Identification of two groups of leghemoglobin genes in alfalfa (Medicago sativa) and a study of their expression during root nodule development

Abstract

Differential screening of an alfalfa root nodule cDNA library with either root or nodule mRNA resulted in the isolation of two groups of leghemoglobin cDNA which differ significantly in sequence. Analysis of one member of each group revealed a divergence within the coding region of 15% at the nucleotide level and 14% at the amino acid level. The 3' non-coding sequences are 25% divergent but are highly conserved over a stretch of 54 nucleotides which contains two sequence motifs common to leghemoglobin genes from other plant species. Southern blotting analysis with exon-specific probes has shown that there are approximately twice as many leghemoglobin gene copies in the alfalfa genome corresponding to one type of cDNA as compared with the other. Using the same criterium of DNA sequence relatedness these two distinct groups of leghemoglobin genes have also been identified in the genomes of the diploid annual Medicago truncatula and the closely related genus, Melilotus. Transcripts corresponding to both groups of leghemoglobin genes are first detected in alfalfa nodules 9-10 days after Rhizobium inoculation. Thereafter, mRNA levels increase rapidly and synchronously, reaching a maximum approximately 2 days later. There is a 2-3 fold difference in the steady-state levels of the two mRNA populations and this is maintained throughout the subsequent two weeks of nodule growth. The absence of any detectable transcription during the early stages of nodule development and the apparent co-ordinate expression of leghemoglobin genes in alfalfa contrasts with the situation in soybean and suggests that important differences in leghemoglobin gene regulation exist between these two distantly related legume species.

A possible explanation for the multiple polyadenylation sites in transcripts coding for a winged-bean leghemoglobin

“…Multiple polyadenylation sites were reported only in one case (two sites, Barker et al 1988), but this variation could not be explained by our model. Interestingly, the Lupinus LB2 leghemoglobin mRNA has an unusually long 3'end (291 nucleotides).…”

“…Multiple polyadenylation sites of specific mRNAs have often been described for both animals and plants (Sasavage et al 1982;LeMeur et al 1984;Dean et al 1986;Barker et al 1988), and this feature might be underestimated. The overall process of mRNA polyadenylation seems to be similar in plants and animals, but the mechanisms and apparatus are not identical (Hunt et al 1987;Joshi 1987).…”

Rhizobium meliloti elicits transient expression of the early nodulin gene ENOD12 in the differentiating root epidermis of transgenic alfalfa.

“…The isolation and purification of high molecular weight genomic DNA from leaves of M. truncatula cv Jemalong, M. sativa cv Gemini, and Pisum sativum cv Rondo were carried out as described in Barker et al (1988). Total RNA was extracted from M. truncatula nodules harvested at various times after inoculation with R. meliloti according to Lullien et al (1987).…”

Sequence analysis of alfalfa (Medicago sativa) leghemoglobin cDNA and genomic clones

“…As a first step toward understanding the structure, organization and regulation of the alfalfa Lb gene family, the DNA sequence of 7 new cDNA and 2 genomic clones has been determined by the method of Sanger et al [ 19]. These sequences are compared with each other with 4 previously published CDNA sequences [ 1,2,9].The alignment of nucleotide sequences shown in Fig. 1 facilitates the comparison of the coding and 3' untranslated regions of all known alfalfa Lb clones.…”