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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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Comparison of the  pHc (Fig. 7 a) and the extracellular proton fluxes (Fig. 7  b) in two tubes with matching sizes, growth rates,  length, and extracellular pH.  A close correlation is observable between the cytosolic alkaline band and the patterns  of proton efflux, in which  both roughly correspond to  the domain occupied by the  clear zone. Again the tip inverted cone is clearly visible  in the pHc picture. Although  these patterns were obtained  in different tubes, they show  a close correlation, suggesting that the elevation of pHc  in the clear zone may correspond (at least partly) to an  active proton efflux in the  same area.
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Figure 7: Comparison of the pHc (Fig. 7 a) and the extracellular proton fluxes (Fig. 7 b) in two tubes with matching sizes, growth rates, length, and extracellular pH. A close correlation is observable between the cytosolic alkaline band and the patterns of proton efflux, in which both roughly correspond to the domain occupied by the clear zone. Again the tip inverted cone is clearly visible in the pHc picture. Although these patterns were obtained in different tubes, they show a close correlation, suggesting that the elevation of pHc in the clear zone may correspond (at least partly) to an active proton efflux in the same area.

Mentions: The correlation of the intracellular and extracellular patterns was then established, as shown in Fig. 7. Simultaneous imaging and vibrating probe analysis, due to technical limitations, has not been possible. Therefore, correlations were made through the comparison of two different pollen tubes, which were chosen with similar growing and structural characteristics (growth medium, length, growth rate, tube diameter, and clear zone extension). Six tubes were compared with very similar characteristics. A correlation of the one depicted in Fig. 7 revealed that the alkaline band corresponded generally to the proton efflux domain.


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)

Comparison of the  pHc (Fig. 7 a) and the extracellular proton fluxes (Fig. 7  b) in two tubes with matching sizes, growth rates,  length, and extracellular pH.  A close correlation is observable between the cytosolic alkaline band and the patterns  of proton efflux, in which  both roughly correspond to  the domain occupied by the  clear zone. Again the tip inverted cone is clearly visible  in the pHc picture. Although  these patterns were obtained  in different tubes, they show  a close correlation, suggesting that the elevation of pHc  in the clear zone may correspond (at least partly) to an  active proton efflux in the  same area.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132912&req=5

Figure 7: Comparison of the pHc (Fig. 7 a) and the extracellular proton fluxes (Fig. 7 b) in two tubes with matching sizes, growth rates, length, and extracellular pH. A close correlation is observable between the cytosolic alkaline band and the patterns of proton efflux, in which both roughly correspond to the domain occupied by the clear zone. Again the tip inverted cone is clearly visible in the pHc picture. Although these patterns were obtained in different tubes, they show a close correlation, suggesting that the elevation of pHc in the clear zone may correspond (at least partly) to an active proton efflux in the same area.
Mentions: The correlation of the intracellular and extracellular patterns was then established, as shown in Fig. 7. Simultaneous imaging and vibrating probe analysis, due to technical limitations, has not been possible. Therefore, correlations were made through the comparison of two different pollen tubes, which were chosen with similar growing and structural characteristics (growth medium, length, growth rate, tube diameter, and clear zone extension). Six tubes were compared with very similar characteristics. A correlation of the one depicted in Fig. 7 revealed that the alkaline band corresponded generally to the proton efflux domain.

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