Polarized subcellular activation of Rho proteins by specific ROPGEFs drives pollen germination in Arabidopsis thaliana
Fig 3
The C-terminus of GEF8 and GEF9 is necessary and sufficient for protein accumulation.
(A) AlphaFold2 protein structure prediction of full-length GEF8 and ROP1 (Rank 1, predicted separately), matched on the PRONE8-ROP4 double-dimer structure (PDB: 2NTY). Different angles are shown, and the terminal regions are indicated by different colors. (B) Schemes of GEF protein structure and investigated truncation constructs, with variable N and C terminal domains indicated as in (A). (C–E) Protein localization of different mutant constructs during pollen germination. Timepoint 0 corresponds to the beginning of pollen tube emergence, arrowheads mark the site of pollen emergence, and asterisks mark localization around the sperm cells. (C) Truncation constructs mCit-GEF8ΔN, mCit-GEF8ΔC, mCit-GEF9ΔN, and mCit-GEF9ΔC in Col-0. (D) Domain swap constructs of GEF12 with alternative C-terminal domain, mCit-GEF12GEF8C in gef8-cΔ1 and mCit-GEF12GEF9C in Col-0. (E) Phosphorylation site mutations of GEF8-S518 to a phospho-mimic (mCit-GEF8S518D) and phospho-dead variant (mCit-GEF8S518A) variant expressed in gef8-cΔ1. All scale bars represent 10 µm. (F, G) Quantification of measurements shown in C–E). (F) Maximum polarization value per pollen grain before germination. Letters above plots show significantly different groups according to a one-way ANOVA with Tukey’s test (p < 0.05) with mCit-GEF8 (dotted line, group a) as a reference. (G) Portion of pollen with a polarized signal (polarization frequency) before germination. The dotted line indicates mCit-GEF8 frequency. The asterisks indicate a significant difference to mCit-GEF8 or mCit-GEF9 according to a Χ2-test (p < 0.05). (H) Pollen germination efficiency of in vitro germinated pollen 4 h after imbibition on pollen germination media. Each point represents one replicate with more than 50 pollen grains. Groups of statistically significant differences according to a one-way ANOVA with Tukey’s test (p < 0.05) are indicated with letters. For underlying data of all quantification see S1 Data.