Research on legume nodule metabolism has contributed greatly to our knowledge of primary carbon and nitrogen metabolism in plants in general, and in symbiotic nitrogen fixation in particular. However, most previous studies focused on one or a few genes/enzymes involved in selected metabolic pathways in many different legume species. We utilized the tools of transcriptomics and metabolomics to obtain an unprecedented overview of the metabolic differentiation that results from nodule development in the model legume, Lotus japonicus. Using an array of more than 5000 nodule cDNA clones, representing 2500 different genes, we identified approximately 860 genes that were more highly expressed in nodules than in roots. One-third of these are involved in metabolism and transport, and over 100 encode proteins that are likely to be involved in signalling, or regulation of gene expression at the transcriptional or post-transcriptional level. Several metabolic pathways appeared to be co-ordinately upregulated in nodules, including glycolysis, CO(2) fixation, amino acid biosynthesis, and purine, haem, and redox metabolism. Insight into the physiological conditions that prevail within nodules was obtained from specific sets of induced genes. In addition to the expected signs of hypoxia, numerous indications were obtained that nodule cells also experience P-limitation and osmotic stress. Several potential regulators of these stress responses were identified. Metabolite profiling by gas chromatography coupled to mass spectrometry revealed a distinct metabolic phenotype for nodules that reflected the global changes in metabolism inferred from transcriptome analysis.

Gene expression in response to Fe deficiency was analyzed in Arabidopsis roots and shoots through the use of a cDNA collection representing at least 6,000 individual gene sequences. Arabidopsis seedlings were grown 1, 3, and 7 d in the absence of Fe, and gene expression in roots and shoots was investigated. Following confirmation of data and normalization methods, expression of several sequences encoding enzymes known to be affected by Fe deficiency was investigated by microarray analysis. Confirmation of literature reports, particularly for changes in enzyme activity, was not always possible, but changes in gene expression could be confirmed. An expression analysis of genes in glycolysis, the tricarboxylic acid cycle, and oxidative pentose phosphate pathway revealed an induction of several enzymes within 3 d of Fe-deficient growth, indicating an increase in respiration in response to Fe deficiency. In roots, transcription of sequences corresponding to enzymes of anaerobic respiration was also induced, whereas in shoots, the induction of several genes in gluconeogenesis, starch degradation, and phloem loading was observed. Thus, it seemed likely that the energy demand in roots required for the Fe deficiency response exceeded the capacity of oxidative phosphorylation, and an increase in carbon import and anaerobic respiration were required to maintain metabolism.

An array of 2,304 cDNA clones derived from nitrogen-fixing nodules of Lotus japonicus was produced and used to detect differences in relative gene transcript abundance between nodules and uninfected roots. Transcripts of 83 different genes were found to be more abundant in nodules than in roots. More than 50 of these have never before been identified as nodule-induced in any species. Expression of 36 genes was detected in nodules but not in roots. Several known nodulin genes were included among the nodule-induced genes. Also included were genes involved in sucrose breakdown and glycolysis, CO2 recycling, and amino acid synthesis, processes that are known to be accelerated in nodules compared with roots. Genes involved in membrane transport, hormone metabolism, cell wall and protein synthesis, and signal transduction and regulation of transcription were also induced in nodules. Genes that may subvert normal plant defense responses, including two encoding enzymes involved in detoxification of active oxygen species and one that may prohibit phytoalexin synthesis, were also identified. The data represent a rich source of information for hypothesis building and future exploration of symbiotic nitrogen fixation.

Background: The information theoretic concept of mutual information provides a general framework to evaluate dependencies between variables. In the context of the clustering of genes with similar patterns of expression it has been suggested as a general quantity of similarity to extend commonly used linear measures. Since mutual information is defined in terms of discrete variables, its application to continuous data requires the use of binning procedures, which can lead to significant numerical errors for datasets of small or moderate size.