Development of the reproductive population of nitrogen-fixing symbiotic bacteria during growth and senescence of ‘indeterminate’ legume root nodules

The spatial and temporal growth dynamics of the reproductive population of N2-fixing rhizobial symbionts within indeterminate root nodules is poorly understood, yet this process is essential for rhizobia to benefit from symbiosis and to recolonize soil after nodule senescence. Legumes that form indeterminate nodules, such as Medicago sativa (alfalfa) have a continuously-growing meristem at the nodule tip, which contains the youngest tissue. As these nodules develop, the plant imposes terminal differentiation on the N2-fixing population of the symbiont Sinorhizobium meliloti (Sm1021) while an actively-dividing population grows in the ‘infection zone’ just behind the meristem. Therefore, unlike symbionts of determinate-nodulating legumes, for example soybean, the portion of the alfalfa Sm1021 population that engages in N2-fixation cannot resume cell division and repopulate the soil. Using a reporter of Sm1021 cell division, we found that the dividing population increases progressively throughout nodule tissue beginning at 7 weeks-post-inoculation. Dissection of nodules and isolation of reproductive Sm1021 from sections of different ages showed that actively-dividing cells are distributed throughout the nodule at 3-4 months, but at 12 months are confined mostly to the highly-branched meristematic tips, with older sections collapsed and containing few reproductive Sm1021. This suggests that reproductive bacteria may be able to emerge from the root-proximal ‘saprophytic zone’ while the tip of the nodule is still engaging in N2-fixation. Surprisingly, we found that surface-sterilized nodules yield an order of magnitude fewer reproductive Sm1021 and that permeability to surface-sterilizing agents correlates with the presence of a gap in the suberin layer at the meristem.