Cadmium isotope fractionation reveals genetic variation in Cd uptake and translocation by Theobroma cacao and role of natural resistance-associated macrophage protein 5 and heavy metal ATPase-family transporters

Abstract

In response to new European Union regulations, studies are underway to mitigate accumulation of toxic cadmium (Cd) in cacao (Theobroma cacao, Tc). This study advances such research with Cd isotope analyses of 19 genetically diverse cacao clones and yeast transformed to express cacao natural resistance-associated macrophage protein (NRAMP5) and heavy metal ATPases (HMAs). The plants were enriched in light Cd isotopes relative to the hydroponic solution with Δ 114/110 Cd tot-sol = −0.22 ± 0.08‰. Leaves show a systematic enrichment of isotopically heavy Cd relative to total plants, in accord with closed-system isotope fractionation of Δ 114/110 Cd seq-mob = −0.13‰, by sequestering isotopically light Cd in roots/stems and mobilisation of remaining Cd to leaves. The findings demonstrate that (i) transfer of Cd between roots and leaves is primarily unidirectional; (ii) different clones utilise similar pathways for Cd sequestration, which differ from those of other studied plants; (iii) clones differ in their efficiency of Cd sequestration. Transgenic yeast that expresses TcNRAMP5 (T. cacao natural resistance-associated macrophage gene) had isotopically lighter Cd than did cacao. This suggests that NRAMP5 transporters constitute an important pathway for uptake of Cd by cacao. Cd isotope signatures of transgenic yeast expressing HMA-family proteins suggest that they may contribute to Cd sequestration. The data are the first to record isotope fractionation induced by transporter proteins in vivo.

Changes of Cadmium Storage Forms and Isotope Ratios in Rice During Grain Filling

“…Cd was strongly retained in the roots that where enriched in light isotopes compared to the shoots ( Figure 2D ). The same pattern was also observed in wheat, barley, and cacao seedlings ( Wiggenhauser et al, 2016 ; Imseng et al, 2019 ; Moore et al, 2020 ). For hydroponically grown rice seedlings that were exposed for 3 days to 0.1 μM Cd, the isotope ratios between root and leaves were isotopically not distinguishable in wild-type plants, but a slight enrichment of heavy isotopes was observed in leaves when the root vacuolar transporter OsHMA3 was overexpressed ( Zhang et al, 2021 ).…”

“…Binding of Cd to organic ligands may impact the transfer of metals through membranes ( Zhao et al, 2016 ). In a next step, Cd storage forms could be coupled with membrane transporters by comparing wild-type and mutant plants that lack or overexpress specific membrane transporters ( Cadiou et al, 2017 ; Pokharel et al, 2018 ; Lei et al, 2020 ; Moore et al, 2020 ; Wiggenhauser et al, 2021 ; Zhang et al, 2021 ).…”

“…This isotope fractionation then leads to small but distinguishable isotope ratios in different plant tissues. For nonredox-sensitive elements such as Cd and Zn, membrane transport ( John et al, 2007 ; Köbberich and Vance, 2017 ; Moore et al, 2020 ) and binding of the metals to organic and inorganic ligands are key processes for isotope fractionation ( Jouvin et al, 2009 ; Fujii et al, 2014 ; Yang et al, 2015 ; Marković et al, 2017 ; Guinoiseau et al, 2018 ). Experimentally and theoretical determined isotope fractionation factors revealed that Cd bound to S is lighter than hydrated Cd and hydrated Cd is lighter than Cd bound to O and N groups of inorganic and organic ligands ( Yang et al, 2015 ; Guinoiseau et al, 2018 ; Zhao et al, 2021 ).…”

“…In addition, processes that control the mobility of metals in living organisms can be also partly kinetically controlled through, e.g., fast and unidirectional membrane transport ( Köbberich and Vance, 2017 ). Such a kinetically controlled Cd isotope fractionation may occur during Cd uptake into plants and leads to an enrichment of light isotopes compared to soil solution or nutrient solution in hydroponics ( Wei et al, 2016 ; Imseng et al, 2019 ; Moore et al, 2020 ; Zhang et al, 2021 ).…”

Rootstock-Mediated Genetic Variance in Cadmium Uptake by Juvenile Cacao (Theobroma cacao L.) Genotypes, and Its Effect on Growth and Physiology

“…Transporter gene families like zinc-iron permease (ZIP), natural resistance-associated macrophage proteins (NRAMPs), and heavy metal transporting ATPases (HMAs) have been associated with uptake and translocation of Cd in plants ( Vanderschueren et al, 2021 ). According to some studies, TcNRAMP5 may play a role in the regulation of Cd uptake in cacao plants ( Ullah et al, 2018 ; Moore et al, 2020 ), whereas HMA-family proteins may contribute to Cd sequestration ( Moore et al, 2020 ). However, the information regarding the specific role of transporter genes in cacao is still limited, as well as the regulatory mechanisms of Cd translocation to the shoot.…”