Reference genes selection and normalization of oxidative stress responsive genes upon different temperature stress conditions in Hypericum perforatum L

PLoS One. 2014 Dec 11;9(12):e115206. doi: 10.1371/journal.pone.0115206. eCollection 2014.

Abstract

Reverse transcription-quantitative real-time PCR (RT-qPCR) is a widely used technique for gene expression analysis. The reliability of this method depends largely on the suitable selection of stable reference genes for accurate data normalization. Hypericum perforatum L. (St. John's wort) is a field growing plant that is frequently exposed to a variety of adverse environmental stresses that can negatively affect its productivity. This widely known medicinal plant with broad pharmacological properties (anti-depressant, anti-tumor, anti-inflammatory, antiviral, antioxidant, anti-cancer, and antibacterial) has been overlooked with respect to the identification of reference genes suitable for RT-qPCR data normalization. In this study, 11 candidate reference genes were analyzed in H. perforatum plants subjected to cold and heat stresses. The expression stability of these genes was assessed using GeNorm, NormFinder and BestKeeper algorithms. The results revealed that the ranking of stability among the three algorithms showed only minor differences within each treatment. The best-ranked reference genes differed between cold- and heat-treated samples; nevertheless, TUB was the most stable gene in both experimental conditions. GSA and GAPDH were found to be reliable reference genes in cold-treated samples, while GAPDH showed low expression stability in heat-treated samples. 26SrRNA and H2A had the highest stabilities in the heat assay, whereas H2A was less stable in the cold assay. Finally, AOX1, AOX2, CAT1 and CHS genes, associated with plant stress responses and oxidative stress, were used as target genes to validate the reliability of identified reference genes. These target genes showed differential expression profiles over time in treated samples. This study not only is the first systematic analysis for the selection of suitable reference genes for RT-qPCR studies in H. perforatum subjected to temperature stress conditions, but may also provide valuable information about the roles of genes associated with temperature stress responses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cold Temperature
  • Gene Expression Regulation, Plant
  • Hot Temperature
  • Hypericum / genetics*
  • Oxidative Stress / genetics*
  • Plant Proteins / biosynthesis*
  • Plant Proteins / genetics
  • Stress, Physiological / genetics*

Substances

  • Plant Proteins

Grants and funding

This work was supported by the Portuguese Foundation for Science and Technology within the frame of the project PTDC/AGR-GPL/099263/2008 and the program POPH – Operational Program for Human Potential (Ciência 2007 to BA and Ciência 2008 to HC: C2008-UE/ICAM/06). This work is funded by FEDER Funds through the Operational Program for Competitiveness Factors – COMPETE, and National Funds through FCT under the Strategic Projects PEst-C/AGR/UI0115/2011 and PEst-OE/AGR/UI0115/2014. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.