ROP1 Signaling in Oscillatory Pollen Tubes Growth

1. ROP1 Signaling in Oscillatory Pollen Tubes Growth Speaker: Chun-hui Cai

2. Outline • Oscillatory pollen tube growth • Tip-localized F-actin and calcium gradient • ROP1 and its roles in pollen tube growth • ROP1 dependent tip localized tip calcium concentration and tip F-actin dynamics • ROP1 activates two counteracting downstream pathways • ROP1 oscillatory activation leads the oscillatory polarized growth • Discussion

3. Oscillatory pollen tube growth • Pollen tube – slender tubular outgrown from a pollen grain that penetrates the ovule and releases male gametes • Tip-growing pollen tube provides a good model system to study polar and oscillatory growth.

4. Oscillatory pollen tube growth • Pollen tube shows a dramatic rapid elongation of cell length without further cell division and differentiation. • The rapid growth appears to be facilitated by growth oscillation typically with period of tens of seconds. • Recent studies suggest a positive correlation between oscillation and the rate of tip growth. • Oscillatory growing Lily pollen tubes grow faster than the ones without clear oscillation. • The faster growing pollen tubes generally exhibit the shorter periods of oscillation

5. Oscillatory pollen tube growth • Many cellular events associated and highly correlated with tip growth oscillate with the same period of growth rate, including tip-localized F-actin, membrane trafficking, intracellular ion gradient and ion influxes at the tip. • In particular, tip F-actin dynamics and tip-focused calcium gradient have been shown to control tip growth.

6. Tip-localized F-actin • A highly dynamic form of F-actin is localized to the extreme apex (cortical actin filaments). • Tip F-actin oscillation peaks ahead of pollen tube elongation rate. • Dynamic tip F-actin may play a crucial role in oscillatory pollen tube growth. • Actin-disrupting drugs such LatB and cytochalasins effectively block tip growth. • Diffuse dynamic F-actin is present at the tip of growing sites of cells that do not undergo tip growth. • F-actin dynamics may drive the membrane fusion, requiring both inhibition and stimulation during the process, leading to pollen tube growth.

8. Tip-localized calcium • Pollen tube exhibit a tip-focused intracellular calcium gradient that can be very steep. • Calcium levels in lily pollen tubes can reach as high as 3000-5000nM at the extreme apex and fall off sharply to basal levels 100-200nM within 20µm. • Calcium gradients oscillate in phase with but a few seconds behind growth peaks (~40 degrees). • Tip-focused extracellular calcium influx are responsible for the formation of cytosolic calcium gradient, though the underlying mechanisms are poorly understood. • The LaCl3, calcium channel blocker, cause the decrease of tip calcium concentration and further exert an inhibitory effect on pollen tube growth. • Tip-focused calcium gradients are most probably required for exocytosis in pollen tubes based on its well known roles on regulating exocytosis. • Tip-focused calcium may also act as a negative regulator of tip F-actin disassembly by activating actin depolymerization agent.

9. ROP1 and its roles in pollen tube growth • Rop acts as a central switch to control the tip-focused calcium gradient and dynamic F-actin which are required by the pollen tube growth. • Rop (Rho-related GTPase from plants) is a plant-specific subfamily of the RHO family of G protein that include Cdc42, Rac and Rho subfamilies from animals and fungi. • In particular pollen-specific ROP1, probably together with two other closedly related and functional redundant ROPs (ROPs and ROP5), has been shown to be an essential regulator of pollen tube tip growth. • ROP1 is preferentially localized to the apical region of the pollen tube plasma membrane and cycles between GTP-bound active form and GDP-bound inactive status which is critical for normal tip growth.

10. ROP1 and its roles in pollen tube growth • ROP1 regulates pollen germination and pollen tube growth • Pollen tube elongation was inhibited by microinjected anti-Rop1 antibodies or expression of dominant negative (DN) mutants of ROP1 – DN-rop1. • High levels of DN-rop1 expression inhibited pollen germination, whereas overexpression (OX) of wild-type ROP1 promotes pollen germination. • Low levels of ROP1 OX promote tube elongation, whereas high levels caused depolarized growth. • Expression of constitutive active (CA) mutants of ROP1 or ROP5 caused much more severe depolarization of pollen tube growth • Localized recruitment and activation of ROP1 to the PM is critical for the establishment of the tip growth domain

12. ROP1 dependent tip F-actin dynamics • Tip localized ROP1 signaling regulates the dynamics of tip F-actin. • Inactivation of ROP1 by overexpression of RopGAP1 or DN-rop1 reduce or eliminate tip F-actin. • ROP1 overexpression caused the formation of a network of actin filaments at the tip and abnormal tranverse actin bands just behind the extreme , due to the stabilization of the dynamic tip F-actin. • These abnormal forms of F-actin were suppressed by RopGAP1 or AtGDI1 overexpression, which also suppress depolarized ROP1 localization and activation. • LatB treatments suppressed abnormal F-actin and depolarized growth induced by ROP1 OX and recovered normal dynamics of tip F-actin.

13. ROP1 dependent tip-focused calcium gradient • ROP1 regulates the formation of tip-focused calcium gradient • Tip-focused calcium gradients were eliminated within 1-2 min after microinjection of an anti-Rop1Ps antibody into pea pollen tubes (the timing coincides with that of antibody induced growth arrest). • High levels of external calcium concentration suppressed the growth inhibition caused by DN-rop1 expression in transgenic Arabidopsis pollen tubes, whereas the same concentration of extracellualar calcium inhibited wild-type pollen tube growth. • Active ROPs are localized in the apical dome of the PM as tip-high gradient, which appears to correspond to the localization of intracellualar gradients.

14. ROP1 activates two counteracting downstream pathways • ROP1 activate both the assembly of tip F-actin and the formation of tip-focused calcium gradient. • Tip F-actin and tip-focused calcium oscillate in different phases (tip F-actin peaks leads tip-focused calcium by ~110 degrees). ROP1 activates calcium oscillation and actin dynamics via two different pathways involving the direct targets of tip-localized ROP1: RIC3 and RIC4.

15. ROP1 activates two counteracting downstream pathways • RIC3 and RIC4 are two distinct ROP1 target proteins and have distinct subcellular localization. • RIC3 and RIC4 are two distinct ROP1 targets. • RICs – ROP-interactive CRIB(Cdc42/Rac interactive binding)-containing proteins. • Among nine RICs expressed in A. thaliana pollen, only RIC3 and RIC4 OX induced depolarized growth. • In vivo ROP1 interaction with RIC3 or RIC4 was detected using fluorescence resonance energy transfer (FRET) analysis of tobacco pollen tubes coexpressing CFPROP1 and YFP-RIC3 or YFP-RIC4 fusion genes. • The CFP-ROP1/YFP-RIC4 FRET signals were preferentially distributed to the apical region of the PM, similar to the GFP-RIC4 localization. • CFP-ROP1/YFP-RIC3 FRET signals were slightly stronger in the apical region of the PM compared with the cytoplasm, whereas GFP-RIC3, barely detected in the tube PM, is preferentially localized to the apical region of the cytoplasm.

17. ROP1 activates two counteracting downstream pathways • RIC4 promotes the assembly of apical F-actin. • RIC4 OX convert the dynamic tip F-actin into a dense F-actin network and resulted in a significantly greater amount of F-actin at the tip than the control tubes. • LatB and AtPFN3, actin depolymerization agent, recovered normal apical F-actin and tip growth in the majority of RIC4-overexpressing tubes, 85% (n=52) and 80% (n=50) respectively. • RIC4 OX disrupted the oscillation of normal tip F-actin; LatB recovered the oscillation of tip F-actin in RIC4-overexpressing tubes, 92% (n=51). • Loss-of-function (LOF) ric4 mutants containing tubes were significantly shorter and were more sensitive to growth inhibition caused by LatB than wild-type tubes. • WT A. thaliana pollen tubes did not contain as conspicuous F-actin at the tip as do tobacco pollen tubes. This finding could be explained by greater actin disassembly and/or less actin assembly in A. thaliana pollen tubes, which is consistent with their slower growth, as compared with tobacco pollen tubes. • Low levels of RIC4 OX increased tip F-actin accumulation, promoted pollen tube growth, and reduced LatB sensitivity, which suggests that actin assembly is a rate-limiting step in the elongation of WT A.thaliana pollen tubes.

18. ROP1 activates two counteracting downstream pathways • RIC3 regulates calcium signaling and promotes the accumulation of tip-localized calcium by modulating the extracellular calcium influx. • Both EGTA (calcium-chelating agent) and LaCl3 (which blocks PM-localized inward calcium channels) suppressed RIC3 OX induced growth inhibition and actin reorganization. • LOF ric3 inhibited tube growth at lower [Ca2+]ex (0.5-5mM) but promoted growth at high level [Ca2+]ex (10mM) where the optimal [Ca2+]ex condition is 5mM for WT tubes elongation. • RIC3 OX promoted tube elongation at 0.5 mM [Ca2+]ex, but inhibited growth at 2 mM or higher [Ca2+]ex, where the optimal [Ca2+]ex condition is 5mM for WT tubes elongation. • LaCl3, calcium channel blocker, treatments shift the optimal growth condition from 5 to 10mM [Ca2+]ex. • LaCl3-treated RIC3-overexpressing tubes mimicked the Ca2+ response of WT tubes in the absence of LaCl3. • LaCl3 suppressed the ability of high [Ca2+]ex to rescue pollen tube elongation inhibited by LOF ric3. • Pollen bombarded with LAT52:RIC3 was cultured at different [Ca2+]ex levels. At 0.1mM, untransformed pollen did not germinate, whereas 100% of LAT52:RIC3 pollen grains germinated. At 1mM [Ca2+]ex, 70% of untransformed pollen germinated, but few LAT52:RIC3 pollen grains germinated (9%). • RIC3 OX led to a depolarized calcium gradient and disrupted apical growth

19. ROP1 activates two counteracting downstream pathways • RIC3 and RIC4 counteract to control actin dynamics and apical growth • ROP1 activate two pathways: • A RIC4 pathway promoting apical F-actin assembly. • A RIC3 pathway that modulates [Ca2+]ex influx required to generate tip-focused calcium gradient. • ROP1-dependent tip F-actin and tip-focused calcium gradient oscillate with the same period but in the different phases, suggesting a possible functional coordination between the RIC3 and RIC4 pathways. • Cooverexpression of RIC3 and RIC4 resulted in the recovery of tip growth and normal dynamics of tip F-actin. • A balance between RIC3 and RIC4 is critical for efficient tip growth • When either RIC3 or RIC4 was overexpressed alone, the tube length was only half that of WT. • With fixed RIC4 DNA amount and RIC3 DNA amount increased. With RIC3/RIC4=1:2, optimal growth occurs. • Pollen tubes from individual LOF ric3 or ric4 mutants were much shorter than WT tubes at 5mM optimal [Ca2+]ex. • Pollen tubes from double mutants grew almost as long as did WT tubes. Elongation of these tubes showed similar responses to [Ca2+]ex levels as that of WT tubes and F-actin was also normal

21. ROP1 activates two counteracting downstream pathways • RIC3 acts through Ca2+ to promote the disassembly of RIC4-dependent F-actin. • Ca2+ activates several actin disassembly factors, e.g., profilin and gelsolin-like proteins. • Elevation of intracellular calcium levels causes dramatic actin disassembly in poppy pollen tubes. • Raising [Ca2+]ex from 0.01 to 1 mM effectively recovered normal tip growth and the dynamics of apical F-actin in RIC4-overexpressing tobacco pollen tubes. • LaCl3 treatments of tubes cooverexpressing RIC3 and RIC4 induced growth depolarization and the formation of a stable apical F-actin network as found in untreated tubes overexpressing RIC4 alone.

22. ROP1 activates two counteracting downstream pathways • RIC4 acts through F-actin to counteract the RIC3 pathway • GFP-RIC3 and RIC4 cooverexpressing tubes showed polarized growth, but LatB-treated tubes cooverexpressing GFP-RIC3 and RIC4 exhibited growth depolarization identical to that of GFP-RIC3–overexpressing tubes

24. ROP1 oscillatory activation leads the oscillatory polarized growth • ROP1 activity oscillates with same growth frequency with growth oscillation, and leads growth both spatially and temporally. • RIC4 exhibits ROP1 activation-dependent localization to the apical region and can be used as a marker for both the localization and activation of ROP1 activity. • RIC4 localization to the apical cap oscillates ahead of tip growth and tip-localized actin microfilaments oscillates in a similar phase with RIC4 localization • The ROP1 oscillation was ahead of growth rate oscillation by 18s in a typical 70s-period oscillation, ~90 degrees phase shift ahead of growth rate oscillation. • The phase shift of tip-localized F-actin assembly to the associated growth pulse is slightly less than 90 degrees.

25. Oscillations of the growth rate, I-avg-pm and I-avg-cyto of GFP-RIC4ΔC. I-avg-pmoscillations are 10-20 s ahead of growth rate oscillation (asterisksand arrows, ~90degrees ahead of growth). We analyzed 8 representativetubes and obtained the samephase relation between oscillatory GFP-RIC4ΔC localization to the tip and the tip growth(average 87 ± 17 degrees, n=8). Breaks in the curves are because of repositioning thetube that had grown out of the frame.

26. A typical oscillation of tip-localized F-actin compared to growth rate oscillation. Theoscillations of tip-localized F-actin were about 0-30 s ahead of the growth rateoscillationsof ~ 110-s period. The higher level of F-actin accumulation (GFP-mTalin intensity) in thetip is associated with relatively lower growth rate (arrows). We obtained similar resultsfrom 7 individual tubes with oscillatory tip growth and F-actin assembly at the tip. The tiplocalizedF-actin oscillation is on an average 70 ± 17 degrees ahead of tip growthoscillation.

27. Discussion • Why overexpression of ROP1 leads to abnormal actin assembly similar to RIC4 OX but not calcium elevation? • Different binding affinity? • Optimal concentration ratio of RIC4/RIC3=2:1. • RIC4 is preferentially localized to the apical region of PM where the ROP1 is localized, while RIC3 is preferentially localized to the apical region of cytoplasm. • The phenomenon may explain why increase in the ROP1 localized activation almost immediately activate tip F-actin assembly and depress tip-focused calcium accumulation.

28. Discussion • The most important players? ROP1, RIC3, RIC4, tip-localized F-actin, tip-focused calcium gradient, calcium influx, exocytosis/membrane fusion which can be coupled with growth rate, actin depolymerization agent. What is the way they interplay with each other that leads to oscillatory cellular events and polar growth?

29. Discussion • An early actin-mediated feed forward regulation would allow tip-localized ROP activity torapidly reach a peak, which corresponds to a delayed build-up of the tip-focused [Ca2+]cytgradient. The delay in the increase in [Ca2+]cyt may be explained by a requirement for a longcascade of RIC3-mediated signaling events leading to [Ca2+]cyt accumulation. The rise of[Ca2+]cyt above a threshold level would lead to the fall in the tip-localized ROP activity. As described below, this hypothesis is strongly supported by our data described hereand previous work.

30. Discussion • There is emerging evidence that ROP is also subject to feed forward regulation in its control of polarized growth of pollen tubes. (Wu, Hwang, and Yang, unpublished) • Overexpression of ROPs or expression of constitutively active rop mutant caused depolarization of tip growth and eliminated oscillatory growth. The depolarization of growth is apparently caused by caused by a feed forward-mediated lateral amplification of ROP activation. (Wu, Hwang, and Yang, unpublished) • RIC4 overexpression caused depolarized growth due to stabilization of tip actin microfilaments. Promotion of actin depolymerizaton at the tip, e.g., by LatB treatments, recovered polarized growth both in ROP1-overexpressing and RIC4-overepxressing tubes, implicating ROP/RIC4-dependent actin polymerization in ROP feed-forward regulation. We found that LatB treatment significantly reduced the peak of GFP-RIC4ΔC localization to the tip, which is associated with peak assembly of tip F-actin. • LatB treatment significantly reduced the peak of GFP-RIC4ΔC localization to the tip. Tip-localized assembly of actin filaments contributes to maintaining a high level of ROP1 activity through feed forward loop?

31. Discussion • The slow accumulation of [Ca2+]cyt following ROP activation at the tip andthe counteraction of Ca2+ on the RIC4 pathway led us to speculate that as[Ca2+]cyt increases above a certain threshold, it may stimulate the down-regulation of ROPactivity. Ca2+ is reported to affect negative regulators of animalRas-superfamily GTPases. Due to technical difficulties insimultaneously measuring ROP activity and modifying [Ca2+]cyt , we tested the general effectof La3+ on tip-localized ROP activity. These experiments suggest that proper formation ofthe [Ca2+]cyt gradient is necessary for the oscillation of tip-localized ROP activity and tipgrowth. • The regulation of ROP activity in oscillatory pollen tube growth by [Ca2+]cyt increase involves both calcium promotion of actin disassembly and a more direct calcium regulation of the cycling between GTP- and GDP-bound forms of ROP. • In animal cells, several regulators of Rassuperfamily small GTPases, including guanine nucleotide exchange factors (GEFs) andGTPase activating proteins (GAPs), are reported to either contain binding motifs for Ca2+ orCa2+ sensors or to be regulated by Ca2+ dependent modification. • There are also reportsshowing Ca2+-dependent phosphorylation of RhoGDI alpha and p115RhoGEF

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