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Selection of Reference Genes for Normalizing Gene Expression During Seed Priming and Germination Using qPCR in Zea mays and Spinacia oleracea

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Abstract

Quantitative real-time RT-PCR (qPCR) has been widely used to investigate gene expression during seed germination, a process involving seed transition from dry/physiologically inactive to hydrated/active state. This transition may result in altered expression of many housekeeping genes (HKGs), conventionally used as internal controls, thereby posing a challenge about selection of HKGs in such scenarios. The objectives of this study included identifying valid reference genes for seed priming and germination studies, both of which involve the transition of seed hydration status, and assessing whether or not findings derived from the “seed model” used in this study would also be applicable to other plant species. Eight commonly used HKGs were evaluated in maize seeds during hydropriming and germination. Using Bestkeeper, geNorm, and NormFinder, we provided a rank of stability for these HKGs. Actdf, UBQ, βtub, 18S, Act, and GAPDH were adjudged as valid internal controls by geNorm and NormFinder. Under the second objective, we conducted a case study with spinach seeds collected during osmopriming and germination. Our results indicate that the conclusions derived from maize were applicable to spinach as well, in that 18S exhibited greater expression stability than GAPDH in osmoprimed and germinated seeds; this held true even under stress conditions. While both of these genes were rejected by BestKeeper, we found that 18S exhibited stable expression when “dry” and “hydrated” seeds were analyzed as separate data sets. Although this approach precludes the comparison between “hydrated” and “dry” seeds, it still provides effective comparison among samples of same hydration status.

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Abbreviations

HKG:

Housekeeping genes

18S:

18S rRNA

αtub:

alpha-Tubulin

βtub:

belta 2-Tubulin

Actdf:

Actin depolymerizing factor

Act:

Actin

GAPDH:

Glyceraldehydes-3-phosphate dehydrogenase

Glub:

Globulin

UBQ:

Ubiquitine

References

  • Anderson 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  Google Scholar 

  • Argyropoulos D, Psallida C, Spyropoulos CG (2006) Generic normalization method for real-time PCR application for the analysis of the mannanase gene expressed in germinating tomato seed. FEBS J 273:770–777

    Article  PubMed  CAS  Google Scholar 

  • Association of Official Seed Analysts (AOSA) (2010) Rules for testing seeds. Association of Official Seed Analysts, Ithaca, NY

    Google Scholar 

  • Bewley JD, Black M (1994) Seeds: physiology of development and germination, 2nd edn. Plenum, New York

    Google Scholar 

  • Blödner C, Goebel C, Feussner I, Gatz C, Polle A (2007) Warm and cold parental reproductive environments affect seed properties fitness, and cold responsiveness in Arabidopsis thaliana progenies. Plant Cell Environ 30:165–175

    Article  PubMed  Google Scholar 

  • Brunner AM, Yakovlev IA, Strauss SH (2004) Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4:14

    Article  PubMed  Google Scholar 

  • Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  PubMed  CAS  Google Scholar 

  • Chang Li, Wu L, Chen Y, Ku L, Yang S, Zhang S, Wang X, Wei X (2011) Expression and functional analysis of the ZCN1 (ZmTFL1) gene, a TERMINAL FLOWER 1 homologue that regulates the vegetative to reproductive transition in maize. Plant Mol Biol Rep. doi:10.1007/s11105-011-0371-2

  • Chen K, Arora R, Arora U (2010) Osmopriming of spinach (Spinacia oleracea L. cv. Bloosdale) seeds and germination performance under temperature and water stress. Seed Sci Technol 38:45–57

    Google Scholar 

  • Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible W (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17

    Article  PubMed  CAS  Google Scholar 

  • Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A (2004) Validation of housekeeping genes for normalization RNA expression in real-time PCR. Biotechniques 37:112–117

    PubMed  CAS  Google Scholar 

  • Gallego-Bartolomé J, Minguet EG, Marín JA, Prat S, Blázquez MA, Alabadí D (2010) Transcriptional diversification and functional conservation between DELLA proteins in Arabidopsis. Mol Biol Evol 27:1247–1256

    Article  PubMed  Google Scholar 

  • Gong Q, Li P, Ma S, Rupassara SI, Bohnert H (2005) Salinity stress adaptation competence in the extremophile Thellungiella halophila in comparison with its relative Arabidopsis thaliana. Plant J 44:826–839

    Article  PubMed  CAS  Google Scholar 

  • González-Verdejo CI, Die JV, Nadal S, Jiménez-Marín A, Moreno MT, Román B (2008) Selection of housekeeping genes for normalization by real-time RT-PCR: Analysis of Or-MYB1 gene expression in Orobanche ramose development. Anal Biochem 379:38–43

    Google Scholar 

  • Gore MA, Chia J-M, Elshire RJ, Sun Q, Ersoz ES, Hurwitz BL, Peiffer JA, McMullen MD, Grills GS, Ross-lbarra J, Ware DH, Buckler ES (2009) A first-generation haplotype map of maize. Science 20:1115–1117

    Article  Google Scholar 

  • Guénin S, Mauriat M, Pelloux J, Wuytswinkel OV, Bellini C, Gutierrez L (2009) Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J Exp Bot 60:487–493

    Article  PubMed  Google Scholar 

  • Gupta N, Gupta AK, Singh NK, Kumar A (2011) Differential expression of PBF Dof transcription factor in different tissues of three finger millet genotypes differing in seed protein content and color. Plant Mol Biol Rep 29:69–76

    Article  CAS  Google Scholar 

  • Kawade K, Masuda K (2009) Transcriptional control of two ribosome-inactivating protein genes expressed in spinach (Spinacia oleracea) embryos. Plant Physiol Biochem 47:327–334

    Article  PubMed  CAS  Google Scholar 

  • Li Q-F, Samuel SM, Sun D-Y, Yuan H-X, Yu M-H, Gu Q, Liu Q (2010) Validation of candidate reference genes for the accurate normalization of real-time quantitative RT-PCR data in rice during seed development. Plant Mol Biol Rep 28:49–57

    Article  Google Scholar 

  • Liu Y-Z, Dong T, Lei Y, Deng X-X, Gu Q-Q (2011) Isolation of a polygalacturonase gene from Citrus sinensis fruit and its expression relative to fruit mastication trait, fruit development, and calcium or boron treatments. Plant Mol Biol Rep 29:51–59

    Article  CAS  Google Scholar 

  • Rivera-Vega L, Mamidala P, Koch JL, Mason ME, Mittapalli O (2011) Evaluation of reference genes for expression studies in Ash (Fraxinus spp.). Plant Mol Biol Rep. doi:10.1007/s11105-011-0340-3

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

    Article  PubMed  Google Scholar 

  • McGinn PJ, Morel FMM (2008) Expression and inhibition of the carboxylating and decarboxylating enzymes in the photosynthetic C4 pathway of marine diatoms. Plant Physiol 146:300–309

    Article  PubMed  CAS  Google Scholar 

  • Narsai R, Ivanova A, Ng S, Whelan J (2010) Defining reference genes in Oryza sativa using organ, development, biotic and abiotic transcriptome datasets. BMC Plant Biol 10:56

    Article  PubMed  Google Scholar 

  • Naydenov NG, Khanam S, Siniauskaya M, Nakamura C (2010) Profiling of mitochondrial transcriptome in germinating wheat embryos and seedlings subjected to cold, salinity and osmotic stresses. Genes Genet Syst 85:31–42

    Article  PubMed  CAS  Google Scholar 

  • Nicot N, Hausman J-F, Hoffman 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  PubMed  CAS  Google Scholar 

  • Nonogaki H, Bassel GW, Bewley JD (2010) Germination—still a mystery. Plant Sci 179:574–581

    Article  CAS  Google Scholar 

  • Peng Y, Arora R, Li G, Wang X, Fessehaie A (2008) Rhododendron catawbiense plasma membrane intrinsic proteins are aquaporins, and their over-expression compromises constitutive freezing tolerance and cold acclimation ability of transgenic Arabidopsis plants. Plant Cell Environ 31:1285–1289

    Article  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  PubMed  CAS  Google Scholar 

  • Qi J, Yu S, Zhang F, Shen X, Zhao X, Yu Y, Zhang D (2010) Reference gene selection for real-time quantitative polymerase chain reaction of mRNA transcript levels in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Plant Mol Biol Rep 28:597–604

    Article  CAS  Google Scholar 

  • Sawada Y, Aoki M, Nakaminami K et al (2009) Phytochrome- and gibberellins-mediated regulation of abscisic acid metabolism during germination of photoblastic lettuce seeds. Plant Physiol 146:1386–1396

    Article  Google Scholar 

  • Sawada Y, Aoki M, Nakaminami K, Mitsuhashi W, Tatematsu K, Kushiro T, Koshiba T, Kamiya Y, Inoue Y, Nambara E, Toyomasu T (2008) Phytochrome- and gibberellins-mediated regulation of abscisic acid metabolism during germination of photoblastic lettuce seeds. Plant Physiol 146:1386–1396

    Article  PubMed  CAS  Google Scholar 

  • Schnable PS et al (2009) The B73 Maize genome: complexity, diversity, and dynamics. Science 326:1112–1115

    Article  PubMed  CAS  Google Scholar 

  • Shou H (2003) Crop improvement through genetic engineering: development of transformation technologies and production of stress tolerant transgenic crops. PhD dissertation, Iowa State University, Ames, IA

  • Shou H, Bordallo P, Wang K (2004) Expression of the Nicotiana protein kinase (NPK1) enhanced drought tolerance in transgenic maize. J Exp Bot 55:1013–1019

    Article  PubMed  CAS  Google Scholar 

  • Si Y, Zhang C, Meng S, Dane F (2009) Gene expression changes in response to drought stress in Citrullus colocynthis. Plant Cell Rep 28:997–1009

    Article  PubMed  CAS  Google Scholar 

  • Soderlund C, Descour A, Kudrna D, Bomhoff M, Boyd L, Currie J, Angelova A, Collura D, Wissotski M, Ashley E, Morrow D, Fernandes J, Walbot V, Yu Y (2009) Sequencing, Mapping, and Analysis of 27,455 maize full-length cDNAs. PLoS Genet 5:e1000740

    Article  PubMed  Google Scholar 

  • Soeda Y, Konings MCJM, Vorst O, van Houwelingen AMML, Stoopen GM, Maliepaard CA, Koddle J, Bino RJ, Groot SPC, van der Geest AHM (2005) Gene expression programs during Brassica oleracea seed maturation, osmopriming, and germination are indicators of progression of the germination process and the stress tolerance level. Plant Physiol 137:354–368

    Article  PubMed  CAS  Google Scholar 

  • Tong Z, Gao Z, Wang F, Zhou J, Zhang Z (2009) Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Mol Biol 10:71

    Article  PubMed  Google Scholar 

  • Tyler L, Thomas SG, Hu J et al (2004) DELLA proteins and gibberellins-regulated seed germination and floral development in Arabidopsis. Plant Phyisol 135:1008–1019

    Article  CAS  Google Scholar 

  • Van der Geest AHM (2002) Seed Genomics: germinating opportunities. Seed Sci Res 12:145–153

    Article  Google Scholar 

  • Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, Paepe AD, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:1–12

    Article  Google Scholar 

  • Vandesompele J, Kubista M, Pfaffl MW (2009) Reference gene validation software for improved normalization. In: Logan J, Edwards K, Saunders N (eds) Real-time PCR: current technology and applications. Caister Academic, UK, pp 47–63

    Google Scholar 

  • Varier A, Vari AK, Dadlani M (2010) The subcellular basis of seed priming. Curr Sci 99:450–456

    CAS  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  PubMed  CAS  Google Scholar 

  • Wohlbach DJ, Quirino BF, Sussman MR (2008) Analysis of the Arabidopsis histidine kinase ATHK1 reveals a connection between vegetative osmotic stress sensing and seed maturation. Plant Cell 20:1101–1117

    Article  PubMed  CAS  Google Scholar 

  • Yamauchi Y, Ogawa M, Kuwahara A, Hanada A, Kamiya Y, Yamaguchi S (2004) Activation of gibberellin biosynthesis and response pathways by low temperature during imbibition of Arabidopsis thaliana seeds. Plant Cell 16:357–378

    Article  Google Scholar 

  • Yang C, Li A, Zhao Y, Zhang Z, Zhu Y, Tan X, Geng S, Guo H, Zhang X, Kang Z, Mao L (2011a) Overexpression of a wheat CCaMK gene reduces ABA sensitivity of Arabidopsis thaliana during seed germination and seedling growth. Plant Mol Biol Rep 29:681–692

    Article  CAS  Google Scholar 

  • Yang G, Zhou R, Tang T, Chen X, Ouyang J, He L, Li W, Chen S, Guo M, Li X, Zhong C, Shi S (2011b) Gene expression profiles in response to salt stress in Hibiscus tiliaceus. Plant Mol Biol Rep 29:609–617

    Article  Google Scholar 

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Acknowledgments

This paper of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project no. 3601 was supported by Hatch Act and State of Iowa funds. We thank Dr. Manjit Misra (Director, Seed Science Center), Plant Science Institute, and the Department of Horticulture at Iowa State University for their financial assistance to support this work.

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Authors and Affiliations

  1. Department of Horticulture, Iowa State University, Ames, IA, 50011-1100, USA

    Keting Chen & Rajeev Arora

  2. Seed Science Center, Iowa State University, Ames, IA, 50011-1100, USA

    Anania Fessehaie

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  1. Keting Chen
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Correspondence to Rajeev Arora.

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Chen, K., Fessehaie, A. & Arora, R. Selection of Reference Genes for Normalizing Gene Expression During Seed Priming and Germination Using qPCR in Zea mays and Spinacia oleracea . Plant Mol Biol Rep 30, 478–487 (2012). https://doi.org/10.1007/s11105-011-0354-x

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