Identification of mechanisms for cold tolerance in Helleborus orientalis Lam.

摘要

Damage caused by late winter and early spring freezing temperatures affects the commercial values of most plants. Helleborus orientalis Lam. (Lenten-rose) is an evergreen ornamental that can withstand multiple freezing-thawing cycles without encountering significant damage. The goal of this research project was to identify molecular mechanisms and genes in flowers buds of Lenten-rose that provide protection from tissue damage caused by low temperature fluctuations.Lenten-rose flower buds used in this study were collected on March 9 th, 2008 following a freezing event. During that day, the temperature remained at -5°C. On March 10th, the temperature was above 0°C and by the 14th, it was above 10°C.Collected flower buds were flash frozen in liquid nitrogen and kept frozen until processed. The cDNA was isolated from each flower bud sample and transcript populations of cDNA from these samples were compared using a fluorescent cDNA differential display method. Differentially expressed cDNA fragments were identified, cloned, sequenced and their putative functions were determined using bioinformatics analysis. The expression of each clone was validated using quantitative RT-PCR analysis.Through these analyses, it was discovered that the gene expression patterns in H. orientalis flower tissue changed after being subjected to freezing, thawing, and dehydration. Response and tolerance mechanisms observed included changes in sugar and secondary protection mechanisms, protease activity, photosynthetic machinery, signal transduction and cell growth. When these clones were tested against both the nucleotide and the protein databases, few clones had match sequences in the databases. The lack of matches in the databases indicated that the flower bud of Lenten-rose contained cold-responsive genes that were not reported. In general, expression levels were lower for most genes isolated from flower tissues that had been frozen prior to collection. Some genes had no expression or reduced expression when the ambient temperature increased from -5°C to 10°C. Results obtained through these analyses are providing basic information on molecular survival mechanisms in Lenten-rose that allow their flower bud tissue to survive freezing. For the development of cold resistant plants, these genes could potentially be introduced into susceptible species to improve their tolerance or resistance to freezing and thawing cycles.