Stable Isotope Fractionation of Metals and Metalloids in Plants: A Review

Abstract

This work critically reviews stable isotope fractionation of essential (B, Mg, K, Ca, Fe, Ni, Cu, Zn, Mo), beneficial (Si), and non-essential (Cd, Tl) metals and metalloids in plants. The review (i) provides basic principles and methodologies for non-traditional isotope analyses, (ii) compiles isotope fractionation for uptake and translocation for each element and connects them to physiological processes, and (iii) interlinks knowledge from different elements to identify common and contrasting drivers of isotope fractionation. Different biological and physico-chemical processes drive isotope fractionation in plants. During uptake, Ca and Mg fractionate through root apoplast adsorption, Si through diffusion during membrane passage, Fe and Cu through reduction prior to membrane transport in strategy I plants, and Zn, Cu, and Cd through membrane transport. During translocation and utilization, isotopes fractionate through precipitation into insoluble forms, such as phytoliths (Si) or oxalate (Ca), structural binding to cell walls (Ca), and membrane transport and binding to soluble organic ligands (Zn, Cd). These processes can lead to similar (Cu, Fe) and opposing (Ca vs. Mg, Zn vs. Cd) isotope fractionation patterns of chemically similar elements in plants. Isotope fractionation in plants is influenced by biotic factors, such as phenological stages and plant genetics, as well as abiotic factors. Different nutrient supply induced shifts in isotope fractionation patterns for Mg, Cu, and Zn, suggesting that isotope process tracing can be used as a tool to detect and quantify different uptake pathways in response to abiotic stresses. However, the interpretation of isotope fractionation in plants is challenging because many isotope fractionation factors associated with specific processes are unknown and experiments are often exploratory. To overcome these limitations, fundamental geochemical research should expand the database of isotope fractionation factors and disentangle kinetic and equilibrium fractionation. In addition, plant growth studies should further shift toward hypothesis-driven experiments, for example, by integrating contrasting nutrient supplies, using established model plants, genetic approaches, and by combining isotope analyses with complementary speciation techniques. To fully exploit the potential of isotope process tracing in plants, the interdisciplinary expertise of plant and isotope geochemical scientists is required.

Keywords: fractionation; metalloids; metals; multiple-collector-ICP-MS; plant uptake; process tracing; stable isotopes; translocation.

Figure 1

Overview of physiological processes potentially causing isotope fractionation. (A) In shoots, isotope fractionation may occur during short distance transport across membranes, during long-distance transport in xylem (unidirectional transport) or in the phloem (bidirectional transport), due to metabolic functionalization (e.g., enzyme activation, ligand exchange), element storage or compartmentalization. Major physiological processes in roots are nutrient uptake and short distance transport across membranes. Elements are taken up from the rhizosphere and translocated via the xylem to sink tissue (young leaves or generative tissue, such as fruits, grains, or pods). Elements may also be re-translocated via the phloem from source tissue (e.g., old leaves) to sink tissue. (B) Simplified model of symplastic (blue), apoplastic (red) versus transcellular (orange) element transport routes in plant roots. Cell-to-cell transport in the symplastic pathway occurs via plasmodesmata. In the apoplastic pathway elements travel in the cell wall and the intercellular space. The plasma membrane must be crossed to exit the root cortex and enter the stele and the vasculature system. (C) Elements may be bound to soil particles in the rhizosphere or to the negatively charged cell wall prior to uptake across the plasma membrane mediated by transport proteins (e.g., via carriers and channels). In the cytoplasm, elements may form complexes with enzymes and substrates—for example, ATP activation through Mg-ATP complexation (Adapted from Marschner and Marschner, 2012; Taiz et al., 2015). For glossary of terms and processes see Table 3.

Figure 2

A few studies have investigated B isotope fractionation in plants. Bell pepper has been most systematically investigated. (1) Due to lacking B mass balances, Δ¹¹B_plant-source is not known, but it can be semi-quantitatively shown that (2) shoots are enriched in heavy isotopes compared to roots. (3) Within plants, leaves are enriched in heavy B isotopes compared to stems while leaves can be heavier and lighter than reproductive organs. (A) At equilibrium, boric acid (B(OH)₃) is isotopically heavier than borate (B(OH)₄⁻) which may contribute to the isotope fractionation between root and shoot. (B) The integration of borate into cell walls may cause the enrichment of heavy isotopes in leaves compared to stems.

Figure 3

Mg isotope fractionation in plants has been studied in trees and cereals. Exemplified for cereals: (1) Plants are enriched in heavy isotopes compared to the Mg source while (2) shoots tend to be enriched in light isotopes compared to roots. (3) Grains are enriched in heavy isotopes compared to stems and leaves. (A) Heavy Mg isotopes preferentially bind onto negatively charged surfaces in the root apoplast. (B) Binding of Mg to membrane transporters that are active at low Mg supply may induce a shift towards heavy isotopes during plant uptake and root-to-shoot transport compared to Mg transport at regular (or high) Mg supply. (C) It is assumed that the root contains a heavy Mg pool that is bound to organic ligands (Mg-L) while ionic Mg²⁺ is enriched in light isotopes that may be preferentially transported toward the shoots. (D) During grain filling, organic ligands that contain Mg degrade (e.g., chlorophyll) and release heavy Mg which may lead to a preferential re-translocation of heavy isotopes from senescent tissues.

Figure 4

Si isotope fractionation in plants exemplified for cereals. (1) Root uptake of Si leads in most cases to an enrichment of light isotopes while (2) no consistent pattern of Si isotope fractionation has been observed between root and shoot. (3) Within the shoot, plants become significantly enriched in heavy isotopes along the transpiration stream leading to a strong enrichment of heavy isotopes in husks and grains of rice. (A) The preferential uptake of light isotopes has been ascribed to a non-membrane protein-mediated transfer of Si (non-Si accumulators) and a membrane protein-mediated transfer of Si (LSi1) in Si-accumulating plants. Both processes may be driven by Si diffusion that favors light Si isotopes. (B) Precipitation of light Si isotopes into phytoliths (SiO₂) results in an enrichment of heavy isotopes in the Si pool (H₄³⁰SiO₄) and thereby in an enrichment of heavy isotopes along the transpiration stream.

Figure 5

Ca isotope fractionation in plants exemplified for beans. (1) Root uptake of Ca leads to an enrichment of light isotopes. (2) In most cases, shoots are enriched in heavy isotopes compared to roots. (3) Leaves are preferentially enriched in light isotopes compared to stems while reproductive organs are mostly lighter than leaves. The preferential uptake of light isotopes has been ascribed to (A) isotope differences in hydrated Ca species with distinct number of water molecules and (B) the preferential sorption of light Ca isotopes to negatively charged surfaces in the root apoplast. Within the plant, (C) lighter isotopes may precipitate with oxalate (⁴⁰Ca) and (D) are structurally bound in cell walls to pectins (RCOO⁻ groups). The retention of light Ca isotopes leads to a higher mobility of heavy Ca isotopes within plants.

Figure 6

Fe isotope fractionation in plants for Fe acquisition strategy I and II plants. (1) Fe isotope fractionation between the Fe source and the plant strongly depends on the Fe source (i.e., Fe-EDTA in hydroponics, bulk soil, or soil extract). In most cases, Fe acquisition by plants leads to a stronger enrichment of light isotopes in strategy I plants (e.g., tomato and beans) compared to strategy II plants. (2) The Fe isotope fractionation between shoot and root highly depends on the root Fe pools analyzed since significant fractionation can occur between precipitated Fe in the root apoplast compared to Fe that is taken up into the symplast and not deposited in the root apoplast. (3) Reproductive organs tend to be enriched in light isotopes in strategy I plants while no such fractionation step occurs in strategy II plants. (A) The Fe reduction from Fe(III) to Fe(II) prior to membrane transport may explain the successive enrichment of light isotopes from soil via roots and leaves to reproductive organs in strategy I plants. (B) Fe reoxidation from soluble Fe(II) to insoluble Fe(III) in the root apoplast can lead to an enrichment of light Fe isotope in the soluble Fe fraction. (C) In strategy II plants, the mobilization of Fe(III) from the mineral lattice of the soil by phytosiderophores (L for ligand, e.g., deoxymugineic acid) does not require a Fe reduction step and leads, if at all, to small Fe isotope fractionation. (D) Within the shoot, ligand (L) exchange of Fe (e.g., nicotianamine, citrate, and ferritin) that can include Fe redox changes may further fractionate Fe isotopes.

Figure 7

Nickel isotope ratios have been mostly measured in Ni hyperaccumulating plants. (1) Root uptake leads to an enrichment of light Ni isotopes in plants. (2) From roots to shoots as well as (3) within shoots, though no clear pattern of isotope fractionation has been identified so far. (A) It is hypothesized that the enrichment of light isotopes during plant uptake of Ni is induced by low-affinity transport. High Ni uptake rates in hyperaccumulator plants may induce a depletion of Ni in the proximity of the membrane transporters and induce a Ni diffusion which leads to additional enrichment of light isotopes during uptake. (B) Within the shoot, Ni speciation to organic ligands (L), such as citrate, may be excluded as major factor controlling Ni isotope fractionation.

Figure 8

Cu isotope fractionation in plants exemplified for tomato plants. (1) Root uptake can lead to an enrichment of heavy or light Cu isotopes. The isotope fractionation during root uptake is more negative in Fe strategy I plants compared to strategy II plants. At low Cu supply light isotopes were preferentially taken up compared to heavy isotopes at high supply likely due to a shift from active to passive uptake. (2) Shoots can be enriched in light and heavy isotopes compared to roots. The enrichment of light isotopes increases with organ height and Cu availability. (3) Cu isotopes fractionate in shoots of Fe strategy I plants is stronger than in strategy II plants. (A) Cu reduction induced by root reductases favors light Cu isotopes, (B) Cu membrane transport can favor light Cu isotopes, and (C) Cu binding to S to detoxify Cu in the root at high Cu supply may contribute to the retention of light isotopes in roots. (D) Cu binding to O ligands in the root apoplast should retain heavy Cu in the roots.

Figure 9

Zn isotope fractionation has been reported in several plant species (see review of Caldelas and Weiss, 2017 and references therein). (1,2) In most cases, light Zn isotopes are preferentially taken up by plants and transported from root to shoot. (3) Within the shoots, leaves are enriched in light isotopes compared to stems and reproductive organs are enriched in light isotopes compared to the remaining shoot or senescent tissues. (A) The uptake of light Zn isotopes has been ascribed to diffusion, Zn speciation in solution, and stripping of the hydration shell prior to membrane transport. A set of studies provides robust evidence, that at low Zn supply, Zn complexation to organic ligands (L), such as phytosiderophores followed by the uptake of the Zn-phytosiderophores leads to a shift toward heavy isotopes in cereals. During membrane transport, the enrichment of light isotopes is stronger at regular than at low Zn supply. (B) Within the root, binding of Zn to O/N donors of organic ligands in the cytosol and vacuole as well as diffusion of the Zn in the apoplast and symplast toward the xylem may control the preferential transport light isotopes from roots to shoots. (C) The enrichment of light Zn isotopes in reproductive plant organs is induced by the strong retention of heavy isotopes in mature leaves or senescing tissues likely induced binding of Zn to O donors of organic ligands in the apoplast, cytosol, or vacuole.

Figure 10

Cadmium isotope fractionation in plants exemplified for cereals and cacao. (1) Root uptake leads to an enrichment of light isotopes in plants. (2) Shoots are mostly enriched in heavy isotopes compared to roots. (3) In cereals, grains are enriched in heavy isotopes compared to the remaining shoot while in cacao, the beans tend to be enriched in light isotopes compared to other shoot parts. (A) Root membrane transport of NRAMP5 induces an enrichment or depletion in light Cd isotopes during uptake. (B) Light isotopes are preferentially sorbed to the negatively charged surfaces in the root apoplast. (C) Light Cd isotopes are sequestrated in root vacuoles via tonoplast proteins. (D) Chelation of Cd by thiols (Cd-S) contributes to the sequestration of light Cd in roots. (E) The non-membrane bound protein CAL1 that preferentially binds light Cd with thiols in the xylem parenchyma cells and transports light Cd into the xylem. (F) Xylem to phloem transfer in the nodes favors heavy Cd isotopes through transport by OsHMA2 and LCT. (G) In cacao, given that beans are enriched in light isotopes compared to leaves, the processes transporting and loading Cd into beans may differ from cereals.