The soybean GmN6L gene encodes a late nodulin expressed in the infected zone of nitrogen-fixing nodules.

Previously, we determined the N-terminal amino acid sequences of a number of putative peribacteroid membrane proteins from soybean. Here, we report the cloning of a gene, GmN6L, that encodes one of these proteins. The protein encoded by GmN6L is similar in sequence to MtN6, an early nodulin expressed in Medicago truncatula roots in response to infection by Sinorhizobium meliloti. The GmN6L gene was strongly expressed in mature nodules but not in other plant organs. GmN6L protein was first detected 2 weeks after inoculation with Bradyrhizobium japonicum and was limited to the infected zone of nodules. GmN6L protein was found in symbiosomes isolated from mature soybean nodules, both as a soluble protein and as a peripheral membrane protein bound to the peribacteroid membrane. These data indicate that GmN6L is a late nodulin, which is not involved in the infection process. Homology between GmN6L and FluG, a protein involved in signaling in Aspergillus nidulans, suggests that GmN6L may play a role in communication between the host and microsymbionts during symbiotic nitrogen fixation.

Novel aspects of symbiotic nitrogen fixation uncovered by transcript profiling with cDNA arrays.

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.

Differentiation of plant cells during symbiotic nitrogen fixation.

Nitrogen-fixing symbioses between legumes and bacteria of the family Rhizobiaceae involve differentiation of both plant and bacterial cells. Differentiation of plant root cells is required to build an organ, the nodule, which can feed and accommodate a large population of bacteria under conditions conducive to nitrogen fixation. An efficient vascular system is built to connect the nodule to the root, which delivers sugars and other nutrients to the nodule and removes the products of nitrogen fixation for use in the rest of the plant. Cells in the outer cortex differentiate to form a barrier to oxygen diffusion into nodules, which helps to produce the micro-aerobic environment necessary for bacterial nitrogenase activity. Cells of the central, infected zone of nodules undergo multiple rounds of endoreduplication, which may be necessary for colonisation by rhizobia and may enable enlargement and greater metabolic activity of these cells. Infected cells of the nodule contain rhizobia within a unique plant membrane called the peribacteroid or symbiosome membrane, which separates the bacteria from the host cell cytoplasm and mediates nutrient and signal exchanges between the partners. Rhizobia also undergo differentiation during nodule development. Not surprisingly, perhaps, differentiation of each partner is dependent upon interactions with the other. High-throughput methods to assay gene transcripts, proteins, and metabolites are now being used to explore further the different aspects of plant and bacterial differentiation. In this review, we highlight recent advances in our understanding of plant cell differentiation during nodulation that have been made, at least in part, using high-throughput methods.

A sucrose transporter, LjSUT4, is up-regulated during Lotus japonicus nodule development.

LjSUT4, encoding a putative sucrose transporter, was identified in a Lotus japonicus nodule cDNA library. The deduced amino acid sequence showed a high degree of identity with sucrose transporters from other plants. Semi-quantitative RT-PCR analysis demonstrated that the L. japonicus SUT4 gene was expressed at high levels in both roots and nodules. In situ hybridization revealed that, in young nodules, SUT4 mRNA transcripts are present in vascular bundles, inner cortex and both infected and uninfected cells while, in mature nodules, accumulation of transcripts was restricted only in vascular bundles and the inner cortex. The results indicated that LjSUT4 codes for a putative sucrose transporter, and its expression pattern suggests a possible shift in the mechanism of sugar transport during nodule development. The role of this polypeptide in sucrose transport and metabolism is discussed.

Global changes in transcription orchestrate metabolic differentiation during symbiotic nitrogen fixation in Lotus japonicus.

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.