AtPRK2 Promotes ROP1 Activation via RopGEFs in the Control of Polarized Pollen Tube Growth

Abstract

The ROP1 GTPase-based signaling network controls tip growth in Arabidopsis pollen tubes. Our previous studies imply that ROP1 might be directly activated by RopGEF1, which belongs to a plant-specific family of Rho guanine nucleotide exchange factors (RopGEFs) and in turn may be activated by an unknown factor through releasing RopGEF1's auto-inhibition. In this study, we found that RopGEF1 forms a complex with ROP1 and AtPRK2, a receptor-like protein kinase previously shown to interact with RopGEFs. AtPRK2 phosphorylated RopGEF1 in vitro and the atprk1,2,5 triple mutant showed defective pollen tube growth, similar to the phenotype of the ropgef1,9,12,14 quadruple mutant. Overexpression of a dominant negative form of AtPRK2 (DN-PRK2) inhibited pollen germination in Arabidopsis and reduced pollen elongation in tobacco. The DN-PRK2-induced pollen germination defect was rescued by overexpressing a constitutively active form of RopGEF1, RopGEF1(90-457), implying that RopGEF1 acts downstream of AtPRK2. Moreover, AtPRK2 increased ROP1 activity at the apical plasma membrane whereas DN-PRK2 reduced ROP1 activity. Finally, two mutations at the C-terminal putative phosphorylation sites of RopGEF1 (RopGEF1S460A and RopGEF1S480A) eliminated the function of RopGEF1 in vivo. Taken together, our results support the hypothesis that AtPRK2 acts as a positive regulator of the ROP1 signaling pathway most likely by activating RopGEF1 through phosphorylation.

Novel ABP1-TMK auxin sensing system controls ROP GTPase-mediated interdigitated cell expansion in Arabidopsis

“…Several recent studies on the mechanism of pollen tube growth and ROS-mediated polarized root hair development showed that diverse RLKs can directly interact with and phosphorylate RopGEFs, and/or act upstream of ROPs. [30][31][32][33][34] A yeast two-hybrid screen showed that the tomato pollenspecific RLKs LePRK1 and LePRK2 interact with a tomato RopGEF homolog, which led to further work on a physical and functional interaction between the pollen-specific Arabidopsis RopGEF12 and a LePRK2 homolog AtPRK2a. 30,31 A series of biochemical and overexpression experiments suggest that this interaction recruits RopGEF12 to the plasma membrane and releases the inhibition of the C-terminal region on the GEF activity via PRK2a-mediated phosphorylation.…”

“…More recent work regarding AtPRK2 uncovers that this pollen-specific RLK regulate both RopGEF1 and ROP1 activities via forming a complex with them, which makes a conclusion that RopGEF1 bridges the signaling event from AtPRK2 to ROP1 to control polarized pollen tube growth in Arabidopsis. 34 Also, several members of the Catharanthusroseus RLK (CrRLK)-related family were identified as upstream regulators of RopGEFs. 32,35 CrRLK FERONIA (FER) acts as an upstream regulator of RAC/ROP2 signaling pathway most likely by interacting with RopGEF1.…”

RLKs orchestrate the signaling in plant male-female interaction

“…This finding confirms that the dissociated LePRK1 and LePRK2 both function during pollen tube growth within the pistil, possibly in response to different ligands. On the contrary, another aspect making the signaling pathway more complex is that PRKs share overlapping function as evidenced by the aggravated shortened pollen tubes in mutants depleted with multiple PRKs (Chang et al, 2012).…”

“…In Arabidopsis, several members of PRK homologs also regulate pollen tube growth through RopGEF (Zhang and McCormick, 2007;Chang et al, 2012). Similar to the effect of excessive LePRK2 or KPP, over-expression of AtPRK2 or AtGEF causes ectopic GEF activity at a broad range on the plasma membrane of the tube tip, leading to pollen tube depolarization.…”

“…Intriguingly, the kinase activity of PRK6 is not required for the pollen tube perception of LURE1 but essential for the pollen tube growth (Takeuchi and Higashiyama, 2016). Previous studies shows that GEF1 phosphorylation by PRK2 was essential for its full function (Chang et al, 2012). It raises the query that if the kinase-dependent GEF activation is common for all PRK family member or just specific to PRK2.…”