Synchronous expression of leghaemoglobin genes inMedicago truncatula during nitrogen-fixing root nodule development and response to exogenously supplied nitrate

Abstract

Two leghaemoglobin genes from the diploid, autogamousMedicago truncatula (Mtlb1 andMtlb2) have been cloned and their nucleotide sequences determined. The deduced amino acid sequences encoded by these two genes differ significantly (18%), confirming that they belong to different sub-groups ofMedicago leghaemoglobin genes [2]. RNAse protection experiments have been used to show that both genes are transcriptionally active, and are expressed specifically in the nitrogen-fixing root nodule ofM. truncatula. WhilstMtlb1 mRNA is present at approximatively 3-fold higher steady-state levels thanMtlb2 mRNA, the transcription of both genes is triggered concomitantly during nodule development (5 days after inoculation withRhizobium meliloti), and the ratio of the steady-state levels of the two mRNA species remains constant throughout nodule maturation. When the growth medium of nodulatedM. truncatula is supplemented with 5 mM KNO₃ over a period of 2–3 days there is a progressive drop in specific nitrogen fixation activity to only 20–25% of the original level. This is accompanied with a parallel and synchronous reduction in the quantities of mRNA corresponding to bothMtlb1 andMtlb2. By contrast, the expression of the nodule parenchyma-specific geneENOD2 is not significantly modified following nitrate treatment, clearly demonstrating differences in tissue-specific gene regulation in response to combined nitrogen.