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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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Effect of BCECF-dextran intracellular concentration on the resolution of  pHc. The intracellular concentration was estimated by  in vitro/in vivo calibration  methods (see Materials and  Methods). Only when an estimated concentration of  less than 1.0 μM was obtained could some differentiation of the pHc be observed  (b). In tubes injected to final  concentrations of less than  0.5 μM, e.g., 0.3 μM (c), a  stable spatial pattern of pHc  was always observed, with  a distinct alkaline band  roughly located over the  clear zone area and an acidic  tip, often appearing with the  shape similar to the inverted  cone of vesicles located in  close vicinity to the tip. The  rest of the tube cytosol was  neutral.Figure 4. Comparison of two growing tubes with different diameters (a and b) and a nongrowing tube (c). Although all growing tubes,  regardless of the tube diameter and growth speed, exhibit an alkaline band, only growing tubes show an acidic tip. Note how the alkaline  band extends to the apex in nongrowing tubes (c).
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Figure 3: Effect of BCECF-dextran intracellular concentration on the resolution of pHc. The intracellular concentration was estimated by in vitro/in vivo calibration methods (see Materials and Methods). Only when an estimated concentration of less than 1.0 μM was obtained could some differentiation of the pHc be observed (b). In tubes injected to final concentrations of less than 0.5 μM, e.g., 0.3 μM (c), a stable spatial pattern of pHc was always observed, with a distinct alkaline band roughly located over the clear zone area and an acidic tip, often appearing with the shape similar to the inverted cone of vesicles located in close vicinity to the tip. The rest of the tube cytosol was neutral.Figure 4. Comparison of two growing tubes with different diameters (a and b) and a nongrowing tube (c). Although all growing tubes, regardless of the tube diameter and growth speed, exhibit an alkaline band, only growing tubes show an acidic tip. Note how the alkaline band extends to the apex in nongrowing tubes (c).

Mentions: The results above show that significant extracellular fluxes occur, and define specific domains of proton dynamics along the tube. In some instances, especially at low pHs (∼5.0), the extracellular expression of these fluxes outside the cell is a measurable gradient which, at the extreme of some apical influx peaks, attained values of ∼0.5 pH U over 10 μm. Despite the fact that the intracellular buffering is dramatically different than the weakly buffered germination milieu in which these gradients are formed and detected, it became mandatory to determine if this pattern was in some way expressed inside the tube. We selected BCECF-dextran because it is the most widely used pH probe and has been applied in a number of systems with reliable and reproducible results. Furthermore, the dextran ensures that any pattern that arises is not dependent on organelle sequestration. The first injections were made with a pipette concentration of ∼10 mg/ml. When introduced into the cell at this concentration, streaming was abolished at the point of injection and tube growth halted. As the dye diffused along the tube one could observe a progressive inhibition of cytoplasmic streaming with complete blockage after 15–30 min, and no apparent recovery. It followed that at this concentration BCECF-dextran exerted a pronounced deleterious effect, especially on streaming and growth. Using lower concentrations (1 mg/ml) we found that pollen tubes were relatively unaffected in the growth rates or any other structural feature. However, no gradients or hot spots of pH could be observed (Fig. 3 a). Since under these conditions the signal generated was very high, requiring the use of several neutral density filters and exposures of just a few milliseconds, and since at higher concentrations deleterious effects of the probe have been observed, we tried even lower concentrations of BCECF-dextran to fully explore the camera sensitivity and dynamic range. The concentration of the injection solution was then lowered to 0.7 and then 0.5 mg/ml, again with minimal injection quantities. Below this range, edge artifacts, i.e., optical gradients after ratio provoked by insufficient concentration of the dye on the edges, became evident. Within this range, however, an alkaline gradient became evident in the apical region (Fig. 3 b) and finally, in the optimized conditions for system sensitivity and dynamics, in which there were minimal or no neutral density filters, and in which exposures lasted a few hundred milliseconds, a clear pattern began to emerge that consisted of an alkaline band at the base of the clear zone and an acidic gradient at the extreme apex (Fig. 3 c).


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)

Effect of BCECF-dextran intracellular concentration on the resolution of  pHc. The intracellular concentration was estimated by  in vitro/in vivo calibration  methods (see Materials and  Methods). Only when an estimated concentration of  less than 1.0 μM was obtained could some differentiation of the pHc be observed  (b). In tubes injected to final  concentrations of less than  0.5 μM, e.g., 0.3 μM (c), a  stable spatial pattern of pHc  was always observed, with  a distinct alkaline band  roughly located over the  clear zone area and an acidic  tip, often appearing with the  shape similar to the inverted  cone of vesicles located in  close vicinity to the tip. The  rest of the tube cytosol was  neutral.Figure 4. Comparison of two growing tubes with different diameters (a and b) and a nongrowing tube (c). Although all growing tubes,  regardless of the tube diameter and growth speed, exhibit an alkaline band, only growing tubes show an acidic tip. Note how the alkaline  band extends to the apex in nongrowing tubes (c).
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Related In: Results  -  Collection

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Figure 3: Effect of BCECF-dextran intracellular concentration on the resolution of pHc. The intracellular concentration was estimated by in vitro/in vivo calibration methods (see Materials and Methods). Only when an estimated concentration of less than 1.0 μM was obtained could some differentiation of the pHc be observed (b). In tubes injected to final concentrations of less than 0.5 μM, e.g., 0.3 μM (c), a stable spatial pattern of pHc was always observed, with a distinct alkaline band roughly located over the clear zone area and an acidic tip, often appearing with the shape similar to the inverted cone of vesicles located in close vicinity to the tip. The rest of the tube cytosol was neutral.Figure 4. Comparison of two growing tubes with different diameters (a and b) and a nongrowing tube (c). Although all growing tubes, regardless of the tube diameter and growth speed, exhibit an alkaline band, only growing tubes show an acidic tip. Note how the alkaline band extends to the apex in nongrowing tubes (c).
Mentions: The results above show that significant extracellular fluxes occur, and define specific domains of proton dynamics along the tube. In some instances, especially at low pHs (∼5.0), the extracellular expression of these fluxes outside the cell is a measurable gradient which, at the extreme of some apical influx peaks, attained values of ∼0.5 pH U over 10 μm. Despite the fact that the intracellular buffering is dramatically different than the weakly buffered germination milieu in which these gradients are formed and detected, it became mandatory to determine if this pattern was in some way expressed inside the tube. We selected BCECF-dextran because it is the most widely used pH probe and has been applied in a number of systems with reliable and reproducible results. Furthermore, the dextran ensures that any pattern that arises is not dependent on organelle sequestration. The first injections were made with a pipette concentration of ∼10 mg/ml. When introduced into the cell at this concentration, streaming was abolished at the point of injection and tube growth halted. As the dye diffused along the tube one could observe a progressive inhibition of cytoplasmic streaming with complete blockage after 15–30 min, and no apparent recovery. It followed that at this concentration BCECF-dextran exerted a pronounced deleterious effect, especially on streaming and growth. Using lower concentrations (1 mg/ml) we found that pollen tubes were relatively unaffected in the growth rates or any other structural feature. However, no gradients or hot spots of pH could be observed (Fig. 3 a). Since under these conditions the signal generated was very high, requiring the use of several neutral density filters and exposures of just a few milliseconds, and since at higher concentrations deleterious effects of the probe have been observed, we tried even lower concentrations of BCECF-dextran to fully explore the camera sensitivity and dynamic range. The concentration of the injection solution was then lowered to 0.7 and then 0.5 mg/ml, again with minimal injection quantities. Below this range, edge artifacts, i.e., optical gradients after ratio provoked by insufficient concentration of the dye on the edges, became evident. Within this range, however, an alkaline gradient became evident in the apical region (Fig. 3 b) and finally, in the optimized conditions for system sensitivity and dynamics, in which there were minimal or no neutral density filters, and in which exposures lasted a few hundred milliseconds, a clear pattern began to emerge that consisted of an alkaline band at the base of the clear zone and an acidic gradient at the extreme apex (Fig. 3 c).

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