Cold responsive EARLI1 type HyPRPs improve freezing survival of yeast cells and form higher order complexes in plants
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Plants have large families of proteins sharing a conserved eight-cysteine-motif (8CM) domain. The biological functions of these proteins are largely unknown. EARLI1 is a cold responsive Arabidopsis gene that encodes a hybrid proline-rich protein (HyPRP) with a three-domain architecture: a putative signal peptide at the N-terminus, a proline-rich domain (PRD) in the middle, and an 8CM domain at the C-terminus. We report here that yeast cells expressing different EARLI1 genes had significantly higher rates of freezing survival than empty-vector transformed controls. Arabidopsis plants with knocked down EARLI1 genes had an increased tendency for freezing-induced cellular damage. EARLI1-GFP fluorescence in transgenic plants and immunoblot analyses using protoplasts suggested cell wall localization for EARLI1 proteins. Immunoblot analyses showed that EARLI1 proteins form higher order complexes in plants, and that the PRD is a soluble and the 8CM an insoluble protein domain. We propose that EARLI1 proteins have a bimodular architecture in which the PRD may interact with the cell wall and the 8CM domain with the plasma membrane to protect the cells during freezing stress.
KeywordsCold acclimation Cysteines EARLI1 Freezing tolerance Yeast
Cauliflower mosaic virus
Columbia ecotype of Arabidopsis
Hybrid proline-rich protein
Lipid transfer protein
We are grateful to J. Anderson (Marquette University, Milwaukee, WI, USA) and members of his lab for help with yeast experiments; to J. Bubier and G. T. Hayman (Marquette University) for generating EARLI1 knockdown lines; to M. Nagao (Iwate University, Morioka, Japan) for help with the electrolyte leakage assays; and to Y. Pan (Marquette University) for protoplast preparation. The phosphorimager was provided by NSF Instrumentation grant No. D.B.I. 0100667. This research was supported by NRI Competitive Grant No. 2001-35100-10688 from the US Department of Agriculture.
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