Physiological and genetic changes during natural senescence of Medicago truncatula root nodules

Under nitrogen-limiting conditions, legumes are able to form a symbiotic interaction with bacteria of the Rhizobiaceae family to produce root nodules. These new root organs satisfy plant nitrogen needs by reducing atmospheric nitrogen to ammonium. However, the senescence of these organs disturbs the assimilation of nitrogen. In this study, we present different histological, biochemical, and genetic markers of the natural nodule senescence in Medicago truncatula over a 10-week period following bacterial inoculation. During aging the length and the weight of nodules increased, whereas the nitrogen-fixing capacity of the nodules decreased. The development of the nodule senescence zone correlated with a reduction in leghemoglobin levels without significant reduction in the total protein concentration of the nodule. In contrast, no difference in glutathione and homoglutathione concentration was detected at the onset of senescence at 6 and 8 weeks after bacterial infection. Furthermore, we observed a significant decrease in the relative transcription levels of Nodule Cysteine Rich 001, MtLb1, sucrose synthase 1, thioredoxin S1 and thioredoxin S2 genes, which are involved in nodule development and functioning, thus demonstrating that natural senescence impacts the transcription of genes involved in the expansion and the metabolism of the nitrogen-fixing zone. Finally, the induction of amine oxidase and cysteine protease CP6 transcription was unstable, suggesting that these two genes are related to senescence but are not robust gene markers of the natural senescence process. Considered together, our results define novel biochemical and genetic markers for natural nodule senescence and show that leghemoglobin gene transcription and protein concentration are robust markers that closely correlate with nitrogen fixation efficiency.