FERONIA and microtubules independently contribute to mechanical integrity in the Arabidopsis shoot

Abstract

To survive, cells must constantly resist mechanical stress. In plants, this involves the reinforcement of cell walls, notably through microtubule-dependent cellulose deposition. How wall sensing might contribute to this response is unknown. Here, we tested whether the microtubule response to stress acts downstream of known wall sensors. Using a multistep screen with 11 mutant lines, we identify FERONIA (FER) as the primary candidate for the cell's response to stress in the shoot. However, this does not imply that FER acts upstream of the microtubule response to stress. In fact, when performing mechanical perturbations, we instead show that the expected microtubule response to stress does not require FER. We reveal that the feronia phenotype can be partially rescued by reducing tensile stress levels. Conversely, in the absence of both microtubules and FER, cells appear to swell and burst. Altogether, this shows that the microtubule response to stress acts as an independent pathway to resist stress, in parallel to FER. We propose that both pathways are required to maintain the mechanical integrity of plant cells.

Fig 1. Pavement cell shape in receptor-like kinase mutants.

(A) Representative images of Col-0, WS-4, fer-4, the1-6, and bot1-7 pavement cells. Samples were PI stained, and cell contours were extracted with MorphoGraphX and projected in 2D. Scale = 100 μm. (B) Relative contribution of 27 shape descriptors to pavement cell shape, as assessed by principal component analysis. (C) Circularity (violin plots) of pavement cells and p-values of Dunn tests for the WT (in blue, Col-0 and WS-4), for the microtubule regulator mutants (in orange, nek6-1, spr2-2, tua3, tua4, tua5, and bot1-7) and the receptor-like kinase mutants (in pink, tfr1-1, cvy1-1, fer-4, herk1-1, herk2-1, the1-4, the1-6, wak1-1, wak2-1, wak3-1, wak4-1, and mik2-1). (D) Percentage of increase or decrease in pavement cell circularity from the WT (in blue, Col-0 and WS-4), for the microtubule regulator mutants (in orange, nek6-1, spr2-2, tua3, tua4, tua5, and bot1-7) and the receptor-like kinase mutants (in pink, tfr1-1, cvy1-1, fer-4, herk1-1, herk2-1, the1-4, the1-6, wak1-1, wak2-1, wak3-1, wak4-1, and mik2-1). All underlying data can be found in S1 Data. PI, propidium iodide; WT, wild type.

Fig 2. Impact of isoxaben on hypocotyl length in receptor-like kinase mutants.

(A) Representative images of Col-0, WS-4, fer-4, the1-,6 and bot1-7 etiolated seedlings grown with or without 1 nM isoxaben. Scale = 1 cm. (B) Hypocotyl length index (violin plot): distribution of isoxaben-grown hypocotyl length, normalized relative to the DMSO-grown ones. p-values of Wilcoxon–Mann–Whitney test for the WT (in blue, Col-0 and WS-4), the microtubule regulator (in orange, nek6-1, spr2-2, tua3, tua4, tua5, and bot1-7), and the receptor-like kinase mutants (in pink, tfr1-1, cvy1-1, fer-4, herk1-1, herk2-1, the1-4, the1-6, wak1-1, wak2-1, wak3-1, wak4-1, and mik2-1). (C) Deviation of hypocotyl length index. The WT accessions (Col-0 and WS-4) are labeled in blue. The microtubule regulator mutants (nek6-1, spr2-2, tua3, tua4, tua5, and bot1-7) are labeled in orange. The receptor-like kinase mutants (tfr1-1, cvy1-1, fer-4, herk1-1, herk2-1, the1-4, the1-6, wak1-1, wak2-1, wak3-1, wak4-1, and mik2-1) are labeled in pink. All underlying data can be found in S2 Data. WT, wild type.

Fig 3. The fer phenotype can be partially rescued on 2.5% agar.

(A) Representative confocal images of Col-0 and fer-4 etiolated hypocotyls, from seedlings grown on a medium containing 1 nM isoxaben with 0.7% or 2.5% agar (propidium iodide staining). Scale = 100 μm. (B) Bursting index (violin plot) and p-values of Wilcoxon–Mann–Whitney tests in Col-0 and fer-4 etiolated hypocotyls grown on a medium containing 1nM isoxaben with 0.7% or 2.5% agar. (C) Representative confocal images of Col-0 and fer-4 pavement cells, from seedlings grown on a medium containing 0.7% or 2.5% agar at t = 4 DAG and t = 12 DAG (propidium iodide staining). Scale = 100 μm. (D) Bursting index (violin plot) and p-values of Wilcoxon–Mann–Whitney tests in Col-0 and fer-4 pavement cells, from seedlings grown on a medium containing 0.7% or 2.5% agar at t = 4 DAG and t = 12 DAG. (E) Representative images of Col-0 and fer-4 cotyledons, grown on a medium containing 0.7% or 2.5% agar at t = 4 DAG, t = 8 DAG, and t = 12 DAG. Scale = 1 mm. (F) Cotyledon area (violin plot) and p-values of Wilcoxon–Mann–Whitney tests in Col-0 and fer-4 seedlings, grown on a medium containing 0.35%, 0.7%, 1.25%, or 2.5% agar at t = 4 DAG, t = 8 DAG, and t = 12 DAG. All underlying data can be found in S3 Data.

Fig 4. Cortical microtubule alignment in fer after ablation.

All seedlings were grown on 2.5% agar. (A–D) Representative confocal images of pPDF1::mCit-MBD (A, B) and fer-4 pPDF1::mCit-MBD (C, D) pavement cells, immediately after an ablation (t0, A, C) and 7 hours later (t7h, B, D). The red bars indicate the average orientations of cortical microtubule arrays, and the length of the red bars is proportional to the anisotropy of the cortical microtubule arrays (using ImageJ FibrilTool). Scale = 50 μm. (E) Anisotropy (violin plot) of cortical microtubule arrays and p-values of Wilcoxon–Mann–Whitney tests in cells surrounding the ablation site in pPDF1::mCit-MBD and fer-4 pPDF1::mCit-MBD pavement cells, immediately after an ablation (t0) and 7 hours later (t7h). (F–I) Cortical microtubule orientations (polar plots) and p-values of Wilcoxon–Mann–Whitney tests in cells surrounding the ablation site in pPDF1::mCit-MBD (F, G) and fer-4 pPDF1::mCit-MBD (H, I) pavement cells, immediately after an ablation (t0, F, H)) and 7 hours later (t7h, G, I). All underlying data can be found in S4 Data.

Fig 5. FER and microtubules independently contribute to the response to mechanical stress.

(A) Basal lobe width (violin plot) of pavement cells and p-values of Dunn tests for the WT (Col-0, WS-4), katanin mutant (bot1-7), and fer-4. Seedlings were grown on 0.8% agar. (B) Representative confocal images of pPDF1::mCit-MBD and fer-4 pPDF1::mCit-MBD hypocotyls grown on 0.7% and 2.5% agar with and without 5 μm of oryzalin. Scale bar = 50 μm. (C) Bursting index (violin plot) and p-values of Wilcoxon–Mann–Whitney tests in pPDF1::mCit-MBD and fer-4 pPDF1::mCit-MBD hypocotyls grown on 0.7% or 2.5% agar, with and without 5 μm of oryzalin. (D) In the WT (bottom), cells resist mechanical stress (red arrows) by 2 independent pathways (microtubule-dependent cellulose synthesis and FER-dependent wall reinforcement). In absence of both FER and microtubules (top), cells deform like passive matter and ultimately burst. All underlying data can be found in S5 Data. FER, FERONIA; WT, wild type.