Endophytic bacteria discovered in oil body organelles of the liverworts Marchantia polymorpha and Radula complanata

Abstract

Premise: Interactions between endophytic microbes and bryophytes have been understudied. The liverwort oil body has also remained poorly understood since its discovery, and modern studies have failed to ascertain its function and composition. Many liverwort species possess oil bodies with conspicuous granules of unknown structure. We surveyed these granular liverwort oil bodies for the presence of bacteria to improve upon the understanding of liverworts, their oil bodies, and bacterial endophytes in nonvascular land plants.

Methods: Wild-collected specimens from living samples of Marchantia polymorpha and Radula complanata were stained with SYTO-13 and RADA to determine the presence or absence of bacteria within their oil bodies. Samples stained with calcofluor white, SYTO-13, and RADA were observed with confocal fluorescent microscopy for presence of nucleic acids and bacterial peptidoglycan cell walls within oil bodies.

Results: We discovered large masses of bacteria within the oil bodies of M. polymorpha and R. complanata based on the presence of stained nucleic acids and peptidoglycans localized to the oil body "granules". Such bacteria were present in all oil bodies of the two species.

Conclusions: These newly discovered intraorganellar bacteria correspond to the previously described "granules" of oil bodies. The existence of granular oil bodies in many liverwort species implies that this endophytic association may not be isolated to species investigated here. Assessments of additional liverwort species for presence and identity of oil body bacteria are needed to understand this intriguing association in one of the oldest land plant lineages.

Keywords: Marchantia polymorpha; Marchantiaceae; Marchantiophyta; Radula complanate; Radulaceae; endophytic bacteria; endosymbiosis; intracellular bacteria; liverwort oil body; liverworts.

Figure 1

Light (A–C, I–K) and confocal (D–H, L–P) micrographs of oil bodies in living cells of Radula complanata (Young 568) (A–H, scale bars: 10 µm) and Marchantia polymorpha (Young 566) (I–P, scale bars: 20 µm). (A, I) Unstained tissue exhibiting typical color. (B, J) Blue to purple color in bacterial cell walls from Gentian violet staining. (C, K) Grayscale, z‐stacks of oil bodies emphasizing bacterial morphology for comparison with confocal images D and L. (C) Calcoflour white fluorescing blue in plant cell walls after calcoflour white staining with no autofluorescence of oil body or bacteria. (E, F, M, N) Calcoflour white and SYTO‐13 staining with blue fluorescence in plant cell walls and green fluorescence from nucleic acids (presumably bacterial DNA) from the oil body surface and in cross section. (G, H, O, P) RADA staining with red fluorescence of peptidoglycan in bacterial cell walls shown from the oil body surface and in cross section.