The pattern of leghaemoglobin (Lb) gene expression within the indeterminate nitrogen-fixing root nodule of Medicago species such as alfalfa has been examined by means of ‘in-situ’ hybridization. Hybridization of alfalfa nodule sections with radiolabelled Lb RNA probes clearly shows that Lb gene expression is triggered in a single cell layer just prior to the start of the nitrogen fixation zone. This cell layer is part of a unique region (interzone II-III) in which invaded cells are rich in amyloplasts. Lb transcripts can only be detected in cells which have been invaded by the microsymbiont Rhizobium. The conclusion that Lb gene activation precedes the start of nitrogen fixation is further supported by the observation that Lb transcripts are already present in immature non-nitrogen-fixing nodules of alfalfa. The addition of 5 mM KNO3 to the growth medium of nodulated M. truncatula (a diploid autogamous species of Medicago) results in a significant drop in both nitrogen-fixing activity and nodular Lb mRNA levels. Short periods of exposure to nitrate (24 h) lead to the disappearance of Lb transcripts from the proximal region of the hybridizing zone, whilst longer exposures (72 h) lead to a marked decrease in the intensity of the hybridization signal within the amyloplast-rich interzonal region.
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