Oscillations in Pollen Tube Growth Oscillations in Pollen Tube Growth & &

ROP Signaling Network ROP Signaling Network

XIA, FanXIA, Fan

IntroductionIntroduction ObservationObservation of of

oscillations in pollen tube:oscillations in pollen tube: SpontaneousSpontaneous Positive correlation between Positive correlation between

oscillation and the rate of oscillation and the rate of growth.growth.

Phase relationshipPhase relationship

MechanismMechanism underlying underlying these oscillations:these oscillations:

The oscillations with a The oscillations with a period of minutes or shorter period of minutes or shorter can are unlikely to involve can are unlikely to involve periodic changes in gene periodic changes in gene expression.expression.

ROP1 signaling networkROP1 signaling network plays a key role.plays a key role.

0°: tip F-actin & ROP1 activity

90°: Growth rate

120°: Calcium concentration210°: Calcium

influx

ROP1 Signaling NetworkROP1 Signaling Network

Jae-Ung Hwang et al., 2005

ROP1ROP1 Location of the active Location of the active

ROP1 establish the tip ROP1 establish the tip growth domain.growth domain.

RecruitmentRecruitment: A tip-localized : A tip-localized recruitment factor and a tip-recruitment factor and a tip-localized Rop sequestering localized Rop sequestering factor (AtGDI1).factor (AtGDI1).

MaintenanceMaintenance: active ROP1 : active ROP1 promotes the recruitment to promotes the recruitment to the apical PM region.the apical PM region.

Oscillation of ROP1 activityOscillation of ROP1 activity

RIC4 localization to the apical cap, i.e., the apparent tip-localized ROP1 activity, is oscillatory and leads growth bursts by 90°.

Experiment RIC4 localization to the PM is the direct result of its binding to the active form of ROP1. RIC4∆C loses the effort function of RIC4 in the promotion of F-actin assembly, but faithfully reports its interaction with active ROP1. Intensity of RIC4∆C at PM reflects the activity of ROP1.

Result

RIC3 and RIC4RIC3 and RIC4 RICs: ROP-interactive CRIB-containing proteins.RICs: ROP-interactive CRIB-containing proteins. In pollen tubes, only RIC3 and RIC4 OX induced In pollen tubes, only RIC3 and RIC4 OX induced

depolarized growth.depolarized growth.o Structurally differentStructurally differento Two different pathwaysTwo different pathways

Interaction with ROP1Interaction with ROP1o Only interact with active formOnly interact with active formo Recruit from the cytoplasm to the PMRecruit from the cytoplasm to the PMo While RIC4 is tightly associated with PM domain, RIC3 is primarily While RIC4 is tightly associated with PM domain, RIC3 is primarily

cytoplasmic. RIC3 interacts with ROP1 in transient and dynamic cytoplasmic. RIC3 interacts with ROP1 in transient and dynamic manner, as AtGDI1.manner, as AtGDI1.

RIC4 downstream pathwayRIC4 downstream pathway Similar to ROP1OX, RIC4OX induced dense F-Similar to ROP1OX, RIC4OX induced dense F-

actin network and depolarization. actin network and depolarization. LatB could recover the oscillation of tip F-actin in LatB could recover the oscillation of tip F-actin in

RIC4OX tubes.RIC4OX tubes. RIC4 promotes the actin assembly.RIC4 promotes the actin assembly.

RIC3 downstream pathwayRIC3 downstream pathway Similar to high [CaSimilar to high [Ca2+2+]]exex, RIC3OX induced loss of , RIC3OX induced loss of

F-actin and protrusion of the axial actin cables F-actin and protrusion of the axial actin cables toward the extreme apex.toward the extreme apex.

EGTA and LaClEGTA and LaCl33 could recover RIC3OX induced could recover RIC3OX induced

growth inhibition and actin reorganization.growth inhibition and actin reorganization. RIC3 affects [CaRIC3 affects [Ca2+2+]]exex influx. influx.

Counteract between the two pathwaysCounteract between the two pathways RIC3OX and RIC4OX resulted in the recovery of RIC3OX and RIC4OX resulted in the recovery of

growth.---antagonistic effectgrowth.---antagonistic effect Optimal ratio Optimal ratio RIC3/RIC4 = 1:2RIC3/RIC4 = 1:2 Not due to the competition to bind to ROP1 but Not due to the competition to bind to ROP1 but

due to downstream targets.due to downstream targets.o Different location of actionDifferent location of actiono RIC3 acts through CaRIC3 acts through Ca2+2+ to promote the disassembly of RIC4- to promote the disassembly of RIC4-

dependent F-actin. dependent F-actin. o RIC4 acts through F-actin to counteract the RIC3 pathway.RIC4 acts through F-actin to counteract the RIC3 pathway.

F-actin feedback to ROP1 activityF-actin feedback to ROP1 activity LatB treatments recovered the polarized growth in both LatB treatments recovered the polarized growth in both

ROP1OX and RIC4OX.ROP1OX and RIC4OX. LatB treatments significantly reduced the peak of GFP-LatB treatments significantly reduced the peak of GFP-

RIC4RIC4∆C localization/ROP1 activity.∆C localization/ROP1 activity. Mechanism unknown.Mechanism unknown. Analog in other system.Analog in other system.

[Ca[Ca2+2+]]cyccyc feedback to ROP1 activity feedback to ROP1 activity Possible down regulation above threshold.Possible down regulation above threshold. Mechanism unknown.Mechanism unknown. Common interaction between Ca2+ and GEFs, GAPs Common interaction between Ca2+ and GEFs, GAPs

and GDIs.and GDIs.

Quantitative ModelingQuantitative Modeling Target:Target:o Oscillation with proper phase relationship Oscillation with proper phase relationship

between ROP activity, F-actin and [Cabetween ROP activity, F-actin and [Ca2+2+]]cyc.cyc.

o Mutant experiment (OX elimination of Mutant experiment (OX elimination of oscillation)oscillation)