Molecular and biochemical characterization of an aminoalcoholphosphotransferase (AAPT1) from Brassica napus: effects of low temperature and abscisic acid treatments on AAPT expression in Arabidopsis plants and effects of over-expression of BnAAPT1 in transgenic Arabidopsis.
Aminoalcoholphosphotransferases (AAPT, EC 2.7.8.1 and EC 2.7.8.2) catalyze the transfer of CDP-aminoalcohols to sn-1, 2 diacylglycerol (DAG) to form phosphatidylaminoalcohols with the release of CMP. The Brassica napus L. AAPT1 gene (designated BnAAPT1) was identified from cDNA libraries of seedlings and developing seeds. Functional characterization was accomplished by heterologous expression of BnAAPT1 in a yeast strain deficient in AAPT activities. BnAAPT1 exhibited a greater preference for utilizing CDP-choline as a substrate with Vmax of 35 [14C]phosphatidylcholine nmol h(-1) mg(-1) protein and apparent Km of 32 microM while CDP-ethanolamine had a Vmax of 13 [14C]phosphatidylethanolamine nmol h(-1) mg(-1)protein and an apparent Km of 127 microM. The enzyme was activated by Mg2+, Mn2+ and phospholipid mixtures, and inhibited by Ca2+. A CDP-alcohol phosphotransferase motif, Asp99-Gly100-(X2)-Ala103-Arg104-(X8)-Gly113-(X3)-Asp117-(X3)-Asp121, was completely conserved in BnAAPT1 and its catalytic role was confirmed by scanning alanine mutagenesis. Over-expression of BnAAPT1 under the control of the double 35S promoter in transgenic Arabidopsis thaliana (L.) Heynh. plants led to elevated levels of the corresponding transcript and enzyme activity. In four of the high over-expression transgenic lines, phospholipid and fatty acid composition analyses revealed that chloroplastidic and extrachloroplastidic membranes isolated from transgenic leaves had about a 25% increase in phosphatidylcholine and in the proportions of polyunsaturated fatty acids [18:2+18:3], relative to the control. There were also consistent, but small differences observed in the proportions of 18:3 in transgenic green siliques and in 20:1 in mature transgenic seeds of these lines. Induction of Arabidopsis AAPT transcription in response to (+)-abscisic acid and low-temperature treatments, and the cold tolerance in BnAAPT1 transgenic seedlings implies that AAPT may play a role in resistance to damage at low growth temperatures.
Qi
Qungang
Q
Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada.
Huang
Yong-fen
YF
Cutler
Adrian J
AJ
Abrams
Suzanne R
SR
Taylor
David C
DC
D000040:Abscisic Acid / Q000494:pharmacology*; D000222:Adaptation, Physiological / Q000235:genetics; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029688:Brassica napus / Q000201:enzymology* / Q000235:genetics; D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003080:Cold Temperature; D017124:Conserved Sequence / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D002798:Diacylglycerol Cholinephosphotransferase / Q000235:genetics / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D004982:Ethanolaminephosphotransferase / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000032:analysis; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D008055:Lipids / Q000032:analysis; D008969:Molecular Sequence Data; D009154:Mutation; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D005227:Fatty Acids; D008055:Lipids; D010940:Plant Proteins; D000040:Abscisic Acid; D010770:Phosphotransferases; C423366:aminoalcoholphosphotransferase; D004982:Ethanolaminephosphotransferase; D002798:Diacylglycerol Cholinephosphotransferase