Evaluation of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Soybean Tissues under Various Abiotic Stress Conditions

Dung Tien Le,Truyen N Quach,Juan J Gutierrez-Gonzalez,Rie Nishiyama,Satish K Guttikonda,Babu Valliyodan,Chien Van Ha,Donavan L Aldrich,Lam-Son Phan Tran,Yasuko Watanabe,Henry T Nguyen,Christian Schönbach

doi:10.1371/journal.pone.0046487

Dung Tien Le, Truyen N Quach + Show 10 more

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https://doi.org/10.1371/journal.pone.0046487

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Abstract

Quantitative RT-PCR can be a very sensitive and powerful technique for measuring differential gene expression. Changes in gene expression induced by abiotic stresses are complex and multifaceted, which make determining stably expressed genes for data normalization difficult. To identify the most suitable reference genes for abiotic stress studies in soybean, 13 candidate genes collected from literature were evaluated for stability of expression under dehydration, high salinity, cold and ABA (abscisic acid) treatments using delta CT and geNorm approaches. Validation of reference genes indicated that the best reference genes are tissue- and stress-dependent. With respect to dehydration treatment, the Fbox/ABC, Fbox/60s gene pairs were found to have the highest expression stability in the root and shoot tissues of soybean seedlings, respectively. Fbox and 60s genes are the most suitable reference genes across dehydrated root and shoot tissues. Under salt stress the ELF1b/IDE and Fbox/ELF1b are the most stably expressed gene pairs in roots and shoots, respectively, while 60s/Fbox is the best gene pair in both tissues. For studying cold stress in roots or shoots, IDE/60s and Fbox/Act27 are good reference gene pairs, respectively. With regard to gene expression analysis under ABA treatment in either roots, shoots or across these tissues, 60s/ELF1b, ELF1b/Fbox and 60s/ELF1b are the most suitable reference genes, respectively. The expression of ELF1b/60s, 60s/Fbox and 60s/Fbox genes was most stable in roots, shoots and both tissues, respectively, under various stresses studied. Among the genes tested, 60s was found to be the best reference gene in different tissues and under various stress conditions. The highly ranked reference genes identified from this study were proved to be capable of detecting subtle differences in expression rates that otherwise would be missed if a less stable reference gene was used.

Highlights

The study of plant adaptive responses to abiotic stresses, such as drought, high salinity and cold stress, is a rapidly growing field of research due largely to its immense impact on global food supply
The strategy conceived for determining effective reference genes in soybean began with a screening of the candidate genes whose expression were commonly stable across the major stressors, including dehydration, salt and cold stresses, which are often encountered by soybean plants [1,3]
Gene expression in response to abiotic stress has been known to be regulated in ABAdependent and/or ABA-independent manner [39,51,52,53]; we included ABA treatment into our study

Summary

Introduction

The study of plant adaptive responses to abiotic stresses, such as drought, high salinity and cold stress, is a rapidly growing field of research due largely to its immense impact on global food supply. Abiotic stressors are the adverse environmental conditions which are unfavorable to plant growth and include such circumstances as flooding, extreme temperatures, high soil salinity, and drought These stressors can have detrimental effects on plants which generally result in major yield losses for the economically important crops, including soybean [1,2,3,4,5]. Plants have always evolved under the highly selective pressure of these stresses and have established exceedingly complex and broad-stroked genetic and molecular mechanisms to survive the adverse conditions imposed by them It has been demonstrated in many instances that many of the primary mechanisms plants use to cope with abiotic stress are not constitutively active throughout their lifetime, but are induced at a transcriptional level when these stresses are present [6,9,11]. The ability to perform highthroughput profiling sensitive enough to detect subtle changes in the expression levels of a magnitude of target genes is a valuable tool for research focused on abiotic stress-induced gene expression [1,10,19]

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