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

['Alice Malivert', 'Laboratoire De Reproduction Et Développement Des Plantes', 'Université De Lyon', 'Ucb Lyon', 'Ens De Lyon', 'Inrae', 'Cnrs', 'Lyon', 'Özer Erguvan', 'Antoine Chevallier']

Date: 2021-12

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.

Funding: This work was supported by the European Research Council (ERC-2013-CoG-615739 ’MechanoDevo’ to O.H). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Malivert et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Although they are significantly less stiff, other wall components contribute to wall properties. In particular, pectins can partially rescue defects in cellulose synthesis in young cell walls. For instance, isoxaben treatment, which inhibits cellulose deposition through the internalization of CESA complexes, leads to thicker walls that are enriched in pectin [ 15 ]. Similarly, in young hypocotyls, pectin polarities precede the formation of mechanically anisotropic walls [ 16 ]. In contrast to cellulose deposition, pectin, as well as all other matrix components, are secreted to the cell wall [ 6 , 7 ]. Therefore, in principle, this provides an alternative mechanism for the cell to resist wall tension or damage. As for microtubules, the related mechanotransduction pathway is largely unknown. Yet, over the past decade, Catharanthus roseus Receptor-Like Kinases (CrRLKs) have emerged as key players. Although the link with mechanical stress remains to be formally established, THESEUS1 (THE1) has been implicated in the wall integrity pathway [ 17 – 19 ]. Based on defective root growth behavior on stiff interface, calcium signaling, pH response, and TOUCH gene expression, FERONIA (FER) has emerged as a candidate mechanosensor [ 20 ]. FER can sense the status of the cell wall, notably when salinity rises, through pectin binding [ 21 ]. It was recently proposed that FER also monitors microtubule behavior through a cascade involving Rho GTPases (ROP6) and the microtubule severing protein katanin [ 22 ]. Here, through a reverse genetic screen on wall sensors and using a suite of mechanical tests, we show that our best wall sensor candidate FER is not required for the microtubule response to stress, further suggesting that the microtubule response to stress can be more autonomous than anticipated. We also reveal that FER-dependent wall integrity pathway depends on wall tension and that both FER and the microtubule response to stress contribute to wall integrity.

Plant cell walls are composed of load-bearing cellulose microfibrils, tethered by a matrix made of polysaccharides and structural proteins [ 6 , 7 ]. The deposition of cellulose microfibrils is generally guided by cortical microtubules [ 8 ]. Beyond the average stiffness, the orientation of cellulose microfibrils controls the mechanical anisotropy of the wall. There is now ample evidence showing that cortical microtubules align with maximal tensile stress directions in the wall [ 9 , 10 – 13 ]. This provides a feedback loop in which shape and growth, whether at the individual cell or whole organ scale, prescribes a pattern of stress, to which cells resist by reinforcing their walls along maximal tensile stress directions [ 12 , 14 ].

All living organisms use mechanical forces as instructive cues during their development [ 1 , 2 ]. They also share a common mechanical property: Cells are pressurized by osmotic pressure and thus experience cortical tension. Osmotic pressure in plants is several orders of magnitude higher than that of animal cells, and it is counterbalanced by stiff cell walls [ 3 ]. Regulating the mechanical properties of cell walls, through the perception of wall tension and integrity, is thus crucial for plant growth and development [ 4 , 5 ].

Results

Altered pavement cell shape as a proxy for defective response to mechanical stress We first used a morphometric proxy to test the involvement of wall sensors in the microtubule response to stress. The jigsaw puzzle shape of Arabidopsis pavement cells has been proposed to be actively maintained and amplified by the microtubule response to mechanical stress. Indeed, necks in such cells prescribe highly anisotropic tensile stresses locally, to which microtubule arrays and thus cellulose deposition align [14,23,24]. We reasoned that the shape of pavement cells could be used in a mutant screen as a proxy for defects in that mechanical feedback loop. In past research, such screens have targeted the intracellular biochemical cues behind cell–cell coordination [25] and the contribution of cell wall properties in cell shape [26]. Whether wall sensors are involved in pavement cell shape remains ill described. Here, we focused on mutants impaired in receptor-like kinases that are highly expressed in the epidermis and aerial parts of the plant during early development and that exhibit an established link with the cell wall (and their closest homologs), namely feronia (fer), theseus1 (the1), theseus1/feronia-related1 (tfr1; at5g24010), curvy1 (cvy1), hercules receptor kinase 1 (herk1), herk2, mdis1-interacting receptor-like kinase2 (mik2-1), wall-associated kinase 1 (wak1), wak2, wak3, and wak4 (see S1 Table). We imaged and quantified the pavement cell shapes in receptor-like kinase candidate mutants, with the aim to select the ones with the strongest cell shape defects (Fig 1A). PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3001454.g001 To do so, we extracted the epidermal signal and used PaCeQuant to segment pavement cells and measure 27 shape descriptors (see Materials and methods [27] (S1A Fig)). To select the most discriminating PaCeQuant shape descriptor, we first performed a principal component analysis on our data. We compared the contribution of each parameter to the 2 axes with the most variation (Fig 1B). The cell perimeter was first, followed by the convex hull perimeter, the convex hull area, the cell area, the nonlobe area, and the longest path length, then by circularity (for relevant definition, see S1B Fig). To confirm that such shape descriptors are pertinent, and knowing that cortical microtubules are well-known pavement cell shape regulators, we used mutants with microtubule defects as positive controls. We performed the same analysis on 2 loss-of-function mutants: nek6 is impaired in a tubulin kinase which depolymerizes microtubules, and the mutant exhibits an enhanced microtubule response to stress [28]; bot1 is impaired in the microtubule severing protein katanin and exhibits a reduced response to stress [29]. We also included lines with tubulin mutations affecting microtubule dynamics (tua3D205N, tua4S178Δ, and tua6A281T referred as tua3, tua4, and tua5 in the following [30,31]) and spr2 with a reported enhanced cortical microtubule response to stress [32] but also ambivalent regulatory role in microtubule severing depending on tissue [33–35]. We found that cell perimeter, convex hull perimeter, convex hull area, cell area, and the longest path length descriptors were not sufficient to discriminate the pavement cell shape phenotype of those microtubules regulators (cell perimeter: p spr2-2 = 0.07; p tua3 = 0.018; p tua4 = 0.4; convex hull perimeter: p tua3 = 0.39; p tua5 = 0.011; convex hull area: p tua3 = 0.5; p tua5 = 0.03; cell area: p tua3 = 0.06; p tua5 = 0.07; longest path length: p tua3 = 0.28; p tua4 = 0.1; n spr2-2 = 262; n tua3 = 204; n tua4 = 230; n tua5 = 297; n Col-0 = 428). By contrast, all microtubule regulator mutant lines tested exhibited a defect in nonlobe area and circularity (nonlobe area: p nek6-1 = 0.009; p spr2-2 < 10−3; p tua3 = 0.003; p tua4 < 10−3; p tua5 < 10−3; p bot1-7 < 10−3; circularity: p nek6-1 < 10−3; p spr2-2 < 10−3; p tua3 < 10−3; p tua4 < 10−3; p tua5 < 10−3; p bot1-7 < 10−3, n nek6-1 = 183; n spr2-2 = 262; n tua3 = 204; n tua4 = 230; n tua5 = 297; n bot1-7 = 252; n Col-0 = 428; n WS-4 = 294, Fig 1C, S1C Fig). A total of 9 of the 11 receptor-like kinase mutants exhibited a nonlobe area significantly different from that of the wild type (WT). tfr1-1, fer-4, herk1-1, herk2-1, and wak4-1 displayed a nonlobe area significantly higher than that of the WT (p tfr1-1 < 10−3; p fer-4 < 10−3; p herk1-1 < 10−3; p herk2-1 < 10−3; p wak4-1 = 0.007; n tfr1-1 = 244; n fer-4 = 321; n herk1-1 = 225; n herk2-1 = 238; n wak4-1 = 204; n Col-0 = 428, S1C Fig), while the nonlobe area of cvy1-1, the1-6, wak1-,1 and mik2-1 was significantly lower than that of the WT (p cvy1-1 < 10−3; p the1-6 < 10−3; p wak1-1 < 10−3; p mik2-1 < 10−3; n cvy1-1 = 277; n the1-6 = 174; n wak1-1 = 295; n mik2-1 = 177; n Col-0 = 428). Only wak2-1 and wak3-1 displayed nonlobe area values that were nonsignificantly different from that of the WT (p wak2-1 = 0.1; p wak3-1 = 0.03; n wak2-1 = 271; n wak3-1 = 202; n Col-0 = 428). Thus, nonlobe area is not a discriminant parameter in our screen. We decided to study the next most variable parameter with defects in known microtubule regulator lines—circularity—for the rest of the analysis, justifying a posteriori a common choice in the literature on pavement cell shape [36]. Among the receptor-like kinase mutants tested, the pavement cells in fer-4, the1-6, and wak2-1 were significantly more circular than the WT supporting the hypothesis that the corresponding proteins could contribute to the microtubule response to stress in pavement cells (p fer-4 < 10−3; p the1-6 < 10−3; p wak2-1 = 0.002; n fer-4 = 321; n the1-6 = 174; n wak2-1 = 271; n Col-0 = 428; Fig 1C). wak1-1 also exhibited increased pavement cell circularity, albeit much less significantly (p wak1-1 = 0.04; n wak1-1 = 295; n Col-0 = 428). Only mik2-1 exhibited a significantly decreased pavement cell circularity (p mik2-1 < 10−3; n mik2-1 = 177; n Col-0 = 428), while tfr1-1 exhibited a tendency toward a decreased pavement cell circularity (p tfr1-1 = 0.03; n tfr1-1 = 244; n Col-0 = 428). Pavement cells in all the other receptor-like kinase mutants (cvy1-1, herk1-1; herk2-1; wak3-1; wak4-1) displayed a circularity comparable to that of the WT (p cvy1-1 = 0.5; p herk1-1 = 0.2; p herk2-1 = 0.47; p wak3-1 = 0.47; p wak4-1 = 0.5; n cvy1-1 = 277; n herk1-1 = 225; n herk2-1 = 238; n wak3-1 = 202; n wak4-1 = 204; n Col-0 = 428). To distinguish the relative contributions of the most affected mutants, we quantified the deviation of circularity from the WT. Among all the receptor-like kinases tested, the candidate mutants with the largest defect in circularity when compared to the WT were fer-4 (consistent with previously published results, [37]) and, to a lesser extent, the1-6 (Fig 1D). fer-4 cells were 30% more circular and the1-6 cells were 7% more circular than the WT, values that were comparable to that of microtubule regulator mutants, such as spr2-2 or bot1-7 (Fig 1D). Note that the same trend was obtained when considering solidity, another parameter often used to characterize lobe formation [38] (S1D Fig). Note also that while circularity and solidity can be impacted when stress response levels change, they do not necessarily directly scale with stress response level. Different shapes can lead to similar circularity and solidity values (e.g., see last figure); nevertheless, this cell shape-based screening allowed us to identify the RLK mutants with the most affected pavement cell shape, possibly through a defective microtubule response to stress. Because fer-4 stands out, this first screening suggests that FER could play a role in the microtubule response to mechanical stress.

Differential response of receptor-like kinase mutants to isoxaben To challenge the results from this initial screen, we next used a well-established protocol to mechanically perturb cell walls. Isoxaben inhibits the biosynthesis of cellulose [39], and thus weakens the wall. In past work, such treatment were shown to induce a hyperalignment of cortical microtubules at the shoot apical meristem and in cotyledon pavement cells [14,40], consistent with a response to increased tensile stress levels in the cell wall. Note that isoxaben can also trigger other responses, including reactive oxygen species (ROS) production, lignification, and changes in gene expression [17]. Thus, depending on time and dose, isoxaben may also ultimately reduce stress level [41]. Here, we use this drug as a screening tool, complementary to the pavement cell shape screen, to identify mutants insensitive or hypersensitive to mechanical perturbation and which are thus likely to be defective in mechanosensing. We grew the seedlings in a medium containing 1 nM isoxaben or the same volume of DMSO, in the dark (Fig 2A). We then measured the length of the etiolated hypocotyls 4 days after germination. After isoxaben treatment, 4-day-old WT seedlings exhibited a shorter hypocotyl (by 41% for Col-0, 34% for WS-4, n Col-0 DMSO = 281, n Col-0 iso = 271, n WS-4 DMSO = 127, n WS-4 iso = 129). To compare WT and mutants, we normalized the obtained distribution of lengths to the same mean and standard deviation as the control, thus providing a hypocotyl length index (Fig 2B, S2 Fig). Treated mutants with a relatively shorter hypocotyl than the treated WT were labeled more sensitive to isoxaben, whereas treated mutants with a relatively longer hypocotyl than the treated WT were labeled less sensitive than the WT. PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3001454.g002 A total of 10 out of the 11 receptor-like kinase mutants studied were less sensitive than the WT (tfr1-1, cvy1-1, herk1-1, herk2-1, the1-6, wak1-1, wak2-1, wak3-1, wak4-1, mik2-1; p tfr1-1 < 10−3; p cvy1-1 < 10−3; p herk1-1 < 10−3; p herk2-1 < 10−3; p the1-6 < 10−3; p wak1-1 < 10−3; p wak2-1 < 10−3; p wak3-1 < 10−3; p wak3-1 < 10−3; p mik2-1 < 10−3; n tfr1-1 DMSO = 80; n tfr1-1 iso = 83; n cvy1-1 DMSO = 109; n cvy1-1 iso = 115; n herk1-1 DMSO = 82; n herk1-1 iso = 70; n herk2-1 DMSO = 88; n herk2-1 iso = 106; n the1-6 DMSO = 57; n the1-6 iso = 91; n wak1-1 DMSO = 116; n wak1-1 iso = 89; n wak2-1 DMSO = 63; n wak2-1 iso = 78; n wak3-1 DMSO = 99; n wak3-1 iso = 89; n wak4-1 DMSO = 103; n wak4-1 iso = 86; n mik2-1 DMSO = 84; n mik2-1 iso = 96; n Col-0 DMSO = 281; n Col-0 iso = 271), whereas fer-4 was significantly more sensitive (p fer-4 < 10−3; n fer-4 DMSO = 104; n fer-4 iso = 90) than the WT (Fig 2B). When plotting the deviation of each mutant from the WT phenotype, it appeared that among all the receptor-like kinase tested, fer-4 and the1-6 were the most affected mutants in their response to isoxaben, albeit in opposite trend: fer-4 hypocotyl were more sensitive to isoxaben, whereas the1-6 were less sensitive to isoxaben (Fig 2C). These results indicate that all the receptor-like kinases tested might be involved in the cellular response to a mechanical stress, with FER and THE1 having the most clear-cut, and opposing, response. To check whether these defects could be related to the microtubule response to stress, we performed the same analysis on microtubule regulator mutants. bot1-7 (in WS-4 ecotype) was more sensitive to the isoxaben treatment than the WT (p bot1-7 < 10−3; n bot1-7 DMSO = 99; n bot1-7 iso = 96; n WS-4 DMSO = 127; n WS-4 iso = 129) and thus fell in the same cluster as fer-4. The nek6-1 mutant exhibited the same isoxaben sensitivity as the WT (p nek6-1 = 0.1; n nek6-1 DMSO = 91; n nek6-1 iso = 104; n Col-0 DMSO = 281; n Col-0 iso = 271) (Fig 2B). The spr2-2 mutant was significantly less sensitive than the WT (p spr2-2 < 10−3; n spr2-2 DMSO = 99; n spr2-2 iso = 85; n Col-0 DMSO = 281; n Col-0 iso = 271) and thus fell in the same cluster as the1-6. Last, tua3, tua4, and tua5 were significantly less sensitive than the WT (p tua3 < 10−3; p tua4 < 10−3; p tua5 < 10−3; n tua3 DMSO = 93; n tua3 iso = 90; n tua4 DMSO = 83; n tua4 iso = 108; n tua5 DMSO = 72; n tua5 iso = 66; n Col-0 DMSO = 281; n Col-0 iso = 271). Altogether, our primary screening approach using both pavement cell shape analysis and isoxaben sensitivity test as a combined proxy for the microtubule response to mechanical stress highlights FER as a primary candidate. So far, our data are also consistent with the proposed scenario in which FER and katanin belong to the same pathway [22], but these remain primary screening approaches that do not directly test the involvement of our main candidate in the microtubule response to mechanical stress. Based on this screening, in the following we decided to focus on FER to investigate its involvement in the mechanical integrity of the shoot and to directly test its contribution to the microtubule response to mechanical stress.

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