1. Nutritional Zn requirements in plants and a narrow range of permissive Zn concentrations

Minimum required concentrations of the micronutrient zinc (Zn) in living tissues of land plants are around 30 μM, corresponding to 20 mg kg⁻¹ dry biomass (Krämer, Reference Krämer2010, Reference Krämer2024; Marschner & Marschner, Reference Marschner and Marschner2012; Sinclair & Krämer, Reference Sinclair and Krämer2012; Stanton et al., Reference Stanton, Sanders, Krämer and Podar2022). Plants experience Zn toxicity generally above ~300 mg kg⁻¹ dry biomass (450 μM). Thus, the range of tissue Zn levels permissive for plant growth is narrow by comparison to most other mineral nutrients, although plants store considerable amounts of Zn in harmless form inside the vacuoles (usually ~100 μM, see below) and extracellularly bound to cell walls, where they are separated from the most sensitive metabolic pathways. Plants can face Zn deficiency because of its generally low abundance in soils, with totals between 10 and 300, and an average of around 55 mg Zn kg⁻¹ (Noulas et al., Reference Noulas, Tziouvalekas and Karyotis2018). More importantly, Zn²⁺ cations are poorly soluble, resulting in low Zn concentrations of ~1 μM in the soil solution from which plants take up nutrients (Krämer, Reference Krämer2024; Marschner & Marschner, Reference Marschner and Marschner2012). When present in excess, Zn can cause toxicity directly, and also indirectly through nutrient imbalances arising from the competition between Zn²⁺ and other divalent cations for uptake by roots and within the plant (Krämer, Reference Krämer2024). There are well-known examples of soils containing excess, toxic levels of Zn, and of Zn-deficient soils, as well as of plant species, ecotypes and crop varieties capable of growing on such soils (Alloway, Reference Alloway2009; Ernst, Reference Ernst1974; Marschner & Marschner, Reference Marschner and Marschner2012). Moreover, even in common plants including the major reference species, researchers have characterized considerable Zn-related plasticity comprising responses to both Zn deficiency and Zn excess (Assunção, Reference Assunção2022; Sinclair & Krämer, Reference Sinclair and Krämer2012). This implies that all land plants encounter non-optimal Zn levels internally during their life cycle and externally in their roots’ microenvironment. The latter is likely to reflect heterogeneity and dynamics in soil composition at multiple scales (Stark, Reference Stark, Caldwell and Pearcy1994). In natural settings, the activities of symbiotic and associated microbes contribute to root Zn acquisition and the exclusion of excess Zn (Gonzalez-Guerrero et al., Reference Gonzalez-Guerrero, Escudero, Saez and Tejada-Jimenez2016). Most of our present knowledge of the involved molecular mechanisms was acquired by studying plants in isolation, and yet, it evidently has major predictive relevance also for plants cultivated in the soil in the presence of microbes (Sinclair & Krämer, Reference Sinclair and Krämer2012; Stanton et al., Reference Stanton, Sanders, Krämer and Podar2022; Thiébaut & Hanikenne, Reference Thiébaut and Hanikenne2022). In light of fluctuations in both environmental Zn availability and internal Zn demands, plant Zn homoeostasis has the roles of preventing Zn toxicity and of maintaining the functionality of all Zn-requiring proteins. A second, broader biological role of Zn homoeostasis in general Zn²⁺-dependent signalling and regulation is discussed in this article, and a third role in mineral co-option (such as elemental defence) has recently been addressed elsewhere (Krämer, Reference Krämer2024).

2. Distinctive biological chemistry of the micronutrient Zn provided opportunities for evolutionary innovations

The transition metal element Zn, which is positioned in the d-block of the periodic table, occurs exclusively in the +II oxidation state in biology (Fraústo da Silva & Williams, Reference Fraústo da Silva and Williams2001; Krezel & Maret, Reference Krezel and Maret2016). Adding on to the low solubility of many Zn^II salts, such as phosphates and oxides, the Zn²⁺ cation acts as a strong Lewis acid that establishes exceptionally stable high-affinity coordinative bonds in solution to form complexes with a large variety of ligands acting as free electron pair donors, by comparison to Mg²⁺ and Ca²⁺ (Box 1.1) (Fraústo da Silva & Williams, Reference Fraústo da Silva and Williams2001). The so-called chelation of Zn²⁺, which is coordinative Zn²⁺ binding to the free electron pairs of several donor atoms of the same ligand molecule, can form particularly stable complexes – a characteristic of the divalent cations of transition metal elements, as well as of the alkaline earth metals Mg²⁺ and Ca²⁺. When a metal cation undergoes complex formation in the presence of a ligand, this decreases the ‘chemically active’ concentration of the metal cation interacting merely with surrounding water molecules (‘free aqueous’, ‘hydrated’; Me ⁿ⁺ _aq, e.g., Zn²⁺ _aq). As a result, another additional ligand or binding partner will tend to bind lower net total concentrations of the metal at chemical equilibrium.

After binding to a ligand metabolite or a protein, the dissociation of Zn²⁺ to form the hydrated cation occurs far more slowly than for Ca²⁺, for example (dissociation rate constant k _off is around five orders of magnitude smaller, based on approximated generalized rate constants) (Fraústo da Silva & Williams, Reference Fraústo da Silva and Williams2001). Zn²⁺ can displace not only Ca²⁺ and Mg²⁺ but even other transition metal micronutrient cations, such as Fe²⁺, Mn²⁺ and Co²⁺, from solvent-exposed binding sites of proteins and bound ligands of any chemical nature, following the Irving–Williams series (Box 1.2) (Fraústo da Silva & Williams, Reference Fraústo da Silva and Williams2001). The stability of Zn complexes is generally the largest for the binding of Zn²⁺ involving thiol groups, followed by free electron pairs of nitrogen atoms and, subsequently, oxygen atoms contained in ligands (Williams, Reference Williams2012). Moreover, compared to Fe²⁺, Mn²⁺ and Co²⁺, the increase in stability of a complex formed with Zn²⁺ is generally the largest for thiol, intermediate for nitrogen, and the smallest for oxygen coordination (Williams, Reference Williams2012).

As a strong Lewis acid, Zn²⁺ is highly effective in polarizing bound ligands (Box 1.1). Its preferred coordination geometry is tetrahedral, but more flexible than that of other transition metal cations (Krezel & Maret, Reference Krezel and Maret2016; Stanton et al., Reference Stanton, Sanders, Krämer and Podar2022). Whereas dissociative ligand exchange via a free hydrated aqueous Zn²⁺ cation (Zn²⁺ _aq) intermediate occurs exceedingly slowly, associative ligand exchange, that is an exchange of ligands directly on the metal without any formation of Zn²⁺ _aq as an intermediate, can occur rapidly, in the order of 10⁶ times faster (Box 1.3) (Costello et al., Reference Costello, Fenselau and Franklin2011; Foster et al., Reference Foster, Osman and Robinson2014; Heinz et al., Reference Heinz, Kiefer, Tholey and Adolph2005; Krezel & Maret, Reference Krezel and Maret2016). Thus, associative ligand exchange is now thought to be of central relevance also for the integration of Zn into apo-metalloproteins (protein metalation) and not only for Zn-mediated catalysis in vivo. In summary, the biological chemistry of Zn²⁺ is distinct from that of other nutrient and non-nutrient transition metal cations, and it differs decisively from that of alkali and alkaline earth metal cations including Ca²⁺.

3. The functions of Zn in the plant metalloproteome

Of the 27,500 protein-coding loci of Arabidopsis, about 1,900 proteins (7%) were estimated to be Zn metalloproteins (Andreini et al., Reference Andreini, Banci, Bertini and Rosato2006, Reference Andreini, Bertini and Rosato2009; Zhang & Krämer, Reference Zhang and Krämer2018) – a list that has to be considered preliminary. These proteins comprise enzymes that depend on Zn²⁺ as a catalytic cofactor participating directly in substrate conversion or as a structural cofactor conferring the functional three-dimensional tertiary structure of a protein (Type I metalloproteins; Box 1.4). Proteins mediating transmembrane Zn transport or Zn binding directly in the operational maintenance of plant Zn homoeostasis are also addressed as Zn metalloproteins (Type II; Box 1.4). In comparison to metals with similar chemical properties, Zn²⁺-mediated catalysis is faster and is more versatile, thus explaining the use of Zn as a cofactor of enzymes across all functional classes (Fraústo da Silva & Williams, Reference Fraústo da Silva and Williams2001). Its high binding affinities and the absence of redox transitions under biologically relevant conditions may explain the exceptionally widespread use of Zn²⁺ as a structural cofactor, such as in Zn finger domains which often mediate protein–DNA or protein–protein interactions.

Comparisons among organisms suggest an increasing use of Zn²⁺ with evolution, from 5% to 6% of bacterial and archaeal proteomes to around 9% in eukaryotes (Andreini et al., Reference Andreini, Bertini and Rosato2009). Early during the evolution of life, Zn was immobilized in the form of insoluble sulphides and, thus, was unavailable. With ~10⁸-fold increased levels of Zn (to around 10 nM) in the oceans upon the oxygenation of the Earth’s atmosphere, organisms apparently evolved to gradually replace other metals, in particular Ni²⁺, Co²⁺ and Fe²⁺, by Zn²⁺ as a catalytic cofactor in a variety of enzymatic functions (Fraústo da Silva & Williams, Reference Fraústo da Silva and Williams2001; Krämer, Reference Krämer2024). In addition, novel protein functions in transcriptional regulation and protein–protein interactions increasingly made use of Zn in the evolution of eukaryotes and multicellular organisms, alongside an overall elevated bioavailability of Zn on land. Compared to our knowledge of type I and type II metalloproteins, which remains sketchy and relies heavily on in silico predictions, our knowledge of type III Zn metalloproteins (Zn²⁺ sensors; Box 1.4) in plants is even less developed because of their poor conservation across the kingdoms of life (Andreini et al., Reference Andreini, Banci, Bertini and Rosato2006, Reference Andreini, Bertini and Rosato2009; Krämer, Reference Krämer2024). Type III metalloproteins are characterized by the presence of facultative metal binding sites, which confer alternate protein functional states in the metal-bound and -unbound forms. Important examples of Zn²⁺ sensors in plants are the basic leucine zipper proteins AtbZIP19 and AtbZIP23, two partly redundant activators of the transcription of genes encoding Zn²⁺ transporters and chelator biosynthetic enzymes constituting a subset of the known transcriptional Zn deficiency responses (Assunção et al., Reference Assunção, Herrero, Lin, Huettel, Talukdar, Smaczniak and Aarts2010; Lilay et al., Reference Lilay, Persson, Castro, Liao, Alexander, Aarts and Assunção2021) (Figure 1a). These proteins contain a Zn²⁺-binding motif in the N-terminal region preceding the DNA-binding motif and the leucine zipper, and only their apo-form is capable of activating transcription (Assunção, Reference Assunção2022; Lilay et al., Reference Lilay, Castro, Campilho and Assunção2018). In addition, the root high-affinity Fe uptake protein iron-regulated transporter 1 (IRT1) could be considered another example of a type III Zn metalloprotein. The binding of Zn²⁺, which is a secondary substrate of the transporter, to a cytosolic loop of IRT1 triggers its own phosphorylation, polyubiquitination-mediated removal from the plasma membrane and degradation (Dubeaux et al., Reference Dubeaux, Neveu, Zelazny and Vert2018).

Figure 1. Cellular zinc homoeostasis in plants. (a) Cellular Zn homoeostasis and the cytosolic Zn buffer. (b) Cytosolic buffered available metal cation concentrations in Salmonella enterica (grey line around symbols) and in Arabidopsis thaliana (for Zn²⁺ only, green line around symbol). [Zn^II _b], the buffered available Zn²⁺ concentration, is dependent on the metal buffer. Cytosolic Zn is present in the form of Zn complexes of low-molecular-mass ligands, such as glutathione (GSH) to form [Zn(GS)₂], nicotianamine ([ZnNA]), phytochelatins ([ZnPC]), histidine ([ZnHis]) (ligand biosynthesis not shown), as well as Zn-binding sites of type I Zn metalloproteins (not shown) and metallothionein (MT) (a). Dashed arrow indicates the movement of mRNAs (drawn line ending in AAA, with names) of bZIP19/23 target genes out of the nucleus under Zn deficiency (a). C, plastid; Cit, citrate; ER, endoplasmatic reticulum; G, Golgi apparatus; L, ligand forming Zn complex; M, mitochondrium; P, peroxisome; PD: plasmodesma; X, phosphate or other anion forming Zn-precipitates. Yellow stripe-like (YSL) and natural resistance associated macrophage protein (NRAMP) family proteins can transport Zn, but critical roles in Zn homoeostasis have not yet been demonstrated for them in Arabidopsis. They might alternatively mediate Zn influx into the cytosol across other membranes, such as the plasma membrane. See text for other abbreviations and descriptions; more details have been described elsewhere (Sinclair & Krämer, Reference Sinclair and Krämer2012).

Of the 1,921 Zn metalloproteins (Zhang & Krämer, Reference Zhang and Krämer2018) identified based on known Zn-binding motifs (Andreini et al., Reference Andreini, Bertini and Rosato2009), proteomics of Arabidopsis rosette leaves identified 636 (Mergner et al., Reference Mergner, Frejno, List, Papacek, Chen, Chaudhary and Küster2020a), as can be established by comparing published datasets. Furthermore, approximated relative numbers of Zn-metalloprotein molecules were about 10%–20% higher in the oldest leaf than in the youngest leaf. According to proteomics-based abundance estimates, carbonic anhydrases, proteases, Calvin–Benson–Bassham cycle and central metabolism enzymes were among the top 15 most abundant Zn metalloproteins in leaves, with an apparent predominance of plastid-localized proteins (Table 1, similar when using leaf data from Mergner et al., Reference Mergner, Frejno, Messerer, Lang, Samaras, Wilhelm and Küster2020b). Cytosolic ribosomal Zn metalloproteins were among the most abundant proteins in pollen, roots and the shoot apical meristem, for example (Mergner et al., Reference Mergner, Frejno, Messerer, Lang, Samaras, Wilhelm and Küster2020b).

Table 1 The top 15 most abundant Zn proteins in leaves of Arabidopsis

^a https://suba.live/; cyt, cytosol; gol, Golgi apparatus; mt, mitochondria; per, peroxisomes; pl, plastids; pm, plasma membrane.

^b Numbers are mio. TMT (tandem mass tags), as a preliminary approximation of abundance (Mergner et al., Reference Mergner, Frejno, Messerer, Lang, Samaras, Wilhelm and Küster2020b).

^c Calvin–Benson–Bassham Cycle.

^d Central metabolism.

^e Kmiec et al., (Reference Kmiec, Teixeira, Berntsson, Murcha, Branca, Radomiljac and Glaser2013).

^f Splicing of Group II introns, relevant for chloroplast ribosome.

4. Protein metalation: how Zn²⁺ cations reach their cognate binding sites

Given the comparably high binding affinity of Zn²⁺ for a variety of ligands, the questions arise of how it is incorporated (exclusively) in its cognate binding sites of newly synthesized apo-metalloproteins and how the cell prevents the binding of other metals, in particular Cu (Box 1.2). Extrapolating from studies in bacteria and mammalian cells, our present concept of protein metalation in plants is now based entirely on associative Zn^II transfer (see above; Box 1.3) (Costello et al., Reference Costello, Fenselau and Franklin2011; Foster et al., Reference Foster, Young, Chivers and Robinson2022b; Krämer, Reference Krämer2024). A Zn-trafficking metallochaperone protein, in analogy with the role of Cu-metallochaperones in the metalation of apo-Cu-metalloproteins, is only known for one single Zn-dependent protein of plants to date (Pasquini et al., Reference Pasquini, Grosjean, Hixson, Nicora, Yee, Lipton and Blaby-Haas2022). Instead, the metalation of Zn-dependent apo-metalloproteins is now thought to occur co- or post-translationally from the cellular metal buffer via rapid ligand exchange reactions (Box 1.5) (Foster et al., Reference Foster, Young, Chivers and Robinson2022b; Krezel & Maret, Reference Krezel and Maret2016). The metal buffer consists of labile-bound Zn^II and other transition metals and an overall excess of ligands and solvent-accessible metal-binding sites provided by a complex mix of metabolites, inorganic compounds, protein surfaces and other macromolecules, which can chelate Zn²⁺ (Figure 1a) (Foster et al., Reference Foster, Young, Chivers and Robinson2022b; Osman & Robinson, Reference Osman and Robinson2023). In bacteria, intracellular buffered available Zn²⁺ concentrations are thus maintained in the femtomolar to picomolar range (2 pM in aerobically cultivated Escherichia coli), which allows the adequate metalation of Zn and other metalloproteins (Box 1.5, Figures 1b and 2a) (Foster et al., Reference Foster, Young, Chivers and Robinson2022b; Outten & O’Halloran, Reference Outten and O'Halloran2001). This implies that there are no free hydrated Zn²⁺ ions in the bacterial cytoplasm statistically, despite total intracellular Zn concentrations in the sub-millimolar range (Foster et al., Reference Foster, Clough, Aki, Young, Clarke and Robinson2022a; Outten & O’Halloran, Reference Outten and O'Halloran2001). In Arabidopsis, total intracellular Zn concentrations are thought to be at similar levels and cytosolic buffered available Zn²⁺ levels were reported at 400 pM under standard conditions and 2 nM under excess-Zn conditions – at least 100-fold higher than in bacteria and similar to levels around 100 pM reported in mammalian cells (Figure 1a, b) (Krämer, Reference Krämer2024; Lanquar et al., Reference Lanquar, Grossmann, Vinkenborg, Merkx, Thomine and Frommer2014; Qin et al., Reference Qin, Miranda, Stoddard, Dean, Galati and Palmer2013). However, the availability of only a single published study on a land plant to date, and the use of different methods limit the comparability with bacterial studies. Plant vacuoles, which contain ~100 μM Zn, constitute a large potential cellular reservoir for the dynamic entry of metal ions into, and removal from, the cytosolic metal buffer – a feature that differs strongly from many other organisms (Lanquar et al., Reference Lanquar, Grossmann, Vinkenborg, Merkx, Thomine and Frommer2014, Reference Lanquar, Ramos, Lelievre, Barbier-Brygoo, Krieger-Liszkay, Krämer and Thomine2010). Experimental procedures involving the destruction of plant cells inevitably permit the redistribution of metal cations among all accessible binding sites in aqueous suspensions or solutions and are thus unable to provide any information on Zn localization, binding or speciation in vivo.

Figure 2. Model of the metalation of Zn metalloproteins. (a–c) Expectations of cytosolic protein metalation under normal conditions (a), excess Zn conditions (b), and conditions of a more oxidized redox state, for example, under oxidative stress (c). Apo-metalloproteins (left) undergo metalation from the Zn buffer by associative ligand exchange (middle, shown for the ligand GSH as an example) to generate the Zn-bound forms (right, blue shapes) (a–c). The darker end of the arrows and the progressively darker shapes indicate the direction during metalation, whereby each step is reversible; important equilibria are emphasized by dotted arrows (a–c). Protein conformational changes can render the bound Zn solvent-inaccessible, removing it from the buffer (upper right blue shape in a, shown again in b and c). Red vertical arrows show the direction of changes when cytosolic Zn levels increase (b), and when cytosolic GSH is oxidized to GSSG (c), and red arrowheads emphasize that metalation processes are generally favoured (b, c). This is predicted to result in the binding of Zn²⁺ to labile binding sites on additional proteins (green shapes) including type III metalloproteins which can activate signalling and regulation (b, c). Note that a more oxidized redox state can also result in the formation of disulphide bridges among thiol groups of Zn-binding sites, thus strongly decreasing their affinity for Zn²⁺ (not shown).

5. Metal buffering in plant cells highlights that a more oxidized redox state can generate an internal burst in available Zn ²⁺

With low-millimolar concentrations, a major contribution of glutathione (GSH), γ-glutamyl-cysteinyl-glycine (γ-ECG), to the cytosolic metal buffer appears feasible (Figures 1a and 2a) (apparent pK _d of the Zn(GS)₂ complex ~8.1 at pH 7.7; Box 1.2) (Koffler et al., Reference Koffler, Bloem, Zellnig and Zechmann2013; Krezel & Maret, Reference Krezel and Maret2016; Meyer et al., Reference Meyer, May and Fricker2001). Plant metallothionein proteins, a family of small cysteine-rich proteins capable of metal binding, and other proteins containing surface thiols are likely to make notable contributions to the cytosolic metal buffer (Hassinen et al., Reference Hassinen, Tervahauta, Schat and Kärenlampi2011; Hübner & Haase, Reference Hübner and Haase2021). As a result of the preference of Zn for thiol-containing ligands including GSH, they are thought to contribute overproportionately to the intracellular buffering of Zn²⁺, compared to the buffering of Fe²⁺ and Mn²⁺, for example. We would expect that any decrease in total cytosolic thiol concentrations, either through their oxidation or through a decrease in total thiol levels, leads to increased buffered available cytosolic concentrations of Zn²⁺, similar to a situation in which excess Zn levels are present in the cytosol (Box 1.5, Figure 2a–c). Under conditions of oxidative stress, we would thus expect at least a transient homoeostatic downregulation of known transcriptional Zn deficiency markers in Arabidopsis. Concordant with the transition to a more sufficient cellular Zn status, transcript levels of the bZIP19/bZIP23 target gene ZIP3 are downregulated in roots of wild-type Arabidopsis plants after 2 h of oxidative stress (Figure 1a) (Lehmann et al., Reference Lehmann, Schwarzländer, Obata, Sirikantaramas, Burow, Olsen and Laxa2009), as well as in GSH-deficient mutants by comparison to the wild type (Schnaubelt et al., Reference Schnaubelt, Queval, Dong, Diaz-Vivancos, Makgopa, Howell and Foyer2015). This highlights the potential relevance of changes in redox state for the homoeostasis of Zn in plants. Beyond this, transient increases in intracellular buffered available Zn²⁺ concentrations could act as a signal contributing to the regulation of a broader range of cellular processes by altering the functional state of hitherto unidentified type III metalloproteins through the binding of Zn²⁺ to facultative binding sites (Box 1.4, Figure 2a–c). This possibility expands the physiological scope for possible Zn²⁺-dependent signalling, which could be triggered in plant cells through several processes. As is well-known, it could be initiated by the increased influx of Zn into the cytosol upon release from the vacuole or a sudden increase in extracellular bioavailable Zn levels (Figures 1a and 2b). Alternatively, it could be activated during episodes of a more oxidized redox state, which can result from cellular stress, dynamic changes in metabolism, or as a response to a variety of abiotic or biotic stimuli (Mittler et al., Reference Mittler, Zandalinas, Fichman and Van Breusegem2022) (Figure 2c). Beyond this, also a decrease in pH, predicted to result in the protonation of free electron pairs of important Zn ligands including GSH, could potentially lead to an increase in Zn²⁺ availability. Despite a growing body of literature in the animal field on signalling and regulatory roles of Zn²⁺ outside maintaining metal homoeostasis alone, they are yet to be experimentally examined in plants (Assunção, Reference Assunção2022; Clemens, Reference Clemens2022; Earley et al., Reference Earley, Mendoza, Tan and Kornfeld2021; Hübner & Haase, Reference Hübner and Haase2021; Krämer, Reference Krämer2024; Maret, Reference Maret2019).

Based on the stability constants of Zn complexes of phytochelatins (PCs), which increase with increasing lengths of the (γ-EC) _n G peptide (n = 2–5), it was proposed that PC2 ((γ-EC)₂G) contributes to the cytosolic metal buffer in plants cultivated under standard conditions (in the absence of a metal excess) (Luczkowski et al., Reference Luczkowski, Leszczynska, Watly, Clemens and Krezel2024). The authors proposed that the stability of Zn complexes with PC2 (apparent pK _d ~7.3 at pH 7.4; Box 1.2), with PC2 levels of 5–30 nmol g⁻¹ fresh biomass (i.e., up to around 30 μM in fresh tissues) in Arabidopsis roots, could contribute to cytosolic Zn buffering. Nicotianamine concentrations in Arabidopsis tissues are in a similar range as PC2 levels, and NA is thought to contribute to the cytosolic metal buffer (ca. 25–140 nmol g⁻¹ fresh biomass (Klatte et al., Reference Klatte, Schuler, Wirtz, Fink-Straube, Hell and Bauer2009; Pianelli et al., Reference Pianelli, Mari, Marques, Lebrun and Czernic2005; Weber et al., Reference Weber, Harada, Vess, Roepenack-Lahaye and Clemens2004), pK _d = 15.4 (von Wirén et al., Reference Von Wirén, Klair, Bansal, Briat, Khodr, Shioiri and Hider1999) and apparent pK _d ~10.48 at pH 7.7 (Krezel & Maret, Reference Krezel and Maret2016)). Yet, we are presently lacking important information on the cytosolic concentrations of these low-molecular-mass chelators and their subcellular distribution between the cytosol, the vacuole and intracellular vesicles, for instance (Chao et al., Reference Chao, Wang, Chen, Zhang, Wang, Song and Chao2021; Haydon et al., Reference Haydon, Kawachi, Wirtz, Hillmer, Hell and Krämer2012; Song et al., Reference Song, Park, Mendoza-Cozatl, Suter-Grotemeyer, Shim, Hortensteiner and Martinoia2010).

Plant cells must also accomplish and maintain protein metalation in cellular compartments other than the cytosol. Work in mammalian model systems suggested buffered available Zn²⁺ concentrations of 0.3 nM in mitochondria and >5 nM in the endoplasmatic reticulum (ER) (Chabosseau et al., Reference Chabosseau, Tuncay, Meur, Bellomo, Hessels, Hughes and Rutter2014; Clemens, Reference Clemens2022). The latter is consistent with a more oxidized redox state of GSH in the ER than in the cytosol of plant cells (Schwarzländer et al., Reference Schwarzländer, Fricker, Müller, Marty, Brach, Novak and Meyer2008). Under Zn deficiency, the possibility of maintaining higher buffered available Zn²⁺ concentrations in the ER may help to maintain a rate of root-to-shoot Zn flux critical for survival (see below).

6. A brief overview of Zn acquisition and homoeostasis in Arabidopsis

The uptake of Zn into the root symplasm of Arabidopsis and other plants is thought to occur via redundantly acting Zn²⁺ transporters of the Zn-regulated transporter, IRT-like protein (ZIP) family, which are capable of mediating cellular Zn uptake when produced in the yeast Saccharomyces cerevisiae (Figure 1a) (Grotz et al., Reference Grotz, Fox, Connolly, Park, Guerinot and Eide1998; Lee et al., Reference Lee, Lee, Ricachenevsky, Punshon, Tappero, Salt and Guerinot2021). Yet, this model is not unequivocally supported by reverse genetic evidence. An increased vacuolar accumulation of Zn in a quadruple irt3 zip4 zip6 zip9 mutant and irt3 zip4 zip6/zip9 triple mutants of Arabidopsis thaliana implies additional or other transporters in root Zn uptake, and it could alternatively reflect a role of these transporters in the remobilization of Zn from pools stored in the vacuoles of root cortex cells (Lee et al., Reference Lee, Lee, Ricachenevsky, Punshon, Tappero, Salt and Guerinot2021; Robe et al., Reference Robe, Lefebvre-Legendre, Cleard and Barberon2024). Additional unidentified Zn transporters of plants might have dual substrates or transport Zn complexes. For example, the high-affinity inorganic phosphate uptake system PHO84 of S. cerevisiae was proposed to additionally mediate the uptake of Mn, Zn and other metals (Jensen et al., Reference Jensen, Ajua-Alemanji and Culotta2003). The closest plant homologues of ScPHO84 are in the phosphate transporter family of major facilitator superfamily proteins (Nino-Gonzalez et al., Reference Nino-Gonzalez, Novo-Uzal, Richardson, Barros and Duque2019).

Zinc transport into vacuoles is mediated by metal transport/tolerance protein 1 (MTP1) and MTP3 of the so-called cation diffusion facilitator family, as well as heavy metal ATPase 3 (HMA3; note that Col-0 is an hma3 mutant) (Figure 1a) (Arrivault et al., Reference Arrivault, Senger and Krämer2006; Desbrosses-Fonrouge et al., Reference Desbrosses-Fonrouge, Voigt, Schröder, Arrivault, Thomine and Krämer2005; Morel et al., Reference Morel, Crouzet, Gravot, Auroy, Leonhardt, Vavasseur and Richaud2009). MTP1 appears to be highly expressed in growing tips, whereas both MTP3 and HMA3 are upregulated in the roots of plants exposed to Fe deficiency and excess Zn. Transport of nicotianamine into the vacuoles via zinc-induced facilitator 1 (ZIF1) enhances vacuolar Zn storage and can contribute to immobilizing Zn in the roots (Haydon & Cobbett, Reference Haydon and Cobbett2007; Haydon et al., Reference Haydon, Kawachi, Wirtz, Hillmer, Hell and Krämer2012). The function of ZIF1 illustrates the principle of ‘Zn trapping’ through the localized accumulation of a chelator, which can have a major impact on Zn partitioning within the plant. With similar effects, silicates, phosphates and polyphosphates such as phytate (inositol hexakisphosphate, IP6), for example, have been implicated in the formation of Zn precipitates, mostly extracellularly and inside vacuoles (Bouain et al., Reference Bouain, Shahzad, Rouached, Khan, Berthomieu, Abdelly and Rouached2014; Neumann & zur Nieden, Reference Neumann and zur Nieden2001).

ZIF1 transcript levels respond in a similar manner as those of MTP3 and HMA3. Root cortex cells appear to be important in Zn storage and immobilization based on the localization of promoter activities of HMA3, MTP3 and ZIF1. Only the Fe deficiency responsiveness of HMA3 and MTP3, but not that of ZIF1 transcript levels, depends on upstream regulator of IRT1 (URI) and Fe deficiency-induced transcription factor 1 (FIT1), central transcriptional activators of Fe deficiency responses (Colangelo & Guerinot, Reference Colangelo and Guerinot2004; Kim et al., Reference Kim, LaCroix, Gerber and Guerinot2019; Mai et al., Reference Mai, Pateyron and Bauer2016). The similarities between the responses of roots to excess Zn and Fe deficiency highlight the ability of Zn to interfere with Fe nutrition and the relevance of the high-affinity Fe uptake system IRT1 for the influx of Zn²⁺ into root cells as a secondary substrate (Figure 1a). Consistent with the disruption of Fe nutrition by exposure to excess Zn, brutus-like 1 (btsl1) btsl2 double mutants are more Zn-tolerant than wild-type Arabidopsis plants. This has been attributed to aspects of a general upregulation of Fe deficiency responses in these mutants (Stanton et al., Reference Stanton, Rodriguez-Celma, Krämer, Sanders and Balk2023).

Nicotianamine, when not sequestered in vacuoles, appears to be important in the radial symplastic cell-to-cell movement of Zn in roots towards the stele, where Zn²⁺ cations are exported into the xylem by the metal pumps HMA4 and HMA2 (Figure 1a) (Deinlein et al., Reference Deinlein, Weber, Schmidt, Rensch, Trampczynska, Hansen and Clemens2012; Haydon et al., Reference Haydon, Kawachi, Wirtz, Hillmer, Hell and Krämer2012; Hussain et al., Reference Hussain, Haydon, Wang, Wong, Sherson, Young and Cobbett2004). These two metal pumps act partially redundantly. Whereas HMA4 transcripts are constitutively present in A. thaliana roots at low levels, the levels of HMA2 transcripts are upregulated under Zn deficiency (Sinclair et al., Reference Sinclair, Senger, Talke, Cobbett, Haydon and Krämer2018). Compared to A. thaliana, strongly elevated HMA4 transcript levels in Arabidopsis halleri are necessary for naturally selected Zn hyperaccumulation, the accumulation of extraordinarily high Zn levels in shoots of this species (Hanikenne et al., Reference Hanikenne, Talke, Haydon, Lanz, Nolte, Motte and Krämer2008; Krämer, Reference Krämer2010). The high rates of HMA4-dependent Zn²⁺ export from the root symplasm into the xylem were shown to cause the secondary homoeostatic upregulation of the levels of Zn deficiency-responsive bZIP19/bZIP23 target transcripts in roots of A. halleri, presumably as a result of Zn depletion in root cells (Hanikenne et al., Reference Hanikenne, Talke, Haydon, Lanz, Nolte, Motte and Krämer2008). This is a second example of how changing a single process in the Zn homoeostasis network can strongly affect Zn fluxes in plants (see ZIF1 above).

ZIP family transporters are thought to contribute to xylem unloading in the shoot by mediating Zn uptake into the adjacent cells. The molecular mechanisms involved in the distribution of Zn across the leaf blade are poorly understood. HMA4 is likely to contribute to this at least in A. halleri, as well as in A. thaliana under specific conditions such as pathogen attack, in the context of elemental defences against biotic stress (Escudero et al., Reference Escudero, Sánchez, Abreu, Sopeña-Torres, Makarovsky-Saavedra, Bernal and Jordá2022; Hanikenne et al., Reference Hanikenne, Talke, Haydon, Lanz, Nolte, Motte and Krämer2008; Krämer, Reference Krämer2024).

Zn deficiency responses are regulated in a cell-autonomous manner by the Zn-sensing transcription factors bZIP19 and bZIP23 (Figure 1a), which may also act as heterodimers and have partially redundant functions (Assunção, Reference Assunção2022; Kimura et al., Reference Kimura, Vaattovaara, Ohshita, Yokoyama, Yoshida, Hui and Fukao2023; Sinclair et al., Reference Sinclair, Senger, Talke, Cobbett, Haydon and Krämer2018). Targets with known roles in Zn homoeostasis are primarily subsets of ZIP transporter- and nicotianamine synthase-encoding genes (Assunção et al., Reference Assunção, Herrero, Lin, Huettel, Talukdar, Smaczniak and Aarts2010). In addition, there is a systemically regulated Zn deficiency response in roots controlled by the Zn status of rosette leaves, which includes the upregulation of transcript levels of MTP2 and HMA2 (Sinclair et al., Reference Sinclair, Senger, Talke, Cobbett, Haydon and Krämer2018). Both of the encoded proteins contribute to enhancing root-to-shoot Zn flux. It appears that MTP2 acts by transporting Zn²⁺ into the ER (Figure 1a), suggesting that radial cell-to-cell transport of Zn in roots occurs within desmotubules under Zn deficiency, which will require further study.

Both HMA2 and HMA4 also act in the export of Zn from the maternal seed coat for its translocation to the developing embryo (Olsen et al., Reference Olsen, Hansen, Larue, Osterberg, Hoffmann, Liesche and Palmgren2016). MTP2 promoter activity and transcripts are also found in developing seeds of Zn-deficient plants. In addition to ZIP family proteins, natural resistance associated macrophage protein (NRAMP) and yellow stripe-like (YSL) family proteins are candidate membrane transporters for contributing to the remobilization of Zn²⁺ from vacuoles or other cellular compartments (Figure 1a), and also for mediating Zn influx across the plasma membrane. HMA1 is thought to export Zn from the chloroplast stroma to protect chloroplasts from Zn toxicity (Figure 1a) (Kim et al., Reference Kim, Choi, Segami, Cho, Martinoia, Maeshima and Lee2009). Although a number of critical Zn metalloproteins localize to plastids and mitochondria, we do not presently know through which transporters Zn enters these organelles.

Basal Zn tolerance (Clemens, Reference Clemens2001) involves Zn-binding PCs ((γ-EC) _n G, n = 2–11). PCs are synthesized from GSH by PC synthase enzymes, which are activated by metal binding (Tennstedt et al., Reference Tennstedt, Peisker, Böttcher, Trampczynska and Clemens2009). The proposed roles of cysteine-rich extracellular plant defensins in Zn-binding and Zn tolerance in plants are less well understood (Shahzad et al., Reference Shahzad, Ranwez, Fizames, Marques, Le Martret, Alassimone and Gosti2013). Apoplastically localized cysteine-rich defensin-like (DEFL) proteins have been implicated in root Zn deficiency responses (Kimura et al., Reference Kimura, Vaattovaara, Ohshita, Yokoyama, Yoshida, Hui and Fukao2023; Niehs et al., Reference Niehs, Rajniak, Johnson, Wengier and Sattely2024). DEFL202, 203 and 206–208 are transcriptionally upregulated under Zn deficiency and – different from the wild type – defl202 defl203 double mutants fail to attenuate root growth under Zn deficiency (Kimura et al., Reference Kimura, Vaattovaara, Ohshita, Yokoyama, Yoshida, Hui and Fukao2023).

Arabidopsis de-etiolated by zinc (dez, or trichome birefringence, tbr) and overly zinc sensitive 2 (ozs2, or pectin methylesterase 3, pme3) mutants are Zn hypersensitive, and this was demonstrated to be related to reduced contents or accessibility of de-methyl esterified pectin in the cell wall (Sinclair et al., Reference Sinclair, Larue, Bonk, Khan, Castillo-Michel, Stein and Krämer2017; Weber et al., Reference Weber, Deinlein, Fischer, Rogowski, Geimer, Tenhaken and Clemens2013). The extracellular binding of Zn²⁺ to cell walls thus contributes to basal Zn tolerance. Recently identified natural variants of TBR may relate to this, but we presently do not know whether this natural genetic variation is associated with variation in bioavailable Zn levels in the soils of origin of these Arabidopsis accessions (Zhong et al., Reference Zhong, Zhang, Wei, Platre, He, Zhang and Busch2024). The current status of knowledge suggests that the main proteins and processes of Zn homoeostasis are conserved across flowering plants, including also rice, for example (Stanton et al., Reference Stanton, Sanders, Krämer and Podar2022).

In summary, there is plenty of scope for research towards an improved understanding of Zn homoeostasis in plants. Future research will address the nature, abundance and subcellular localization of Zn metalloproteins, protein metalation, the cellular dynamics of Zn availability, as well as aim to identify Zn²⁺-sensing proteins and their roles in Zn²⁺-dependent signalling and regulation, both within and beyond Zn homoeostasis alone. In addition to the role of Zn in its metalloproteome, Zn can be co-opted for other functions such as elemental defence (see Krämer, Reference Krämer2024). Scientific progress in plant Zn biology will contribute to increasing yield, quality and stress resilience of crops.