A distinct mechanism regulating a pollen-specific guanine nucleotide exchange factor for the small GTPase Rop in Arabidopsis thaliana

Abstract

Rop/Rac small GTPases are central to diverse developmental and cellular activities in plants, playing an especially important role in polar growth of pollen tubes. Although it is established that a class of plant-specific RopGEFs promotes the activity of Rop/Rac through the catalytic PRONE (Plant-specific Rop nucleotide exchanger) domain, not much is known about how RopGEF function is controlled to allow a spatiotemporally regulated Rop activity. To understand such a process in pollen, we performed functional analysis with a pollen-specific RopGEF, AtRopGEF12. Overexpression of AtRopGEF12 had minimal phenotypic effects, whereas overexpression of a C-terminally truncated version disturbed tube growth, suggesting that the C terminus was inhibitory to GEF function. In contrast to non-pollen-expressed RopGEFs, pollen-expressed RopGEFs have conserved C termini. A phospho-mimicking mutation at an invariant serine within the C terminus of AtRopGEF12 resulted in loss of the C-terminal inhibition, suggesting that phosphorylation regulates GEF activity in vivo. The PRONE domain of AtRopGEF12 (PRONE12) was not sufficient to induce isotropic tube growth. We used mbSUS to show that AtRopGEF12 interacts with an Arabidopsis pollen receptor kinase AtPRK2a through its C terminus, and BiFC to show that they interact in pollen tubes. Coexpression of AtRopGEF12 and AtPRK2a caused isotropic growth reminiscent of that seen upon overexpression of a constitutively active (CA) Rop. Coexpression of AtPRK2a with an Nterminally truncated AtRopGEF12 did not induce isotropic growth, indicating a positive role for the N-terminal domain. Our results suggest a mechanism by which the noncatalytic domains of pollenspecific/enriched RopGEFs regulate PRONE function, leading to polarized pollen tube growth.phosphorylation ͉ pollen tube growth ͉ receptor kinase

Whole Genome Analysis of Gene Expression Reveals Coordinated Activation of Signaling and Metabolic Pathways during Pollen-Pistil Interactions in Arabidopsis

“…Other examples include the RopGEFs known to interact with leucinerich repeat (LRR) kinases during pollen tube growth in the pistil (Kaothien et al, 2005). In our data sets, we identified two pollen-specific RopGEFs (Zhang and McCormick, 2007), RopGEF12 (At1g79860) and Rop-GEF11 (At1g52240), both of which are up-regulated during pollen tube growth (cluster 9). Interestingly, two out of six up-regulated LRR kinases are preferentially expressed in pollen and follow a similar regulatory pattern (cluster 9).…”

“…Interestingly, two out of six up-regulated LRR kinases are preferentially expressed in pollen and follow a similar regulatory pattern (cluster 9). One is the ortholog of LePRK2 (for pollen receptor-like kinase), AtPRK2a (At2g07040), recently found to interact with RopGEF12 (Zhang and McCormick, 2007). The second pollen LRR (At1g49490; Fig.…”

Intercellular communication in Arabidopsis thaliana pollen discovered via AHG3 transcript movement from the vegetative cell to sperm

“…AHG3 was amplified from Col-0 genomic DNA and cloned into pENTR-D/TOPO, then transferred into a plant expression destination vector modified from pB7FWBG2 (37) (35S promoter removed). The AHG3 CDS was amplified from pollen cDNA with the primers specified in Table S1 and cloned into pENTR-D/TOPO (Invitrogen), then transferred into a plant expression destination vector modified from pZY06 (38). The resulting plasmid was introduced into qrt1 plants (39) by Agro-infiltration (40).…”

The Small GTPase ROP10 of Medicago truncatula Is Required for Both Tip Growth of Root Hairs and Nod Factor-Induced Root Hair Deformation

“…Various regulators that modulate the shuttling between inactive and active forms of ROP GTPases have been identified, including guanine nucleotide exchange factors (RopGEFs) that stimulate GDP-to-GTP exchange to activate ROP GTPases, ROP GTPase-activating proteins (RopGAPs) that accelerate GTP hydrolysis, and guanine nucleotide dissociation inhibitors (RhoGDIs) that inhibit GDP release, thus shifting ROP GTPases toward the inactive state. Some of these regulators have been shown to modulate ROP GTPase activity in pollen tubes and root hairs (Carol et al, 2005;Gu et al, 2006;Zhang and McCormick, 2007;Hwang et al, 2008Hwang et al, , 2010Riely et al, 2011).…”