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Homocitrate is a component of the iron-molybdenum cofactor in nitrogenase, where nitrogen fixation occurs. NifV, which encodes homocitrate synthase (HCS), has been identified from various diazotrophs but is not present in most rhizobial species that perform efficient nitrogen fixation only in symbiotic association with legumes. Here we show that the FEN1 gene of a model legume, Lotus japonicus, overcomes the lack of NifV in rhizobia for symbiotic nitrogen fixation. A Fix(-) (non-fixing) plant mutant, fen1, forms morphologically normal but ineffective nodules. The causal gene, FEN1, was shown to encode HCS by its ability to complement a HCS-defective mutant of Saccharomyces cerevisiae. Homocitrate was present abundantly in wild-type nodules but was absent from ineffective fen1 nodules. Inoculation with Mesorhizobium loti carrying FEN1 or Azotobacter vinelandii NifV rescued the defect in nitrogen-fixing activity of the fen1 nodules. Exogenous supply of homocitrate also recovered the nitrogen-fixing activity of the fen1 nodules through de novo nitrogenase synthesis in the rhizobial bacteroids. These results indicate that homocitrate derived from the host plant cells is essential for the efficient and continuing synthesis of the nitrogenase system in endosymbionts, and thus provide a molecular basis for the complementary and indispensable partnership between legumes and rhizobia in symbiotic nitrogen fixation.

The first kinetically stabilized germanethiones (germanium-sulfur double-bond species) and germaneselones (germanium-selenium double-bond species) were synthesized. Dechalcogenation of the novel 1,2,3,4,5-tetrachalcogenagermolane Tbt(Tip)GeX 4 (Tbt ) 2,4,6-tris[bis(trimethylsilyl)methyl]phenyl, Tip ) 2,4,6-triisopropylphenyl, X ) S or Se) with 3 molar equiv of triphenylphosphine gave diaryl-substituted germanethione Tbt(Tip)GedS 1a and germaneselone Tbt(Tip)GedSe 2a as orange-yellow crystals and red crystals, respectively, which were unstable in air but thermally quite stable. Chalcogenation of germylene Tbt(Dis)Ge: resulted in the isolation of the corresponding germanethione Tbt(Dis)GedS 1b and germaneselone Tbt(Dis)GedSe 2b (Dis ) bis(trimethylsilyl)methyl). The molecular structures of germanethione 1a and germaneselones 2a,b determined by X-ray crystallographic analysis indicate that they have trigonal planar geometies around the germanium atom and a remarkably shorter Ge-S (2.049(3) Å for 1a) or Ge-Se bond lengths (2.180(2) Å for 2a and 2.173(3) Å for 2b), compared with that of typical single bonds. The doublebond characters are also reflected in their spectral data such as UV/vis (1a, 450 nm; 1b, 432 nm; 2a, 519 nm; 2b, 492 nm), Raman (1a, 521 cm -1 ; 1b, 512 cm -1 ; 2a, 381 cm -1 ; 2b, 386 cm -1 ), and 77 Se NMR (2a, 941 ppm; 2b, 872 ppm). The germanethiones and germaneselones underwent [2 + 2] (with phenyl isothiocyanate), [3 + 2] (with mesitonitrile oxide), and [4 + 2] cycloadditions (with 1,3-dienes) to give the corresponding adducts in high yields. The regioselectivities of the [4 + 2] cycloadditions were examined using unsymmetrical dienes such as isoprene and interpreted in terms of the interaction between LUMO of the GedX unit and HOMO of the diene part.

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