–266.

Blumwald E. (2000) Sodium transport and salt tolerance in plants.Current Opinion in Cell Biology 12, 431–434.

Boston R.S., Viitanen P.V. & Vierling E. (1996) Molecular chaper-ones and protein folding in plants. Plant Molecular Biology 32,191–222.

Bradford M. (1976) A rapid and sensitive method for the quanti-fication of microgram quantities of protein utilizing the principleof protein-dye binding. Analytical Biochemistry 72, 248–254.

Bray E.A. (1997) Plant responses to water deficit. Trends in PlantScience 2, 48–54.

Breiman A., Fawcett T.W., Ghirardi M.L. & Mattoo A.K. (1992)Plant organelles contain distinct peptidyl prolyl cis, trans-isomerases. Journal of Biological Chemistry 267, 21293–21296.

Chen A.P., Wang G.L., Qu Z.L., Lu C.X., Liu F. & Xia G.X. (2007)Ectopic expression of ThCYP1, a stress-responsive cyclophilingene from Thellungiella halophila, confers salt tolerance infission yeast and tobacco cells. Plant Cell Reports 26, 237–245.

Chen W.Q.J. & Zhu T. (2004) Networks of transcription factorswith roles in environmental stress response. Trends in PlantScience 9, 591–596.

Chen W., Provart N.J., Glazebrook J., et al. (2002) Expressionprofile matrix of Arabidopsis transcription factor genes suggeststheir putative functions in response to environmental stresses.The Plant Cell 14, 559–574.

Chou I.T. & Gasser C.S. (1997) Characterization of the cyclophilingene family of Arabidopsis thaliana and phylogenetic analysis ofknown cyclophilin proteins.Plant Molecular Biology 35, 873–892.

Dobson C.M. (2004) Principles of protein folding, misfoldingand aggregation. Seminars in Cell & Developmental Biology 15,3–16.

Dominguez-Solis J.R., He Z., Lima A., Ting J., Buchanan B.B. &Luan S. (2008) A cyclophilin links redox and light signals tocysteine biosynthesis and stress responses in chloroplasts. Pro-ceedings of the National Academy of Sciences of the United Statesof America 105, 16386–16391.

Dwivedi R.S., Breiman A. & Herman E.M. (2003) Differentialdistribution of the cognate and heat-stress-induced isoforms ofhigh Mr cis-trans prolyl peptidyl isomerase (FKBP) in the cyto-plasm and nucleoplasm. Journal of Experimental Botany 54,2679–2689.

Faure J.D., Gingerich D. & Howell S.H. (1998) An Arabidopsisimmunophilin AtFKBP12 binds to AtFIP37 (FKBP interactingprotein) in an interaction that is disrupted by FK506. The PlantJournal 15, 783–789.

Ferreira P.A., Nakayama T.A., Pak W.L. & Travis G.H. (1996)Cyclophilin-related protein RanBP2 acts as chaperon for red/green opsin. Nature 383, 637–640.

Freskgard P.O., Bergenhem N., Jonsson B.H., Svensson M. & Carls-son U. (1992) Isomerase and chaperone activity of prolylisomerase in the folding of carbonic anhydrase. Science 258,466–468.

Fu A., He Z., Cho H.S., Lima A., Buchanan B.B. & Luan S. (2007)A chloroplast cyclophilin functions in the assembly and mainte-nance of photosystem II in Arabidopsis thaliana. Proceedings ofthe National Academy of Sciences of the United States of America104, 15947–15952.

Galat A. (2003) Peptidyl prolyl cis/trans isomerases (immunophil-ins): biological diversity-targets-functions. Current Topics inMedicinal Chemistry 3, 1315–1347.

Gething M.J. & Sambrook J. (1992) Protein folding in the cell.Nature 355, 33–45.

Gisbert C., Rus A.M., Bolarin M.C., Lopez-coronado J.M., Arril-laga I., Montesinos C., Caro M., Serrano R. & Moreno V. (2000)

The yeast HAL1 gene improves salt tolerance of transgenictomato. Plant Physiology 123, 393–402.

Godoy A.V., Lazzaro A.S., Casalongue C.A. & San Segundo B.(2000) Expression of a Solanum tuberosum cyclophilin gene isregulated by fungal infection and abiotic stress conditions. PlantScience 152, 123–134.

Gullerova M., Barta A. & Lorkovic Z.J. (2006) AtCyp59 is a mul-tidomain cyclophilin from Arabidopsis thaliana that interactswith SR proteins and the C-terminal domain of the RNA poly-merase II. RNA 12, 631–643.

Handschumacher R.E., Harding M.W., Rice J. & Drugge R.J.(1984) Cyclophilin: a specific cytosolic binding protein forcyclosporine A. Science 226, 544–546.

Harrar Y., Bellini C. & Faure J.D. (2001) FKBPs: at the crossroads of folding and transduction. Trends in Plant Science 6,426–431.

Hasegawa P.M., Bressan R.A., Zhu J.K. & Bohnert H.J. (2000)Plant cellular and molecular responses to high salinity. AnnualReview of Plant Biology 51, 463–499.

Hoagland D.R. & Arnon D.I. (1938) The water culture method forgrowing plants without soil. California Experimental Station Cire71, 34–39.

Horowitz D.S., Lee E.J., Mabon S.A. & Misteli T. (2002) A cyclo-philin functions in pre-mRNA splicing. EMBO Journal 21, 470–480.

Knight H. & Knight M.R. (2001) Abiotic stress signaling pathways:specificity and cross-talk. Trends in Plant Science 6, 262–267.

Kong H.Y., Lee S.C. & Hwang B.K. (2001) Expression of peppercyclophilin gene is differentially regulated during the pathogeninfection and abiotic stress conditions. Physiological andMolecular Plant Pathology 59, 189–199.

Kullertz G., Liebau A., Rucknagel P., Schierhorn A., Diettrich B.,Fischer G. & Luckner M. (1999) Stress-induced expression ofcyclophilins in proembryonic masses of Digitalis lanata does notprotect against freezing/thawing stress. Planta 208, 599–605.

Kumari S., Singh P., Singla-Pareek S.L. & Pareek A. (2009) Heter-ologous expression of a salinity and developmentally regulatedrice cyclophilin gene (OsCyp2) in E. coli and S. cerevisiae conferstolerance towards multiple abiotic stresses. Molecular Biotech-nology 42, 195–204.

Lazof D.B. & Bernstein N. (1999) The NaCl induced inhibition ofshoot growth; the case for distributed nutrition with special con-sideration of calcium. Advance Botany Research 29, 113–189.

Lippuner V., Chou I.T., Scott S.V., Ettinger W.F., Theg S.M. &Gasser C.S. (1994) Cloning and characterization of chloroplastand cytosolic forms of cyclophilin from Arabidopsis thaliana.Journal of Biological Chemistry 269, 7863–7868.

Luan S., Lane W.S. & Schreiber S.L. (1994) pCyP B: a chloroplast-localized, heat shock-responsive cyclophilin from fava bean. ThePlant Cell 6, 885–892.

Mahalakshmi S., Christopher G.S., Reddy T.P., Rao K.V. & ReddyV.D. (2006) Isolation of a cDNA clone (PcSrp) encoding serine-rich-protein from Porteresia coarctata T. and its expression inyeast and finger millet (Eleusine coracana L.) affording salttolerance. Planta 224, 347–359.

Marivet J., Frendo P. & Burkard G. (1992) Effects of abioticstresses on cyclophilin gene expression in maize and bean andsequence analysis of bean cyclophilin cDNA. Plant Science 84,171–178.

Marivet J., Margis-Pinheiro M., Frendo P. & Burkard G. (1994)Bean cyclophilin gene-expression during plant development andstress conditions. Plant Molecular Biology 26, 1181–1189.

Mera-Zepeda L.A., Baudo M.M., Palva E.T. & Heino P. (1998)Isolation and characterization of a cDNA corresponding to astress-activated cyclophilin gene in Solanum commersonii.Journal of Experimental Botany 49, 1451–1452.



Miernyk J.A. (1999) Protein folding in the plant cell. Plant Physi-ology 121, 695–703.

Mishra R.N., Reddy P.S., Nair S., Markandeya G., Reddy A.R.,Sopory S.K. & Reddy M.K. (2007) Isolation and characterizationof expressed sequence tags (ESTs) from subtracted cNDAlibraries of Pennisetum glaucum seedlings. Plant MolecularBiology 64, 713–732.

Murashige T. & Skoog F.A. (1962) Revised medium for rapidgrowth and bioassays with tobacco tissue cultures. Plant Physi-ology 15, 473–497.

Nene Y.L. & Sheila V.K. (1990) Geography and importance. InPigeonpea (eds Y.L. Nene, S.D. Hall & V.K. Sheila), pp. 1–14.CAB Intl, Wallingford, UK.

Nigam N., Singh A., Sahi C., Chandramouli A. & Grover A. (2008)SUMO-conjugating enzyme (Sce) and FK506-binding protein(FKBP) encoding rice (Oryza sativa L.) genes: genome-wideanalysis, expression studies and evidence for their involvementin abiotic stress response. Molecular Genetics and Genomics 279,371–383.

Oh K.C.H., Ivanchenko M.G., White T.J. & Lomax T.L. (2006) Thediageotropica gene of tomato encodes a cyclophilin: a novelplayer in auxin signaling. Planta 224, 133–144.

Priyanka B., Sekhar K., Reddy V.D. & Rao K.V. (2010a) Expressionof pigeonpea hybrid- proline-rich protein encoding gene(CcHyPRP) in yeast and Arabidopsis affords multiple abioticstress tolerance. Plant Biotechnology Journal 8, 76–87.

Priyanka B., Sekhar K., Sunitha T., Reddy V.D. & Rao K.V. (2010b)Characterization of expressed sequence tags (ESTs) of pigeon-pea (Cajanus cajan L.) and functional validation of selectedgenes for abiotic stress tolerance in Arabidopsis thaliana.Molecular Genetics and Genomics 283, 273–287.

Queitsch C., Hong S.W., Vierling E. & Lindquist S. (2000) Heatshock protein 101 plays a crucial role in thermotolerance inArabidopsis. The Plant Cell 12, 479–492.

Romano P.G.N., Horton P. & Gray J.E. (2004a) The Arabidopsiscyclophilin gene family. Plant Physiology 134, 1268–1282.

Romano P.G.N., Edvardsson A., Ruban A.V., Andersson B., VenerA.V., Gray J.E. & Horton P. (2004b) Arabidopsis AtCYP20-2is a light-regulated cyclophilin-type peptidyl-prolyl cis-transisomerase associated with the photosynthetic membranes. PlantPhysiology 134, 1244–1247.

Romano P., Gray J., Horton P. & Luan S. (2005) Plant immunophi-lins: functional versatility beyond protein maturation. New Phy-tologist 6, 753–769.

Rycyzyn M.A. & Clevenger C.V. (2002) The intranuclear prolactin/cyclophilin B complex as a transcriptional inducer. Proceedingsof the National Academy of Sciences of the United States ofAmerica 99, 6790–6795.

Sambrook J. & Russell D.W. (2001) Molecular Cloning a Labora-tory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY, USA.

Schiene-Fischer C. & Yu C. (2001) Receptor accessory foldinghelper enzymes: the functional role of peptidyl prolyl cis/transisomerase. FEBS Letters 495, 1–6.

Scholander P.F., Hammel H.T., Hemmingsen E.A. & BradstreetE.D. (1964) Hydrostatic pressure and osmotic potential in leavesof mangroov and some other plants. Proceedings of the NationalAcademy of Sciences of the United States of America 52, 119–125.

Scholze C., Petersom A., Diettrich B. & Luckner M. (1999) Cyclo-philin isoforms from Digitalis lanata: sequences and expressionduring embryogenesis and stress. Plant Physiology 155, 212–219.

Schreiber S.L. (1991) Chemistry and biology of the immuno-philins and their immunosuppressive ligands. Science 251, 283–287.

Sharma A.D. & Singh P. (2003) Comparative studies on drought-induced changes in peptidyl prolyl cis-trans isomerase activity indrought-tolerant and susceptible cultivars of Sorghum bicolor.Current Science 84, 911–918.

Shaw P.E. (2007) Peptidyl-prolyl cis/trans isomerases and transcrip-tion: is there a twist in the tail? EMBO Reports 8, 40–45.

Shi H., Quintero F.J., Pardo J.M. & Zhu J.K. (2002) The putativeplasma membrane Na+/H+ antiporter SOS1 controls long-distance Na+ transport in plants. The Plant Cell 14, 465–477.

Shi H., Lee B.H., Wu S.J. & Zhu J.K. (2003) Over-expressionof a plasma membrane Na+/H+ antiporter gene improves salttolerance in Arabidopsis thaliana. Nature Biotechnology 21, 81–85.

Skruzny M., Ambrozkova M., Fukova I., Martinkova K.,Blahuskova A., Hamplova L., Puta F. & Folk P. (2001) Cyclophi-lins of a novel subfamily interact with SNW/SKIP coregulator inDictyosteleum discoidum and Schizosaccharomyces pombe. Bio-chimica et Biophysica Acta 1521, 146–151.

Sreenivasulu N., Sopory S.K. & Kavi Kishore P.B. (2007) Decipher-ing the regulatory mechanisms of abiotic stress tolerance inplants by genomic approaches. Gene 388, 1–13.

Sykes K., Gething M.J. & Sambrook J. (1993) Proline isomerasesfunction during heat shock. Proceedings of the NationalAcademy of Sciences of the United States of America 90, 5853–5857.

Topfer R., Matzeit V., Gronenborn B., Schell J. & Steinbiss H.H.(1987) A set of plant expression vectors for transcriptional andtranslational fusions. Nucleic Acids Research 15, 5890.

Umezawa T., Fujita M., Fujita Y., Yamaguchi-Shinozaki K. &Shinozaki K. (2006) Engineering drought tolerance in plants:discovering and tailoring genes to unlock the future. CurrentOpinion in Biotechnology 17, 113–122.

Wang P. & Heitman J. (2005) The cyclophilins. Genome Biology 6,226.

Wood Z.A., Schroder E., Robin H.J. & Poole L.B. (2003) Structure,mechanisms and regulation of peroxiredoxins. Trends in Bio-chemical Sciences 28, 32–40.

Yang W.M., Inouye C.J. & Seto E. (1995) Cyclophilin A andFKBP12 interact with YY1 and alter its transcriptional activity.Journal of Biological Chemistry 270, 15187–15193.

Zhu J.K. (2003) Regulation of ion homeostasis under salt stress.Current Opinion in Plant Biology 6, 441–445.

Received 12 January 2010; received in revised form 6 March 2010;accepted for publication 8 March 2010

SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article:

Figure S1. PCR and GUS expression of control and CcCYPtransgenic lines (a) PCR analysis for the presence of gusAin wild-type (WT), control (C) and CcCYP transgenics(CC1, CC2, CC3 and CC4). (b) PCR–Southern blot of gusAin wild-type, control and CcCYP transgenic lines. (c) GUSexpression in control (C1 and C2) and CcCYP transgenics.(d) PCR analysis for the presence of CcCYP (~500 bp). Mrepresents 1 kb DNA ladder; P represents positive control.Figure S2. Effect of CcCYP in transgenic Arabidopsis linessubjected to drought, salt and cold stresses. Two-week-oldseedlings of control (C) and transgenics (CC2 and CC4)subjected to (a) 0.3 m mannitol, (b) 0.1 m NaCl and (c) cold(4 °C) stresses for 15 d.



Figure S3. Effect of mannitol, NaCl and cold stresses oncontrol and CcCYP transgenics of Arabidopsis. Two-week-old seedlings of control and CcCYP transgenics (CC2 andCC4) were subjected to 0.3 m mannitol, 0.1 m NaCl and cold(4 °C) stresses for 15 d. Data on (a) survival rate, (b) totalbiomass and (c) root length were recorded after 15 d stresstreatment. In each treatment, 20 seedlings of controland transgenic lines were used. Bar represents mean andI represents SE from three independent experiments.** and * indicate significant differences in comparisonwith the control at P < 0.01 and P < 0.1, respectively. FWrepresents fresh weight; WS represents without stress.Figure S4. Effect of heat stress (42 °C) on hypocotyl elon-gation in control and CcCYP transgenic plants. Seeds ger-minated for 3 d at normal temperature (20 � 1 °C) wereplaced sequentially at 37 °C for 11/2 h, 20 � 1 °C for 2 h and42 °C for 2 h. Later, treated seedlings were grown at normal

temperature (20 � 1 °C) for 3 d in dark for recovery. (a)Hypocotyl elongation in seedlings subjected to recoveryafter heat treatment. (b) Effect of heat stress on hypocotyllength. Bar represents mean and I represents SE from threeindependent experiments. In each experiment, 20 seedlingsof control and two transgenics were used for measurementof hypocotyls length. ** and * indicate significant dif-ferences in comparison with the control at P < 0.01 andP < 0.1, respectively. C represents vector transformedcontrol; CC2 and CC4 represent two independent CcCYPtransgenic lines.

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting materials sup-plied by the authors. Any queries (other than missing mate-rial) should be directed to the corresponding author for thearticle.



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