# Evidence of cadmium transport via the phloem in cacao seedlings

Hester Blommaert, Clara de Meese, Matthias Wiggenhauser, Géraldine

Sarret, Erik Smolders

► To cite this version:

Hester Blommaert, Clara de Meese, Matthias Wiggenhauser, Géraldine Sarret, Erik Smolders. Evidence of cadmium transport via the phloem in cacao seedlings. Plant and Soil, 2024, 10.1007/s11104-024-06753-0. hal-04763016

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# Evidence of cadmium transport via the phloem in cacao seedlings

Hester Blommaert* · Clara De Meese ·
Matthias Wiggenhauser · Géraldine Sarret ·
Erik Smolders

Received: 8 February 2024 / Accepted: 16 May 2024
© The Author(s) 2024

## Abstract

*Background and aims* It is unclear if cadmium (Cd) is loaded into cacao beans directly from the roots and stem or rather via reallocation from leaves and stem via the phloem. Here, a split-root experiment with a stable isotope ¹⁰⁸Cd tracer was set up to determine the short-term circulation in the vegetative tissues of Cd in *Theobroma cacao* L. seedlings.

*Methods* Roots of cacao seedlings were split into two parts, each growing in separate containers. The compartments contained an equal volume of nutrient solution with a low-dose Cd concentration of 20 nM, and were either (+) or not (-) enriched in stable ¹⁰⁸Cd.

Responsible Editor: Jian Feng Ma.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s11104-024-06753-0.

H. Blommaert · G. Sarret
Université Grenoble Alpes, Université Savoie Mont Blanc, CNRS, IRD, Univ. Gustave Eiffel, ISTerre, Grenoble, France
e-mail: hester.blommaert@gmail.com

G. Sarret
e-mail: Geraldine.sarret@univ-grenoble-alpes.fr

C. De Meese · E. Smolders
Division of Soil and Water Management, Department of Earth and Environmental Sciences, KU Leuven, Louvain, Belgium
e-mail: Clarademeese@gmail.com

Seedlings were grown for two weeks and periodically harvested.

*Results* On day 1 after labelling, there was a rise in the ¹⁰⁸Cd isotopic abundance (IA) in the roots in the labelled compartment whereas the ¹⁰⁸Cd IA was unaffected in other plant parts. After 7 days, about 75% of the tracer ¹⁰⁸Cd in the plant had been translocated to the shoots and the largest fraction of tracer was recovered in the stem. The ¹⁰⁸Cd IA in the roots in the non-labelled compartment rose after day 11, demonstrating that Cd was phloem mobile and reallocated to developing tissues. The isotopic abundance of ¹⁰⁸Cd in developing leaves was similar to that in the older leaves, suggesting that Cd originated from leaves rather than directly from roots.

*Conclusions* We corroborated Cd pathways previously studied in mature cacao trees and demonstrated that Cd is reallocated to sink organs via the phloem.

E. Smolders
e-mail: Erik.smolders@kuleuven.be

M. Wiggenhauser (✉)
Institute of Agricultural Sciences, ETH Zurich, Eschikon
33, CH-8315 Lindau, Switzerland
e-mail: matthias.wiggenhauser@usys.ethz.ch
**Keywords** Low-dose · Stable isotope labelling · *Theobroma cacao* L. · Split-root design · Uptake · Reallocation · Xylem

**Introduction**

Chronic Cd exposure has been related to adverse health effects, such as renal tubular dysfunction and osteomalacia (FAO/WHO 2010). For the non-smoking population, dietary intake is the main route of Cd exposure. After staple foods like cereals and potato tubers, it has been estimated that chocolate and chocolate products contribute significantly to the total dietary Cd exposure in the European population (EFSA 2012). To protect consumers, regulators worldwide have established limits on Cd concentrations in chocolate and cacao powders between 0.10 and 0.80 mg Cd kg⁻¹ (Mercosur 2011; Ministry of Health of the Russian Federation 2011; European Commission 2014; Food Standards Australia New Zealand 2017; Codex Alimentarius Commission 2018). These regulations prompted research to reduce Cd concentrations in cacao beans, which are the major ingredient for chocolate and cacao powders (Meter et al. 2019; Vanderschueren et al. 2021). A major bottleneck that hampers the development of these mitigation strategies, is that the knowledge about the circulation of Cd in cacao is small compared to that in other plant species, notably regarding the transport of Cd towards the beans.

The circulation of Cd in mature trees has been studied but remains elusive. Based on existing knowledge from other plants than cacao plants, we know that Cd could be loaded into the beans (seeds) by potentially two pathways: a direct translocation or a reallocation from deposits in vegetative tissues (Mallard et al. 2015). In cacao, the first pathway may be a direct translocation from roots and branches via the transpiration stream (xylem) to the beans, with a possible xylem-to-phloem transfer in the rays of the branches or in the transpiring tissues surrounding the bean (pod husk and testa). The second possible pathway is that Cd is reallocated from vegetative stores, like the leaves. Reallocation from leaves to beans requires that Cd is mobile in the phloem (White and Brown 2010). In cacao, the first study on Cd pathways suggested that a substantial amount of Cd could be directly loaded from roots and branches into beans with a xylem-to-phloem transfer in the woody parts,

without passage through the leaves (Engbersen et al. 2019). This conclusion was based on differences in Cd distribution among 11 cultivars. Their suggestion implied that reallocation from leaves through the phloem was rather limited for the loading of Cd in the beans. A later study with isotope data at natural abundance corroborated that Cd in the beans could be loaded from the phloem tissues of the branches (Blommaert et al. 2022). They observed no isotope fractionation between the branches and beans, whilst the isotopic signature of Cd in the leaves was heavier than in the branches and beans. The absence of isotopic fractionation between the branches and beans can indicate a direct translocation from the branch phloem to the beans, while Cd gets more and more depleted in light isotopes during its translocation via the xylem to the leaves. Yet, a follow-up study that used pulse-labelling of intact trees in the field, suggested that Cd in the beans is rather reallocated from a vegetative store of Cd in the tree than by direct transport from the roots (Vanderschueren et al. 2023). This is because the ¹⁰⁸Cd isotope tracer that was applied to the soil arrived significantly later in the beans than in the leaves, which are fed directly by the transpiration stream. However, it remained unclear if the source of Cd was a reallocation from the leaves or if bean Cd was rather loaded from a stock in the branches. Despite the abovementioned studies, it remains thus unclear how exactly Cd circulates in cacao, and more particularly if Cd can be reallocated from vegetative stores via the phloem to developing organs like the beans or if it is rather retained in the leaves.

Isotope tracing using stable isotopes or radioisotopes is an interesting tool to study the short-term circulation of elements in plants. In the case of Cd, isotope tracing has been used to infer Cd pathways in agricultural crops (Page and Feller 2005; Yan et al. 2019, 2023) and hyperaccumulators (Wei et al. 2014). For example, in wheat, the use of the radioisotope ¹⁰⁹Cd as a tracer showed a strong initial retention of Cd in the roots and corresponding slow release of ¹⁰⁹Cd into the shoots (Page and Feller 2005). On the contrary, in the hyperaccumulator *S. nigrum*, ¹⁰⁹Cd was rapidly transported to the shoots due to an efficient loading into the xylem in the roots (Wei et al. 2014). Once, in the leaves it was not or slowly redistributed via the phloem within the shoots. Thus, the comparison of these studies illustrates that the mobility of Cd in the phloem affects
the efficiency of reallocation and varies largely among plant families (Page and Feller 2015; Stercke-man and Thomine 2020). Today, there are no studies that document the short-term circulation and phloem mobility of Cd in cacao, which could infer our under-standing of how Cd is taken up and distributed, and if it can be reallocated from the leaves to developing organs.

Split-root experiments have been used in the past to reveal the circulation of trace elements in plants (Mclaughlin et al. 2000; Haslett et al. 2001; Qin et al. 2013; Page and Feller 2015; Valentinuzzi et al. 2020; Wei et al. 2022). This experimental design involves dividing the root system into two equivalent segments, each grow-ing in distinct compartments. By introducing an isotope tracer to one compartment, the subsequent circulation of this tracer throughout the plant can be precisely tracked. Tracer movement from the labelled to the non-labelled root compartment can only occur via the phloem, either by transport through the phloem between root segments or by a downward flux of Cd from the shoot (Welch et al. 1999). Hence, such design can advance greatly our understanding of how Cd circulates in cacao.

This study was set up to identify the circulation of Cd in cacao, and if there is Cd reallocation via the phloem. To this end, we used a split-root approach with cacao seedlings. In this setup, the nutrient solu-tion of one root compartment was labelled with ¹⁰⁸Cd stable isotope to trace the fate of ¹⁰⁸Cd in the plant.

## Materials and methods

### Experimental design

A split-root hydroponic system was set up with five-week-old *Theobroma cacao* L. seedlings. The roots were divided into two separate containers (Figure S1). The containers had an equal volume (330 mL) of nutrient solution either enriched with the stable iso-tope ¹⁰⁸Cd (+) or not (-). There were three different treatments; (-/-), (+/-), and (+/+) in treatment (+/-), the nutrient solution in the root+compartment was labelled with ¹⁰⁸Cd as a tracer. The other compartment (root -) had an identical solution, but no ¹⁰⁸Cd tracer. Due to the labelling, the Cd concentrations increased in the (+) treatments by 8%, compared to those in the non-labelled (-) nutrient solution (as detailed in Sect. “Cadmium stable isotope labelling”). In treatment (-/-),

both root compartments contained the nutrient solu-tion without a ¹⁰⁸Cd tracer. In (+/+) treatment, both compartments were labelled with ¹⁰⁸Cd tracer. The non-labelled and fully labelled solutions served as refer-ence treatments. The experiment took fourteen days, in this period, neither the nutrient nor the ¹⁰⁸Cd label-ling solutions were replaced. At days 0, 1, 7, 11, and 14, four plants per treatment were harvested according to a sampling strategy shown in Table 1. In total, the experiment consisted of n=40 split-root seedlings.

### Plant growth

Three-week-old seedlings (from the genetic group *Trinitario*) were purchased from a nursery (Cacao-bomen.nl, Harmelen, The Netherlands), where they germinated from seed. Upon arrival in the laboratory facilities in Leuven (Belgium), the seedlings were removed from their nursery substrate. Their roots were washed by immersion in deionized water to remove any adhering substrate particles. The seedlings were transplanted into 9-L vessels with continuously aer-ated nutrient solution for a two-week preculture in hydroponics before the split-root experiment started. The nutrient solution contained: Ca(NO₃)₂, 2.05 mM; MgSO₄, 0.50 mM; KNO₃, 1.24 mM; KH₂PO₄, 90 µM; H₃BO₃, 15 µM; (NH₄)₆Mo₇O₂₄·4H₂O, 0.10 µM; MnSO₄·H₂O, 4.81 µM; CuSO₄·5H₂O, 1.42 µM; ZnSO₄·7H₂O, 5.80 µM; NaCl, 25 µM; MES (2-(*N*-morpholino)ethanesulfonic acid) 2 mM; NaOH, 1 mM; FeNaEDTA, 10 µM. Cadmium was added as Cd(NO₃)₂·4H₂O together with nitrilotriacetic acid (NTA (50 µM)) (total Cd 19.6 nM). The pH of the nutrient solution was measured at the beginning of the preculture and at the beginning of the experi-ment. The pH was on average 6.1, and was maintained

<table><caption>**Table 1** Experimental design and sampling timeline after transfer of seedlings to the experimental solutions (day 0)</caption><thead><tr><th colspan="5">Sampling time (days after transfer to solution)</th></tr><tr><th>0</th><th>1</th><th>7</th><th>11</th><th>14</th></tr></thead><tbody><tr><td>(-/-)</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>(+/-)</td><td>(+/-)</td><td>(+/-)</td><td>(+/-)</td></tr><tr><td></td><td>(+/-+)</td><td>(+/-+)</td><td>(+/-+)</td><td>(+/-+)</td></tr></tbody></table>

Split-root compartments were either both labelled with ¹⁰⁸Cd (+/+), one labelled (+/-) or not labelled (-/-). All other solution components were equal between the compartments. Each treatment had n=4 biological replicates

<figure><img src="image_2.png" /></figure>
within 0.1 pH units with the MES pH buffer. Speciation calculations predicted that most Cd was complexed with EDTA (37%) and NTA (61%) ligands (Visual Minteq version 3.1). This complexation ensured that free $Cd^{2+}$ was buffered at low activity (0.38 nM) and environmentally relevant concentrations. Yet, it should be noted that during the experiment, the pH was not monitored and Cd speciation could have potentially changed.

The culture vessels with the seedlings were installed in a growth chamber with a constant air humidity of 70% and a 12 h/12 h (25 °C/20 °C) day/night cycle with 200 µmol m⁻² s⁻¹ light intensity. The vessels were covered with aluminium foil to exclude light to prevent algal growth. After two weeks of pre-culture, individual plants were transferred from the 9 L containers to the split root device (Figure S1) of twice 330 mL of continuously aerated solution. The lateral roots were gently split and each part was transferred into separate compartments (as described in Saiz-Fernández et al. 2021). During the course of the experiment, the nutrient solution (with the $^{108}$Cd tracer) was not replaced and consequently, the volume of the nutrient solution was reduced because of evapotranspiration. Therefore, on day 7, deionized water was added to each compartment to restore the initial volume of 330 mL. The Cd concentration was not monitored during the experiment but the mass balance calculations based on the net Cd uptake in the plant suggest that almost all Cd was taken up during the experiment (details not shown).

## Cadmium stable isotope labelling

A stock solution (6.3 mg Cd L⁻¹) with an isotopic abundance of $^{108}$Cd of 71.1% was prepared from an enriched $^{108}$Cd stable isotope (Trace Sciences International, Delaware, USA) (for details on the preparation, see Argüello et al. (2020); Vanderschueren et al. (2023)). In Table S1, the isotopic abundance of natural Cd and the enriched Cd sources is depicted (Berglund and Wieser 2011). For the preparation of the labelled (enriched in $^{108}$Cd) nutrient solution (+), 260 µL of the enriched stock solution was added to 9 L of nutrient solution (19.6 nM Cd) and well mixed. The final (measured) $^{108}$Cd isotopic abundance in the (+) nutrient solution was 6.51% (Table S1). In the labelled nutrient solutions, the Cd concentrations increased (21.3 nM) compared to those in the non-labelled (-)

nutrient solution (8% difference). Depending on the treatment, the compartments were filled with either $^{108}$Cd enriched (+) or natural Cd (-) nutrient solution. The isotope labelling experiment started at day 0 when the roots were immersed in the nutrient solution.

## Sample collection

After each harvest (days 0, 1, 7, 11, and 14 (Table 1)), the plant organs were gently separated into the two root parts, crown (which is the interface between the split roots and the stem), stem, leaves, and newly developing leaves if available (Figure S2). Special care was taken to ensure that there was no cross-contamination from one root compartment to the other. The roots were immersed in a $CaCl_2$ solution (2 mM) for 20 min to remove apoplastically bound Cd. All parts of the plant were rinsed in baths with deionized water and dried with paper towels to remove excess water. All plant tissues were oven-dried overnight (60 °C) and weighed for mass balance calculations. A subsample of each sample was ground into a powder with a mill, or with a mortar and pestle when the dry mass was not sufficient for the mill. Crowns were not included for Cd and $^{108}$Cd measurements.

## Chemical analyses

Leaves and stems (400 ± 20 mg) were acid digested with 8 mL $HNO_3$ (NORMATOM® 67–69% w/w, VWR International) in a microwave digestion system (MARS 6, CEM, Matthews, NC, USA). The dry mass of the roots and new leaves was not sufficient for microwave digestion that requires > 200 mg DW. Therefore, roots and new leaves (50 mg) were digested with 2 mL $HNO_3$ in an open block digestion with 10 mL glass tubes.

Cadmium isotopes ($^{108}$Cd, $^{111}$Cd) were measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Agilent 7700x, Agilent Technologies, collision cell ORS3), with He as the collision gas. $^{111}$Cd is commonly the preferred isotope for Cd detection with ICP-MS as it has almost no isobaric interferences (Reh kamper et al. 2012).

The mass bias of the isotopic ratio (IR) $^{108}$Cd/$^{111}$Cd was determined during two different ICP-MS measurement sessions. A Cd single element standard (10 µg Cd L⁻¹, Certipur Merck 119,777) was measured and the measured IR
(IR<sub>measured</sub>) was compared with the theoretical IR (IR<sub>theory</sub>) to obtain the mass bias correction factor f<sub>MB</sub> (Nolan et al. 2004).

$$f_{MB} = \frac{IR_{theory}}{IR_{measured}}$$

The mass bias factor was 1.11 in the first measurement session and 1.18 in the second session. For each session, the corresponding value was selected as the linear mass bias correction factor. The limits of quantification (LOQs) were determined as ten times the standard deviation of the measured signal in all procedural blanks (n=22) and were 15 counts per second (cps) for <sup>108</sup>Cd and 42 cps for <sup>111</sup>Cd (expressed as signal). The signal in the measured plant samples in our study was on average 868 cps for <sup>108</sup>Cd and 4019 cps for <sup>111</sup>Cd. The IA (isotopic abundance) of <sup>108</sup>Cd was determined on the mass-bias corrected isotopic ratio of <sup>108</sup>Cd/<sup>111</sup>Cd and the isotopic ratios of all other Cd isotopes relative to <sup>111</sup>Cd;

$$^{108}\text{Cd IA} = \frac{\left( \frac{^{109}\text{Cd}}{^{111}\text{Cd}} \right)_{\text{measured}}}{\left( \frac{^{109}\text{Cd}}{^{111}\text{Cd}} \right)_{\text{measured}} + \frac{^{110}\text{Cd}}{^{111}\text{Cd}} + 1 + \frac{^{112}\text{Cd}}{^{111}\text{Cd}} + \frac{^{113}\text{Cd}}{^{111}\text{Cd}} + \frac{^{114}\text{Cd}}{^{111}\text{Cd}} + \frac{^{116}\text{Cd}}{^{111}\text{Cd}}}$$

Only the <sup>108</sup>Cd and <sup>111</sup>Cd isotopes were measured, thus we used the isotope ratios of <sup>106</sup>Cd, <sup>110</sup>Cd, <sup>112</sup>Cd, <sup>113</sup>Cd, <sup>114</sup>Cd and <sup>116</sup>Cd relative to <sup>111</sup>Cd at natural abundance (Berglund and Wieser 2011). This decision was justified because the change in the isotopic ratios of these isotopes with respect to <sup>111</sup>Cd was negligible at the maximum IA of <sup>108</sup>Cd (6.51%) in this study (Sect. 1.1 in SI and Table S1). The analytical quality of the ICP-MS <sup>108</sup>Cd measurements was assessed by measuring a series of 1 µg Cd L<sup>-1</sup> solutions with increasing <sup>108</sup>Cd IA. These solutions were prepared by mixing a certified reference Cd solution with the <sup>108</sup>Cd stock solution used for labeling. The measured IA was compared with the theoretical IA and the data closely followed the 1:1 line, which indicated accurate IA measurements (see SI (Vanderschueren et al. 2023)). The average ± SD of the measured <sup>108</sup>Cd IA in the plant material grown without <sup>108</sup>Cd isotope in nutrient solution (n=28) was 0.93 ± 0.19% (theoretical natural abundance <sup>108</sup>Cd: 0.89%). The precision (SD) and accuracy (mean) of the natural abundance depended on the sample matrix (roots: 1.00 ± 0.20% (n=12), leaves: 0.91 ± 0.17 (n=8), stems: 0.84 ± 0.05% (n=8)).

Cadmium concentration measurements with ICP-MS typically use one isotope only (here: <sup>111</sup>Cd) and are calibrated with calibration standards at natural abundance. The <sup>111</sup>Cd was at non-natural abundance in the labelled (+) treatments. The Cd concentrations were not corrected for the change in isotopic abundance of <sup>111</sup>Cd in the samples at non-natural abundance. This decision was justified because the difference in isotope abundance of <sup>111</sup>Cd between natural abundance samples (<sup>111</sup>Cd IA=12.80%) and samples that were maximally enriched with <sup>108</sup>Cd (<sup>111</sup>Cd IA=12.15%) was negligible for our experiment (Table S1). Plant-certified reference materials were included in each ICP-MS measurement session. The % total Cd recoveries ranged from 86–95% (min–max) with on average 90% for NIST-1573a Tomato Leaves (certified concentration: 1.52 ± 0.07 mg Cd kg<sup>-1</sup>), n=8 and from 91–100% with on average 95% for BCR-679 White Cabbage (certified concentration: 1.66 ± 0.07 mg Cd kg<sup>-1</sup>), n=2.

## Calculations and data analysis

Mass balances combined with isotope abundances allowed us to calculate the fraction of the <sup>108</sup>Cd tracer in each plant organ at the different harvest times. The total Cd in each organ i (roots, crown, stem, leaf, new leaf) was calculated by multiplying the Cd concentration measured with the biomass of that organ. The sum of total Cd in each organ i represents the total Cd taken up by the plant. Crown Cd concentrations (not measured) were assumed to be similar to the stem Cd concentrations.

$$\text{Total Cd}_i(\text{ng}) = \text{Cd}_i(\text{mg kg}^{-1} \text{DW}) * \text{biomass}_i(\text{mg DW})$$

The total Cd in each organ i was multiplied with the <sup>108</sup>Cd IA corrected for the natural <sup>108</sup>Cd in that organ to obtain the mass of tracer derived Cd in that tissue.

$$^{108}\text{Cd, tracer}_i(\text{ng}) = \text{total Cd}_i(\text{ng})[^{108}\text{Cd}_i \text{IA} - ^{108}\text{Cd}_{i,nat} \text{IA}]$$

With <sup>108</sup>Cd<sub>i,nat</sub> IA the average natural abundance measured in organ i in the (-/-) treatment. The mass of the tracer <sup>108</sup>Cd in each organ i was summed to determine the total <sup>108</sup>Cd in the different plants (<sup>108</sup>Cd, tracer<sub>plant</sub> (ng)). Subsequently, the <sup>108</sup>Cd in each organ i was divided by the total tracer <sup>108</sup>Cd in the plant to obtain the fraction of tracer in each plant organ.
$$f_{\text{tracer}} = {}^{108}\text{Cd}, \text{tracer}_i(\text{ng})/ {}^{108}\text{Cd}, \text{tracer}_{\text{plant}}(\text{ng})$$

All statistical analyses were performed with the software JMP-Pro version 17.0. The mean and standard deviation (SD) reported throughout the study were calculated with biological replicates (n=4), unless otherwise specified. One-way analysis of variance (ANOVA) was conducted in the (+/-) and (+/+) treatments, to determine if there were differences in ¹⁰⁸Cd isotopic abundances among organs on the same day or differences. Differences were tested by Student's t-test or Tukey's HSD post-hoc test at *p* < 0.05 significance level. For the root of the non-labelled compartment, leaf, and stem, differences in ¹⁰⁸Cd isotopic abundances for the same organ compared to the first day (control) were tested with Dunnett's post-hoc test. All data were checked to comply with ANOVA assumptions (Table S2). The normal distribution of residuals was tested with the Shapiro-Wilk test and the equality of variances with the O'Brien test. If the equality of variances was not met, the Welch test was used instead of ANOVA.

## Results

### Plant growth

A one-way ANOVA revealed that total plant biomass was similar among the different treatments (-/-), (+/-), (+/+) at day fourteen (*p*=0.07). Thus, labelling of the nutrient solution with ¹⁰⁸Cd did not affect total

plant biomass (DW) (Figure S3). Hence, at each harvest day, we grouped the biomasses of all treatments (-/-), (-/+), and (+/+) to infer plant growth. The total plant biomass approximately doubled between day 0 (850±160 mg (mean±SD, n=4) and day 14 (1630±260 mg, n=12). At day 0, the largest biomass fraction was in the leaves (50±5% of DW, mean±SD of all treatments (n=4)), followed by the stem (37±4%), crown (10±2%), and roots (root mass fraction in one compartment was on average 1.5±1%). At the end of the experiment, the stem was the largest organ (38±1% (n=12)), followed by the leaves (33±2%), crown (16±1%), roots (13±1%), and newly developing leaves (2±1%). These new leaves started to develop on day 7 of the experiment.

### Cadmium concentrations in plant organs

Cadmium concentrations were measured separately in the root tissues from each compartment, leaves, stem and, if available, newly developing leaves (Table 2). Regardless of the treatment, there were no differences in Cd concentrations between the roots of the two compartments, except for day 7 in treatment (+/+), where the root Cd concentration was significantly higher in one root compartment (2.87±1.25 mg kg⁻¹) than in the other root compartment (1.26±0.11 mg kg⁻¹) (*p*=0.04) (data not shown). Therefore, the Cd concentration measurements in these two root compartments were grouped for simplicity (Table 2).

Before the start of the experiment, the seedlings had already grown for two weeks in a preculture

<table><caption>**Table 2** Cd concentrations (mg kg⁻¹ DW⁻¹) in different organs at each sampling day (t)</caption><caption>Cd concentrations (mg kg<sup>-1</sup>)</caption><thead><tr><th rowspan="2">Day</th><th rowspan="2">n</th><th colspan="2">Roots</th><th colspan="2">Leaf</th><th colspan="2">New leaf</th><th colspan="2">Stem</th></tr><tr><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th></tr></thead><tbody><tr><td>0</td><td>5</td><td><strong>1.52</strong></td><td>0.52</td><td>4</td><td><strong>0.55</strong></td><td>0.10</td><td>0</td><td>4</td><td><strong>1.07</strong></td><td>0.54</td></tr><tr><td>1</td><td>16</td><td><strong>1.78</strong></td><td>1.06</td><td>8</td><td><strong>0.50</strong></td><td>0.10</td><td>0</td><td>8</td><td><strong>0.90</strong></td><td>0.23</td></tr><tr><td>7</td><td>16</td><td><strong>1.66</strong></td><td>1.08</td><td>8</td><td><strong>0.85</strong></td><td>0.18</td><td>1</td><td><strong>1.34</strong></td><td>8</td><td><strong>1.61</strong></td><td>0.40</td></tr><tr><td>11</td><td>14</td><td><strong>0.88</strong></td><td>0.90</td><td>8</td><td><strong>1.00</strong></td><td>0.16</td><td>8</td><td><strong>1.51</strong></td><td>0.48</td><td><strong>1.46</strong></td><td>0.26</td></tr><tr><td>14</td><td>22</td><td><strong>0.99</strong></td><td>0.76</td><td>12</td><td><strong>0.90</strong></td><td>0.33</td><td>7</td><td><strong>0.89</strong></td><td>0.19</td><td><strong>1.02</strong></td><td>0.40</td></tr></tbody></table>

The Cd concentrations of both root compartments and of the different treatments ((-/-), (-/+), and (+/+)) were grouped together
nutrient solution that contained 19.6 nM Cd. Hence, at day 0, there was already an accumulation of Cd in the roots (1.52±0.52 mg kg⁻¹), leaves (0.55±0.10 mg kg⁻¹) and stem (1.07±0.54 mg kg⁻¹). At day 14, the Cd concentrations were similar in all organs (Table 2). At the end of the experiment, the total plant Cd uptake did not differ significantly among the three treatments (1.15±0.54 µg (-/-), 1.59±0.43 µg (+/-), and 1.90±0.07 µg Cd (+/+)) (F(2, 9)=3.51, *p*=0.08). We can thus conclude that the isotope enrichment in the (+) nutrient solution did not affect the uptake of Cd. This result is logical given the fact that the isotope labelling enriched total Cd from 19.6 to 21.3 nM, i.e. an enrichment in total Cd of only 8%. The fraction of total Cd in each organ remained similar over the course of the experiment (Fig. 1) and ranged from 5 to 11% in the roots, from 12 to 20% in the crown (under the assumption that the crown contains the same Cd concentration as the stem), from 26 to 35% in the leaves and from 39 to 46% in the stem. The newly developing leaves contained about 4% of all plant Cd.

Circulation of ¹⁰⁸Cd tracer in the split-root cacao plants

The (+/-) treatment revealed a clear increase in ¹⁰⁸Cd IA in the labelled root compartment (+) (4.70±0.93%) at day 1 compared to the natural abundance (0.89%) (Fig. 2, Table S3). In addition, the ¹⁰⁸Cd IA did not change in all other organs. Thus, the uptake of the ¹⁰⁸Cd tracer was already observed within 24 h and accumulated in this period primarily in the root of the labelled compartment (+) (¹⁰⁸Cd fraction, labelled roots, day 1=66%) (Fig. 3). At day 7, the ¹⁰⁸Cd IA increased in the stem (2.04±0.76%) compared to day 1 (Table S3). Most of the ¹⁰⁸Cd tracer was translocated to the shoot organs and accumulated in the stem (43% in (+/-)) (Fig. 3). In contrast, the ¹⁰⁸Cd IA in the roots of the non-labelled compartment was near natural abundance which showed that the tracer was not yet transported to the other root compartment. At day 11, the ¹⁰⁸Cd IA in the roots of the non-labelled compartment (1.73±0.23%) increased above the natural abundance at day 1 (0.98±0.19%). The

<figure><img src="image_3.png" /><figcaption>**Fig. 1** Mass fraction of total Cd in different organs (Cd<sub>organ</sub> (ng)/Cd<sub>plant</sub> (ng)*100%) as a function of time (day). Crown Cd concentrations were not measured but were treated as equal to the stem Cd concentrations. The partitioning of Cd in the organs remained similar over the course of the experiment. The error bars represent the standard error of the mean (n=4 for crown, leaves, and stem and n=8 for roots)</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2** The isotopic abundance ($^{108}$Cd IA) (%) for the (+/-) and (+/+) treatment as a function of time after labelling one root compartment with $^{108}$Cd. Error bars represent the standard error of the mean (n=4 or n=8 in case of the labelled roots). The results of statistical tests are shown on day 11 (indicated by the blue boxes) (Table S3). Letters a and b denote different statistical groups. In the (+/-) treatment, at day 11, the $^{108}$Cd IA in the non-labelled roots (-) differs significantly from $^{108}$Cd</figcaption></figure>

IA in the non-labelled roots (-) at day 1. This indicates that there is a transport of Cd via the phloem to the non-labelled root part. In the (+/+) treatment, the newly developing leaves have the same $^{108}$Cd IA as the leaves and stems and differ from the roots, which indicates that the developing leaves are rather fed from the leaves/stems via reallocation than via direct root uptake

<figure><img src="image_5.png" /><figcaption>**Fig. 3** The fractions of the $^{108}$Cd tracer (%) in each organ relative to the total tracer quantity of $^{108}$Cd in the plant. The error bars represent the standard error of the mean (n=4 or n=8</figcaption></figure>

in the case of the labelled roots (+) in the (+/+) treatment). The calculation of the fraction $^{108}$Cd tracer is explained in the *Materials and methods* section
newly developing leaves (2.50±0.41%) had an
¹⁰⁸Cd IA similar to that of the stem (2.61±0.24%),
and leaves (2.22±0.25%). In addition, the ¹⁰⁸Cd
IA in the labelled root decreased after day 7, likely
because of reallocation of natural Cd from e.g.,
the non-labelled root compartment and the shoot.
Finally, on day 14, there were no significant dif-
ferences in ¹⁰⁸Cd IA among stem, leaves, and new
leaves as well as in the labelled and non-labelled
roots (Table S3). The fraction of ¹⁰⁸Cd tracer indi-
cated that most of the ¹⁰⁸Cd tracer was accumulating
in the stem (around 46%), and the leaves (21%).

In the (+/+) treatment, the general trends in the
circulation of the ¹⁰⁸Cd tracer were similar to those
observed in the (+/-) treatment (Fig. 2, Table S3).
At day 1, the roots had already absorbed ¹⁰⁸Cd
(¹⁰⁸Cd IA in roots: 3.83 ± 0.83%) and accumulated
most of the tracer ¹⁰⁸Cd in the plant (Fig. 3). The
other organs had a ¹⁰⁸Cd IA around natural abun-
dance at day 1. At day 7, the ¹⁰⁸Cd IA in the roots
(6.06 ± 1.30%) was similar to the ¹⁰⁸Cd IA of the
nutrient solution (6.51%). The increase of ¹⁰⁸Cd
IA in the stem (3.73 ± 0.34%) was faster than that
in the leaves (1.93 ± 0.56%). Thus, most of the
translocated ¹⁰⁸Cd tracer was accumulating in the
stem (54%, Fig. 3). The new leaves sampled on
day 11 had an isotopic abundance intermediate
between the stem and leaves (4.08 ± 0.86%) and
differed significantly from the ¹⁰⁸Cd IA of the roots
(p = 0.05). At day 11, the isotopic abundances of
the leaves and stem in the (+/+) treatments dou-
bled compared to those in the (+/-) treatments.
At day 14, there were no significant differences in
¹⁰⁸Cd IA among the different organs. At the end of
the experiment, the distribution of the ¹⁰⁸Cd tracer
was very similar to the distribution of the total
Cd (Fig. 1). At day 14, the new leaves contained
on average 2.4% of the total ¹⁰⁸Cd in the plant, the
roots 10%, the crown 20%, the leaves 24%, and the
stem 43%.

Discussion

Efficient root-to-shoot translocation and accumulation
in the stem

The leaf Cd concentrations in the plant organs were in
a similar range as the average leaf Cd concentration

observed in a meta-analysis in cacao plantations in
Latin America (2.6 mg kg⁻¹ dry weight, n=762)
(Vanderschueren et al. 2021). The concentrations
in this study were also below the critical Cd toxic-
ity levels that were proposed for various crops in
mature leaves (6–19 mg Cd kg⁻¹ (Krämer 2010) and
5–30 mg Cd kg⁻¹ (Kabata-Pendias 2010). The Cd
dose of ca. 20 nM Cd in hydroponic solution can thus
be considered as environmentally relevant.

During the experiment, the Cd mass fraction dis-
tribution (%) remained constant. Most Cd accumu-
lated in the shoots and only a minor fraction remained
in the roots (Fig. 1). The fraction of the freshly added
¹⁰⁸Cd tracer was logically larger in the labelled roots
at the beginning of the experiment than the fraction
in the shoot organs (Fig. 3). At the end of the experi-
ment, most ¹⁰⁸Cd tracer had been translocated to the
shoots. Cadmium was thus only slightly retained in
the roots. An experiment in a similar time frame with
wheat and white lupin showed that Cd was only slightly
translocated from the roots to the shoots, and primar-
ily retained in the roots (Page and Feller 2005; Riesen
and Feller 2005). That translocation behavior denotes
an excluder strategy (Sterckeman and Thomine 2020),
while we observed in this study that cacao follows more
of a hyperaccumulator strategy in terms of the high
root-to-shoot translocation (van der Ent et al. 2013).

Cadmium is known to use nutrient pathways. Once
Cd is transferred in the xylem sap of root plants, it
migrates towards the shoots thanks to the transpira-
tion flow, and it can be deposited along this pathway
in xylem cell wall and parenchyma cells (Stercke-
man and Thomine 2020). This deposition is more
pronounced in cereals like rice, where Cd has been
shown to accumulate at the shoot base and in nodes
(Fujimaki et al. 2010), than in Cd hyperaccumulators,
where Cd is efficiently transferred to shoots via the
xylem (Ueno et al. 2008; Hu et al. 2013). In rice, it
has been shown that Cd reaches the sink organs after
xylem-to-phloem transfer in the nodes (Fujimaki
et al. 2010). In the present study, most of the ¹⁰⁸Cd
tracer accumulated in the stem and crown (Fig. 3).
This result suggests that Cd could be sequestered in
the stem during its translocation to the transpiring
leaves. Alternatively, this result might suggest that
part of Cd was reallocated from the leaves to the stem.
The sequestration of Cd in the stem on the one hand,
could be driven by interactions of Cd with cell walls
in the xylem and/or selective removal from the xylem
to the phloem by an exchange in the rays/nodes (Fuji-
maki et al. 2010; Page and Feller 2015). The reallo-
cation of Cd from leaves to stem on the other hand,
would require transport by the phloem. The efficient
root-to-shoot translocation, and possible sequestra-
tion in stems observed in this study correspond well
with observations in mature cacao trees in the field
where branches were a main organ for Cd accumula-
tion (Engbersen et al. 2019; Blommaert et al. 2022).

Cadmium transport via the phloem to roots and newly
developing leaves

The isotopic labelling split-root setup was used to
precisely identify short-term Cd circulation fluxes
in the (+/-) treatment. In the non-labelled roots,
the ¹⁰⁸Cd IA increased after day 11 and the ¹⁰⁸Cd
tracer fractions in the non-labelled roots increased
to 6% at the end of the experiment (Fig. 2). Along
the same lines, the ¹⁰⁸Cd IA in the labelled roots
decreased between day 7- 14 after an initial rise

at day 1, suggesting transport of natural Cd in the
opposite direction. This transport can only occur
via the phloem, either by transport through the
phloem between root segments or by a downward
flux of Cd from the shoot to the roots (Welch
et al. 1999). Consequently, this result evidenced
the mobility of Cd in the phloem of cacao seed-
lings. Moreover, the earlier rise in ¹⁰⁸Cd IA in the
shoot compared to that in the non-labelled roots
suggests that Cd was first transported to the shoot
and then reallocated to the roots (Figs. 2 and 4).
The reallocation of Cd from the shoots to the roots
was small (6%) compared to the root-to-shoot
translocation (> 75%) and was of a similar extent
as observed in wheat and white lupin (Page and
Feller 2005; Riesen and Feller 2005; Page et al.
2006).

Because the mature leaves did not gain much
biomass over the course of the experiment (Fig-
ure S3), we consider them to be a net phloem
source. This means that they are fed mainly by the

**Fig. 4** A model split-root
hydroponic system with
cacao seedlings. In treat-
ment (+/-), the nutrient
solution in the + compart-
ment was labelled with sta-
ble isotope ¹⁰⁸Cd as a tracer.
The non-labelled compart-
ment (-) had an identical
solution, with a similar Cd
concentration but no ¹⁰⁸Cd
tracer. The results of our
study indicated that the
¹⁰⁸Cd absorbed in the roots
is readily transported via
xylem (blue) to the aerial
organs, where it accumu-
lates primarily in the stems.
A minor contribution of
¹⁰⁸Cd was reallocated to
the roots via the phloem
(red). Cadmium in the new
developing leaves origi-
nated from reallocation via
the phloem from the stems,
crowns and/or leaves rather
than from direct root uptake
via the xylem

<figure><img src="image_6.png" /></figure>
xylem sap. The fact that the ¹⁰⁸Cd tracer was not detected in the mature leaves after one day of treatment, suggests that Cd translocation is slower than the transpiration flow in the xylem. This phenomenon has previously been observed in rice (Fujimaki et al. 2010), and is in line with the pronounced Cd accumulation in the stem as discussed above. On the contrary, in a study of the hyperaccumulator S. nigrum, the root-to-shoot translocation was rapid and large fractions of Cd accumulated in leaves and stems (Wei et al. 2014). Additionally, there was only a poor reallocation from leaves and stems to other organs via the phloem. Thus, in the present study, Cd shows a slower xylem mobility in cacao than the Cd hyperaccumulator S. nigrum.

The newly developing leaves had a ¹⁰⁸Cd IA that was similar compared to the stem and leaves, and significantly different from the roots (Fig. 2, Table S3 (+/+) treatment, day 11). These results indicate that the majority of Cd in the new leaves was reallocated from leaves/stems and/or crowns, as opposed to directly transported from roots. This observation is consistent with the classical representation of newly developing leaves as a phloem sink due to their need for nutrients for growth (Page and Feller 2015).

In conclusion, our experiment demonstrated an efficient transfer of Cd from the root to the stem, probably through the xylem, and a reduced xylem mobility in the shoots, probably related to a progressive deposition of Cd in the stem. We moreover evidenced the phloem mobility of Cd towards the roots and young leaves. Thus, in terms of Cd accumulation strategy, we suggest that the circulation of Cd in cacao lies between that of the hyperaccumulator S. Nigrum and agricultural crops such as wheat and white lupin. This is in line with the interpretations from a meta-study with mature cacao trees, that compared the uptake potential and partitioning of cacao with other plants, and that suggested to term cacao a moderate Cd accumulator (Vanderschueren et al. 2021).

## Conclusions and perspectives

In conclusion, this study evidenced that Cd was mobile in the phloem of cacao seedlings that were grown in environmentally relevant Cd conditions. Through the phloem, Cd was translocated from shoot tissues to the roots and newly developing leaves. The dominant transport pathway of Cd

was nonetheless the transpiration pathway (xylem) from roots to leaves, with a large Cd accumulation in the stems (Fig. 4). Limitations of this study are that short-term labelling results strongly depend on the developmental stage of the plant at the time of labelling, and that the seedlings do not reach a reproductive stage for the development of beans. Yet, this simplified system helped to corroborate the Cd uptake, distribution and redistribution patterns that have previously been observed in mature trees. We confirmed that there is a high Cd translocation to the above-ground tissues and that the stems (or branches in mature trees) are an important organ for the accumulation of Cd. In addition to the more direct transport pathway of Cd from roots and stems to the beans (a sink organ) as proposed by previous studies (Engbersen et al. 2019; Blommaert et al. 2022), this study shows that there is also Cd reallocation via the phloem from the stems, crowns and/or mature leaves to sink organs such as new leaves and roots.

**Acknowledgements** We want to thank the staff at the laboratory of the Division of Soil and Water, and in particular Maarten Everaert, Dries Grauwels, Kristin Coorevits, Simon De Waelheyns, and Ruth Vanderschueren. Finally, we want to express gratitude to the two reviewers for their time and effort to review and improve the manuscript.

**Author contributions** HB, GS, MW, and, ES authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by HB, CDM and ES. The first draft of the manuscript was written by HB and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

**Funding** Open access funding provided by Swiss Federal Institute of Technology Zurich This work was financially supported by the French National Research Agency program 'Investissements d'avenir' (ANR-15- IDEX-02), and in the CNRS/INSU/EC2CO project CACAO. HB and GS (ISTerre) are part of Labex OSUG (ANR10 LABX56). GS, HB and ES are working in the framework of the Program Hubert Curien "TOURNESOL" 2020-2021 (project n° 44274TC). HB, MW, GS, and ES are members of the COST Action CA19116 PLANTMETALS (COST, European Cooperation in Science and Technology, www.cost.eu).

**Data availability** The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

## Declarations

**Competing interests** The authors have no relevant financial or non-financial interests to disclose.
**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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