Ahn Chang Sook, Pai Hyun-Sook
Department of Biology, Yonsei University, Seoul, 120-749, Korea.
Plant Mol Biol. 2008 Mar;66(5):503-17. doi: 10.1007/s11103-007-9286-0. Epub 2008 Jan 8.
Isoprenoid biosynthesis in plants occurs by two independent pathways: the cytosolic mevalonate (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. In this study, we investigated the cellular effects of depletion of IspE, a protein involved in the MEP pathway, using virus-induced gene silencing (VIGS). The IspE gene is preferentially expressed in young tissues, and induced by light and methyl jasmonate. The GFP fusion protein of IspE was targeted to chloroplasts. Reduction of IspE expression by VIGS resulted in a severe leaf yellowing phenotype. At the cellular level, depletion of IspE severely affected chloroplast development, dramatically reducing both the number and size of chloroplasts. Interestingly, mitochondrial development was also impaired, suggesting a possibility that the plastidic MEP pathway contributes to mitochondrial isoprenoid biosynthesis in leaves. A deficiency in IspE activity decreased cellular levels of the metabolites produced by the MEP pathway, such as chlorophylls and carotenoids, and stimulated expression of some of the downstream MEP pathway genes, particularly IspF and IspG. Interestingly, the IspE VIGS lines had significantly increased numbers of cells of reduced size in all leaf layers, compared with TRV control and other VIGS lines for the MEP pathway genes. The increased cell division in the IspE VIGS lines was particularly pronounced in the abaxial epidermal layer, in which the over-proliferated cells bulged out of the plane, making the surface uneven. In addition, trichome numbers dramatically increased and the stomata size varied in the affected tissues. Our results show that IspE deficiency causes novel developmental phenotypes distinct from the phenotypes of other MEP pathway mutants, indicating that IspE may have an additional role in plant development besides its role in isoprenoid biosynthesis.
胞质甲羟戊酸（MVA）途径和质体甲基赤藓糖醇磷酸（MEP）途径。在本研究中，我们利用病毒诱导基因沉默（VIGS）研究了参与MEP途径的IspE蛋白缺失对细胞的影响。IspE基因在幼嫩组织中优先表达，并受光和茉莉酸甲酯诱导。IspE的绿色荧光蛋白融合蛋白定位于叶绿体。通过VIGS降低IspE的表达导致严重的叶片黄化表型。在细胞水平上，IspE的缺失严重影响叶绿体发育，显著减少叶绿体的数量和大小。有趣的是，线粒体发育也受到损害，这表明质体MEP途径可能参与叶片中线粒体类异戊二烯的生物合成。IspE活性的缺乏降低了MEP途径产生的代谢物的细胞水平，如叶绿素和类胡萝卜素，并刺激了一些MEP途径下游基因的表达，特别是IspF和IspG。有趣的是，与TRV对照和MEP途径基因的其他VIGS株系相比，IspE VIGS株系所有叶层中细胞大小减小的细胞数量显著增加。IspE VIGS株系中增加的细胞分裂在叶背表皮层尤为明显，其中过度增殖的细胞从平面突出，使表面不平。此外，受影响组织中的毛状体数量显著增加，气孔大小也有所不同。我们的结果表明，IspE缺乏会导致与其他MEP途径突变体表型不同的新的发育表型，这表明IspE除了在类异戊二烯生物合成中的作用外，可能在植物发育中还具有额外的作用。
