百脉根血红素加氧酶LjHO1维持根瘤高效固氮的分子机制研究

As the prosthetic group or cofactor of leghemoglobins and other functional proteins, heme biosynthesis is dramatically enhanced to stimulate nitrogen fixation in legume nodules. However, misaccumulation of free heme can lead to cellular damage and inhibit nitrogen fixation activity. Therefore, coordinated synthesis and degradation of heme is essential to efficient symbiotic nitrogen fixation in nodules. In plants, heme oxygenases decompose heme and release iron. The model legume Lotus japonicus encodes two HOs in the nuclear genome, but only LjHO1 contains the conserved amino acid essential for heme degradation. LjHO1 was specifically expressed in uninfected cells of nodule. However, the molecular mechanism of LjHO1 in degrading infected-cells-derived heme and in maintaining nitrogen fixation efficiency is largely unknown.. In this project, we utilize the ho1 mutant and compare the phenotypic differences of nitrogen fixation by cell-specific expression of LjHO1 in genetically complemented lines. Biochemical approaches will be carried out to quantify the distribution patterns of heme and iron in both infected and uninfected cells. By combining cell isolation/separation with transcriptome sequencing, key genes involved in heme iron recycling in nodules will be identified and further characterized. Abiotic stress treatments are also performed to compare nitrogen fixation efficiency in wild type and the ho1 mutant nodules and to validate the contribution of heme degradation products (i.e., biliverdin) to abiotic stress responses in nodules. Those proposed experiments will provide a systematic view of the functional diversity of LjHO1 in nodules. . The implementation of this project can provide novel insights into the intercellular transport of heme from infected cells to uninfected cells. It is also expected to reveal the biological significance of heme degradation within uninfected cells pertaining to efficient nodule nitrogen fixation.

豆科植物根瘤中大量合成血红素，作为豆血红蛋白等功能蛋白的辅基参与固氮。血红素的异常积累会破坏细胞结构并抑制固氮活性，因此血红素的降解对根瘤固氮至关重要。血红素加氧酶(HO)分解血红素并释放铁，百脉根编码两个HOs，仅LjHO1含有代谢血红素的保守氨基酸。LjHO1在根瘤非侵染细胞中特异表达，但该蛋白降解侵染细胞中血红素并维持根瘤固氮效率的分子机制很不清楚。本项目利用获得的ho1突变体，通过遗传互补比较LjHO1细胞特异性表达对固氮活性的影响；利用生化技术鉴定血红素及铁离子在侵染细胞和非侵染细胞中分布的差异；分离两类细胞并结合转录组测序鉴定根瘤中参与血红素铁循环的基因网络；非生物胁迫处理并比较突变体固氮活性的变化及血红素代谢产物在根瘤逆境胁迫响应中的功能，从而系统了解LjHO1在根瘤中功能的多样性。本项目的实施可以阐明血红素的细胞间转运并揭示非侵染细胞中血红素降解对根瘤高效固氮的重要意义。

豆科植物与根瘤菌形成的共生固氮系统是最为高效的生物固氮的方式，在固氮根瘤中，豆血红蛋白是最为丰富的一类蛋白，其结合的血红素与氧气可逆结合为固氮酶发挥正常功能提供了必要的低氧环境。迄今仍然缺乏对血红素分子在根瘤中的转运途径、代谢通路、信号传递等过程的系统理解。本项目以豆科植物百脉根为研究对象，阐明质体中血红素小分子合成在转录后水平的系统调控途径，并鉴定到GluTR, FLU等关键调控因子；发现百脉根中存在两种血红素加氧酶LjHO1和LjHO2，其中仅LjHO1具有降解血红素的酶学活性；该基因在成熟根瘤及根瘤衰老过程中表达显著增强，蛋白定位于非侵染细胞的质体中；ho1突变体根瘤固氮活性下降，血红素降解及胆绿素的积累受到显著抑制，并且突变体中活性氧含量显著增加，黑暗及高硝酸盐等环境胁迫条件下突变体根瘤固氮活性相比野生型根瘤下降更为明显，这些结果表明LjHO1通过降解血红素维持了根瘤的高效固氮。本项目另外优化了针对共生根瘤中植物基因组及根瘤菌基因敲除的技术体系，通过显微操作分离根瘤中的侵染细胞和非侵染细胞并进行单细胞测序，鉴定到多个差异表达基因，这些基因大部分为本研究首次鉴定发现，包括相关转录因子及转运蛋白。对这些基因的RNAi抑制及共生表型分析发现，多个基因抑制后表现为结瘤数下降、固氮活性显著降低甚至形成白瘤或绿瘤表型。这些基因的鉴定为深入研究根瘤中信号转导、血红素及其他代谢物的跨膜转运及稳态调控机制等奠定了基础。