105 PCR-Based Cloning of an α-Galactosidase Gene from Deacclimated Petunia (Petunia x Hybrida) J.C. Pennycooke 1 , M.L. Jones 2 , R. Vepachedu 1 and C. Stushnoff 1a Department of Horticulture and Landscape Arch., Colorado State University, Fort Collins, CO 80523, USA 1 ; Email: stushnof@lamar.colostate.edu 1a Department of Horticulture and Crop Science, Ohio State University, Wooster, OH 44691, USA 2 Keywords: α-galactosidase; cloning; deharden; petunia; RFO catabolism; RT-PCR Abstract Previous studies of plant cold hardiness have focused mainly on the cold acclimation response, while studies on the deacclimation process have been limited. Alpha-galactosidase a key catabolic enzyme of the raffinose family oligosaccharides (RFO), cleaves the terminal-linked moiety from galactose-containing oligosaccha- rides. A cDNA clone petgal, was isolated from Petunia x hybrida Mitchell leaf RNA by RT-PCR using degenerate oligosaccharide primers designed to amplify the mature α-galactosidase protein. The putative α-galactosidase cDNA sequence has high nucleotide sequence homology (>80%) to other known plant α-galactosidases. Southern blot analysis suggests that α-galactosidase represents a single gene family. Based on the amino acid alignment of the petunia α-galactosidase enzyme with other known α-galactosidases, it appears that the protein is conserved among species. Up- regulation of the α-galactosidase gene in response to deacclimation duration and temperature, suggests that warm temperature may regulate RFO catabolism by increasing the transcription of the α-galactosidase gene. INTRODUCTION Previous biochemical and physiological studies have demonstrated the accumulation of specific metabolites such as the raffinose family oligosaccharides (RFO) upon low temperature exposure (Bachmann et al., 1994). These studies of plant tolerance to low temperatures have focused primarily on the cold acclimation response, the process by which plants increase their tolerance to freezing in response to low non-freezing temperatures (Guy, 1990), while studies on the deacclimation process have been largely neglected. Alpha-galactosidase (α-Gal), the key enzyme involved in the breakdown of RFOs, cleaves the terminal-linked moiety from galactose-containing oligosaccharides. Castonguay and Nadeau (1998) showed that α-Gal increased activity during spring dehardening at the time when RFO levels decreased rapidly. α-Gal is widely distributed in microorganisms, humans and plants and cDNAs have been cloned from several sources including yeast (Liljestrom, 1985), human (Bishop et al., 1986), and tomato seed (Feurtado et al., 2001). This study reports on the cloning, sequence, and characterization of a cDNA clone encoding the petunia α-Gal to deduce its role in cold deacclimation in plants. MATERIALS AND METHODS Plant Materials and Treatments Petunia x hybrida ‘Mitchell’ plants were grown from seed and maintained in a greenhouse at 25 °C with a 16/8 h photoperiod. Eight-week old plants were transferred to a growth chamber to induce cold acclimation by incubating at 15 °C for 7 d, 10 °C for 7 d, 5 °C for 7 d and subsequently at 3 °C for 3 d with a 12/12 h photoperiod under cool white fluorescent light at 60 µmole m -2 s -1 . Plants were deacclimated by incubating previously cold acclimated plants at 25 °C with a 16/8 h photoperiod for varying periods of time. In a separate experiment plants were deacclimated for 1 h at various temperatures Proc. XXVI IHC – Environmental Stress Eds. K.K. Tanino et al. Acta Hort. 618, ISHS 2003 Publication supported by Can. Int. Dev. Agency (CIDA)