Molecular and physiological analyses of a temperature dependent alpha-galactosidase in Petunia x Hybrida 'Mitchell'

Raffinose family oligosaccharides (RFO) have been implicated in the acquisition of tolerance to low temperature stresses. Alpha-galactosidase (α-Gal) is a key catabolic enzyme of RFO involved in the cold hardiness pathway, cleaving the terminal-linked moiety from galactose-containing oligosaccharides.; This research was based on the primary hypothesis that as cold acclimation occurs, specific soluble sugars increase and as tissues deacclimate, the sugar levels decrease. Based on this, a second hypothesis was formulated; that down regulation of the α-Gal gene may be an important element in the accumulation or maintenance of RFO levels that are required to enhance freezing tolerance. The objectives were to determine the role of α-Gal in deacclimation and in freezing tolerance. This was accomplished by examining α-Gal activity and transcript accumulation during raffinose catabolism and overexpression and downregulation of the α-Gal gene in petunia.; A cDNA clone petgal, was isolated from Petunia × hybrida cv Mitchell RNA by RT-PCR using degenerate oligosaccharide primers designed to amplify the α-Gal cDNA. The putative α-Gal cDNA sequence has high nucleotide sequence homology (>80%) to other known α-Gals. Southern blot analysis suggests that α-Gal represents a single gene family. This study showed a comprehensive analysis of petgal expression including non tissue-specific expression, no developmental regulation and expression in response to increased temperature. Increases in α-Gal transcript one hour after deacclimation correspond with increases in α-Gal activity suggesting that warm temperature may regulate RFO catabolism by increasing the transcription of the α-Gal gene.; To examine the relationship between endogenous sugars and freezing stress, the expression of α-Gal was modified in transgenic petunia. The α-Gal cDNA from tomato seed under the control of the Figwort Mosaic Virus promoter was introduced into petunia using the Agrobacterium tumefaciens-mediated transformation.; Overexpression of the α-Gal gene inhibited low temperature tolerance when compared to antisense petunia lines suggesting that α-Gal plays a major role in low temperature tolerance. The combination of molecular and physiological approaches demonstrated the role of raffinose in low temperature stress. Through antisense technology α-Gal was shown to be an essential component of the cold hardiness pathway by providing a direct route to modify raffinose accumulation in target tissues needed for freezing stress tolerance. (Abstract shortened by UMI.)...