The molecular identity of the characean OH− transporter: a candidate related to the SLC4 family of animal pH regulators

Characeae are closely related to the ancient algal ancestors of all land plants. The long characean cells display a pH banding pattern to facilitate inorganic carbon import in the acid zones for photosynthetic efficiency. The excess OH−, generated in the cytoplasm after CO2 is taken into the chloroplasts, is disposed of in the alkaline band. To identify the transporter responsible, we searched the Chara australis transcriptome for homologues of mouse Slc4a11, which functions as OH−/H+ transporter. We found a single Slc4-like sequence CL5060.2 (named CaSLOT). When CaSLOT was expressed in Xenopus oocytes, an increase in membrane conductance and hyperpolarization of resting potential difference (PD) was observed with external pH increase to 9.5. These features recall the behavior of Slc4a11 in oocytes and are consistent with the action of a pH-dependent OH−/H+ conductance. The large scatter in the data might reflect intrinsic variability of CaSLOT transporter activation, inefficient expression in the oocyte due to evolutionary distance between ancient algae and frogs, or absence of putative activating factor present in Chara cytoplasm. CaSLOT homologues were found in chlorophyte and charophyte algae, but surprisingly not in related charophytes Zygnematophyceae or Coleochaetophyceae.

Fluid-phase and membrane markers reveal spatio-temporal dynamics of membrane traffic and repair in the green alga Chara australis

We investigated the mechanisms and the spatio-temporal dynamics of fluid-phase and membrane internalization in the green algaChara australisusing fluorescent hydrazides markers alone, or in conjunction with styryl dyes.Using live-cell imaging, immunofluorescence and inhibitor studies we revealed that both fluid-phase and membrane dyes were actively taken up into the cytoplasm by clathrin-mediated endocytosis and stained various classes of endosomes including brefeldin A- and wortmannin-sensitive organelles (trans-Golgi network and multivesicular bodies). Uptake of fluorescent hydrazides was poorly sensitive to cytochalasin D, suggesting that actin plays a minor role in constitutive endocytosis inCharainternodal cells. Sequential pulse-labelling experiments revealed novel aspects of the temporal progression of endosomes inCharainternodal cells. The internalized fluid-phase marker distributed to early compartments within 10 min from dye exposure and after about 30 min, it was found almost exclusively in late endocytic compartments. Notably, fluid cargo consecutively internalized at time intervals of more than 1h, was not targeted to the same vesicular structures, but was sorted into distinct late compartments. We further found that fluorescent hydrazide dyes distributed not only to rapidly recycling endosomes but also to long-lived compartments that participated in plasma membrane repair after local laser injury. Our approach highlights the benefits of combining different fluid-phase markers in conjunction with membrane dyes in simultaneous and sequential application modus for investigating vesicle traffic, especially in organisms, which are still refractory to genetic transformation like characean algae.

A revision of Chara sect. Protochara, comb. et stat. nov. (Characeae: Charophyceae)

A revision of a group of ecorticate species of Chara is presented, on the basis of fresh, pressed and spirit-preserved material. The following seven species are recognised, characterised by a very simple morphology, with few or inconspicuous accessory cells (cortication, stipulodes, bract cells, bracteoles) and large gametangia: Chara australis R.Br., C. lucida (A.Braun) Casanova & Karol comb et. stat. nov., C. porteri Casanova, sp. nov., C. protocharoides Casanova & Karol, nom. nov. (=Protochara australis Womersley & Ophel) and C. stuartiana (Kütz.) Casanova & Karol comb. et. stat. nov. from Australia, and C. corallina Klein ex Willd. and C. wallichii A.Braun from Asia. A new section, Chara subg. Charopsis sect. Protochara (Womersley & Ophel) Casanova & Karol, comb. et stat. nov., is erected to accommodate these taxa, formerly placed in sect. Charopsis.