Plasma Membrane Domains Participate in pH Banding of Chara Internodal Cells

Abstract

We investigated the identity and distribution of cortical domains, stained by the endocytic marker FM 1-43, in branchlet internodal cells of the characean green algae Chara corallina and Chara braunii. Co-labeling with NBD C6-sphingomyelin, a plasma membrane dye, which is not internalized, confirmed their location in the plasma membrane, and co-labelling with the fluorescent pH indicator Lysotracker red indicated an acidic environment. The plasma membrane domains co-localized with the distribution of an antibody against a proton-translocating ATPase, and electron microscopic data confirmed their identity with elaborate plasma membrane invaginations known as charasomes. The average size and the distribution pattern of charasomes correlated with the pH banding pattern of the cell. Charasomes were larger and more frequent at the acidic regions than at the alkaline bands, indicating that they are involved in outward-directed proton transport. Inhibition of photosynthesis by DCMU prevented charasome formation, and incubation in pH buffers resulted in smaller, homogenously distributed charasomes irrespective of whether the pH was clamped at 5.5 or 8.5. These data indicate that the differential size and distribution of charasomes is not due to differences in external pH but reflects active, photosynthesis-dependent pH banding. The fact that pH banding recovered within several minutes in unbuffered medium, however, confirms that pH banding is also possible in cells with evenly distributed charasomes or without charasomes. Cortical mitochondria were also larger and more abundant at the acid bands, and their intimate association with charasomes and chloroplasts suggests an involvement in carbon uptake and photorespiration.

Molecular Analysis and Localization of CaARA7 a Conventional RAB5 GTPase from Characean Algae

“…Immunofluorescence of internodal cells with the anti‐CaARA7 revealed only few punctate structures with a size of up to one µm in the cortex and located between charasomes and the stationary chloroplasts (Figure A–D). Charasomes are convoluted regions of the plasma membrane and are required for efficient photosynthesis‐dependent carbon uptake . Considerably more CaARA7‐positive organelles were present in the endoplasm (Figure E) consistent with the distribution of MVEs (see below).…”

“…The cytoplasm of Chara internodal cells consists of a stationary cortex, containing files of helically aligned chloroplasts ( Figure 4A-D) and the streaming endoplasm ( Figure 4E-G). Immunofluorescence of internodal cells with the anti-CaARA7 revealed only few punctate structures with a size of up to one μm in the cortex and located between charasomes and the stationary chloroplasts ( Figure 4A are required for efficient photosynthesis-dependent carbon uptake (31). Considerably more CaARA7-positive organelles were present in the endoplasm ( Figure 4E) consistent with the distribution of MVEs (see below).…”

“…Detection was carried out using CDP star detection kit (New England Biolabs) and a LAS 3000 mini imaging system (Fujifilm). The immunofluorescence procedure was as described in (31). For localization of ARA7-labelled organelles the polyclonal anti-CaARA7 (see above) was used at dilutions between 1:500 and 1:750 and combined with an Alexa Fluor 488-conjugated anti-rabbit IgG (Invitrogen; diluted 1:1000) or with an Alexa Fluor 546-conjugated anti-rabbit IgG (Thermo Fisher; diluted 3:1000).…”

Heterotrimeric G-proteins in green algae

“…12 Aquatic tissue was harvested and lyophilized for 16 h and subsequently ground in liquid N 2 . Fifty mg algal dry mass was used for acidic hormone quantification.…”

Characterization of the Heterotrimeric G-Protein Complex and Its Regulator from the Green Alga Chara braunii Expands the Evolutionary Breadth of Plant G-Protein Signaling

“…Chara braunii cultures (kindly provided by Dr. Ilse Foissner) were cultivated in distilled water on a sand/soil/peat mixture at room temperature and a 14/10-h light/dark cycle as described previously (Schmölzer et al, 2011).…”