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Research article

Salinity and drought tolerant OsACA6 enhances cold tolerance intransgenic tobacco by interacting with stress-inducible proteins

Kazi Md. Kamrul Huda, Mst. Sufara Akhter Banu, Sandep Yadav, Ranjan Kumar Sahoo,Renu Tuteja, Narendra Tuteja*

International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India

a r t i c l e i n f o

Article history:Received 28 February 2014Accepted 3 June 2014Available online 21 June 2014

Keywords:Ca2þATPasesCold stressInteracting partnersRiceReactive oxygen speciesTobacco

a b s t r a c t

Plant Ca2þATPases regulate many signalling pathways which are important for plant growth, develop-ment and abiotic stress responses. Our previous work identified that overexpression of OsACA6 conferssalinity and drought tolerance in tobacco. In the present work we report, the function of OsACA6 in coldstress tolerance in transgenic tobacco plants. The expression of OsACA6 was induced by cold stress. Thepromoter-GUS fusion analyses in the different tissues of transgenic tobacoco confirmed that OsACA6promoter is cold stress-inducible. Transgenic tobacco plants overexpressing OsACA6 exhibited coldtolerance compared to the wild type (WT) controls. The enhanced tolerance was confirmed by pheno-typic analyses as well as by measuring germination, survival rate, chlorophyll content, cell membranestability, malondialdehyde and proline content. Compared to the WT, the expression of catalase, ascor-bate peroxidase and superoxide dismutase increased in the OsACA6 overexpressing plants, which wasinversely correlated with the levels of H2O2 in the transgenic lines. We also identified interacting proteinsof OsACA6 by using yeast two-hybrid screening assay. Most of the interacting partners of OsACA6 areassociated with the widespread biological processes including plant growth, development, signalling andstress adaptation. Furthermore, we also confirmed that OsACA6 is able to self-interact. Overall, theseresults suggest that OsACA6 plays an important role in cold tolerance at least in part, by regulatingantioxidants-mediated removal of reactive oxygen species or by interacting with different calcium signaldecoders including calmodulin-like proteins (CaM) calcium/calmodulin dependent protein kinases(CDPKs) and receptor-like protein kinases (RLKs).

© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

The temperature is one of the major factors affecting nutrientdistribution, growth, and development of plants (Pereira da Cruzet al., 2013). Cold sensitive plants are readily injured or killed atlow and non-freezing temperatures while other plants (low tem-perature areas) are more tolerant to freezing by a process known ascold acclimation. Plants respond to cold stress by a series of phys-iological, metabolic, morphological and molecular changes that arederived from selective increase or decrease in the biosynthesis of alarge number of distinct proteins/enzymes. The cold stress inducestransient Ca2þ influx into the cell cytoplasm and therefore affectscalcium signalling (Huda et al., 2013a). As the cold responsepathway involves influx of Ca2þ from the apoplast, it can be

speculated that efflux likewise occurs to the apoplast by plasmamembrane located pumps or antiporters (Huda et al., 2013a).

Ca2þ plays a crucial role in the normal cellular function. Theplasma membrane Ca2þATPases (PM Ca2þATPases), maintain thecellular [Ca2þ]cyt levels by removing excess Ca2þ to the extracellularenvironment (Reddy et al., 2011). Apart from their traditional role,emerging evidence also suggests that PM Ca2þATPases are involvedin abiotic stress responses (Qudeimat et al., 2008; Huda et al.,2013b) and widespread physiological roles by regulatingCa2þ-triggered signal transduction pathways (Wang et al., 2011;Lucca and Le�on, 2012; Huda et al., 2013a). During cold stress, spe-cific Ca2þ signal is perceived by putative sensor proteins that detectphysical phase transitions of the plasma membrane (PM) due totemperature fluctuation. The PM is a major platform for signalperception and transduction and its close relationship with plantcytoskeleton suggests that microtubules and microfilaments aredownstream targets of various signalling pathways including coldstress (Thion et al., 1996). PM Ca2þATPases activate different

* Corresponding author. Tel.: þ91 11 26742357; fax: þ91 11 26742316.E-mail address: narendra@icgeb.res.in (N. Tuteja).

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downstream Ca2þ decoding elements such as CaM, CBLs and pro-tect plants from a wide range of environmental stresses (Reddyet al., 2011). In winter wheat, the stability and activity ofCa2þATPase increases in response to low temperature (Sun et al.,1998).

In non-plant system, it has been reported that PM Ca2þATPasesperform many specialized functions via interactions between theirintracellular domains and partner proteins. Indeed, PMCa2þATPases interact with PDZ domain-containing proteinsincluding guanylate kinase, CLP36 protein, Naþ/Hþ exchanger reg-ulatory factor- 2, scaffold protein Ania-3/Homer (Holton et al.,2010), the calcium/calmodulin-dependent serine protein kinase(Schuh et al., 2003), calcium-dependent phosphatase calcineurin(Buch et al., 2005) and 14-3-3 protein (Linde et al., 2008). Thus,interactome of PM Ca2þATPases have been shown to downregulatethe enzymatic activity of various important signaling partner pro-teins that play very pivotal roles in the regulation of numeroussignal transduction pathways.

Therefore understanding the mechanism by which plantsrespond to cold stress is of utmost importance for basic as well asapplied researches. It has been reported that the mRNA abundanceof AtACA8 and AtACA10 changes during low temperatures, whichmay be responsible for Ca2þ efflux during cold stress (George et al.,2008). Previously, we reported that PM localized P-type IIBCa2þATPase (OsACA6) provided salinity and drought stress toler-ance in transgenic tobacco by regulating the expression of stress-responsive genes (Huda et al., 2013b). However, the role ofOsACA6 in cold stress tolerance has not been reported so far. In thepresent study, we report that OsACA6 overexpressing transgenictobacco plants are also tolerant to cold stress in addition to salinityand drought stresses. Our results show that overexpression ofOsACA6 acts as a positive regulator of cold tolerance in transgenictobacco by modulating antioxidants-mediated H2O2 scavenging.We have also identified the novel interactors of OsACA6 by usingyeast two-hybrid system. Interestingly, some interacting partnersof OsACA6 have roles in abiotic stress tolerance including coldstress.

2. Methods

2.1. Plant materials and treatments

The rice cultivar IR64 and homozygous OsACA6 overexpressing(T2) transgenic tobacco lines (Huda et al., 2013b) were used forexpression analysis. Rice plants were grown at room temperatureand the transgenic tobacco lines were grown in the glasshouse at26±2 �C.

The two-week old rice seedlings were transferred to incubatorsat 4 �C and samples were collected at designated time intervals (0,1, 2, 3, 6, 12 and 24h). The expression of antioxidant enzymesincluding SOD (superoxide dismutase), APX (ascorbate peroxidase)and CAT (catalase) were measured in the WT and transgenic to-bacco plants with (4 �C) or without cold stress (untreated control).For GUS expression in OsACA6 promoter driven transgenic plant,leaf, shoot and root were separated and was also incubated at 4 �Cfor 24 h, prior to staining with X-Gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid).

2.2. Analysis of gene expression by quantitative real-time PCR

The quantitative real-time PCR was performed to analyze theexpression of OsACA6 in rice as well as the expression of antioxidantenzymes (SOD, APX and CAT) in transgenic tobacco lines. For this,total RNA was isolated from stressed and unstressed samples andcDNA was prepared using AccuScript™ High Fidelity 1st Strand

cDNA Synthesis kit (Agilent Technologies, USA) as per manufac-turer's instructions. The qRT-PCR analysis was performed by apreviously described method (Huda et al., 2013c) using real timespecific primers (Supplementary Table I) and rice a-tubulin and b-actin was used to normalize the expression in rice and transgenictobacco lines respectively (Supplementary Table I). Biological andtechnical replicates of each sample were taken for the analysis.

2.3. Promoter isolation and analysis

The promoter of OsACA6 (1478 bp) was PCR amplified from ricegenomic DNA by using promoter specific primers (SupplementaryTable I). The sequence was analysed by using DNAMAN software(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

2.4. Plasmids construction

The promoter of OsACA6was cloned into BamHI/HindIII sites of apromoter less vector pCAMBIA-1391Z (http://www.cambia.org)before GUS coding sequence for plant transformation. For yeasttwo-hybrid assay, full-length cDNA of OsACA6was amplified from arice cDNA library by PCR using gene specific primers(Supplementary Table I) and cloned into SalI site of the TA cloningvector (pGEMT, Promega, Madison, WI, USA) and verified bysequencing. The OsACA6-pGEMT plasmid was digested with SalIand ligated into pGBKT7 vector (Clontech, California, USA) to createOsACA6-pGBKT7. The cDNA encoding full-length of OsACA6,OsCaMBP (rice calmodulin binding protein, LOC_Os01g04280.1),OsRK2 (rice receptor-like protein kinase 2, LOC_Os10g03420.1) andOsDR (rice dirigent, LOC_Os07g01680.1) were also PCR amplifiedfrom rice cDNA library using gene specific primers (SupplementaryTable I) and cloned into pGADT7 vector (Clontech, California, USA)for the creation of OsACA6- pGADT7, OsCaMBP-pGADT7, OsRK2-pGADT7 and OsDR-pGADT7 constructs.

2.5. Plant transformation

The standard tobacco transformation method was used for thegeneration of OsACA6 promoter expressing transgenic tobaccoplants. For the positive control, plants expressing CaMV 35S pro-moter fused to GUS were used while WT plants were used as anegative control. Transgenic lines were confirmed by PCR usingpromoter-specific primers (Supplementary Table I).

2.6. Histochemical staining

Leaves, shoots, roots and flowers were vacuum infiltrated for15 min in the GUS reaction mixture containing 2 mM 5-bromo-4-chloro-3-indolyl- b-D-glucuronide (X-Gluc) and 100 mM sodiumphosphate buffer as described earlier (Jefferson et al., 1987) andincubated at 37 �C overnight. The reaction was stopped by adding75% ethanol, and the pigments and chlorophylls were removed byrepeated ethanol washing. The b-Glucuronidase activity was alsoquantified by fluorometric GUS assays and expressed as picomolesof 4-MU (methylumbelliferone) per minute per milligram of pro-tein. The accumulation of H2O2 was detected by histochemicalstaining using the diaminobenzidine (DAB) method.

2.7. Phenotypic analysis

Homozygous T2 transgenic tobacco lines (L7, L19 and L21) re-ported in a previous study (Huda et al., 2013b) was used forphenotypic analysis in response to cold stress. For germinationassay, the seeds of transgenic and WT plants were exposed on MSmedia and then kept at 4 �C for 12d then transferred to 25 �C for

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remaining 14d and germination was monitored and scored. In anadditional experiment, the germinating seeds of WT and OsACA6transgenic lines were placed on plate containingMSmedia and firstkept vertically under 4 �C for 3d and again placed on 26±2 �C inculture room for 2d. This process was repeated for 21d. The phys-iological changes were monitored and root length and fresh weightwere also recorded. In another experiment, 21d old transgenic andWT seedlings were exposed to 4 �C for 10d and then kept undernormal greenhouse conditions. During treatment, morphologicalchanges were detected and photographed when variations wereapparent and finally the survival rate was recorded. Leaf discs wereexcised from young and healthy leaves of same age from transgenicand WT plants and floated on water, kept at 4 �C and room tem-perature (control) respectively. When the stress induced bleachingwas observed, the samples were used for determination of the totalchlorophyll content.

2.8. Determination of membrane damages, H2O2 and prolinecontent

The plant cell membrane stability (CMS) was determined withthe help of a conductivity meter (Cond 3150i/SET, Germany) byusing the formula CMS (%) ¼ (1e initial electrical conductivity/electrical conductivity after boiling) X 100. 14d old seedlings oftransgenics and WT plants were incubated at 4 �C. When signs ofstress began to appear on WT plants, seedlings were removed andthoroughly rinsed with double distilled water (ddH2O) prior toimmersion in 20 ml ddH2O at room temperature. After 2 h theinitial conductivities of the solutions were recorded. Samples werethen boiled for 30 min, cooled at room temperature and the finalconductivities were measured. In another experiment, 14d oldseedlings of transgenics and WT plants were incubated at 4 �C for5d and the level of lipid peroxidation (malondialdehyde) and pro-line content was measured by a previously described method (Garget al., 2012). The level of H2O2 was assessed using the methoddescribed earlier (Velikova et al., 2000).

2.9. Yeast two-hybrid assay and screening

A rice cDNA library was constructed according to the protocol ofclontech (California, USA). After amplification, the titer of the li-brary was determined which was approximately 6 � 108 cfu/ml.The rice cDNA library in GAL4 activation domain vector pGADT7was screened to identify the putative interacting partners ofOsACA6. For yeast two-hybrid assay, the plasmids of OsACA6-pGBKT7 and rice cDNA library-pGADT7 were introduced intoyeast strain AH109 using the lithium acetate method and trans-formants were selected onto 2 drop-out (DO) (media lacking Leu-Trp-), 3DO (media lacking Leu-Trp-His-) and 4DO (media lackingLeu- Trp- His- Ade-) media in order to screen the interacting col-onies. For one to one interaction assays, the plasmids of OsACA6-pGBKT7/OsACA6-pGADT7, OsACA6-pGBKT7/OsCaMBP-pGADT7,OsACA6-pGBKT7/OsRK2-pGADT7 and OsACA6-pGBKT7/OsDR-pGADT7 were also transformed into the same yeast strain. Thecolonies, which survived on 4DO plates were further confirmed bythe b-galactosidase expression/filter-lift assays.

2.10. Statistical analysis

Data were analyzed by one-way variance (ANOVA) using g SPSS(12.0 Inc., USA) to determine the least significant difference (LSD)for the significant data to identify the differences in the meanamong the treatments. The means were separated by Duncan'smultiple range tests (DMRT). Different letters indicate means thatwere statistically different at P < 0.05.

3. Results

3.1. Expression and OsACA6 promoter-GUS fusion analysis inresponse to cold stress

To examine the expression pattern of OsACA6 under cold stress,we performed real-time PCR. Cold stress induced transcripts ofOsACA6 significantly increased ~24.44 folds after 1 h and upto ~35.18folds expressionwas observed at 12 h after initiation of the treatment(Fig. 1(A)). Although at some time points, a decline was observed butthe expression of OsACA6 was still significantly higher(~14.15e~21.02 folds increased) as compared to that of untreatedcontrols (Fig. 1(A)). To observe the expression patterns of the pro-moter of OsACA6 in vivo, transgenic tobaccos were generated byexpressing theGUS reporter gene driven byOsACA6 promoter of 1478bp in length. GUS staining and fluorometric quantification assays oftransgenic tobacco plants showed strongGUS signals in all the tissuesexamined, including leaf, shoot and roots (Fig.1(BeC)) in response tocold stress, indicating that OsACA6 is a stress inducible gene.

3.2. Overexpression of OsACA6 increases cold tolerance in tobacco

To investigate the function of OsACA6, transgenic tobacco lineswere generated by overexpressing OsACA6 under control of a CaMV35S promoter. The stable integration of transgenic lines selected inthis study was confirmed by PCR and Southern blot analysis in ourearlier report (Huda et al., 2013b). In the same report, we have alsoshown that OsACA6 was expressed at higher levels in transgenictobacco as compared to the WT plants (Huda et al., 2013b).

To compare the germination efficiency, seeds of WT and OsACA6transgenic lines were grown on MS media and kept under cold(4 �C) condition. The seeds of transgenic lines started to germinateafter 8d with a dramatically lower germination rate but nogermination was observed for WT seeds up to 12d (Fig. 2(A)).However, when the same plate was kept on 25 �C, seeds of WTstarted to germinate at 14d and the rate of germination continuedto increase until it peaked at 65%. At the same time point more than85% seeds of the transformed plants germinated (Fig. 2(A)). Inanother experiment, germinating seeds of transformed lines andWT plants were transferred to MSmedium and kept at 4 �C for 21d.After 21d, most of the transgenic seedlings showed vigorousgrowth performance (higher root and shoot growth) but WT plantsstopped growing, became yellowish and finally died (Fig. 2(B)).Furthermore, OsACA6 transformed plants possess significantlylonger root system and produce more fresh weight compared tothat of WT plants (Fig. 2(CeD)). These results suggest that coldstress had no clear effect on transgenic lines.

To investigate the effect of OsACA6 overexpression on cold toler-ance, 21d old transgenic and WT seedlings were exposed to 4 �C for10d and then grown under normal greenhouse conditions. Pheno-typically, most of the transgenic plants were green and regrown,whereas the WT plants showed severe chlorosis and wilting(Fig. 2(E)). The survival percentage under cold stress also showeddramatic difference between WT and transgenic plants (Fig. 2(F)).The leaf disks of OsACA6 transformed and WT plants were alsofloated separately on water (as control), and cold (4 �C) condition.Cold stress-induced loss of chlorophyll was lower in transgenic linesas compared to WT plants (Fig. 2(GeH)). Thus, OsACA6 expressingtransgenic plants showed high tolerance to cold stress.

3.3. Determination of cell membrane stability (CMS),malondialdehyde (MDA), H2O2 and proline content

It is evident from literature that under low temperature,plasma membranes of tobacco is disturbed; as a result electrical

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conductivity is increased in various tissues. The WT and OsACA6transformed tobacco plants were stressed at 4 �C for 10d and CMSwas measured. A clear difference in the CMS was found after 10dexposure to 4 �C (Fig. 3(A)). The CMS percentage of the WT to-bacco reached to 29% whereas more than 60% CMS was noticed forOsACA6 transformed plants (Fig. 3(A)). After cold treatment (10d),stress induced damage was also assessed by quantifying theaccumulation of MDA, a product of lipid peroxidation (Fig. 3(B)).Under normal condition, no significant changes in the levels ofMDA content was noticed for WT and OsACA6 transformed plant(Fig. 3(B)). After cold stress, MDA content was significantlyincreased in the WT plants which cause more membrane damagesleading to plant death. However, in response to cold stress thetransgenic lines maintained the MDA level similar to unstressedcontrol plants; and therefore they coped up with stress-induceddamages leading to survival of the transgenic plants (Fig. 3(B)).The H2O2 content significantly increased in the WT plants aftercold treatment. However, in transgenic lines H2O2 content wassignificantly lower as compared to WT plants signifying thatexpression of OsACA6 restricted the oxidative damage in thetransgenic lines (Fig. 3(C)). Free proline was also measured and theWT plants showed significantly reduced accumulation of freeproline on cold (4 �C) exposure, while OsACA6 expressing trans-genic tobacco plants accumulated more proline (Fig. 3(D)). Overall,OsACA6 transgenic plants have strong capacity to tolerate coldstress.

3.4. Analysis of antioxidant enzymes expression and ROS levels inthe transgenic plants

It is well known that superoxide dismutase (SOD), ascorbateperoxidase (APX) and catalase (CAT) play key roles in scavenging ofcellular H2O2, produced by reactive oxygen species (ROS) duringstress conditions (Gill and Tuteja, 2010). To test this hypothesis, weperformed qRT-PCR to determine whether OsACA6 overexpressionaffects antioxidant-mediated ROS scavenging under cold stress intransgenic plants. The expression of SOD, APX and CAT weremeasured in the WT and transgenic tobacco plants with (4 �C) orwithout cold stress (untreated control). Under normal conditions,no significant difference was detected in the expression of SOD, APXand CAT in OsACA6 overexpressing tobacco plants compared to theWT plants. The results show that the expression of CAT (~1.86 fold),APX (~1.76 fold) and SOD (~1.83 fold) was significantly increased inthe transgenic plants as compared to WT plants (Fig. 4(AeC)). Theaccumulation of H2O2 in the leaves after cold stress was alsoexamined by histochemical staining with 3, 3’-diaminobenzidine(DAB) and image analysis. Brown-colored polymeric oxidationproducts were visualized in both the tobacco leaves but the in-tensity in the WT was markedly stronger as compared to OsACA6transgenic plants, suggesting that accumulation of H2O2 was alle-viated during cold stress (Fig. 4(DeE)). Taken together, these resultsshow that OsACA6 regulates the antioxidant-mediated ROSdetoxification in transgenic plants.

Fig. 1. Expression and OsACA6 promotereGUS analysis in response to cold stress. A) Cold stress- induced expression analysis of OsACA6. Two-week-old rice seedlings wereexposed to cold stress. The 2�DDCT method was used to measure relative expression of target genes in stressed and nonstressed leaves. B) OsACA6 promoter-GUS analysis in differenttissues of transgenic tobacco in response to cold stress. C) Quantification of OsACA6 promoter-GUS activity in response to cold stress. Values are means ± standard errors from threeindependent measurements. For positive and negative controls 35S-GUS and WT were used separately. Values are means ± standard errors from three independent measurements.Different letters on the top of bars indicate significant differences at P < 0.05 level as determined by Duncan's multiple range test (DMRT).

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3.5. Identification of novel OsACA6 interactors using the yeast two-hybrid system

To identify the interacting partners of OsACA6, we extensivelyscreened rice cDNA library cloned in pGADT7 vector via a yeasttwo-hybrid system using OsACA6 as bait. The transformants wereselected on synthetic 4 dropout (SD)/eLeu, -Trp, -His and -Ade totest the expression of the reporter gene (Fig. 5, first panel). A total of13 colonies exhibited b-galactosidase activity, demonstrating theexpression of reporter gene (Fig. 5, last panel). All the b-galactosi-dase positive clones were amplified using AD specific primers andsequenced. The sequences were further analyzed for homology(TGIR database) and the genes showing high similarity with theirputative functions are shown in Table 1.

To further confirm the interaction, the full-length cDNAencoding OsCaMBP, OsRK2 and OsDR were amplified, cloned intopGADT7 vector and used as prey for yeast one- to- one interactionassays. The b-galactosidase activity showed the expression of re-porter genes in yeast cell cotransformed with OsACA6 intoOsCaMBP, OsRK2, OsDR and positive control while the empty yeastcells failed to express the reporter (Supplementary Fig. 1 (AeC)lower panel). However, OsACA6 exhibited strong interaction withOsCaMBP and OsRK2 as compared to OsDR (Supplementary Fig. 1(AeC) lower panel). These results indicate that OsACA6 indeed in-teracts with OsCaMBP, OsRK2, and OsDR in the yeast two-hybridsystem.

To test the self-interaction, the plasmids of OsACA6-pGBKT7þOsACA6-pGADT7, OsACA6-pGBKT7 (NC1) and OsACA6-pGADT7

(NC2) were co-transformed into the yeast strain AH109. Yeast cellscarrying the plasmids of OsACA6-pGBKT7þ OsACA6-pGADT7 andGb-pGADT7þGb-pGBKT7 exhibited b-galactosidase activity, indi-cating the expression of reporter genes (Supplementary Fig. 2(AeC) last panel). No blue coloration was observed in any of thenegative controls. These results confirm that the full-lengthOsACA6 is able to self-interact in the yeast two-hybrid system.

4. Discussion

It has been reported that the expression pattern of differentCa2þATPase genes including PCA1, GmACA1, AtACA8, AtACA9 andOsACA6 was increased during abiotic stress (Qudeimat et al., 2008;George et al., 2008; Huda et al., 2013b). All these findings clearlydemonstrated the function of Ca2þATPases in abiotic stress relatedpathways. The Ca2þATPases are known to be transcriptionallyregulated by cold treatment (George et al., 2008). Likewise, theexpression of OsACA6 was continuously induced by cold stress,implying a possible role of this gene in mediating the response tocold stress. Furthermore, in transgenic tobacco lines carryingOsACA6 promoter-GUS fusion construct tissue specific high GUSexpression was observed. This result indicates that OsACA6 mightbe regulating the upstream regulators which ultimately control thecold stress inducible signalling networks.

A prolonged high level of cytosolic Ca2þ is harmful for cellularactivity, resulting in structural damage as well as metabolicdysfunction. To overcome this, a membrane bound active Ca2þ

transport system, such as Ca2þATPases, is required tomaintain Ca2þ

Fig. 2. Cold tolerance assay of transgenic tobacco plants overexpressing OsACA6. A) Germination of OsACA6 transgenic and WT plants in response to cold (4 �C) treatment. B)Cold stress response of seedling growth. C) Comparison of root measurements. D) Comparison of Fresh weights of 21-day-old seedlings. E) Phenotypes of OsACA6 transgenic linesand WT following cold stress. F) The survival rate of OsACA6 transgenic and WT plants was recorded after treatment. G) Leaf disk assay for stress tolerance in transgenic tobaccoplants following cold stress. Disks floated on water and kept on 4 �C showed chlorophyll retention in transgenic lines, whereas WT plants showed bleaching of leaf disks comparedwith the untreated control (water). H) Quantification of chlorophyll content from the leaf disk assay. Values are means ± SE, and each experiment was performed in triplicate.Different letters on the top of bars indicate significant differences at P < 0.05 level as determined by Duncan's multiple range test (DMRT).

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homeostasis. It was observed that the cold stress causes rapid in-duction of OsACA6 mRNA levels; therefore to elucidate the roles ofOsACA6 in cold tolerance transgenic tobacco plants were generated.The overexpression of OsACA6 in tobacco conferred enhanced

tolerance to cold stress as observed by phenotypic analysisincluding germination, root length, fresh weight, survival rate andchlorophyll content. These data suggest that OsACA6 might play apositive regulatory role in cold tolerance. Previous transgenic

Fig. 4. Expression analysis of antioxidant enzymes and detection of H2O2 in transgenic tobacco plants in response to cold stress. Comparison of normalized expression of A)Catalase (CAT), B) Ascorbate peroxidase (APX) and C) Superoxide dismutase (SOD) in absence or presence of cold stress in OSACA6 transgenic and WT plants. D) Detection of stress-induced H2O2 production by staining with diaminobenzidine. E) Quantification of H2O2 production in leaf as determined by ImageJ software. Means ± SE are the average of the threeindependent biological replicates and each biological replicate possesses three technical replicates. Different letters on the top of bars indicate significant differences at P < 0.05level as determined by Duncan's multiple range test (DMRT).

Fig. 3. Effect of OsACA6 overexpression on membrane damage and proline content in transgenic tobacco under cold stress. A) Comparison of cell membrane stability ofOsACA6 transgenic plants and controls following cold stress. Twenty separate plants were used. B) Levels of lipid peroxidation expressed in terms of MDA content. C) Changes inhydrogen peroxide (H2O2) content. D) Changes in the level of proline accumulation. Values are means ± SE from three independent experiments. Different letters on the top of barsindicate significant differences at P < 0.05 level as determined by Duncan's multiple range test (DMRT).

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studies of various Ca2þATPAses demonstrated the involvement ofthese gene families in many fundamental cellular processes(George et al., 2008; Wang et al., 2011; Lucca and Le�on, 2012) andabiotic stress tolerance (Qudeimat et al., 2008; Huda et al., 2013a).The gene knock out studies of AtACA10 have indicated its role incold tolerance (George et al., 2008). However, further experimentssuch as OsACA6 RNAi/mutant andmicroarray analysis are needed toelucidate the molecular mechanism of OsACA6 mediated coldtolerance.

The evaluation of membrane damage is a fundamental aspect ofplant cold stress tolerance/sensibility assessment. These analysesinclude lipid peroxidation (Zhang et al., 2011; Yang et al., 2012) andcell membrane stability (Mao et al., 2012) which is used to identifymembrane damage indirectly. In the present study the higher CMSand reduced MDA levels in transgenic tobacco plants suggest thatless membrane damage under cold stress was due to the over-expression of the OsACA6 gene. Proline acts as an effective metab-olite to overcome the adverse impacts of abiotic stresses includingcold (Nounjan et al., 2012; Huang et al., 2012). It may also functionin the stabilization and protection of the cellular membranes,

enzymes and proteins (Hoque et al., 2008). Proline content hasbeen associated with cold tolerance in transgenic plants (Zhanget al., 2011; Huang et al., 2012). Increased proline content inOsACA6 transgenic plants under cold stress also supports the pre-vious reports. All these findings indicate that OsACA6 could conferprotection to the cell membrane damage and provide enhancedcold tolerance in transgenic tobacco but the molecular mechanismby which the overexpression of OsACA6 enhances the protection ofcellular membranes against cold stress remains to be elucidated.

Reactive oxygen species (ROS) produce toxic compounds andcauses damage to lipids, proteins and nucleic acids (Gill and Tuteja,2010; Suzuki et al., 2012). Under abiotic stresses, overproduction ofROS induces oxidative stress causing lipid peroxidation leading tomembrane damage (Miller et al., 2010). So the plants require tomaintain a balance between production and scavenging. Theenzymatic and non-enzymatic antioxidant defense machinery al-lows plants to protect cells from the oxidative stress by scavengingROS (Gill and Tuteja, 2010; Miller et al., 2010). Previous reports haveshown that ROS accumulation is associated with the cold stress(Suzuki and Mittler, 2006) and an effective ROS removing capacity

Fig. 5. Yeast twoehybrid assay for identification of OsACA6 interactor. A) Yeast cells growth on 4DO media (media lacks of Leu- Trp- His- Ade-). B) Colonies grown on normalyeast growth (YPD) media. C) b-galactosidase activity (filter-lift assay) N: negative control (empty pGADT7 vector transformed in yeast).

Table 1Putative interacting partners of OsACA6 identified by yeast two hybrid screening.

S. No. Accession no Interacting partners Putative function

12. LOC_Os04g32850.3 Basic proline-rich protein Development, abiotic stress tolerance and microRNA biogenesis31. LOC_Os10g35300.1 Dof zinc finger domain containing protein Transcriptional regulators in plant growth and development34. LOC_Os07g01680.1 Dirigent, putative, expressed Plant disease resistance response37. LOC_Os12g05040.1 Heavy-metal-associated domain-containing protein Metal ion binding40 LOC_Os02g42314.2 Ubiquitin-conjugating enzyme BR-mediated salt stress response45 LOC_Os03g21710.1 WRKY79 Transcriptional regulators in plants, response to biotic and abiotic stresses46. LOC_Os01g04280.1 Calmodulin binding protein Plant growth, development and response to environmental stimuli48 LOC_Os01g07520.1 Proline-rich family protein Development, abiotic stress tolerance and microRNA biogenesis49. LOC_Os06g50030.1 CAMK includes calcium/calmodulin dependent

protein kinasesPlant signalling and stress tolerance

50. LOC_Os10g03420.1 Receptor-like protein kinase 2 precursor Regulators of cell wall function57 LOC_Os03g63710.2 GTPase-activating protein, putative Plant defence responses62. LOC_Os03g57940.1 CK1 includes the casein kinase 1 Cell growth and shape formation63. LOC_Os01g08160.1 MYB family transcription factor Regulatory networks controlling development, metabolism and responses

to biotic and abiotic stresses

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is generated under the cold stress. SOD, APX and CAT are theantioxidant enzymes, which play crucial roles in scavenging ofH2O2. Higher expression of SOD, APX and CAT was detected inoverexpressing plants, indicating that OsACA6 might efficientlymodulate antioxidant defence network in transgenic plants. Sub-sequently, OsACA6 transgenic lines accumulated lower amounts ofH2O2 compared to the WT plants. It is well known that excess ofH2O2 causes detrimental effect on cellular functions in the plantcells. Overall, these findings indicate that OsACA6 transgenic linessuffered from less oxidative stress under cold conditions, which isconsistent with the higher CMS and proline and reduced MDAlevels. Thus, overexpression of OsACA6 provides cold tolerance atleast in part by regulating enzymatic antioxidant mediated ROSscavenging in the transgenic plants.

To the best of our knowledge in plants so far, there are no reportsthe interactome of PM Ca2þATPases. In this study, we have identi-fied some novel OsACA6 interacting partner proteins by using yeasttwo-hybrid screening, which includes calmodulin binding protein(CaM), receptor-like protein kinase 2 (RK2), calcium/calmodulindependent protein kinases (CDPK), GTPase-activating protein,dirigent, transcription factors (WRKY, MYB) and some other pro-teins (Table 1). All the putative interactors of OsACA6 have beenreported to play multiple roles in plant including growth, devel-opment and cellular metabolism. These interactors also function inresponses to biotic and abiotic stresses including cold stress. Earlier,in non-plant system it has been reported that PM Ca2þATPasesinteract with the calmodulin dependent serine protein kinase,calcium-sensitive proteins, calcineurin, endothelial nitric oxidasesynthase and, neuronal nitric oxide synthase (Holton et al., 2010).Therefore, it appears that OsACA6 can have various interactingpartners with distinct biochemical properties, which makes it

possible to modulate more diverse cellular and physiologicalprocesses.

Ca2þ plays a vital role in mediating the multitude of abiotic andbiotic stimuli by triggering unique Ca2þ signatures which allowplants to adapt to the changing environment (Dodd et al., 2010;Kudla et al., 2010). Ca2þdependent activation of cellular processesoccurs via different intracellular Ca2þ-binding proteins includingCaM, CDPK and RK2 which in turn regulate the activity of manydownstream target proteins (Reddy et al., 2011; Frey et al., 2012).CaM-like proteins activate the Ca2þATPases and thus play role as akey regulator of Ca2þ homeostasis (Boursiac and Harper, 2007). Ithas also been reported that PM Ca2þATPase interacts with multipleRKs suggesting its involvement in the regulation of RK-mediatedsignalling pathways (Frey et al., 2012). In plants, it has been re-ported that CDPKs act as a sensor in response to abiotic stresses,including cold stress (Chinnusamy et al., 2004). Our yeast twohybrid and one to one interaction studies indeed confirm thatOsACA6, interacts with CaM, RK2 and dirigent proteins. Themembrane-bound Ca2þATPase showed a correlation in betweenCa2þ transport and the molecular interaction of Ca2þATPasemonomers (Nagata et al., 1997). It is also reported that the ATPaseactivity of Ca2þATPase does not depend on monomeric interactionbetween Ca2þATPase (Takeda and Kawamura, 2002). The hetero-dimeric forms of Ca2þATPase do not transport Ca2þ or hydrolyseATP indicating that it requires an interaction between functionalCa2þATPase monomers (Ushimaru and Fukushima, 2008). Our re-sults suggest that the functional unit of Ca2þATPase is either adimer or oligomer, which is required for Ca2þ transport or hydro-lysis of ATP.

Cold stress tolerance is controlled by complex mechanismsinvolving many changes, including activation of calcium signal

Fig. 6. Hypothetical model for OsACA6 mediated cold stress adaptation. Cold stress induces an increase in cytosolic Ca2þ. Plasma membrane bound OsACA6 provide cellularhomeostasis by removing excess Ca2þ to the extracellular environment. The Overexpression of OsACA6 also switches off the ROS by controlling the antioxidants defence network. Byinteracting with CaM binding proteins OsACA6 controls numerous cellular functions, such as metabolism, the cytoskeleton, ion balance and protein modifications. It also regulatesthe expression of cold inducible genes by binding with transcription factors resulting in enhanced cold responses. By interacting with CDPKs PRPs and many other proteins, OsACA6upregulates the expression of protective proteins, participates in the modification of the cell wall components and thus provide cold tolerance. Overall, the cold response involvesthe coordination action of many gene products, which may cross-talk with each other. CaM: Calmodulin binding proteins; CDPKs: calmodulin dependent protein kinases; PRPs:proline-rich proteins, ROS: reactive oxygen species; DREBs: the DREBs pathway; CBF: CBF cold response pathway; MYB: MYB transcription factor; CRT: C-repeat elements; DRE:dehydration-responsive elements; LTRE: low-temperature-responsive element.

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decoders, accumulation of compatible solutes and modulations inthe membrane lipid composition. Calmodulin-like proteins (CaM)and calcium/calmodulin dependent protein kinases (CDPKs) arewell characterized calcium signal decoders in cold acclimation(Ludvig et al., 2004; Reddy et al., 2011). In this study, OsACA6modulated cold-induced Ca2þ signal or signature by interactingwith calmodulin binding proteins. Previously it was shown that theCaM-like proteins activate the autoinhibited Ca2þATPases (Boursiacand Harper, 2007) and reversible phosphorylation seems to be amechanism of post-translational regulation of Ca2þATPases. CaMhas a positive regulatory role under low temperature (Reddy et al.,2011) and overexpression of CaM increases cold tolerance in to-bacco (Na et al., 2013). The changes in the expression of CDPK geneshave been correlated in different species upon exposure to coldstress (Martin and Busconi, 2001). OsACA6 also interacts withCDPKs, implying a role in mediating Ca2þ signals during acquisitionof cold or chilling tolerance. Furthermore, OsACA6 interacts withtwo proline-rich proteins (PRPs) and these proteins have been re-ported to be accumulated in response to various stresses includingwounding and infection (Gothandam et al., 2010). Insolubilizationof the PRPs might help in the formation of protein/carbohydrate orprotein/protein linkages within the cell wall that enhance the sta-bility of the extracellular matrix (Gothandam et al., 2010). Theinteraction of OsACA6 with PRPs and its overexpression mightresult in modification of the integrity of plant cell wall and thestructural maintenance of the organ under cold stress and there-fore, may indirectly enhance cold tolerance in transgenic plants.Combining all these observations, a hypothetical model for OsACA6mediated cold stress tolerance is proposed in Fig. 6. Cellular Ca2þ

and ROS production increases in response to cold stress. Theoverexpression of OsACA6 increases Ca2þ efflux and antioxidantdefence, thus providing cellular homeostasis and cold tolerance.The overexpression of OsACA6 facilitated Ca2þ/CaM binding whichdirectly controls cellular metabolism, ionic balance and modifica-tion of proteins thereby providing improved tolerance against coldstress. It might regulates the expression of cold-inducible genes bybinding with the nuclear localized various transcription factorssuch as MYB, DREBs CBF and plays an indirect role in cold adap-tation. PM localized OsACA6, also interacts with CDPKs, PRPs andmany other proteins, and finally upregulates the expression ofnumerous protective proteins by triggering different signallingpathways in response to cold stress.

5. Conclusion

In this study, we report that OsACA6 interacts with wide rangeof calcium sensors and other proteins that produce a specific Ca2þ

signature which ultimately regulates the plant growth, develop-ment and environmental stress responses including cold stress. Theoverexpression of OsACA6 also plays a positive role in cold stresstolerance. The activation of antioxidant-mediated H2O2 scavengingand interaction with other proteins by OsACA6 are possiblyresponsible for the enhanced cold tolerance in transgenic tobacco.Efforts are going on to understand the regulatory mechanisms ofcold tolerance as well as biological function of OsACA6 mediatedinteraction in plants.

Acknowledgements

This work was financially supported by the Department of Sci-ence and Technology and Department of Biotechnology, Govern-ment of India and International Centre for Genetic Engineering andBiotechnology (New Delhi, India). The authors thank ICGEB forawarding ‘The Arturo Falaschi ICGEB Pre-doctoral Fellowship’ toK.M.K.H and M.S.A.B.

List of abbreviations

APX Ascorbate peroxidaseCaM Calmodulin-like proteinsCAT CatalaseCDPKs Calcium/calmodulin dependent protein kinasesCMS Cell membrane stabilityDAB 3, 3’-diaminobenzidineMDA MalondialdehydePM Plasma membraneRLKs Receptor-like protein kinasesROS Reactive oxygen speciesSOD Superoxide dismutase

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.plaphy.2014.06.007.

Authors' contributions

KMKH and NT conceived and designed the experiments. KMKH,MSAB, SY and RKS performed the experiments. KMKH, MSAB andNT analyzed the data. KMKH, RT and NT wrote the manuscript.

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