Metal Homeostasis in Land Plants: A Perpetual Balancing Act Beyond the Fulfilment of Metalloproteome Cofactor Demands.

One of life's decisive innovations was to harness the catalytic power of metals for cellular chemistry. With life's expansion, global atmospheric and biogeochemical cycles underwent dramatic changes. Although initially harmful, they permitted the evolution of multicellularity and the colonization of land. In land plants as primary producers, metal homeostasis faces heightened demands, in part because soil is a challenging environment for nutrient balancing. To avoid both nutrient metal limitation and metal toxicity, plants must maintain the homeostasis of metals within tighter limits than the homeostasis of other minerals. This review describes the present model of protein metalation and sketches its transfer from unicellular organisms to land plants as complex multicellular organisms. The inseparable connection between metal and redox homeostasis increasingly draws our attention to more general regulatory roles of metals. Mineral co-option, the use of nutrient or other metals for functions other than nutrition, is an emerging concept beyond that of nutritional immunity. Expected final online publication date for the Annual Review of Plant Biology, Volume 75 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Effects of metal amendment and metalloid supplementation on foliar defences are plant accession-specific in the hyperaccumulator Arabidopsis halleri.

Soil pollution by metals and metalloids as a consequence of anthropogenic industrialisation exerts a seriously damaging impact on ecosystems. However, certain plant species, termed hyperaccumulators, are able to accumulate extraordinarily high concentrations of these metal(loid)s in their aboveground tissues. Such hyperaccumulation of metal(loid)s is known to act as a defence against various antagonists, such as herbivores and pathogens. We investigated the influences of metal(loid)s on potential defence traits, such as foliar elemental, organic and mechanical defences, in the hyperaccumulator plant species Arabidopsis halleri (Brassicaceae) by artificially amending the soil with common metallic pollutants, namely cadmium (Cd) and zinc (Zn). Additionally, unamended and metal-amended soils were supplemented with the metalloid silicon (Si) to study whether Si could alleviate metal excess. Individuals originating from one non-/low- and two moderately to highly metal-contaminated sites with different metal concentrations (hereafter called accessions) were grown for eight weeks in a full-factorial design under standardised conditions. There were significant interactive effects of metal amendment and Si supplementation on foliar concentrations of certain elements (Zn, Si, aluminium (Al), iron (Fe), potassium (K) and sulfur (S), but these were accession-specific. Profiles of glucosinolates, characteristic organic defences of Brassicaceae, were distinct among accessions, and the composition was affected by soil metal amendment. Moreover, plants grown on metal-amended soil contained lower concentrations of total glucosinolates in one of the accessions, which suggests a potential trade-off between inorganic defence acquisition and biosynthesis of organic defence. The density of foliar trichomes, as a proxy for the first layer of mechanical defence, was also influenced by metal amendment and/or Si supplementation in an accession-dependent manner. Our study highlights the importance of examining the effects of co-occurring metal(loid)s in soil on various foliar defence traits in different accessions of a hyperaccumulating species.

BRUTUS-LIKE (BTSL) E3 ligase-mediated fine-tuning of Fe regulation negatively affects Zn tolerance of Arabidopsis.

The mineral micronutrients zinc (Zn) and iron (Fe) are essential for plant growth and human nutrition, but interactions between the homeostatic networks of these two elements are not fully understood. Here we show that loss of function of BTSL1 and BTSL2, which encode partially redundant E3 ubiquitin ligases that negatively regulate Fe uptake, confers tolerance to Zn excess in Arabidopsis thaliana. Double btsl1 btsl2 mutant seedlings grown on high Zn medium accumulated similar amounts of Zn in roots and shoots to the wild type, but suppressed the accumulation of excess Fe in roots. RNA-sequencing analysis showed that roots of mutant seedlings had relatively higher expression of genes involved in Fe uptake (IRT1, FRO2, and NAS) and in Zn storage (MTP3 and ZIF1). Surprisingly, mutant shoots did not show the transcriptional Fe deficiency response which is normally induced by Zn excess. Split-root experiments suggested that within roots the BTSL proteins act locally and downstream of systemic Fe deficiency signals. Together, our data show that constitutive low-level induction of the Fe deficiency response protects btsl1 btsl2 mutants from Zn toxicity. We propose that BTSL protein function is disadvantageous in situations of external Zn and Fe imbalances, and formulate a general model for Zn-Fe interactions in plants.

Arabidopsis nicotianamine synthases comprise a common core-NAS domain fused to a variable autoinhibitory C terminus.

Nicotianamine synthase (NAS) catalyzes the biosynthesis of the low-molecular-mass metal chelator nicotianamine (NA) from the 2-aminobutyrate moieties of three SAM molecules. NA has central roles in metal nutrition and metal homeostasis of flowering plants. The enzymatic function of NAS remains poorly understood. Crystal structures are available for archaeal and bacterial NAS-like proteins that carry out simpler aminobutanoyl transferase reactions. Here, we report amino acids essential for the activity of AtNAS1 based on structural modeling and site-directed mutagenesis. Using a newly developed enzyme-coupled continuous activity assay, we compare differing NAS proteins identified through multiple sequence alignments and phylogenetic analyses. In most NAS of dicotyledonous and monocotyledonous plants (class Ia and Ib), the core-NAS domain is fused to a variable C-terminal domain. Compared to fungal and moss NAS that comprise merely a core-NAS domain (class III), NA biosynthetic activities of the four paralogous Arabidopsis thaliana NAS proteins were far lower. C-terminally trimmed core-AtNAS variants exhibited strongly elevated activities. Of 320 amino acids of AtNAS1, twelve, 287-TRGCMFMPCNCS-298, accounted for the autoinhibitory effect of the C terminus, of which approximately one-third was attributed to N296 within a CNCS motif that is fully conserved in Arabidopsis. No detectable NA biosynthesis was mediated by two representative plant NAS proteins that naturally lack the C-terminal domain, class Ia Arabidopsis halleri NAS5 and Medicago truncatula NAS2 of class II which is found in dicots and diverged early during the evolution of flowering plants. Next, we will address a possible posttranslational release of autoinhibition in class I NAS proteins.

Energy status-promoted growth and development of Arabidopsis require copper deficiency response transcriptional regulator SPL7.

Copper (Cu) is a cofactor of around 300 Arabidopsis proteins, including photosynthetic and mitochondrial electron transfer chain enzymes critical for adenosine triphosphate (ATP) production and carbon fixation. Plant acclimation to Cu deficiency requires the transcription factor SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE7 (SPL7). We report that in the wild type (WT) and in the spl7-1 mutant, respiratory electron flux via Cu-dependent cytochrome c oxidase is unaffected under both normal and low-Cu cultivation conditions. Supplementing Cu-deficient medium with exogenous sugar stimulated growth of the WT, but not of spl7 mutants. Instead, these mutants accumulated carbohydrates, including the signaling sugar trehalose 6-phosphate, as well as ATP and NADH, even under normal Cu supply and without sugar supplementation. Delayed spl7-1 development was in agreement with its attenuated sugar responsiveness. Functional TARGET OF RAPAMYCIN and SNF1-RELATED KINASE1 signaling in spl7-1 argued against fundamental defects in these energy-signaling hubs. Sequencing of chromatin immunoprecipitates combined with transcriptome profiling identified direct targets of SPL7-mediated positive regulation, including Fe SUPEROXIDE DISMUTASE1 (FSD1), COPPER-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR1 (CITF1), and the uncharacterized bHLH23 (CITF2), as well as an enriched upstream GTACTRC motif. In summary, transducing energy availability into growth and reproductive development requires the function of SPL7. Our results could help increase crop yields, especially on Cu-deficient soils.

Translational fidelity and growth of Arabidopsis require stress-sensitive diphthamide biosynthesis.

Diphthamide, a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR2 (eEF2), is the human host cell-sensitizing target of diphtheria toxin. Diphthamide biosynthesis depends on the 4Fe-4S-cluster protein Dph1 catalyzing the first committed step, as well as Dph2 to Dph7, in yeast and mammals. Here we show that diphthamide modification of eEF2 is conserved in Arabidopsis thaliana and requires AtDPH1. Ribosomal -1 frameshifting-error rates are increased in Arabidopsis dph1 mutants, similar to yeast and mice. Compared to the wild type, shorter roots and smaller rosettes of dph1 mutants result from fewer formed cells. TARGET OF RAPAMYCIN (TOR) kinase activity is attenuated, and autophagy is activated, in dph1 mutants. Under abiotic stress diphthamide-unmodified eEF2 accumulates in wild-type seedlings, most strongly upon heavy metal excess, which is conserved in human cells. In summary, our results suggest that diphthamide contributes to the functionality of the translational machinery monitored by plants to regulate growth.

Effects of 4-Br-A23187 on Bacillus subtilis cells and unilamellar vesicles reveal it to be a potent copper ionophore.

Ionophores are small molecules or peptides that transport metal ions across biological membranes. Their transport capabilities are typically characterized in vitro using vesicles and single ion species. It is difficult to infer from these data which effects ionophores have on living cells in a complex environment (e.g., culture medium), since net ion movement is influenced by many factors including ion composition of the medium, concentration gradients, pH gradient, and protein-mediated transport processes across the membrane. To gain insights into the antibacterial mechanism of action of the semisynthetic polyether ionophore 4-Br-A23187, known to efficiently transport zinc and manganese in vitro, we investigated its effects on the gram-positive model organism Bacillus subtilis. In addition to monitoring cellular ion concentrations, the physiological impact of treatment was assessed on the proteome level. 4-Br-A23187 treatment resulted in an increase in intracellular copper levels, the extent of which depended on the copper concentration of the medium. Effects of copper accumulation mirrored by the proteomic response included oxidative stress, disturbance of proteostasis, metal and sulfur homeostasis. The antibiotic effect of 4-Br-A23187 is further aggravated by a decrease in intracellular manganese and magnesium. A liposome model confirmed that 4-Br-A23187 acts as copper ionophore in vitro.

Elemental bioimaging of Zn and Cd in leaves of hyperaccumulator Arabidopsis halleri using laser ablation-inductively coupled plasma-mass spectrometry and referencing strategies.

The spatial distribution of Zn and Cd in leaves of the heavy metal hyperaccumulator species Arabidopsis halleri, a land plant in the Brassicaceae family of angiosperms, is determined by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Detected intensities of nuclides of the environmental pollutants Zn and Cd are referenced to nuclides of the naturally abundant elements C, Mg, P, Ca, and Rb as internal standards, in order to compensate for widespread experimental issues in whole-leaf laser ablation. Referencing occurs by dividing the signal intensity of the analyte by the corresponding intensity of the internal standard. In order to avoid large quotients that occur during division by small numbers, quotients of pixels for which the internal standard is no higher than the background are set to zero. The effects of referencing on a loss of laser focus, overlapping layers of leaf tissue and cell damage within the imaged leaf tissue are addressed specifically. It is reported that referencing to 25Mg, 31P, 44Ca or 85Rb can skew the results of Zn and Cd distribution because of their different ion mobility within leaves or other element-specific effects. This is particularly valid in the leaf venation and in regions of leaves where cell damage has occurred. Considering all aspects, 13C was found to be best suited among the investigated elements for referencing of leaves, because it stabilizes the resulting distributions of Zn and Cd even in samples affected by experimental issues.

A two-step adaptive walk rewires nutrient transport in a challenging edaphic environment.

Most well-characterized cases of adaptation involve single genetic loci. Theory suggests that multilocus adaptive walks should be common, but these are challenging to identify in natural populations. Here, we combine trait mapping with population genetic modeling to show that a two-step process rewired nutrient homeostasis in a population of Arabidopsis as it colonized the base of an active stratovolcano characterized by extremely low soil manganese (Mn). First, a variant that disrupted the primary iron (Fe) uptake transporter gene (IRT1) swept quickly to fixation in a hard selective sweep, increasing Mn but limiting Fe in the leaves. Second, multiple independent tandem duplications occurred at NRAMP1 and together rose to near fixation in the island population, compensating the loss of IRT1 by improving Fe homeostasis. This study provides a clear case of a multilocus adaptive walk and reveals how genetic variants reshaped a phenotype and spread over space and time.

Arabidopsis thaliana Zn2+-efflux ATPases HMA2 and HMA4 are required for resistance to the necrotrophic fungus Plectosphaerella cucumerina BMM.

Zinc is an essential nutrient at low concentrations, but toxic at slightly higher ones. It has been proposed that hyperaccumulator plants may use the excess zinc to fend off pathogens and herbivores. However, there is little evidence of a similar response in other plants. Here we show that Arabidopsis thaliana leaves inoculated with the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM) accumulate zinc and manganese at the infection site. Zinc accumulation did not occur in a double mutant in the zinc transporters HEAVY METAL ATPASE2 and HEAVY METAL ATPASE4 (HMA2 and HMA4), which has reduced zinc translocation from roots to shoots. Consistent with a role in plant immunity, expression of HMA2 and HMA4 was up-regulated upon PcBMM inoculation, and hma2hma4 mutants were more susceptible to PcBMM infection. This phenotype was rescued upon zinc supplementation. The increased susceptibility to PcBMM infection was not due to the diminished expression of genes involved in the salicylic acid, ethylene, or jasmonate pathways since they were constitutively up-regulated in hma2hma4 plants. Our data indicate a role of zinc in resistance to PcBMM in plants containing ordinary levels of zinc. This layer of immunity runs in parallel to the already characterized defence pathways, and its removal has a direct effect on resistance to pathogens.

Root-to-shoot iron partitioning in Arabidopsis requires IRON-REGULATED TRANSPORTER1 (IRT1) protein but not its iron(II) transport function.

IRON-REGULATED TRANSPORTER1 (IRT1) is the root high-affinity ferrous iron (Fe) uptake system and indispensable for the completion of the life cycle of Arabidopsis thaliana without vigorous Fe supplementation. Here we provide evidence supporting a second role of IRT1 in root-to-shoot partitioning of Fe. We show that irt1 mutants overaccumulate Fe in roots, most prominently in the cortex of the differentiation zone in irt1-2, compared to the wild type. Shoots of irt1-2 are severely Fe-deficient according to Fe content and marker transcripts, as expected. We generated irt1-2 lines producing IRT1 mutant variants carrying single amino-acid substitutions of key residues in transmembrane helices IV and V, Ser206 and His232, which are required for transport activity in yeast. Root short-term 55 Fe uptake rates were uninformative concerning IRT1-mediated transport. Overall irt1-like concentrations of the secondary substrate Mn suggested that the transgenic Arabidopsis lines also remain incapable of IRT1-mediated root Fe uptake. Yet, IRT1S206A partially complements rosette dwarfing and leaf chlorosis of irt1-2, as well as root-to-shoot Fe partitioning and gene expression defects of irt1-2, all of which are fully complemented by wild-type IRT1. Taken together, these results suggest a regulatory function for IRT1 in root-to-shoot Fe partitioning that does not require Fe transport activity of IRT1. Among the genes of which transcript levels are partially dependent on IRT1, we identify MYB DOMAIN PROTEIN10, MYB DOMAIN PROTEIN72 and NICOTIANAMINE SYNTHASE4 as candidates for effecting IRT1-dependent Fe mobilization in roots. Understanding the biological functions of IRT1 will help to improve Fe nutrition and the nutritional quality of agricultural crops.

Zinc in plants: Integrating homeostasis and biofortification.

Zinc plays many essential roles in life. As a strong Lewis acid that lacks redox activity under environmental and cellular conditions, the Zn2+ cation is central in determining protein structure and catalytic function of nearly 10% of most eukaryotic proteomes. While specific functions of zinc have been elucidated at a molecular level in a number of plant proteins, wider issues abound with respect to the acquisition and distribution of zinc by plants. An important challenge is to understand how plants balance between Zn supply in soil and their own nutritional requirement for zinc, particularly where edaphic factors lead to a lack of bioavailable zinc or, conversely, an excess of zinc that bears a major risk of phytotoxicity. Plants are the ultimate source of zinc in the human diet, and human Zn deficiency accounts for over 400 000 deaths annually. Here, we review the current understanding of zinc homeostasis in plants from the molecular and physiological perspectives. We provide an overview of approaches pursued so far in Zn biofortification of crops. Finally, we outline a "push-pull" model of zinc nutrition in plants as a simplifying concept. In summary, this review discusses avenues that can potentially deliver wider benefits for both plant and human Zn nutrition.

Corrigendum: Involvement of Arabidopsis Multi-Copper Oxidase-Encoding LACCASE12 in Root-to-Shoot Iron Partitioning: A Novel Example of Copper-Iron Crosstalk.

[This corrects the article DOI: 10.3389/fpls.2021.688318.].

Constitutively enhanced genome integrity maintenance and direct stress mitigation characterize transcriptome of extreme stress-adapted Arabidopsis halleri.

Heavy metal-rich toxic soils and ordinary soils are both natural habitats of Arabidopsis halleri, a diploid perennial and obligate outcrosser in the sister clade of the genetic model plant Arabidopsis thaliana. The molecular divergence underlying survival in sharply contrasting environments is unknown. Here we comparatively address metal physiology and transcriptomes of A. halleri originating from the most highly heavy metal-contaminated soil in Europe, Ponte Nossa, Italy (Noss), and from non-metalliferous (NM) soils. Plants from Noss exhibit enhanced hypertolerance and attenuated accumulation of cadmium (Cd), and their transcriptomic Cd responsiveness is decreased, compared to plants of NM soil origin. Among the condition-independent transcriptome characteristics of Noss, the most highly overrepresented functional class of 'meiotic cell cycle' comprises 21 transcripts with elevated abundance in vegetative tissues, in particular Argonaute 9 (AGO9) and the synaptonemal complex transverse filament protein-encoding ZYP1a/b. Increased AGO9 transcript levels in Noss are accompanied by decreased long terminal repeat retrotransposon expression. Similar to Noss, plants from other highly metalliferous sites in Poland and Germany share elevated somatic AGO9 transcript levels in comparison to plants originating from NM soils in their respective geographic regions. Transcript levels of Iron-Regulated Transporter 1 (IRT1) are very low and transcript levels of Heavy Metal ATPase 2 (HMA2) are strongly elevated in Noss, which can account for its altered Cd handling. We conclude that in plants adapted to the most extreme abiotic stress, broadly enhanced functions comprise genes with likely roles in somatic genome integrity maintenance, accompanied by few alterations in stress-specific functional networks.

Involvement of Arabidopsis Multi-Copper Oxidase-Encoding LACCASE12 in Root-to-Shoot Iron Partitioning: A Novel Example of Copper-Iron Crosstalk.

Numerous central biological processes depend on the participation of the essential elements iron (Fe) or copper (Cu), including photosynthesis, respiration, cell wall remodeling and oxidative stress protection. Yet, both Fe and Cu metal cations can become toxic when accumulated in excess. Because of the potent ligand-binding and redox chemistries of these metals, there is a need for the tight and combined homeostatic control of their uptake and distribution. Several known examples pinpoint an inter-dependence of Fe and Cu homeostasis in eukaryotes, mostly in green algae, yeast and mammals, but this is less well understood in multicellular plants to date. In Arabidopsis, Cu deficiency causes secondary Fe deficiency, and this is associated with reduced in vitro ferroxidase activity and decreased root-to-shoot Fe translocation. Here we summarize the current knowledge of the cross-talk between Cu and Fe homeostasis and present a partial characterization of LACCASE12 (LAC12) that encodes a member of the multicopper oxidase (MCO) protein family in Arabidopsis. LAC12 transcript levels increase under Fe deficiency. The phenotypic characterization of two mutants carrying T-DNA insertions suggests a role of LAC12 in root-to-shoot Fe partitioning and in maintaining growth on Fe-deficient substrates. A molecular understanding of the complex interactions between Fe and Cu will be important for combating Fe deficiency in crops and for advancing biofortification approaches.

Chloroplast Ribosomes Interact With the Insertase Alb3 in the Thylakoid Membrane.

Members of the Oxa1/YidC/Alb3 protein family are involved in the insertion, folding, and assembly of membrane proteins in mitochondria, bacteria, and chloroplasts. The thylakoid membrane protein Alb3 mediates the chloroplast signal recognition particle (cpSRP)-dependent posttranslational insertion of nuclear-encoded light harvesting chlorophyll a/b-binding proteins and participates in the biogenesis of plastid-encoded subunits of the photosynthetic complexes. These subunits are cotranslationally inserted into the thylakoid membrane, yet very little is known about the molecular mechanisms underlying docking of the ribosome-nascent chain complexes to the chloroplast SecY/Alb3 insertion machinery. Here, we show that nanodisc-embedded Alb3 interacts with ribosomes, while the homolog Alb4, also located in the thylakoid membrane, shows no ribosome binding. Alb3 contacts the ribosome with its C-terminal region and at least one additional binding site within its hydrophobic core region. Within the C-terminal region, two conserved motifs (motifs III and IV) are cooperatively required to enable the ribosome contact. Furthermore, our data suggest that the negatively charged C-terminus of the ribosomal subunit uL4c is involved in Alb3 binding. Phylogenetic analyses of uL4 demonstrate that this region newly evolved in the green lineage during the transition from aquatic to terrestrial life.

Arabidopsis halleri shows hyperbioindicator behaviour for Pb and leaf Pb accumulation spatially separated from Zn.

Lead (Pb) ranks among the most problematic environmental pollutants. Background contamination of soils is nearly ubiquitous, yet plant Pb accumulation is barely understood. In a survey covering 165 European populations of the metallophyte Arabidopsis halleri, several field samples had indicated Pb hyperaccumulation, offering a chance to dissect plant Pb accumulation. Accumulation of Pb was analysed in A. halleri individuals from contrasting habitats under controlled conditions to rule out aerial deposition as a source of apparent Pb accumulation. Several elemental imaging techniques were employed to study the spatial distribution and ligand environment of Pb. Regardless of genetic background, A. halleri individuals showed higher shoot Pb accumulation than A. thaliana. However, dose-response curves revealed indicator rather than hyperaccumulator behaviour. Xylem sap data and elemental imaging unequivocally demonstrated the in planta mobility of Pb. Highest Pb concentrations were found in epidermal and vascular tissues. Distribution of Pb was distinct from that of the hyperaccumulated metal zinc. Most Pb was bound by oxygen ligands in bidentate coordination. A. halleri accumulates Pb whenever soil conditions render Pb phytoavailable. Considerable Pb accumulation under such circumstances, even in leaves of A. thaliana, strongly suggests that Pb can enter food webs and may pose a food safety risk.

Do Arabidopsis Squamosa promoter binding Protein-Like genes act together in plant acclimation to copper or zinc deficiency?

The genome of Arabidopsis thaliana encodes approximately 260 copper (Cu)-dependent proteins, which includes enzymes in central pathways of photosynthesis, respiration and responses to environmental stress. Under Cu-deficient growth conditions, Squamosa promoter binding Protein-Like 7 (SPL7) activates the transcription of genes encoding Cu acquisition systems, and it mediates a metabolic reorganization to economize on Cu. The transcription factor SPL7 groups among comparably large proteins in the SPL family, which additionally comprises a second group of small SPL proteins targeted by miRNA156 with roles in plant development. SPL7 shares extended regions of sequence homology with SPL1 and SPL12. Therefore, we investigated the possibility of a functional overlap between these three members of the group of large SPL family proteins. We compared the spl1 spl12 double mutant and the spl1 spl7 spl12 triple mutant with both the wild type and the spl7 single mutant under normal and Cu-deficient growth conditions. Biomass production, chlorophyll content and tissue elemental composition at the seedling stage, as well as plant and flower morphology during reproductive stages, confirmed the involvement of SPL7, but provided no indication for important roles of SPL1 or SPL12 in the acclimation of Arabidopsis to Cu deficiency. Furthermore, we analyzed the effects of zinc (Zn) deficiency on the same set of mutants. Different from what is known in the green alga Chlamydomonas reinhardtii, Arabidopsis did not activate Cu deficiency responses under Zn deficiency, and there was no Cu overaccumulation in either shoot or root tissues of Zn-deficient wild type plants. Known Zn deficiency responses were unaltered in spl7, spl1 spl12 and spl1 spl7 spl12 mutants. We observed that CuZnSOD activity is strongly downregulated in Zn-deficient A. thaliana, in association with an about 94% reduction in the abundance of the CSD2 transcript, a known target of miR398. However, different from the known Cu deficiency responses of Arabidopsis, this Zn deficiency response was independent of SPL7 and not associated with an upregulation of MIR398b primary transcript levels. Our data suggest that there is no conservation in A. thaliana of the crosstalk between Zn and Cu homeostasis mediated by the single SPL family protein CRR1 of Chlamydomonas. In the future, resolving how the specificity of SPL protein activation and recognition of target gene promoters is achieved will advance our understanding of the specific functions of different SPL family proteins in the regulation of either Cu deficiency responses or growth and development of land plants.

Real-time whole-plant dynamics of heavy metal transport in Arabidopsis halleri and Arabidopsis thaliana by gamma-ray imaging.

Heavy metals such as zinc are essential for plant growth, but toxic at high concentrations. Despite our knowledge of the molecular mechanisms of heavy metal uptake by plants, experimentally addressing the real-time whole-plant dynamics of heavy metal uptake and partitioning has remained a challenge. To overcome this, we applied a high sensitivity gamma-ray imaging system to image uptake and transport of radioactive 65Zn in whole-plant assays of Arabidopsis thaliana and the Zn hyperaccumulator Arabidopsis halleri. We show that our system can be used to quantitatively image and measure uptake and root-to-shoot translocation dynamics of zinc in real time. In the metal hyperaccumulator Arabidopsis halleri, 65Zn uptake and transport from its growth media to the shoot occurs rapidly and on time scales similar to those reported in rice. In transgenic A. halleri plants in which expression of the zinc transporter gene HMA4 is suppressed by RNAi, 65Zn uptake is completely abolished.

Convergent evolution in Arabidopsis halleri and Arabidopsis arenosa on calamine metalliferous soils.

It is a plausible hypothesis that parallel adaptation events to the same environmental challenge should result in genetic changes of similar or identical effects, depending on the underlying fitness landscapes. However, systematic testing of this is scarce. Here we examine this hypothesis in two closely related plant species, Arabidopsis halleri and Arabidopsis arenosa, which co-occur at two calamine metalliferous (M) sites harbouring toxic levels of the heavy metals zinc and cadmium. We conduct individual genome resequencing alongside soil elemental analysis for 64 plants from eight populations on M and non-metalliferous (NM) soils, and identify genomic footprints of selection and local adaptation. Selective sweep and environmental association analyses indicate a modest degree of gene as well as functional network convergence, whereby the proximal molecular factors mediating this convergence mostly differ between site pairs and species. Notably, we observe repeated selection on identical single nucleotide polymorphisms in several A. halleri genes at two independently colonized M sites. Our data suggest that species-specific metal handling and other biological features could explain a low degree of convergence between species. The parallel establishment of plant populations on calamine M soils involves convergent evolution, which will probably be more pervasive across sites purposely chosen for maximal similarity in soil composition. This article is part of the theme issue 'Convergent evolution in the genomics era: new insights and directions'.