Hostname: page-component-7dd5485656-glrdx Total loading time: 0 Render date: 2025-10-31T17:34:42.198Z Has data issue: false hasContentIssue false
  • English
  • Français

Identification of reference genes for gene expression studies during seed germination and seedling establishment in Ricinus communis L.

Published online by Cambridge University Press:  23 September 2014

Paulo R. Ribeiro ,
Bas J. W. Dekkers ,
Luzimar G. Fernandez ,
Renato D. de Castro ,
Wilco Ligterink  and
Henk W. M. Hilhorst
Paulo R. Ribeiro*
Affiliation:
Wageningen Seed Lab, Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands Laboratório de Bioquímica, Biotecnologia e Bioprodutos, Departamento de Biofunção, Universidade Federal da Bahia, Reitor Miguel Calmon s/n, 40160-100 Salvador, Brazil
Bas J. W. Dekkers
Affiliation:
Wageningen Seed Lab, Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands Molecular Plant Physiology, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
Luzimar G. Fernandez
Affiliation:
Laboratório de Bioquímica, Biotecnologia e Bioprodutos, Departamento de Biofunção, Universidade Federal da Bahia, Reitor Miguel Calmon s/n, 40160-100 Salvador, Brazil
Renato D. de Castro
Affiliation:
Laboratório de Bioquímica, Biotecnologia e Bioprodutos, Departamento de Biofunção, Universidade Federal da Bahia, Reitor Miguel Calmon s/n, 40160-100 Salvador, Brazil
Wilco Ligterink*
Affiliation:
Wageningen Seed Lab, Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
Henk W. M. Hilhorst
Affiliation:
Wageningen Seed Lab, Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
*
*Correspondence Fax: +31317 418094 E-mails: paulodc3@gmail.com; wilco.ligterink@wur.nl;
*Correspondence Fax: +31317 418094 E-mails: paulodc3@gmail.com; wilco.ligterink@wur.nl;
Get access Rights & Permissions [Opens in a new window]

Abstract

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is an important technology to analyse gene expression levels during plant development or in response to different treatments. An important requirement to measure gene expression levels accurately is a properly validated set of reference genes. In this context, we analysed the potential use of 17 candidate reference genes across a diverse set of samples, including several tissues, different stages and environmental conditions, encompassing seed germination and seedling growth in Ricinus communis L. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. GeNorm and Normfinder indicated that ACT, POB and PP2AA1 comprise the optimal combination for normalization of gene expression data in inter-tissue (heterogeneous sample panel) studies. We also describe the optimal combination of reference genes for a subset of root, endosperm and cotyledon samples. In general, the most stable genes suggested by GeNorm are very consistent with those indicated by NormFinder, which highlights the strength of the selection of reference genes in our study. We also validated the selected reference genes by normalizing the expression levels of three target genes involved in energy metabolism with the reference genes suggested by GeNorm and NormFinder. The approach used in this study to identify stably expressed genes, and thus potential reference genes, was applied successfully for R. communis and it provides important guidelines for RT-qPCR studies in seeds and seedlings for other species (especially in those cases where extensive microarray data are not available).

Keywords

castor beangene expressionGeNormnormalizationNormFinderquantitative real-time polymerase chain reaction

Information

Type
Research Papers
Information
Seed Science Research , Volume 24 , Issue 4 , December 2014 , pp. 341 - 352
Check for updates
Copyright
Copyright © Cambridge University Press 2014 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Article purchase

Temporarily unavailable

References

Andersen, C.L., Jensen, J.L. and Ørntoft, T.F. (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 Research 64, 5245–5250.CrossRefGoogle Scholar
Anjani, K. (2012) Castor genetic resources: a primary gene pool for exploitation. Industrial Crops and Products 35, 1–14.CrossRefGoogle Scholar
Arroyo-Caro, J.M., Chileh, T., Kazachkov, M., Zou, J., Alonso, D.L. and García-Maroto, F. (2013) The multigene family of lysophosphatidate acyltransferase (LPAT)-related enzymes in Ricinus communis. Cloning and molecular characterization of two LPAT genes that are expressed in castor seeds. Plant Science 199–200, 29–40.Google Scholar
Artico, S., Nardeli, S.M., Brilhante, O., Grossi-de-Sa, M.F. and Alves-Ferreira, M. (2010) Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data. BMC Plant Biology 10, 49.Google Scholar
Cagliari, A., Margis-Pinheiro, M., Loss, G., Mastroberti, A.A., de Araujo Mariath, J.E. and Margis, R. (2010) Identification and expression analysis of castor bean (Ricinus communis) genes encoding enzymes from the triacylglycerol biosynthesis pathway. Plant Science 179, 499–509.CrossRefGoogle ScholarPubMed
Cesar, A.d.S. and Batalha, M.O. (2010) Biodiesel production from castor oil in Brazil: a difficult reality. Energy Policy 38, 4031–4039.CrossRefGoogle Scholar
Chen, G.Q., Turner, C., He, X., Nguyen, T., McKeon, T.A. and Laudencia-Chingcuanco, D. (2007) Expression profiles of genes involved in fatty acid and triacylglycerol synthesis in castor bean (Ricinus communis L.). Lipids 42, 263–274.CrossRefGoogle ScholarPubMed
Chileh, T., Esteban-García, B., Alonso, D.L. and García-Maroto, F. (2010) Characterization of the 11S globulin gene family in the castor plant Ricinus communis L. Journal of Agricultural and Food Chemistry 58, 272–281.Google Scholar
Conceicao, M.M., Candeia, R.A., Silva, F.C., Bezerra, A.F., Fernandes, V.J. and Souza, A.G. (2007) Thermo analytical characterization of castor oil biodiesel. Renewable & Sustainable Energy Reviews 11, 964–975.Google Scholar
Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K. and Scheible, W.R. (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in arabidopsis. Plant Physiology 139, 5–17.CrossRefGoogle ScholarPubMed
Dekkers, B.J.W., Willems, L., Bassel, G.W., Van Bolderen-Veldkamp, R.P.M., Ligterink, W., Hilhorst, H.W.M. and Bentsink, L. (2012) Identification of reference genes for RT-qPCR expression analysis in Arabidopsis and tomato seeds. Plant and Cell Physiology 53, 28–37.Google Scholar
De Oliveira, L.A., Breton, M.C., Bastolla, F.M., Camargo, S.D.S., Margis, R., Frazzon, J. and Pasquali, G. (2012) Reference genes for the normalization of gene expression in eucalyptus species. Plant and Cell Physiology 53, 405–422.Google Scholar
Doyle, E.A., Lane, A.M., Sides, J.M., Mudgett, M.B. and Monroe, J.D. (2007) An α-amylase (At4g25000) in Arabidopsis leaves is secreted and induced by biotic and abiotic stress. Plant, Cell and Environment 30, 388–398.Google Scholar
Eastmond, P.J. (2004) Cloning and characterization of the acid lipase from Castor beans. Journal of Biological Chemistry 279, 45540–45545.Google Scholar
Fan, Z., Li, J., Lu, M., Li, X. and Yin, H. (2013) Overexpression of phosphoenolpyruvate carboxylase from Jatropha curcas increases fatty acid accumulation in Nicotiana tabacum . Acta Physiologiae Plantarum 35, 2269–2279.Google Scholar
Foyer, C.H., Neukermans, J., Queval, G., Noctor, G. and Harbinson, J. (2012) Photosynthetic control of electron transport and the regulation of gene expression. Journal of Experimental Botany 63, 1637–1661.CrossRefGoogle ScholarPubMed
Gong, Q., Li, P., Ma, S., Indu Rupassara, S. and Bohnert, H.J. (2005) Salinity stress adaptation competence in the extremophile Thellungiella halophila in comparison with its relative Arabidopsis thaliana . Plant Journal 44, 826–839.Google Scholar
Gu, K., Yi, C., Tian, D., Sangha, J.S., Hong, Y. and Yin, Z. (2012) Expression of fatty acid and lipid biosynthetic genes in developing endosperm of Jatropha curcas . Biotechnology for Biofuels 5, 47.Google Scholar
Hellemans, J., Mortier, G., De Paepe, A., Speleman, F. and Vandesompele, J. (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biology 8, R19.Google Scholar
Jarosová, J. and Kundu, J.K. (2010) Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR. BMC Plant Biology 10, 146.Google Scholar
Le, D.T., Aldrich, D.L., Valliyodan, B., Watanabe, Y., van Ha, C., Nishiyama, R., Guttikonda, S.K., Quach, T.N., Gutierrez-Gonzalez, J.J., Tran, L.S.P. and Nguyen, H.T. (2012) Evaluation of candidate reference genes for normalization of quantitative RT-PCR in soybean tissues under various abiotic stress conditions. PLoS ONE 7, 9.Google Scholar
Li, H.L., Zhang, L.B., Guo, D., Li, C.Z. and Peng, S.Q. (2012) Identification and expression profiles of the WRKY transcription factor family in Ricinus communis . Gene 503, 248–253.Google Scholar
Li, X.S., Yang, H.L., Zhang, D.Y., Zhang, Y.M. and Wood, A.J. (2012) Reference gene selection in the desert plant Eremosparton songoricum . International Journal of Molecular Science 13, 6944–6963.Google Scholar
Loss-Morais, G., Turchetto-Zolet, A.C., Etges, M., Cagliari, A., Körbes, A.P., Maraschin, F.S., Margis-Pinheiro, M. and Margis, R. (2013) Analysis of castor bean ribosome-inactivating proteins and their gene expression during seed development. Genetics and Molecular Biology 36, 74–86.CrossRefGoogle ScholarPubMed
Maciel, F.M., Salles, C.M.C., Retamal, C.A., Gomes, V.M. and Machado, O.L.T. (2011) Identification and partial characterization of two cysteine proteases from castor bean leaves (Ricinus communis L.) activated by wounding and methyl jasmonate stress. Acta Physiologiae Plantarum 33, 1867–1875.CrossRefGoogle Scholar
Nolan, T., Hands, R.E. and Bustin, S.A. (2006) Quantification of mRNA using real-time RT-PCR. Nature Protocols 1, 1559–1582.CrossRefGoogle ScholarPubMed
O'Leary, B., Fedosejevs, E.T., Hill, A.T., Bettridge, J., Park, J., Rao, S.K., Leach, C.A. and Plaxton, W.C. (2011) Tissue-specific expression and post-translational modifications of plant- and bacterial-type phosphoenolpyruvate carboxylase isozymes of the castor oil plant, Ricinus communis L. Journal of Experimental Botany 62, 5485–5495.Google Scholar
Pettengill, E.A., Parmentier-Line, C. and Coleman, G.D. (2012) Evaluation of qPCR reference genes in two genotypes of Populus for use in photoperiod and low-temperature studies. BMC Research Notes 5, 366.Google Scholar
Pfaffl, M.W., Tichopad, A., Prgomet, C. and Neuvians, T.P. (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnology Letters 26, 509–515.Google Scholar
Popovici, V., Goldstein, D.R., Antonov, J., Jaggi, R., Delorenzi, M. and Wirapati, P. (2009) Selecting control genes for RT-QPCR using public microarray data. BMC Bioinformatics 10, 42.Google Scholar
Rapacz, M., Stepień, A. and Skorupa, K. (2012) Internal standards for quantitative RT-PCR studies of gene expression under drought treatment in barley (Hordeum vulgare L.): the effects of developmental stage and leaf age. Acta Physiologiae Plantarum 34, 1723–1733.Google Scholar
Rocha, A.J., Soares, E.L., Costa, J.H., Costa, W.L.G., Soares, A.A., Nogueira, F.C.S., Domont, G.B. and Campos, F.A.P. (2013) Differential expression of cysteine peptidase genes in the inner integument and endosperm of developing seeds of Jatropha curcas L. (Euphorbiaceae). Plant Science 213, 30–37.Google Scholar
Salimon, J., Noor, D.A.M., Nazrizawati, A.T., Firdaus, M.Y.M. and Noraishah, A. (2010) Fatty acid composition and physicochemical properties of Malaysian castor bean Ricinus communis L. seed oil. Sains Malaysiana 39, 761–764.Google Scholar
Sánchez-García, A., Moreno-Pérez, A.J., Muro-Pastor, A.M., Salas, J.J., Garcés, R. and Martínez-Force, E. (2010) Acyl-ACP thioesterases from castor (Ricinus communis L.): an enzymatic system appropriate for high rates of oil synthesis and accumulation. Phytochemistry 71, 860–869.CrossRefGoogle ScholarPubMed
Severino, L.S. and Auld, D.L. (2013) A framework for the study of the growth and development of castor plant. Industrial Crops and Products 46, 25–38.Google Scholar
Severino, L.S., Auld, D.L., Baldanzi, M., Candido, M.J.D., Chen, G., Crosby, W., Tan, D., He, X.H., Lakshmamma, P., Lavanya, C., Machado, O.L.T., Mielke, T., Milani, M., Miller, T.D., Morris, J.B., Morse, S.A., Navas, A.A., Soares, D.J., Sofiatti, V., Wang, M.L., Zanotto, M.D. and Zieler, H. (2012) A review on the challenges for increased production of castor. Agronomy Journal 104, 853–880.Google Scholar
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. and Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3, 7.Google Scholar
Wei, W., Dai, X., Wang, Y., Chuan, Y., Gou, C.B. and Chen, F. (2010) Cloning and expression analysis of 1-i-myo-inositol-1-phosphate synthase gene from Ricinus communis L. Zeitschrift für Naturforschung–Section C Journal of Biosciences 65 C, 501–507.Google Scholar
Xu, Y.H., Liu, R., Yan, L., Liu, Z.Q., Jiang, S.C., Shen, Y.Y., Wang, X.F. and Zhang, D.P. (2012) Light-harvesting chlorophyll a/b-binding proteins are required for stomatal response to abscisic acid in Arabidopsis. Journal of Experimental Botany 63, 1095–1106.Google Scholar
Supplementary material: Image

Ribeiro Supplementary Material

Figure S1

Download Ribeiro Supplementary Material(Image)
Image 1.1 MB
Supplementary material: File

Ribeiro Supplementary Material

Table S1

Download Ribeiro Supplementary Material(File)
File 12.2 KB