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Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip.

Feijó JA, Sainhas J, Hackett GR, Kunkel JG, Hepler PK - J. Cell Biol. (1999)

Bottom Line: Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 microM or greater, will dissipate the gradient, possibly through shuttle buffering.The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins.These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.

View Article: PubMed Central - PubMed

Affiliation: Department Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, P-1749-016 Lisboa, Portugal. jose.feijo@fc.ul.pt

ABSTRACT
Using both the proton selective vibrating electrode to probe the extracellular currents and ratiometric wide-field fluorescence microscopy with the indicator 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran to image the intracellular pH, we have examined the distribution and activity of protons (H+) associated with pollen tube growth. The intracellular images reveal that lily pollen tubes possess a constitutive alkaline band at the base of the clear zone and an acidic domain at the extreme apex. The extracellular observations, in close agreement, show a proton influx at the extreme apex of the pollen tube and an efflux in the region that corresponds to the position of the alkaline band. The ability to detect the intracellular pH gradient is strongly dependent on the concentration of exogenous buffers in the cytoplasm. Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 microM or greater, will dissipate the gradient, possibly through shuttle buffering. The apical acidic domain correlates closely with the process of growth, and thus may play a direct role, possibly in facilitating vesicle movement and exocytosis. The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins. These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.

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Detailed real-time exploration of the  currents around the tip and subtip areas of a  tube ∼1.0-mm long and grown at pH 6.5. Continuous vibration of the probe at the closest possible point (less than 2 μm) of the growing tip was  achieved by very small steps mimicking the tube  advance. Under these conditions a sustained oscillatory pattern of the tip proton influx was observed, sometimes during more than an hour, in  which the influx could be seen to change from  nearly 0 to 0.4 pmol/cm2/s. From time 2,800 s the  direction of the probe vibration was orthogonally changed, and the probe left stationery as  the tube was allowed to grow along it (see inset  with tube tip). In this circumstance a profile of  the proton flux domains underneath the tip was  observed without changing the probe position.  The physical location of the probe related to the  tip is assigned in A and B. As soon as the probe  slips out of the tip dome a sharp reversal of the flux occurs and remains as an efflux over a distance that roughly correlates with the extent of the clear zone. Another reversal of the flux is then observed in the rest of the tube, which then evolves to a pattern of small, less  defined effluxes. Background reference measurements are included for both directions of probe vibration showing that the bath background fluxes are insignificant in relation to the signal.
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Figure 2: Detailed real-time exploration of the currents around the tip and subtip areas of a tube ∼1.0-mm long and grown at pH 6.5. Continuous vibration of the probe at the closest possible point (less than 2 μm) of the growing tip was achieved by very small steps mimicking the tube advance. Under these conditions a sustained oscillatory pattern of the tip proton influx was observed, sometimes during more than an hour, in which the influx could be seen to change from nearly 0 to 0.4 pmol/cm2/s. From time 2,800 s the direction of the probe vibration was orthogonally changed, and the probe left stationery as the tube was allowed to grow along it (see inset with tube tip). In this circumstance a profile of the proton flux domains underneath the tip was observed without changing the probe position. The physical location of the probe related to the tip is assigned in A and B. As soon as the probe slips out of the tip dome a sharp reversal of the flux occurs and remains as an efflux over a distance that roughly correlates with the extent of the clear zone. Another reversal of the flux is then observed in the rest of the tube, which then evolves to a pattern of small, less defined effluxes. Background reference measurements are included for both directions of probe vibration showing that the bath background fluxes are insignificant in relation to the signal.

Mentions: Germinating pollen grains and short tubes (<300 μm) were shown to follow much the same pattern described for total electric currents, i.e., the grain is the only source and the tube is the sink (Feijó et al., 1994b). This pattern was confirmed in the present study. However, tubes were followed to longer lengths, revealing a new, emerging pattern that is depicted in Fig. 1. From 600–800 μm on, we detected a new membrane domain, roughly corresponding to the clear zone, that effluxes. These fluxes, which in some instances attained values (per area unit) similar to those detected in the grain, seemed to be stable, or at least did not exhibit distinct temporal changes. It follows that at this length two closed loops of proton circulation emerged, one around the grain, and another around the growing tip. The bulk of the flux source and sink range to ∼150–200 μm in both cases. Although the absolute values of the flux showed some variability from cell to cell, this pattern was stable, being confirmed in a significant number of growing tubes (n = 13). Between the two loops there was another membrane domain where only very small fluxes were observed. Some of these fluxes revealed significant deviations from the background noise but they cannot be compared in magnitude with the signals detected around the grain and the tip. In the transition domain area in which reversal of the flux occurs, no significant fluxes could be detected. This pattern seems to reflect a gradual, rather than specifically localized, change from one membrane domain to another. Longer tubes (up to 3 mm) were also investigated and again the closed loop at the tip was always detectable (see Fig. 2). Yet two other trends could be also characterized: first, whereas the efflux in the clear zone remained stable, the grain efflux decreased from a certain point on (>2 mm); second, alternate banding of effluxes and influxes arose along the tube in between the tip loop and the grain loop. The size of these alternating bands was not completely regular, and, as in the transition zone in Fig. 1, the magnitude of the fluxes was always one order of magnitude lower than the one driven by the tip and the grain.


Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip.

Feijó JA, Sainhas J, Hackett GR, Kunkel JG, Hepler PK - J. Cell Biol. (1999)

Detailed real-time exploration of the  currents around the tip and subtip areas of a  tube ∼1.0-mm long and grown at pH 6.5. Continuous vibration of the probe at the closest possible point (less than 2 μm) of the growing tip was  achieved by very small steps mimicking the tube  advance. Under these conditions a sustained oscillatory pattern of the tip proton influx was observed, sometimes during more than an hour, in  which the influx could be seen to change from  nearly 0 to 0.4 pmol/cm2/s. From time 2,800 s the  direction of the probe vibration was orthogonally changed, and the probe left stationery as  the tube was allowed to grow along it (see inset  with tube tip). In this circumstance a profile of  the proton flux domains underneath the tip was  observed without changing the probe position.  The physical location of the probe related to the  tip is assigned in A and B. As soon as the probe  slips out of the tip dome a sharp reversal of the flux occurs and remains as an efflux over a distance that roughly correlates with the extent of the clear zone. Another reversal of the flux is then observed in the rest of the tube, which then evolves to a pattern of small, less  defined effluxes. Background reference measurements are included for both directions of probe vibration showing that the bath background fluxes are insignificant in relation to the signal.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132912&req=5

Figure 2: Detailed real-time exploration of the currents around the tip and subtip areas of a tube ∼1.0-mm long and grown at pH 6.5. Continuous vibration of the probe at the closest possible point (less than 2 μm) of the growing tip was achieved by very small steps mimicking the tube advance. Under these conditions a sustained oscillatory pattern of the tip proton influx was observed, sometimes during more than an hour, in which the influx could be seen to change from nearly 0 to 0.4 pmol/cm2/s. From time 2,800 s the direction of the probe vibration was orthogonally changed, and the probe left stationery as the tube was allowed to grow along it (see inset with tube tip). In this circumstance a profile of the proton flux domains underneath the tip was observed without changing the probe position. The physical location of the probe related to the tip is assigned in A and B. As soon as the probe slips out of the tip dome a sharp reversal of the flux occurs and remains as an efflux over a distance that roughly correlates with the extent of the clear zone. Another reversal of the flux is then observed in the rest of the tube, which then evolves to a pattern of small, less defined effluxes. Background reference measurements are included for both directions of probe vibration showing that the bath background fluxes are insignificant in relation to the signal.
Mentions: Germinating pollen grains and short tubes (<300 μm) were shown to follow much the same pattern described for total electric currents, i.e., the grain is the only source and the tube is the sink (Feijó et al., 1994b). This pattern was confirmed in the present study. However, tubes were followed to longer lengths, revealing a new, emerging pattern that is depicted in Fig. 1. From 600–800 μm on, we detected a new membrane domain, roughly corresponding to the clear zone, that effluxes. These fluxes, which in some instances attained values (per area unit) similar to those detected in the grain, seemed to be stable, or at least did not exhibit distinct temporal changes. It follows that at this length two closed loops of proton circulation emerged, one around the grain, and another around the growing tip. The bulk of the flux source and sink range to ∼150–200 μm in both cases. Although the absolute values of the flux showed some variability from cell to cell, this pattern was stable, being confirmed in a significant number of growing tubes (n = 13). Between the two loops there was another membrane domain where only very small fluxes were observed. Some of these fluxes revealed significant deviations from the background noise but they cannot be compared in magnitude with the signals detected around the grain and the tip. In the transition domain area in which reversal of the flux occurs, no significant fluxes could be detected. This pattern seems to reflect a gradual, rather than specifically localized, change from one membrane domain to another. Longer tubes (up to 3 mm) were also investigated and again the closed loop at the tip was always detectable (see Fig. 2). Yet two other trends could be also characterized: first, whereas the efflux in the clear zone remained stable, the grain efflux decreased from a certain point on (>2 mm); second, alternate banding of effluxes and influxes arose along the tube in between the tip loop and the grain loop. The size of these alternating bands was not completely regular, and, as in the transition zone in Fig. 1, the magnitude of the fluxes was always one order of magnitude lower than the one driven by the tip and the grain.

Bottom Line: Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 microM or greater, will dissipate the gradient, possibly through shuttle buffering.The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins.These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.

View Article: PubMed Central - PubMed

Affiliation: Department Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, P-1749-016 Lisboa, Portugal. jose.feijo@fc.ul.pt

ABSTRACT
Using both the proton selective vibrating electrode to probe the extracellular currents and ratiometric wide-field fluorescence microscopy with the indicator 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran to image the intracellular pH, we have examined the distribution and activity of protons (H+) associated with pollen tube growth. The intracellular images reveal that lily pollen tubes possess a constitutive alkaline band at the base of the clear zone and an acidic domain at the extreme apex. The extracellular observations, in close agreement, show a proton influx at the extreme apex of the pollen tube and an efflux in the region that corresponds to the position of the alkaline band. The ability to detect the intracellular pH gradient is strongly dependent on the concentration of exogenous buffers in the cytoplasm. Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 microM or greater, will dissipate the gradient, possibly through shuttle buffering. The apical acidic domain correlates closely with the process of growth, and thus may play a direct role, possibly in facilitating vesicle movement and exocytosis. The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins. These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.

Show MeSH
Related in: MedlinePlus