Poster: Lipids & related molecules
Abs #
306: Molecular cloning, expression during cold acclimation and subcellular localization of an aminoalcoholphosphotransferase (TaAAPT1) for phospholipids biosynthesis from Triticum aestivum
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Presenter: |
Sutoh, Keita , skeita@naro.affrc.go.jp |
Authors | Sutoh, Keita (A) Sakaki, Takeshi (B) Imai, Ryozo (A) | | Affiliations: |
(A): Winter Stress Labolatory, National Agricultural Research Center for Hokkaido Region, NARO
(B): Department of Bioscience and Technology, Hokkaido Tokai University
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Aminoalcoholphosphotransferases (AAPT, EC 2.7.8.1 and EC 2.7.8.2) are involved in the three-step nucleotide pathway for the synthesis of the abundant membrane lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). AAPT utilizes diacylglycerols and cytidine diphosphate (CDP)-aminoalcohols as substrates. Molecular and biochemical characterization of plant AAPT has not been described until recently (Qungang et al., Planta, 217: 547-558, 2003). No study about subcellular localization of AAPT is reported. We cloned a cDNA for AAPT (TaAAPT1) from a wheat crown cDNA library. The deduced amino acid sequence of TaAAPT1 had a high similarity to each of the two well-characterized AAPT sequences from yeast, cholinephosphotransferase and ethanolaminephosphotransferase. RNA blot analysis showed that TaAAPT1 mRNA levels increased in crown tissue during cold acclimation. The pattern of the accumulation was similar to that of WECT1 which is involved in the second step of nucleotide pathway, suggesting activation of the nucleotide pathway during cold acclimation. TaAAPT1 was fused with the green fluorescent protein (GFP) and transiently expressed in wheat leaves and shoots. Fluorescence and confocal laser scanning microscopy images of wheat leaves and shoots revealed that the TaAAPT::GFP fusion protein is localized in the nuclear envelope membrane and ER near nucleus. This suggests that TaAAPT1 is involved in the synthesized PE and/or PC close to the nuclear envelope membrane and ER for cold tolerance. Enzymatic characterization including substrate specificity of a recombinant TaAAPT1 is underway.
