拟南芥含hemerythrin结构域的RING型E3泛素连接酶BTS参与铁稳态调控及其分子机制的研究

Iron is an essential micronutrient for both plants and animals. Our previous work screened an Arabidopsis mutant which is more tolerant under iron-deficient conditions. By map-based cloning, we identified the mutant gene is BRUTUS(BTS) and named this mutant as bts-2. In our previous study, we found that bts-2 exhibited severe dwarf and chlorosis in young leaves,although with accumulated high level of Fe in various organs when cultivated in soil in normal conditions. Interestingly, the ultrastructure of chloroplasts and microassay indicated that the chlorosis of leaves in bts-2 was probably caused by the reduced Fe in mesophyll cells rather than oxidative damage. Furthermore, by slicing layers for leaves, Fe was accumulated mainly in the phloem, suggesting the remobilization of Fe from the phloem to the mesophyll cells may be disturbed. On this basis, we hypothesized BTS may play an important role in iron absorbtion and transport. To study the mechanism of BTS in regulating the iron homeostasis, firstly we would like to investigate the iron content in chloroplasts to sudy the mechanism of leaf chlorosis at physiological levels. Furthermore, more work will be needed to investigate its interaction proteins and the possible ubiquitination signal pathway in regulating the iron homeostasis at the molecular level, and the homeostasis of BTS itself under different iron contents via hemerythrin domains. Our work would illuminate the function and mechanism of BTS in maintaining the iron homeostasis in plants.

铁是植物和动物等有机体必不可少的微量元素。我们通过图位克隆，筛选并鉴定到一个拟南芥耐受缺铁突变体bts-2。初步研究发现，bts-2植株虽然过量吸收铁，然而新生叶片却表现为铁缺乏导致的的叶绿体发育异常及黄化，有趣的是，突变体中铁大量堆积于韧皮部，提示铁向新生叶片细胞内的转运异常。鉴于BTS蛋白是一个预测的RING型E3泛素连接酶，并含6个hemerythrin结构域，其可能介导一种新型铁稳态调控机制，值得深入研究。因此，本项目拟先利用遗传学手段及生化检测，验证BTS负调控铁吸收及其突变所导致叶片黄化的生物学机制；通过互作分子的筛选，进而研究可能的泛素化修饰机制在铁稳态调控中的作用；进一步，探讨BTS蛋白自身是否通过hemerythrin结构域的作用受到铁稳态通路的反馈调节。本项目通过对BTS蛋白生理功能及其分子机制的深入研究，将有助于进一步阐明植物铁稳态的调控机制。

铁是植物和动物等有机体必不可少的微量元素，对于植物的正常生长发育及人体生理功能的维持至关重要。植物是人类摄取铁元素的重要食用来源，因此，系统地了解植物的铁吸收运输及其稳态调控机制，对于农作物铁营养价值的改良具有非常重要的意义。本项目的研究目标是在遗传、生理以及分子水平上阐述E3泛素连接酶BTS参与拟南芥铁稳态调控的具体作用机制。我们通过对BTS干涉植株及bts-2突变体的表型分析，发现其表现出明显的缺铁耐受及铁过量的表型，从而确证了BTS负调控拟南芥铁吸收的生物学功能。进一步通过遗传学分析，发现两个IVc亚组bHLH转录因子bHLH104与ILR3可能作为BTS的下游成员，受到BTS负调控。ilr3-2 bts-2及bhlh104-2 bts-2双突植株都能够部分回补bts-2突变体的铁过量吸收的表型。进一步的生理学及生化研究表明，bhlh104突变体与野生型相比，其缺铁条件下根表皮细胞氢质子泵活性及Fe(III)螯合还原酶活性明显降低，提示bHLH104的缺失导致植株铁吸收能力的下降。分析根中铁吸收和转运相关基因的表达谱，发现与野生型相比，bhlh104突变体中铁吸收及运输相关基因的转录本水平显著降低，提示bHLH104可调控一系列铁相关基因的表达。相反，bHLH104过表达植株则相较于野生型表现出明显的耐受缺铁表型，在正常土培条件下，植株各器官均积累过量的铁。此外，bHLH104过表达能够明显增强铁吸收相关基因的表达。这些结果都进一步提示bHLH104是铁吸收的正调控因子。对ILR3的生物学功能分析表明，ILR3同样在铁稳态调控过程中发挥重要功能，其缺失突变体表现出对缺铁环境的敏感，而过表达ILR3则能够显著促进植株对铁的吸收。遗传学功能分析提示ILR3与bHLH104二者协同作用，通过转录因子间的级联调控模式参与拟南芥铁稳态的调控。以上发现对于进一步解析植物的铁稳态调控机制具有重要的理论意义。