The role of Lotus japonicus thioesterases in lipid metabolism and carbon exchange with the arbuscular mycorrhizal fungus Rhizophagus irregularis

豆科植物(如莲花、紫花苜蓿)与丛枝菌根真菌(如不规则根腐病菌)发生共生相互作用。真菌提供矿物质，特别是磷酸盐，以换取植物根部的代谢物。以前的实验表明，这种真菌从根中吸收己糖。食根菌的转录组和全基因组序列是可用的。这些测序工作的一个主要结果是，我们后来才认识到，噬根虫缺乏从头合成脂肪酸的基因，因此它不能从乙酰辅酶A中产生脂肪酸。出于这个原因，植物产生的脂类可能作为碳源转移到真菌中。莲花和紫花苜蓿的几个突变体最近被发现，它们的基因可能涉及脂类从根到植物表面或到真菌的转移。我们将集中于编码酰基-ACP硫代酯酶(FAT)的莲花基因，这些基因参与调节植物中脂肪酸的从头合成。系统发育分析表明，莲花含有4个脂肪基因(FATA、FatB、FATC、FatD)。FATC的表达是在菌斑形成过程中诱导的。在本项目中，我们将在大肠杆菌中表达莲花中所有四种硫代酯酶的cDNA，并确定它们的底物特异性。将从插入突变体群体中分离相应的莲花突变体(FATA、FatB、FATC、FATD)，并测定它们在不同器官中的脂肪酸组成。将研究突变体的菌丝化效率，特别是FATC的菌丝效率。我们将测量脂肪前体并进行脂肪标记实验，以跟踪脂肪酸从植物根部到真菌的流量。在合作中，我们将测量其他影响脂质代谢的Medicago突变株的血脂。这些实验将有助于揭示脂类在建立丛枝菌根相互作用中的作用。

Plants of the Fabaceae (e.g. Lotus japonicus, Medicago truncatula) undergo symbiotic interactions with arbuscular mycorrhizal fungi (e.g. Rhizophagus irregularis). The fungus provides minerals, in particular phosphate, in exchange for metabolites from the plant root. Previous experiments suggested that the fungus takes up hexoses from the roots. Transcriptome and full genome sequences are available for Rhizophagus. A major result of these sequencing efforts which was recognized only later by us was the fact that Rhizophagus is devoid of genes for de novo fatty acid synthesis and that it is therefore incapable of producing fatty acids from acetyl-CoA. For this reason, it is possible that the plant produces lipids which are transferred to the fungus as a carbon source. Several mutants of Lotus and Medicago were recently identified which are affected in genes possibly involved in the transfer of lipids from the root to the plant surface or to the fungus. We will focus on Lotus genes encoding acyl-ACP thioesterases (Fat) which are involved in regulating fatty acid de novo synthesis in plants. Phylogenetic analyses revealed that Lotus contains four Fat genes (FatA, FatB, FatC, FatD). Expression of FatC is induced during mycorrhization. In this project, we will express the cDNAs for all four thioesterases from Lotus in Escherichia coli and determine their substrate specificities. The corresponding Lotus mutants (fatA, fatB, fatC, fatD) will be isolated from insertional mutant populations and their fatty acid compositions in different organs will be measured. The mycorrhization efficiency of the mutants, in particular of fatC, will be studied. We will measure lipid precursors and perform lipid labeling experiments to follow the flux of fatty acids from the plant root to the fungus. In collaboration, we will measure lipids in additional Medicago mutant lines affected in lipid metabolism. These experiments will be instrumental to unravel the role of lipids for the establishment of arbuscular mycorrhiza interactions.