Padham Anita K, Hopkins Marianne T, Wang Tzann-Wei, McNamara Linda M, Lo Maisie, Richardson Lynn G L, Smith Matthew D, Taylor Catherine A, Thompson John E
Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
Plant Physiol. 2007 Mar;143(3):1372-84. doi: 10.1104/pp.106.090811. Epub 2007 Jan 26.
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.
以拟南芥衰老莲座叶的RNA为模板，通过逆转录-聚合酶链反应获得了与拟南芥（Arabidopsis thaliana）基因At2g31690对应的全长cDNA，该基因在GenBank中被注释为推定的三酰甘油（TAG）脂肪酶。发现同源蛋白含有脂肪酶活性位点序列，相应的重组蛋白被证明能够使TAG脱酯。体外叶绿体导入试验表明该脂肪酶靶向叶绿体。用异硫氰酸荧光素标记的脂肪酶特异性抗体处理莲座叶组织的共聚焦显微镜证实了这一点，结果显示脂肪酶蛋白与质体球中性脂质共定位。蛋白质免疫印迹分析表明，该脂肪酶在根、花序茎、花、角果和叶中表达，并且在衰老的莲座叶组织中强烈上调。通过在组成型启动子的调控下以反义方向表达At2g31690 cDNA，获得了脂肪酶蛋白水平受到抑制的转基因植物。转基因植物与野生型植物同时抽薹开花，但严重发育不良并表现出莲座叶衰老延迟。此外，发育不良的生长表型与叶绿体形态不规则相关。转基因植物的叶绿体结构变形，类囊体丰度降低且异常堆叠，并且比野生型植物的叶绿体含有更多的质体球中性脂质。这些观察结果共同表明，这种TAG脂肪酶可能通过动员质体球TAG的脂肪酸在维持叶绿体的结构完整性中发挥作用。
