# Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al

Harinderbir Kaur, Jean-marie Teulon, Christian Godon, Thierry Desnos, Shu-wen W Chen, Jean-luc Pellequer

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Harinderbir Kaur, Jean-marie Teulon, Christian Godon, Thierry Desnos, Shu-wen W Chen, et al. Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al. Plant, Cell and Environment, 2024, 47 (2), pp.574-584. 10.1111/pce.14744. hal-04355569v2

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# Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al

Harinderbir Kaur<fnref n="1" /> ID

Jean-Marie Teulon<fnref n="1" />

Christian Godon<fnref n="2" /> ID

Thierry Desnos<fnref n="2" /> ID

Shu-wen W. Chen<fnref n="1" />,<fnref n="3" /> ID

Jean-Luc Pellequer<fnref n="1" /> ID

### Correspondence

Jean-Luc Pellequer, Institut de Biologie Structurale, Grenoble, 38000, France.

Email: Jean-luc.pellequer@ibs.fr

### Funding information

Agence Nationale de la Recherche; European Union's Horizon 2020 research and innovation programme, Grant/Award Number: 812772

### Abstract

The plasticity and growth of plant cell walls (CWs) remain poorly understood at the molecular level. In this work, we used atomic force microscopy (AFM) to observe elastic responses of the root transition zone of 4-day-old *Arabidopsis thaliana* wild-type and *almt1*-mutant seedlings grown under Fe or Al stresses. Elastic parameters were deduced from force-distance curve measurements using the trimechanic-3PCS framework. The presence of single metal species Fe<fnref n="2" />⁺ or Al<fnref n="3" />⁺ at 10 µM exerts no noticeable effect on the root growth compared with the control conditions. On the contrary, a mix of both the metal ions produced a strong root-extension arrest concomitant with significant increase of CW stiffness. Raising the concentration of either Fe<fnref n="2" />⁺ or Al<fnref n="3" />⁺ to 20 µM, no root-extension arrest was observed; nevertheless, an increase in root stiffness occurred. In the presence of both the metal ions at 10 µM, root-extension arrest was not observed in the *almt1* mutant, which substantially abolishes the ability to exude malate. Our results indicate that the combination of Fe<fnref n="2" />⁺ and Al<fnref n="3" />⁺ with exuded malate is crucial for both CW stiffening and root-extension arrest. However, stiffness increase induced by single Fe<fnref n="2" />⁺ or Al<fnref n="3" />⁺ is not sufficient for arresting root growth in our experimental conditions.

### KEYWORDS

atomic force microscopy (AFM), elasticity, nanoindentation, primary root, trimechanic-3PCS

## 1 | INTRODUCTION

The cell wall (CW) of land plants has been depicted as a highly intertwining architecture consisting of cellulose microfibrils, hemi-cellulose and pectin (Carpita & Gibeaut, 1993), which compose the three major components of the primary CW. Cellulose microfibrils are the stiffest component, playing a load-bearing role (Bashline et al., 2014; Bidhendi & Geitmann, 2016; Cosgrove, 2005, 2018).

Their orientation creates a mechanical anisotropy, restricting cell expansion in the microfibril direction (Majda et al., 2017). Hemi-celluloses (xyloglucan chains) bind to cellulose microfibrils using hydrogen bonds (Valent & Albersheim, 1974); they also bind covalently to pectin (Bauer et al., 1973), a network made of matrix pectin polysaccharides and soluble proteins (Kerr & Bailey, 1934). Water is also a major constituent of primary CWs (Gaff & Carr, 1961), up to 65% (Jackman & Stanley, 1995), and an essential element for

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2023 The Authors. *Plant, Cell & Environment* published by John Wiley & Sons Ltd.

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<footnote n="1">¹Univ. Grenoble Alpes, CEA, CNRS, IBS, Grenoble, France</footnote>

<footnote n="2">²Aix Marseille Université, CEA, CNRS, BIAM, Saint Paul-Lez-Durance, Cadarache, France</footnote>

<footnote n="3">³Rue Cyprien Jullin, Vinay, France</footnote>
chemical reactions within the CW. The thickness of the primary CW was suggested to be around 80–100 nm for meristematic and parenchymatous cells, in accordance with a layered structure of cellulose microfibrils with a layer spacing of ~20–40 nm (McCann et al., 1990). However, the accurate thickness measurement of external primary CWs remains challenging, roughly estimated as ~0.1–1 µm (Derbyshire et al., 2007).

Cell growth is characterized by an irreversible increase in cell volume and surface area, concomitant with a CW loosening. The complexity of CW growth results in poorly known pathways and mechanisms that control root CW plasticity (Somssich et al., 2016). Upon various environmental stresses, a reduction of cell growth associated with CW stiffening is a well-known phenomenon observed in plants (Schopfer, 2006), tightly linked to dynamic behaviours of primary CWs. It has been proposed that strain-stiffening limits growth and restricts organ bulging (Kierzkowski et al., 2012). During plant growth, some cells enlarge their volumes by 10–1000 times (Cosgrove, 1997), with this growth regulated by external stimuli such as temperature, light, water, xenobiotics and internal factors like growth hormones (Preston & Hepton, 1960). The cessation of coleoptile growth was attributed to the loss of CW plasticity but not to turgor pressure which implicates an increase of CW stiffness (Kutschera, 1996). One pioneering work on CW nanomechanics used atomic force microscopy (AFM) to observe stiffness heterogeneity in the meristem surfaces at regional, cellular and even subcellular levels (Milani et al., 2011). AFM has been shown to be powerful for stiffness measurements on plant tissues (Cuadrado-Pedetti et al., 2021; Milani et al., 2014; Peaucelle et al., 2011). For characterizing the nano-stiffness of a sample in response to a given stress, AFM nanoindentation provides a promising strategy of detecting changes in physico-chemical properties of cellular or tissue surfaces on a nanoscale.

Recently, stiffening of seedling roots has been observed within 30 min after exposition to iron stress (Balzergue et al., 2017). In a condition of low phosphate, low pH (<6) and the presence of iron, a primary root extension arrest (REA) was observed and a signalling pathway involving STOP1 and ALMT1 proteins was found to inhibit CW expansion (Balzergue et al., 2017; Mora-Macias et al., 2017). STOP1 abundance in the nucleus of the plant cell was found to be controlled by the presence of iron (Fe) and aluminum (Al) metals, both of which induced malate exudation through the ALMT1 channel (Godon et al., 2019; Le Poder et al., 2022). Although Fe is a fundamental nutrient for plants, a defense mechanism somehow occurs in a Fe-rich environment, implying that an excess of Fe is deleterious to plants (Oliveira de Araujo et al., 2020). The deleterious effect of Fe is linked with the ferritin capacity of plant cell for storing free reactive iron (Ravet et al., 2009) instead of being driven to the vacuole (Hirsch et al., 2006; Ward et al., 2008). Indeed, ferritin encapsulates the Fe³⁺ cation after oxidizing Fe²⁺ before storage (Macara et al., 1972). In bean roots, the apoplast provides a storage space for Fe³⁺, where it could be extracted for nutrition use in case of iron deficiency (Bienfait et al., 1985). The *Arabidopsis lpr1/lpr2* mutants lack the capability of oxidizing Fe²⁺ to Fe³⁺ and were shown

to reduce the amount of iron in the apoplast, exhibiting a Fe-insensitive phenotype in low-phosphate conditions (Svistoonoff et al., 2007).

Inhibition of root elongation is a well-known plant response to the tolerance of Al (Clarkson, 1965), especially at low pH (Bian et al., 2013). Al toxicity resides in its cationic binding to negatively charged sites (membranes, proteins, saccharides) available in the root (Nichol et al., 1993). One creep-extension analysis showed that Al accumulation in the CW provoked a reduction of CW extensibility in wheat roots (Ma et al., 2004). Within 1 h of Al supply, callose deposition was observed in the root tip of soybean seedlings (Wissemeier et al., 1992). In addition to callose deposition, the main physiological mechanism of Al tolerance is the exclusion of Al from the root apex (Kochian et al., 2015), where Al usually accumulates in the root apex symplast and the apoplast (Delhaize & Ryan, 1995) and binds directly to negatively charged pectins of the CW of root border cells (Yang et al., 2016). This exclusion is accomplished by exudation of organic acids (Miyasaka et al., 1991) such as malate and citrate (Liu et al., 2009). In cultured tobacco, Al accumulation in plant cell walls was found to depend on the presence of ferrous iron (Fe²⁺) (Chang et al., 1999). However, unlike Al, Fe does not stimulate malate excretion (Delhaize et al., 1993).

Fe²⁺ in phosphate-deficient conditions is able to arrest primary root growth (Abel, 2011; Godon et al., 2019). Potential harmfulness of excessive Fe to cells is attributed to ROS (reactive oxygen species) production either by the Fenton (involving Fe²⁺) or by the Haber-Weiss reactions (Fe³⁺) (Gill & Tuteja, 2010). Concentration above 40 µM of mixed Fe with Al resulted in a drastic reduction of root length, likely through the ROS production (Cakmak & Horst, 1991). The presence of Fe²⁺ in *Arabidopsis* roots stimulates ROS production with peroxidase activity (Balzergue et al., 2017; Müller et al., 2015; Naumann et al., 2022), particularly together with the class III peroxidase to stiffen and loosen the plant CWs (Francoz et al., 2015; Passardi et al., 2005; Wolf & Hofte, 2014). In grass, peroxidase activity was linked to leaf growth arrest and CW cross-linking (MacAdam & Grabber, 2002); in rice, peroxidase was found to be present in coleoptile growth arrest of shoots with increased ferulic and diferulic acids (Wakabayashi et al., 2012); similar findings were obtained for maize (Uddin et al., 2014). However, the causality between CW stiffness and REA remains to be elucidated.

To investigate Al and Fe effects on physiology and morphology of growing roots, we performed AFM indentations on *Arabidopsis* seedling roots under Fe and Al stresses using various metal concentrations and compositions. The present research provides a link between measurements of the structural stiffness (Chen et al., 2023) with stress effects of metal ions on root growth. The recently developed trimechanic-3PCS framework was used throughout this study (Chen et al., 2023). The correspondence between the variations in the magnitude of elasticity parameters and the length of seedling roots under these stress conditions, our understanding of the molecular mechanisms of CW stiffening and root growth as a function of metal ion concentration can be improved.
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2 | MATERIALS AND METHODS

2.1 | Seedling growth and manipulation

The experimental specimens are *Arabidopsis thaliana* L. (Heynh.) lines of Columbia (Col-0) or the Coler105 background as specified in (Bonnot et al., 2016). The production of *almt1*51 mutant was previously described (Balzergue et al., 2017). Seeds were surface sterilized by 70% ethanol +0.05% SDS for 1 min, followed by washing twice with 95% ethanol for 1 min each time and drying in a laminar airflow hood. To alleviate gravitropism effects on seedling growth such as inducing root wriggling or waving by growing vertically in a Petri dish, the seeds sown on Day 0 were placed in a 24-well crystallization plate (VDX plate HR3-140, Hampton Research). Plates were placed in a growing chamber (IPP100+ incubator; Memmert, Fisher Scientific) for 4 days with a 16-h photoperiod with 24°C/21°C day/night, respectively. During the 4 days, seedlings grew under the -Pi condition (no phosphate added) in the nutrient solution. The chemical content of the agar presently used is particularly poor in phosphate and metals, as determined by ICP analysis (Mercier et al., 2021), which is different from the agar used in our previous study (Balzergue et al., 2017).

After 4 days, seedlings were transferred into 60-mm agar Petri
dishes in the -Pi condition while supplemented with or without
10 or 20 µM of FeCl₂ and/or AlCl₃ for 2 h. Then, seedlings were
transferred from the agar plates to a glass slide for AFM
nanoindentation experiments and classical force-distance curves
were collected within 30 min after mounting the samples on the
glass slide. In brief, a thin layer of silicone, NuSil MED1-1356
(NuSil Technology LLC), was spread on the glass slide as described
(Kaur, Godon, et al., 2023; Kaur, Teulon, et al., 2023). Partial
polymerization was allowed for a few seconds before the root was
laid over the silicone. Then, several thin silicone bands were
stretched using a syringe needle to fasten the root over all its
length except the transition zone, which is located about 500 µm
from the root apex (Figure 1). To prevent drying, a droplet of the
growth medium (without the agar powder) was deposited to cover
the entire seedling. The mounted seedling was positioned under
the AFM for data acquisition (Figure 1).

## 2.2 | Length measurement of primary root

The root lengths were measured on Day 6 after sowing with
seedlings directly deposited in the Petri dishes. The photos were
taken with a standard phone camera and the root lengths in the
photos were measured using the NeuronJ plugin (Meijering
et al., 2004) of ImageJ software (Schneider et al., 2012) with a
5 mm grid paper for length calibration. Snapshots from NeuronJ
root tracing were saved in the PNG format and data were plotted
using GraphPad Prism 5.0.

<figure><img src="image_5.png" /></figure>

**FIGURE 1** Principle of nanomechanical measurement of seedling roots with atomic force microscopy. A seedling root (R) is deposited on a microscope slide using silicon glue (N, for Nusil). A fastening band of silicon is seen near the tip of the root (T). The thickness of the fastening band must be thin enough to avoid hindering the AFM support (S), but thick enough to withstand the bending of the root tip. The root is placed under the AFM cantilever (C) as observed by the AFM optical camera. The triangular shaped cantilever (200 µm long) was placed 500 µm away from the root tip in the transition zone where nanoindentation measurements proceeded (as shown). The seedling root and the AFM cantilever are placed within a liquid environment (growth solution, see Supplementary file). AFM, atomic force microscopy.

**2.3 | Nanoindentation experiments with atomic force microscopy**

Force-distance (F-D) curves were obtained using an AFM Dimension
3100 (Bruker) with a nanoscope five controller running the
Nanoscope 7.3 software. Triangular pyrex nitride cantilever with
pyramidal tips of a max nominal radius 10 nm, a half-opening angle of
35°, and a nominal spring constant k = 0.08 N/m were used (PNP-
TR; NanoWorld AG).

Calibration of photodiode sensitivity was done first using the approach-retract curve in air on the glass substrate followed by a thermal tuning to determine the cantilever spring constant (Kaur, Teulon, et al., 2023). The determined spring constants were about 0.08 ± 0.01 N/m. In case of a large divergence, the cantilever was manually readjusted inside the probe holder and the calibration was repeated (Schillers et al., 2017). Then the photodiode sensitivity was performed again in a liquid medium with an average value of 65 nm/V. In our case, a SUM value of 3.5–4 V was usually achieved with PNP-TR cantilevers. The engaging deflection setpoint was kept at 2.5 V while the initial vertical deflection on the photodiode was set to 0 V. For performing the indentation experiment, a ramp size of 3 µm, a scan rate of 0.5 Hz, and 4096 data points per curve were set.
Trigger was set off and no trigger value was used, implying that z-start value for the ramp at each new engagement may need adjustments. To limit the maximal force during the measurement, a range of 25-40 nN was usually adopted for F-D data values.

The glass slide with a glued seedling (Figure 1) was positioned under the AFM cantilever with the help of an AFM optical camera. Due to the large motorized sample stage, the glass slide was adjusted in such a way that the cantilever could be positioned perpendicularly at the longitudinal middle of the glued root. The target working area, the transition zone, was 500 µm away from the root apex, almost twice the length of PNP cantilever.

## 2.4 | Hierarchical statistics and reproducibility of experiments

Owing to the roughness of root surfaces, indentations were performed at various locations in a matrix form. Different sizes of matrices were used: 5 × 2, 4 × 3, 4 × 4, representing 10, 12 or 16 indenting nodes. The optimal distance between nodes was 5 µm. Each node was formed of a submatrix with 3 × 3 or 2 × 2 F-D curves spaced by 50 nm in X and Y directions. Most of the presented results were obtained with a 4 × 4 node matrix of a 2 × 2 submatrix. Measurements from various forms of matrices were merged altogether. It usually took 25 min to record a full set of F-D curves for a single seedling root; the manipulation time was kept as short as possible to avoid additional stress effects.

For each stress condition, experiments were repeated three to five times. Each experiment involved two to four plants. The robustness of our protocol was confirmed by reproducible results from experiments repeated in a remote institute with another AFM instrument (Nanowizard IV, JPK-Bruker). Here, we considered all the measurements on one plant as one independent experiment. To synthesize the overall measurements into one comprehensive result for elasticity of the plant, hierarchical statistics were adopted. Explicitly, each elasticity parameter of one plant was obtained by averaging all the collected data (with 3 × 3 or 2 × 2 submatrices) of a node, then subsequently averaged over all the nodes of the plant. For one stress condition, at least 10 plants were analyzed (n ≥ 10).

Regarding the reproducibility of results, two criteria were imposed: a valid node should have more than half of its F-D curves within 2 sigma from the mean; a valid plant needs at least half of its measured nodes valid. The distribution of elastic parameters from all nodes of a given stress condition was most often log-normal. Therefore, we computed geometric means for the average value of elastic parameters of a plant. We also applied nonparametric Mann-Whitney t tests to evaluating the statistical significance of these parameters among different stress conditions using a null hypothesis that assumes no difference on average among these conditions. A *p* value was calculated using Graphpad Prism 5.0 with an α-threshold of 0.01. The box-and-whiskers plots were drawn using Graphpad Prism 8.

## 2.5 | Characterization of plant elasticity by the trimechanic-3PCS framework

The trimechanic-3PCS framework (Chen et al., 2023) allows us to investigate the variation of stiffness with varied depth for biomaterials of heterogeneous elasticity responding to an external force. For a depth of indentation trajectory exhibiting a linear-elasticity behaviour, this theory states that the responding force $F_T$ of that depth zone can be expressed as a linear combination of three force components: $F_C$, $F_H$ and $F_S$. In this work, the elasticity parameters of the very surface of CWs, that is, the first depth zone with depth $Z_1$, are of concern.

The three force types ($F_C$, $F_H$ and $F_S$) govern three modes of restoration mechanics, namely, depth impact, Hookean and tip-shape nanomechanics, respectively. The contributions (or strengths) of the three nanomechanics to the overall response are represented by the spring constants ($k_C$, $k_H$, $k_S$) of three parallel-connected spring (3PCS) analogs. The stiffness is defined as $k_T = k_H + k_S$. Another important elastic parameter is $r_S = k_S/k_T$, which quantifies penetration ease of the material and the composition of responding nanomechanics; it can represent material rigidity or deformability. Moreover, the $F_S$-deduced effective Young's modulus, $\hat{E} = E/(1 - \eta^2)$ with E the Young's modulus and η the Poisson's ratio, represents the intrinsic property of elasticity. The calculations of these parameters were detailed previously (Chen et al., 2023).

## 3 | RESULTS

To evaluate the relationship between root extension and root cell wall elasticity, we used AFM to perform vertical indentations on surfaces of living plant roots. We analyzed elastic responses of the CWs of Arabidopsis seedlings in the presence of metallic stresses, either Fe or Al alone or both combined. We used a robust measurement protocol to obtain reproducible and reliable results (Kaur, Teulon, et al., 2023). Seeds are sown on Day 0 on a 24-well crystallization plate, a key step that reduces the waving effect of seedling growth. On Day 4, seedlings are transferred on a 25 mm agar plate with or without metals (Fe, Al or Fe/Al) for 2 h. Then, seedlings are removed from the agar plates, deposited on a glass slide over a specific pressure sensitive adhesive (see Section 2), and quickly covered with growth media to avoid stress due to air exposure. Force-distance curves are measured using AFM. Because of the heterogeneity of seedling CW surfaces, we used the recently developed trimechanics-3PCS framework for interpreting force-distance curves (Chen et al., 2023). The trimechanics-3PCS framework allows the extraction of both stiffness and elasticity along the depth of indentation.

### 3.1 | WT seedling root lengths and elasticity in the absence of metals

The nonstressed (control) systems were characterized as the seedling roots grown without supplemented metals (Fe0Al0) with or without a
transfer step. In our nomenclature, metal stresses are given for both Fe and Al in µM; thus, Fe0Al0 indicates that 0 µM of FeCl2 and 0 µM of AlCl3 were added during the transfer step. Nanoindentation experiments were performed on these nonstressed seedlings 2 h later after being transferred, or immediately in case of no transfer. According to *p* values with the significance threshold *α* = 0.01, no single group in the control systems significantly distinguishes itself from the others (Supporting Information S2: Figure 1). These results demonstrate that the transfer of seedlings do not alter the properties of the cell wall. In this work, negative control data includes only Fe0Al0 with the transfer step.

The average of root length obtained on Day 6 (4 days growth + 2 days after transferring from the Fe0Al0 condition) is 25.0 mm (Table 1). Averaged elasticity parameters are listed in Table 2. The results show that the effective Young modulus *Ê* for Fe0Al0 is about 54 kPa, the stiffness *k*<sub>T</sub> ~ 4.3 10<sup>-3</sup> N/m, and indentation depth Z<sub>1</sub> ~ 150 nm.

## 3.2 | WT seedling root lengths and elasticity in the presence of metals

Several combinatorial concentrations of Fe and Al were applied to the test on the growth and CW stiffening of seedling roots.

<table><caption>**TABLE 1** The average of seedling root length for all study systems.</caption><thead><tr><th>Plant type</th><th>Stress conditions</th><th>n</th><th>Length (mm)</th></tr></thead><tbody><tr><td rowspan="7">WT</td><td>Fe0Al0</td><td>19</td><td>25.0 ± 3.1</td></tr><tr><td>Fe0Al10</td><td>18</td><td>27.6 ± 3.1</td></tr><tr><td>Fe10Al0</td><td>17</td><td>26.8 ± 3.3</td></tr><tr><td>Fe10Al10</td><td>19</td><td>13.4 ± 1.3</td></tr><tr><td>Fe0Al20</td><td>26</td><td>26.8 ± 2.8</td></tr><tr><td>Fe20Al0</td><td>24</td><td>25.1 ± 2.2</td></tr><tr><td>Fe10Al10+P</td><td>27</td><td>25.0 ± 2.9</td></tr><tr><td rowspan="2">almt1</td><td>ALMT1_Fe0Al0</td><td>9</td><td>23.5 ± 2.1</td></tr><tr><td>ALMT1_Fe10Al10</td><td>9</td><td>22.0 ± 4.9</td></tr></tbody></table>

<table><thead><tr><th></th><th>n</th><th>Z<sub>1</sub> (nm)</th><th>Ê (kPa)</th><th>r<sub>s</sub></th><th>k<sub>T</sub> (10<sup>-3</sup> N/m)</th></tr></thead><tbody><tr><td>Fe0Al0</td><td>11</td><td>147 ± 55</td><td>53.9 ± 21.8</td><td>0.78 ± 0.05</td><td>4.30 ± 1.16</td></tr><tr><td>Fe0Al10</td><td>10</td><td>150 ± 62</td><td>51.7 ± 30.9</td><td>0.74 ± 0.07</td><td>4.26 ± 1.73</td></tr><tr><td>Fe10Al0</td><td>14</td><td>153 ± 41</td><td>58.4 ± 50.4</td><td>0.75 ± 0.08</td><td>4.94 ± 3.51</td></tr><tr><td>Fe10Al10</td><td>11</td><td>127 ± 57</td><td>105 ± 52</td><td>0.81 ± 0.09</td><td>8.89 ± 8.63</td></tr><tr><td>Fe0Al20</td><td>15</td><td>136 ± 41</td><td>76.9 ± 39.4</td><td>0.81 ± 0.05</td><td>5.72 ± 2.18</td></tr><tr><td>Fe20Al0</td><td>11</td><td>119 ± 29</td><td>106 ± 42</td><td>0.83 ± 0.06</td><td>7.51 ± 3.77</td></tr><tr><td>Fe10Al10+P</td><td>8</td><td>162 ± 25</td><td>59.6 ± 27.6</td><td>0.81 ± 0.06</td><td>5.37 ± 1.97</td></tr></tbody></table>

Systematically, the seedling roots were placed under metal stress for 2 h. These stress conditions were prepared with 10 µM of FeCl2 or 10 µM of AlCl3, or mixing both, and labelled as Fe10Al0, Fe0Al10 and Fe10Al10, respectively. No REA is observed in either Fe0Al10 or Fe10Al0 whereas a full REA is observed in the Fe10Al10 condition (Figure 2a), see Table 1 for root lengths.

The results from nanoindentation experiments in 10 µM metal conditions are shown in Figure 2. The hierarchical averages of their elasticity parameters are presented in Table 2. The elastic behaviours of conditions Fe10Al0 and Fe0Al10 exhibit no distinction from Fe0Al0; with an average *Ê* of about 55 kPa. It indicates that the total amount of metal ions at 10 µM changes little in the effective Young's modulus, stiffness or indentation depth compared to no metal at all. However, the elasticity of roots grown with mixed Fe and Al (Fe10Al10) yields a value of 127 kPa for the effective Young's modulus *Ê*, a significant increase in CW stiffness. However, although the averaged *k*<sub>T</sub> of 8.89 10<sup>-3</sup> N/m for Fe10Al10 is about double that of all the conditions of a single metal element at 10 µM (cf. 4–5 10<sup>-3</sup> N/m in Table 2), there is no statistical significance likely due to the very large standard deviation for Fe10Al10 (Figure 2c).

We further explored the concentration impact of metal ions by doubling the concentration from 10 to 20 µM. We found that Fe20Al0 displays a significantly higher *Ê* and *k*<sub>T</sub> than the control systems, while Fe0Al20 exhibits a moderate effect (Figure 2). The results show that the average of *Ê* and *k*<sub>T</sub> have a similar value between Fe20Al0 and Fe10Al10 conditions (Table 2) whereas the corresponding values of Fe0Al20 are intermediate. Very interestingly, doubling the cationic concentration of single metal does not provoke the occurrence of REA (Table 1).

## 3.3 | *almt1* mutant seedling root lengths and elasticity in the presence of metals

Unlike WT seedling roots, no REA phenotype was found from *almt1* mutants in the Fe10Al10 condition (Table 1, Figure 3). The elasticity parameters for *almt1* mutant seedlings grown in Fe0Al0 and Fe10Al10 conditions are listed in Table 3. No significant difference was found in the magnitudes of *Ê* and *k*<sub>T</sub> between the two stress conditions (Figure 3). Moreover, these values are comparable to WT in Fe0Al0 (Tables 2 and 3). This implies that without exuded malate

<caption>**TABLE 2** Elastic properties of WT seedling roots.</caption>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**FIGURE 2** Box-and-whiskers plots of elastic parameters of WT seedling roots in various stress metal concentration. (a) Average root lengths measured on Day 6. (b) Effective Young's modulus (Ê) in the kPa unit. (c) Stiffness measure, k<sub>T</sub>, in the N/m unit.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**FIGURE 3** Box-and-whiskers plots of elastic properties of *almt1* mutant roots in comparison with WT (cf. Figure 2) in two stressed conditions, FeOAl0 and Fe10Al10. (a) Average root lengths measured on Day 6. (b) Effective Young's modulus, Ê, (in kPa). (c) Presentation of k<sub>T</sub> in the N/m unit.</figcaption></figure>

<table><caption>**TABLE 3** Elastic properties of *almt1* mutant seedling roots.</caption><thead><tr><th></th><th>n</th><th>Z<sub>1</sub> (nm)</th><th>Ê (kPa)</th><th>r<sub>s</sub></th><th>k<sub>T</sub> (10<sup>-3</sup> N/m)</th></tr></thead><tbody><tr><td>FeOAl0</td><td>14</td><td>162 ± 79</td><td>63.5 ± 50.4</td><td>0.79 ± 0.08</td><td>4.6 ± 2.0</td></tr><tr><td>Fe10Al10</td><td>11</td><td>133 ± 28</td><td>58.3 ± 36.5</td><td>0.74 ± 0.08</td><td>4.8 ± 2.8</td></tr></tbody></table>
the two metal ions cannot exert substantial effects on elastic responses of mutant roots.

## 4 | DISCUSSION

The present results show that the elastic responses of external epidermal cell walls of Arabidopsis seedling roots to external forces vary in terms of concentration and composition of Fe and Al metal ions. It indicates that elasticity of plant CW is sensitive and can be used as to assess abiotic stresses on plant growth and stiffening. However, unexpectedly, the stiffening and the phenotype of seedling roots such as REA are not directly correlated.

### 4.1 | Root extension arrest (REA) and metallic stress

The root lengths of Arabidopsis seedlings were measured from the root tip to the cotyledon base (Supporting Information S2: Figure 2). Among all the stress conditions (Fe10Al0, Fe0Al10, Fe20Al0, Fe0Al20, and Fe10Al10), we observed the REA phenotype appeared only in the WT roots grown in the Fe10Al10 condition (Table 1). It is surprising that no REA was observed with doubled concentrations of single metal species (either Fe or Al). This reveals that the excess of a single metal species did not trigger the occurrence of REA. To ascertain that the REA phenotype is only due to the mixture of the two metal species, we carried out an experiment in a condition with the same metal ingredients and 500 µM phosphate (Pi). Phosphate is known for binding cations (Foy et al., 1978) but does not completely abolish the entry of metals into seedling roots (Balzergue et al., 2017). Results show no REA in the presence of Pi (Table 2, Supporting Information S2: Figure 5).

To further resolve the origin of REA occurrence, the WT results were compared with those of the *almt1* mutant. Lacking the malate-transporter ALMT1, the *almt1* mutant is strongly altered in exuding malate, a small organic anion known to chelate Fe³⁺ and Al³⁺. The root growth of *almt1* mutant was known to be insensitive to Fe²⁺ (under -Pi condition) and exhibited no REA phenotype (Balzergue et al., 2017; Mora-Macias et al., 2017). The absence of REA phenotype was explained as a consequence of reduced accumulation of iron in the apoplast due to a dramatic decrease of malate exudation. From our results, the mixed Fe and Al stress also lacks the ability to stimulate REA in the *almt1* mutants, and these mixed metal cations act like single metal ions of 10 µM in WT roots. In other words, without the malate exudation, the mixed Al and Fe are no longer growth inhibitors, leading to a normal growth phenotype. It further suggests that trapping metal ions by malate molecules is a key step to promote REA in WT. Taken together, these data suggest that the factors to simulate REA include the amount of metal ions, the composition of metal species and the exudation of malate.

### 4.2 | Metallic stress and elasticity of living seedling roots

When the interlaced architecture of CWs is perturbed by metal ions, the bonding modes are accordingly adjusted; these changes can be reflected by altered elastic responses. It is noteworthy that the used AFM indenting tip has a small apex (~10 nm radius), enabling us to sense structural strengths of different layers of constituents in primary CWs such as cellulose microfibrils. Applying the trimechanic-3PCS framework to data analysis, the elasticity parameters defined therein helped us to differentiate elastic properties modulated by various stressed environments. The force decomposition of the theory reveals that the *F*<sub>S</sub>-deduced *Ê* is a sensitive parameter to varying metal content in the growth medium (Table 2). The change in penetration ease *r*<sub>S</sub> underlies the varying modes of nanomechanics and network bonding of CW architecture under different stresses (Chen et al., 2023). The *r*<sub>S</sub> parameter is provided only by the trimechanic-3PCS framework and cannot be accessed by the conventional methods (Hermanowicz et al., 2014). This *r*<sub>S</sub> parameter can also represent the deformability of the indented root.

According to comparable *r*<sub>S</sub> values of WT roots in Fe0Al0, Fe10Al0 and Fe0Al10 conditions, the bonding properties of CW structure are inferred to be alike. However, with higher concentration of metal ions (Fe20Al0, Fe0Al20 and Fe10Al10), an increase in *r*<sub>S</sub> is observed (*r*<sub>S</sub> > 0.8, Table 2). It follows that in all these conditions of high metal concentration, the bonding properties of CW are different from that of low metal concentration. As already demonstrated, Al binds directly to negatively charged pectins of CWs and provokes a reduction in CW extensibility (Ma et al., 2004; Yang et al., 2016). In addition, expression profiling experiments suggested that pectins do bind with Fe (Hoehenwarter et al., 2016), which therefore, like Al, changes the bonding elasticity of the external primary cell wall. It is noteworthy that elastic parameters presented here are referred to the indentation depth of about 150 nm, which most likely corresponds to the pectin constituents of CW (as opposed to cellulose microfibrils). Thus, the increased stiffness of CW for seedlings grown from Fe20Al0 and Fe0Al20 conditions likely involves the binding of Fe and Al to the pectin components of CW.

From the results of Z₁, *Ê* or *k*<sub>T</sub>, the increase of the total amount of metal ions is closely related to CW stiffening. At 10 µM of either iron or aluminum, the elastic properties of WT roots are similar to that of the control that contains no metal ions. At 20 µM (regardless of metal composition), the parameters *Ê*, *k*<sub>T</sub> and *r*<sub>S</sub> increase while Z₁ slightly reduces; see the results from Fe10Al10, Fe20Al0 and Fe0Al20 in Table 2. However, the Fe20Al0 and Fe0Al20 (single metal species) conditions exhibit no REA phenotype. Thus, the increase of CW stiffness is not causal or not sufficient to trigger REA, at least not in the conditions of our experiments, that is, short-term treatment.

### 4.3 | Root extension arrest and CW stiffening

The REA phenotype induced by the Al stress is multifactorial and its mechanism remains largely unknown (Kochian et al., 2015). However,
<figure><img src="image_9.png" /></figure>

from our previous work and others, the REA phenotype due to Fe stress is documented in its initial steps of Fe redox cycle that produces ROS in the CW and promotes peroxidase-dependent cell wall stiffening in the transition zone (Balzergue et al., 2017; Naumann et al., 2022). The major tolerance mechanism of Al toxicity is through the stimulation of the expression of the ALMT1 gene (Godon et al., 2019), which encodes a malate transporter (ALMT1 [Sasaki et al., 2004]). The rate-limiting step in this mechanism is the transport of organic acids rather than the cellular synthesis of these molecules (Ryan et al., 2001). Indeed, Al³⁺ binding to the extracellular face of the ALMT1 channel opens the channel thereby stimulating the exudation of malate (Wang et al., 2022).

We have shown that the CW stiffness increases without REA at high Fe²⁺ concentrations (≥20 µM) for seedling roots grown from an agar medium with poor phosphate and other metals, probably reflecting a lack of ROS production (Figure 4). In the ferrous state, the Fe ion has multiple possible outcomes: adsorbed by the cell via its importing receptor, chelated with some organic acids in the CW or oxidized to a ferric ion that

may nonspecifically bind to pectins of the CW. However, at 20 µM Fe²⁺, none of these outcomes are important enough to form the necessary redox condition for REA occurrence. At the same concentration, Al³⁺ activates the exudation of malate that chelates Al to move it out of the root. It is known that Al³⁺ ions have a high affinity for pectic molecules of the cell wall and this binding is considered a major cause of Al³⁺ toxicity (Horst et al., 2010). Furthermore, within a few minutes Al³⁺ inhibits wall loosening in the elongation zone (Hajiboland et al., 2023). The Al³⁺ ions unbound to malate molecules in the CW then bind to negatively-charged pectins, leading to an increase of stiffness though without REA occurrence (Figure 4). The stress effect of the co-presence of Fe and Al highlights the importance of malate accumulated in the apoplast. A current model postulates that, in combination with the apoplastic ferroxidase LPR1, malate-Fe³⁺ complexes trigger ROS in the apoplast (Naumann et al., 2022). Based on this model, our results show that Al³⁺ increases exudation of malate in the apoplast, thereby accumulating Fe in the apoplast followed by an accumulation of ROS to end up with a root extension arrest (Figure 4).

<figure><img src="image_10.png" /><figcaption>**FIGURE 4** Model explaining the effects of Fe and Al on CW stiffening and root extension. Left panel shows a reconstituted picture of an Arabidopsis primary root tip; the square indicates part of the epidermis in the transition zone, where AFM measures were performed in this work. The top to bottom panels explain the phenomena that occur, depending on the Fe²⁺ and Al³⁺ content of the Pi-poor culture medium. Top panel: the Fe²⁺ ions enter the apoplast of the cell wall (CW, in light grey background colour), which subsequently can cross the plasma membrane (PM, in light tan colour) through an unknown transporter (not presented here for clarity) and activates the STOP1-ALMT1 signalling (not shown), or accumulate into the apoplast in complex with constitutively present small organic acids like malate (M). The ALMT1 transporter exports malate from the cytosol (CYT, light blue background) to the apoplast. The accumulation of Fe cations, possibly in the Fe³⁺ state (darker green on the bottom left) binds to pectins (curled red lines), thereby increasing CW stiffness without triggering the root extension arrest (REA). Middle panel: the Al³⁺ ions enter the CW and activate the transcription of ALMT1 (not shown) and the opening of ALMT1 transporter, thereby releasing malate in the apoplast. The accumulation of Al³⁺ leads to a modest increase of CW stiffness mostly of pectins, but without REA. Bottom panel: the combination of Fe²⁺ and Al³⁺ results in a large release of malate and a high accumulation of ROS-promoting iron-malate complexes in the apoplast. These ROS concomitantly increase CW stiffness (besides the effect of metals on pectins) and strongly prevents root extension. CW, cell wall; CYT, cytoplasm; M, malate; PM, plasma membrane; REA, root extension arrest; -Pi, phosphate-poor medium; +Fe, adding Fe²⁺ in the medium; +Al, adding Al³⁺ in the medium. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>
Our AFM study shows that aluminum acts rapidly on parietal stiffness and the synergy with iron amplifies this inhibition. These results provide a better understanding of the interaction of iron and aluminum found in acidic soils. This work is a step forward to decipher the interplay of metal bioavailability in low phosphate conditions. It may also provide future route to develop and grow breeding targets best adapted to Al³⁺ stress.

5 | CONCLUSIONS

Root extension arrest was observed from *A. thaliana* WT seedlings only stressed by a mix of 10 µM FeCl₂ and 10 µM AlCl₃ in a low phosphate agar medium. This REA is concomitant with a stiffening of the external primary cell walls. However, single metals, even at a higher concentration (20 µM), did not induce REA despite an increase in CW stiffness. Thus, the increase in the stiffness of CW may have independent origins: one associated with the binding of metals to pectin components of CW, and another associated with the redox cycle that produces ROS in the CW and promotes the peroxidase-dependent stiffening of CW. Consequently, the REA occurs in a balance of metabolic events (chemical and/or mechanical) that depends upon a change in the contribution of each factors including the chelating effect of malate in the combined Fe-Al stress.

ACKNOWLEDGEMENTS

IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). This work acknowledges the AFM platform at the IBS. Acknowledgement to the ANR project BioPhyt -18-CE20-0023-03 and the support of the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 812772, Project Phys2-BioMed. We thank Dr. Anne-Emmanuelle Foucher (IBS/EPIGEN) for critical improvements of the plant growth protocol. We also thank Isabel Ayala and Lionel Imbert (IBS/NMR) for their support in lab experiments. We are grateful to Chloe Zubieta for improving the manuscript.

DATA AVAILABILITY STATEMENT

Data available on request from the authors.

ORCID

Harinderbir Kaur http://orcid.org/0000-0002-2418-2449

Christian Godon http://orcid.org/0000-0002-1535-9855

Thierry Desnos http://orcid.org/0000-0002-6585-1362

Shu-wen W. Chen http://orcid.org/0000-0003-4205-4931

Jean-Luc Pellequer http://orcid.org/0000-0002-8944-2715

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SUPPORTING INFORMATION

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How to cite this article: Kaur, H., Teulon, J.-M., Godon, C., Desnos, T., Chen, S.-w. W. & Pellequer, J.-L. (2024) Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al. *Plant, Cell & Environment*, 47, 574–584.
https://doi.org/10.1111/pce.14744