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Symbiotic nitrogen fixation (SNF) in legume nodules is a highly energy demanding process, fuelled by plant-supplied carbohydrates mainly in the form of sucrose. In this study, we have combined molecular and biochemical approaches in order to study the spatial and temporal organisation of sucrose metabolism in nitrogen-fixing nodules of the model legume Lotus japonicus, with an emphasis on the neglected role of alkaline/neutral invertase. For this purpose, a full-length cDNA clone coding for an alkaline/neutral invertase isoform, termed LjInv1, was identified in a L. japonicus mature nodule cDNA libraries. Alkaline/neutral invertase activity was also found to be the predominant invertase activity in mature nodules. Real-time reverse-transcription polymerase chain reaction analysis was used in order to study the temporal expression patterns of LjInv1 in parallel with genes encoding acid invertase and sucrose synthase (SuSy) isoforms, and enzymes involved in the subsequent hexose partitioning including hexokinase, phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI). The spatial organisation of sucrose metabolism was studied by in situ localisation of LjInv1 transcripts and alkaline/neutral invertase activity, and SuSy protein during nodule development. Furthermore, the spatial organisation of hexose metabolism was investigated by histochemical localisation of hexokinase, PGM and PGI activities in mature nodules. The results considered together indicate that alkaline/neutral invertase could contribute to both the Glc-1-P and Glc-6-P pools in nodules, fuelling both biosynthetic processes and SNF. Furthermore, transcript profiling analysis revealed that genes coding for hexokinase and putative plastidic PGM and PGI isoforms are upregulated during the early stages of nodule development, while the levels of transcripts corresponding to cytosolic PGM and PGI isoforms remained similar to uninfected roots, indicating a possible role of LjInv1 in producing hexoses for starch production and other biosynthetic processes in developing nodules.

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The biosynthesis of the polyamines spermidine (Spd) and spermine (Spm) from putrescine (Put) is catalysed by the consequent action of two aminopropyltransferases, spermidine synthase (SPDS EC: 2.5.1.16) and spermine synthase (SPMS EC: 2.5.1.22). Two cDNA clones coding for SPDS and SPMS homologues in the nitrogen-fixing nodules of the model legume Lotus japonicus were identified. Functionality of the encoded polypeptides was confirmed by their ability to complement spermidine and spermine deficiencies in yeast. The temporal and spatial expression pattern of the respective genes was correlated with the accumulation of total polyamines in symbiotic and non-symbiotic organs. Expression of both genes was maximal at early stages of nodule development, while at later stages the levels of both transcripts declined. Both genes were expressed in nodule inner cortical cells, vascular bundles, and central tissue. In contrast to gene expression, increasing amounts of Put, Spd, and Spm were found to accumulate during nodule development and after maturity. Interestingly, nodulated plants exhibited systemic changes in both LjSPDS and LjSPMS transcript levels and polyamine content in roots, stem and leaves, in comparison to uninoculated plants. These results give new insights into the neglected role of polyamines during nodule development and symbiotic nitrogen fixation (SNF).

Plant ammonium transporters of the AMT1 family are involved in N-uptake from the soil and ammonium transport, and recycling within the plant. Although AMT1 genes are known to be expressed in nitrogen-fixing nodules of legumes, their precise roles in this specialized organ remain unknown. We have taken a reverse-genetic approach to decipher the physiological role of LjAMT1;1 in Lotus japonicus nodules. LjAMT1;1 is normally expressed in both the infected zone and the vascular tissue of Lotus nodules. Inhibition of LjAMT1;1 gene expression, using an antisense gene construct driven by a leghemoglobin promoter resulted in a substantial reduction of LjAMT1;1 transcript in the infected tissue but not the vascular bundles of transgenic plants. As a result, the nitrogen-fixing activity of nodules was partially impaired and nodule number increased compared to control plants. Expression of LjAMT1;1-GFP fusion protein in plant cells indicated a plasma-membrane location for the LjAMT1;1 protein.Taken together, the results are consistent with a role of LjAMT1;1 in retaining ammonium derived from symbiotic nitrogen fixation in plant cells prior to its assimilation.

The cis/trans isomerization of the peptide bond preceding proline is an intrinsically slow process, although important in many biological processes in both prokaryotes and eukaryotes. In vivo, this isomerization is catalyzed by peptidyl-prolyl cis/transisomerases (PPIases). Here, we present the molecular and biochemical characterization of parvulin-type PPIase family members of the model legume Lotus japonicus, annotated as LjPar1, LjPar2, and LjPar3. Although LjPar1 and LjPar2 were found to be homologous to PIN1 (Protein Interacting with NIMA)-type parvulins and hPar14 from human, respectively, LjPar3 represents a novel multidomain parvulin, apparently present only in plants, that contains an active carboxyl-terminal sulfurtransferase domain. All Lotus parvulins were heterologously expressed and purified from Escherichia coli, and purified protein verification measurements used a liquid chromatography-mass spectrometry-based proteomic method. The biochemical characterization of the recombinant Lotus parvulins revealed that they possess PPIase activity toward synthetic tetrapeptides, although they exhibited different substrate specificities depending on the amino acid amino terminal to proline. These differences were also studied in a structural context using molecular modeling of the encoded polypeptides. Real-time reverse transcription-polymerase chain reaction revealed that the three parvulin genes of Lotus are ubiquitously expressed in all plant organs. LjPar1 was found to be up-regulated during the later stages of nodule development. Subcellular localization of LjPar-enhanced Yellow Fluorescence Protein (eYFP) fusions expressed in Arabidopsis (Arabidopsis thaliana) leaf epidermal cells revealed that LjPar1-and LjPar2-eYFP fusions were localized in the cytoplasm and in the nucleus, in contrast to LjPar3-eYFP, which was clearly localized in plastids. Divergent substrate specificities, expression profiles, and subcellular localization indicate that plant parvulin-type PPIases are probably involved in a wide range of biochemical and physiological processes.

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