Plant Mol Biol Rep DOI 10.

1007/s11105-011-0360-z

Characterization and Functional Validation of Tobacco PLC Delta for Abiotic Stress Tolerance
Manas Kumar Tripathy & Wricha Tyagi & Mamta Goswami & Tanushri Kaul & Sneh Lata Singla-Pareek & Renu Deswal & Malireddy K. Reddy & Sudhir K. Sopory

# Springer-Verlag 2011

Abstract The role of plant phospholipase C-mediated signaling has been implicated in various phases of plant growth and development. In this study, we report on the isolation and characterization of phospholipase C from tobacco and demonstrate that transcripts of phospholipase C are up-regulated in responses to drought and salt stress. These responses are likely by abscisic acid (ABA). Transgenic tobacco plants overexpressing the phospholipase C protein were found to tolerate higher levels of drought and also salinity stress. This tolerance could be mediated by the regulation of genes downstream to phospholipase mediated signaling. As a demonstration, when tested the transgenic plants showed higher transcript of heat shock factor NtHSF2, heat shock protein HSP70-3 and an AP2 domain transcription factor. Also the transgenic

plants showed higher accumulation of sodium in older leaves compared to the young leaves. The present report is the first to demonstrate the role of phospholipase C in salinity stress tolerance. Keywords Salinity . Drought . Phospholipase C . Tobacco . Signaling . Abscisic acid

Introduction Phosphatidylinositols (PI) are a class of membrane phospholipids that are substrates for phosphoinositide-specific phospholipase C (PI-PLC). PI-PLC catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to generate inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (Testerink and Munnik 2005). Despite the emerging evidence of the presence of PI-PLC in higher plants (Hirayama et al. 1995, 1997; Shi et al. 1995; Kopka et al. 1998; Venkataraman et al. 2003; Zhai et al. 2005), the role played by PI-PLCs in various pathways involved in growth and development is still emerging. Transcript analyses in PI-PLCs have given an indication that PI-PLCs might be playing a role in light and abiotic stress signalling pathway (Tuteja and Sopory 2008). Transgenic Arabidopsis plants expressing the AtPLC1 antisense gene accumulated lower IP3 concentrations after being treated with ABA and showed decreased induction of ABA- and stress-responsive genes compared to such induction in control plants (Sanchez and Chua 2001). Study on loss-of-function mutation in the FRY1 (encoding an inositol polyphosphate 1-phosphatase that is involved in the catabolism of IP3) identified in Arabidopsis has provided the first genetic evidence that phosphoinositols mediate gene regulation by cold, drought, and salt stress, as well as by ABA (Xiong et al. 2001). The fry1 mutation

Electronic supplementary material The online version of this article (doi:10.1007/s11105-011-0360-z) contains supplementary material, which is available to authorized users. M. K. Tripathy : W. Tyagi : M. Goswami : T. Kaul : S. L. Singla-Pareek : M. K. Reddy : S. K. Sopory (*) Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India e-mail: sopory@mail.jnu.ac.in S. K. Sopory e-mail: sopory@icgeb.res.in M. K. Tripathy : R. Deswal Department of Botany, University of Delhi, Delhi 110007, India Present Address: S. K. Sopory Jawaharlal Nehru University, New Delhi 110067, India

Plant Mol Biol Rep

phenotype showed enhanced accumulation of IP3. These results strongly support an association between PI signaling cascade and stress response in higher plants.The expression of the ZmPLC gene in roots is up-regulated under conditions of high salt, dehydration, cold or low osmotic stress conditions (Zhai et al. 2005). In potato leaves, three StPLC genes are differentially expressed after wounding or during drought stress. The StPLC1 exhibited strong reduction of its transcript level whereas StPLC2 was equally strongly induced in response to both stresses with only minor alterations in the expression of StPLC3 (Kopka et al. 1998). Transgenic maize expressing ZmPLC1 sense gene improved drought tolerance and the transgenic plants showed higher relative water content, better osmotic adjustment, increased photosynthesis rates, lower percentage of ion leakage and less lipid membrane peroxidation and higher grain yield than the WT (Wang et al. 2008). The mechanism could be via release of calcium which acts as a molecular switch to trigger downstream signalling events (Parre et al. 2007). Although previous studies on plant PI-PLCs have been performed, a detailed analysis on any isoform has not been carried out. We have isolated and characterized the expression pattern of NtPLC1 under stress. The cytoplasmic localization of phospholipase C1 has been established and our results also shed light on the role of phospholipase C1 in response of abiotic stress, as its overexpression in tobacco plants conferred increased tolerance not only to drought but also to salinity stress.

Isolation of NtPLC1 cDNAs The cDNA library was constructed, starting from 5 g of mRNA isolated from salinity- treated plants of N. tabaccum, in the UNIZAP vector using a cDNA synthesis kit (Stratagene) according to the manufacturers protocol. The PstIXhoI fragment of a soybean cDNA for phospholipase C (Shi et al. 1995) was labelled (Gibco BRL) to a specific activity of >1 108 cpm/g DNA using 32 P[dCTP]. The cDNA library was screened and many independent plaques that produced strong hybridization signals were observed. From these, two independent clones with the largest insert size were selected. The insert DNAs were excised as pBluescript SK+ phagemids and sequenced completely. The cDNA sequence has been deposited in the NCBI/GenBank/DDBJ database under the Accession No. AF223351. Tobacco Plant Transformation The complete ORF of NtPLC1 cDNA (1.7 kb) was PCR amplified by using PLC-F (5-GCCTCTAGAATGTCGAG ACAGACGTACG-3) starting from the translation initiation site and PLC-R (5GCCTCTAGAGACAAATTCGA AACGCATAA-3) primers designed to create an XbaI site at the 3 end (underlined above) next to the translation termination codon and the same was confirmed by sequencing. The amplified fragment was cloned into pBI121 (Clontech) to create pBI-NtPLC1 (Fig. 4a). This vector contains NtPLC1 and GUS (uidA) under a single 35S CaMV promoter; however, a stop codon has been inserted between NtPLC1 and the reporter gene to avoid translational fusions. The pBI-NtPLC1 also carries the nptII (kanamycin) gene as a selectable marker. The NtPLC1 construct was introduced into tobacco via Agrobacterium-mediated leaf disc transformation method (Horsch et al. 1985). Transgenic plants were selected on 50 g/ml kanamycin and several lines of transgenic plants were obtained. RNA Isolation and cDNA Synthesis Total RNA from plant tissues was isolated using Trizol (Sigma, USA) according to the manufacturers protocol. Approximately 20 g of total RNA was used to synthesize first-strand cDNA using the cDNA synthesis kit (Fermentas, USA). Genomic DNA PCR and Southern Analysis Genomic DNA was isolated by grinding leaf tissue followed by extraction using the CTAB (N-acetyl-N,N,Ntrimethylammonium bromide) method (Murray and

Materials and Methods Plant Materials Sterilized seeds of Nicotiana tabaccum cv. Xanthi were allowed to germinate in wet germination paper roll for 5 days at 26C, and were then grown up to 35 days in Hoaglands solution at 14/10-h light/dark cycle. For salinity stress, Nicotiana seedlings were treated with 250 mM NaCl in Hoagland solution for 4 and 24 h. For ABA treatment, Nicotiana seedlings were sprayed with 50 M ABA solution for 4 and 24 h and cold (4C) treatment was given for 4 and 24 h. After treatments, the seedlings were frozen in liquid nitrogen and processed for RNA isolation using Trizol (Sigma, USA) according to the manufacturers protocol to analyze the expression of NtPLC 1 transcript. To monitor salinity and mannitol stress effects on NtPLC1 overexpressing plants, 10-day-old WT and kanamycinpositive T1 transgenic seedlings were transferred to Murashige and Skoog medium (Caisson) supplemented with 100, 200, 250 mM NaCl and 200, 400 mM mannitol, under culture room conditions. The length of root and shoot of seedlings surviving under stress was scored in 40 days.

Plant Mol Biol Rep

Thompson 1980). PCR was performed by using the forward primer PLC-F (5-ATGTCGAGACAGACGTACAG-3) and PLC-R (5-TTAGACAAATTCGAAACGCATA-3), as the reverse primer. PCR conditions were 94C for 1 min, 53C for 1 min and 72C for 2 min. For Southern analysis, 20 g of genomic DNA from transgenic tobacco lines was digested with XbaI separately, electrophoresed, and blotted on Hybond N+ Nylon membranes (Amersham Pharmacia). Radiolabelled 1.7-kb complete ORF of NtPLC1 cDNA was used as a probe. Hybridization and washing were carried out at 65C as described previously (Reddy et al. 1999). The blots were autoradiographed. Quantitative Real-Time PCR

injection after 1 month, the blood was collected after 2 weeks and serum separated. Protein Extraction, SDS-PAGE and Immunoblotting Total proteins from the plant tissue were isolated as reported previously (Pandey et al. 2002) and 20 g of the soluble proteins were separated by 12% SDS-PAGE. NtPLCI antibodies (1:200 dilution) were used as primary antibody while peroxidase-conjugated anti-rabbit IgG antibodies as secondary antibodies (Amersham Biosciences, USA). Western blot analysis was performed in an ECL protein detection system (Amersham, USA) according to the manufacturers instructions. Immunofluorescence Staining and Confocal Microscopy

First-strand cDNA(s) prepared from RNA isolated from tobacco seedlings that are exposed to various stress treatments and from T1 transgenic plants (S10 and S11) seedlings was used as template and PCR was performed using the isoform specific primers for stress sample and some selected genes such as NtHSF2 (Accession No. AB014484), HSP70-3 (Accession No. AY372071) and AP2 domain TF (Accession No. AJ299252) for T1 transgenic plants (S10 and S11). Tobacco Actin gene was used as internal control. For Heat shock factor NtHSF2, primers sequence is (F5-AGAGTTGGATG A ATCTAC A AG T TG -3 and R 5 -CACTGTCTA CTGGTTTGCTTTC-3), Heat shock protein HSP70-3 (F5- GGTCCAGGAAGCAGAGAAG-3 and R5-GAG CATCATCATCCATAGGTG-3), Signal transduction AP2 domain (F5-AATACAGAGGAATAAGGCAGAGAC-3 and R5-CTCAGCAGCGGGCATTTC-3). PCR reaction mixture consisted of 1X PCR buffer, 200 M of each of dNTPs, and 150 ng of each of the gene specific primers, in the presence of 1 SYBR Green dye in a 50 l reaction. For analyzing NtPLC1 transcript under various conditions, the PCR conditions were as follows: 94C for 1 min; 53C for 1 min; 72C for 1 min for 30 cycles, and for selected genes 94C for 1 min; 55C for 1 min; 72C for 1 min for 30 cycles. At the end of the PCR cycles, the products were analysed through a meltcurve analysis to check the specificity of PCR amplification. Three biological repeats were done for each sample, and data were analyzed by REST 2009 software. Production of Polyclonal Antibodies Against NtPLC1 The 67-kDa purified recombinant polypeptide of NtPLC1 (Fig. 1a, lane 3) was mixed with an equal amount of Freunds complete adjuvant (Sigma, USA). The emulsified sample was injected subcutaneously into a New Zealand white rabbit. The rabbit was given a booster

Exponentially growing tobacco BY2 suspension cells were fixed, permeabilized according to Proust et al. (1999) and layered onto poly-L-lysine-coated cover slips and the cells immunostained with NtPLCI specific primary antibody in 1:100 dilution and Alexa fluor 488-labelled secondary antibody (Molecular Probes, USA) in 1:1000 dilution. The cells were counterstained with DAPI (0.2 g/ml) for 10 min just before mounting the slide in antifade solution (Fluroguard, Bio-Rad, USA). Confocal microscopy (Radiance 2100, Bio-Rad) was performed as described (Singh et al. 2003). Leaf Disc Assay for Tolerance Against Salinity Stress Healthy and fully expanded leaves (of similar age) from WT and transgenic plants (60 days old) were briefly washed in deionized water and leaf discs of 1-cm diameter were punched out and floated in a 6-ml solution of NaCl (100 and 200 mM for 72 h) or sterile-distilled water (which served as experimental control). The chlorophyll content was measured as described (Arnon 1949). The experiment was repeated thrice with two different transgenic lines. Endogenous Ion Content Determination Mature WT and transgenic plants grown under water and in 200 mM NaCl in a greenhouse for 150 days were used. Roots, old leaves, young leaves, and stems were collected from three different plants of each type and thoroughly rinsed in deionized water, and the fresh weight of each sample was determined. The samples were dried at 70C for 48 h, and the dry weight of each sample was measured. The material was ground to a fine powder and digested in concentrated HNO3 overnight at 120C. Samples were then dissolved in HNO3/HClO4 (1:1, vol/vol) at 220C, resus-

Plant Mol Biol Rep

a
Nt At St Ps Gm Os Nt At St Ps Gm Os Nt At St Ps Gm Os Nt At St Ps Gm Os Nt At St Ps Gm Os MS---RQTYRVCFCFRRRFRVVAAEAPADVKNLFNRYS--DNGVMNAENLQRFLIEVQ-KEENASLEDAQGIMN---NLHDLKILNIFHRRGLHLDAFFKYLFA-DINPPINPKRG-IHHDMNE 113 MK----ESFKVCFCCVRNFKVKSSEPPEEIKNLFHDYS--QDDRMSADEMLRFVIQVQ-GETHADINYVKDIFHR------LKHHGVFHPRGIHLEGFYRYLLS-DFNSPLPLTRE-VWQDMNQ 109 MS---KQTYRIC-CFQRKFKLKEAEAPDEIKDLFERFS--ENGIMTAEHLCKFLKDVQ-GEENVTKEEAETVMESALKLVHEHLNIVFHRKGLNLDGFFRYLFS-DLNVSISTDKK-VHHDMTA 115 MASKQKQTYSVCFCCRRRFKLGISEAPSQIRELYHNYSD-ESAIMTASHLQRFLIEVQ-GDENITENEAQSIIDG------HKHLSIFHRRGLNLESFFKFLFS-DNNAPLLASRG-VHQVMSL 114 MTS--KQTYSVCFCWRRRFKLALAEAPSEIKTLFEEYS--ENEFMTPSHLKRFLVEVQ-RQEKATEEDAQAIIDS------FRHFPR-RGAGLNLETFFKYLFS-DDNPPLLPSHG-VHHDMTL 110 MG-----TYKCCLIFKRRFRWNDAPPPDDVRALFANHSAGGGPHMAADGLRAYLQATGTRTATWTWSGWWSRSGSCRGAAGASRGWGGHSHSCTVDDFHRFLFSHELNPPIRHGQGQVHHDMAA 119 * .. * * *. . * .. *. * * . .. . .. * ..* . . * . . . . * X-domain (109-251 AA) PLSHYFIYTGHNSYLTGNQLSSDCSDVPIIQALGRGVRVIELDIWPNSAKDDVEVLHGGTLTTPVALIKCLRSIKEHAFTVSEYPVVITLEDHLTPDLQAKVAEIT-QTFGDMLFSP-DSCLKN 235 PLSHYFLYTGHNSYLTGNQLNSNSSIEPIVKALRNGVRVIELDLWPNSSGKEAEVRHGGTLTSREDLQKCLNVVKENAFQVSAYPVVLTLEDHLTPILQKKVAKMVSKTFGGSLFQCTDETTEC 233 PLSHYFIYTSHNTYLTGNQLNSDCSDVPIIKALQQGVRVIELDMWPNSSKDNVDILHGGTLTPPVELIQCLKSIKEHAFVASEYPVIITLEDHLTPDLQAKAAEMVTQVFGDILFTCGAECLSE 239 PLSHYYIHTGHNSYLTGNQLSSDCSDAPIIVALQRGVRVIELDIWPNGSKDDIEVLHGRTLTTPVALIKCLRSIKEYAFVASEYPVVITLEDHLTPDLQAKVAQMVTQTFGDILFCPSSESLKE 238 PLSHYFIYTGHNSYLTGNQLSSDCSDVPIINALKRGVRVIELDIWPNASKDNIDVLHGRTLTTPVELIRCLRSIKDHAFVASEYPVVITLEDHLTPDLQAKVAEMVTETFGDLLFTPNSESVKE 234 PLSHYFIYTGHNSYLTGNQLSSDCSDLPIIRALQRGVRVIELDMWPNSSKDDISILHGRTPTTPVSLLKCLLSIKEHAFEASPYPVIITLEDHLTPDLQDKAAKMVLEVFGDILYYPDKDHLKE 243 *****.. * **.******* * .* **. ** ********.*** . . ** * * * .** .*. ** * ***..******** ** * * . ** *. FPSPESLKRRVLISTKPPKEYLQAKEVK-------EKD----SKKGTESPDTEARGREVSDLK-ARYND-KDDSDDGAGVEDDESD--EGDPNSQQNVAPEYKCLIAIHAGKGKGGLSDWLRVD 344 FPSPESLKNKILISTKPPKEYLQTQISK------------------GSTTDESTRAKKISDAEEQVQEE------------------------DEESVAIEYRDLISIHAGNRKGGLKNCLNGD 315 FPSPESLKGRIIISTKPPKEYLESKKPS-------EKDN--GSQKGKKSSEEKAWGAEISDLS-QKMIA-YSENKDNGECQDDEADSHHENPNIQQNIAPEYKHLIAIQAGKSKGPTSEWLTVD 352 FPSPDSLKRRIIISTKPPKEYLEAKEVQ-------EKE---ELTKGKSSGDEEAWGKEVPSLRGGTISD-YKNNDDDDEDDLNEEEDSEEAEKSRQNGSGEYRRLIAIHAGKPKGGLVEGLKVD 351 FPSPESLKKRIIISTKPPKEYLEAKEKEKGDDSQHEKEKGDDSQHGKALGEDEAWGKEVPSLKGGTIED-YKDYNVD--EDLNDEEEFDESDKSHHNEAPEYRRLIAIHAGKPKGGLAECLKVD 355 FPSPQDLKGRVLLSTKPPKEYLQAKDGN---------AATIKEDAKAAATDDAAWGKEVPDIHSQIHSATKHDQREDDDDTDEDEDDEEEEQKMQQHLAPQYKHLITIKAGKPKGTLLDALQSD 358 **** ** ....*********... . . . . .*. **.*.** ** * * Y-domain (322-438 AA) PDKVRRLSLSEQELGKAVVTHGKEIIRFTQRNLLRIYPKGIRFDSSNYNPFVAWTHGAQMVHH-MQGYGRSLWLMHGMFRSNGGCGYVKKPDILLKAGPNNQIFDPEAN-LPVKTTLKVTVFMG 466 PNRVIRLSMSEQWLETLAKTRGPDLVKFTQRNLLRIFPKTTRFDSSNYDPLVGWIHGAQMVAFNMQSHGRYLWMMQGMFKANGGCGYVKKPDVLLSNGPEGEIFDPCSQNLPIKTTLKVKIYTG 439 PIKVKRISLNEEKLINVALNHGKDLIRFTQRNLLRIYPKGMRVDSSNYNPLMGWMHGAQMVAFNMQGHGRPLWLMQGMFKANGGCGYVKKPELLLKTDANNEVHDPKRL-LSVKTTLKVKVYMG 475 PDKVRRLSLSESQLEKAAETYGKEIVRFTQRNILRVYPKGTRITSSNYNPLIGWMHGAQMVAFNMQGYGRSLWLMQGMFKANGGCGFVKKPDFLLKTGPNNEVFDPKAS-LPLKTTLKVTVYMG 474 PDKVRRLSLSEQQLEKAAINHGQQIVRFTQRNILRVYPKGTRIDSSNYNPLIGWMHGAQMVAFNMQGYGRSLWLMHGMFRANGGCGYVKKPNFLLETGPDDEVFNPKAK-LPVKTTLKVTVYMG 478 PEKVRRLSLSEQQLAKLADHHGTEIVRFTQRNLLRIYPKGTRVTSSNYNPFLGWVHGAQMVAFNMQGYGRALWLMHGFYKANGGCGYVKKPDFLMQTDP--EVFDPKKS-LSSKKTLKVKVYMG 479 * .* *.*. * * ** ** **.*.* ...*****.**** *. .. * * * **** .. * * ...*****.**..** * **** * . * ****** C2-domain (458-566 AA) EGWYIDFKHTHFDAYTPPDFYAKIGIAGVPADNVMKKTKTLEDMDTPTWDEKFEFPLTVPELALLRVEVHEYDMSEKDDFAGQTCLPVSELRQGIRAVSLHDRKGEKYNSVKLLMRFEFV-- 586 EGWNMDFPLDHFDRYSPPDFYAKVGIAGVPLDTASYRTEIDKDEWFPIWDKEFEFPLRVPELSLLCITVKDYDSNTQNDFAGQTCFPLSEVRPGIRAVRLHDRAGEVYKHVRLLMRFVLEPR 561 KGWHLDFKRTHFDAYSPPDFYVKIGIAGVAADSRVKKTKAIEDNWIPIWNDEFEFPLTVPELALLRVEVHEYDMSEIDDFGGQTCIPVSELRTGIRAVPIYNEKGEKYPSVKLLMRFEFVK- 596 EGWYYDFDHTHFDQFSPPDFYARVGIAGVPFDTIMKKTKTVEDSWLPSWNEVFEFPLSVPELALLRIEVHEYDMSEKDDFGGQTCLPVWELRTGIRAVPLHSRKGDKYNNVKLLMRFEFI-- 594 EGWYYDFKHTHFDQYSPPDFYTRVGIAGVPNDTIMKRTKAIEDNWLPTWNEVFEFPLTVPELALLRIEVHEYDMSEKDDFGGQACLPIWELRSGIRAIPLHSQKGDKYNTVKLLMRFEFINN 600 DGWRMDFTQTHFDQYSPPDFYARVGIAGVPADSVMKRTRAIEDNWVPVWEEDFTFKLTVPEIALLRVEVHEYDMSEKDDFGGQTVLPVSELIPGIRAVALHDRKGIKLNNVKLLMRFEFE-- 599 ** ** *** ..***** ..***** * .* * * * * * * ***..** . *..** ** **. *. *. ****. . * *.*****

b
0.016

0.276 0.047 0.008 0.241 0.111 0.124 0.163 0.194

AtPLC PsPLC GmPLC NtPLC StPLC OsPLC

Fig. 1 a Comparison of the deduced amino-acid sequence of phospholipase C from N. tabacum (Accession No. AAF33823) with A. thaliana (Accession No. NP568881), S. tuberosum (Accession No. CAA63777), P. sativum (Accession No. CAA75546), O. sativa (Accession No. AAK01711), G. max (Accession No. AAA74441). Conserved regions overlined represent the structural organization of

NtPLC and its various domains. Stars are identical and dots are similar amino acids mentioned under the sequences. b Dendrogram showing the phylogenetic relationship between phospholipase C sequences characterized from different plant species. The tree was constructed based on the amino acid sequences deduced from phospholipase C gene sequences reported from different plants

pended in 5% (vol/vol) HNO3, and analyzed for sodium (Na+) content by using simultaneous inductively coupled argon-plasma emission spectrometry (ICP trace analyzer; Labtam, Australia). Statistical Analysis of Root and Shoot Length

was done using CLUSTALW using MacVector suite of programmes.

Result and Discussion Sequence and Phylogenetic Analysis of NtPLC1 Data of six plants of WT and transgenic lines for each stress experiment were collected. The mean values and standard deviations were calculated. To represent the root and shoot length data for control, NaCl and mannitol tolerance assays, % was used. During calculation, the values of WT were taken as 100%, the value of S10, S11 transgenic lines in% calculated with respect to WT. Sequence Analysis Most of the routine nucleotide sequence analyses were performed using MacVector 12.01. Homology searches were done using FASTA and multiple sequence alignment Screening of the N. tabaccum cDNA library with the heterologous phospholipase C cDNA from soybean as a probe resulted in the isolation of many independent positive plaques. Upon sequencing, the N. tabaccum cDNA with the largest insert was found to show homology to PI-PLC genes. Our work on tobacco (unpublished data) revealed that at least two isoforms exist. In this study, detailed analysis has been done on isoform 1, NtPLC1 (Accession No. AF223351).The total length of the cDNA was 2,098 bp, comprising an ORF of 1,761 bp, with an untranslated sequence of 105 bp at the 5 end and 232 bp at the 3 end, and encodes a protein of 586 amino acids with

Plant Mol Biol Rep

an apparent molecular weight of 67 kDa and an estimated isoelectric point of 6.14. The multiple alignment of the deduced amino acid sequence revealed that the primary structure of NtPLC1 is similar to other known plant phospholipases (Fig. 1a) and shows the presence of functionally important conserved X, Y and C2 domains. The phylogenetic tree shows that NtPLC is close to StPLC and lies on the same branch (Fig. 1b). Purification and Localization of Recombinant Tobacco NtPLC1 The complete ORF of NtPLC1 was successfully overexpressed in prokaryotic expression system (Fig. 2a, lane 2). The recombinant protein was purified to homogeneity (Fig. 2a, lane 3) and antibodies raised (Fig. 2b). Immunofluorescence staining and confocal microscopic observations revealed that the NtPLC1 is localized to the cytoplasm (Fig. 2c). This is consistent with the role of phospholipase C in signal transduction pathway occurring in the cytosol. As this PLC protein from tobacco exhibits characteristics of both subgroups, may be the soluble form (group 1) is the predominant form as far as the localization is concerned. In vivo targeting experiment using a PLCgreen fluorescent fusion protein showed that in Vigna radiata L. (mung bean) the delta-isoform, Vr-PLC3 was localized primarily in the plasma membrane of the Arabidopsis protoplast (Kim et al. 2004). In the case of soybean, the delta isoform (PI-PLC1) was localized in the cytosol and plasma membrane (Shi et al. 1995). Expression of NtPLC1 Under Various Abiotic Stresses We used real-time PCR technology to study the expression pattern of the NtPLC1 isoform, in response to salinity stress, cold and also following external application of ABA. An attempt was made to design forward primer from conserved region and a specific reverse primer for the NtPLC1 isoform using Oligo 4 software. This software yielded only one NtPLC1 isoform-specific unique reverse primer from the 3 UTR region (due to the very high level of similarities between the two isoforms). Therefore, primers common to both the isoforms were designed. In response to 4-h treatment with 250 mM NaCl, the expression level of NtPLC1 showed a 1.34-fold increase in the transcript level, while in the 24-h treatment a 1.5-fold increase in the transcript was seen (Fig. 3). It has been reported that there is a considerable overlap among different signal transduction pathway in response to various abiotic stresses (Seki et al. 2002). The expression levels of NtPLC1 increased nearly 2.1-fold when the seedlings were exposed to low temperature for 4 h and 1.8Fig. 2 a Purification of recombinant NtPLC1 from E. coli. SDS-PAGE profile of protein at different stages. Lane 1 uninduced; lane 2 induced, lane 3 purified protein eluted from Ni-NTA column. The arrow on the right shows the size of the purified NtPLC1 protein. b Purified protein recognized by the polyclonal antibodies raised in rabbit. c Subcellular localization of phospholipase C 1 in tobacco BY2 cells by using indirect immunofluorescence microscopy. I Image of cell stained with DAPI, II immunofluorescently stained cell, III phase-contrast image of cell, IV superimposed image of cell. The arrow indicates the nucleus

fold in 24 h (Fig. 3). Recently, 58 genes that are regulated by temperature downshift via PLC activity have been identified by analyzing the transcriptome of cold-treated Arabidopsis thaliana suspension cells in the presence of U73122 (inhibitor of the PLC/diacylglycerol kinase pathway). These results support the idea that the PLC pathway is upstream of signaling pathway that leads to the activation of the cold response (Vergnolle et al. 2005). Several abiotic stresses such as salt, drought, low temperature, salt-alkaline, CdCl2 are mediated via ABA

Plant Mol Biol Rep

Fig. 3 Effect of salinity, ABA and cold treatment on transcript level of NtPLC1 in shoots. Quantification of mRNA levels was achieved by using quantitative real-time PCR. The expression levels of the gene was normalised with respect to the internal control actin. The relative values were calculated based on the mean of three Ct values from three biological replicates for each sample (250 mM NaCl, 50 M ABA, cold (4C) treatment for 0, 4 and 24 h). The figure in the X-axis represents NaCl, ABA, cold stress treatments given for a period of 0, 4, and 24 h. The Y-axis represents relative transcript level under corresponding stress treatment

transgenic plants and grew them to maturity; however, only seven different plants (S1, S5, S6, S7, S10, S11, and S12) were randomly selected for subsequent analysis. The morphological and growth characteristics of T0 plants were similar to the WT plants. The stable integration was confirmed by hybridization of the XbaI-digested DNA with the NtPLC1 probe, and a single band of 1.7 kb, corresponding to the NtPLC1 cDNA, was observed (Fig. 4c). Immunoblot of the T0 transgenics by using NtPLC1 antibodies identified a 67-kDa band corresponding to the tobacco protein in different transgenic lines (Fig. 4d). Tobacco Plants Overexpressing NtPLC1 Shows Salinity and Mannitol Tolerance To determine whether NtPLC1-imparted salinity and drought tolerance is functionally and genetically stable, the T1 progeny were characterized. Seeds from the T0 plants, when plated on to kanamycin-containing medium, segregated in the expected 3:1 ratio of Kanr/Kans.

(Seki et al. 2002; Alam et al. 2010; Gao et al. 2010; Jellouli et al. 2010; Wang et al. 2010). The transcript levels of NtPLC1 were 2-fold up-regulated in response to 50 M ABA treatment when tobacco seedlings were treated for 4 h and 1.8-fold in 24 h (Fig. 3). Earlier, the transcript level of mung bean delta-isoform, Vr-PLC3, was found to be induced in salt stress in ABA-independent manner (Kim et al. 2004). Analysis of changes in inositol 1,4,5-trisphosphate [Ins (1,4,5) P3] content in response to hyperosmotic shock or salinity in A. thaliana T87 cultured cells suggest the involvement of PI-PLC and Ins(1,4,5)P3 in an ABA-independent manner (Takahashi et al. 2001). Arabidopsis phospholipase C1 (encoded by AtPLC1) has been shown to play a role in secondary ABA responses (Sanchez and Chua 2001). Our study shows that the NtPLC1 is salinity and cold regulated and ABA treatment could mimic stress treatment by increasing the steady-state transcript levels of NtPLC1 in shoots. Overexpression of NtPLC1 in Tobacco The results from the real-time PCR study indicated NtPLC1s role in stress physiology. To verify this, we overexpressed the complete ORF of NtPLC1 gene in tobacco plants by using Agrobacterium-mediated transformation (Fig. 4a). The NtPLC1 gene driven by the 35S promoter was cloned in pBI 121 vector (Fig. 4a). The preliminary screening of kanamycin-resistant putative tobacco transformants was followed by genomic PCR in the GUS positive plants by PCR using PLC-F and PLC-R as primers (Fig. 4b). We selected 20 independently transformed T0

Fig. 4 Analysis of transgenic plants (T0) to confirm the integration and expression of the gene. a Schematic representation of the pBINtPLC construct used to overexpress NtPLC1 cDNA in tobacco plants. bd The WT and NtPLC T0 transgenic lines were analyzed by the following methods. Analysis was performed by genomic PCR (b), Southern blots (c) and Western blot (d) analysis using anti-NtPLC1 antibodies. The size of the band is indicated by arrows. c Southern blot showing the presence of the gene NtPLC1. DNA was digested with XbaI

Plant Mol Biol Rep

Fig. 5 Representative pictures to show relative salinity and mannitol tolerance of WT and kanamycin-positive T1- NtPLC1tobacco transgenic plants. Seedling growth comparison of WT and T1 transgenic lines (S10 and S11) of 40-day-old seedlings on a control,

b 100 mM NaCl, c 200 mM NaCl, d 250 mM NaCl and e 200 mM mannitol, f 400 mM mannitol. The graphs represent the mean value and SD over six replicates for shoot and root length of seedlings

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Growth, being the endpoint of metabolic process, is often used as a parameter to assess tolerance. Kanamycin resistance T1 generation seedlings and WT were germinated on various concentrations of NaCl, mannitol and their growth was monitored for 40 days (Fig. S1). The transgenics and WT plants showed comparable growth when grown on medium without NaCl and mannitol (Fig. 5a). When exposed to 100, 200 and 250 mM NaCl and 200, 400 mM mannitol, the WT plants showed drastic reduction in growth, whereas the transgenic lines tolerated this level of salinity and mannitol (Fig. 5bf). Some growth parameters such as length of shoots and length of roots were measured after 40 days of growth. Under control con-

Fig. 7 Na+ content (calculated as percent dry weight [DW] of the tissue) in various tissues of the phospholipase C delta transgenic plants (S10, S11) grown under the continued presence of 200 mM NaCl. In the histogram, S10 and S11 are indicated by white and black colour as shown. For each determination, roots, old leaf (fourth leaf from the bottom), young leaf (second leaf from the top), and stem were collected from three different plants of both transgenic lines. Values are the mean standard deviation (n=3). Similar data for WT plants could not be obtained as these plants did not grow further in the presence of 200 mM NaCl. However, the relative values for Na+ in the WT plants grown in water were found to be 0.2% in roots, 0.18% in old leaf, 0.1% in young leaf, and 0.5% in stem

ditions, S10 shows 51.78% increase in shoot length, 150.45% increase in root length and S11 shows 16.07% increase in shoot length, 48.64% increase in root length as compared to WT. Under 100, 200 and 250 mM NaCl stress, S10 showed a 32.07% to 64% increase in shoot length and a 40.12% to 73.46% increase in root length, whereas S11 showed a 5.66% to 24% increase in shoot length and a 20.07% to 75.51% increase in root length as compared to WT. Under 200 and 400 mM mannitol stress, S10 showed a 31.57% to 15.62% increase in shoot length and a 134.42%

Fig. 6 Leaf disc senescence assay for salinity tolerance in transgenic tobacco plants (T1). a Representative picture to show phenotypic differences in leaf discs of WT and transgenic plants (S10 and S11) after incubation in 100 and 200 mM solutions of NaCl for 72 h is shown. b Amount of chlorophyll content (g/g of fresh weight) in NaCl-treated (100, 200 mM) leaf discs of WT and transgenic plants (S10, S11). Discs floated in water served as the experimental control. The standard deviation in each case is represented by the vertical bar in each graph. Note the difference in retention of chlorophyll in WT and transgenic plants

Fig. 8 Relative transcript level of three genes in NtPLC1 tobacco transgenic plants (S10 and S11) under control condition as revealed by quantitative RT-PCR analyses.The relative values were calculated based on the mean of three Ct values from three biological replicates for each sample. The figure on the X-axis represents three different genes such as NtHSF2, HSP70-3, AP2 domain TF. The Y-axis represents relative transcript level of S10, S11in respect to WT under three different genes. The WT value was treated as 0

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to 66.66% increase in root length, whereas S11 showed a 26.31% to 28.12% increase in shoot length and a 17.21% to 94.44% increase in root length as compared to WT. It was previously reported that enhanced expression of phospholipase C 1 improves drought tolerance (Wang et al. 2008). We also found its role in tolerance to salinity stress. Therefore, salt tolerance levels of T1 transgenic plants were additionally assessed by leaf disc senescence assay. Leaf discs from T1 transgenic and WT tobacco plants were floated separately on 100, 200 mM NaCl and H2O (control) for 72 h and their chlorophyll contents were measured. Compared to 10% in wild type, the transgenic lines retained up to 7684% of their chlorophyll contents at salt concentrations of 200 mM NaCl. Salinity-induced loss of chlorophyll was lower in NtPLC1 overexpressing lines compared with those from the WT plants. This provides support for a positive relationship between the overexpression of NtPLC1 and tolerance to salinity stress (Fig. 6). To examine the basic mechanism resulting in salt tolerant phenotypes of NtPLC1 overexpressing lines, we measured the endogenous Na+ levels in root, stem and leaves of T1 plants growing in the presence of 200 mM and absence of NaCl. The Na+ concentration was lower in young leaves and roots compared with mature leaves. Most of the Na+ got accumulated in the older leaves of the transgenic plants (Fig. 7).This finding suggests that the old leaves seem to function as ion sinks, thus keeping the young leaves essentially free from the additional uptake of Na+ ions (Singla-Pareek et al. 2003). Quantitative RT-PCR Analysis of Plants Overexpressing NtPLC1 To understand the stress tolerance mechanism, transgenics were studied for the expression of some stress target genes like heat shock protein a protein involved in signal transduction response in two T1 transgenic lines (S10 and S11). The two genes encoding for heat shock factor and heat shock protein-70 (NtHSF2, HSP70-3) showed higher expression. The AP2domain containing transcription factor also showed higher expression in both transgenic plants (Fig. 8). This result shows the role of heat shock factor, heat shock protein and AP2 domain containing transcription factor in salinity and drought tolerance mechanism (Wang et al. 2003; Wang et al. 2010; Fu et al. 2010).

using immunofluorescence and confocal microscopy, tobacco phospholipase C was localized to the cytoplasm of BY2 cells. The NtPLC1 transgenic plants shows tolerance to salinity and drought stress due to higher expression of downstream genes and better ion homeostasis machinery.

Acknowledgements This work was supported by Department of Biotechnology, Govt. of India (Indo-Belarus) project. For providing the confocal microscope facility (Welcome Grant) and technical help, we are thankful to Dr. Shahid Jameel and Ms. Charu, respectively. We also thank Drs. V. Rajamani, J.K. Tripathi (Jawaharlal Nehru University, New Delhi) for help with ionic measurements.

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