Symbiotic vesicle ultrastructure in high pressure-frozen, freeze-substituted actinorhizae

Abstract

Using tissue stained en bloc with chromic acid or tissue prepared by high pressure-freezing and freeze-substitution, it was possible to analyze quantitatively the ultrastructure of symbiotic vesicle envelopes (SVE) inAlnus serrulata, Ceanothus americanus, Elaeagnus umbellata, andMyrica cerifera. The lamina measured about 4.7 nm in thickness in thin section. Despite diverse symbiotic vesicle morphology, the SVE thickness was similar in all of these symbioses: 36–71 nm, which corresponded to 6–15 laminae based on counts of chromic acid-stained SVEs. This similarity in structure suggests that a similar environmental signal regulates envelope thickness in the different root nodules. Based on previous studies, this is likely to be pO2. Three types of envelope morphologies were distinguished: (1) theAlnus-type (as inAlnus andElaeagnus), which had localized thickenings around the vesicle and had thickest dimensions over the stalk; (2) theCeanothus-type. characterized as a relatively uniform envelope over both vesicle and attached hypha, and (3) theMyrica-type, which had no stalk region and a basal SVE thickness of about six laminae throughout except where localized thickening occurred. Localized thickening of the SVE resulted from extra numbers of laminae being deposited, generally over regions where septa contacted the edge of the vesicle. Freeze-substituted symbiotic vesicles had a variety of novel structures that are poorly preserved in chemically-fixed tissue. A paracrystalline body inAlnus symbiotic vesicles may be composed of particles that also exist free in the symbiotic vesicle cytoplasm. In addition, a previously unknown complex at the base of theAlnus-type symbiotic vesicle and within its stalk was evident in freeze-substituted tissues.