Data_Sheet_1_Differential Regulation of an OsIspH1, the Functional 4-Hydroxy-3-Methylbut-2-Enyl Diphosphate Reductase, for Photosynthetic Pigment Biosynthesis in Rice Leaves and Seeds.xlsx

The methylerythritol 4-phosphate (MEP) pathway is responsible for providing common precursors for the biosynthesis of diverse plastidial terpenoids, including chlorophylls, carotenoids, and phytohormones, in plants. In rice (Oryza sativa), the last-step genes encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase [HDR/isoprenoid synthesis H (IspH)] have been annotated in two genes (OsIspH1 and OsIspH2) in the rice genome. The spatial transcript levels indicated that OsIspH1 is highly expressed in all tissues at different developmental stages, whereas OsIspH2 is barely expressed due to an early stop in exon 1 caused by splicing error. OsIspH1 localized into plastids and osisph1, a T-DNA inserted knockout mutant, showed an albino phenotype, indicating that OsIspH1 is the only functional gene. To elucidate the role of OsIspH1 in the MEP pathway, we created two single (H145P and K407R) and double (H145P/K407R) mutations and performed complementation tests in two hdr mutants, including Escherichia coli DLYT1 strains and osisph1 rice plants. The results showed that every single mutation retained HDR function, but a double mutation lost it, proposing that the complementary relations of two residues might be important for enzyme activity but not each residue. When overexpressed in rice plants, the double-mutated gene, OsIspH1MUT, reduced chlorophyll and carotenoid biosynthesis in the leaves and seeds. It confirmed the crucial role of OsIspH1 in plastidic terpenoid biosynthesis, revealing organ-specific differential regulation of OsIspH1 in rice plants.

甲基赤藓糖醇4-磷酸（methylerythritol 4-phosphate, MEP）通路负责为植物体内多种质体类萜类化合物的生物合成提供共同前体，此类化合物涵盖叶绿素、类胡萝卜素与植物激素。在水稻（Oryza sativa）基因组中，编码4-羟基-3-甲基-2-丁烯基二磷酸还原酶[HDR/异戊二烯合成H（isoprenoid synthesis H, IspH）]的通路末步基因已被注释为两个拷贝，分别为OsIspH1与OsIspH2。组织转录水平检测结果显示，OsIspH1在水稻不同发育阶段的所有组织中均呈高表达；而OsIspH2几乎不表达，原因是其第1外显子因剪接错误产生了提前终止密码子。OsIspH1定位于质体；T-DNA插入敲除突变体osisph1呈现白化表型，这表明OsIspH1是唯一具备功能的基因拷贝。为阐明OsIspH1在MEP通路中的功能，我们构建了两个单点位突变体（H145P与K407R）与一个双点位突变体（H145P/K407R），并分别在两种hdr突变体中开展互补实验——这两种突变体分别为大肠杆菌DLYT1菌株与水稻osisph1植株。实验结果显示，两个单点位突变体均保留了HDR的酶活性，而双点位突变体则丧失了该功能；这提示两个氨基酸残基间的协同作用对酶活性至关重要，而单个残基的改变并不足以影响酶活。将双点位突变基因OsIspH1MUT在水稻植株中过表达后，叶片与种子中的叶绿素及类胡萝卜素生物合成水平均显著下降。该结果证实了OsIspH1在质体类萜类化合物生物合成中的关键作用，并揭示了水稻中OsIspH1存在器官特异性差异调控机制。