Plant Cell Rep (2005) 23:764–769 DOI 10.1007/s00299-004-0883-y PHYSIOLOGY AND BIOCHEMISTRY Can-kui Zhang · Ping Lang · Fenny Dane · Robert C. Ebel · Narendra K. Singh · Robert D. Locy · William A. Dozier Cold acclimation induced genes of trifoliate orange (Poncirus trifoliata) Received: 30 June 2004 / Revised: 25 August 2004 / Accepted: 26 August 2004 / Published online: 22 September 2004 # Springer-Verlag 2004 Abstract Commercial citrus varieties are sensitive to low temperature. Poncirus trifoliata is a close relative of Cit- rus species and has been widely used as a cold-hardy rootstock for citrus production in low-temperature envi- ronments. mRNA differential display-reverse transcrip- tion (DDRT)-PCR and quantitative relative-RT-PCR were used to study gene expression of P. trifoliata under a gradual cold-acclimation temperature regime. Eight up- regulated cDNA fragments were isolated and sequenced. These fragments showed high similarities at the amino acid level to the following genes with known functions: betaine/proline transporter, water channel protein, aldo- keto reductase, early light-induced protein, nitrate trans- porter, tetratricopeptide-repeat protein, F-box protein, and ribosomal protein L15. These cold-acclimation up-regu- lated genes in P. trifoliata are also regulated by osmotic and photo-oxidative signals in other plants. Keywords Cold acclimation · Differential display · Gene expression · Poncirus trifoliata · Quantitative relative reverse transcription (RT)-PCR Introduction Environmental factors such as freezing, drought, and high salt levels affect the growth, productivity, and distribution of plants (Kasuga et al. 1999). Plants have evolved a wide variety of mechanisms that allow them to thrive in hostile environments, albeit their ability to survive in adverse environments varies greatly (Garwe et al. 2003). The distribution of many temperate fruit crops is restricted by low-temperature conditions (Owens et al. 2002). Low temperatures can cause extracellular ice formation and damage to the cell-membrane (Gilmour et al. 1988) transporters and receptor proteins (Hazel 1995). Many plants can acclimate to freezing by a pre-expo- sure to low but nonfreezing temperatures (Fowler and Thomashow 2002). The cold acclimation process requires the synthesis of new proteins (Tseng and Li 1991), al- terations in lipid and carbohydrate composition, the ac- cumulation of compatible osmolytes such as proline, be- taine, and soluble sugars (Bohnert et al. 1995; Lynch and Steponkus 1987; Thomashow 1994), and the activation of ion channels (Knight et al. 1996). Several plant genes induced by low temperature have been identified in al- falfa (Wolfraim et al. 1993), Arabidopsis thaliana (Gil- mour et al. 1992), and strawberry (Yubero-Serrano et al. 2003). A low-temperature-responsive dehydrin-like pro- tein, wcor410, is involved in cryoprotection of the plasma membrane against freezing or dehydration stress (Dany- luk et al. 1994). Several cold-regulated (COR) proteins from different plants are involved in membrane stability (Thomashow 1998). COR proteins are hydrophilic, and it has been suggested that they protect cells from low- temperature, water-deficit, and high-salt stress condi- tions (Artus et al. 1996; Steponkus et al. 1998). C-repeat binding factor 1 (CBF1) is the DNA binding protein isolated from A. thaliana. The binding of this transcrip- tion factor to the C-repeat/dehydration response element (CRT/DRE-motif) can activate the co-ordinate expression of several COR genes (Jaglo-Ottosen et al. 1998). Ex- pression of CBF1 in transgenic plants resulted in en- The first two authors have contributed equally to this work. The nucleotide sequences reported in this paper have been sub- mitted to GenBank under accession numbers of CN779663 (P1), CN779664 (P2), CN779665 (P3), CN779666 (P4), CN779667 (P5), CN779668 (P6), CN779669 (P7) and CN779670 (P8). Communicated by J.C. Register III C.-k. Zhang · P. Lang · F. Dane ( ) ) · R. C. Ebel · W. A. Dozier Department of Horticulture, Auburn University, Auburn, AL, 36849, USA e-mail: danefen@auburn.edu Tel.: +1-334-8443046 Fax: +1-334-8443131 N. K. Singh · R. D. Locy Department of Biological Sciences, Auburn University, Auburn, AL, 36849, USA