Planta

, Volume 227, Issue 6, pp 1343–1349 | Cite as

Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations

  • Tony Remans
  • Karen Smeets
  • Kelly Opdenakker
  • Dennis Mathijsen
  • Jaco Vangronsveld
  • Ann Cuypers
Original Article

Abstract

Accurate quantification by real-time RT-PCR relies on normalisation of the measured gene expression data. Normalisation with multiple reference genes is becoming the standard, but the best reference genes for gene expression studies within one organism may depend on the applied treatments or the organs and tissues studied. Ideally, reference genes should be evaluated in all experimental systems. A number of candidate reference genes for Arabidopsis have been proposed, which can be used as a starting point to evaluate their expression stability in individual experimental systems by available computer algorithms like geNorm and NormFinder. Using this approach, we identified the best three reference genes from a set of ten candidates, which included three traditional “housekeeping” genes, for normalisation of gene expression when roots and leaves of Arabidopsis thaliana are exposed to cadmium (Cd) and copper (Cu). The expression stabilities of AT5G15710 (F-box protein), AT2G28390 (SAND family protein) and AT5G08290 (mitosis protein YLS8) were the highest when considering the effect to the roots and shoots of Cd and Cu treatments. Even though the effect of Cd and excess Cu on the plants is very different, the same best reference genes were identified when considering Cd or Cu treatments separately. This suggests that these three genes may also be suitable when studying the gene expression after exposure of Arabidopsis thaliana to increased concentrations of other metals.

Keywords

Cadmium Copper Metal stress Reference genes Housekeeping genes Real-time PCR qBase geNorm NormFinder 

Abbreviations

Cd

Cadmium

Cu

Copper

ROS

Reactive oxygen species

Supplementary material

425_2008_706_MOESM1_ESM.pdf (29 kb)
Average expression stability (M) and pairwise variation (V) graphs obtained from the geNorm algorithm using input of all samples (biological replicates of each treatment/organ combination entered separately). (PDF 29 kb)
425_2008_706_MOESM2_ESM.pdf (29 kb)
Average expression stability (M) and pairwise variation (V) graphs obtained from the geNorm algorithm using averaged input per treatment/organ combination for different data sets. (PDF 29 kb)
425_2008_706_MOESM3_ESM.pdf (12 kb)
"NormFinder" output for the selected data sets, obtained by using expression values for individual samples as input (A), or by using an averaged input per treatment/organ combination (B). (PDF 12 kb)

References

  1. Andersen CL, Jensen JL, Ørntoft TF (2004) Normalisation of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalisation, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250PubMedCrossRefGoogle Scholar
  2. Baryla A, Carrier P, Franck F, Coulomb C, Sahut C, Havaux M (2001) Leaf chlorosis in oilseed rape plants (Brassica napus) grown on cadmium-polluted soil: causes and consequences for photosynthesis and growth. Planta 212:696–709PubMedCrossRefGoogle Scholar
  3. Becher M, Talke IN, Krall L, Krämer U (2004) Cross-species microarray transcript profiling reveals high constitutive expression of metal homeostasis genes in shoots of the zinc hyperaccumulator Arabidopsis halleri. Plant J 37:251–268PubMedCrossRefGoogle Scholar
  4. Bovet T, Eggmann M, Meylan-Bettex J, Polier P, Kammer E, Marin U, Feller E, Martinoia E (2003) Transcript levels of AtMRPs after cadmium treatment: induction of AtMRP3. Plant Cell Environ 26:371–381CrossRefGoogle Scholar
  5. Brunner AM, Yakovlev IA, Strauss SH (2004) Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4:14PubMedCrossRefGoogle Scholar
  6. Cagnac O, Bourbouloux A, Chakrabarty D, Zhang MY, Delrot S (2004) AtOPT6 transports glutathione derivatives and is induced by primisulfuron. Plant Physiol 135:1378–1387PubMedCrossRefGoogle Scholar
  7. Chaoui A, El Ferjani E (2005) Effects of cadmium and copper on antioxidant capacities, lignification and auxin degradation in leaves of pea (Pisum sativum L.) seedlings. C R Biol 328:23–31PubMedCrossRefGoogle Scholar
  8. Cuypers A, Vangronsveld J, Clijsters H (2000) Biphasic effect of copper on the ascorbate–glutathione pathway in primary leaves of Phaseolus vulgaris seedlings during the early stages of metal assimilation. Physiol Plant 110:512–517CrossRefGoogle Scholar
  9. Cuypers A, Vangronsveld J, Clijsters H (2002) Peroxidases in roots and primary leaves of Phaseolus vulgaris copper and zinc phytotoxicity: a comparison. J Plant Physiol 159:869–876CrossRefGoogle Scholar
  10. Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible W-R (2005) Genome-wide identification and testing of superior reference genes for transcript normalisation in Arabidopsis. Plant Physiol 139:5–17PubMedCrossRefGoogle Scholar
  11. Fargasova A (2001) Phytotoxic effects of Cd, Zn, Pb, Cu and Fe on Sinapis alba L. seedlings and their accumulation in roots and shoots. Biol Plant 44:471–473CrossRefGoogle Scholar
  12. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8(2):R19PubMedCrossRefGoogle Scholar
  13. Herbette S, Taconnat L, Hugouvieux V, Piette L, Magniette MLM, Cuine S, Auroy P, Richaud P, Forestier C, Bourguignon J, Renou J-P, Vavasseur A, Leonhardt N (2006) Genome-wide transcriptome profiling of the early cadmium response of Arabidopsis roots and shoots. Biochimie 88:1751–1765PubMedCrossRefGoogle Scholar
  14. Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Comm 345:646–651PubMedCrossRefGoogle Scholar
  15. Jonak C, Nakagami H, Hirt H (2004) Heavy metal stress. Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium. Plant Physiol 136:3276–3283PubMedCrossRefGoogle Scholar
  16. Maksymiec W, Krupa Z (2006) The effects of short-term exposition to Cd, excess Cu ions and jasmonate on oxidative stress appearing in Arabidopsis thaliana. Environ Exp Bot 57:187–194CrossRefGoogle Scholar
  17. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498PubMedCrossRefGoogle Scholar
  18. Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalisation in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914PubMedCrossRefGoogle Scholar
  19. Ortega-Villasante C, Rellan-Alvarez R, Del Campo FF, Carpena-Ruiz RO, Hernandez LE (2005) Cellular damage induced by cadmium and mercury in Medicago sativa. J Exp Bot 56:2239–2251PubMedCrossRefGoogle Scholar
  20. Pandey N, Sharma CP (2002) Effect of heavy metals Co2+, Ni2+ and Cd2+ on growth and metabolism of cabbage. Plant Sci 163:753–758CrossRefGoogle Scholar
  21. 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 26:509–15PubMedCrossRefGoogle Scholar
  22. Prasad SM, Zeeshan M (2005) UV-B radiation and cadmium-induced changes in growth, photosynthesis, and antioxidant enzymes of cyanobacterium Plectonema boryanum. Biol Plant 49:229–236CrossRefGoogle Scholar
  23. 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 4:367–373Google Scholar
  24. Rodriguez-Serrano M, Romero-Puertas MC, Zabalza A, Corpas FJ, Gomez M, Del Rio LA, Sandalio LM (2006) Cadmium effect on oxidative metabolism of pea (Pisum sativum L.) roots. Imaging of reactive oxygen species and nitric oxide accumulation in vivo. Plant Cell Environ 29:1532–1544PubMedCrossRefGoogle Scholar
  25. Romero-Puertas MC, Rodriguez-Serrano M, Corpas FJ, Gomez M, Del Rio LA, Sandalio LM (2004) Cadmium-induced subcellular accumulation of O2 and H2O2 in pea leaves. Plant Cell Environ 27:1122–1134CrossRefGoogle Scholar
  26. Smeets K, Cuypers A, Lambrechts A, Semane B, Hoet P, Van Laere A, Vangronsveld J (2005) Induction of oxidative stress and antioxidative mechanisms in Phaseolus vulgaris after Cd application. Plant Physiol Biochem 43:437–444PubMedCrossRefGoogle Scholar
  27. Smeets K, Ruytinx J, Van Belleghem F, Semane B, Lin D, Vangronsveld J, Cuypers A (2007) Critical evaluation and statistical validation of a hydroponic culture system for Arabidopsis thaliana. Plant Physiol Biochem. doi:10.1016/j.plaphy.2007.09.014
  28. Sunkar R, Kapoor A, Zhu JK (2006) Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance. Plant Cell 18:2051–2065PubMedCrossRefGoogle Scholar
  29. Talke IN, Hanikenne M, Kramer U (2006) Zinc-dependent global transcriptional control, transcriptional deregulation, and higher gene copy number for genes in metal homeostasis of the hyperaccumulator Arabidopsis halleri. Plant Physiol 142:148–167PubMedCrossRefGoogle Scholar
  30. Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A, Heinen E (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75: 291–295PubMedCrossRefGoogle Scholar
  31. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalisation of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):research0034.1–0034.11CrossRefGoogle Scholar
  32. Weber M, Trampczynska A, Clemens S (2006) Comparative transcriptome analysis of toxic metal responses in Arabidopsis thaliana and the Cd2+-hypertolerant facultative metallophyte Arabidopsis halleri. Plant Cell Environ 29:950–963PubMedCrossRefGoogle Scholar
  33. Yeh CM, Chien PS, Huang HJ (2007) Distinct signalling pathways for induction of MAP kinase activities by cadmium and copper in rice roots. J Exp Bot 58:659–671PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Tony Remans
    • 1
  • Karen Smeets
    • 1
  • Kelly Opdenakker
    • 1
  • Dennis Mathijsen
    • 1
  • Jaco Vangronsveld
    • 1
  • Ann Cuypers
    • 1
  1. 1.Environmental Biology Hasselt UniversityDiepenbeekBelgium

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