Abstract
Ammopiptanthus mongolicus, the only evergreen broadleaf shrub endemic to the northwest desert of China, is a valuable species for plant abiotic stress research. No report has so far described the selection of reference genes to get stringent normalization for qPCR in A. mongolicus. This work identified reliable reference genes for normalization of qPCR data in A. mongolicus under abiotic stresses from 14 reference gene candidates (UBQ, Tub1, Tub2, Abc1, Ubc1, Ubc2, Ubc4, Ubc5, eIF1, eIF2, eIF3, eIF4, EF1, EF2), and used the most suitable combination of reference genes to normalize the expression profiles of seven ROS-scavenging enzyme genes (AmSOD, AmAPX, AmGPX, AmCAT, AmGLR, AmPrx, and AmTrx). We set a series of 22 experimental samples covering the control and different time points under cold, dry, salt, and heat stresses. According to geNorm and NormFinder, the combination of eIF1 and eIF3 was best for accurate normalization across all the treatments, confirmed by normalizing qPCR data with AmHsp90. In contrast, these data show that Tub1, Abc1, and EF1 are not suitable reference gene candidates. After being normalized against eIF1 and eIF3, the seven ROS-scavenging enzyme genes exhibited differentially up- or down-regulated expression patterns. AmSOD and AmGPX were up-regulated by all four treatments, indicating that they may participate in an anti-oxidative mechanism under abiotic stresses in A. mongolicus. AmCAT exhibited a much higher expression level than AmAPX, AmPrx, and AmGPX, suggesting a principle role in detoxifying excessive H2O2. AmSOD, AmGPX and AmAPX showing the most abundant transcripts under heat, AmCAT and AmGLR under drought, and AmPrx under salt, were observed. Expression patterns of the seven ROS-scavenging enzyme genes suggest different antioxidant protection roles of these genes under abiotic stresses. These results are valuable for future research on gene expression and abiotic stress tolerance in A. mongolicus.
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References
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(15):5245–5250
Borges A, Tsai SM, Caldas DG (2011) Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses. Plant Cell Rep. doi:10.1007/s00299-011-1204-x
Brunner AM, Yakovlev IA, Strauss SH (2004) Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4:14
Bunnel TM, Burbach BJ, Shimizu Y, Ervasti JM (2011) β-Actin specifically controls cell growth, migration and the G-actin pool. Mol Biol Cell 22:4047–4058
Cortleven A, Remans T, Brenner WG, Valcke R (2009) Selection of plastid- and nuclear-encoded reference genes to study the effect of altered endogenous cytokinin content on photosynthesis genes in Nicotiana tabacum. Photosynth Res 102:21–29
de Almeida MR, Ruedell CM, Ricachenevsky FK, Sperotto RA, Pasquali G, Fett-Neto AG (2010) Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globules Labill. BMC Mol Biol 11:73
de Oliveira Hiragi C, Miranda-Vilela AL, Rocha DM, de Oliveira SF, Hatagima A, de Nazaré Klautau-Guimarães M (2011) Superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferases M1 and T1 gene polymorphisms in three Brazilian population groups. Genet Mol Biol 34:11–18
Fernandez P, Di Rienzo JA, Moschen S, Dosio GA, Aguirrezábal LA, Hopp HE, Paniego N, Heinz RA (2011) Comparison of predictive methods and biological validation for qPCR reference genes in sunflower leaf senescence transcript analysis. Plant Cell Rep 30:63–74
Frank G, Pressman E, Ophir R, Althan L, Shaked R, Freedman M, Shen S, Firon N (2009) Transcriptional profiling of maturing tomato (Solanum lycopersicum L.) microspores reveals the involvement of heat shock proteins, ROS scavengers, hormones, and sugars in the heat stress response. J Exp Bot 60:3891–3908
Gachon C, Mingam A, Charrier B (2004) Real-time PCR: what relevance to plant studies. J Exp Bot 55:1445–1454
Gechev TS, Van Breusegem F, Stone JM, Denev I, Laloi C (2006) Reactive oxygen species as signals that modulate plant stress responses and programmed cell death. Bioessays 28:1091–1101
Guénin S, Mauriat M, Pelloux J, Van Wuytswinkel O, 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
Gutierrez L, Mauriat M, Guénin S (2008) The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol 6:609–618
Hong SY, Seo PJ, Yang MS, Xiang F, Park CM (2008) Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biol 8:112
Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalisation: strategies and considerations. Genes Immun 6(4):279–284
Huis R, Hawkins S, Neutelings G (2010) Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biol 10:71
Jarošová J, Kundu JK (2010) Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR. BMC Plant Biol 10:146
Jian B, Liu B, Bi Y, Hou W, Wu C, Han T (2008) Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol 9:59
Kim BR, Hy N, Kim SU, Kim SI, Chang YJ (2003) Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. Biotechnol Lett 25(21):1869–1872
Lee JM, Roche JR, Donaghy DJ, Thrush A, Sathish P (2010) Validation of reference genes for quantitative RP-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.). BMC Mol Biol 11:8
Libauh M, Thibivilliers S, Bilgin DD, Radwan O, Benitez M, Clough SJ, Stacey G (2008) Identification of four soybean reference genes for gene expression normalization. Plant Genome 1:44–54
Liu M, Chen Y, Lu C (2007) Cold acclimation induced accumulation of phenolic compounds and freezing tolerance in Ammopiptanthus mongolicus. For Stud China 9(3):203–207
Liu R, Liu M, Liu J, Chen Y, Chen Y, Lu C (2010) Heterologous expression of an Ammopiptanthus mongolicus late embryogenesis abundant protein gene (AmLEA) enhances Escherichia coli viability under cold and heat stress. Plant Growth Regul 60:163–168
Migdal C, Serres M (2011) Reactive oxygen species and oxidative stress. Med Sci 27:405–412
Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410
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 normalization in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45
Selim M, Legay S, Berkelmann-Löhnertz B, Langen G, Kogel KH, Evers D (2012) Identification of suitable reference genes for real-time RT-PCR normalization in the grapevine-downy mildew pathosystem. Plant Cell Rep 31(1):205–216
Silveira ED, Alves-Ferreira M, Guimarães LA, da Silva FR, Carneiro VT (2009) Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha. BMC Plant Biol 9:84
Stürzenbaum SR, Cater S, Morgan AJ, Kille P (2001) Earthworm pre-procarboxypeptidase: a copper responsive enzyme. Biometals 14:85–94
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
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 3:1–11
Virdi AS, Pareek A, Singh P (2011) Evidence for the possible involvement of calmodulin in regulation of steady state levels of Hsp90 family members (Hsp87 and Hsp85) in response to heat shock in sorghum. Plant Signal Behav 6:393–399
Xu SJ, An LZ, Feng H (2002) The seasonal effects of water stress on Ammopiptanthus mongolicus in a desert environment. J Arid Environ 51:437–447
Yokawa K, Kagenishi T, Kawano T, Mancuso S, Baluška F (2011) Illumination of Arabidopsis roots induces immediate burst of ROS production. Plant Signal Behav 6:1457–1461
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This work was financially supported by the National Natural Science Foundation of China (Grant No. 30700559, 30671476) and Natural Science Foundation of Beijing (Grant No. 6112016).
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J. Shi and M. Liu contributed equally to this work.
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Shi, J., Liu, M., Shi, J. et al. Reference gene selection for qPCR in Ammopiptanthus mongolicus under abiotic stresses and expression analysis of seven ROS-scavenging enzyme genes. Plant Cell Rep 31, 1245–1254 (2012). https://doi.org/10.1007/s00299-012-1245-9
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DOI: https://doi.org/10.1007/s00299-012-1245-9