Measurement and manipulation of cytosolic pH in polarizing zygotes

The role of cytosolic pH (pHc) in establishment and expression of developmental polarity was examined in zygotes of the brown alga Pelvetia. pHc was measured and manipulated at specific developmental stages during the first zygotic cell cycle. pHc was measured using pH-sensitive microelectrodes and by confocal ratio imaging of dextran-conjugated SNARF 1 (dc SNARF 1) loaded cells. The two techniques yielded very similar values of pHc in the cellular cortex, but ratio imaging was not effective in measuring endoplasmic pHc values. As zygotes formed a developmental axis, cortical pHc decreased abruptly by approximately 0.1 units, and a small but significant difference in pHc was detected at the thallus and rhizoid poles. The cortical cytosol was relatively acidic at the presumptive rhizoid pole. The magnitude of the pHc difference increased following initiation of rhizoid growth. pHc was manipulated by treating zygotes with membrane-permeant weak acids (propionic and benzoic acid) or bases (methylamine and procaine), which effectively clamp pHc to specific values in a concentration-dependent manner. pHc values in treated zygotes were measured for each concentration of acid or base, and a dose response curve was generated. Zygotes in which pHc had been clamped were examined for their ability to form a developmental axis and to initiate rhizoid outgrowth (germination). Both developmental processes were inhibited by relatively small (0.2-0.3 pH units) perturbations of pHc. The permissive ranges of pHc were slightly different, germination (permissive pHc range-pH 7.0 to 7.7) being more acid tolerant than axis formation (permissive pHc range-7.2 to 7.8).