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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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Correlation between pHc oscillations in the alkaline  band and growth rate on the same tube. At the low probe intracellular concentrations used, the time interval (25 s) shown represents the best possible compromise between fading and time-resolution. Although not allowing precise correlation, oscillatory  variations (i.e., with at least three full cycles) of pHc in the alkaline band were detected, with periods ranging from 25 to 55 s. In  most of these the correlation of pHc at the alkaline band and the  growth rate showed the trend depicted in this plot, i.e., when  growth goes up, the pH goes down (or, in other words, proton  concentration goes up). At this time resolution, however, a significant portion of the wave function is lost and precise correlation  is not possible.
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Figure 12: Correlation between pHc oscillations in the alkaline band and growth rate on the same tube. At the low probe intracellular concentrations used, the time interval (25 s) shown represents the best possible compromise between fading and time-resolution. Although not allowing precise correlation, oscillatory variations (i.e., with at least three full cycles) of pHc in the alkaline band were detected, with periods ranging from 25 to 55 s. In most of these the correlation of pHc at the alkaline band and the growth rate showed the trend depicted in this plot, i.e., when growth goes up, the pH goes down (or, in other words, proton concentration goes up). At this time resolution, however, a significant portion of the wave function is lost and precise correlation is not possible.

Mentions: Since most of the tubes over 800 μm display oscillatory proton influxes at the tip and oscillatory growth rates, we then tried to follow the variations of pH during growth. A typical result is displayed in Fig. 8. Two features emerge from these sequences: first, there is a periodic change in pHc, especially expressed in the alkaline band, and second, the shape and position of the acidic tip also changes, from plano-convex when the alkaline band pH is higher, to an inverted, or bi-convex cone when the pH decreases (see figure legend for details). Furthermore, in the final two frames, where the tube bends, the acidic tip is also asymmetric with the lowest pH region corresponding to the direction of pollen tube elongation. The deformation of the acidic tip would then be consistent with a flux of protons entering through the extreme apex, which would lower pHc in the alkaline band. Further analysis of this correlation was assayed and the variations of the alkaline band pH were plotted together with the growth rate (see Fig. 12). The use of diluted probe solutions renders the injected cells susceptible to photobleaching, restricting the time resolution to 15–20 s. The sequence plotted had an interval of 16.5 s between consecutive frames. Thus, although precise correlation is difficult, the global trend shows that when pHc increases in the alkaline band, the growth rates decrease, and conversely, when pHc decreases (or in other words, proton concentration increases) the growth rates increase. Taken together, the results are globally consistent with the model of a constitutive alkaline band that is, at least partly, generated by proton extrusion, and a slightly acidic tip that is, at least partly, dependent upon localized proton influx. Moreover, pollen tube elongation appears to be correlated with the concentration of protons in the extreme apex.


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)

Correlation between pHc oscillations in the alkaline  band and growth rate on the same tube. At the low probe intracellular concentrations used, the time interval (25 s) shown represents the best possible compromise between fading and time-resolution. Although not allowing precise correlation, oscillatory  variations (i.e., with at least three full cycles) of pHc in the alkaline band were detected, with periods ranging from 25 to 55 s. In  most of these the correlation of pHc at the alkaline band and the  growth rate showed the trend depicted in this plot, i.e., when  growth goes up, the pH goes down (or, in other words, proton  concentration goes up). At this time resolution, however, a significant portion of the wave function is lost and precise correlation  is not possible.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 12: Correlation between pHc oscillations in the alkaline band and growth rate on the same tube. At the low probe intracellular concentrations used, the time interval (25 s) shown represents the best possible compromise between fading and time-resolution. Although not allowing precise correlation, oscillatory variations (i.e., with at least three full cycles) of pHc in the alkaline band were detected, with periods ranging from 25 to 55 s. In most of these the correlation of pHc at the alkaline band and the growth rate showed the trend depicted in this plot, i.e., when growth goes up, the pH goes down (or, in other words, proton concentration goes up). At this time resolution, however, a significant portion of the wave function is lost and precise correlation is not possible.
Mentions: Since most of the tubes over 800 μm display oscillatory proton influxes at the tip and oscillatory growth rates, we then tried to follow the variations of pH during growth. A typical result is displayed in Fig. 8. Two features emerge from these sequences: first, there is a periodic change in pHc, especially expressed in the alkaline band, and second, the shape and position of the acidic tip also changes, from plano-convex when the alkaline band pH is higher, to an inverted, or bi-convex cone when the pH decreases (see figure legend for details). Furthermore, in the final two frames, where the tube bends, the acidic tip is also asymmetric with the lowest pH region corresponding to the direction of pollen tube elongation. The deformation of the acidic tip would then be consistent with a flux of protons entering through the extreme apex, which would lower pHc in the alkaline band. Further analysis of this correlation was assayed and the variations of the alkaline band pH were plotted together with the growth rate (see Fig. 12). The use of diluted probe solutions renders the injected cells susceptible to photobleaching, restricting the time resolution to 15–20 s. The sequence plotted had an interval of 16.5 s between consecutive frames. Thus, although precise correlation is difficult, the global trend shows that when pHc increases in the alkaline band, the growth rates decrease, and conversely, when pHc decreases (or in other words, proton concentration increases) the growth rates increase. Taken together, the results are globally consistent with the model of a constitutive alkaline band that is, at least partly, generated by proton extrusion, and a slightly acidic tip that is, at least partly, dependent upon localized proton influx. Moreover, pollen tube elongation appears to be correlated with the concentration of protons in the extreme apex.

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