Oscillatory growth of pollen tubes has been correlated with oscillatory influxes of the cations Ca(2+), H(+), and K(+). Using an ion-specific vibrating probe, a new circuit was identified that involves oscillatory efflux of the anion Cl(-) at the apex and steady influx along the tube starting at 12 microm distal to the tip. This spatial coupling of influx and efflux sites predicts that a vectorial flux of Cl(-) ion traverses the apical region. The Cl(-) channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid completely inhibited tobacco pollen tube growth at 80 and 20 microM, respectively. Cl(-) channel blockers also induced increases in apical cell volume. The apical 50 micro m of untreated pollen tubes had a mean cell volume of 3905 +/- 75 microm(3). DIDS at 80 microM caused a rapid and lethal cell volume increase to 6206 +/- 171 microm(3), which is at the point of cell bursting at the apex. DIDS was further demonstrated to disrupt Cl(-) efflux from the apex, indicating that Cl(-) flux correlates with pollen tube growth and cell volume status. The signal encoded by inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P(4)] antagonized pollen tube growth, induced cell volume increases, and disrupted Cl(-) efflux. Ins(3,4,5,6)P(4) decreased the mean growth rate by 85%, increased the cell volume to 5997 +/- 148 microm(3), and disrupted normal Cl(-) efflux oscillations. These effects were specific for Ins(3,4,5,6)P(4) and were not mimicked by either Ins(1,3,4,5)P(4) or Ins(1,3,4,5,6)P(5). Growth correlation analysis demonstrated that cycles of Cl(-) efflux were coupled to and temporally in phase with cycles of growth. A role for Cl(-) flux in the dynamic cellular events during growth is assessed. Differential interference contrast microscopy and kymographic analysis of individual growth cycles revealed that vesicles can advance transiently to within 2 to 4 microm of the apex during the phase of maximally increasing Cl(-) efflux, which temporally overlaps the phase of cell elongation during the growth cycle. In summary, these investigations indicate that Cl(-) ion dynamics are an important component in the network of events that regulate pollen tube homeostasis and growth.

A coherent picture of pollen tube growth is beginning to emerge that couples ion dynamics with biochemical, biophysical and cytological processes in ordered and controlled feedback circuits that define the nature of polarized apical growth. It is a paradox, however, that complete understanding of the mechanical forces that drive cell elongation in this system still remains to be fully achieved. The results of our recent studies to characterize Cl– ion dynamics during apical growth in tobacco pollen tubes led us to re-examine this question in the light of a possible force-generating role provided by hydrodynamic flow. Previously we found that oscillatory Cl– efflux from the apex is closely coupled to oscillatory growth and the cell volume of the apical domain. Cl– influx occurs in a region of the tube that is distal to the clear zone; hence, a vectorial flow of anion traverses the apical domain and fluxes out of the tip with oscillatory dynamics. Because of the effects that this could induce on charge and osmotic potentials, water could potentially flow through the apical domain, linked to the flux of Cl–. This conjecture is consistent with studies in other plant cells that demonstrate a pivotal role for flux through anion channels in the control or normalization of osmotic status. In the current report, the relationship between Cl– efflux oscillations and the physical characteristics of the apical dome during oscillatory growth is examined in closer detail. Evidence is presented that shows a cyclic deformation of the extreme apex occurs during the growth pulse and is correlated with cyclic Cl– efflux. In addition, there is a dramatic increase in the number and density of clear thread-like zones traversing the apical plasma membrane during the process of tip elongation. Possible functional roles of Cl– flux and hydrodynamics are discussed in the context of what drives tip elongation during cycles of pollen tube growth.

The occurrence of oscillatory behaviours in living cells can be viewed as a visible consequence of stable, regulatory homeostatic cycles. Therefore, they may be used as experimental windows on the underlying physiological mechanisms. Recent studies show that growing pollen tubes are an excellent biological model for these purposes. They unite experimental simplicity with clear oscillatory patterns of both structural and temporal features, most being measurable during real-time in live cells. There is evidence that these cellular oscillators involve an integrated input of plasma membrane ion fluxes, and a cytosolic choreography of protons, calcium and, most likely, potassium and chloride. In turn, these can create positive feedback regulation loops that are able to generate and self-sustain a number of spatial and temporal patterns. Other features, including cell wall assembly and rheology, turgor, and the cytoskeleton, play important roles and are targets or modulators of ion dynamics. Many of these features have similarities with other cell types, notably with apical-growing cells. Pollen tubes may thus serve as a powerful model for exploring the basis of cell growth and morphogenesis. BioEssays 23:86–94, 2001. © 2001 John Wiley & Sons, Inc.

In this paper, we report direct measurement of an influx of extracellular Ca 2+ induced by gamete fusion in flowering plants. This result was obtained during maize in vitro fertilization with the use of an extracellular Ca 2+ -selective vibrating probe. Ca 2+ influx recorded at the surface of isolated egg cells, with or without adhesion of a male sperm cell, was close to zero and stable over time. Gamete fusion, however, triggered a Ca 2+ influx in the vicinity of the sperm entry site with a delay of 1.8 ± 0.6 sec. The Ca 2+ influx spread subsequently through the whole egg cell plasma membrane as a wavefront, progressing at an estimated rate of 1.13 μm⋅sec −1 . Once established, Ca 2+ influx intensities were sustained, monotonic and homogeneous over the whole egg cell, with an average peak influx of 14.92 pmol⋅cm −2 ⋅sec −1 and an average duration of 24.4 min. The wavefront spread of channel activation correlates well with the cytological modifications induced by fertilization, such as egg cell contraction, and with the cytosolic Ca 2+ ( c [Ca 2+ ]) elevation previously reported. Calcium influx was inhibited effectively by gadolinium, possibly implicating mechanosensitive channels. Furthermore, artificial influxes created by incubation with Ca 2+ ionophores mimicked some aspects of egg activation. Taken together, these results suggest that, during fertilization in higher plants, gamete membrane fusion starts the first embryonic events by channel opening and Ca 2+ influx. In turn, c [Ca 2+ ] may work as a trigger and possibly a space and time coordinator of many aspects of egg activation.