Enzyme inhibitor studies reveal complex control of methyl-D-erythritol 4-phosphate (MEP) pathway enzyme expression in Catharanthus roseus.
Enzyme inhibitor studies reveal complex control of methyl-D-erythritol 4-phosphate (MEP) pathway enzyme expression in Catharanthus roseus.
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DOI：
10.1371/journal.pone.0062467
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Rausch T
中科院分区：
文献类型：
--
作者：
Han M;Heppel SC;Su T;Bogs J;Zu Y;An Z;Rausch T
在长春花中，单萜部分对单萜吲哚生物碱（MIA）的形成具有很强的通量控制作用。单萜的合成依赖于甲基-D-β 4-磷酸（MEP）途径。在这里，我们已经探讨了这一途径的调节响应于发展和环境的线索，并响应于特定的酶抑制剂。对于MEP途径进入酶1-脱氧-D-木酮糖5-磷酸合酶（DXS），一个新的（I型）DXS亚型，CrDXS 1，已被克隆，这与以前的报告II型CrDXS，不转录激活的转录因子ORCA 3。已分别使用酶抑制剂异恶草酮（5-ketochlomazone的前体，DXS的抑制剂）和磷咪霉素（脱氧木酮糖5-磷酸还原异构酶（DXR）的抑制剂）探索了MEP途径对代谢扰动的调节。未开花植物的嫩叶暴露于这两种抑制剂，采用非侵入性体内技术。DXS（3种异构体），DXR和羟甲基丁烯二磷酸合酶（HDS）的转录本和蛋白质进行了监测，并在分离的叶绿体蛋白质稳定性。DXS 1的转录抑制剂，而转录DXS 2A和B，DXR和HDS异恶草酮治疗后增加，但几乎没有受到影响，磷霉素治疗。DXS蛋白的积累响应于这两种抑制剂，而DXR和HDS蛋白的影响较小。磷霉素诱导的DXS蛋白的积累表明大量的转录后调节。此外，磷霉素有效地保护DXR在植物和分离的叶绿体中的降解。因此，我们的研究结果表明，DXR蛋白质的稳定性可能会受到底物结合的影响。总之，本研究结果为DXS在C.玫瑰花响应MEP途径扰动。
In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation.