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

Abstract

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 ([Ca 2 ؉ ] c ). In this paper, we studied the activity of a Ca 2 ؉ -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 [Ca 2 ؉ ] c induced by photolysis of caged Ca 2 ؉ 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 Ca 2 ؉ -mediated exocytosis and may act to regulate Ca 2 ؉ channel activity. INTRODUCTIONSuccessful fertilization in the angiosperms depends on pollen tubes finding their way to the ovule. Pollen tubes frequently change their direction of growth until they finally reach the micropyle. Previously (Malhó et al., 1994Malhó and Trewavas, 1996), we have shown that the distribution of cytosolic free calcium ([Ca 2 ϩ ] c ) in the apical region and Ca 2 ϩ influx by asymmetric activity of Ca 2 ϩ channels play crucial roles in this process. However, the downstream processes involved in signal transduction are not known (Feijó et al., 1995;Trewavas and Malhó, 1997).Phosphorylation cascades are known to be a major part of signaling pathways in plant cells, particularly those involving Ca 2 ϩ (Trewavas and Malhó, 1997). However, methods for imaging protein kinase in living cells are limited. Olds et al. (1995) used the fluorescent dye NBD-phorbol acetate to visualize the activation of protein kinase C (PKC) in living cells. This dye interacts directly with the protein, and the authors showed that when the cells were incubated in a solution of low-dye concentration, they remained viable and performed their physiological functions; continued protein synthesis and turnover might prevent the inactivation of significant amounts of the protein kinase by interaction with the dye. However, NBD-phorbol acetate is a single-wavelength dye, which makes quantitation difficult because it was designed primarily to label PKC. Despite the importance of this enzyme in the regulation of animal cell metabolism, direct sequence equivalents in plant cells have yet to be identified. Nevertheless, there is evidence for lipid-activated kinases that might represent functional equivalents (Nanmori et al., 1994; AboEl-Saad and Wu, 1995;Sokolova et al., 1997;Subramaniam et al., 1997). The loading of fluorescently labeled peptides (syntide-2) recently has been used to map the distribution of a membe...

Control of Pollen Tube Tip Growth by a Rop GTPase–Dependent Pathway That Leads to Tip-Localized Calcium Influx

“…Tip-localized Ca 2ϩ -dependent protein kinase activity could be a key factor involved in this connection (Moutinho et al, 1998). It is possible that a component downstream of Rop1At could directly activate plasma membrane Ca 2ϩ channels.…”

“…Thus, tip growth may involve a mechanism that couples the site of Ca 2 ϩ influx with the rate of influx. A tip-localized Ca 2 ϩ -dependent protein kinase activity has been suggested to regulate localized Ca 2 ϩ changes (Moutinho et al, 1998). However, how the tip-focused Ca 2 ϩ gradient is established and how the site and rate of extracellular Ca 2 ϩ influx are regulated are not known.…”

Imaging of the Yellow Cameleon 3.6 Indicator Reveals That Elevations in Cytosolic Ca2+ Follow Oscillating Increases in Growth in Root Hairs of Arabidopsis

“…Indeed, the structures of the apical actin cytoskeleton and its regulatory proteins (villins, gelsolins, and actin-depolymerizing factors; Smertenko et al, 1998;Tominaga et al, 2000;Allwood et al, 2001;Ketelaar et al, 2003;Fan et al, 2004) are thought to be regulated by the tip-high Ca 21 (Yokota et al, 2005). Similarly, there is evidence that annexins (Blackbourn et al, 1992;Clark et al, 1992;Carroll et al, 1998), phosphoinositide metabolism (Preuss et al, 2006), and calmodulin and protein kinases (Moutinho et al, 1998;Yoon et al, 2006) also play roles in sustaining tip growth and are regulated via the cytosolic Ca 21 gradient.…”

Ca2+-Dependent Protein Kinase11 and 24 Modulate the Activity of the Inward Rectifying K+ Channels inArabidopsisPollen Tubes

“…The tip [Ca 2+ ] cyt oscillates at same frequency as pollen tube growth rate (Holdaway-Clarke et al, 1997;Messerli and Robinson, 1997;Messerli et al, 2000). Some Ca 2+ binding proteins, such as calmodulins, calciumdependent protein kinases (CDPKs), and cytoskeletal proteins, sense Ca 2+ signals and trigger downstream responses during pollen germination and pollen tube growth (Moutinho et al, 1998;Snowman et al, 2002;Harper et al, 2004;Rato et al, 2004;Myers et al, 2009). In particular, CDPKs are emerging as important regulators of pollen tube growth.…”