1. Oscillations in Pollen Tube Growth & ROP Signaling Network Oscillations in Pollen Tube Growth & ROP Signaling Network XIA, Fan

2. Introduction Observation of oscillations in pollen tube: Observation of oscillations in pollen tube:  Spontaneous  Positive correlation between oscillation and the rate of growth.  Phase relationship Mechanism underlying these oscillations: Mechanism underlying these oscillations:  The oscillations with a period of minutes or shorter can are unlikely to involve periodic changes in gene expression.  ROP1 signaling network plays a key role. 0°: tip F-actin & ROP1 activity 90 ° : Growth rate 120 ° : Calcium concentration 210 ° : Calcium influx

4. ROP1 Signaling Network ROP1 Signaling Network Jae-Ung Hwang et al., 2005 ROP1 ROP1  Location of the active ROP1 establish the tip growth domain.  Recruitment: A tip- localized recruitment factor and a tip-localized Rop sequestering factor (AtGDI1).  Maintenance: active ROP1 promotes the recruitment to the apical PM region.

6. Oscillation of ROP1 activity Oscillation 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

7. RIC3 and RIC4 RIC3 and RIC4  RICs: ROP-interactive CRIB-containing proteins.  In pollen tubes, only RIC3 and RIC4 OX induced depolarized growth. o Structurally different o Two different pathways  Interaction with ROP1 o Only interact with active form o Recruit from the cytoplasm to the PM o While RIC4 is tightly associated with PM domain, RIC3 is primarily cytoplasmic. RIC3 interacts with ROP1 in transient and dynamic manner, as AtGDI1.

10. RIC4 downstream pathway RIC4 downstream pathway  Similar to ROP1OX, RIC4OX induced dense F- actin network and depolarization.  LatB could recover the oscillation of tip F-actin in RIC4OX tubes.  RIC4 promotes the actin assembly.

11. RIC3 downstream pathway RIC3 downstream pathway  Similar to high [Ca 2+ ] ex, RIC3OX induced loss of F-actin and protrusion of the axial actin cables toward the extreme apex.  EGTA and LaCl 3 could recover RIC3OX induced growth inhibition and actin reorganization.  RIC3 affects [Ca 2+ ] ex influx.

12. Counteract between the two pathways Counteract between the two pathways  RIC3OX and RIC4OX resulted in the recovery of growth.---antagonistic effect  Optimal ratio RIC3/RIC4 = 1:2  Not due to the competition to bind to ROP1 but due to downstream targets. o Different location of action o RIC3 acts through Ca 2+ to promote the disassembly of RIC4- dependent F-actin. o RIC4 acts through F-actin to counteract the RIC3 pathway.

13. F-actin feedback to ROP1 activity F-actin feedback to ROP1 activity  LatB treatments recovered the polarized growth in both ROP1OX and RIC4OX.  LatB treatments significantly reduced the peak of GFP- RIC4∆C localization/ROP1 activity.  Mechanism unknown.  Analog in other system. [Ca 2+ ] cyc feedback to ROP1 activity [Ca 2+ ] cyc feedback to ROP1 activity  Possible down regulation above threshold.  Mechanism unknown.  Common interaction between Ca2+ and GEFs, GAPs and GDIs.

14. Quantitative Modeling Quantitative Modeling  Target: o Oscillation with proper phase relationship between ROP activity, F-actin and [Ca 2+ ] cyc. o Mutant experiment (OX elimination of oscillation)