Plant Science 236 (2015) 103–115 Contents lists available at ScienceDirect Plant Science j ourna l ho me pa ge: www.elsevier.com/locate/plantsci Quantification of expression of dehydrin isoforms in the desiccation tolerant plant Craterostigma plantagineum using specifically designed reference genes Valentino Giarola a,1 , Dinakar Challabathula a,b,1 , Dorothea Bartels a,∗ a Institute of Molecular Physiology and Biotechnology of Plants, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany b Department of Life Sciences, School of Basic and Applied Sciences, Central University of Tamil Nadu, Thiruvarur, India a r t i c l e i n f o Article history: Received 30 December 2014 Received in revised form 16 March 2015 Accepted 20 March 2015 Available online 28 March 2015 Keywords: Dehydrins Desiccation tolerance Reference genes Quantitative expression RT-qPCR a b s t r a c t Craterostigma plantagineum is a desiccation tolerant resurrection plant. Many genes are induced during desiccation. Dehydrins are a group of dehydration-induced genes present in all higher plants. The cur- rent study aims at classifying the most abundantly expressed dehydrin genes from vegetative tissues of C. plantagineum and quantifying their expression. To identify variations between dehydrin isoforms at different stages of desiccation and rehydration by RT-qPCR, the target mRNA requires an accurate and reliable normalization. Previously we reported that RNAs from leaves and roots of C. plantagineum are not degraded during desiccation and subsequent rehydration thus allowing the use of RT-qPCR to test the stability of reference genes. The expression stability of eight candidate reference genes was tested in leaves, roots and callus. These genes were ranked according to their stability of gene expression using GeNorm PLUS and RefFinder. The most consistently expressed reference genes in each tissue were identi- fied and used to normalize gene expression data. Dehydrin isoforms were divided in three groups based on the expression level during the desiccation process in three different tissues (leaves, roots and callus). © 2015 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Response of plants to dehydration is a complex process mainly dependent on the modulation of transcriptional activity of stress- related genes. Gene expression analysis is important to understand the metabolic pathways and complex regulatory networks that underlie development and survival under stress conditions in plants. Several of the dehydration-induced genes encode proteins that play an important role in stress protection. Late embryo- genesis abundant (LEA) genes are a group of these genes that are abundantly expressed in response to dehydration/desiccation. Dehydrins, group II LEA proteins, occur ubiquitously in higher plants and are hydrophilic and thermostable proteins with a high number of polar and charged amino acids [1,2]. Dehydrin genes are organized in small gene families with variable number of mem- bers: Arabidopsis has ten, rice has eight, barley has thirteen and ∗ Corresponding author at: Institute of Molecular Physiology and Biotechnology of Plants University of Bonn, Kirschallee 1, D-53115 Bonn, Germany. Tel.: +49 228 73 2070; fax: +49 228 73 1697. E-mail address: dbartels@uni-bonn.de (D. Bartels). 1 These authors contributed equally to this work. populus has three dehydrin genes [3–6]. Despite the first descrip- tion of dehydrin genes more than 25 years ago, the biochemical function is not yet understood. Several hypotheses have been put forward to explain how dehydrins may contribute to cellular pro- tection [1,2]. Genes encoding these proteins are expressed in seeds during late embryogenesis and in different vegetative tissues like leaves, roots, vascular tissues, stems etc., subjected to dehydration, low temperature or high salt conditions [1,3,7,8]. The presence of several conserved amino acid motifs often organized in repeats is a distinctive feature of dehydrins. The presence of the 15 amino acid long K-segment (EKKGIMDKIKEKLPG), the seven amino acid long N-terminal Y-segment [(V/T)D(E/Q)YGNP] and the poly-serine S- segment (SDSSSSSSS) are the characteristic features of dehydrins [9,10]. All dehydrins contain at least one copy of the K-segment with a varied composition of Y and S segments [10]. The dehy- drins can be divided into five subgroups K n , SK n , Y n SK n , Y n K n and K n S based on the conserved segments. Dehydrins are localized in different organelles, and are differentially expressed throughout development and under stress conditions [1]. Recently Candat et al. [11] compared the sub-cellular protein localization prediction of 10 web-based tools with the experimental GFP or RFP localization results of 51 LEA proteins from Arabidopsis thaliana. The results of their study suggested PProwler [12] as the most efficient predictor for protein localization studies. http://dx.doi.org/10.1016/j.plantsci.2015.03.014 0168-9452/© 2015 Elsevier Ireland Ltd. All rights reserved.