Characterization of a plastid triacylglycerol lipase from Arabidopsis.
Full-length cDNA corresponding to Arabidopsis (Arabidopsis thaliana) gene At2g31690, which has been annotated in GenBank as a putative triacylglycerol (TAG) lipase, was obtained by reverse transcription-polymerase chain reaction using RNA from senescing rosette leaves of Arabidopsis as a template. The cognate protein was found to contain the lipase active site sequence, and corresponding recombinant protein proved capable of deesterifying TAG. In vitro chloroplast import assays indicated that the lipase is targeted to chloroplasts. This was confirmed by confocal microscopy of rosette leaf tissue treated with fluorescein isocyanate-labeled, lipase-specific antibody, which revealed that lipase protein colocalizes with plastoglobular neutral lipids. Western-blot analysis indicated that the lipase is expressed in roots, inflorescence stems, flowers, siliques, and leaves and that it is strongly up-regulated in senescing rosette leaf tissue. Transgenic plants with suppressed lipase protein levels were obtained by expressing At2g31690 cDNA in antisense orientation under the regulation of a constitutive promoter. Transgenic plants bolted and flowered at the same time as wild-type plants, but were severely stunted and exhibited delayed rosette senescence. Moreover, the stunted growth phenotype correlated with irregular chloroplast morphology. The chloroplasts of transgenic plants were structurally deformed, had reduced abundance of thylakoids that were abnormally stacked, and contained more plastoglobular neutral lipids than chloroplasts of wild-type plants. These observations collectively indicate that this TAG lipase plays a role in maintaining the structural integrity of chloroplasts, possibly by mobilizing the fatty acids of plastoglobular TAG.
Padham
Anita K
AK
Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
Hopkins
Marianne T
MT
Wang
Tzann-Wei
TW
McNamara
Linda M
LM
Lo
Maisie
M
Richardson
Lynn G L
LG
Smith
Matthew D
MD
Taylor
Catherine A
CA
Thompson
John E
JE
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D016373:DNA, Antisense / Q000378:metabolism; D008049:Lipase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014280:Triglycerides / Q000378:metabolism
D029681:Arabidopsis Proteins; D016373:DNA, Antisense; D014280:Triglycerides; C521254:At2g31690 protein, Arabidopsis; D008049:Lipase