Pea Late Nodulin Genes Characterized By A Signal Peptide And Conserved Cysteine Residues

T. Kato1, N. Suganuma1, K. Kawashima1, M. Miwa1, Y. Mimura1, M. Tamaoki1, H. Kouchi2

'Dept Life Science, Aichi University of Education, Kariya, Aichi 448-8542, Japan 2Dept Appl. Physiology, Nat'l Inst. Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan

1. Introduction

The legume-Rhizobium symbiosis is established by coordinated expression of genes of both partners. Dozens of plant genes, which are exclusively expressed in nodules and termed nodulin genes, have been isolated and are presumed to be involved in symbiosis. A few, such as leghemoglobin (Lb), uricase and glutamine synthetase, have been characterized, though functions of most nodulin genes remain unclear. We previously isolated a set of nodulin genes from pea (Pisum sativum) and compared their expression in effective Sparkle nodules and ineffective nodules induced on the pea mutant E135 (syml3) (Suganuma et al. 1995). The E135 mutant forms ineffective nodules in which nitrogenase proteins are synthesized, but lack activity. In this type of nodule, most nodulin genes are expressed. However, expression of six of them is decreased in the El 35 nodules, indicating that the functions of those genes may be related to nitrogen fixation. One of them, PsN5, is the Lb gene, and further characterization showed that this type of Lb has a higher oxygen-binding affinity and is expressed in the central tissue of the nodules (Kawashima et al. 2001). These results suggested that this Lb is more effective for nitrogen fixation. Here, we investigated the primary structures of the remaining five nodulin genes whose expression is reduced in El 35 nodules, PsNl, PsN6, PsN314, PsN335 and PsN466, and their temporal and spatial expression patterns during nodule development.

2. Results and Discussion

Determination of the nucleotide sequences showed their deduced amino acid sequences were homologous to the early nodulins PsENOD3/14 and the late nodulin PsNOD6. They all encoded small polypeptides containing a putative signal peptide and two pairs of cysteine clusters, such as Cys-X5-Cys and Cys-X4-Cys. Hydropathy analysis showed that the amino N-terminal regions are hydrophobic. A small gene family encoding small polypeptides of late nodulins containing a putative signal peptide and two pairs of cysteine clusters has been recently reported in Vicia faba (Frühling et al. 2000). Five nodulin genes characterized in this study appeared to belong to the same gene family. Multiple bands were detected in each gene by genomic Southern analysis. Northern analysis showed that they all were exclusively expressed in nodules and were in a similar way to the Lb gene during nodule development. They were expressed from the interzone II-III to the distal part of nitrogen-fixing zone in effective nodules, like the Lb gene. However, in ineffective El35 nodules, their expression regions were narrower than those in effective nodules. These results indicated that these nodulins are abundant in nodules and that their continuous expression during nodule development is closely associated with nitrogen-fixing activity.

3. References

Frühling M et al. (2000) Plant Sei. 152, 67-77 Kawashima K et al. (2001) Plant Physiol. 125, 641-651 Suganuma N et al. (1995) Plant Mol. Biol. 28, 1027-1038

4. Acknowledgements

This work was supported by Special Coordination Funds of the Science and Technology Agency of the Japanese government.

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