Description:

The long standing interest of my laboratory (Robinson) is the mechanism of generation and maintenance of polarity. The growing pollen tube is an extreme example. Lilly pollen tubes grow at the rate of 0.2 µm/s by mobilizing the cellular machinery to insert new membrane and cell wall material in a precisely defined region at the growing tip. This growth is not steady; instead, it oscillates between times of rapid growth and very slow growth with a period of about 40 s. It is known that a tip-focused gradient of Ca²⁺ is necessary for growth, and the steepness of the gradient also oscillates with the same period as the growth rate oscillations. The use of self-referencing ion selective (Seris) probes has shown that there are oscillatory influxes of Ca²⁺, K⁺ and H⁺ that have the same periodicity as growth, but are out of phase with growth oscillations. More recently, claims of Cl^- efflux have been reported by others. This was a surprising result, as pollen tube growth is completely independent of external Cl-, raising the question of how the very large efflux of Cl^- could be maintained. Furthermore, their findings raised important osmotic issues about how the necessary turgor pressure could be maintained in the face of the massive loss of an osmotically active ion. Growth involves the continuous uptake of water, which dilutes the cellular solutes so the system faces the task of adding solute. The efflux of Cl^- through a channel would also be expected to have significant electrical consequences, which have not been detected.

Progress:

We decided to re-investigate this problem, as it seemed important to verify that data interpretation was as claimed. We carried out a careful evaluation of the performance of the Cl^- ISE, including dynamic testing of the temporal response of the electrodes to step changes in Cl^- activity in the presence of various interferents. We also used the electrodes to measure putative Cl^- fluxes from growing pollen tubes and employed ion chromatography to measure the reserves of Cl^- carried by pollen grains. The conclusion from these experiments was that the putative Cl^- efflux was an artifact caused by the substantial changes in pH due to oscillatory proton influx. It was found that the phase of the putative Cl^- efflux matched the phase of H⁺ influx and was not in phase with growth oscillations.

We conclude that 1) that the selectivity of the Cl^- anion exchanger is only an order magnitude for Cl^- over any other anions in the culture medium or that may reasonably be found in a plant cell
2) that the Cl^- anion exchanger is not inhibited by the Cl^- channel blockers Tamoxifen, NPPB or Niflumic acid but does detect DIDS in a concentration dependent manner that blinds it to changes in Cl^- concentration.

This important correction to the plant physiology literature could not have happened without the resources of the BRC. The careful characterization of the properties of the Cl^- ISE will be useful to all physiologists, regardless of whether they work on plant or animal systems.