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. 2020 Mar 12;117(13):7494–7503. doi: 10.1073/pnas.2000100117

Fig. 4.

Fig. 4.

LRX8 functional mechanism requires homodimerization to control pollen tube growth. (A) Close-up view of the dimer interface in LRX2. Details of the interactions between the two protomers (depicted in blue and gray) are highlighted. The Cys (C138) mediating the disulfide bond formation is depicted in yellow. The ionic and hydrogen bonds are shown as dotted lines (in magenta). (B) Side surface representation of LRX2 colored according to LRX family sequence conservation. The black rectangle depicts the LRX dimer interface. (C) The dimer interface of LRX proteins is stabilized by a central disulfide bridge. SEC runs of wild-type LRX8–RALF4, LRX8C157A–RALF4, LRX8Y87A.A133F–RALF4, and LRX8C157A.Y87A.A133F–RALF4. Peaks 1 and 2 correspond to the dimeric and monomeric forms of LRX8, respectively. (D) SEC–MALS summary table for the LRX8C157A–RALF4 and LRX8C157A.Y87A.A133F–RALF4 complexes in solution at pH 5.0, along with the predicted molecular weights. The values indicated in the table are the mean ± SD of two independent measurements. (E) ITC of LRX8C157A.Y87A.A133F vs. RALF4folded. The table summarizes the biophysical values obtained. Kd, (dissociation constant), binding stoichiometries (N), and thermodynamic parameters (ΔH). (F) Shift of the monomer–dimer equilibrium under reducing conditions in solution. LRX8Y87A.A133F–RALF4 run in the absence (orange) and presence (purple) of TCEP. (G) Complementation assays of lrx8,9,10,11 with LRX8 oligomeric mutants. (G, Left) Seeds per silique of WT, lrx8,9,10,11, pLRX8::LRX8WT lines 1 and 2, pLRX8::LRX8Y87A.A133F and pLRX8::LRX8C157A.Y87A.A133F in the lrx8,9,10,11 quadruple mutant background. Data are means ± SEM of 10 siliques. For LRX8 mutant transgenic lines, 16 independent lines were analyzed. (Scale bar, 10 µm.) (G, Right) Siliques images of the corresponding lines. (H) Working model and open questions about RALF-mediated autocrine signaling during pollen tube growth. Physical interactions are depicted by double arrows and genetic relationships by single arrows. Straight lines indicate what is known, and dotted lines indicate speculative relationships. Affinity ranges are marked next to the interaction arrows. Legends of the different components are shown on Top.