Tip Localized Active ROP1 Couples the Spatial and Temporal Control of Pollen Tube Elongation

The preferential localization of ROP1 to the apex and the effect of ROP1 activation on both tip growth and growth polarity suggest that localized ROP1 activation in the tip is not only critical for growth control, but may also define growth polarity or the site for tip growth. A recent investigation of in vivo ROP1 activity using a live GFP-based ROP1 activity marker supports this hypothesis. An active ROP1 reporter was developed based on RIC4 (Hwang et al. 2005). RIC4 is a downstream effector molecule of ROP1 (Wu et al. 2001; Gu et al. 2005). It preferentially binds to the GTP-bound active ROP1 both in vitro and in vivo (Gu et al. 2005). A mutant of RIC4, in which C-terminal 21-

amino acids behind the CRIB motif were deleted (RIC4AC), retained the full capacity of RIC4 to specifically bind to the active form of ROP1 but did not alter pollen tube growth when transiently expressed in tobacco pollen tubes (Hwang et al. 2005). Visualization of GFP-RIC4AC reveals that active ROP1 forms a tip-high gradient in the extreme of the pollen tube plasma membrane (PM), termed the apical cap of active ROP1, which apparently corresponds

Fig. 1 The oscillatory tip-localized ROP1 activity coordinates two downstream pathways, [Ca2+]c dynamics and F-actin assembly. a A typical round of tip-localized ROP1 oscillation of - 60-70 s period. In vivo ROP1 activity was visualized by GFP-RIC4AC and the average intensity of GFP-RIC4AC localized to the tip apical PM oscillates with the tip growth rate. Numbers at the top right indicate the elapsed time (seconds) from the beginning (0 s). b Quantitative data showing that a tip-localized ROP1 activity increase leads the burst of tip growth, supporting the control of tip growth by active ROP1. The graph was reproduced from Hwang et al. (2005) with permission of MBC (This needs to be obtained). The average activity of ROP1 in the tip apical PM was quantified by measuring the average intensity of GFP-RIC4AC localized to the tip PM (I-avg-PM). I-avg-PM oscillates 10-20 s ahead of tip growth rate, whereas the amount of GFP-RIC4AC in the tip (I-avg-c) remains relatively constant with only minor fluctuations. c A cartoon showing the phase relations between tip growth, tip-localized ROP1 (RIC4) activity, F-actin assembly, and [Ca2+]c increase. Tip-localized ROP1 activity oscillation is on average ~ 90° ahead of tip growth. Two downstream targets of active ROP1 appear to be activated differentially: F-actin assembly is stimulated early, which is in similar phase with ROP1 activation, while the [Ca2+]c increase is stimulated late (~ 40° behind tip growth)

to the active growth domain (Fig. 1a). The tip-localized ROP1 activity is very dynamic in normal growing pollen tubes and was found to oscillate with the same frequency as the growth rate (Hwang et al. 2005; Fig. 1). Tip-localized ROP1 activity starts increasing about 10-20 s ahead of the tip growth rate increase in a 70-s period of oscillation (i.e., 90° ahead of growth). This phase relationship is different from oscillations of ion fluxes such as Ca2+, Cl-, K+, and H+, which appear to be associated with the growth or post-growth events (Holdaway-Clarke et al. 1997; Messerli et al. 1999, 2000). The temporal relation between ROP1 activation and growth thus supports a crucial role of active ROP1 in pollen tubes (Fig. 1c).

The formation of the apical cap of active ROP1 at the presumed active growth site indicates that ROP1 activation may be spatially regulated to de-

Fig. 2 The tip-localized ROP1 activation defines the growth polarity. a Representative pollen tube images with tip-localized ROP1 activity. The pollen tube grew in a rapid oscillatory mode of 40-60-s period. Numbers at the top right indicate the elapsed time (seconds) from the beginning (0 s). The repositioning of the active ROP1 apical cap is indicated by arrows, which point in the future growth direction. The position of the tip apex at the previous time point is outlined with white dots. b A cartoon showing the spatiotemporal regulation of tip growth by tip-localized ROP1. The red lines indicate the amount and distribution of active ROP1 in the tip apical PM. One round of oscillation is divided into six phases (1-6): (1) tip-localized active ROP1 at the peak; (2) growth burst with tip active ROP1 amount being decreased; (3) interpulse state of ROP1 oscillation with growth rate being decreased; ROP1 activity starts increasing again from the minimum level; (4) ROP1 activity increases, pointing in new growth direction; the growth rate is in the minimum; (5) the new growth polarity is stabilized with increased ROP1 activity; ROP1 activity is in the maximum with growth rate being increased slowly; and (6) tip growth bursts toward new growth direction defined by active ROP1

Fig. 2 The tip-localized ROP1 activation defines the growth polarity. a Representative pollen tube images with tip-localized ROP1 activity. The pollen tube grew in a rapid oscillatory mode of 40-60-s period. Numbers at the top right indicate the elapsed time (seconds) from the beginning (0 s). The repositioning of the active ROP1 apical cap is indicated by arrows, which point in the future growth direction. The position of the tip apex at the previous time point is outlined with white dots. b A cartoon showing the spatiotemporal regulation of tip growth by tip-localized ROP1. The red lines indicate the amount and distribution of active ROP1 in the tip apical PM. One round of oscillation is divided into six phases (1-6): (1) tip-localized active ROP1 at the peak; (2) growth burst with tip active ROP1 amount being decreased; (3) interpulse state of ROP1 oscillation with growth rate being decreased; ROP1 activity starts increasing again from the minimum level; (4) ROP1 activity increases, pointing in new growth direction; the growth rate is in the minimum; (5) the new growth polarity is stabilized with increased ROP1 activity; ROP1 activity is in the maximum with growth rate being increased slowly; and (6) tip growth bursts toward new growth direction defined by active ROP1

fine the region of the plasma membrane for growth. This notion is supported by a tight correlation between the distribution of GFP-RIC4AC in the apical region of the plasma membrane and spatial changes in pollen tube growth. In pollen tubes overexpressing ROP1 or RIC4, an increase in the size of the GFP-RIC4/RIC4AC-containing apical cap was associated with an increase in tube width (Wu et al. 2001; Gu et al. 2005; G Wu et al. unpublished data). Conversely, a reduction of tube width was correlated with a decrease in the size of the apical cap in tubes expressing ROP negative regulators (G Wu et al. unpublished data). In reorienting pollen tubes, the GFP-RIC4AC apical cap was relocated to the future growth site before observable growth direction occurred (Hwang et al. 2005). Tobacco pollen tubes treated with 0.5 nM latrunculin B (LatB) displayed transient growth retardation and then resumed the oscillatory growth in a new direction (Hwang et al. 2005). The apical cap of GFP-RIC4AC relocation toward a future growth direction was detected clearly before the growth surge (Fig. 2). Consistent with the effects of ROP 1 inactivation and overactivation on pollen tube growth, these observations strongly support the hypothesis that the spatiotemporal dynamics of tip-localized ROP activation couples the spatial and temporal regulation of tip growth (Fig. 2b). The apical cap of ROP1 activity can thus be considered as a dynamic growth organizer in time and space, which predicts the timing of the next growth surge and the position or direction of new growth. Given that Rho GTPases have been established as an integrator of different upstream signals, it is reasonable to speculate that ROP1 can integrate various signals from female tissues that regulate and guide pollen tube growth as the pollen tube is targeted to the ovule for fertilization (see the chapter by Johnson and Lord, this volume). Therefore, it is not surprising that the study of pollen tube signaling has been focused on two most interesting questions: (1) how the spatiotemporal dynamics of ROP1 activity is regulated and (2) how the localized ROP1 activity signals to localized growth.

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