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ENVIRONMENTAL STRESS AND ADAPTATION

Down-Regulating -Galactosidase Enhances Freezing Tolerance in Transgenic Petunia¹

Department of Horticulture and Landscape Architecture, Colorado State University, Fort Collins, Colorado 80523 (J.C.P., C.S.); and Department of Horticulture and Crop Science, The Ohio State University/Ohio Agricultural Research and Development Center, Wooster, Ohio 44691 (M.L.J.)

-Galactosidase (-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the -1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of -Gal was modified in transgenic petunia (Petunia x hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that -Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of -Gal mRNAs. -Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from –4°C for cold-acclimated wild-type plants to –8°C for the most tolerant antisense line. Down-regulating -Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the -Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with -Gal provides an additional method for improving the freezing tolerance of plants.

¹ This work was supported in part by The Colorado Institute of Research in Biotechnology, by the Ohio Floriculture Foundation, and by funds from the Colorado Agricultural Experiment Station (project nos. 690 and 738).

^* Present address: Department of Horticulture and Crop Science, The Ohio State University/Ohio Agricultural Research and Development Center, 1680 Madison Ave, Wooster, OH 44691.

^* Corresponding author; e-mail stushnof{at}lamar.colostate.edu; fax 970–491–7745.

Received March 31, 2003; returned for revision May 8, 2003; accepted July 1, 2003.

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