Functional Characterization of the Arabidopsis β-Ketoacyl-Coenzyme A Reductase Candidates of the Fatty Acid Elongase
Functional Characterization of the Arabidopsis β-Ketoacyl-Coenzyme A Reductase Candidates of the Fatty Acid Elongase
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DOI：
10.1104/pp.109.137497
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发表时间：
2009-07-01
期刊：
影响因子：
7.4
通讯作者：
Kunst, Ljerka
中科院分区：
文献类型：
--
作者：
Beaudoin, Frederic;Wu, Xianzhong;Kunst, Ljerka
在植物中，超长链脂肪酸(VLCFAs；>；18碳)是鞘脂、三酰甘油、角质蜡和黄油的前体。VLCFA是由多蛋白膜结合的脂肪酸延长系统合成的，该系统催化四个连续的酶反应：缩合、还原、脱水和二次还原。对拟南芥基因组的生物信息学研究发现，有两个序列与YBR159w编码酿酒酵母β-酮酰基还原酶(KCR)同源，KCR在VLCFA延伸过程中催化第一次还原。表达分析表明，AtKCR1和AtKCR2基因在角果、花、花序茎、叶和发育中的胚胎中都有转录，但在根中只有AtKCR1的转录本。荧光蛋白标记的AtKCR1和AtKCR2定位于内质网，即脂肪酸伸长的部位。酵母ybr159 Delta突变体的互补实验表明，这两种KCR蛋白是不同的，只有AtKCR1能够恢复与天然酵母KCR基因相似的异源延伸酶活性。对AtKCR1和AtKCR2插入突变体的分析表明，AtKCR1功能的丧失会导致胚胎死亡，这不能通过AtKCR1启动子的AtKCR2表达来挽救。相反，AtKCR2基因的破坏没有明显的表型效应。综上所述，这些结果表明，只有AtKCR1是参与微粒体脂肪酸伸长的功能性KCR亚型。为了研究AtKCR1在胚胎后发育中的作用，建立了针对AtKCR1的RNA干扰和过表达的转基因株系。这些品系的形态和生化特征证实，抑制的KCR活性导致角质层蜡质负荷的减少，并影响神经鞘脂脂、种子三酰甘油和根部甘油脂的VLCFA组成，这在植物中证明了KCR参与了为所有这些不同类别的脂类提供VLCFA的延伸反应。
In plants, very-long-chain fatty acids (VLCFAs; >18 carbon) are precursors of sphingolipids, triacylglycerols, cuticular waxes, and suberin. VLCFAs are synthesized by a multiprotein membrane-bound fatty acid elongation system that catalyzes four successive enzymatic reactions: condensation, reduction, dehydration, and a second reduction. A bioinformatics survey of the Arabidopsis (Arabidopsis thaliana) genome has revealed two sequences homologous to YBR159w encoding a Saccharomyces cerevisiae beta-ketoacyl reductase (KCR), which catalyzes the first reduction during VLCFA elongation. Expression analyses showed that both AtKCR1 and AtKCR2 genes were transcribed in siliques, flowers, inflorescence stems, leaves, as well as developing embryos, but only AtKCR1 transcript was detected in roots. Fluorescent protein-tagged AtKCR1 and AtKCR2 were localized to the endoplasmic reticulum, the site of fatty acid elongation. Complementation of the yeast ybr159 Delta mutant demonstrated that the two KCR proteins are divergent and that only AtKCR1 can restore heterologous elongase activity similar to the native yeast KCR gene. Analyses of insertional mutants in AtKCR1 and AtKCR2 revealed that loss of AtKCR1 function results in embryo lethality, which cannot be rescued by AtKCR2 expression using the AtKCR1 promoter. In contrast, a disruption of the AtKCR2 gene had no obvious phenotypic effect. Taken together, these results indicate that only AtKCR1 is a functional KCR isoform involved in microsomal fatty acid elongation. To investigate the roles of AtKCR1 in postembryonic development, transgenic lines expressing RNA interference and overexpression constructs targeted against AtKCR1 were generated. Morphological and biochemical characterization of these lines confirmed that suppressed KCR activity results in a reduction of cuticular wax load and affects VLCFA composition of sphingolipids, seed triacylglycerols, and root glycerolipids, demonstrating in planta that KCR is involved in elongation reactions supplying VLCFA for all these diverse classes of lipids.