# Cadmium allocation to grains depends on sulfur nutrition in durum wheat

Agathe Vidal, Valentin Dubois, Sophie Brunel-Muguet, Jean-Christophe

Avice, Christophe Nguyen, Jean-Yves Cornu

► To cite this version:

Agathe Vidal, Valentin Dubois, Sophie Brunel-Muguet, Jean-Christophe Avice, Christophe Nguyen, et al.. Cadmium allocation to grains depends on sulfur nutrition in durum wheat. Journal of Agriculture and Food Research, 2025, 23, 10.1016/j.jafr.2025.102235. hal-05206126

HAL Id: hal-05206126

https://hal.science/hal-05206126v1

Submitted on 11 Aug 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# Cadmium allocation to grains depends on sulfur nutrition in durum wheat

Agathe Vidal<sup>a</sup>, Valentin Dubois<sup>b</sup>, Sophie Brunel-Muquet<sup>b ID</sup>, Jean-Christophe Avice<sup>b ID</sup>, Christophe Nguyen<sup>a ID</sup>, Jean-Yves Cornu<sup>a,* ID</sup>

## ARTICLE INFO

### Keywords:

- Low-exposure Cd

- Cereal

- Cd-S interaction

- Detoxification

- Remobilization

- Food safety

## ABSTRACT

The aim of this study was to determine if the allocation of cadmium (Cd) to grains depends on sulfur (S) nutrition in durum wheat. Durum wheat was grown in semi-hydroponics with 100 nM Cd (Cd+) or at a background concentration of Cd (Cd-). Starting from stem elongation, the plants exposed to 100 nM Cd received a standard supply of S (S100), a reduced supply of S (S20) or were subjected to S deprivation (S0). Plants were sampled at the early milk development stage (Z71) and the early ripening stage (Z91) to assess differences in plant growth, Cd uptake and partitioning among organs, as well as net remobilization of Cd in the different treatments. Durum wheat growth was not affected by exposure to 100 nM Cd, and the root-to-shoot translocation of Cd was more restricted in the Cd+ treatment than in the Cd-treatment. Reducing or stopping the supply of S did not affect durum wheat growth but enhanced S remobilization from almost all plant parts, including the roots. Stopping the supply of S resulted in a 35 % increase in the concentration of Cd in the grains. The proportional decrease in the concentrations of S and Cd in the roots of S-deficient plants between Z71 and Z91 suggests that Cd remobilization is associated with S remobilization in the roots of durum wheat. Sulfur deficiency promoted Cd accumulation in durum wheat grains likely due to enhanced remobilization of S and Cd in the roots of S-deficient plants.

## 1. Introduction

Cadmium (Cd) is a highly toxic trace metal that is naturally present in agricultural soils, and threatens the safety of food products made from cereals. As substantiated by the French national biomonitoring program [1], human exposure to Cd is a cause for concern and, in the non-smoking population, mainly results from the consumption of cereal products, particularly among children. Since 1993, the concentration of Cd in cereal grains grown in Europe for human consumption has been regulated by regulation EC 315/93 and its amendments. Among cereal crops, durum wheat (*Triticum turgidum* L. subsp. durum) is the species that accumulates the highest concentrations of Cd in its grains [2], on average 3 to 4 times more than bread wheat [3]. Annual surveys of the quality of French wheat harvests showed that on average, 5 % of durum wheat contained concentrations of Cd above the European limit of 0.18 µg g⁻¹ fresh weight (FW) (EC 2023/915), and up to 13 % in French administrative departments where the availability of Cd in soil is high (source Arvalis-Institut du Végétal, https://www.arvalis.fr/technical-institute). There is thus a need to reduce the accumulation of Cd in durum wheat grains for sanitary reasons but also to avoid economic

losses due to non-compliance with the regulations.

One strategy to minimize the Cd load in durum wheat grains is to use "low-Cd" cultivars, i.e. cultivars with limited ability to accumulate Cd in their grains. This approach, which has been used in Canada since the 1990s [4], revealed that in Canadian lines, the concentration of Cd in the grain can vary by up to a factor 4, depending on whether or not the cultivar carries or not the functional allele of the gene *TdHMA3-B1*, that codes for a metal-transporting protein and leads to the accumulation of Cd in the root vacuoles [5]. However, because no specific selection was undertaken in France, many French durum wheat cultivars are "high-Cd" since they do not carry the functional allele of *TdHMA3-B*, including the most widely cultivated ones [6]. Another strategy to minimize the Cd load in durum wheat grains is to adapt mineral fertilization, for instance the supply of nitrogen, silicon or sulfur, since Cd is known to interact with these three elements in the soil and/or on its way to the grain once in the plant [7]. Given that regulations aimed at limiting S emissions by industry (e.g. Kyoto 1997) have resulted in S deficiency in agricultural soils that is often corrected by S fertilization, studying the impact of sulfur (S) fertilization is of particular interest. Furthermore, sulfur is essential for achieving quality objectives in

<footnote><sup>a</sup> ISPA, Bordeaux Sciences Agro, INRAE, Villenave d'Ornon, 33140, France</footnote>

<footnote><sup>b</sup> UMR INRAE/UNICAEN 950 EVA, Université de Caen Normandie, SFR Normandie Végétal (FED 4277), 14032, Caen, France</footnote>

<footnote>* Corresponding author.
E-mail address: jean-yves.cornu@inrae.fr (J.-Y. Cornu).</footnote>
cereals: optimal S nutrition enhances the technological quality of flours through the positive impact it has on the protein content and quality in grains, and consequently on the rheological properties of dough [8].

In *planta*, S-Cd interaction has been the subject of numerous studies because (i) as a soft Lewis acid, Cd has a high affinity for soft Lewis bases such as S ligands, and (ii) the vast majority of Cd hyperaccumulating plants (e.g. *N. caerulescens*, *A. halleri*) are Brassicaceae, i.e. species with high S requirements. These studies have highlighted the important role played by S ligands in Cd tolerance and (hyper)accumulation at the cellular level [9,10]. For example, glutathione (GSH) plays a central role in plant tolerance to Cd due to its antioxidant properties and its ability to complex Cd in the cytosol [11]. In the same way, phytochelatins (PCs) play a key role in Cd detoxification and (hyper)accumulation through their involvement in Cd transfer and storage in the vacuole [12]. Hence, a plant well supplied with sulfur is assumed to be more tolerant to Cd [13], and, in the case of non-hyperaccumulating species, to sequester Cd more efficiently in its roots [14,15] than a S-deficient plant. Conversely, a plant exposed to Cd is supposed to take up more S than a plant not exposed to Cd in order to increase the supply of GSH for PC biosynthesis [16,17]. However, S-Cd interaction in plants has most often been described in contexts where S and/or Cd are highly concentrated, i.e. in Cd hyperaccumulating plants, S-rich plants, in the case of excessive exposure to Cd, and/or luxury S fertilization. A better understanding is thus required of the extent to which the S-Cd interaction described in *planta* at these "high" doses of S and/or Cd prevails at "lower" levels, such as those encountered in soils used to grow cereals.

The aim of this study was to test the hypothesis of an effect of S nutrition on Cd allocation to grains in durum wheat, at Cd concentrations representative of those observed in cereal cultivation. Our objectives were to investigate (i) if exposure to Cd affects the growth and uptake + partitioning among organs of Cd and S in durum wheat, (ii) if a reduction in S nutrition increases the accumulation of Cd in grains, and (iii) in the case of an increase in the accumulation of Cd in grains, if this is due to lower sequestration of Cd in roots and/or to higher remobilization of Cd to grains in S-deficient plants.

## 2. Materials and methods

### 2.1. Experimental design

To investigate the impact of S nutrition on the allocation of Cd to grains in durum wheat (*Triticum turgidum* L. subsp. durum (Desf.) Husn.), the French cultivar *Sculptur* (RAGT Semences), which does not have the low-Cd accumulating allele at *Cdu1* [6], was grown in semi-hydroponics with 100 nM Cd (Cd+) to reproduce the concentration of Cd found in the pore water of contaminated agricultural soils [18]. The choice of a semi-hydroponic system is explained by the need to finely control the concentrations of S and Cd to which the plants were exposed, and to constrain the growth of the root system using a solid phase. One fourth of the plants that received a standard supply of S were grown at a background concentration of Cd (Cd-) to assess the response of durum wheat to low-exposure Cd in terms of growth and Cd + S partitioning among organs. Starting at the stem elongation stage (Zadok stage 30), plants grown with 100 nM Cd received either a standard supply of sulfur (S100, corresponding to 0.5 mM of MgSO₄), or a reduced supply (S20, corresponding to 20 % of S100) or were subjected to sulfur deprivation (S0, no MgSO₄). The plants were sampled at two stages: the early milk development stage (Z71) and the early ripening stage (Z91) to assess dry matter content and the concentrations of S, Cd and Zn in the plant organs.

### 2.2. Plant culture

Seeds of durum wheat (*Triticum durum* cv. *Sculptur*) were germinated in the dark at 25 °C for three days. When their first leaf emerged (Z11), the seedlings were placed in tubes (30 cm height x 8 cm diameter; 2

seedlings per tube) with a drainage hole at the bottom filled with a mix of quartz sand and perlite (1:1, v/v) and transferred to a greenhouse. Nutrient solution was supplied to plants using a drip system that delivered 30 mL to each tube at 6-h intervals. The solution was recycled via the drain hole to a stock tank, in which the nutrient solution was renewed every week to limit variations in its composition. Plants were grown at 20–22 °C/15–18 °C day/night temperature under natural light supplemented by sodium lamps (MASTER GreenPower T400W, Philips) functioning 10 h a day with photosynthetically active radiations of 350–400 µmol m⁻² s⁻¹ at canopy height. Until tillering, all the plants were supplied with a 25 % Hoagland's nutrient solution consisting of 1.25 mM Ca(NO₃)₂, 1.25 mM KNO₃, 0.5 mM MgSO₄, 0.25 mM KH₂PO₄, 200 µM FeNaEDTA, 14 µM H₃BO₃, 5 µM MnSO₄, 3 µM ZnSO₄, 0.7 µM CuSO₄, 0.7 µM (NH₄)₆Mo₇O₂₄ and 0.1 µM CoCl₂. At tillering (Z20), three fourths of the plants were grown with 100 nM Cd(NO₃)₂ (Cd+) while the remaining fourth (Cd-) were grown with a background concentration of Cd (i.e. below 2 nM). When the stem started to elongate (Z30), the plants grown with 100 nM Cd were supplied with different concentrations of MgSO₄ to modify their S status: 0.5 mM MgSO₄ for S100, 0.1 mM MgSO₄ for S20 and no MgSO₄ for S0. A low concentration of SO₄ (i.e. 8.7 µM) remained present in the nutrient solution at S0 as Cu, Mn, and Zn were added as sulfate salts. To avoid Mg²⁺ depletion in S20 and S0, a solution of MgCl₂ was used to adjust the supply of Mg at 0.5 mM in both treatments. Under each treatment, the plants were sampled at the early milk development stage (Z71) and the early ripening stage (Z91), corresponding to respectively, 43 days and 97 days, after the beginning of the S treatment. Four independent replicates, each consisting in one tube and two individual plants, were sampled under each treatment and at each development stage.

### 2.3. Plant sampling and analysis

After sampling, the plants were divided into roots, internodes, nodes, senescent leaves, photosynthetic leaves and heads. Photosynthetic leaves were those with less than 50 % of their surface in senescence. All the plant organs were washed twice with deionized water before being freeze-dried for 30 h. After freeze-drying, the heads of the plants sampled at Z91 were separated into grains, bracts and rachis. All the dried organs were weighed to determine the dry weight (DW), and then ground to a fine powder using an ultra-centrifugal mill (ZM 200, Retsch) and a mixer mill (MM 200, Retsch) before further analyses. Digestion was performed as follows: 40 mg of dried powder was added to 2 mL of a HNO₃/H₂O₂ mix (4:1, v/v) in a 50 mL Teflon® tube and digested in a microwave digestion system (Multiwave ECO, Anton Paar) according to the procedure described in Maignan et al. [19]. The recovery rate of this digestion procedure was checked using a certified sample of citrus leaves (NISTSRM 1572) and was above 95 % for Cd. Total concentrations of S, Cd and Zn in the plant organs were determined by high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS, Element 2, Thermo Scientific). For HR-ICP-MS, all the samples were spiked with two internal-standard solutions of gallium and rhodium, and each element was quantified using external standard calibration curves.

### 2.4. Statistical analysis

Data were processed using © R 4.1.2 statistical software [20]. The root sequestration factor of Cd (RSF) was determined by calculating the ratio of the concentration of Cd in the roots to the concentration of Cd in the shoots. When necessary, data were log-transformed to achieve normal residual distribution and equal variance. Analysis of variance (ANOVA) and Dunnett's test (at 5 % significance level) were performed to identify significant differences between Cd treatments (Cd + vs Cd-) in plants that received a standard supply of S (S100), and between S treatments (S20 vs S100 and S0 vs S100) in plants grown with 100 nM Cd (Cd+). Pearson's correlation test was performed to test the linear relationship between the concentration of S and Cd (Fig. 4a) and
between the concentration of S and Zn (Fig. 4b) in the roots at S0 and S20.

# 3. Results

## 3.1. Plant growth

No visual symptom of Cd toxicity such as leaf chlorosis was observed in plants grown with 100 nM Cd. However, when exposed to 100 nM Cd, plants underwent accelerated senescence of their leaves during the early stage of grain filling, as evidenced by the higher (p < 0.05) percentage of senescent leaf at Cd+ (i.e. 36 %) compared to Cd- (i.e. 21 %) in plants sampled at Z71 (Table 1). Plant biomass was not negatively affected by exposure to 100 nM Cd. The dry weight of plants (Fig. 1a) and of most of the plant organs (Table 1) was even slightly higher (10 % on average) in the Cd + treatment compared to Cd-. This difference was observed at both sampling stages but was only significant (p < 0.05) in nodes sampled at Z71. No visual symptom of S deficiency such as paler green leaves was observed, even in plants subjected to S deprivation. At Z71 and Z91, the dry weight of plants (Fig. 1b) and of all plant organs except nodes (Table 1) was the same at S100, S20 and S0, meaning that plant growth was not affected (p > 0.05) by either partial or total reduction in the supply of S from Z30 until maturity.

## 3.2. Sulfur concentration

Regardless of the stage at which the plants were sampled, the level of exposure to Cd and the S treatment, the concentration of S in plant organs followed the order leaf > stem (node and internode) > roots, and the leaf S concentration was above the S deficiency threshold of 2 mg g⁻¹ reported for wheat (Arvalis-Institut du Végétal) (Table 2). The concentration of S was lower in plants exposed to Cd than in the controls (Cd-) (Fig. 2a), notably at Z71 when the difference between the two Cd exposures was significant (p < 0.05) in most plant organs, and averaged 15 % (Table 2). As expected, the reduction in the supply of sulfate led to a decrease in the concentration of S in the plants (Fig. 2b) and in most plant organs (Table 2), but only at Z91. At this stage, the concentration of S in the roots, stem (node and internode), leaf and bract was lower (p < 0.05) in the S0 treatment (−39 % on average) and S20 (−29 % on average) than in the S100 treatment.

Conversely, there was no significant difference (p > 0.05) in the concentration of S in the grains among the three S treatments, and the concentration of S in the rachis was significantly higher (p < 0.05) at S0 compared to at S100. The concentration of S decreased between Z71 and Z91 in almost all vegetative organs at S0 and S20, for example by 40 % at S0 and by 37 % at S20 in roots, whereas no decrease in the concentration of S between Z71 and Z91 was observed at S100 (Table 2).

## 3.3. Cadmium concentration

The concentration of Cd was higher (p < 0.05) in plants exposed to Cd than in the controls (Cd-), by an average factor of 9.5 at Z71 and 6.9 at Z91 (Fig. 3a, Table 3). At 100 nM Cd (Cd+), the concentration of Cd in the leaves was less than 5 µg g⁻¹ DW, which is the critical leaf concentration for Cd toxicity reported in non-tolerant crop plants [21], and the concentration of Cd in grains exceeded 0.18 µg g⁻¹ FW (Fig. 3a), which is the regulatory limit set by the European Union for durum wheat (EC 2023/915). The distribution of Cd within the plant differed between the two levels of exposure to Cd, notably the partitioning of Cd between roots and shoots. The root sequestration factor of Cd (RSF) increased (p < 0.05) with the increase in the level of exposure to Cd, for instance from 0.8 in the controls to 4.2 at 100 nM Cd at Z71 (Table 3). There was no significant difference among the three S treatments (p > 0.05) in the average concentration of Cd in the plants at Z71, whereas the average concentration of Cd in the plants at Z91 was 20 % higher in the S0 treatment than in the S100 treatment (Fig. 3b). The concentration of Cd in the grains at Z91 was on average 35 % higher (p < 0.05) in the S0 treatment than in the S100 treatment (Fig. 3c). Among the other plant organs, only the concentration of Cd in the roots decreased with the decrease in the supply of S, although the difference was only significant (p < 0.05) at Z71 and between S0 and S100 (Fig. 3d). The concentration of Cd decreased between Z71 and Z91 in the roots and, to a lesser extent, in the nodes, in all three S treatments. In roots, the decrease in the concentration of Cd between the two stages was on average the same at S0, S20 and S100 and was around 40 % (Table 3).

<caption>**Table 1**</caption>

Dry weight (DW) of the whole plant, root, stem (internode and node), leaf, spikelet, rachis, bract and grain, and percentage of senescent leaf, at the early milk development stage (Z71) and the early ripening stage (Z91) in durum wheat. Durum wheat (cv. *Sculptur*) was grown in semi-hydroponics at a background concentration of Cd (Cd-.) or with 100 nM Cd (Cd+), and received a standard supply of S (S100), a reduced supply of S (S20) or was subjected to S deprivation (S0) starting at the stem elongation stage (Z30).

<table><thead><tr><th rowspan="3"></th><th colspan="4">Plants harvested at Z71</th><th colspan="4">Plants harvested at Z91</th></tr><tr><th colspan="2">Cd-</th><th colspan="2">Cd+</th><th colspan="2">Cd-</th><th colspan="2">Cd+</th></tr><tr><th>S100</th><th>S100</th><th>S20</th><th>S0</th><th>S100</th><th>S100</th><th>S20</th><th>S0</th></tr></thead><tbody><tr><td>g DW</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Whole plant</td><td>9.91</td><td>10.99</td><td>9.66</td><td>10.29</td><td>11.45</td><td>12.93</td><td>12.28</td><td>12.81</td></tr><tr><td>Root</td><td>1.51</td><td>1.65</td><td>1.33</td><td>1.61</td><td>0.88</td><td>0.90</td><td>0.96</td><td>0.90</td></tr><tr><td>Stem</td><td>3.07</td><td>3.49</td><td>3.02</td><td>3.28</td><td>2.06</td><td>2.35</td><td>2.39</td><td>2.43</td></tr><tr><td>Internode</td><td>2.93</td><td>3.32</td><td>2.86</td><td>3.13</td><td>1.95</td><td>2.21</td><td>2.25</td><td>2.28</td></tr><tr><td>Node</td><td>0.13</td><td>0.22</td><td>0.17</td><td>0.15*</td><td>0.11</td><td>0.14</td><td>0.14</td><td>0.15</td></tr><tr><td>Leaf</td><td>1.41</td><td>1.55</td><td>1.35</td><td>1.34</td><td>1.17</td><td>1.31</td><td>1.32</td><td>1.32</td></tr><tr><td>Spikelet</td><td>3.91</td><td>4.31</td><td>3.96</td><td>4.06</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>Rachis</td><td>na</td><td>na</td><td>na</td><td>na</td><td>0.14</td><td>0.15</td><td>0.15</td><td>0.18</td></tr><tr><td>Bract</td><td>na</td><td>na</td><td>na</td><td>na</td><td>1.41</td><td>1.50</td><td>1.42</td><td>1.62</td></tr><tr><td>Grain</td><td>na</td><td>na</td><td>na</td><td>na</td><td>5.80</td><td>6.71</td><td>6.28</td><td>6.59</td></tr><tr><td>% senescent leaf</td><td>21 (5)</td><td>36 (5)</td><td>27 (6)</td><td>30 (3)</td><td>80 (5)</td><td>84 (2)</td><td>87 (4)</td><td>83 (2)</td></tr></tbody></table>

Values are means of four independent replicates.

For the percentage of senescent leaf, values into brackets stand for one standard deviation.

For plants grown with 100 nM Cd (Cd+), (*), *, ** and *** indicate that the mean value at S20 or S0 differed significantly from that in the control (S100) at a probability level of p < 0.1, p < 0.05, p < 0.01 and p < 0.001, respectively.

For plants grown with 100 nM Cd (Cd+) that received a standard supply of S (S100), values are underlined when they are significantly different (p < 0.05) than those obtained under the Cd-treatment.

na: not available.
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Dry weight of durum wheat plants measured 43 days (Z71) and 97 days (Z91) after the beginning of the S treatment. Durum wheat (cv. *Sculptur*) was grown in semi-hydroponics at a background concentration of Cd (Cd-) or with 100 nM Cd (Cd+), and received a standard supply of S (S100), a reduced supply of S (S20) or was subjected to S deprivation (S0) starting at the stem elongation stage (Z30). For a given harvest stage, (*), *, ** and *** indicate in (a) that the dry weight of plants grown with 100 nM Cd (Cd+) differed significantly from that measured in the control (Cd-); and in (b) that the dry weight of plants grown with a reduced supply of S (S20) or subjected to S deprivation (S0) differed significantly from that measured in plants grown with a standard supply of S (S100), at a probability level of p < 0.1, p < 0.05, p < 0.01 and p < 0.001, respectively. Data are mean values ± one standard deviation calculated from the four replicates per treatment. ns: non-significant.</figcaption></figure>

**Table 2**

Sulfur concentration in the whole plant, root, stem (internode and node), leaf, spikelet, rachis, bract and grain, at the early milk development stage (Z71) and the early ripening stage (Z91) in durum wheat. Durum wheat (cv. *Sculptur*) was grown in semi-hydroponics at a background concentration of Cd (Cd-) or with 100 nM Cd (Cd+), and received a standard supply of S (S100), a reduced supply of S (S20) or was subjected to S deprivation (S0) starting at the stem elongation stage (Z30).

<table><thead><tr><th rowspan="2"></th><th colspan="4">Plants harvested at Z71</th><th colspan="4">Plants harvested at Z91</th></tr><tr><th>Cd-<br>S100</th><th>Cd+<br>S100</th><th>S20</th><th>S0</th><th>Cd-<br>S100</th><th>Cd+<br>S100</th><th>S20</th><th>S0</th></tr></thead><tbody><tr><td>mg g<sup>-1</sup> DW</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Whole plant</td><td>2.54</td><td><u>2.16</u></td><td>2.23</td><td>1.87</td><td>2.25</td><td>2.02</td><td>1.55**</td><td>1.48***</td></tr><tr><td>Root</td><td>1.99</td><td><u>1.81</u></td><td>1.87</td><td>1.61</td><td>1.61</td><td>1.74</td><td>1.17*</td><td>0.96**</td></tr><tr><td>Stem</td><td>2.28</td><td><u>1.64</u></td><td>1.76</td><td>1.40</td><td>2.76</td><td>2.42</td><td>2.06</td><td>1.28***</td></tr><tr><td>Internode</td><td>2.16</td><td><u>1.56</u></td><td>1.65</td><td>1.30</td><td>2.78</td><td>2.39</td><td>2.03</td><td>1.27***</td></tr><tr><td>Node</td><td>4.96</td><td><u>3.28</u></td><td>3.74</td><td>3.33</td><td>2.41</td><td>2.84</td><td>2.49</td><td>1.50*</td></tr><tr><td>Leaf</td><td>5.61</td><td><u>4.84</u></td><td>4.75</td><td>4.47</td><td>6.03</td><td><u>5.04</u></td><td>2.17**</td><td>3.31(*)</td></tr><tr><td>Spikelet</td><td>1.86</td><td><u>1.75</u></td><td>1.85</td><td>1.51</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>Rachis</td><td>na</td><td>na</td><td>na</td><td>na</td><td>2.39</td><td>1.33</td><td>1.13</td><td>3.67*</td></tr><tr><td>Bract</td><td>na</td><td>na</td><td>na</td><td>na</td><td>1.84</td><td><u>1.38</u></td><td>1.22</td><td>0.95**</td></tr><tr><td>Grain</td><td>na</td><td>na</td><td>na</td><td>na</td><td>1.50</td><td>1.47</td><td>1.38</td><td>1.30</td></tr></tbody></table>

Values are means of four independent replicates.

For plants grown with 100 nM Cd (Cd+), (*), *, ** and *** indicate that the mean value at S20 or S0 differed significantly from that in the control (S100) at a probability level of p < 0.1, p < 0.05, p < 0.01 and p < 0.001, respectively.

For plants grown with 100 nM Cd (Cd+) that received a standard supply of S (S100), values are underlined when they are significantly different (p < 0.05) than those obtained under the Cd-treatment.

na: not available.

## 3.4. Zinc concentration

Durum wheat likely did not suffer either from Zn deficiency or from Zn excess in our experiment since the concentration of Zn in the leaf ranged from 20 to 30 µg g⁻¹ DW among treatments (Table 4) and these concentrations are within the range of optimal growth reported for crop plants [22]. There was no significant difference (p > 0.05) in the concentration of Zn in plant organs between the two levels of exposure to Cd except in stem at Z71, and almost no significant difference (p > 0.05) among the three S treatments, at either Z71 or Z91 (Table 4). Unlike Cd, the concentration of Zn in grains was on average the same (p > 0.05) in the S0 and S100 treatments, and the concentration of Zn in the roots increased between Z71 and Z91.

## 3.5. Relationships between the concentration of S, Cd and Zn in roots at S0 and S20

To test the relationships between the changes in the concentrations of S, Cd and Zn in roots during grain filling under S limitation, the concentrations of Cd (Fig. 4a) and Zn (Fig. 4b) were plotted against the concentration of S in plants grown at S0 and S20. Cd in the roots was positively correlated with S in the roots (r = 0.80, p = 0.001) and the slope of the relationship did not differ significantly (p > 0.05) between S0 and S20, as evidenced by the overlap of the corresponding 95 % confidence intervals. By contrast, Zn in the roots was negatively correlated with S in the roots (r = -0.76, p = 0.002) and the slope of the relationship differed significantly (p < 0.05) between the S0 and S20 treatments.
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Sulfur concentration in durum wheat plants measured 43 days (Z71) and 97 days (Z91) after the beginning of the S treatment. Durum wheat (cv. *Sculptur*) was grown in semi-hydroponics at a background concentration of Cd (Cd-) or with 100 nM Cd (Cd+), and received a standard supply of S (S100), a reduced supply of S (S20) or was subjected to S deprivation (S0) starting at the stem elongation stage (Z30). For a given harvest stage, (*), *, ** and *** indicate in (a) that the concentration of S in plants grown with 100 nM Cd (Cd+) differed significantly from that measured in the control (Cd-); and in (b) that the concentration of S in plants grown with a reduced supply of S (S20) or subjected to S deprivation (S0) differed significantly from that measured in plants grown with a standard supply of S (S100), at a probability level of p < 0.1, p < 0.05, p < 0.01 and p < 0.001, respectively. Data are mean values ± one standard deviation calculated from the four replicates per treatment. ns: non-significant.</figcaption></figure>

## 4. Discussion

It is difficult to assess the representativeness of plant exposure to Cd under hydroponic conditions compared with plant exposure to Cd in soil, as it depends not only on the concentration of Cd (total or free) in solution but also on other parameters, including the presence of Cd-complexing agents in the nutrient solution, its degree of agitation, the renewal rate used to buffer variations in its composition, and the presence of a solid phase to constrain the growth of the root system. In our opinion, the best evaluation criterion remains the concentration of Cd in plant organs, and in particular in grains, which are the organs that are consumed and in which the level of Cd accumulation is the subject of regulation. In this respect, the concentrations of Cd in durum wheat grains measured in this experiment (0.01 µg g⁻¹ DW in control plants, and from 0.3 to 0.4 µg g⁻¹ DW in plants grown with 100 nM Cd) are close to those encountered in agricultural soils [4,23], even more than in recent studies by our team on the same subject [24,25], and can therefore be described as realistic and “low” compared with the level of exposure to Cd tested in many hydroponic studies on Cd [26].

### 4.1. Response of durum wheat to low exposure to Cd

Increasing background Cd to 100 nM Cd in the nutrient solution had no negative effect on the growth of durum wheat, as evidenced by the absence of leaf chlorosis and by the similar plant dry weight observed at both Cd concentrations at the early ripening stage (Z91). Combined with the fact that the concentration of Cd in the leaf at 100 nM Cd was lower than the critical leaf concentration for Cd toxicity reported in non-tolerant crop plants [21], this result suggests that there is probably no negative effect of Cd on durum wheat growth at concentrations of Cd in grain ranging from 0.009 to 0.35 µg g⁻¹ FW. Therefore, Cd is likely not toxic to durum wheat in situations in which the level of Cd in harvested grain slightly exceeds the European regulation (grain Cd > 0.18 µg g⁻¹ FW). In the study by Yan et al. [25] using the same durum wheat cultivar (i.e. *Sculptur*), a 14 % decrease in plant growth was observed between a treatment in which the concentration of Cd in grain was 0.035 µg g⁻¹ FW and a treatment in which the concentration of Cd in grain was 1.2 µg g⁻¹ FW. This suggests that the critical concentration of Cd in the grain resulting in toxicity is between 0.35 and 1.2 µg g⁻¹ FW in durum wheat,

although a Cd concentration of 1.2 µg g⁻¹ FW in grain is not usually encountered in durum wheat grown in agricultural soils in France [23].

Unexpectedly, a slight plant growth promoting effect of Cd was detected at 100 nM based on the higher dry weight of most plant organs in the Cd + treatment compared to in the Cd-treatment, although the difference between the two levels of exposure to Cd was only significant (p < 0.05) for nodes. This suspected hormetic effect of Cd has been reported in many plants including Cd hyperaccumulators for which a physiological role of Cd is sometimes proposed [27], and at concentrations that are sometimes much higher than 100 nM [28]. Nevertheless, to our knowledge, this is the first time it is proposed for durum wheat. Hormesis is attributed to the positive effect that some defense mechanisms, such as enhanced antioxidant machinery and altered detection and signaling, may have on plant growth when exposed to a sub-toxic dose of a contaminant [28]. Hormesis could also explain the early leaf senescence observed at Cd + assuming the synthesis of hormones such as abscisic acid (ABA) is induced by exposure to 100 nM Cd. However, this hypothesis implies that early leaf senescence that may be caused by Cd-induced hormonal modulation, has no deleterious effect on biomass production.

In addition, our results showed that Cd transfer to the shoots was more restricted at 100 nM Cd than at background Cd, as evidenced by 5 fold higher RSF at Cd + compared to Cd-. The restricted root-to-shoot translocation of Cd observed at 100 nM Cd is in line with the results of numerous studies in which Cd translocation was lower at higher Cd exposure due to the stimulated maturation of root apoplastic barriers [29] and/or to the promoted sequestration of Cd in root vacuoles [30], which both restrict the movement of Cd to the root xylem and its further transport upwards to aboveground organs. This interpretation implies that at least some of these Cd detoxification processes, which are usually observed at high levels of Cd exposure (µM range), were already active at 100 nM. In this respect, the studies by Andresen et al. [31,32] showed that in soybean plants, the synthesis of Cd detoxifying ligands was stimulated at 140 nM Cd, supporting the hypothesis of a role for Cd detoxification processes in the enhanced sequestration of Cd in roots we observed at 100 nM. Finally, our results suggest that the activation of these Cd detoxification processes at 100 nM Cd had no detrimental metabolic cost for durum wheat growth in terms of biomass production (see above) and did not enhance the accumulation of S in durum wheat
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Cadmium concentration in durum wheat plants measured 43 days (Z71) and 97 days (Z91) after the beginning of the S treatment. Durum wheat (cv. *Sculptur*) was grown in semi-hydroponics at a background concentration of Cd (Cd-) or with 100 nM Cd (Cd+), and received a standard supply of S (S100), a reduced supply of S (S20) or was subjected to S deprivation (S0) starting at the stem elongation stage (Z30). For a given harvest stage, (*), *, ** and *** indicate in (a) that the concentration of Cd in plants grown with 100 nM Cd (Cd+) differed significantly from that measured in the control (Cd-); and in (b, c, d) that the concentration of Cd in plants grown with a reduced supply of S (S20) or subjected to S deprivation (S0) differed significantly from that measured in plants grown with a standard supply of S (S100), at a probability level of p < 0.1, p < 0.05, p < 0.01 and p < 0.001, respectively. In (c), the concentration of Cd in grains is expressed in µg g⁻¹ fresh weight assuming a residual grain moisture content of 15 % at Z91, and the red line represents the European regulatory limit for Cd in durum wheat (EC 1323/2021), i.e. 0.18 µg Cd g⁻¹ FW. Data are mean values ± one standard deviation calculated from the four replicates per treatment. ns: non-significant.</figcaption></figure>

organs (either roots or shoots), contrary to what was shown at a high level of Cd exposure in *A. thaliana* [16]. In most plant organs, the concentration of S was even lower at 100 nM Cd, notably in nodes where the hormesis effect was most pronounced, suggesting rather a dilution of S in plants exposed to Cd.

## 4.2. Reduced S supply promoted S remobilization and increased Cd accumulation in grains

Reducing the supply of S starting at the stem elongation stage (Z30) had a late and limited effect on the accumulation of S in durum wheat. At Z71, the concentration of S in plant organs was on average the same in the S0, S20 and S100 treatments, regardless of the plant organ concerned. By contrast, at the early ripening stage (Z91), the concentration of S in roots, stem and leaves was lower in the S0 and S20 treatments compared to in the S100 treatment, which suggests that the reduced S supply impacted the uptake and the accumulation of S in plant organs predominantly during grain filling. The reduction in the supply of S did not lead to S deficiency even in plants subjected to S deprivation from Z30, as evidenced by their leaves, which remained green, and whose S concentration remained above the S deficiency threshold of 2 mg g⁻¹, at

Z91. The late and limited effect of S supply on S accumulation in durum wheat likely resulted from the high S concentration of the nutrient solution supplied to the plants until Z30, and perhaps from imperfect washing of the sand-perlite mix at Z30 in the S0 and S20 treatments. The high foliar concentration of S at S100 (>5 mg g⁻¹) supports the hypothesis of luxury consumption of S at the start of vegetative growth, and of a large stock of S in the plants at Z30. The hypothesis of luxury consumption of S until Z30 is in agreement with a recent study by our team [33], which shows that over 30 % of the total S is stored as sulfate in durum wheat leaves with a concentration of S of 3 mg g⁻¹. Efficient recycling of this initial S stock is the most likely reason why plants subjected to a reduction in S or to complete deprivation starting at Z30 were able to cover the demand for S of their developing organs, and to maintain plant growth and grain production at a similar level to that of plants grown at S100. The driving force behind S recycling would be its remobilization from leaves, stems and roots [34,35] because their S concentration decreased between Z71 and Z91 at S0 and S20. In comparison, we observed no decrease in the concentration of S between Z71 and Z91 in roots, stems and leaves of plants grown at S100, indicating no net remobilization of S from these organs at this high level of S supplied. Moreover, the fact that the concentration of S in grains was similar in all
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Relationship between (a) the concentration of S and the concentration of Cd, and (b) the concentration of S and the concentration of Zn, in roots of durum wheat grown with a reduced supply of S (S20) or subjected to S deprivation (S0), harvested at the early milk development stage (S71) and at the early ripening stage (Z91). In (a), the grey dotted line represents the best linear fit obtained between root S and root Cd for plants grown at S0 and S20. In (b), the pale green dotted line represents the best linear fit obtained between root S and root Zn for plants grown at S20, and the dark green dotted line the best linear fit obtained between root S and root Zn for plants grown at S0.</figcaption></figure>

<caption>**Table 3**</caption>

Cadmium concentration in the whole plant, root, stem (internode and node), leaf, spikelet, rachis, bract and grain, and root sequestration factor (RSF), at the early milk development stage (Z71) and the early ripening stage (Z91) in durum wheat. Durum wheat (cv. *Sculptur*) was grown in semi-hydroponics at a background concentration of Cd (Cd-) or with 100 nM Cd (Cd+), and received a standard supply of S (S100), a reduced supply of S (S20) or was subjected to S deprivation (S0) starting at the stem elongation stage (Z30).

<table><thead><tr><th rowspan="3"></th><th colspan="4">Plants harvested at Z71</th><th colspan="4">Plants harvested at Z91</th></tr><tr><th>Cd-</th><th>Cd+</th><th colspan="2">S20</th><th>Cd-</th><th>Cd+</th><th colspan="2">S20</th></tr><tr><th>S100</th><th>S100</th><th>S20</th><th>S0</th><th>S100</th><th>S100</th><th>S20</th><th>S0</th></tr></thead><tbody><tr><td>μg g<sup>-1</sup> DW</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Whole plant</td><td>0.11 (0.003)</td><td><u>1.05</u> (0.05)</td><td>1.02 (0.06)</td><td>0.93 (0.09)</td><td>0.15 (0.01)</td><td><u>1.04</u> (0.17)</td><td>1.03 (0.10)</td><td>1.25* (0.09)</td></tr><tr><td>Root</td><td>0.09 (0.01)</td><td><u>2.99</u> (0.42)</td><td>2.70 (0.35)</td><td>2.20* (0.24)</td><td>0.12 (0.01)</td><td><u>1.79</u> (0.59)</td><td>1.27 (0.18)</td><td>1.23 (0.13)</td></tr><tr><td>Stem</td><td>0.08 (0.01)</td><td><u>0.98</u> (0.04)</td><td>1.02 (0.09)</td><td>0.94 (0.17)</td><td>0.21 (0.02)</td><td><u>1.57</u> (0.22)</td><td>1.66 (0.52)</td><td>2.05 (0.46)</td></tr><tr><td>Internode</td><td>0.07 (0.01)</td><td><u>0.94</u> (0.05)</td><td>0.95 (0.08)</td><td>0.89 (0.18)</td><td>0.21 (0.02)</td><td><u>1.57</u> (0.24)</td><td>1.68 (0.53)</td><td>2.09 (0.49)</td></tr><tr><td>Node</td><td>0.16 (0.04)</td><td><u>1.95</u> (0.41)</td><td>2.31 (0.33)</td><td>1.93 (0.24)</td><td>0.16 (0.03)</td><td><u>1.59</u> (0.18)</td><td>1.44 (0.39)</td><td>1.48 (0.32)</td></tr><tr><td>Leaf</td><td>0.41 (0.06)</td><td><u>1.21</u> (0.23)</td><td>1.10 (0.07)</td><td>1.12 (0.07)</td><td>0.60 (0.06)</td><td><u>2.36</u> (0.78)</td><td>1.85 (1.07)</td><td>2.32 (0.21)</td></tr><tr><td>Spikelet</td><td>0.03 (0.003)</td><td><u>0.31</u> (0.08)</td><td>0.42(*) (0.07)</td><td>0.36 (0.06)</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>Rachis</td><td>na</td><td>na</td><td>na</td><td>na</td><td>1.08 (1.03)</td><td>1.15 (0.67)</td><td>2.13(*) (0.14)</td><td>2.55* (0.68)</td></tr><tr><td>Bract</td><td>na</td><td>na</td><td>na</td><td>na</td><td>0.17 (0.01)</td><td><u>1.80</u> (0.49)</td><td>2.08 (0.26)</td><td>2.43(*) (0.28)</td></tr><tr><td>Grain</td><td>na</td><td>na</td><td>na</td><td>na</td><td>0.01 (0.003)</td><td><u>0.30</u> (0.02)</td><td>0.31 (0.05)</td><td>0.41** (0.02)</td></tr><tr><td>RSF</td><td>0.81</td><td><u>4.11</u></td><td>3.48</td><td>3.10*</td><td>0.77</td><td><u>1.84</u></td><td>1.29</td><td>1.01(*)</td></tr></tbody></table>

Values are means of four independent replicates. Values into brackets stand for one standard deviation.

For plants grown with 100 nM Cd (Cd+), (*), *, ** and *** indicate that the mean value at S20 or S0 differed significantly from that in the control (S100) at a probability level of p < 0.1, p < 0.05, p < 0.01 and p < 0.001, respectively.

For plants grown with 100 nM Cd (Cd+) that received a standard supply of S (S100), values are underlined when they are significantly different (p < 0.05) than those obtained under the Cd-treatment.

na: not available.

three S treatments suggests that most of the S remobilized in S-deprived plants was allocated to the grains.

Reducing the S supply starting at the stem elongation stage enhanced the accumulation of Cd in grains in durum wheat. Indeed, the concentration of Cd in grains at the early ripening stage was on average 35 % higher in the S0 treatment than in the S100 treatment. There are several reasons for the enhanced accumulation of Cd in grains at S0, linked to various processes in which S and Cd interact *in planta*. The first reason is an increased uptake of Cd during grain filling in S-deprived plants. This hypothesis is supported by the average concentration of Cd in plants at Z91, which was 20 % higher in the S0 treatment than in the S100 treatment. However, it assumes that (at least some) physiological responses to S deficiency [36] could increase the uptake of Cd by durum wheat, which, to our knowledge, has not been evidenced so far except perhaps under iron shortage conditions [37]. The second reason for the enhanced accumulation of Cd in grains at S0 is a higher allocation of absorbed Cd to the grains in S-deprived plants. In this respect, the fact that the roots are the only organ whose Cd concentration tended to decrease with a decreasing supply of S, although not always significantly, suggests a link between the increased accumulation of Cd in grains and the decreased accumulation of Cd in roots in S-deprived plants. One hypothesis is that the sequestration of Cd in roots was impaired in S-deprived plants due to a lack of sulfur ligands able to bind Cd. This hypothesis is consistent with the fact that in our experiment Cd detoxification processes were likely active at 100 nM Cd (see above), but implies an increased concentration of Cd in all aboveground organs in S-deprived plants, including leaves and stem that is not supported by our data (Table 3). Another hypothesis is that Cd remobilization from roots was enhanced in S-deprived plants due to the enhanced remobilization of S. This hypothesis is consistent with studies on durum wheat that described roots as a net source of S [34] and as a net source of Cd [25] for the grains, and is further supported by the proportional decrease in the concentrations of S and Cd in the roots of S-deprived plants over grain filling (Fig. 4a). This hypothesis assumes that S remobilization is a driving force of Cd remobilization in the roots of durum wheat, based on the fact that S and Cd share the same storage locations (e.g. the vacuole) and/or are chemically bound (e.g. as PCs-Cd) in root cells. To compare with, the decrease in the concentration of S over grain filling was not associated with a decrease in the concentration of Zn in the roots of S-deprived plants (Fig. 4b). Hence, the remobilization of S is likely a
<caption>**Table 4**</caption>

Zinc concentration in the whole plant, root, stem (internode and node), leaf, spikelet, rachis, bract and grain, at the early milk development stage (Z71) and the early ripening stage (Z91) in durum wheat. Durum wheat (cv. *Sculptur*) was grown in semi-hydroponics at a background concentration of Cd (Cd-) or with 100 nM Cd (Cd+), and received a standard supply of S (S100), a reduced supply of S (S20) or was subjected to S deprivation (S0) starting at the stem elongation stage (Z30).

<table><thead><tr><th rowspan="2"></th><th colspan="4">Plants harvested at Z71</th><th colspan="4">Plants harvested at Z91</th></tr><tr><th>Cd-<br>S100</th><th>Cd+<br>S100</th><th>S20</th><th>S0</th><th>Cd-<br>S100</th><th>Cd+<br>S100</th><th>S20</th><th>S0</th></tr></thead><tbody><tr><td>μg g<sup>-1</sup> DW</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Whole plant</td><td>28.9</td><td>24.4</td><td>25.3</td><td>22.4</td><td>27.6</td><td>26.6</td><td>22.8</td><td>23.2</td></tr><tr><td>Root</td><td>22.3</td><td>18.4</td><td>19.2</td><td>18.7</td><td>57.9</td><td>48.9</td><td>25.3*</td><td>28.4(*)</td></tr><tr><td>Stem</td><td>16.8</td><td><u>8.9</u></td><td>8.1</td><td>6.2</td><td>11.5</td><td>10.5</td><td>11.3</td><td>9.9</td></tr><tr><td>Internode</td><td>16.1</td><td><u>8.6</u></td><td>7.6</td><td>6.0</td><td>11.6</td><td>10.7</td><td>11.5</td><td>10.1</td></tr><tr><td>Node</td><td>32.3</td><td><u>10.9</u></td><td>15.7</td><td>9.8</td><td>9.6</td><td>7.4</td><td>8.6</td><td>7.5</td></tr><tr><td>Leaf</td><td>30.3</td><td>30.4</td><td>29.1</td><td>30.2</td><td>28.7</td><td>29.8</td><td>20.6**</td><td>30.7</td></tr><tr><td>Spikelet</td><td>39.8</td><td>37.2</td><td>39.1</td><td>34.4</td><td>na</td><td>na</td><td>na</td><td>na</td></tr><tr><td>Rachis</td><td>na</td><td>na</td><td>na</td><td>na</td><td>25.0</td><td>19.0</td><td>21.3</td><td>27.9</td></tr><tr><td>Bract</td><td>na</td><td>na</td><td>na</td><td>na</td><td>31.3</td><td>30.7</td><td>29.8</td><td>33.3</td></tr><tr><td>Grain</td><td>na</td><td>na</td><td>na</td><td>na</td><td>28.2</td><td>28.2</td><td>26.0</td><td>23.4</td></tr></tbody></table>

Values are means of four independent replicates.

For plants grown with 100 nM Cd (Cd+), (*), *, ** and *** indicate that the mean value at S20 or S0 differed significantly from that in the control (S100) at a probability level of p < 0.1, p < 0.05, p < 0.01 and p < 0.001, respectively.

For plants grown with 100 nM Cd (Cd+) that received a standard supply of S (S100), values are underlined when they are significantly different (p < 0.05) than those obtained under the Cd-treatment.

na: not available.

driver of the remobilization of Cd but not of the remobilization Zn in the roots of durum wheat, which could be due to the higher binding affinity of sulfur ligands such as PCs for Cd than for Zn [38]. It is worth noting that the second hypothesis does not explain why a net remobilization of Cd was observed without a net remobilization of S in the roots of plants in the S100 treatment. To explain this, we must assume that there were two Cd pools in the roots of durum wheat: a first Cd pool whose remobilization was not linked to that of S, and a second Cd pool whose remobilization was associated with that of S. The allocation to the grains of the second Cd pool is supposed higher than the allocation to the grains of the first Cd pool, possibly because sulfur ligands (e.g. GSH, PCs) play an important role in the long-distance transport of Cd in planta [39], as they may prevent fixation of Cd (as free Cd ion) onto xylem vessels and may increase the solubility and the mobility of Cd in the phloem [39, 40].

## 5. Conclusion

Exposure of durum wheat to 100 nM Cd did not penalize plant growth but resulted in concentrations of Cd in the grains that did not comply with European regulations. This study thus further highlights that the problem with Cd in agriculture is not phytotoxicity and yield loss, but sanitary quality and compliance with regulations that apply to the harvested grain. Sulfate deprivation starting at stem elongation increased the concentration of Cd in grains by 35 %, highlighting a potential link between S status and Cd allocation to grains in durum wheat. The suspected process is an associated remobilization of S and Cd in the roots, and more intense remobilization of these two elements towards the grains in S-deprived plants. Because this link suggests a potential effect of S fertilization on the sanitary quality of durum wheat grains with respect to Cd, it requires further investigation. The first step would be to precisely quantify the fluxes of S and Cd remobilized from roots, for example by isotope labeling, to determine whether they respond similarly, in intensity and kinetics, to S deprivation. The second step would be to visualize potential changes in the localization and the speciation of Cd and S in root cells during grain filling, for instance by µXRF imaging and µXANES analyses [10], to test the hypothesis of their associated remobilization. The last step would be to investigate other potential causes of the effect of a reduction in S on Cd accumulation in grains, for example by monitoring grain size (via thousand kernel weight) and the number of kernels per head, as both these parameters can be sensitive to S deprivation [41] and can alter the elemental

composition of the grain [42].

## CRediT authorship contribution statement

**Agathe Vidal:** Writing – original draft, Visualization, Formal analysis, Data curation. **Valentin Dubois:** Investigation, Formal analysis. **Sophie Brunel-Muguet:** Writing – review & editing, Supervision, Conceptualization. **Jean-Christophe Avice:** Writing – review & editing, Supervision, Methodology, Conceptualization. **Christophe Nguyen:** Writing – review & editing, Supervision, Formal analysis, Data curation, Conceptualization. **Jean-Yves Cornu:** Writing – original draft, Supervision, Funding acquisition, Conceptualization.

## Funding

This work was financially supported by the French National Institute for Agriculture, Food and Environment (INRAE), by the CNRS-INSU EC2CO program under the “CADSOU” project and by the “Nouvelle Aquitaine” regional council (France) under the ESTOCADE project.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors are grateful to Anne-Françoise Ameline (UMR EVA) for technical help in conducting the experiment, and to PLATIN' (PLATeau d'Isotopie de Normandie) core facility for HR-ICP-MS analyses.

## Data availability

Data will be made available on request.

## References

[1] A. Oleko, C. Fillol, A. Saoudi, A. Zeghnoun, M.-L. Bidondo, J. Gane, A. Balicco, Imprénction de la population française par le cadmium, Programme national de biosurveillance, Esteban 2014-2016, Saint-Maurice : Santé publique France, 2021. https://www.santepubliquefrance.fr.
[2] G. Jansson, Cadmium in Arable Crops: the Influence of Soil Factors and Liming, Dissertation, The Swedish University of Agricultural Sciences, Uppsala, 2002.

[3] R.M. Welch, W.A. Norvell, Mechanisms of cadmium uptake, translocation and deposition in plants, in: M.J. McLaughlin, B.R. Singh (Eds.), Cadmium in Soils and Plants, Springer Netherlands, Dordrecht, 1999, pp. 125-150.

[4] S.A. Tittlemier, A. Richter, Cadmium concentrations in Canadian durum exports decreased with the adoption of low accumulating cultivars, Food Addit. Contam. 39 (2022) 1953-1962.

[5] M. Maccaferri, N.S. Harris, S.O. Twardziok, R.K. Pasam, H. Gundlach, M. Spannagl, D. Ormanbekova, T. Lux, V.M. Prade, S.G. Milner, A. Himmelbach, M. Mascher, P. Bagnaresi, P. Faccioli, P. Cozzi, M. Lauria, B. Lazzari, A. Stella, A. Manconi, M. Gnocchi, M. Moscatelli, R. Avni, J. Deek, S. Biyiklioglu, E. Frascaroli, S. Corneti, S. Salvi, G. Sonnante, F. Desiderio, C. Marè, C. Crosatti, E. Mica, H. Özkan, B. Kilian, P. De Vita, D. Marone, R. Joukhadar, E. Mazzucotelli, D. Nigro, A. Gadaleta, S. Chao, J.D. Faris, A.T.O. Melo, M. Pumphrey, N. Pecchioni, L. Milanesi, K. Wiebe, J. Ens, R.P. MacLachlan, J.M. Clarke, A.G. Sharpe, C.S. Koh, K.Y.H. Liang, G.J. Taylor, R. Knox, H. Budak, A.M. Mastrangelo, S.S. Xu, N. Stein, I. Hale, A. Distelfeld, M.J. Hayden, R. Tuberosa, S. Walkowiak, K.F.X. Mayer, A. Ceriotti, C.J. Pozniak, L. Cattivelli, Durum wheat genome highlights past domestication signatures and future improvement targets, Nat. Genet. 51 (2019) 885-895.

[6] S. Zimmerl, J. Lafferty, H. Buerstmayr, Assessing diversity in Triticum durum cultivars and breeding lines for high versus low cadmium content in seeds using the CAPS marker usw47, Plant Breed. 133 (2014) 712-717.

[7] N. Sarwar, Saifullah, S.S. Malhi, M.H. Zia, A. Naem, S. Bibi, G. Farid, Role of mineral nutrition in minimizing cadmium accumulation by plants, J. Sci. Food Agric. 90 (2010) 925-937.

[8] I. Tea, T. Genter, N. Naulet, L.M. Marie, D. Kleiber, Interaction between nitrogen and sulfur by foliar application and its effects on flour bread-making quality, J. Sci. Food Agric. 87 (2007) 2853-2859.

[9] S. Clemens, M.G.M. Aarts, S. Thomine, Plant science: the key to preventing slow cadmium poisoning, Trends Plant Sci. 18 (2013) 92-99.

[10] M.-P. Isaire, S. Huguet, C.-L. Meyer, H. Castillo-Michel, D. Testemale, D. Vantelon, P. Saumitou-Laprade, N. Verbruggen, G. Sarret, Evidence of various mechanisms of Cd sequestration in the hyperac accumulator Arabidopsis halleri, the non- accumulator Arabidopsis lyrata, and their progenies by combined synchrotron-based techniques, J. Exp. Bot. 66 (2015) 3201-3214.

[11] Z. Fu, Y. Yao, M.Z. Ul Haq, Y. Liu, D. Yang, H. Yang, Y. Wu, Glutathione's role in mitigating cadmium stress in Pogostemon cablin: insights from combined transcriptomic and metabolomic approaches, J. Hazard. Mater. 491 (2025) 137921.

[12] I.V. Seregin, A.D. Kozhevnikova, Phytochelatins: Sulfur-containing metal(loid)-chelating ligands in plants, Int. J. Mol. Sci. 24 (2023) 2430.

[13] G. Na, D.E. Salt, The role of sulfur assimilation and sulfur-containing compounds in trace element homeostasis in plants, Environ. Exp. Bot. 72 (2011) 18-25.

[14] T.S. Liang, H. Ding, G.D. Wang, J.Q. Kang, H.X. Pang, J.Y. Lv, Sulfur decreases cadmium translocation and enhances cadmium tolerance by promoting sulfur assimilation and glutathione metabolism in Brassica chinensis L., Ecotoxicol. Environ. Saf. 124 (2016) 129-137.

[15] D. Zhang, G. Du, D. Chen, G.L. Shi, W. Rao, X. Li, Y. Jiang, S.L. Liu, D.C. Wang, Effect of elemental sulfur and gypsum application on the bioavailability and redistribution of cadmium during rice growth, Sci. Total Environ. 657 (2019) 1460-1467.

[16] S. Herbette, L. Taconnat, V. Hugouvieux, L. Piette, M.L.M. Magniette, S. Cuine, P. Auroy, P. Richaud, C. Forestier, J. Bourguignon, J.P. Renou, A. Vavasseur, N. Leonhardt, Genome-wide transcriptome profiling of the early cadmium response of Arabidopsis roots and shoots, Biochimie 88 (2006) 1751-1765.

[17] C. Lancilli, B. Giacomini, G. Lucchini, J.C. Davidian, M. Cocucci, G.A. Sacchi, F. F. Nocito, Cadmium exposure and sulfate limitation reveal differences in the transcriptional control of three sulfate transporter (Sultr1;2) genes in Brassica juncea, BMC Plant Biol. 14 (2014) 132.

[18] S. Sauvé, W.A. Norvell, M. McBride, W. Hendershot, Speciation and complexation of Cd in extracted soil solutions, Environ. Sci. Technol. 34 (2000) 291-296.

[19] V. Maignan, B. Bernay, P. Géliot, J.-C. Avice, Biostimulant effects of glutacetine® and its derived formulations mixed with N fertilizer on post-heading N uptake and remobilization, seed yield, and grain quality in winter wheat, Front. Plant Sci. 11 (2020) 607615.

[20] R Core Team, R, A Language and Environment for Statistical Computing, R foundation for statistical computing, Vienna, 2022.

[21] P.J. White, P.H. Brown, Plant nutrition for sustainable development and global health, Ann. Bot. 105 (2010) 1073-1080.

[22] H. Marschner, Mineral Nutrition of Higher Plants, second ed., Academic Press, London, 1995.

[23] C. Nguyen, A. Roucou, G. Grignon, J.-Y. Cornu, B. Méléard, Efficient models for predicting durum wheat grain Cd conformity using soil variables and cultivars, J. Hazard. Mater. 401 (2021) 12313.

[24] B.F. Yan, C. Nguyen, O.S. Pokrovsky, F. Candaudap, C. Coriou, S. Bussière, T. Robert, J.-Y. Cornu, Contribution of remobilization to the loading of cadmium in durum wheat grains: impact of post-anthesis nitrogen supply, Plant Soil 424 (2018) 591-606.

[25] B.F. Yan, C. Nguyen, O.S. Pokrovsky, F. Candaudap, C. Coriou, S. Bussière, T. Robert, J.-Y. Cornu, Cadmium allocation to grains in durum wheat exposed to low Cd concentrations in hydroponics, Ecotoxicol. Environ. Saf. 184 (2019) 109592.

[26] A. van der Ent, P.M. Kopittke, H. Schat, R.L. Chaney, Hydroponics in physiological studies of trace element tolerance and accumulation in plants focusing on metallophytes and hyperac accumulator plants, Plant Soil 501 (2024) 573-594.

[27] E.J. Calabrese, E. Agathokleous, Accumulator plants and hormesis, Environ. Pollut. 274 (2021) 116526.

[28] M.E.A. Carvalho, P.R.C. Castro, R.A. Azevedo, Hormesis in plants under Cd exposure: from toxic to beneficial element, J. Hazard. Mater. 384 (2020) 121434.

[29] A. Lux, M. Martinka, M. Vaculik, P.J. White, Root responses to cadmium in the rhizosphere: a review, J. Exp. Bot. 62 (2011) 21-37.

[30] F.F. Nocito, C. Lancilli, B. Dendena, G. Lucchini, G.A. Sacchi, Cadmium retention in rice roots is influenced by cadmium availability, chelation and translocation, Plant Cell Environ. 34 (2011) 994-1008.

[31] E. Andresen, L. Lyubenova, T. Hubácek, S.N.H. Bokhari, S. Matousková, A. Mijiovovich, J. Rohovec, H. Küpper, Chronic exposure of soybean plants to nanomolar cadmium reveals specific additional high-affinity targets of cadmium toxicity, J. Exp. Bot. 71 (2020) 1628-1644.

[32] E. Andresen, J.J. Flores-Sanchez, D. Brückner, S.N.H. Bokhari, G. Falkenberg, H. Küpper, Sublethal and lethal Cd toxicity in soybean roots specifically affects the metabolome, Cd binding to proteins and cellular distribution of Cd, J. Hazard. Mater. 442 (2023) 130062.

[33] A. Vidal, C. Nguyen, S. Brunel-Muguet, J.-C. Avice, A.-F. Ameline, J.-Y. Cornu, Sulfur deficiency affects the accumulation and the allocation to shoots of cadmium and of some macro- (P, K, Ca, Mg) and micro-nutrients (Cu, Mn, Mo, Zn) in durum wheat at heading, Environ. Sci. Pollut. Res. 32 (2025) 14114-14126.

[34] P. Borpatragohain, T.J. Rose, L. Liu, B.J. Barkla, C.A. Raymond, G.J. King, Remobilization and fate of sulphur in mustard, Ann. Bot. 124 (2019) 471-480.

[35] J.M. Monaghan, C.M. Scrimgeour, W.M. Stein, F.J. Zhao, E.J. Evans, Sulphur accumulation and redistribution in wheat (Triticum aestivum): a study using stable sulphur isotope ratios as a tracer system, Plant Cell Environ. 22 (1999) 831-839.

[36] M.J. Hawkesford, Plant responses to sulphur deficiency and the genetic manipulation of sulphate transporters to improve s-utilization efficiency, J. Exp. Bot. 51 (2000) 131-138.

[37] S. Astolfi, S. Celletti, G. Vigani, T. Mimmo, S. Cesco, Interaction between sulfur and iron in plants, Front. Plant Sci. 12 (2021) 670308.

[38] B.H. Cruz, J.M. Díaz-Cruz, C. Ariño, M. Esteban, Complexation of heavy metals by phytochelatins: voltammetric study of the binding of Cd2+ and Zn2+ ions by the phytochelatin (γ-Glu-Cys)3Gly assisted by multivariate curve resolution, Environ. Sci. Technol. 39 (2005) 778-786.

[39] D.G. Mendoza-Cózatl, E. Butko, F. Springer, J.W. Torpey, E.A. Komives, J. Kehr, J. I. Schroeder, Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus: a role for thiol-peptides in the long-distance transport of cadmium and the effect of cadmium on iron translocation, Plant J. 54 (2008) 249-259.

[40] M.-L. Pons, B. Collin, E. Doelsch, P. Chaurand, T. Fehlauer, C. Levard, C. Keller, J. Rose, X-ray absorption spectroscopy evidence of sulfur-bound cadmium in the Cd-hyperac accumulator Solanum nigrum and the non-accumulator Solanum melongena, Environ. Pollut. 279 (2021) 116897.

[41] T. Ausma, V. Bansal, M. Kraaij, A.C.M. Verloop, A. Gasperl, M. Müller, S. Kopriva, L.J. De Kok, C.J. van der Kooi, Floral displays suffer from sulphur deprivation, Environ. Exp. Bot. 192 (2021) 104656.

[42] D.F. Calderini, I. Ortiz-Monasterio, Grain position affects grain macronutrient and micronutrient concentrations in wheat, Crop Sci. 43 (2003) 141-151.