Lotus japonicus contains two distinct ENOD40 genes that are expressed in symbiotic, nonsymbiotic, and embryonic tissues.

ENOD40, an early nodulin gene, has been postulated to play a significant role in legume root nodule ontogenesis. We have isolated two distinct ENOD40 genes from Lotus japonicus. The transcribed regions of the two ENOD40 genes share 65% homology, while the two promoters showed no significant homology. Both transcripts encode a putative dodecapeptide similar to that identified in other legumes forming determinate nodules. Both ENOD40 genes are coordinately expressed following inoculation of roots with Mesorhizobium loti or treatment with purified Nod factors. In the former case, mRNA accumulation could be detected up to 10 days following inoculation while in the latter case the accumulation was transient. High levels of both ENOD40 gene transcripts were found in nonsymbiotic tissues such as stems, fully developed flowers, green seed pods, and hypocotyls. A relatively lower level of both transcripts was observed in leaves, roots, and cotyledons. In situ hybridization studies revealed that, in mature nodules, transcripts of both ENOD40 genes accumulate in the nodule vascular system; additionally, in young seed pods strong signal is observed in the ovule, particularly in the phloem and epithelium, as well as in globular stage embryos.

Phaseolus ENOD40 is involved in symbiotic and non-symbiotic organogenetic processes: expression during nodule and lateral root development.

ENOD40 is an early nodulin gene, recently isolated from legume species forming nodules either after Rhizobium infection or spontaneously. ENOD40 cDNAs from Phaseolus plants were isolated and nucleotide sequence determination revealed 85% and 88.5% homology with the reported soybean cDNA clones. The putative polypeptide deduced coincides with the soybean one but a stop codon, almost in the middle of the respective ORF, renders it much shorter. This polypeptide was overexpressed as a fusion protein in Escherichia coli. Although the spatial expression pattern of the gene in the root pericycle and nodule primordium at early stages of development as well as in the pericycle of the vascular bundles and uninfected cells in mature nodules is comparable to the gene's expression pattern in soybean, differences in developmental regulation are evident. We have shown that ENOD40 transcripts are also detected at very early stages of lateral root development, in the dividing pericycle cells of the root stele that give rise to the lateral root primordia. The presence of Rhizobium causes an enhancement of the gene's expression and also induction of the gene in the vascular tissues of developed lateral roots. Interestingly, a discrimination on the gene's expression level in adventious and acropetal incipient lateral root primordia, emerging in infected and uninfected roots, is observed. This indicates that the gene's product may be involved in the hormonal status of the plant and that ENOD40 may be used as a molecular marker in lateral root initiation.

Expression pattern of uricase II gene during root nodule development in Phaseolus vulgaris.

A Phaseolus vulgaris uricase II cDNA clone has been isolated and sequenced. Comparison on the nucleotide level between this clone and the soybean uricase II clone revealed 88.8% identity. The in situ hybridization technique was used to follow the expression pattern in developing root nodules of Phaseolus vulgaris. The uricase II transcripts were localized only in the uninfected cells of the central tissue and mainly in the periphery of the cell. Uricase II mRNA is first detected in nodules 12 days after infection. A maximum level of transcripts is reached in 21-day-old nodules, followed by a considerable reduction in 28-day-old nodules.

A plant regulator controlling development of symbiotic root nodules.

Symbiotic nitrogen-fixing root nodules on legumes are founded by root cortical cells that de-differentiate and restart cell division to establish nodule primordia. Bacterial microsymbionts invade these primordia through infection threads laid down by the plant and, after endocytosis, membrane-enclosed bacteroids occupy cells in the nitrogen-fixing tissue of functional nodules. The bacteria excrete lipochitin oligosaccharides, triggering a developmental process that is controlled by the plant and can be suppressed. Nodule inception initially relies on cell competence in a narrow infection zone located just behind the growing root tip. Older nodules then regulate the number of nodules on a root system by suppressing the development of nodule primordia. To identify the regulatory components that act early in nodule induction, we characterized a transposon-tagged Lotus japonicus mutant, nin (for nodule inception), arrested at the stage of bacterial recognition. We show that nin is required for the formation of infection threads and the initiation of primordia. NIN protein has regional similarity to transcription factors, and the predicted DNA-binding/dimerization domain identifies and typifies a consensus motif conserved in plant proteins with a function in nitrogen-controlled development.