LEAVES ARE CADMIUM SINKS IN COMPETITION WITH DEVELOPING GRAINS

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Cadmium in soils

Cadmium in soils is generally the main cause and the major source of Cd contamination for crops. The median level of total Cd in the topsoil is 0.15 mg kg-1 in Europe (van der Voet et al., 2013) and 0.2 mg kg-1 in France (Figure 1-2; source Gis Sol-RMQS, data 2011). In France, in agricultural soils, the level of total Cd is a bit greater than the average for all top soils, namely 0.3 mg kg-1 for the median, but could be above 2 mg kg-1 (0.7% of frequency) in some hotspots (Mench and Baize, 2004). The content of soil in Cd increases in the following order: sandy soil < loam soil < clay soil, due to their different physico-chemical characteristics (McLaughlin and Singh, 1999). For instance, the median concentration of total Cd is 0.16 mg kg-1 in sandy soils while it is 0.44 mg kg-1 in clay soils and 0.56 mg kg-1 in heavy clay soils of France (Baize et al., 2007). Moreover, the soil contents in Cd strongly depends on pedogenesis and tends to be higher in sedimentary rocks than in igneous rocks, but the latter one can importantly release Cd (Nagajyoti et al., 2010). Plant roots absorb Cd from the soluble fraction in the soil pore water. The soluble concentration of Cd was found to be a better indicator of Cd in mature durum wheat grains than the total soil Cd content (Viala et al., 2017). In agricultural soils, the concentration of Cd in the pore water is generally in the nanomolar range (< 20 nM). The Cd in the soil pore water depends on the content of Cd that is exchangeable from the soil surfaces, which is depending on the soil physico-chemical properties, including soil pH, clay and organic matter contents, content of manganese oxides, redox potential, and type and content of organic and inorganic complexing ligands (McLaughlin and Singh, 1999; Nolan et al., 2003).
Soil pollution is much more serious in newly industrialized countries, due to a rapid economic growth in combination with a long-period lack of awareness and of technics regarding environmental protection resulting in an inappropriate and poorly implemented environmental policy. For example, in China, the Ministry of Environmental Protection (MEP) reported that 19.4% of the farmlands have been polluted (according to the national standard in soil quality, GB 15618-1995), and 82.8% of them are polluted by heavy metals, Cd being the dominant metal (source MEP, data 2014).

Cadmium in foodstuffs

Food is the principal source (approx. 90%) of Cd exposure for non-smoking population (UNEP, 2010). In 1988, the Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives (JECFA) established a provisional maximum tolerable weekly intake for Cd of 7 μg kg-1 body weight (BW) (FAO/WHO, 1988). This tolerable limit was revised downwards to 5.8 μg kg-1 BW in 2010 (FAO/WHO, 2010). In Europe, in 2011, the European Food Safety Authority (EFSA) even recommended a lower weekly intake of 2.5 μg kg-1 BW in view of the up-to-date knowledge about chronic Cd toxicity (EFSA, 2011).
In average, the Cd exposure of the European population is close to the tolerable level, especially in some subgroups such as children and vegetarians, for which the levels of exposure are more frequently exceeded (Clemens et al., 2013). In the French population, the tolerable weekly intake value set by the EFSA is exceeded by 0.6% of the adults and by 14.9% of the children (Arnich et al., 2012). Meanwhile, the risk of Cd exposure increases with the deficiency of micronutrients, which is prevalent in developing regions, especially regarding iron (Fe) for young women. Fe deficiency in human, for example, may cause the upregulation of the divalent metal transporter 1 (DMT1) which also non-specifically mediates Cd2+ absorption and thereby can increase the accumulation of Cd (Vahter et al., 2007). It is clearly a necessity of reducing dietary Cd exposure to improve the health of general population.
Cereals are staple foodstuffs. In Europe, about a quarter of the dietary exposure to Cd is due to cereals (Figure 1-3) (EFSA, 2012). In some Asian regions, such as the southern China and Japan, cereals could even account for more than 40% of Cd dietary intake (Gao et al., 2006; UNEP, 2010). The European Union has set the threshold for the maximum levels of Cd in cereals: 0.2 mg kg-1 fresh weight (FW, i.e. the weight at harvest or to be consumed) for bran, germ, wheat and rice, and 0.1 mg kg-1 FW for other cereals (EC, 2008). However, following the EFSA recommendation of lowering the weekly intake of Cd from food, strong debates hold as to lower these threshold levels in foodstuffs, especially in cereals. A project was established by the Directorate-General for Health and Food Safety (DG SANCO) of the European Commission to lower the regulatory limit for Cd in durum wheat from 0.2 to 0.15 mg kg-1 (DGSanco, 2011). Reducing the accumulation of Cd in the edible part of cereal crops (i.e. grains) is a major issue and perhaps the most efficient way to protect the general population from Cd poisoning (Clemens et al., 2013).

Design of the experiment

To investigate the intraspecific variability of grain Cd accumulation in durum wheat ( Tritium turgidum L. subsp. durum (Desf.) Husn.), eight French widely cultivated spring cultivars were grown in the presence of Cd, with five replicates per cultivar. The cultivars differed in their stem height (from very short to short) and in their grain size (from medium to very big), but also in precocity a nd in their grain protein content ( Table 2 1 ). None possess the allele responsible for the low Cd trait at Cdu1 Zimmerl et al. al., 2014 )). Two Canadian lines Kyle and Strongfield ) with long stems Table 2 1 ) were included in the experimental design. One has the low Cd accumulating allele at Cdu1 (i.e. Strongfield ) and was used as a reference for the low Cd accumulation trait. The plants were sam pled at the early ripening stage (Zadoks Stage 87 89), which corresponds to 68 to 82 days of Cd exposure after transplanting. To ensure a constant level of exposure to Cd and to allow sampling of the entire root system, the plants were grown in hydroponics . The concentration of Cd 2+ in the nutrient solution was fixed at 2 nM to reproduce the level of exposure to Cd found in low to moderately contaminated agricultural soils Sauvé et al. al., 2000.

Plant sampling and analyses

First, the root systems were immersed for 10 min at 4 °C in 5 L of 5 mM CaCl 2 to desorb root apoplastic Cd Buckley et al. al., 2010 )). Second, the plants were separated into roots, stems, leaves and heads. The leaves and stems were washed in two successive baths of deionized water and the heads were separated into grains and bracts+rachis (B+R). The grains were weighed fresh to determin e kernel residual water (KRW) at harvest. All the plant organs were dried at 50 °C, weighed and milled (Retsch PM 400) before wet digestion, according to the procedure described in Laporte et al. 2015 )). The concentration of Cd in the plant organs was determined by HR ICP MS (Element XR, Therm o Scientific) at the GET laboratory in Toulouse (http://www.get.obs mip.fr/). The detection limit of the Element XR in the plant digests was around 0.5 ppt for Cd. A grain ionomic profile was established based on the concentrations of K, P, Ca, Mg, Na dete rmined by ICP OES (ACTIVA, Horiba Jobin Yvon) and Mn, Fe, Co, Ni, Cu, Zn, Mo, Cr determined by ICP MS (7700x, Agilent Technologies), by the central analytical service of the University of Basque Country http://www.ehu.eu s ). The concentrations of N in the grain (multiplied by 5.7 to obtain the protein level) and C were assayed with an elemental analyzer (Flash EA1112, ThermoFisher), according to the Dumas method. The yield components TKW (thousand kernel weight) and KPH (k ernels per head) were calculated for each plant from the weight and the number of grains collected on the heads of the four tillers.

Determination of the root cation exchange capacity

The cation exchange capacity (CEC) of the roots was determined in all durum wheat cultivars according to a procedure adapted from Guigues et al. 2014 )). Briefly, 50 mg of dried roots were shaken for 30 min on a roller in 15 mL of 10 mM CuSO 4 . The suspension was centrifuged for 5 min at 10 000 g and the pellet was rinsed three times with 50 mL of 0.1 mM CuSO min at 10 000 g and the pellet was rinsed three times with 50 mL of 0.1 mM CuSO44. The roots . The roots were then shaken for 20 min on a roller in 40 mL of 0.1 M HNOwere then shaken for 20 min on a roller in 40 mL of 0.1 M HNO33. The suspension was finally . The suspension was finally centrifuged for 5 min at 10centrifuged for 5 min at 10 000 g 000 g before flamebefore flame–AAS determination of the Cu concentration in the AAS determination of the Cu concentration in the filtrate.filtrate.

Plant growth, biomass partitioning and grain yield

No visual symptom of Cd toxicity and/or mineral deficiency was observed, whatever the cultivar considered. The cumulative thermal time recorded at head emergence ranged from 544 to 793 GDD depending on the cultivar ( Suppl. Table S 2 2 ). The kernel residual water (KRW) in all the cultivars at harvest was close to 40%, showing that the grains had reached their physiological maturity. The French lines differed significantly in growth, biomass partitioning and gra in yield Table 2 2 ). Plant dry weight (DW) ranged from 12.1 g in Sculptur up to 29.8 g in Miradou x. The aboveground–root biomass ratio (AR) ranged from 3.0 in root biomass ratio (AR) ranged from 3.0 in DakteDakter to 7.3 in r to 7.3 in SculpturSculptur. Aboveground, . Aboveground, grains were the main biomass compartment in all cultivars. However, betweengrains were the main biomass compartment in all cultivars. However, between–organ partitioning organ partitioning of aboveground dry matter (DM) varied from one cultivar to anotherof aboveground dry matter (DM) varied from one cultivar to another. . NeferNefer, , PharaonPharaon and and SculpturSculptur lines allocated the highest proportion of aboveground DM to the grain (about 60%) while lines allocated the highest proportion of aboveground DM to the grain (about 60%) while DakterDakter and and MiradouxMiradoux allocated the highest proportion of aboveground DM to the leaves + stems (about allocated the highest proportion of aboveground DM to the leaves + stems (about 40%). The proportion of aboveground DM40%). The proportion of aboveground DM allocated to the bracts+rachis was very similar in all allocated to the bracts+rachis was very similar in all cultivars and averaged 12%. The grain yield ranged from 1.2 g per head in cultivars and averaged 12%. The grain yield ranged from 1.2 g per head in DakterDakter up to 2.8 g per up to 2.8 g per head in head in MiradouxMiradoux, as a result of a between, as a result of a between–cultivar variation in both the number of kernels per head cultivar variation in both the number of kernels per head (K(KPH) and the thousand kernel weight (TKW). Indeed, KPH ranged from 27 to 47 and TKW PH) and the thousand kernel weight (TKW). Indeed, KPH ranged from 27 to 47 and TKW from 44 to 71 g (from 44 to 71 g (Suppl. Table Suppl. Table SS22–22). As expected from their reported grain size (). As expected from their reported grain size (Table Table 22–11), TKW ), TKW was lowest in was lowest in SculpturSculptur and highest in and highest in MiradouxMiradoux..

Table of contents :

CHAPTER 1 GENERAL INTRODUCTION
1.1 CADMIUM AND HUMAN
1.1.1 Cadmium poisoning
1.1.2 Anthropogenic cadmium contamination
1.1.3 Cadmium in soils
1.1.4 Cadmium in foodstuffs
1.2 DURUM WHEAT
1.2.1 The plant of durum wheat
1.2.2 Durum wheat production and consumption
1.2.3 Cadmium in durum wheat
1.3 PHYSIOLOGY OF CADMIUM ALLOCATION TO GRAINS
1.3.1 Root uptake and short-distance transport to the stele
1.3.2 Long-distance transport and partitioning
1.3.3 Remobilization from vegetative organs to developing grains
1.3.4 Loading and storage in grains
1.4 WAYS TO MINIMIZE THE LEVEL OF CADMIUM IN DURUM WHEAT-BASED FOODS
1.4.1 Agriculture practices
1.4.2 Post-harvest practices
1.5 STRUCTURE AND AIMS OF THE THESIS
CHAPTER 2 VARIABILITY IN GRAIN CADMIUM CONCENTRATION AMONG DURUM WHEAT CULTIVARS: IMPACT OF ABOVEGROUND BIOMASS PARTITIONING
ABSTRACT
2.1 INTRODUCTION
2.2 MATERIALS AND METHODS
2.2.1 Design of the experiment
2.2.2 Plant culture
2.2.3 Plant sampling and analyses
2.2.4 Determination of the root cation exchange capacity
2.2.5 Modelling between-cultivar variability in grain cadmium
2.2.6 Data processing
2.3 RESULTS
2.3.1 Plant growth, biomass partitioning and grain yield
2.3.2 Grain cadmium
2.3.3 Plant cadmium
2.3.4 Predictive model for grain cadmium concentration
2.3.5 Correlations between grain cadmium and other grain characteristics
2.4 DISCUSSION
2.4.1 Variability in grain cadmium within French lines was not explained by uptake and root sequestration of cadmium
2.4.2 Impact of the grain yield
2.4.3 Impact of the aboveground partitioning of biomass
2.4.4 Co-accumulation of cadmium with manganese, phosphorus and zinc in the grain
2.5 CONCLUSIONS
2.6 ACKNOWLEDGMENTS
2.7 SUPPLEMENTARY MATERIAL
CHAPTER 3 CONTRIBUTION OF REMOBILIZATION TO THE LOADING OF CADMIUM IN DURUM WHEAT GRAINS: IMPACT OF POST-ANTHESIS NITROGEN SUPPLY
ABSTRACT
3.1 INTRODUCTION
3.2 MATERIALS AND METHODS
3.2.1 Experimental design
3.2.2 Plant culture
3.2.3 Cadmium isotope labeling
3.2.4 Plant sampling and analysis
3.2.5 Data analysis
3.2.6 Statistical analysis
3.3 RESULTS
3.3.1 Plant growth
3.3.2 Grain characteristics
3.3.3 Concentrations of nitrogen and cadmium in vegetative organs
3.3.4 Post-anthesis uptake and apparent remobilization of nitrogen
3.3.5 Post-anthesis uptake and remobilization of cadmium
3.4 DISCUSSION
3.4.1 Contribution of cadmium remobilization under standard nitrogen supply
3.4.2 Source of cadmium remobilization
3.4.3 Cadmium remobilization as a senescent-independent process?
3.4.4 Impact of post-anthesis nitrogen-deprivation
3.4.5 Between-cultivar differences
3.5 CONCLUSION
3.6 ACKNOWLEDGMENTS
3.7 SUPPLEMENTARY MATERIAL
CHAPTER 4 ALLOCATION OF CADMIUM TO GRAINS OF DURUM WHEAT EXPOSED TO 5 OR 100 NM CADMIUM IN HYDROPONICS
ABSTRACT
4.1 INTRODUCTION
4.2 METHODS AND MATERIALS
4.2.1 Experimental design
4.2.2 Plant culture
4.2.3 Cadmium isotope labeling
4.2.4 Plant sampling and analysis
4.2.5 Cadmium accumulation in the first node and in the peduncle
4.2.6 Data processing
4.2.7 Statistical analysis
4.3 RESULTS
4.3.1 Plant growth
4.3.2 Grain characteristics
4.3.3 Cadmium accumulation level in plant tissues
4.3.4 Pre-anthesis uptake and partitioning of cadmium
4.3.5 Post-anthesis uptake and partitioning of cadmium
4.3.6 Cadmium remobilization
4.3.7 Relationships between the grain maturation rate and the dynamics of cadmium post-anthesis
4.4 DISCUSSION
4.4.1 Effect of cadmium exposure on plant growth
4.4.2 Buffer ability of durum wheat in the change of exposure level to cadmium
4.4.3 Effect of earlier maturing of grains
4.5 CONCLUSION
4.6 ACKNOWLEDGMENTS
4.7 SUPPLEMENTARY MATERIAL
CHAPTER 5 IMAGING OF CADMIUM DISTRIBUTION IN MATURE DURUM WHEAT GRAINS USING LASER ABLATION-ICP-MS
ABSTRACT
5.1 INTRODUCTION
5.2 MATERIALS AND METHODS
5.2.1 Grain production
5.2.2 Cryomicrotomy
5.2.3 LA-ICP-MS imaging
5.2.4 Grain dissection and analysis
5.2.5 Budget of element partitioning between grain parts from dissection results
5.2.6 Statistical analysis
5.3 RESULTS
5.3.1 LA-ICP-MS imaging of elements in durum wheat grains
5.3.2 Concentrations of elements in dissected grain parts
5.3.3 Loss of biomass and elements by removing grain parts
5.4 DISCUSSION
5.4.1 Localization of cadmium and essential elements in durum wheat grain assessed by LA-ICP-MS and grain dissection
5.4.2 Localization of cadmium and essential elements in durum wheat grains with respect to the mechanisms of grain filling
5.4.3 Interest of post-harvest treatment for lowering the level of cadmium in durum wheat-derived products
5.5 CONCLUSIONS
5.6 ACKNOWLEDGMENTS
5.7 SUPPLEMENTARY MATERIAL
CHAPTER 6 GENERAL DISCUSSION
6.1 LEAVES ARE CADMIUM SINKS IN COMPETITION WITH DEVELOPING GRAINS
6.2 HALF OF GRAIN CADMIUM ORIGINATES FROM CADMIUM TAKEN UP DURING GRAIN FILLING WHILE THE OTHER HALF FROM CADMIUM REMOBILIZED
6.3 DURUM WHEAT IS SENSITIVE TO CHANGES IN THE LEVEL OF CADMIUM EXPOSURE IN THE RANGE OF THOSE ENCOUNTERED IN AGRICULTURAL CONTEXT
6.4 TOWARDS THE EFFICIENCY AND THE SELECTIVITY OF GRAIN MILLING PROCESS TO LOWER THE CADMIUM CONCENTRATION IN DURUM WHEAT-BASED PRODUCTS
6.5 POSSIBLE LIMITATIONS OF THE THESIS
CHAPTER 7 GENERAL CONCLUSION AND PERSPECTIVES
7.1 CONCLUSIONS
7.2 SUGGESTIONS
7.3 WHAT WOULD NEED TO BE DONE FURTHER?
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