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Reference genes assessment for the seagrass Posidonia oceanica in different salinity, pH and light conditions

Abstract

The stability on the expression level of putative reference genes (RGs) for reverse transcriptase-quantitative PCR (RT-qPCR) was tested in the seagrass Posidonia oceanica for plants collected in different environmental conditions (at different depths and in different salinity and pH). Besides adult plants, seedlings were also used for pH and salinity experiments. The putative RGs encompassed the most frequently used in other species to normalize RT-qPCR in a range of experimental conditions. Assignment of the best RGs has been performed with three Excel-based applets, BestKeeper, geNorm and NormFinder, and the results showed that best RGs may change depending on the experimental conditions tested, plant age and on the software utilized. Preferred sets of RGs are proposed for each of the conditions and a general procedure concerning their selection is discussed. In addition, expression levels of four genes involved in plant response to light, salinity and pH variation have also been evaluated.

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References

  • Alleva K, Marquez M, Villarreal N, Mut P, Bustamante C, Bellati J, Martinez G, Civello M, Amodeo G (2010) Cloning, functional characterization, and co-expression studies of a novel aquaporin (FaPIP2;1) of strawberry fruit. J Exp Bot 61:3935–3945

    Article  CAS  Google Scholar 

  • Andersen CL, Jensen JL, Orntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250

    Article  CAS  Google Scholar 

  • Arnaud-Haond S, Migliaccio M, Diaz-Almela E, Teixeira S, van de Vliet MS, Alberto F, Procaccini G, Duarte CM, Serrao EA (2007) Vicariance patterns in the Mediterranean Sea: east-west cleavage and low dispersal in the endemic seagrass Posidonia oceanica. J Biogeogr 34:963–976

    Article  Google Scholar 

  • Bashitialshaaer R, Persson KM, Aljaradin M (2011) Estimated future salinity in the Arabian Gulf, the Mediterranean Sea and the Red Sea consequences of brine discharge from desalination. Int J of Academic Res 3:133–140

    Google Scholar 

  • Bellati J, Alleva K, Soto G, Vitali V, Jozefkowicz C, Amodeo G (2010) Intracellular pH sensing is altered by plasma membrane PIP aquaporin co-expression. Plant Mol Biol 74:105–118

    Article  CAS  Google Scholar 

  • Belzunce M, Navarro RM, Rapoport HF (2008) Posidonia oceanica seedling root structure and development. Aquat Bot 88:203–210

    Article  Google Scholar 

  • Bergmann N, Winters G, Rauch G, Eizaguirre C, Gu J, Nelle P, Fricke B, Reusch TBH (2010) Population-specificity of heat stress gene induction in northern and southern eelgrass Zostera marina populations under simulated global warming. Mol Ecol 19:2870–2883

    Article  Google Scholar 

  • Biela A, Grote K, Otto B, Hoth S, Hedrich R, Kaldenhoff R (1999) The Nicotiana tabacum plasma membrane aquaporin NtAQP1 is mercury-insensitive and permeable for glycerol. Plant J 18:565–570

    Article  CAS  Google Scholar 

  • Björk M, Short F, McLeod E, Beer S (2008) Managing seagrasses for resilience to climate change. IUCN, Gland, p 56

    Google Scholar 

  • Boudouresque C, Bernard G, Bonhomme P, Charbonnel E, Diviacco G, Meinesz A, Pergent G, Pergent-Martini C, Ruitton S, Tunesi L (2006) Préservation et conservation des herbiers à Posidonia oceanica. Ramonge pub: 1-202. ISBN 2-905540-30-3. www.ramonge.org

  • Boyé H (2008) Water, energy, desalination and climate change in the Mediterranean. http://www.planbleu.org/publications/Regional_study_desalination_EN.pdf

  • Bruno A, Bruno L, Chiappetta A, Giannino D, Bitonti MB (2010) PoCHL P expression pattern in Posidonia oceanica is related to critical light conditions. Mar Ecol Prog Ser 415:61–71

    Article  CAS  Google Scholar 

  • Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193

    Article  CAS  Google Scholar 

  • Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  CAS  Google Scholar 

  • Bustin SA, Beaulieu JF, Huggett J, Jaggi R, Kibenge FSB, Olsvik PA, Penning LC, Toegel S (2010) MIQE precis: practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol 11:74

    Article  Google Scholar 

  • Chaumont F, Barrieu F, Jung R, Chrispeels MJ (2000) Plasma membrane intrinsic proteins from maize cluster in two sequence subgroups with differential aquaporin activity. Plant Physiol 122:1025–1034

    Article  CAS  Google Scholar 

  • Cozza R, Pangaro T (2009) Tissue expression pattern of two aquaporin-encoding genes in different organs of the seagrass Posidonia oceanica. Aquat Bot 91:117–121

    Article  CAS  Google Scholar 

  • Danielson J (2010) Plant major intrinsic proteins. Natural Variation and Evolution Media-Tryck AB, Lund. ISBN 978-91-7422-259-3

    Google Scholar 

  • Duarte C, Marbà N, Santos R (2004) What may cause loss of seagrasses? In: Borum J, Duarte CM, Krause-Jensen D, Greve TM (eds) European seagrasses: an introduction to monitoring and management. The MandMS project, Hillerød, pp 19–23. ISBN: 87-89143-21-3

  • Fernandez-Torquemada Y, Sanchez-Lizaso JL (2005) Effects of salinity on leaf growth and survival of the Mediterranean seagrass Posidonia oceanica (L.) Delile. J Exp Mar Biol Ecol 320:57–63

    Article  Google Scholar 

  • Fetter K, Van Wilder V, Moshelion M, Chaumont F (2004) Interactions between plasma membrane aquaporins modulate their water channel activity. Plant Cell 16:215–228

    Article  CAS  Google Scholar 

  • Fritzmann C, Loewenberg J, Wintgens T, Melin T (2007) State-of-the-art of reverse osmosis desalination. Desalination 216:1–76

    Article  CAS  Google Scholar 

  • Gacia E, Invers O, Manzanera M, Ballesteros E, Romero J (2007) Impact of the brine from a desalination plant on a shallow seagrass (Posidonia oceanica) meadow. Estuar Coast Shelf Sci 72:579–590

    Article  CAS  Google Scholar 

  • Greco M, Chiappetta AAC, Bruno L, Bitonti MBA (2011) In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning. J Exp Bot. doi:10.1093/jxb/err313

    Google Scholar 

  • Gu C, Chen S, Liu Z, Shan H, Luo H, Guan Z, Chen F (2011) Reference gene selection for quantitative real-time PCR in chrysanthemum subjected to biotic and abiotic stress. Mol Biotechnol 49:192–197

    Article  CAS  Google Scholar 

  • Hall-Spencer JM, Rodolfo-Metalpa R, Martin S, Ransome E, Fine M, Turner SM, Rowley SJ, Tedesco D, Buia M-C (2008) Volcanic carbon dioxide vents show ecosystem effects of ocean acidification. Nature 454:96–99

    Article  CAS  Google Scholar 

  • Heckmann LH, Connon R, Hutchinson TH, Maund SJ, Sibly RM, Callaghan A (2006) Expression of target and reference genes in Daphnia magna exposed to ibuprofen. BMC Genomics 7:175

    Google Scholar 

  • Heinen RB, Ye Q, Chaumont F (2009) Role of aquaporins in leaf physiology. J Exp Bot 60:2971–2985

    Article  CAS  Google Scholar 

  • Innocenti AM, Cardilio M, Nicastro S, Rende F (2007) Method to optimize the survival and growth of Posidonia oceanica cuttings. Patent CS2007A000018. Holder: Università della Calabria, Italy. http://www.uibm.gov.it/dati/Codice. aspx?load=info uno&id=1499893&table=Invention

  • Invers O, Zimmerman RC, Alberte RS, Perez M, Romero J (2001) Inorganic carbon sources for seagrass photosynthesis: an experimental evaluation of bicarbonate use in species inhabiting temperate waters. J Exp Mar Biol Ecol 265:203–217. doi:10.1016/S0022-0981(01)00332-X

    Article  CAS  Google Scholar 

  • Lauritano C, Borra M, Carotenuto Y, Biffali E, Miralto A, Procaccini G, Ianora A (2011) First molecular evidence of diatom effects in the copepod Calanus helgolandicus. J Exp Mar Biol Ecol 404:79–86

    Article  CAS  Google Scholar 

  • Li HP, Qin YX, Xiao XH, Tang CR (2011) Screening of valid reference genes for real-time RT–PCR data normalization in Hevea brasiliensis and expression validation of a sucrose transporter gene HbSUT3. Plant Sci 181:132–139

    Article  CAS  Google Scholar 

  • Lovdal T, Lillo C (2009) Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Anal Biochem 387:238–242

    Article  CAS  Google Scholar 

  • Maestrini P, Giordani T, Lunardi A, Cavallini A, Natali L (2004) Isolation and expression of two aquaporin-encoding genes from the marine phanerogam Posidonia oceanica. Plant Cell Physiol 45:1838–1847

    Article  CAS  Google Scholar 

  • Marbà N (2009) Loss of seagrass meadows from the Spanish coast: results of the Praderas project. In: Fundación BBVA (ed). ISBN: 978-84-96515-84: 61-90

  • Marbà N, Duarte CM (2010) Mediterranean warming triggers seagrass (Posidonia oceanica) shoot mortality. Glob Chang Biol. doi:10.1111/j.1365-2486.2009.02130.x16:2366-2375

    Google Scholar 

  • Maroufi A, Van Bockstaele E, De Loose M (2010) Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol Biol 11:15

    Article  Google Scholar 

  • Maurel C (2007) Plant aquaporins: novel functions and regulation properties. FEBS Lett 581:2227–2236

    Article  CAS  Google Scholar 

  • Maurel C, Verdoucq L, Luu D-T, Santoni V (2008) Plant aquaporins: membrane channels with multiple integrated functions. Annu Rev Plant Biol 187:595–624

    Google Scholar 

  • Migliaccio M, De Martino F, Silvestre F, Procaccini G (2005) Meadow-scale genetic structure in Posidonia oceanica. Mar Ecol Prog Ser 304:55–65

    Article  CAS  Google Scholar 

  • Moshelion M, Becker D, Biela A, Uehlein N, Hedrich R, Otto B, Levi H, Moran N, Kaldenhoff R (2002) Plasma membrane aquaporins in the motor cells of Samanea saman: diurnal and circadian regulation. Plant Cell 14:727–739

    Article  CAS  Google Scholar 

  • Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT–PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914

    Article  CAS  Google Scholar 

  • Orth RJ, Carruthers TJB, Dennison WC, Duarte CM, Fourqurean JW, Heck KL Jr, Hughes AR, Kendrick GA, Kenworthy WJ, Olyarnik S, Short FT, Waycott M, Williams SL (2006) A global crisis for seagrass ecosystems. Bioscience 56:987–996

    Article  Google Scholar 

  • Otto B, Uehlein N, Sdorra S, Fischer M, Ayaz M, Belastegui-Macadam X, Heckwolf M, Lachnit M, Pede N, Priem N, Reinhard A, Siegfart S, Urban M, Kaldenhoff R (2010) Aquaporin tetramer composition modifies the function of tobacco aquaporins. J Biol Chem 285:31253–31260

    Article  CAS  Google Scholar 

  • Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST (c)) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 75:30

    Google Scholar 

  • Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515

    Article  CAS  Google Scholar 

  • Procaccini G, Buia M, Gambi M, Perez M, Pergent-Martini C, Pergent G, Romero J (2003) The seagrasses of the Western Mediterranean. In: Green EP, Short FT (eds) World atlas of seagrasses. University of California Press, Berkeley, CA, 8:48–58

  • Procaccini G, Dattolo E, Innocenti AM, Mazzuca S, Cavallini A, Natali LJ, Bayer PG (2010) Differential gene expression profiling under different light conditions in Posidonia oceanica (L.) Delile by SSH analysis. Rapp Comm Int Mer Médit 39:641

    Google Scholar 

  • Ralph PJ, Tomasko D, Moore K, Seddon S, Macinnis-Ng CMO (2006) Human impacts on seagrasses: eutrophication, sedimentation, and contamination. In: Larkum AWD, Orth RJ, Duarte CM (eds) Seagrasses: biology, ecology and conservation. Springer, The Netherlands, pp 567–593

    Chapter  Google Scholar 

  • Ransbotyn V, Reusch TBH (2006) Housekeeping gene selection for quantitative real-time PCR assays in the seagrass Zostera marina subjected to heat stress. Limnol Oceanogr Methods 4:367–373

    Article  CAS  Google Scholar 

  • Ruiz JM, Marin-Guirao L, Sandoval-Gil JM (2009) Responses of the Mediterranean seagrass Posidonia oceanica to in situ simulated salinity increase. Bot Mar 52:459–470

    Article  Google Scholar 

  • Schmidt GW, Delaney SK (2010) Stable internal reference genes for normalization of real-time RT–PCR in tobacco (Nicotiana tabacum) during development and abiotic stress. Mol Genet Genomics 283:233–241

    Article  CAS  Google Scholar 

  • Serra IA, Procaccini G, Intrieri MC, Migliaccio M, Mazzuca S, Innocenti AM (2007) Comparison of ISSR and SSR markers for the analysis of genetic diversity in Posidonia oceanica (L.) Delile. Mar Ecol Prog Ser 338:71–79

    Article  CAS  Google Scholar 

  • Serra IA, Innocenti AM, Di Maida G, Calvo S, Migliaccio M, Zambianchi E, Pizzigalli C, Arnaud-Haond S, Duarte CM, Serrao EA, Procaccini G (2010) Genetic structure in the Mediterranean seagrass Posidonia oceanica: disentangling past vicariance events from contemporary patterns of gene flow. Mol Ecol 19:557–568

    Article  CAS  Google Scholar 

  • Serra IA, Nicastro S, Mazzuca S, Natali L, Cavallini A, Innocenti AM (2011) Response to salt stress in seagrasses: PIP1;1 aquaporin antibody localization in Posidonia oceanica leaves. Aquat Bot. doi:10.1016/j.aquabot.2011.05.008

    Google Scholar 

  • Short FT, Neckles HA (1999) The effects of global climate change on seagrasses. Aquat Bot 63:169–196

    Article  Google Scholar 

  • Sirakov M, Zarrella I, Borra M, Rizzo F, Biffali E, Arnone MI, Fiorito G (2009) Selection and validation of a set of reliable reference genes for quantitative RT–PCR studies in the brain of the Cephalopod Mollusc Octopus vulgaris. BMC Mol Biol 10:70

    Article  Google Scholar 

  • Spinsanti G, Panti C, Lazzeri E, Marsili L, Casini S, Frati F, Fossi CM (2006) Selection of reference genes for quantitative RT–PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies. BMC Mol Biol 7:32

    Article  Google Scholar 

  • Toegel S, Huang WW, Piana C, Unger FM, Wirth M, Goldring MB, Gabor F, Viernstein H (2007) Selection of reliable reference genes for qPCR studies on chondroprotective action. BMC Mol Biol 8:R13

    Article  Google Scholar 

  • Tomasello A, Di Maida G, Calvo S, Pirrotta M, Borra M, Procaccini G (2009) Seagrass meadows at the extreme of environmental tolerance: the case of Posidonia oceanica in a semi-enclosedcoastal lagoon. Mar Ecol Evol Perspect 30:288–300

    Article  Google Scholar 

  • Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT–PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:0034.1–0034.11

    Google Scholar 

  • Wan H, Zhao Z, Qian C, Sui Y, Malik AA, Chen J (2010) Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem 399:257–261

    Article  CAS  Google Scholar 

  • Waycott M, Duarte CM, Carruthers TJB, Orth RJ, Dennison WC, Olyarnik S, Calladine A, Fourqurean JW, Heck KL Jr, Hughes AR, Kendrick GA, Kenworthy WJ, Short FT, Williams SL (2009) Accelerating loss of seagrasses across the globe threatens coastal ecosystems. Proc Nat Acad Sci USA 106:12377–12381

    Article  CAS  Google Scholar 

  • Winters G, Nelle P, Fricke B, Rauch G, Reusch TBH (2011) Effects of a simulated heat wave on photophysiology and gene expression of high- and low-latitude populations of Zostera marina. Mar Ecol Prog Ser 435:83–95

    Article  Google Scholar 

  • Wissler L, Dattolo E, Moore A, Reusch T, Olsen J, Migliaccio M, Bornberg-Bauer E, Procaccini G (2009) Dr. Zompo: an online data repository for Zostera marina and Posidonia oceanica ESTs. Database. doi:10.1093/database/bap009

    Google Scholar 

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Acknowledgments

The authors thank the staff of the Molecular Biology Service of Stazione Zoologica Anton Dohrn for their technical support in laboratory experiments, M. Lorenti and V. Rando for their help in collecting environmental data, M.C. Buia for providing support in the Lacco Ameno sampling site and the anonymous reviewers for their constructive suggestions.

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Correspondence to Gabriele Procaccini.

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Communicated by T. Reusch.

Ilia Anna Serra, Chiara Lauritano share equal responsibility.

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227_2012_1907_MOESM1_ESM.pdf

Online resource Ranking of the best reference genes (RGs) obtained with BestKeeper, geNorm and NormFinder in the different experimental conditions: adults and young exposed to salinity, adults and young exposed to pH, adults exposed to salinity, adults exposed to pH, young exposed to pH, young exposed to salinity, adults exposed to different depths, young exposed to salinity and pH and adults plants exposed to different depths, salinity and pH. a) Results of the BestKeeper analysis on the six putative Reference Genes tested. Number of samples (n), geometric mean of Ct (GM), arithmetic mean of Ct (AM), extreme values of Ct (Min and Max), standard deviation of the Ct (SD), coefficient of variance, expressed as a percentage on the Ct level (CV), extreme values of expression levels, expressed as an absolute x-fold over- or under-regulation coefficient (Min [x-fold] and Max [x-fold]), and standard deviation of the absolute regulation coefficients (SD [± x-fold]), are given. b) The stepwise exclusion of genes that are more variable among samples using the geNorm program. More stable genes are indicated by the arrow; c) Pairwise variation (V) to evaluate the effect of adding another RG to the best couple already analyzed using geNorm (e.g., adding a third gene V2/3, a fourth V3/4, etc.). The inclusion of additional RGs was not required below the cutoff value of 0.15; d) The NormFinder algorithm ranks the candidate RGs according to their expression stability. Lower stability values indicate more stable genes (PDF 328 kb)

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Serra, I.A., Lauritano, C., Dattolo, E. et al. Reference genes assessment for the seagrass Posidonia oceanica in different salinity, pH and light conditions. Mar Biol 159, 1269–1282 (2012). https://doi.org/10.1007/s00227-012-1907-8

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  • DOI: https://doi.org/10.1007/s00227-012-1907-8

Keywords

  • Reference Gene
  • Candidate Reference Gene
  • Salinity Variation
  • Subtractive Suppression Hybridization Library
  • geNorm Analysis