Ca2+-Permeable Channels in the Plasma Membrane of Arabidopsis Pollen Are Regulated by Actin Microfilaments

Abstract

Cytosolic free Ca 21 and actin microfilaments play crucial roles in regulation of pollen germination and tube growth. The focus of this study is to test the hypothesis that Ca 21 channels, as well as channel-mediated Ca 21 influxes across the plasma membrane (PM) of pollen and pollen tubes, are regulated by actin microfilaments and that cytoplasmic Ca 21 in pollen and pollen tubes is consequently regulated. In vitro Arabidopsis (Arabidopsis thaliana) pollen germination and tube growth were significantly inhibited by Ca 21 channel blockers La 31 or Gd 31 and F-actin depolymerization regents. The inhibitory effect of cytochalasin D (CD) or cytochalasin B (CB) on pollen germination and tube growth was enhanced by increasing external Ca 21 . Ca 21 fluorescence imaging showed that addition of actin depolymerization reagents significantly increased cytoplasmic Ca 21 levels in pollen protoplasts and pollen tubes, and that cytoplasmic Ca 21 increase induced by CD or CB was abolished by addition of Ca 21 channel blockers. By using patch-clamp techniques, we identified the hyperpolarization-activated inward Ca 21 currents across the PM of Arabidopsis pollen protoplasts. The activity of Ca 21 -permeable channels was stimulated by CB or CD, but not by phalloidin. However, preincubation of the pollen protoplasts with phalloidin abolished the effects of CD or CB on the channel activity. The presented results demonstrate that the Ca 21 -permeable channels exist in Arabidopsis pollen and pollen tube PMs, and that dynamic actin microfilaments regulate Ca 21 channel activity and may consequently regulate cytoplasmic Ca 21 .The primary function of pollen and pollen tubes is to deliver sperms to egg apparatus for double fertilization that is required for sexual reproduction of flowering plants. Pollen germination and pollen tube growth are a continuous and highly polarized process characteristic of tip growth; thus pollen and pollen tubes provide an ideal model system for the study of cell polarity control and tip growth. It is well known that extracellular Ca 21 is required for pollen germination and tube growth (for review, see Steer and Steer, 1989;Taylor and Hepler, 1997;Malhó , 1998;Franklin-Tong, 1999), which indicates a possible involvement of Ca 21 influx in pollen germination and tube growth. Upon pollen hydration and germination, cytoplasmic Ca 21 concentration ([Ca 21 ] i ) at the germinal aperture where the pollen tube emerges increases to a higher level than other regions, and a tip-focused Ca 21 gradient is then established and sustained while a pollen tube grows forward (Rathore et al., 1991;Pierson et al., 1994;Feijó et al., 1995). Disruption or modification of the Ca 21 gradient inhibits pollen tube growth (Miller et al., 1992) or changes its growth direction (Malhó and Trewavas, 1996). The tip-focused Ca 21 gradient oscillates in the pollen tubes of lily (Lilium longiflorum) and other species (Holdaway-Clarke et al., 1997;Messerli and Robinson, 1997;Messerli et al., 2000). Ca 21 mobilization from intracellular Ca...

Actin filament reorganisation controlled by the SCAR/WAVE complex mediates stomatal response to darkness

“…This offers a potential explanation for the functional relevance of branched F‐actin. It has long been established that the response of membrane‐integrated stress‐responsive calcium channels requires a dynamic actin cytoskeleton (Wang et al ., 2004; Zhang et al ., 2007). It seems increasingly likely that specific actin network configurations contribute to the coupling of physical and physiological factors during guard cell closure dynamics, and our work presents the Arp2/3 complex and its regulators as candidate molecular players in this process.…”

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

“…S3). Alternatively, cytoskeletal elements may play a role in regulating mechanosensitive channel activity, as suggested for pollen tubes (Wang et al, 2004). The relationship between the ROS/ hyperpolarization-activated Ca 21 channel thought to support the gradient in root hairs (Foreman et al, 2003) and such a mechanical response remains to be defined.…”

Cyclic nucleotide-gated channel 18 is an essential Ca 2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

“…cGMP was freshly added each day. A ramp voltage protocol of 2 s duration from −180 mV to +20 mV was applied 1 min after accessing to whole-cell configuration (32 ] cyt levels. Thirty-five pollen tubes were tested for WT, 33 for cngc18-17, and 39 for cngc18-22.…”

“…3 A and B), as previously reported (41). To test whether the point mutations R491Q and R578K impaired the activation of CNGC18 in vivo, we isolated pollen tube protoplasts (46) and measured the whole-cell inward Ca 2+ currents using a patch-clamping technique (32). In comparison with background currents in a cGMP-free condition, large currents were triggered by the intracellular application of 100 μM cGMP in WT (Fig.…”

“…Thus, the Ca 2+ channels are the key components for regulating the Ca 2+ gradients and are consequently essential for pollen tube guidance. Using electrophysiological techniques, inward Ca 2+ currents were observed in both pollen grain and pollen tube protoplasts (31)(32)(33)(34)(35)(36), supporting the presence of plasma membrane Ca 2+ channels in pollen tube tips. Recently, a number of candidate Ca 2+ channels were identified in pollen tubes, including six cyclic nucleotide-gated channels (CNGCs) and two glutamate receptor-like channels (GLRs) in Arabidopsis (37)(38)(39)(40).…”