Ultrastructural characterization of an inner envelope that confines Azolla endosymbionts to the leaf cavity periphery

Abstract

Ultrastructural characteristics of Azolla leaf cavities were examined using transmission electron microscopy. An extracellular inner envelope, which forms a boundary between the central cavity region and the peripheral region containing cyanobacteria and (or) bacteria, was found in all closed leaf cavities of symbiotic and Anabaena-free Azolla species. In the youngest leaf, which is in the process of developing a cavity, a continuous inner envelope was never observed. However, in young leaves that contained closed cavities, and in older leaves, continuous inner envelopes were always observed. The inner envelope may serve to restrict the symbionts to the cavity periphery. The inner envelope was similar in appearance to the outer envelope, which encloses the symbionts and hair cells within leaf cavities. Examination of the inner envelope using three different fixation procedures revealed that it was most distinct in fronds fixed in potassium permanganate, suggesting that it is membranelike in structure. The envelope was occasionally seen folded back upon itself or curled, which is also characteristic of a membranelike structure. Nevertheless, the envelope did not exhibit typical tripartite membrane ultrastructure. The occurrence of the inner envelope in Anabaena-free Azolla indicates that this envelope is of eubacterial or plant origin.