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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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Ratio calculation  and calibration from raw images. (a and b) Typical raw  images of the pH-independent and the pH-dependent  BCECF excitation wavelengths, without background  subtraction. (c) In vitro calibration from raw images as  collected in exactly the same  filter and integration conditions. The left half (D) represents the pH-dependent excitation wavelength and the  right half (I) represents the  pH-independent excitation  wavelength. Calibrations were  made in pseudocytosol medium (see Materials and  Methods) contained 0.35 μM  BCECF-dextran, and an approximate thickness of 15 μm  between slide and coverslip  (as calculated from the volume applied). Note that the  pixel intensity in the independent channel is similar in the three images and similar to the pixel intensity along the midtransect of the pollen tube independent wavelength image. (d) Ratio values as calculated directly from the raw images (a, b, and c) without background subtraction. The lines represent the pixel profiles along the midsection of the tube and their ratio (dependent/independent × 130). The horizontal lines represent  the calibration ratio value calculated by averaging the pixel intensity of square boxes with 2,000 μm2, as in c.Figure 6. Image of a growing BCECF-loaded tube that impacts an unloaded tube (arrow). (a) Mixed fluorescence/transmitted light image shows the relative position of the two tubes. (b) The fluorescence collected in the pH-independent channel and (c) in the pH-dependent channel. The graph plotted in d summarizes the mid line scans of both b and c, their respective ratio (dependent/independent ×  130) and the calibration values collected under the same imaging conditions (see Fig. 5). The two bottom traces correspond to mid line  scans along the unloaded tube (arrows) from the fluorescence images b and c. The slight elevation at 30 μm corresponds to the area  where the tip of the loaded tube abuts the unloaded tube. The mean pixel intensity plotted in the upper and lower traces can be taken as  an indirect measure of the signal/noise ratio of the system used, namely, ∼10-fold. An acidic tip in the loaded tube is not seen because  the tube slowed markedly in growth after striking the unloaded tube.
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Figure 5: Ratio calculation and calibration from raw images. (a and b) Typical raw images of the pH-independent and the pH-dependent BCECF excitation wavelengths, without background subtraction. (c) In vitro calibration from raw images as collected in exactly the same filter and integration conditions. The left half (D) represents the pH-dependent excitation wavelength and the right half (I) represents the pH-independent excitation wavelength. Calibrations were made in pseudocytosol medium (see Materials and Methods) contained 0.35 μM BCECF-dextran, and an approximate thickness of 15 μm between slide and coverslip (as calculated from the volume applied). Note that the pixel intensity in the independent channel is similar in the three images and similar to the pixel intensity along the midtransect of the pollen tube independent wavelength image. (d) Ratio values as calculated directly from the raw images (a, b, and c) without background subtraction. The lines represent the pixel profiles along the midsection of the tube and their ratio (dependent/independent × 130). The horizontal lines represent the calibration ratio value calculated by averaging the pixel intensity of square boxes with 2,000 μm2, as in c.Figure 6. Image of a growing BCECF-loaded tube that impacts an unloaded tube (arrow). (a) Mixed fluorescence/transmitted light image shows the relative position of the two tubes. (b) The fluorescence collected in the pH-independent channel and (c) in the pH-dependent channel. The graph plotted in d summarizes the mid line scans of both b and c, their respective ratio (dependent/independent × 130) and the calibration values collected under the same imaging conditions (see Fig. 5). The two bottom traces correspond to mid line scans along the unloaded tube (arrows) from the fluorescence images b and c. The slight elevation at 30 μm corresponds to the area where the tip of the loaded tube abuts the unloaded tube. The mean pixel intensity plotted in the upper and lower traces can be taken as an indirect measure of the signal/noise ratio of the system used, namely, ∼10-fold. An acidic tip in the loaded tube is not seen because the tube slowed markedly in growth after striking the unloaded tube.

Mentions: A detailed exploration of the tip area in one of these longer tubes (∼1.5 mm) is shown in Fig. 2. The plot shows a real-time trace of the vibrating probe, taken with a frequency of 0.5 Hz and an excursion of 10 μm. Under these operating conditions, and with the software used, a datum was generated every 3.3 s. The measuring routine is shown in the inserted cartoon. The probe was first driven to follow the growing apex. As it is shown, the apical domain possesses well-defined, oscillating peaks of proton influx. These oscillations are sustained during long periods of time with sequences up to 30 min being routinely detected, which exhibit stable period and amplitude. In the experiment shown the tip was followed for ∼10 min, after which the probe's direction was changed 90° and the subapical domains were mapped. A clear-cut reversal of the current was detected about one tube diameter behind the apical dome, which then peaked in the midpoint of the clear zone (point A in Fig. 2). This specific plot was obtained by halting the probe vibration position, and letting the tube grow past the probe. Therefore, the up and down of the trace reflects the flux topography along the tube, as if the tube had not grown, and the probe was continuously translated along the side of the membrane. At about the point where the clear zone ends and the normal streaming of large organelles is observed, the flux direction reverses again to an influx domain (point B in Fig. 2), which in this example, extends ∼100 μm. Along the tube other reversals were then observed, but with much less defined spatial borders, and much lower flux magnitudes. The inserted background references refer to both directions of vibration and clearly show an insignificant noise level, and a very good signal/noise ratio for all measurements plotted in this chart. A spatial representation of a data set similar to this one is shown in Fig. 5 b. It should be stressed that although the lateral fluxes are consistently stable, the vector depicted at the apex is the average of the oscillating flux integrated over 10 min. As will be described, the extracellular proton flux pattern closely matches the adjacent intracellular pattern of pHc.


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)

Ratio calculation  and calibration from raw images. (a and b) Typical raw  images of the pH-independent and the pH-dependent  BCECF excitation wavelengths, without background  subtraction. (c) In vitro calibration from raw images as  collected in exactly the same  filter and integration conditions. The left half (D) represents the pH-dependent excitation wavelength and the  right half (I) represents the  pH-independent excitation  wavelength. Calibrations were  made in pseudocytosol medium (see Materials and  Methods) contained 0.35 μM  BCECF-dextran, and an approximate thickness of 15 μm  between slide and coverslip  (as calculated from the volume applied). Note that the  pixel intensity in the independent channel is similar in the three images and similar to the pixel intensity along the midtransect of the pollen tube independent wavelength image. (d) Ratio values as calculated directly from the raw images (a, b, and c) without background subtraction. The lines represent the pixel profiles along the midsection of the tube and their ratio (dependent/independent × 130). The horizontal lines represent  the calibration ratio value calculated by averaging the pixel intensity of square boxes with 2,000 μm2, as in c.Figure 6. Image of a growing BCECF-loaded tube that impacts an unloaded tube (arrow). (a) Mixed fluorescence/transmitted light image shows the relative position of the two tubes. (b) The fluorescence collected in the pH-independent channel and (c) in the pH-dependent channel. The graph plotted in d summarizes the mid line scans of both b and c, their respective ratio (dependent/independent ×  130) and the calibration values collected under the same imaging conditions (see Fig. 5). The two bottom traces correspond to mid line  scans along the unloaded tube (arrows) from the fluorescence images b and c. The slight elevation at 30 μm corresponds to the area  where the tip of the loaded tube abuts the unloaded tube. The mean pixel intensity plotted in the upper and lower traces can be taken as  an indirect measure of the signal/noise ratio of the system used, namely, ∼10-fold. An acidic tip in the loaded tube is not seen because  the tube slowed markedly in growth after striking the unloaded tube.
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Related In: Results  -  Collection

Show All Figures
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Figure 5: Ratio calculation and calibration from raw images. (a and b) Typical raw images of the pH-independent and the pH-dependent BCECF excitation wavelengths, without background subtraction. (c) In vitro calibration from raw images as collected in exactly the same filter and integration conditions. The left half (D) represents the pH-dependent excitation wavelength and the right half (I) represents the pH-independent excitation wavelength. Calibrations were made in pseudocytosol medium (see Materials and Methods) contained 0.35 μM BCECF-dextran, and an approximate thickness of 15 μm between slide and coverslip (as calculated from the volume applied). Note that the pixel intensity in the independent channel is similar in the three images and similar to the pixel intensity along the midtransect of the pollen tube independent wavelength image. (d) Ratio values as calculated directly from the raw images (a, b, and c) without background subtraction. The lines represent the pixel profiles along the midsection of the tube and their ratio (dependent/independent × 130). The horizontal lines represent the calibration ratio value calculated by averaging the pixel intensity of square boxes with 2,000 μm2, as in c.Figure 6. Image of a growing BCECF-loaded tube that impacts an unloaded tube (arrow). (a) Mixed fluorescence/transmitted light image shows the relative position of the two tubes. (b) The fluorescence collected in the pH-independent channel and (c) in the pH-dependent channel. The graph plotted in d summarizes the mid line scans of both b and c, their respective ratio (dependent/independent × 130) and the calibration values collected under the same imaging conditions (see Fig. 5). The two bottom traces correspond to mid line scans along the unloaded tube (arrows) from the fluorescence images b and c. The slight elevation at 30 μm corresponds to the area where the tip of the loaded tube abuts the unloaded tube. The mean pixel intensity plotted in the upper and lower traces can be taken as an indirect measure of the signal/noise ratio of the system used, namely, ∼10-fold. An acidic tip in the loaded tube is not seen because the tube slowed markedly in growth after striking the unloaded tube.
Mentions: A detailed exploration of the tip area in one of these longer tubes (∼1.5 mm) is shown in Fig. 2. The plot shows a real-time trace of the vibrating probe, taken with a frequency of 0.5 Hz and an excursion of 10 μm. Under these operating conditions, and with the software used, a datum was generated every 3.3 s. The measuring routine is shown in the inserted cartoon. The probe was first driven to follow the growing apex. As it is shown, the apical domain possesses well-defined, oscillating peaks of proton influx. These oscillations are sustained during long periods of time with sequences up to 30 min being routinely detected, which exhibit stable period and amplitude. In the experiment shown the tip was followed for ∼10 min, after which the probe's direction was changed 90° and the subapical domains were mapped. A clear-cut reversal of the current was detected about one tube diameter behind the apical dome, which then peaked in the midpoint of the clear zone (point A in Fig. 2). This specific plot was obtained by halting the probe vibration position, and letting the tube grow past the probe. Therefore, the up and down of the trace reflects the flux topography along the tube, as if the tube had not grown, and the probe was continuously translated along the side of the membrane. At about the point where the clear zone ends and the normal streaming of large organelles is observed, the flux direction reverses again to an influx domain (point B in Fig. 2), which in this example, extends ∼100 μm. Along the tube other reversals were then observed, but with much less defined spatial borders, and much lower flux magnitudes. The inserted background references refer to both directions of vibration and clearly show an insignificant noise level, and a very good signal/noise ratio for all measurements plotted in this chart. A spatial representation of a data set similar to this one is shown in Fig. 5 b. It should be stressed that although the lateral fluxes are consistently stable, the vector depicted at the apex is the average of the oscillating flux integrated over 10 min. As will be described, the extracellular proton flux pattern closely matches the adjacent intracellular pattern of pHc.

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