Molecular and physiological analyses of a temperature dependent α-galactosidase in Petunia x hybrida 'Mitchell'

Abstract

Raffinose family oligosaccharides (RFO) have been implicated in the acquisition of tolerance to low temperature stresses. Studies on the biochemical basis of plant tolerance to low temperatures have focused primarily on the cold acclimation response, while retention and loss of the cold acclimated state have been largely neglected. 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. Petunia was chosen as a model for this research because preliminary results in our laboratory demonstrated petunia’s capability to cold acclimate. 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 x 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 a-Gal cDNA from tomato seed under the control of the Figwort Mosaic Virus promoter was introduced into petunia using the Agrobacterium tumefaciens-mediated transformation. Besides lower germination percentages in transgenic plants, there were no phenotypical differences between wild type and transgenic plants. RNA gel blot analysis demonstrated an increase in α-Gal transcript accumulation in sense plants and a decrease in antisense plants. Antisense inhibition of the α-Gal gene resulted in an accumulation of raffinose and enhanced freezing tolerance of petunia. Among the antisense lines examined, several different levels of freezing tolerances were observed with A105 being the most tolerant line and A91 being the least tolerant line. Freezing stress tolerance was predicted based on the relationship between raffinose accumulation and decreased electrolyte leakage. 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.As an adjunct to this study, phenolic extracts from plants subjected to two different cold acclimation regimes to induce chilling tolerance were analyzed for specific phenolic acids and assessed for their antioxidant capacity. Gentisic acid was induced in significant quantities upon cold acclimation (p < 0.05). The data suggest that phenolic metabolism may be a consequence of cold stress and probably not related to protective functions.Knowledge from this research has important implications whereby petunia may be grown later into fall when frost injury is most likely. But most importantly, the target gene (α-Gal), is not limited to petunia and thus may provide a means of improving the freezing tolerance of other economic crops.