Down-Regulating α-Galactosidase Enhances Freezing Tolerance in Transgenic Petunia

Abstract

␣-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 ϫ 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 Ϫ4°C for cold-acclimated wild-type plants to Ϫ8°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.Many plants increase in low temperature and/or freezing tolerance in response to low nonfreezing temperatures, a phenomenon known as cold acclimation. For decades, the study of low temperature stress has had a primary goal of cataloging and understanding the biochemical and physiological changes occurring during cold acclimation (Levitt, 1980;Guy, 1990;Thomashow, 2001). It is known that the disruption of cellular membranes, particularly the plasma membrane, is the primary site of injury during a freeze-thaw cycle and that this injury usually results from dehydration associated with freezing (Steponkus, 1984). Other consequences of freezeinduced cellular dehydration include the generation of reactive oxygen species that are damaging to other cellular components (McKersie, 1991). As such, plants have developed mechanisms to deal with these multiple stresses.It has long been established that changes in gene expression occur upon exposure to cold acclimation (Guy et al., 1985). In the last decade, extensive research to identify and characterize cold-responsive (COR) genes has been undertaken. Hajela et al. (1990) isolated the COR genes from Arabidopsis that encode polypeptides thought to have protective roles against dehydration. Expression profile experiments in Arabidopsis demonstrated that extensive changes in gene expression occur during cold acclimation and that a substantial number of the genes that are up...

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