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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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Related in: MedlinePlus

pHc time sequence  of a growing pollen tube.  Numbers on top of the tubes  are seconds from the beginning of the sequence. The  mean interval between  frames is 16.5 s. An elevation  of pHc in the alkaline band is  clearly visible at times 33, 83,  and 132 s, reflecting possible  oscillations of pHc in this  area of the cytosol. The images also suggest some variations of the acidic tip, but  these ones proved to be more  difficult to quantify. Also visible is the change of shape of  the acidic tip. At times 17, 50,  and 132 s it has a biconvex  shape. However, at times 33,  83, and 116 s the shape  changes to plano-convex,  and at time 66 s it shows the  typical inverted cone configuration. Frames at 99, 149,  and 165 s show asymmetric  acidic tips which, in the later  two cases, follow the change  in growth direction.Figure 9. Typical result of an  experiment in which growth  was arrested with an osmotic  shock and allowed to restart  after replacement of the normal medium. Numbers represent time in minutes from  the beginning of the sequence. The sequence shows  the moment where growth  restarts (third frame, time 5  min). The alkaline band is  slightly asymmetric, with the  higher values closer to the  half tube from which the new  tip is going to protrude. Before this happens no acidic  tip is clearly discernible. Yet,  when the new tip arises  (fourth and fifth frames, 10–12 min.) it defines a slightly acidic area of cytosol. The alkaline band then follows the acidic tip into the  newly formed tube, which is easy to spot due to its smaller diameter (sixth frame, 15 min) (refer to Fig. 11 for calibration wedge).Figure 10. Comparison of the clear zone extension and the cytosol alkaline band. Three tubes with increasing lengths of their clear zone  are shown in transmitted light (a–c) and the respective image for pHc (a′ to c′). Even though all tubes are growing with similar growth  rates, they possess very different clear zones. Tip a belonged to a very elongated tube (>3.0 mm) and has a clear zone restricted to the  terminal 15 μm, behind which the streaming of large organelles was vigorous. Correspondingly, the alkaline band was more restricted  but the acidic tip appeared normal (compare with Fig. 5 a). Tube b represents the more typical situation, with a clear zone of ∼50 μm.  In these tubes the alkaline band is very well defined, and its length extends to about the point where the streaming of the large organelles terminates. More rarely, tubes with very extended clear zones (sometimes over 200 μm) were also observed, as in c. In this case  the alkaline band is also very well defined, but several hot spots of pH elevation occur along the tube membrane (c′). These elevated regions fade where the streaming terminates. Note that in the three cases shown, the acidic tip remains more or less equivalent in size and  magnitude (refer to Fig. 11 for calibration wedge).Figure 11. Effect of buffer injection on pHc of a growing pollen tube. A standard growing tube was first injected with BCECF-dextran  and imaged (first frame). As expected, it shows the alkaline band and the acidic tip. It was then further injected with Hepes (see Materials and Methods) and followed for determination of the buffer effect. The second frame was obtained immediately after needle withdrawal, and ∼5 min after the buffer injection. Growth rate was nearly the same but the alkaline band seems to have been narrowed. 15  min after injection (third frame), the tube continued to grow, although at a lower rate (from 15.0 to 9.0 μm/min) but the alkaline band is  now either absent or beyond the resolution limit of the imaging system.
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Figure 8: pHc time sequence of a growing pollen tube. Numbers on top of the tubes are seconds from the beginning of the sequence. The mean interval between frames is 16.5 s. An elevation of pHc in the alkaline band is clearly visible at times 33, 83, and 132 s, reflecting possible oscillations of pHc in this area of the cytosol. The images also suggest some variations of the acidic tip, but these ones proved to be more difficult to quantify. Also visible is the change of shape of the acidic tip. At times 17, 50, and 132 s it has a biconvex shape. However, at times 33, 83, and 116 s the shape changes to plano-convex, and at time 66 s it shows the typical inverted cone configuration. Frames at 99, 149, and 165 s show asymmetric acidic tips which, in the later two cases, follow the change in growth direction.Figure 9. Typical result of an experiment in which growth was arrested with an osmotic shock and allowed to restart after replacement of the normal medium. Numbers represent time in minutes from the beginning of the sequence. The sequence shows the moment where growth restarts (third frame, time 5 min). The alkaline band is slightly asymmetric, with the higher values closer to the half tube from which the new tip is going to protrude. Before this happens no acidic tip is clearly discernible. Yet, when the new tip arises (fourth and fifth frames, 10–12 min.) it defines a slightly acidic area of cytosol. The alkaline band then follows the acidic tip into the newly formed tube, which is easy to spot due to its smaller diameter (sixth frame, 15 min) (refer to Fig. 11 for calibration wedge).Figure 10. Comparison of the clear zone extension and the cytosol alkaline band. Three tubes with increasing lengths of their clear zone are shown in transmitted light (a–c) and the respective image for pHc (a′ to c′). Even though all tubes are growing with similar growth rates, they possess very different clear zones. Tip a belonged to a very elongated tube (>3.0 mm) and has a clear zone restricted to the terminal 15 μm, behind which the streaming of large organelles was vigorous. Correspondingly, the alkaline band was more restricted but the acidic tip appeared normal (compare with Fig. 5 a). Tube b represents the more typical situation, with a clear zone of ∼50 μm. In these tubes the alkaline band is very well defined, and its length extends to about the point where the streaming of the large organelles terminates. More rarely, tubes with very extended clear zones (sometimes over 200 μm) were also observed, as in c. In this case the alkaline band is also very well defined, but several hot spots of pH elevation occur along the tube membrane (c′). These elevated regions fade where the streaming terminates. Note that in the three cases shown, the acidic tip remains more or less equivalent in size and magnitude (refer to Fig. 11 for calibration wedge).Figure 11. Effect of buffer injection on pHc of a growing pollen tube. A standard growing tube was first injected with BCECF-dextran and imaged (first frame). As expected, it shows the alkaline band and the acidic tip. It was then further injected with Hepes (see Materials and Methods) and followed for determination of the buffer effect. The second frame was obtained immediately after needle withdrawal, and ∼5 min after the buffer injection. Growth rate was nearly the same but the alkaline band seems to have been narrowed. 15 min after injection (third frame), the tube continued to grow, although at a lower rate (from 15.0 to 9.0 μm/min) but the alkaline band is now either absent or beyond the resolution limit of the imaging system.

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)

pHc time sequence  of a growing pollen tube.  Numbers on top of the tubes  are seconds from the beginning of the sequence. The  mean interval between  frames is 16.5 s. An elevation  of pHc in the alkaline band is  clearly visible at times 33, 83,  and 132 s, reflecting possible  oscillations of pHc in this  area of the cytosol. The images also suggest some variations of the acidic tip, but  these ones proved to be more  difficult to quantify. Also visible is the change of shape of  the acidic tip. At times 17, 50,  and 132 s it has a biconvex  shape. However, at times 33,  83, and 116 s the shape  changes to plano-convex,  and at time 66 s it shows the  typical inverted cone configuration. Frames at 99, 149,  and 165 s show asymmetric  acidic tips which, in the later  two cases, follow the change  in growth direction.Figure 9. Typical result of an  experiment in which growth  was arrested with an osmotic  shock and allowed to restart  after replacement of the normal medium. Numbers represent time in minutes from  the beginning of the sequence. The sequence shows  the moment where growth  restarts (third frame, time 5  min). The alkaline band is  slightly asymmetric, with the  higher values closer to the  half tube from which the new  tip is going to protrude. Before this happens no acidic  tip is clearly discernible. Yet,  when the new tip arises  (fourth and fifth frames, 10–12 min.) it defines a slightly acidic area of cytosol. The alkaline band then follows the acidic tip into the  newly formed tube, which is easy to spot due to its smaller diameter (sixth frame, 15 min) (refer to Fig. 11 for calibration wedge).Figure 10. Comparison of the clear zone extension and the cytosol alkaline band. Three tubes with increasing lengths of their clear zone  are shown in transmitted light (a–c) and the respective image for pHc (a′ to c′). Even though all tubes are growing with similar growth  rates, they possess very different clear zones. Tip a belonged to a very elongated tube (>3.0 mm) and has a clear zone restricted to the  terminal 15 μm, behind which the streaming of large organelles was vigorous. Correspondingly, the alkaline band was more restricted  but the acidic tip appeared normal (compare with Fig. 5 a). Tube b represents the more typical situation, with a clear zone of ∼50 μm.  In these tubes the alkaline band is very well defined, and its length extends to about the point where the streaming of the large organelles terminates. More rarely, tubes with very extended clear zones (sometimes over 200 μm) were also observed, as in c. In this case  the alkaline band is also very well defined, but several hot spots of pH elevation occur along the tube membrane (c′). These elevated regions fade where the streaming terminates. Note that in the three cases shown, the acidic tip remains more or less equivalent in size and  magnitude (refer to Fig. 11 for calibration wedge).Figure 11. Effect of buffer injection on pHc of a growing pollen tube. A standard growing tube was first injected with BCECF-dextran  and imaged (first frame). As expected, it shows the alkaline band and the acidic tip. It was then further injected with Hepes (see Materials and Methods) and followed for determination of the buffer effect. The second frame was obtained immediately after needle withdrawal, and ∼5 min after the buffer injection. Growth rate was nearly the same but the alkaline band seems to have been narrowed. 15  min after injection (third frame), the tube continued to grow, although at a lower rate (from 15.0 to 9.0 μm/min) but the alkaline band is  now either absent or beyond the resolution limit of the imaging system.
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Figure 8: pHc time sequence of a growing pollen tube. Numbers on top of the tubes are seconds from the beginning of the sequence. The mean interval between frames is 16.5 s. An elevation of pHc in the alkaline band is clearly visible at times 33, 83, and 132 s, reflecting possible oscillations of pHc in this area of the cytosol. The images also suggest some variations of the acidic tip, but these ones proved to be more difficult to quantify. Also visible is the change of shape of the acidic tip. At times 17, 50, and 132 s it has a biconvex shape. However, at times 33, 83, and 116 s the shape changes to plano-convex, and at time 66 s it shows the typical inverted cone configuration. Frames at 99, 149, and 165 s show asymmetric acidic tips which, in the later two cases, follow the change in growth direction.Figure 9. Typical result of an experiment in which growth was arrested with an osmotic shock and allowed to restart after replacement of the normal medium. Numbers represent time in minutes from the beginning of the sequence. The sequence shows the moment where growth restarts (third frame, time 5 min). The alkaline band is slightly asymmetric, with the higher values closer to the half tube from which the new tip is going to protrude. Before this happens no acidic tip is clearly discernible. Yet, when the new tip arises (fourth and fifth frames, 10–12 min.) it defines a slightly acidic area of cytosol. The alkaline band then follows the acidic tip into the newly formed tube, which is easy to spot due to its smaller diameter (sixth frame, 15 min) (refer to Fig. 11 for calibration wedge).Figure 10. Comparison of the clear zone extension and the cytosol alkaline band. Three tubes with increasing lengths of their clear zone are shown in transmitted light (a–c) and the respective image for pHc (a′ to c′). Even though all tubes are growing with similar growth rates, they possess very different clear zones. Tip a belonged to a very elongated tube (>3.0 mm) and has a clear zone restricted to the terminal 15 μm, behind which the streaming of large organelles was vigorous. Correspondingly, the alkaline band was more restricted but the acidic tip appeared normal (compare with Fig. 5 a). Tube b represents the more typical situation, with a clear zone of ∼50 μm. In these tubes the alkaline band is very well defined, and its length extends to about the point where the streaming of the large organelles terminates. More rarely, tubes with very extended clear zones (sometimes over 200 μm) were also observed, as in c. In this case the alkaline band is also very well defined, but several hot spots of pH elevation occur along the tube membrane (c′). These elevated regions fade where the streaming terminates. Note that in the three cases shown, the acidic tip remains more or less equivalent in size and magnitude (refer to Fig. 11 for calibration wedge).Figure 11. Effect of buffer injection on pHc of a growing pollen tube. A standard growing tube was first injected with BCECF-dextran and imaged (first frame). As expected, it shows the alkaline band and the acidic tip. It was then further injected with Hepes (see Materials and Methods) and followed for determination of the buffer effect. The second frame was obtained immediately after needle withdrawal, and ∼5 min after the buffer injection. Growth rate was nearly the same but the alkaline band seems to have been narrowed. 15 min after injection (third frame), the tube continued to grow, although at a lower rate (from 15.0 to 9.0 μm/min) but the alkaline band is now either absent or beyond the resolution limit of the imaging system.
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