Pollen Tube Reorientation Research Articles

Regulation of pollen tube polarity

Targeted delivery of immotile sperm through growing pollen tubes is a crucial step in achieving sexual reproduction in angiosperms. Unlike diffuse-growing cells, the growth of a pollen tube is restricted to the very apical region where targeted exocytosis and regulated endocytosis occur. The plant-specific Rho GTPases, Rops, are central organizers for pollen tube polarity. Through effector binding, Rops regulate the tip-focused Ca2+ gradient and the actin cytoskeleton during pollen tube growth. Therefore, understanding the spatiotemporal regulation of Rop activity would reveal how establishment and maintenance of tube polarity as well as re-orientation of the growth axis are accomplished. Recent findings indicated that two feedback loops may be fundamental in maintaining a fine-tuned and dynamic Rop activity. The concerted activities of RopGAP and RopGDI prevent lateral diffusion of activated Rop, restricting Rop activity to the apical plasma membrane. Conversely, pollen receptor kinases (PRKs) and RopGEFs positively feedback regulate Rop activity through protein binding and membrane recruitment. Feedback loops would also be essential for pollen tube re-orientation. Shallow extracellular cues amplified by concerted activities of feedback loops would lead to asymmetric activation of Rop and result in tube re-orientation.

Endo/exocytosis in the pollen tube apex is differentially regulated by Ca2+ and GTPases.

Pollen tube growth relies on an extremely fast delivery of new membrane and wall material to the apical region where growth takes place. Despite the obvious meaning of this fact, the mechanisms that control this process remain very much unknown. It has previously been shown that apical growth is regulated by cytosolic free calcium ([Ca(2+)](c)) so it was decided to test how changes in [Ca(2+)](c) affect endo/exocytosis in pollen tube growth and reorientation. The endo/exocytosis was assayed in living cells using confocal imaging of FM 1-43. It was found that growing pollen tubes exhibited a higher endo/exocytosis activity in the apical region whereas in non-growing cells FM 1-43 is uniformly distributed. During pollen tube reorientation, a spatial redistribution of exocytotic activity was observed with the highest fluorescence in the side to which the cell will bend. Localized increases in [Ca(2+)](c) induced by photolysis of caged Ca(2+) increased exocytosis. In order to find if [Ca(2+)](c) changes were modulating endo/exocytosis directly or through a signalling cascade, tests were conducted to find how changes in GTP levels and GTPase activity (primary regulators of the secretory pathway) affect the apical [Ca(2+)](c) gradient and endo/exocytosis. It was found that increases in GTP levels could promote exocytosis (and growth). Interestingly, the increase in [GTP] did not significantly affect [Ca(2+)](c) distribution, thus suggesting that the apical endo/exocytosis is regulated in a concerted but differentiated manner by the Ca(2+) gradient and the activity of GTPases. Rop GTPases are likely candidates to mediate the Ca(2+)/GTP cross-talk as shown by knock-down experiments in growing pollen tubes.

Endo/exocytosis in the pollen tube apex is differentially regulated by Ca2+ and GTPases

Pollen tube growth relies on an extremely fast delivery of new membrane and wall material to the apical region where growth takes place. Despite the obvious meaning of this fact, the mechanisms that control this process remain very much unknown. It has previously been shown that apical growth is regulated by cytosolic free calcium ([Ca2+]c) so it was decided to test how changes in [Ca2+]c affect endo/exocytosis in pollen tube growth and reorientation. The endo/exocytosis was assayed in living cells using confocal imaging of FM 1‐43. It was found that growing pollen tubes exhibited a higher endo/exocytosis activity in the apical region whereas in non‐growing cells FM 1‐43 is uniformly distributed. During pollen tube reorientation, a spatial redistribution of exocytotic activity was observed with the highest fluorescence in the side to which the cell will bend. Localized increases in [Ca2+]c induced by photolysis of caged Ca2+ increased exocytosis. In order to find if [Ca2+]c changes were modulating endo/exocytosis directly or through a signalling cascade, tests were conducted to find how changes in GTP levels and GTPase activity (primary regulators of the secretory pathway) affect the apical [Ca2+]c gradient and endo/exocytosis. It was found that increases in GTP levels could promote exocytosis (and growth). Interestingly, the increase in [GTP] did not significantly affect [Ca2+]c distribution, thus suggesting that the apical endo/exocytosis is regulated in a concerted but differentiated manner by the Ca2+ gradient and the activity of GTPases. Rop GTPases are likely candidates to mediate the Ca2+/GTP cross‐talk as shown by knock‐down experiments in growing pollen tubes.

Role of 1,4,5-inositol triphosphate-induced Ca 2+ release in pollen tube orientation

Pollen tube reorientation is a dynamic cellular event crucial for successful fertilization. Previously, it was shown that reorientation is preceded by an asymmetric increase of cytosolic free calcium ([Ca2+]c) in the side of the apex to which the cell will bend. In order to find the targets for this signal transduction pathway, the effects of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the reorientation process were analyzed. Ins(1,4,5)P3 was artificially increased in different cell domains by localized photoactivation of caged Ins(1,4,5)P3 and its effects on [Ca2+]c monitored by ion confocal microscopy. It was found that photolysis of caged Ins(1,4,5)P3 in the nuclear or subapical region resulted in a transient increase in [Ca2+]c and reorientation of the growth axis, while photolysis in the apex frequently resulted in disturbed growth or tip bursting. Perfusion of the cells with the Ins(1,4,5)P3 receptor blocker heparin prior to photoactivation inhibited the increase in [Ca2+]c and no reorientation was observed. Ca2+ release from Ins(1,4,5)P3-dependent stores localized in the shank of the tube thus seems to be part of the signal transduction pathway that controls tube guidance, although not the primary stimulus leading to reorientation.

Relocation of a Ca2+-dependent protein kinase activity during pollen tube reorientation

Pollen tube reorientation is a dynamic cellular event that is crucial for successful fertilization. We have shown previously that pollen tube orientation is regulated by cytosolic free calcium ([Ca2+]c). In this paper, we studied the activity of a Ca2+-dependent protein kinase during reorientation. The kinase activity was assayed in living cells by using confocal ratio imaging of BODIPY FL bisindolylmaleimide. We found that growing pollen tubes exhibited higher protein kinase activity in the apical region, whereas nongrowing cells showed uniform distribution. Modification of growth direction by diffusion of inhibitors/activators from a micropipette showed the spatial redistribution of kinase activity to predict the new growth orientation. Localized increases in [Ca2+]c induced by photolysis of caged Ca2+ that led to reorientation also increased kinase activity. Molecular and immunological assays suggest that this kinase may show some functional homology with protein kinase C. We suggest that the tip-localized gradient of kinase activity promotes Ca2+-mediated exocytosis and may act to regulate Ca2+ channel activity.

Relocation of a Ca 2+ -Dependent Protein Kinase Activity during Pollen Tube Reorientation ���

Relocation of a Ca 2+ -Dependent Protein Kinase Activity during Pollen Tube Reorientation ���