An investigation of zinc isotope fractionation in cacao (Theobroma cacao L.) and comparison of zinc and cadmium isotope compositions in hydroponic plant systems under high cadmium stress

This study aims to establish whether zinc (Zn) and cadmium (Cd) share similar physiological mechanisms for uptake and translocation in cacao plants (Theobroma cacao L.). Multiple-collector ICP-MS was used to determine the Zn stable isotope compositions in the roots, stems and leaves of 19 diverse cacao genotypes grown in hydroponics with 20 µmol L−1 CdCl2. Additional plants of one genotype were grown in hydroponic solutions containing lower Cd concentrations (0 and 5 µmol L−1 added CdCl2). Regardless of the Cd concentration used in the exposures, the Zn stable isotope compositions show the same systematic patterns in plant organs, with δ66Znroot > δ66Znstem > δ66Znleaf (δ66Zn denotes relative differences in 66Zn/64Zn ratios in parts per thousand). The mean Zn stable isotope fractionation between the plants and the hydroponic solutions was ε66Znuptake = –1.15 ± 0.36‰ (2SD), indicating preferential uptake of isotopically light Zn by plants from the hydroponic solution. The mean stable isotope fractionation factor associated with translocation of Zn from roots to shoots, ε66Znseq-mob =  + 0.52 ± 0.36‰ (2SD), shows that isotopically heavy Zn is preferentially sequestered in the cacao roots, whilst isotopically light Zn is mobilised to the leaves. A comparison with the Cd stable isotope compositions of the same plants shows that both isotopically light Zn and Cd are preferentially taken up by cacao plants. In contrast to Zn, however, the cacao roots retain isotopically light Cd and transfer isotopically heavy Cd to the leaves.


Table of Contents
. Cacao clone names, accession numbers and donor organizations for the cacao germplasms used in this study.
6 Table S5. Dry weights (g) of the different plant organs treated with 20 µmol L −1 CdCl2. 7 Table S6. Summary of Zn concentration data (mg kg -1 ) for cacao leaves from this study and the literature.

NIST SRM 1570a
Spinach leaf 19 79 ± 6 82 ± 4 b +0.41 ±0.06 -All stable isotope compositions (δ 66 ) are reported relative to JMC-Lyon Zn. n represents the number of individual samples that were analysed. a The δ 66 Zn ± 2SD (‰) is the mean value reported in Archer et al. (2017). b The Zn concentration data outlined in the original certificate. SD represents standard deviation. 2SD for individual samples represents the analytical precision of the Zn stable isotope data determined from multiple analyses of the Zn isotope standard (AA-ETH-Zn) that bracketed the sample runs.
14 -n represents the number of individual samples that were digested and analysed. SD represents standard deviation; 1SD is used for dry weight, f and Zn concentrations ([Zn]) and 2SD for Zn stable isotope compositions (δ 66 Zn). 2SD for individual samples represents the analytical precision of the Zn stable isotope data determined from multiple analyses of the Zn isotope standard that bracketed the sample runs. All Zn stable isotope compositions (δ 66 Zn) are reported relative to JMC-Lyon Zn. a non-italicized values represent isotopic differences, whereas italicized values represent the isotope fractionation factor (ε 66 Zn) for uptake. b mean values. c mean values for rice grown in anaerobic and aerobic soils, respectively. d mean values for wheat grown in soil from two different sites. e range of mean values for flag and senescent leaves for wheat. f mean values for Agrostis capillaris L. grown in soil from two different sites. g represents total Zn concentrations in soils. h represents total Zn concentration in soil leachates or extracts.
Note S1. Depletion of Zn from the hydroponic solutions.
Plants were grown in 3 L hydroponic systems containing 4 plants of the same genotype (see main text for further experimental details). The total Zn mass taken up by one plant from each system was calculated based on the Zn concentrations in each organ and their dry mass. For two of the 19 genotypes (CC 41 and GU 207/H), the total Zn mass was calculated for three plants and an average was taken.
Given the good agreement between the total Zn masses for the replicates of CC 41 and GU 207/H, all plants in each system were assumed to take up the same mass of Zn, and therefore the total Zn mass of one plant was multiplied by four. This total mass of Zn that was removed from the hydroponic solution was then converted to a percentage of the total Zn mass available in the hydroponic systems (equation S1): Hydroponic Zn depletion (%) = " !"#$% '( ") * +%$(#, !"#$% '( -( ./01"+"(-2 ,34,#1$#5 # x 100 (S1) The percentages (21±8%, 1SD) were then used to calculate the initial and final Zn stable isotope compositions of the hydroponic solutions, and ultimately, the Zn stable isotope fractionation factor ε 66 Zn during root uptake (see main text for details).