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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- Cd:
-
Cadmium
- Cu:
-
Copper
- ROS:
-
Reactive oxygen species
References
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 64:5245–5250
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 212:696–709
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 37:251–268
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 26:371–381
Brunner AM, Yakovlev IA, Strauss SH (2004) Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4:14
Cagnac O, Bourbouloux A, Chakrabarty D, Zhang MY, Delrot S (2004) AtOPT6 transports glutathione derivatives and is induced by primisulfuron. Plant Physiol 135:1378–1387
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 328:23–31
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 110:512–517
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 159:869–876
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 139:5–17
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 44:471–473
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):R19
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 88:1751–1765
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 345:646–651
Jonak C, Nakagami H, Hirt H (2004) Heavy metal stress. Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium. Plant Physiol 136:3276–3283
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 57:187–194
Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498
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 56:2907–2914
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 56:2239–2251
Pandey N, Sharma CP (2002) Effect of heavy metals Co2+, Ni2+ and Cd2+ on growth and metabolism of cabbage. Plant Sci 163:753–758
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 26:509–15
Prasad SM, Zeeshan M (2005) UV-B radiation and cadmium-induced changes in growth, photosynthesis, and antioxidant enzymes of cyanobacterium Plectonema boryanum. Biol Plant 49:229–236
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
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 29:1532–1544
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 27:1122–1134
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 43:437–444
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
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 18:2051–2065
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 142:148–167
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: 291–295
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):research0034.1–0034.11
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 29:950–963
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 58:659–671
Acknowledgments
This study was funded by a Research Foundation-Flanders grant (project G.0436.06). Tony Remans is a post-doctoral research fellow of the Research Foundation-Flanders (FWO). Karen Smeets was supported by a PhD grant from Hasselt University (tUl-impulsfinanciering-toxicology). We also thank Carine Put and Ann Wijgaerts for their technical assistance.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
425_2008_706_MOESM1_ESM.pdf
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
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
"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)
Rights and permissions
About this article
Cite this article
Remans, T., Smeets, K., Opdenakker, K. et al. Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta 227, 1343–1349 (2008). https://doi.org/10.1007/s00425-008-0706-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00425-008-0706-4