Figures and data
PME activity is required for RALF1-induced root growth repression
(A-B) Three-day-old wild-type seedlings were subjected for three days to 1 µM RALF1 and/or 2.5 µM EGCG (A). (B) Boxplots depict the root length of wild-type seedlings under different treatments shown in (A).
(C-D) Three-day-old wild-type seedlings were transferred for three days to solvent control, 1 µM RALF1 and/or 15 µM EC (C). (D) Boxplots depict root length of wild-type seedlings under different treatments shown in (C).
(E-F) Confocal microscopy images of the root epidermal cells of 6-day-old wild-type seedlings after 3 h treatment in liquid medium with 50 µM EC/EGCG or solvent control. Seedlings were stained with COS488 to visualize de-methylesterified pectin. (F) Boxplots displaying the probe staining signal intensity shown in (E). Scale bar = 25 µm, n = 8-10 roots per treatment with a total number of 79 - 96 quantified cells.
(G-H) Confocal microscopy images of the root epidermal cells of 6-day-old wild-type and PMEI3-OE seedlings, stained with COS488 to visualize de-methylesterified pectin. (H) Boxplots displaying the probe staining signal intensity shown in (G). Scale bar = 25 µm, n = 9 - 11 roots per treatment with 71 - 77 quantified cells.
(I-J) Three-day-old wild-type, PMEI3-OE and PMEI5-OE seedlings were exposed for three days to 1 µM RALF1 or solvent control (I). (J) Boxplots depict the root length of wild-type compared to PMEI3-OE seedlings of the treatments shown in (I).
(K-L) Three-day-old wild-type seedlings were exposed for three days with 0.5 µM flg22 and 15 µM EGCG or solvent control (K). (L) Boxplots depict the root length of wild-type seedlings under different treatments shown in (K).
Statistical significance was determined by a one-way ANOVA with a Tukey Post Hoc multiple comparisons test (P < 0.05, letters indicate significance categories) (B, D, F, J and L) and a student’s t-test (***p = 0.0001) (H). Boxplots: Box limits are representing the 25th and 75th percentile, the horizontal line represents the median. Whiskers display min. to max. values. Representative experiments are shown. (A, C, I, K) Scale bar = 1 cm, n = 11- 13 roots per treatment/line.
RALF1 requires PME activity to affect cell wall integrity
(A-B) Representative transmission electron microscopy (TEM) images of epidermal root cells treated for 3 h with solvent control and 1 µM RALF1, respectively. Arrowheads indicate RALF1-induced plasma membrane invaginations. Scale bar = 10 µm. (B) shows details, Scale bar = 2 µm.
(C) Confocal microscopy images of the root epidermal cells of 6-day-old LTI6b-YFP expressing seedlings, treated for 3 h with solvent control or 1 µM RALF1. Scale bar = 25 µm.
(D-F) Confocal microscopy images of the root epidermal cells of 6-day-old wild-type (D) and fer-4 mutant (E) seedlings, treated for 3 h with 1 µM RALF1 or solvent control. Seedlings were mounted in propidium iodide to visualize the cell walls. Arrowheads indicate RALF1-induced alterations of the cell wall stain. (F) Graphs represent the width of PI signal per root under different treatments shown in (D, E). Scale bar = 25 µm, n = 9 – 11 roots per treatment with a total number of 49 – 51 quantified cells.
(G-H) Confocal microscopy images (G) and quantification (H) of 6-day-old roots of wild-type seedlings, treated in liquid medium with 1 µM RALF1 and/or 50 µM EGCG as well as solvent control for 3 h. Seedlings were mounted in propidium iodide to visualize the cell walls. Arrowheads indicate cell wall invaginations. Scale bar = 25 µm, n = 8 – 10 roots per treatment with a total number of 44 – 49 quantified cells.
(I-K) Confocal microscopy images (I, J) and quantification (K) of 6-day-old wild-type (I) and PMEI3-OE (J) roots, treated in liquid medium with 1 µM RALF1 or solvent control for 3 h. Seedlings were mounted in propidium iodide to visualize the cell walls. Arrowheads indicate cell wall invaginations. Scale bar = 25 µm, n = 8 – 12 roots per treatment with a total number of 46 – 53 quantified cells.
Statistical significance was determined by a one-way ANOVA with a Tukey Post Hoc multiple comparisons test (P < 0.05, letters indicate significance categories) (H) or a two-way ANOVA with Bonferroni Post Hoc test (**** = P < 0.0001) (F and K). Boxplots: Box limits are representing the 25th and 75th percentile, the horizontal line represents the median. Whiskers display min. to max. values.
PME activity is required for RALF1 signalling output
(A-B) HPTS-based (458/405 ratio) pH assessment of late meristematic root cells of wild-type seedlings treated for 3h with 1 µM RALF1, 50 µM ECGC or both compared to solvent control. Representative confocal images (A) are shown. Scale bar = 10 µm. (B) Boxplots depict mean HPTS 458/405 intensities shown in (A), n = 9 – 11.
(C-D) HPTS-based (458/405 ratio) pH assessment of late meristematic cells in wild-type and PMEI3-OE seedlings treated for 3h with 1 µM RALF1 compared to solvent control. Scale bar = 10 µm. (D) Boxplots depict mean HPTS 458/405 intensities shown in (C), n = 10 – 12.
(F-G) RALF1-induced internalization of FER::FER-GFP in late meristematic epidermal root cells of seedlings treated for 3h with 1 µM RALF1, 50 µM ECGC or both compared to solvent control. Scale bar = 25 µm. (G) Boxplots displaying the mean intracellular GFP signal intensity shown in (F), n = 9 – 12.
(H-J) RALF1-induced internalization of FER in late meristematic epidermal root cells in FER::FER-GFP (H) and in FER::FER-GFP crossed with PMEI3-OE (I) seedlings treated for 3h with 1 µM RALF1 compared to solvent control. Scale bar = 25 µm. (J) Boxplots displaying the intracellular GFP signal intensity shown in (H and I).
Statistical significance was determined by a one-way ANOVA with a Tukey Post Hoc multiple comparisons test (P < 0.05, letters indicate significance categories) (B and G) or a two-way ANOVA with Bonferroni Post Hoc test (**** = P < 0.0001) (D and J). Boxplots: Box limits are representing 25th and 75th percentile, the horizontal line represents the median. Whiskers display min. to max. values.
RALF peptides associate with de-methylesterified oligogalacturonides
(A-B) RALF1-induced internalization of FER::FER-GFP in late meristematic epidermal root cells of six-day-old seedlings treated for 3h with a concentration series of 0.5, 1 and 1.5 µM RALF1 compared to solvent control and co-treated with OGs with a chain length of 25-50 (OG25-50; 50 mg/mL). (B) Boxplots displaying the mean intracellular GFP signal intensity are shown in (A). Scale bar = 25 µm, n = 9 – 12.
(C-D) Biolayer interferometry assays showed the binding between RALF1 and OG25-50 with an Equilibrium dissociation constant (Kd) of 105 nM (C). The association and dissociation constants (kON = 1.02 x 103 M-1 s-1, kOFF _=1.07 x 10-4 s-1) imply high avidity binding (D). Statistical significance was determined by a one-way ANOVA with a Tukey Post Hoc multiple comparisons test (P < 0.05, letters indicate significance categories) (B). Boxplots: Box limits are representing 25th and 75th percentile, the horizontal line represents the median. Whiskers display min. to max. values.
LRX proteins are not essential for the PME-dependent activity of RALF1
(A-B) Confocal microscopy images (A) and quantification (B) representing the average cell wall width per root under different treatments shown in (A), of 6-day-old roots of wild-type seedlings, treated in liquid medium with 1 µM RALF1 and/or 50 µM EGCG as well as solvent control for 3 h. Seedlings were mounted in propidium iodide to visualize the cell walls. Arrowheads indicate cell wall invaginations. Scale bar = 25 µm, n = 10 – 12 roots per treatment with a total number of 44 – 52 quantified cells.
(C-D) Confocal microscopy images (C) and quantification (D) representing the average cell wall width per root under different treatments shown in (C), of 6-day-old roots of lrx1/lrx2/lrx3/lrx4/lrx5 quintuple mutant seedlings, treated in liquid medium with 1 µM RALF1 and/or 50 µM EGCG as well as solvent control for 3 h. Seedlings were mounted in propidium iodide to visualize the cell walls. Arrowheads indicate cell wall invaginations. Scale bar = 25 µm, n = 11 – 12 roots per treatment with a total number of 46 – 53 quantified cells. (E-H) Three-day-old wild-type (E) or lrx1/lrx2/lrx3/lrx4/lrx5 quintuple mutant (G) seedlings transferred for three days in liquid growth medium supplemented with solvent control, 1 µM RALF1 and/or 15 µM EGCG. Seedlings were transferred to solid growth medium just before imaging. (F, H) Boxplots display root length of seedlings under different treatments shown in (E, G). Scale bar = 1 cm, n = 14 – 16 roots per treatment/line.
(I-J) De-methylesterified and hence negatively charged pectin is crucial for the signalling output of positively charged RALF peptides (I). Interference with PME activity, application of free de-methylesterified OGs, or removal of positive charges in RALF leads to abolished RALF1 output signalling (J).
Statistical significance was determined by a one-way ANOVA with a Tukey Post Hoc multiple comparisons test (P < 0.05, letters indicate significance categories) (B, D, F and H). Boxplots: Box limits are representing 25th and 75th percentile, the horizontal line represents the median. Whiskers display min. to max. values. Representative experiments are shown.
The summary figures (Fig. 5I, J) were created using the Concepts App for iOS (Version 6.9.2, TopHatch, Inc. (Turku, Finnland)) and edited using AffinityDesigner (version 1.10, Serif (West Bridgford, UK)) by Ann-Kathrin Rößling.
Cell wall invaginations after RALF1 treatment in a plasma membrane marker
(A) Confocal images of epidermal cells in 6-day-old roots of the plasma membrane marker UBQ10::NPSN12-YFP after 3 h treatment in liquid medium with solvent control or 1 µM RALF1. Scale bar = 25 µm, n = 10 – 13.
RALF34 function is similar to RALF1 in initiating ligand-induced endocytosis in FER
(A-B) Internalization of FER::FER-GFP in late meristematic epidermal root cells of six-day- old seedlings treated for 3h with 1 µM RALF1, 1 µM RALF34 and/or 50 µM EGCG compared to solvent control. (B) Boxplots displaying the mean intracellular GFP signal intensity are shown in (A). Scale bar = 25 µm, n = 10 – 12 roots per treatment with a total number of 41 – 46 quantified cells.
Statistical significance was determined by a one-way ANOVA with a Tukey Post Hoc multiple comparisons test (P < 0.05, letters indicate significance categories) (B, D, F and H). Boxplots: Box limits are representing 25th and 75th percentile, the horizontal line represents the median. Whiskers display min. to max. values. Representative experiments are shown.
Application of free de-methylesterified oligogalacturonides disrupts RALF1 activity at the cell surface
(A-B) Confocal images of 6-day-old wild-type roots after 3 h treatment in liquid medium with solvent control, 50 mg/mL OG10-15, 50 mg/mL OG25-50 and 1 µM RALF1, respectively (A). Seedlings were mounted in propidium iodide to visualize the cell walls. Arrowheads indicate cell wall invaginations. (B) Graphs represent the average cell wall width per root under different treatments shown in (A). Scale bar = 25 µm, n = 8 – 11 roots per treatment with a total number of 46 – 52 quantified cells.
(C-D) RALF1-induced internalization of FER::FER-GFP in late meristematic epidermal root cells of 6-day-old seedlings treated for 3h with solvent control, 50 mg/mL OG10-15, 50 mg/mL OG25-50 and 1 µM RALF1, respectively. (D) Boxplots display the intracellular FER-GFP signal intensities. Scale bar = 25 µm, n = 10 – 11.
Statistical significance was determined by a one-way ANOVA with a Tukey Post Hoc multiple comparisons test (P < 0.05, letters indicate significance categories) (B and D). Boxplots: Box limits represent 25th and 75th percentile, and the horizontal line represents the median. Whiskers display min. to max. values. Representative experiments are shown.
Positive charges in RALF1 are required for its bioactivity
(A-C) Amino acid composition of RALF1 and non-charged RALF1-KR peptides and its respective pH-dependent net charge (B, C).
(D-E) Three-day-old wild-type seedlings were exposed for three days to 1 µM RALF1 or RALF1-KR or solvent control (D). (E) Boxplots display root length of seedlings under different treatments shown in (D). Scale bar = 1 cm, n = 14 – 15.
(F-G) Representative confocal images of 6-day-old wild-type roots after 3 h treatment in liquid medium with 1 µM RALF1 or RALF1-KR or solvent control. Seedlings were mounted in propidium iodide to visualize the cell walls. Arrowheads indicate cell wall invaginations (F).
(G) Graphs represent the average cell wall width per root under different treatments shown in (F). Scale bar = 25 µm, n = 9 – 12.
(H-I) RALF1-induced internalization of FER::FER-GFP in late meristematic epidermal root cells of 6-day-old seedlings treated for 3h with solvent control, 1 µM RALF1 or RALF1-KR. Representative confocal images (H) are shown.. (I) Boxplots display the intracellular GFP signal intensity. Scale bar = 25 µm, n = 11 – 12 roots per treatment with a total number of 50 – 56 quantified cells.
(J) Biolayer interferometry assays showed the absence of binding between RALF1-KR and OG25-50. RALF1 was used as a positive control, exhibiting the binding as appeared in the previous measurements (see Fig. 4D).
Statistical significance was determined by a one-way ANOVA with a Tukey Post Hoc multiple comparisons test (P < 0.05, letters indicate significance categories) (E, G and I). Boxplots: Box limits are representing 25th and 75th percentile, the horizontal line represents the median. Whiskers display min. to max. values. Representative experiments are shown.
