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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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Representative  profile of the proton fluxes  along a growing pollen tube  (∼800-μm long) at pH 6.1.  The pollen grain membrane  always drives significant effluxes of protons, presumably by the activity of proton  translocating ATPases located at the pollen grain  membrane (see Discussion). Immediately after the pore, pollen tubes show a strong influx domain of protons in the proximal 150–200  μm. This influx decreases along the tube and eventually reverses, becoming a clear efflux of protons in the region of the clear zone. Another reversal of the current direction is observed in the apical dome, characterized by a strong point influx at the very tip, with chaotic  small amplitude oscillations for short tubes, or sustained large amplitude oscillations for longer tubes. The clear zone domain of proton  efflux is usually not discernible in small tubes (<500 μm), and becomes increasingly well defined from the size of the one depicted. As  tubes continue to elongate this pattern evolves subsequently to a banding alternation of small efflux and influx domains over distances  of ∼300–400 μm. However the grain + proximal tube and the clear zone + tip-closed loops of proton circulation remain as shown in  this figure. Although these patterns have been confirmed in a significant number of tubes (n = 13) the absolute values shown vary considerably with the bath pH and tube length. Bar, 1.0 pmol/cm2/s; pollen grain and tube not drawn exactly to scale; grain is ∼120 × 80 μm  and tube is ∼18 μm in diameter.
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Figure 1: Representative profile of the proton fluxes along a growing pollen tube (∼800-μm long) at pH 6.1. The pollen grain membrane always drives significant effluxes of protons, presumably by the activity of proton translocating ATPases located at the pollen grain membrane (see Discussion). Immediately after the pore, pollen tubes show a strong influx domain of protons in the proximal 150–200 μm. This influx decreases along the tube and eventually reverses, becoming a clear efflux of protons in the region of the clear zone. Another reversal of the current direction is observed in the apical dome, characterized by a strong point influx at the very tip, with chaotic small amplitude oscillations for short tubes, or sustained large amplitude oscillations for longer tubes. The clear zone domain of proton efflux is usually not discernible in small tubes (<500 μm), and becomes increasingly well defined from the size of the one depicted. As tubes continue to elongate this pattern evolves subsequently to a banding alternation of small efflux and influx domains over distances of ∼300–400 μm. However the grain + proximal tube and the clear zone + tip-closed loops of proton circulation remain as shown in this figure. Although these patterns have been confirmed in a significant number of tubes (n = 13) the absolute values shown vary considerably with the bath pH and tube length. Bar, 1.0 pmol/cm2/s; pollen grain and tube not drawn exactly to scale; grain is ∼120 × 80 μm and tube is ∼18 μm in diameter.

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)

Representative  profile of the proton fluxes  along a growing pollen tube  (∼800-μm long) at pH 6.1.  The pollen grain membrane  always drives significant effluxes of protons, presumably by the activity of proton  translocating ATPases located at the pollen grain  membrane (see Discussion). Immediately after the pore, pollen tubes show a strong influx domain of protons in the proximal 150–200  μm. This influx decreases along the tube and eventually reverses, becoming a clear efflux of protons in the region of the clear zone. Another reversal of the current direction is observed in the apical dome, characterized by a strong point influx at the very tip, with chaotic  small amplitude oscillations for short tubes, or sustained large amplitude oscillations for longer tubes. The clear zone domain of proton  efflux is usually not discernible in small tubes (<500 μm), and becomes increasingly well defined from the size of the one depicted. As  tubes continue to elongate this pattern evolves subsequently to a banding alternation of small efflux and influx domains over distances  of ∼300–400 μm. However the grain + proximal tube and the clear zone + tip-closed loops of proton circulation remain as shown in  this figure. Although these patterns have been confirmed in a significant number of tubes (n = 13) the absolute values shown vary considerably with the bath pH and tube length. Bar, 1.0 pmol/cm2/s; pollen grain and tube not drawn exactly to scale; grain is ∼120 × 80 μm  and tube is ∼18 μm in diameter.
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Related In: Results  -  Collection

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Figure 1: Representative profile of the proton fluxes along a growing pollen tube (∼800-μm long) at pH 6.1. The pollen grain membrane always drives significant effluxes of protons, presumably by the activity of proton translocating ATPases located at the pollen grain membrane (see Discussion). Immediately after the pore, pollen tubes show a strong influx domain of protons in the proximal 150–200 μm. This influx decreases along the tube and eventually reverses, becoming a clear efflux of protons in the region of the clear zone. Another reversal of the current direction is observed in the apical dome, characterized by a strong point influx at the very tip, with chaotic small amplitude oscillations for short tubes, or sustained large amplitude oscillations for longer tubes. The clear zone domain of proton efflux is usually not discernible in small tubes (<500 μm), and becomes increasingly well defined from the size of the one depicted. As tubes continue to elongate this pattern evolves subsequently to a banding alternation of small efflux and influx domains over distances of ∼300–400 μm. However the grain + proximal tube and the clear zone + tip-closed loops of proton circulation remain as shown in this figure. Although these patterns have been confirmed in a significant number of tubes (n = 13) the absolute values shown vary considerably with the bath pH and tube length. Bar, 1.0 pmol/cm2/s; pollen grain and tube not drawn exactly to scale; grain is ∼120 × 80 μm and tube is ∼18 μm in diameter.
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