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United States Department of Agriculture

Agricultural Research Service

Research Project: USING FUNCTIONAL AND APPLIED GENOMICS TO IMPROVE STRESS AND DISEASE RESISTANCE IN FRUIT TREES Title: Using gene fusion constructs to target a peach dehydrin protein to specific cellular compartments

Authors
item Wisniewski, Michael
item Webb, Robert
item Bassett, Carole
item Artlip, Timothy
item Becker, Chelsea - WEST VIRGINIA UNIVERSITY
item Karlson, Dale - WEST VIRGINIA UNIVERSITY

Submitted to: International Symposium on Plant Cold Hardiness
Publication Type: Abstract Only
Publication Acceptance Date: August 2, 2008
Publication Date: August 3, 2008
Citation: Wisniewski, M.E., Webb, R.P., Bassett, C.L., Artlip, T.S., Becker, C., Karlson, D. 2008. Using gene fusion constructs to target a peach dehydrin protein to specific cellular compartments. International Symposium on Plant Cold Hardiness. Saskatoon, Canada. Book of Abstracts. Pg 24.

Technical Abstract: Dehydrins have been commonly associated with the response of a cell to dehydrative stress. They are believed to ameliorate the effects of dehydrative stress by preventing the denaturation of proteins and membranes. Dehydrins are highly hydrophyllic, a property believed to contribute to their ability to stablilize other macromolecules under dehydrative conditions. Despite the abundant literature associating dehydrins with stress tolerance, there have been only a few cases where overexpression of a dehydrin gene has resulted in improved stress tolerance in the transgenic plants. The lack of improvement has been attributed to the need to express several "cold tolerance genes" at the same time. PCA60 is a dehydrin protein identified in bark tissues of peach that responds to both low temperature and water stress. It has been shown to accumulate on a seasonal basis and has been associated with levels of cold hardiness. PCA60 is a Y2K8 dehydrin encoded by PpDhn1. In the present study, we constructed two gene fusion constructs in order to target the overexpressed dehydrin to a specific cellular location. The first gene fusion construct consisted of a chloroplast transit peptide sequence fused to PpDhn1 in order to direct the localization of PCA60 to the chloroplast. The second construct consisted of a portion of a plasma-membrane aquaporin gene fused to PpDhn1 to presumbably target PCA60 to the plasma membrane. Both leader sequences were also fused to GFP in order to facilitate visualizing their localization. The GFP constructs were evaluated in tobacco using biolistics, while the dehydrin fusion constructs were used to transform Arabidopsis. Analysis of the tobacco leaves after particle gun bombardment clearly indicated that the chloroplast transit peptide restricted the localization of the GFP exclusively to the chloroplast. Localization of the GFP using the aquaporin segment was more ambiguous, but GFP appeared to be confined to membranes if not specifically to the plasma membrane. Analysis of non-acclimated Arabidopsis plants (T4) indicated that both gene-fusion constructs improved cold hardiness, as determined by electrolyte leakage, by approximately 2 deg C compared to the wild type plants. Further analysis of the freezing tolerance of transgenic lines under cold acclimated conditions, as well as, further confirmation of dehydrin localization using the gene fusion constructs is in progress.

Last Modified: 11/28/2014
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