Phosphate ions in root-tip dividing cells: a combined trapping and squash method with implications for nuclear transcription

Abstract

We examined the pattern of inorganic orthophosphate (PPi) ion distribution in dividing cells of Zea mays root-tips. Unfixed and paraformaldehyde- or glutaraldehyde-vapor fixed tissues were immersed in lead acetate, glutaraldehyde, and cacodylate buffer to capture PPi as insoluble orthophosphate lead hydroxyapatite. Excess lead ions were removed with sodium citrate, then permeabilized in ammonia. Precipitates were stained with potassium sulfide, washed with distilled water and squashed in a drop of glycerin. The accumulation of PPi ions was cyclic in the cytoplasm during mitosis and they surrounded all chromosomes during metaphase and anaphase. Partition between dividing cells started with a high concentration of PPi ions at sites where plasma membrane and cell walls formed. Small daughter cells and those in G1 phase had PPi concentrated in the nucleolus, with lower levels elsewhere in the nucleus. Later in the cell cycle, there were greater amounts of PPi ions associated with condensed chromatin in larger nuclei. In Xenopus laevis oocytes, PPi was concentrated in the nucleus, mainly in the active central core of multiple nucleoli. These results and others indicate that compartmentalization of PPi occurs in the intact cell and correlates with the rate of transcription in distinct functional domains within the nucleus.