Oscillations in extracellular pH and reactive oxygen species modulate tip growth of Arabidopsis root hairs.

Root hairs show highly localized cell expansion focused to their growing tips. This growth pattern is accomplished through restriction of secretion to the elongating apex and modulation of cell wall properties, with the wall just behind the tip becoming rigidified to resist the lateral expansive forces of turgor. In this report we show that root hairs exhibit oscillating growth that is associated with oscillating increases in extracellular pH and reactive oxygen species (ROS), which lag growth by approximately 7 s. Consistent with a role for these changes in growth control, artificially increasing extracellular pH arrested root hair elongation, whereas decreasing pH elicited bursting at the tip. Similarly, application of exogenous ROS arrested elongation, whereas scavenging of ROS led to root hair bursting. Roots hairs of the root hair-defective rhd2-1 mutant, which lack a functional version of the NADPH oxidase ATRBOH C, burst at the transition to tip growth. This phenotype could be rescued by elevating the pH of the growth medium to >/=6.0. Such rescued root hairs showed reduced cytoplasmic ROS levels and a lack of the oscillatory production of ROS at the tip. However, they exhibited apparently normal tip growth, including generation of the tip-focused Ca(2+) gradient thought to drive apical growth, indicating that ATRBOH C is not absolutely required to sustain tip growth. These observations indicate that root hair elongation is coupled to spatially distinct regulation of extracellular pH and ROS production that likely affect wall properties associated with the polarized expansion of the cell.

Monitoring Cl- movement in single cells exposed to hypotonic solution.

Self-referencing ion--selective electrodes (ISEs), made with Chloride Ionophore I-Cocktail A (Fluka), were positioned 1-3 microm from human embryonic kidney cells (tsA201a) and used to record chloride flux during a sustained hyposmotic challenge. The ISE response was close to Nernstian when comparing potentials (VN) measured in 100 and 10 mM NaCl (deltaVN = 57 +/- 2 mV), but was slightly greater than ideal when comparing 1 and 10 mM NaCl (deltaVN = 70 +/- 3 mV). The response was also linear in the presence of 1 mM glutamate, gluconate, or acetate, 10 microM tamoxifen, or 0.1, 1, or 10 mM HEPES at pH 7.0. The ISE was approximately 3 orders of magnitude more selective for Cl- over glutamate or gluconate but less than 2 orders of magnitude move selective for Clover bicarbonate, acetate, citrate or thiosulfate. As a result this ISE is best described as an anion sensor. The ISE was 'poisoned' by 50 microM 5-nitro-2-(3phenylpropyl-amino)-benzoic acid (NPPB), but not by tamoxifen. An outward anion efflux was recorded from cells challenged with hypotonic (250 +/- 5 mOsm) solution. The increase in efflux peaked 7-8 min before decreasing, consistent with regulatory volume decreases observed in separate experiments using a similar osmotic protocol. This anion efflux was blocked by 10 microM tamoxifen. These results establish the feasibility of using the modulation of electrochemical, anion-selective, electrodes to monitor anions and, in this case, chloride movement during volume regulatory events. The approach provides a real-time measure of anion movement during regulated volume decrease at the single-cell level.

Mitochondrial metabolism reveals a functional architecture in intact islets of Langerhans from normal and diabetic Psammomys obesus.

The cells within the intact islet of Langerhans function as a metabolic syncytium, secreting insulin in a coordinated and oscillatory manner in response to external fuel. With increased glucose, the oscillatory amplitude is enhanced, leading to the hypothesis that cells within the islet are secreting with greater synchronization. Consequently, non-insulin-dependent diabetes mellitus (NIDDM; type 2 diabetes)-induced irregularities in insulin secretion oscillations may be attributed to decreased intercellular coordination. The purpose of the present study was to determine whether the degree of metabolic coordination within the intact islet was enhanced by increased glucose and compromised by NIDDM. Experiments were performed with isolated islets from normal and diabetic Psammomys obesus. Using confocal microscopy and the mitochondrial potentiometric dye rhodamine 123, we measured mitochondrial membrane potential oscillations in individual cells within intact islets. When mitochondrial membrane potential was averaged from all the cells in a single islet, the resultant waveform demonstrated clear sinusoidal oscillations. Cells within islets were heterogeneous in terms of cellular synchronicity (similarity in phase and period), sinusoidal regularity, and frequency of oscillation. Cells within normal islets oscillated with greater synchronicity compared with cells within diabetic islets. The range of oscillatory frequencies was unchanged by glucose or diabetes. Cells within diabetic (but not normal) islets increased oscillatory regularity in response to glucose. These data support the hypothesis that glucose enhances metabolic coupling in normal islets and that the dampening of oscillatory insulin secretion in NIDDM may result from disrupted metabolic coupling.

Measurement of stress-induced Ca(2+) pulses in single aequorin-transformed tobacco cells.

Signaling patterns measured in large cell populations are the sum of differing signals from separate cells, and thus, the detailed kinetics of Ca(2+) pulses can often be masked. In an effort to evaluate whether the cytosolic Ca(2+) pulses previously reported in populations of elicitor- and stress-stimulated tobacco cells accurately represent the pulses that occur in individual cells, a study of single cell Ca(2+) fluxes in stress-stimulated tobacco cells was undertaken. Individual aequorin-transformed cells were isolated from a tobacco suspension culture and placed directly on a sensitive photo-multiplier tube mounted in a dark chamber. Ca(2+)-dependent luminescence was then monitored after stimulation with hypo- or hyper-osmotic shock, cold shock, or defense elicitors (oligogalacturonic acid and harpin). Hypo-osmotic shock induced a biphasic Ca(2+) transient in 67% of the single cells tested that exhibited similar kinetics to the biphasic pulses measured repeatedly in 1ml cell suspensions. In contrast, 33% of the stimulated cells displayed Ca(2+) flux patterns that were not previously seen in cell suspension studies. Additionally, because only 29% of the cells tested responded with measurable Ca(2+) pulses to oligogalacturonic acid and 33% to the harpin protein, we conclude that not all cells in a suspension are simultaneously sensitive to stimulation with defense elicitors. In contrast, all cells tested responded with an immediate Ca(2+) influx after cold or hyperosmotic shock. We conclude that in many cases the Ca(2+) signaling patterns of single cells are accurately represented in the signaling patterns of large populations, but that single cell measurements are still required to characterize the Ca(2+) fluxes of the less prominent cell populations.

Neuronal galvanotropism is independent of external Ca(2+) entry or internal Ca(2+) gradients.

The mechanism by which growing neurites sense and respond to small applied electrical fields is not known, but there is some evidence that the entry of Ca(2+) from the external medium, with the subsequent formation of intracellular Ca(2+) gradients, is important in this process. We have employed two approaches to test this idea. Xenopus spinal neurites were exposed to electrical fields in a culture medium in which Ca(2+) was chelated to very low levels compared to the normal extracellular concentration of 2 mM. In other experiments, loading the neurites with the calcium buffer, 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), disrupted the putative internal Ca(2+) gradients, and the effects on the electrical response were determined. Fields of 100 mV/mm were applied for 12 h, and no difference was detected in the cathodal turning response between the treated neurites and the untreated controls. Using the Differential Growth Index (DGI), an asymmetry index, to quantitate the turning response, we recorded DGIs of -0.64, -0.65, and -0.62 for control cells, cells in Ca(2+)-free medium, and cells preloaded with BAPTA, respectively. Furthermore, we detected an increase in neurite length for those neurons cultured in Ca(2+)-free medium; they were 1.5-1.7 times as long as neurites from neurons cultured in normal Ca(2+) medium. Likewise, we found that BAPTA-loaded neurites were longer than control neurites. Our data indicate that neuronal galvanotropism is independent of the entry of external Ca(2+) or of internal Ca(2+) gradients. Both cell-permeant agonistic and antagonistic analogs of cyclic 3',5'-adenosine monophosphate (cAMP) increased the response to applied electrical fields.

Periodic increases in elongation rate precede increases in cytosolic Ca2+ during pollen tube growth.

Pollen tubes grown in vitro require an intracellular tip-high gradient of Ca2+ in order to elongate. Moreover, after about 2 h in vitro both the tip Ca2+ and the elongation rate of lily tubes begin to oscillate regularly with large amplitudes. This raises the question of the phase relation between these two oscillations. Previous studies lacked the temporal resolution to accurately establish this relationship. We have studied these oscillations with a newly developed, high temporal resolution system and the complementary use of both luminescent and fluorescent calcium reporters. We hereby show that the periodic increases in elongation rate during oscillatory growth of Lilium longiflorum pollen tubes clearly precede those in subtip calcium and do so by 4.1 +/- 0.2 s out of average periods of 38.7 +/- 1.8 s. Also, by collecting images of the light output of aequorin, we find that the magnitude of the [Ca2+] at the tip oscillates between 3 and 10 microM, which is considerably greater than that reported by fluorescent indicators. We propose an explanatory model that features cyclic growth and secretion in which growth oscillations give rise to secretion that is essential for the subsequent growth oscillation. We also critically compile data on L. longiflorum stylar growth rates, which show little variation from in vitro rates of pollen tubes grown in optimal medium.

Pulsatile influxes of H+, K+ and Ca2+ lag growth pulses of Lilium longiflorum pollen tubes.

Fluxes of H+, K+ and Ca2+ were measured with self-referencing ion-selective probes, near the plasma membrane of growing Lilium longiflorum pollen tubes. Measurements from three regions around short, steady-growing tubes showed small, steady influx of H+ over the distal 40 microm and a region of the tube within 50-100 microm of the grain with larger magnitude efflux from the grain. K+ fluxes were immeasurable in short tubes. Measurements of longer tubes that were growing in a pulsatile manner revealed a pulsatile influx of both H+ and K+ at the growing tip. The average fluxes at the cell surface during the peaks of the H+ and K+ pulses were 489+/-81 and 688+/-144 pmol cm-2 second-1, respectively. Growth was measured by tracking the pollen tips with a computer vision system that achieved a spatial resolution of approximately 1/10 pixel. The high spatial resolution enabled the detection of growth, and thus the changes in growth rates, with a temporal sampling rate of 1 frame/second. These data show that the H+ and K+ pulses have a phase lag of 103+/-9 and 100+/-11 degrees, respectively, with respect to the growth pulses. Calcium fluxes were also measured in growing tubes. During steady growth, the calcium influx was relatively steady. When pulsatile growth began, the basal Ca2+ influx decreased and a pulsatile component appeared, superimposed on the reduced basal Ca2+ flux. The peaks of the Ca2+ pulses at the cell surface averaged 38.4+/-2.5 pmol cm-2 second-1. Longer tubes had large pulsatile Ca2+ fluxes with smaller baseline fluxes. The Ca2+ influx pulses had a phase lag of 123+/-9 degrees with respect to the growth pulses.