Expression and molecular analysis of the Arabidopsis DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway
Expression and molecular analysis of the Arabidopsis DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway
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DOI：
10.1104/pp.003798
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发表时间：
2002-08-01
期刊：
影响因子：
7.4
通讯作者：
Campos, N
中科院分区：
文献类型：
--
作者：
Carretero-Paulet, L;Ahumada, I;Campos, N
1-脱氧-D-木酮糖5-磷酸还原异构酶（DXR）催化类异戊二烯生物合成2-C-甲基-D-赤藓糖醇4-磷酸途径的第一个关键步骤。在拟南芥中，DXR由单拷贝基因编码。我们已经克隆了相应于该基因的全长cDNA。迄今为止已知的所有植物DXR序列的比较分析预测了质体的N-末端转运肽，具有保守的切割位点，并且在成熟蛋白的N末端具有保守的富含脯氨酸的区域，这在原核DXR同系物中不存在。我们证明，拟南芥DXR是针对质体和定位到叶绿体的叶细胞。通过用特异性抗体检测，证实了成熟拟南芥DXR中富含脯氨酸的区域的存在。该蛋白质的酶功能的证据是通过补充DXR活性缺陷的大肠杆菌突变体获得的。在拟南芥转基因植物中，DXR启动子驱动的β-葡萄糖醛酸酶的表达模式，以及DXR转录本和蛋白质的组织分布，揭示了DXR基因的发育和环境调控。DXR基因的表达模式与拟南芥1-脱氧-D-木酮糖5-磷酸合酶基因的表达模式相似，但前者受到的限制稍多。这些基因在植物的大多数器官中表达，包括根，在幼苗和花序中表达水平较高。用磷霉素阻断拟南芥幼苗中的2-C-甲基-D-DX 4-磷酸途径导致DXR蛋白的快速积累，而1-脱氧-D-木酮糖5-磷酸合酶蛋白水平没有改变。我们的研究结果与拟南芥DXR基因参与2-C-甲基-D-D-β 4-磷酸途径的控制一致。
1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) catalyzes the first committed step of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis. In Arabidopsis, DXR is encoded by a single-copy gene. We have cloned a full-length cDNA corresponding to this gene. A comparative analysis of all plant DXR sequences known to date predicted an N-terminal transit peptide for plastids, with a conserved cleavage site, and a conserved proline-rich region at the N terminus of the mature protein, which is not present in the prokaryotic DXR homologs. We demonstrate that Arabidopsis DXR is targeted to plastids and localizes into chloroplasts of leaf cells. The presence of the proline-rich region in the mature Arabidopsis DXR was confirmed by detection with a specific antibody. A proof of the enzymatic function of this protein was obtained by complementation of an Escherichia coli mutant defective in DXR activity. The expression pattern of beta-glucuronidase, driven by the DXR promoter in Arabidopsis transgenic plants, together with the tissue distribution of DXR transcript and protein, revealed developmental and environmental regulation of the DXR gene. The expression pattern of the DXR gene parallels that of the Arabidopsis 1-deoxy-D-xylulose 5-phosphate synthase gene, but the former is slightly more restricted. These genes are expressed in most organs of the plant including roots, with higher levels in seedlings and inflorescences. The block of the 2-C-methyl-D-erythritol 4-phosphate pathway in Arabidopsis seedlings with fosmidomycin led to a rapid accumulation of DXR protein, whereas the 1-deoxy-D-xylulose 5-phosphate synthase protein level was not altered. Our results are consistent with the participation of the Arabidopsis DXR gene in the control of the 2-C-methyl-D-erythritol 4-phosphate pathway.