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Different models have been proposed to explain the operation of oxygen diffusion barrier in root nodules of leguminous plants. This barrier participates in protection of oxygen-sensitive nitrogenase, the key enzyme in nitrogen fixation, from inactivation. Details concerning structural and biochemical properties of the barrier are still lacking. Here, the properties of pea root nodule cortical cells were examined under normal conditions and after shoot removal. Microscopic observations, including neutral red staining and epifluorescence investigations, showed that the inner and outer nodule parenchyma cells exhibit different patterns of the central vacuole development. In opposition to the inner part, the outer parenchyma cells exhibited vacuolar shrinkage and formed cell wall infoldings. Shoot removal induced vacuolar shrinkage and formation of infoldings in the inner parenchyma and uninfected cells of the symbiotic tissue, as well. It is postulated that cells which possess shrinking vacuoles are sensitive to the external osmotic pressure. The cells can give an additional resistance to oxygen diffusion by release of water to the intercellular spaces.Immunolocalization studies proved higher expression of endo-β-1,4-glucanases within expanding cells of the outer cortex of pea nodules comparing with nodule endodermis or nodule parenchyma, so it is suggested that (1) endo-glucanases are involved in growth related modifications of cell walls and (2) enlarged cells decrease nodule conductance to oxygen.

Vacuolar organization in the nodule parenchyma is important for the functioning of pea root nodules