Skip to main content
SpringerLink
Log in

Expression of a cyclophilin OsCyp2-P isolated from a salt-tolerant landrace of rice in tobacco alleviates stress via ion homeostasis and limiting ROS accumulation

  • Original Paper
  • Published:
Functional & Integrative Genomics Aims and scope Submit manuscript

Abstract

Cyclophilins are a set of ubiquitous proteins present in all subcellular compartments, involved in a wide variety of cellular processes. Comparative bioinformatics analysis of the rice and Arabidopsis genomes led us to identify novel putative cyclophilin gene family members in both the genomes not reported previously. We grouped cyclophilin members with similar molecular weight and subtypes together in the phylogenetic tree which indicated their co-evolution in rice and Arabidopsis. We also characterized a rice cyclophilin gene, OsCyp2-P (Os02g0121300), isolated from a salinity-tolerant landrace, Pokkali. Publicly available massively parallel signature sequencing (MPSS) and microarray data, besides our quantitative real time PCR (qRT-PCR) data suggest that transcript abundance of OsCyp2-P is regulated under different stress conditions in a developmental and organ specific manner. Ectopic expression of OsCyp2-P imparted multiple abiotic stress tolerance to transgenic tobacco plants as evidenced by higher root length, shoot length, chlorophyll content, and K+/Na+ ratio under stress conditions. Transgenic plants also showed reduced lipid peroxidase content, electrolyte leakage, and superoxide content under stress conditions suggesting better ion homeostasis than WT plants. Localization studies confirmed that OsCyp2-P is localized in both cytosol and nucleus, indicating its possible interaction with several other proteins. The overall results suggest the explicit role of OsCyp2-P in bestowing multiple abiotic stress tolerance at the whole plant level. OsCyp2-P operates via reactive oxygen species (ROS) scavenging and ion homeostasis and thus is a promising candidate gene for enhancing multiple abiotic stress tolerance in crop plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

At:

Arabidopsis thaliana

Cyp:

Cyclophilin

CLD:

Cyclophilin-like domain

PPIase:

Peptidyl-prolyl-cis-trans isomerase

Os:

Oryza sativa

WT:

Wild type

References

  • Ahn JC, Kim DW, You YN, Seok MS, Park JM, Hwang H, Kim BG, Luan S, Park HS, Cho HS (2010) Classification of rice (Oryza sativa L. Japonica nipponbare) immunophilins (FKBPs, CYPs) and expression patterns under water stress. BMC Plant Biol 10:253

    Article  PubMed Central  PubMed  Google Scholar 

  • Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24:1–15

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brazin KN, Mallis RJ, Fulton DB, Andreotti AH (2002) Regulation of the tyrosine kinase Itk by the peptidyl-prolyl isomerase cyclophilin A. Proc Natl Acad Sci U S A 99:1899–1904

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chen AP, Wang GL, Qu ZL, Lu CX, Liu N, Wang F, Xia G (2007) Ectopic expression of ThCYP1, a stress-responsive cyclophilin gene from Thellungiella halophila, confers salt tolerance in fission yeast and tobacco cells. Plant Cell Rep 26:237–245

    Article  CAS  PubMed  Google Scholar 

  • Cho EK, Kim M (2008) A red algal cyclophilin has an effect on development and growth in Nicotiana tabacum. Plant Physiol Biochem 46:868–874

    Article  CAS  PubMed  Google Scholar 

  • Chou IT, Gasser CS (1997) Characterization of the cyclophilin gene family of Arabidopsis thaliana and phylogenetic analysis of known cyclophilin proteins. Plant Mol Biol 35:873–892

    Article  CAS  PubMed  Google Scholar 

  • Cross AR, Segal AW (2004) The NADPH oxidase of professional phagocytes—prototype of the NOX electron transport chain systems. Biochim Biophys Acta 1657:1–22

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Davis AR, Mascolo PL, Bunger PL, Sipes KM, Quinn MT (1998) Cloning and sequencing of the bovine flavocytochrome b subunit proteins, gp91-phox and p22-phox: comparison with other known flavocytochrome b sequences. J Leukoc Biol 64:114–123

    CAS  PubMed  Google Scholar 

  • Ferreira PA, Nakayama TA, Pak WL, Travis GH (1996) Cyclophilin-related protein RanBP2 acts as chaperone for red/green opsin. Nature 383:637–640

    Article  CAS  PubMed  Google Scholar 

  • Galat A (1999) Variations of sequences and amino acid compositions of proteins that sustain their biological functions: an analysis of the cyclophilin family of proteins. Arch Biochem Biophys 371:149–162

    Article  CAS  PubMed  Google Scholar 

  • Glenn EP, Brown JJ, Blumwald E (1999) Salt tolerance and crop potential of halophytes. Crit Rev Plant Sci 18:227–255

    Article  Google Scholar 

  • Godoy AV, Lazzaro AS, Casalongue CA, San Segundo B (2000) Expression of a Solanum tuberosum cyclophilin gene is regulated by fungal infection and abiotic stress conditions. Plant Sci 152:123–134

    Article  CAS  Google Scholar 

  • Haas BJ (2008) Analysis of alternative splicing in plants with bioinformatics tools. Curr Top Microbiol Immunol 326:17–37

    CAS  PubMed  Google Scholar 

  • Handschumacher RE, Harding MW, Rice J, Drugge RJ, Speicher DW (1984) Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226:544–547

    Article  CAS  PubMed  Google Scholar 

  • Harlow E, Lane D (1988) Antibodies; A laboratory manual. Cold Spring Harbor Laboratory Press, New York, New York

    Google Scholar 

  • Horowitz DS, Lee EJ, Mabon SA, Misteli T (2002) A cyclophilin functions in pre-mRNA splicing. EMBO J 21:470–480

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Huang JG, Yang M, Liu P, Yang GD, Wu CA, Zheng CC (2010) Genome-wide profiling of developmental, hormonal or environmental responsiveness of the nucleocytoplasmic transport receptors in Arabidopsis. Gene 451:38–44

    Article  CAS  PubMed  Google Scholar 

  • Iki T, Yoshikawa M, Meshi T, Ishikawa M (2012) Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants. EMBO J 31:267–278

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Jain M, Tyagi AK, Khurana JP (2008) Genome-wide identification, classification, evolutionary expansion and expression analyses of homeobox genes in rice. FEBS J 275:2845–2861

    Article  CAS  PubMed  Google Scholar 

  • Jambunathan N, Penaganti A, Tang Y, Mahalingam R (2010) Modulation of redox homeostasis under suboptimal conditions by Arabidopsis nudix hydrolase 7. BMC Plant Biol 10:173

    Article  PubMed Central  PubMed  Google Scholar 

  • Kang B, Zhang Z, Wang L, Zheng L, Mao W, Li M, Wu Y, Wu P, Mo X (2013) OsCYP2, a chaperone involved in degradation of auxin-responsive proteins, plays crucial roles in rice lateral root initiation. Plant J 74:86–97

    Article  CAS  PubMed  Google Scholar 

  • Karali D, Oxley D, Runions J, Ktistakis N, Farmaki T (2012) The Arabidopsis thaliana immunophilin ROF1 directly interacts with PI(3)P and PI(3,5)P2 and affects germination under osmotic stress. PLoS One 7:e48241

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kim SK, You YN, Park JC, Joung Y, Kim BG, Ahn JC, Cho HS (2012) The rice thylakoid lumenal cyclophilin OsCYP20-2 confers enhanced environmental stress tolerance in tobacco and Arabidopsis. Plant Cell Rep 31:417–426

    Article  CAS  PubMed  Google Scholar 

  • Kong HY, Lee SC, Hwang BK (2001) Expression of pepper cyclophilin gene is differentially regulated during the pathogen infection and abiotic stress conditions. Physiol Mol Plant Pathol 59:189–199

    Article  CAS  Google Scholar 

  • Krummrei U, Bang R, Schmidtchen R, Brune K, Bang H (1995) Cyclophilin-A is a zinc-dependent DNA binding protein in macrophages. FEBS Lett 371:47–51

    Article  CAS  PubMed  Google Scholar 

  • Krzywicka A, Beisson J, Keller AM, Cohen J, Jerka-Dziadosz M, Klotz C (2001) KIN241: a gene involved in cell morphogenesis in Paramecium tetraurelia reveals a novel protein family of cyclophilin-RNA interacting proteins (CRIPs) conserved from fission yeast to man. Mol Microbiol 42:257–267

    Article  CAS  PubMed  Google Scholar 

  • Kumar G, Purty RS, Sharma MP, Singla-Pareek SL, Pareek A (2009) Physiological responses among Brassica species under salinity stress show strong correlation with transcript abundance for SOS pathway-related genes. J Plant Physiol 166:507–520

    Article  CAS  PubMed  Google Scholar 

  • Kumar G, Kushwaha HR, Sabharwal VP, Kumari S, Joshi R, Karan R, Mittal S, Singla-Pareek SL, Pareek A (2012) Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging. BMC Plant Biol 12:107

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kumari S, Panjabi Nee Sabharwal V, Kushwaha HR, Sopory SK, Singla-Pareek SL, Pareek A (2009a) Transcriptome map for seedling stage specific salinity stress response indicates a specific set of genes as candidate for saline tolerance in Oryza sativa L. Funct Integr Genom 9:109–123

  • Kumari S, Singh P, Singla-Pareek SL, Pareek A (2009b) Heterologous expression of a salinity and developmentally regulated rice cyclophilin gene (OsCyp2) in E. coli and S. cerevisiae confers tolerance towards multiple abiotic stresses. Mol Biotechnol 42:195–204

  • Kumari S, Roy S, Singh P, Singla-Pareek SL, Pareek A (2013) Cyclophilins: proteins in search of function. Plant Sign Behav 8:8

    Article  Google Scholar 

  • Kushwaha HR, Singh AK, Sopory SK, Singla-Pareek SL, Pareek A (2009) Genome wide expression analysis of CBS domain containing proteins in Arabidopsis thaliana (L.) Heynh and Oryza sativa L. reveals their developmental and stress regulation. BMC Genomics 10:200

    Article  PubMed Central  PubMed  Google Scholar 

  • Kushwaha HR, Kumar G, Verma PK, Singla-Pareek SL, Pareek A (2011) Analysis of a salinity induced BjSOS3 protein from Brassica indicate it to be structurally and functionally related to its ortholog from Arabidopsis. Plant Physiol Biochem 49:996–1004

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Letunic I, Bork P (2011) Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucleic Acids Res 39:W475–W478

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Leverson JD, Ness SA (1998) Point mutations in v-Myb disrupt a cyclophilin-catalyzed negative regulatory mechanism. Mol Cell 1:203–211

    Article  CAS  PubMed  Google Scholar 

  • Lin DT, Lechleiter JD (2002) Mitochondrial targeted cyclophilin D protects cells from cell death by peptidyl prolyl isomerization. J Biol Chem 277:31134–31141

    Article  CAS  PubMed  Google Scholar 

  • Lippuner V, Chou IT, Scott SV, Ettinger WF, Theg SM, Gasser CS (1994) Cloning and characterization of chloroplast and cytosolic forms of cyclophilin from Arabidopsis thaliana. J Biol Chem 269:7863–7868

    CAS  PubMed  Google Scholar 

  • Marivet J, Margis-Pinheiro M, Frendo P, Burkard G (1994) Bean cyclophilin gene expression during plant development and stress conditions. Plant Mol Biol 26:1181–1189

    Article  CAS  PubMed  Google Scholar 

  • Marivet J, Frendo P, Burkard G (1995) DNA sequence analysis of a cyclophilin gene from maize: developmental expression and regulation by salicylic acid. Mol Gen Genet 247:222–228

    Article  CAS  PubMed  Google Scholar 

  • Mittler R, Vanderauwera S, Gollery M, Van BF (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498

    Article  CAS  PubMed  Google Scholar 

  • Mustafiz A, Singh AK, Pareek A, Sopory SK, Singla-Pareek SL (2011) Genome-wide analysis of rice and Arabidopsis identifies two glyoxalase genes that are highly expressed in abiotic stresses. Funct Integr Gen 11:293–305

    Article  CAS  Google Scholar 

  • Ouyang SQ, Liu YF, Liu P, Lei G, He SJ, Ma B, Zhang WK, Zhang ZS, Chen SY (2010) Receptor-like kinase OsSIK1 improves drought and salt stress tolerance in rice (Oryza sativa) plants. Plant J 62:316–329

    Article  CAS  PubMed  Google Scholar 

  • Pareek A, Singh A, Kumar M, Kushwaha HR, Lynn AM, Singla-Pareek SL (2006) Whole-genome analysis of Oryza sativa reveals similar architecture of two-component signaling machinery with Arabidopsis. Plant Physiol 142:380–397

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Pogorelko GV, Mokryakova M, Fursova OV, Abdeeva I, Piruzian ES, Bruskin SA (2014) Characterization of three Arabidopsis thaliana immunophilin genes involved in the plant defense response against Pseudomonas syringae. Gene 538:12–22

    Article  CAS  PubMed  Google Scholar 

  • Romano PG, Horton P, Gray JE (2004) The Arabidopsis cyclophilin gene family. Plant Physiol 134:1268–1282

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ruan SL, Ma HS, Wang SH, Fu YP, Xin Y, Liu WZ, Wang F, Tong JX, Wang SZ, Chen HZ (2011) Proteomic identification of OsCYP2, a rice cyclophilin that confers salt tolerance in rice (Oryza sativa L.) seedlings when overexpressed. BMC Plant Biol 11:34

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Scholze C, Peterson A, Diettrich B, Luckner M (1999) Cyclophilin isoforms from Digitalis lanata. Sequences and expression during embryogenesis and stress. J Plant Physiol 155:212–219

    Article  CAS  Google Scholar 

  • Sekhar K, Priyanka B, Reddy RD, Rao KV (2010) Isolation and characterization of a pigeonpea cyclophilin (CcCYP) gene, and its over-expression in Arabidopsis confers multiple abiotic stress tolerance. Plant Cell Environ 33:1324–1338

    CAS  PubMed  Google Scholar 

  • Sharma AD, Singh P (2003) Effect of water stress on expression of a 20 kD cyclophilin-like protein in drought susceptible and tolerant cultivars of Sorghum. J Plant Biochem Biotechnol 12:77–80

    Article  CAS  Google Scholar 

  • Sharma P, Jha AB, Dubey RS, Pessarakli M (2012) Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions, J Bot: 217037

  • Shieh BH, Stamnes MA, Seavello S, Harris GL, Zuker CS (1989) The ninaA gene required for visual transduction in Drosophila encodes a homologue of cyclosporin A-binding protein. Nature 338:67–70

    Article  CAS  PubMed  Google Scholar 

  • Singla-Pareek SL, Reddy MK, Sopory SK (2003) Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance. Proc Natl Acad Sci U S A 100:14672–14677

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Smith MR, Willmann MR, Wu G, Berardini TZ, Moller B, Weijers D, Poethig RS (2009) Cyclophilin 40 is required for microRNA activity in Arabidopsis. Proc Natl Acad Sci U S A 106:5424–5429

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Trivedi DK, Yadav S, Vaid N, Tuteja N (2012) Genome wide analysis of Cyclophilin gene family from rice and Arabidopsis and its comparison with yeast. Plant Signal Behav 17:1653–1666

    Article  Google Scholar 

  • Wang C, Jing R, Mao X, Chang X, Li A (2010a) TaABC1, a member of the activity of bc1 complex protein kinase family from common wheat, confers enhanced tolerance to abiotic stresses in Arabidopsis. J Expt Bot 63:1299–1311

    Google Scholar 

  • Wang X, Shi X, Hao B, Ge S, Luo J (2005) Duplication and DNA segmental loss in the rice genome: implications for diploidization. New Phytol 165:937–946

    Article  CAS  PubMed  Google Scholar 

  • Wang Z, Song J, Milne TA, Wang GG, Li H, Allis CD, Patel DJ (2010b) Pro isomerization in MLL1 PHD3-bromo cassette connects H3K4me readout to CyP33 and HDAC-mediated repression. Cell 141:1183–1194

    Article  CAS  PubMed  Google Scholar 

  • Wiederrecht G, Hung S, Chan HK, Marcy A, Martin M, Calaycay J, Boulton D, Sigal N, Kincaid RL, Siekierka JJ (1992) Characterization of high molecular weight FK-506 binding activities reveals a novel FK-506-binding protein as well as a protein complex. J Biol Chem 267:21753–21760

    CAS  PubMed  Google Scholar 

  • Xue T, Wang D, Zhang S, Ehlting J, Ni F, Jakab S, Zheng C, Zhong Y (2008) Genome-wide and expression analysis of protein phosphatase 2C in rice and Arabidopsis. BMC Genomics 9:550

    Article  PubMed Central  PubMed  Google Scholar 

  • Yurchenko V, Zybarth G, O’Connor M, Dai WW, Franchin G, Hao T, Guo H, Hung HC, Toole B, Gallay P, Sherry B, Bukrinsky M (2002) Active site residues of cyclophilin A are crucial for its signaling activity via CD147. J Biol Chem 227:22959–22965

    Article  Google Scholar 

  • Zhiguo EZ, Wang L, Zhou J (2013) Splicing and alternative splicing in rice and humans. BMB Rep 46:439–447

    Article  CAS  Google Scholar 

  • Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6:66–71

    Article  CAS  PubMed  Google Scholar 

  • Zivy M, Thiellement H, De VD, Hofmann JP (1983) Study on nuclear and cytoplasmic genome expression in wheat by two-dimensional gel electrophoresis: 1. First results on 18 alloplasmic lines. Theor Appl Genet 66:1–7

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Work carried out in this paper has been supported by funds available from the Department of Biotechnology, Ministry of Science and Technology, Government of India and IAEA, Vienna. The authors acknowledge AIRF, JNU for confocal microscopy. Award of research fellowship from CSIR to SR and SK is also gratefully acknowledged.

Conflict of interest

The authors declare that there are no conflicts of interest.

Author information

Authors and Affiliations

  1. Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India

    Sumita Kumari, Rohit Joshi, Kushwant Singh, Suchismita Roy & Ashwani Pareek

  2. Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India

    Amit K. Tripathi & Sneh L. Singla-Pareek

  3. Department of Biotechnology, Guru Nanak Dev University, Amritsar, 143005, India

    Prabhjeet Singh

Authors
  1. Sumita Kumari
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rohit Joshi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kushwant Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Suchismita Roy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Amit K. Tripathi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Prabhjeet Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sneh L. Singla-Pareek
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ashwani Pareek
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ashwani Pareek.

Additional information

Sumita Kumari, Rohit Joshi, Khushwant Singh, and Suchismita Roy have equally contributed to this study.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 25 kb)

Supplementary Table 1

(DOCX 11 kb)

Supplementary Table 2

(DOCX 15 kb)

Supplementary Fig. I

Chromosomal distribution and segmental duplication events for cyclophilin genes of a Arabidopsis; with prefix “At” and b rice; with prefix “Os.” Only the chromosomes having cyclophilin genes are shown and their number is indicated at their bottom in Romans. The scale is in megabase (Mb), and the centromeric regions are indicated by circles. The chromosomal positions (Mb) and orientation of each of the cyclophilin genes for Arabidopsis and rice are shown as horizontal bars and arrows, respectively. The cyclophilin genes present on duplicated chromosomal segments are connected by broken lines. (GIF 31 kb)

(TIFF 5242 kb)

Supplementary Fig. II

Phylogenetic tree depicting evolutionary relationship, similarity to cyclophilin subtypes identified in human and the presence of conserved domains among the cyclophilin members from Arabidopsis and rice. The detailed phylogenetic tree depicting evolutionary relationship among cyclophilin members of Arabidopsis and rice is shown in inner circle, while the classification of cyclophilin members of Arabidopsis and rice into various subtypes according to their similarity to subtypes identified for cyclophilin gene members in human is shown in middle circle. The outermost circle depicts the domain architecture and presence of signal peptides, if any, of cyclophilin members of Arabidopsis and rice. NLS nuclear localization signal, RRM RNA recognition motifs, ATPase adenosine tri-phosphatase, UBOX E4 ubiquitination factor, Trans trans domain, TPR tetratrico peptide repeats, CLD cyclophilin-like domain, WD40 beta-transducin repeats, TM transmembrane domain (GIF 56 kb)

(TIFF 14256 kb)

Supplementary Fig. III

Expression profile of cyclophilin genes from MPSS data (name of the MPSS libraries are shown below each heat map) in different tissues/organs of a Arabidopsis and b rice. The description of MPSS libraries is given at the website mentioned in the “Materials and methods” section. The heatmaps have been generated using TIGR MeV software package and represent hierarchical clustering of average log signal values of all cyclophilin genes in various tissues/organs (indicated at the top of each lane). The color bar below represents relative expression values; thereby, green color represents lowest expression levels, black represents medium expression levels, and red signifies highest expression level. (GIF 33 kb)

(TIFF 6532 kb)

Supplementary Fig. IV

Heatmap analyses of Cyp genes from Arabidopsis and rice under stress conditions. a The microarray data for expression of Arabidopsis cyclophilin genes under various abiotic stress conditions such as cold, osmotic, salt, drought, genotoxic, oxidative, UV-B, wounding, and heat stress has been retrieved from TAIR. b The microarray data obtained for expression of rice cyclophilin genes under various abiotic stress conditions such as drought, salt, and cold stress (Rice Oligonucleotide Array Database). Color bar at the right represents expression values in terms of fold change, thereby green color representing lowest expression levels, black medium expression levels, and red signifies highest expression level. (GIF 56 kb)

(TIFF 5096 kb)

Supplementary Fig. V

Confirmation of OsCyp2-P ectopically expressing transgenic tobacco plants. a Schematic diagram of OsCyp2-P gene construct in plant expression vector pCAMBIA1304. b Ethidium bromide gel showing PCR amplicons corresponding to OsCyp2-P and c genomic DNA from WT and OsCyp2-P ectopically expressing transgenic lines (L1, L2, L4, L5) digested with BglII and SpeI run on 0.8 % agarose gel stained with ethidium bromide. d Southern blot of respective lines probed with OsCyp2-P specific probe. Lines L1, L2, L4, L5, and L6 were found to be positive for transgene (GIF 41 kb)

(TIFF 14642 kb)

Supplementary Fig. VI

Bar diagram depicting PPIase activity of WT and transgenic line L5 (showing highest OsCyp2-P protein accumulation) plants. Peptidyl-prolyl-cis-trans isomerases activity was assayed in a coupled reaction with chymotrypsin. The assays were performed at 15 °C for 360 s. The 1-ml assay mixture contained 80 IM N-succinyl-ala-ala-prophe-p-nitroanilidine as test peptide, assay buffer [50 mM HEPES (pH 8.0), 150 mM NaCl, 0.05 % Triton X-100] along with varying concentration of the plant protein from either WT or transgenic line. The reaction was initiated by the addition of chymotrypsin (300 mg/ml) and the change in absorbance at 390 nm was monitored using a spectrophotometer. For calculating the PPIase activity, the difference between the catalyzed and uncatalyzed first-order rate constant, derived from the kinetics of the absorbance change at 390 nm, was multiplied by the amount of substrate in each reaction. (GIF 43 kb)

(TIFF 3053 kb)

Supplementary Fig. VII

Germination assay of OsCyp2-P ectopically expressing transgenic tobacco seeds on MS media supplemented with various stressors. For this purpose, medium was supplemented with either a PEG (5 %) for drought stress or b mannitol (200 mM) for osmotic stress. c Bar diagram showing germination percentage of wild-type and transgenic seedlings (L5) under various stress conditions. Bar diagram depicting d shoot length and e root length of wild-type and transgenic seedlings under control and stress conditions as indicated below the bars. The data represent mean ± SE of three biological replicates (n = 3). Asterisk shows values significantly different from respective WT under same conditions at P < 0.05, by Student’s t test. (GIF 212 kb)

(TIFF 18023 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kumari, S., Joshi, R., Singh, K. et al. Expression of a cyclophilin OsCyp2-P isolated from a salt-tolerant landrace of rice in tobacco alleviates stress via ion homeostasis and limiting ROS accumulation. Funct Integr Genomics 15, 395–412 (2015). https://doi.org/10.1007/s10142-014-0429-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10142-014-0429-5

Keywords

Search

Navigation