JOINTLESS Maintains Inflorescence Meristem Identity in Tomato.

JOINTLESS (J) was isolated in tomato (Solanum lycopersicum) from mutants lacking a flower pedicel abscission zone (AZ) and encodes a MADS-box protein of the SHORT VEGETATIVE PHASE/AGAMOUS-LIKE 24 subfamily. The loss of J function also causes the return to leaf initiation in the inflorescences, indicating a pivotal role in inflorescence meristem identity. Here, we compared jointless (j) mutants in different accessions that exhibit either an indeterminate shoot growth, producing regular sympodial segments, or a determinate shoot growth, due to the reduction of sympodial segments and causal mutation of the SELF-PRUNING (SP) gene. We observed that the inflorescence phenotype of j mutants is stronger in indeterminate (SP) accessions such as Ailsa Craig (AC), than in determinate (sp) ones, such as Heinz (Hz). Moreover, RNA-seq analysis revealed that the return to vegetative fate in j mutants is accompanied by expression of SP, which supports conversion of the inflorescence meristem to sympodial shoot meristem in j inflorescences. Other markers of vegetative meristems such as APETALA2c and branching genes such as BRANCHED 1 (BRC1a/b) were differentially expressed in the inflorescences of j(AC) mutant. We also found in the indeterminate AC accession that J represses homeotic genes of B- and C-classes and that its overexpression causes an oversized leafy calyx phenotype and has a dominant negative effect on AZ formation. A model is therefore proposed where J, by repressing shoot fate and influencing reproductive organ formation, acts as a key determinant of inflorescence meristems.

Reduced expression of bZIP19 and bZIP23 increases zinc and cadmium accumulation in Arabidopsis halleri.

Zinc is an essential micronutrient for all living organisms. When challenged by zinc-limiting conditions, Arabidopsis thaliana plants use a strategy centered on two transcription factors, bZIP19 and bZIP23, to enhance the expression of several zinc transporters to improve their zinc uptake capacity. In the zinc and cadmium hyperaccumulator plant Arabidopsis halleri, highly efficient root-to-shoot zinc translocation results in constitutive local zinc deficiency in roots and in constitutive high expression of zinc deficiency-responsive ZIP genes, supposedly boosting zinc uptake and accumulation. Here, to disrupt this process and to analyze the functions of AhbZIP19, AhbZIP23 and their target genes in hyperaccumulation, the genes encoding both transcriptional factors were knocked down using artificial microRNAs (amiRNA). Although AhbZIP19, AhbZIP23, and their ZIP target genes were downregulated, amiRNA lines surprisingly accumulated more zinc and cadmium compared to control lines in both roots and shoot driving to shoot toxicity symptoms. These observations suggested the existence of a substitute metal uptake machinery in A. halleri to maintain hyperaccumulation. We propose that the iron uptake transporter AhIRT1 participates in this alternative pathway in A. halleri.

The Arabidopsis SR45 splicing factor bridges the splicing machinery and the exon-exon junction complex.

The Arabidopsis splicing factor serine/arginine-rich 45 (SR45) contributes to several biological processes. The sr45-1 loss-of-function mutant exhibits delayed root development, late flowering, unusual numbers of floral organs, shorter siliques with decreased seed sets, narrower leaves and petals, and altered metal distribution. SR45 bears a unique RNA recognition motif (RRM) flanked by one serine/arginine-rich (RS) domain on both sides. Here, we studied the function of each SR45 domains by examining their involvement in: (i) the spatial distribution of SR45; (ii) the establishment of a protein-protein interaction network including spliceosomal and exon-exon junction complex (EJC) components; and (iii) the RNA binding specificity. We report that the endogenous SR45 promoter is active during vegetative and reproductive growth, and that the SR45 protein localizes in the nucleus. We demonstrate that the C-terminal arginine/serine-rich domain is a determinant of nuclear localization. We show that the SR45 RRM domain specifically binds purine-rich RNA motifs via three residues (H101, H141, and Y143), and is also involved in protein-protein interactions. We further show that SR45 bridges both mRNA splicing and surveillance machineries as a partner of EJC core components and peripheral factors, which requires phosphoresidues probably phosphorylated by kinases from both the CLK and SRPK families. Our findings provide insights into the contribution of each SR45 domain to both spliceosome and EJC assemblies.

Altered metal distribution in the sr45-1 Arabidopsis mutant causes developmental defects.

The plant serine/arginine-rich (SR) splicing factor SR45 plays important roles in several biological processes, such as splicing, DNA methylation, innate immunity, glucose regulation, and abscisic acid signaling. A homozygous Arabidopsis sr45-1 null mutant is viable, but exhibits diverse phenotypic alterations, including delayed root development, late flowering, shorter siliques with fewer seeds, narrower leaves and petals, and unusual numbers of floral organs. Here, we report that the sr45-1 mutant presents an unexpected constitutive iron deficiency phenotype characterized by altered metal distribution in the plant. RNA-Sequencing highlighted severe perturbations in metal homeostasis, the phenylpropanoid pathway, oxidative stress responses, and reproductive development. Ionomic quantification and histochemical staining revealed strong iron accumulation in the sr45-1 root tissues accompanied by iron starvation in aerial parts. Mis-splicing of several key iron homeostasis genes, including BTS, bHLH104, PYE, FRD3, and ZIF1, was observed in sr45-1 roots. We showed that some sr45-1 developmental abnormalities can be complemented by exogenous iron supply. Our findings provide new insight into the molecular mechanisms governing the phenotypes of the sr45-1 mutant.

Natural variation of nutrient homeostasis among laboratory and field strains of Chlamydomonas reinhardtii.

Natural variation among individuals and populations exists in all species, playing key roles in response to environmental stress and adaptation. Micro- and macronutrients have a wide range of functions in photosynthetic organisms, and mineral nutrition thus plays a sizable role in biomass production. To maintain nutrient concentrations inside the cell within physiological limits and prevent the detrimental effects of deficiency or excess, complex homeostatic networks have evolved in photosynthetic cells. The microalga Chlamydomonas reinhardtii (Chlamydomonas) is a unicellular eukaryotic model for studying such mechanisms. In this work, 24 Chlamydomonas strains, comprising field isolates and laboratory strains, were examined for intraspecific differences in nutrient homeostasis. Growth and mineral content were quantified in mixotrophy, as full nutrition control, and compared with autotrophy and nine deficiency conditions for macronutrients (-Ca, -Mg, -N, -P, and -S) and micronutrients (-Cu, -Fe, -Mn, and -Zn). Growth differences among strains were relatively limited. However, similar growth was accompanied by highly divergent mineral accumulation among strains. The expression of nutrient status marker genes and photosynthesis were scored in pairs of contrasting field strains, revealing distinct transcriptional regulation and nutrient requirements. Leveraging this natural variation should enable a better understanding of nutrient homeostasis in Chlamydomonas.

Long-term acclimation to cadmium exposure reveals extensive phenotypic plasticity in Chlamydomonas.

Increasing industrial and anthropogenic activities are producing and releasing more and more pollutants in the environment. Among them, toxic metals are one of the major threats for human health and natural ecosystems. Because photosynthetic organisms play a critical role in primary productivity and pollution management, investigating their response to metal toxicity is of major interest. Here, the green microalga Chlamydomonas (Chlamydomonas reinhardtii) was subjected to short (3 d) or chronic (6 months) exposure to 50 µM cadmium (Cd), and the recovery from chronic exposure was also examined. An extensive phenotypic characterization and transcriptomic analysis showed that the impact of Cd on biomass production of short-term (ST) exposed cells was almost entirely abolished by long-term (LT) acclimation. The underlying mechanisms were initiated at ST and further amplified after LT exposure resulting in a reversible equilibrium allowing biomass production similar to control condition. This included modification of cell wall-related gene expression and biofilm-like structure formation, dynamics of metal ion uptake and homeostasis, photosynthesis efficiency recovery and Cd acclimation through metal homeostasis adjustment. The contribution of the identified coordination of phosphorus and iron homeostasis (partly) mediated by the main phosphorus homeostasis regulator, Phosphate Starvation Response 1, and a basic Helix-Loop-Helix transcription factor (Cre05.g241636) was further investigated. The study reveals the highly dynamic physiological plasticity enabling algal cell growth in an extreme environment.

The molecular basis of zinc homeostasis in cereals.

Plants require zinc (Zn) as an essential cofactor for diverse molecular, cellular and physiological functions. Zn is crucial for crop yield, but is one of the most limiting micronutrients in soils. Grasses like rice, wheat, maize and barley are crucial sources of food and nutrients for humans. Zn deficiency in these species therefore not only reduces annual yield but also directly results in Zn malnutrition of more than two billion people in the world. There has been good progress in understanding Zn homeostasis and Zn deficiency mechanisms in plants. However, our current knowledge of monocots, including grasses, remains insufficient. In this review, we provide a summary of our knowledge of molecular Zn homeostasis mechanisms in monocots, with a focus on important cereal crops. We additionally highlight divergences in Zn homeostasis of monocots and the dicot model Arabidopsis thaliana, as well as important gaps in our knowledge that need to be addressed in future research on Zn homeostasis in cereal monocots.

ZRT-IRT-Like PROTEIN 6 expression perturbs local ion homeostasis in flowers and leads to anther indehiscence and male sterility.

Metallic micronutrients are essential throughout the plant life cycle. Maintaining metal homeostasis in plant tissues requires a highly complex and finely tuned network controlling metal uptake, transport, distribution and storage. Zinc and cadmium hyperaccumulation, such as observed in the model plant Arabidopsis halleri, represents an extreme evolution of this network. Here, non-ectopic overexpression of the A. halleri ZIP6 (AhZIP6) gene, encoding a zinc and cadmium influx transporter, in Arabidopsis thaliana enabled examining the importance of zinc for flower development and reproduction. We show that AhZIP6 expression in flowers leads to male sterility resulting from anther indehiscence in a dose-dependent manner. The sterility phenotype is associated to delayed tapetum degradation and endothecium collapse, as well as increased magnesium and potassium accumulation and higher expression of the MHX gene in stamens. It is rescued by the co-expression of the zinc efflux transporter AhHMA4, linking the sterility phenotype to zinc homeostasis. Altogether, our results confirm that AhZIP6 is able to transport zinc in planta and highlight the importance of fine-tuning zinc homeostasis in reproductive organs. The study illustrates how the characterization of metal hyperaccumulation mechanisms can reveal key nodes and processes in the metal homeostasis network.

Zinc deficiency responses: bridging the gap between Arabidopsis and dicotyledonous crops.

Zinc (Zn) deficiency is a widespread phenomenon in agricultural soils worldwide and has a major impact on crop yield and quality, and hence on human nutrition and health. Although dicotyledonous crops represent >30% of human plant-based nutrition, relatively few efforts have been dedicated to the investigation of Zn deficiency response mechanisms in dicotyledonous, in contrast to monocotyledonous crops, such as rice or barley. Here, we describe the Zn requirement and impact of Zn deficiency in several economically important dicotyledonous crops, Phaseolus vulgaris, Glycine max, Brassica oleracea, and Solanum lycopersicum. We briefly review our current knowledge of the Zn deficiency response in Arabidopsis and outline how this knowledge is translated in dicotyledonous crops. We highlight commonalities and differences between dicotyledonous species (and with monocotyledonous species) regarding the function and regulation of Zn transporters and chelators, as well as the Zn-sensing mechanisms and the role of hormones in the Zn deficiency response. Moreover, we show how the Zn homeostatic network intimately interacts with other nutrients, such as iron or phosphate. Finally, we outline how variation in Zn deficiency tolerance and Zn use efficiency among cultivars of dicotyledonous species can be leveraged for the design of Zn biofortification strategies.

Intertwined metal homeostasis, oxidative and biotic stress responses in the Arabidopsis frd3 mutant.

FRD3 (FERRIC REDUCTASE DEFECTIVE 3) plays a major role in iron (Fe) and zinc (Zn) homeostasis in Arabidopsis. It transports citrate, which enables metal distribution in the plant. An frd3 mutant is dwarf and chlorotic and displays a constitutive Fe-deficiency response and strongly altered metal distribution in tissues. Here, we have examined the interaction between Fe and Zn homeostasis in an frd3 mutant exposed to varying Zn supply. Detailed phenotyping using transcriptomic, ionomic, histochemical and spectroscopic approaches revealed the full complexity of the frd3 mutant phenotype, which resulted from altered transition metal homeostasis, manganese toxicity, and oxidative and biotic stress responses. The cell wall played a key role in these processes, as a site for Fe and hydrogen peroxide accumulation, and displayed modified structure in the mutant. Finally, we showed that Zn excess interfered with these mechanisms and partially restored root growth of the mutant, without reverting the Fe-deficiency response. In conclusion, the frd3 mutant molecular phenotype is more complex than previously described and illustrates how the response to metal imbalance depends on multiple signaling pathways.

Adaptation of Arabidopsis halleri to extreme metal pollution through limited metal accumulation involves changes in cell wall composition and metal homeostasis.

Metallophytes constitute powerful models for the study of metal homeostasis, adaptation to extreme environments and the evolution of naturally selected traits. Arabidopsis halleri is a pseudometallophyte which shows constitutive zinc/cadmium (Zn/Cd) tolerance and Zn hyperaccumulation but high intraspecific variability in Cd accumulation. To examine the molecular basis of the variation in metal tolerance and accumulation, ionome, transcriptome and cell wall glycan array profiles were compared in two genetically close A. halleri populations from metalliferous and nonmetalliferous sites in Northern Italy. The metallicolous population displayed increased tolerance to and reduced hyperaccumulation of Zn, and limited accumulation of Cd, as well as altered metal homeostasis, compared to the nonmetallicolous population. This correlated well with the differential expression of transporter genes involved in trace metal entry and in Cd/Zn vacuolar sequestration in roots. Many cell wall-related genes were also more highly expressed in roots of the metallicolous population. Glycan array and histological staining analyses demonstrated that there were major differences between the two populations in terms of the accumulation of specific root pectin and hemicellulose epitopes. Our results support the idea that both specific cell wall components and regulation of transporter genes play a role in limiting accumulation of metals in A. halleri at contaminated sites.

Transcriptional regulation of ZIP genes is independent of local zinc status in Brachypodium shoots upon zinc deficiency and resupply.

The biological processes underlying zinc homeostasis are targets for genetic improvement of crops to counter human malnutrition. Detailed phenotyping, ionomic, RNA-Seq analyses and flux measurements with 67 Zn isotope revealed whole-plant molecular events underlying zinc homeostasis upon varying zinc supply and during zinc resupply to starved Brachypodium distachyon (Brachypodium) plants. Although both zinc deficiency and excess hindered Brachypodium growth, accumulation of biomass and micronutrients into roots and shoots differed depending on zinc supply. The zinc resupply dynamics involved 1,893 zinc-responsive genes. Multiple zinc-regulated transporter and iron-regulated transporter (IRT)-like protein (ZIP) transporter genes and dozens of other genes were rapidly and transiently down-regulated in early stages of zinc resupply, suggesting a transient zinc shock, sensed locally in roots. Notably, genes with identical regulation were observed in shoots without zinc accumulation, pointing to root-to-shoot signals mediating whole-plant responses to zinc resupply. Molecular events uncovered in the grass model Brachypodium are useful for the improvement of staple monocots.

Coordinated homeostasis of essential mineral nutrients: a focus on iron.

In plants, iron (Fe) transport and homeostasis are highly regulated processes. Fe deficiency or excess dramatically limits plant and algal productivity. Interestingly, complex and unexpected interconnections between Fe and various macro- and micronutrient homeostatic networks, supposedly maintaining general ionic equilibrium and balanced nutrition, are currently being uncovered. Although these interactions have profound consequences for our understanding of Fe homeostasis and its regulation, their molecular bases and biological significance remain poorly understood. Here, we review recent knowledge gained on how Fe interacts with micronutrient (e.g. zinc, manganese) and macronutrient (e.g. sulfur, phosphate) homeostasis, and on how these interactions affect Fe uptake and trafficking. Finally, we highlight the importance of developing an improved model of how Fe signaling pathways are integrated into functional networks to control plant growth and development in response to fluctuating environments.

The two copies of the zinc and cadmium ZIP6 transporter of Arabidopsis halleri have distinct effects on cadmium tolerance.

Plants have the ability to colonize highly diverse environments. The zinc and cadmium hyperaccumulator Arabidopsis halleri has adapted to establish populations on soils covering an extreme range of metal availabilities. The A. halleri ZIP6 gene presents several hallmarks of hyperaccumulation candidate genes: it is constitutively highly expressed in roots and shoots and is associated with a zinc accumulation quantitative trait locus. Here, we show that AhZIP6 is duplicated in the A. halleri genome. The two copies are expressed mainly in the vasculature in both A. halleri and Arabidopsis thaliana, indicative of conserved cis regulation, and acquired partial organ specialization. Yeast complementation assays determined that AhZIP6 is a zinc and cadmium transporter. AhZIP6 silencing in A. halleri or expression in A. thaliana alters cadmium tolerance, but has no impact on zinc and cadmium accumulation. AhZIP6-silenced plants display reduced cadmium uptake upon short-term exposure, adding AhZIP6 to the limited number of Cd transporters supported by in planta evidence. Altogether, our data suggest that AhZIP6 is key to fine-tune metal homeostasis in specific cell types. This study additionally highlights the distinct fates of duplicated genes in A. halleri.

Homology modeling and in vivo functional characterization of the zinc permeation pathway in a heavy metal P-type ATPase.

The P1B ATPase heavy metal ATPase 4 (HMA4) is responsible for zinc and cadmium translocation from roots to shoots in Arabidopsis thaliana. It couples ATP hydrolysis to cytosolic domain movements, enabling metal transport across the membrane. The detailed mechanism of metal permeation by HMA4 through the membrane remains elusive. Here, homology modeling of the HMA4 transmembrane region was conducted based on the crystal structure of a ZntA bacterial homolog. The analysis highlighted amino acids forming a metal permeation pathway, whose importance was subsequently investigated functionally through mutagenesis and complementation experiments in plants. Although the zinc pathway displayed overall conservation among the two proteins, significant differences were observed, especially in the entrance area with altered electronegativity and the presence of a ionic interaction/hydrogen bond network. The analysis also newly identified amino acids whose mutation results in total or partial loss of the protein function. In addition, comparison of zinc and cadmium accumulation in shoots of A. thaliana complemented lines revealed a number of HMA4 mutants exhibiting different abilities in zinc and cadmium translocation. These observations could be instrumental to design low cadmium-accumulating crops, hence decreasing human cadmium exposure.

Genetic architecture of a plant adaptive trait: QTL mapping of intraspecific variation for tolerance to metal pollution in Arabidopsis halleri.

Anthropogenic activities are among the main drivers of global change and result in drastic habitat modifications, which represent strong evolutionary challenges for biological species that can either migrate, adapt, or disappear. In this context, understanding the genetics of adaptive traits is a prerequisite to enable long-term maintenance of populations under strong environmental constraints. To examine these processes, a QTL approach was developed here using the pseudometallophyte Arabidopsis halleri, which displays among-population adaptive divergence for tolerance to metallic pollution in soils. An F2 progeny was obtained by crossing individuals from metallicolous and non-metallicolous populations from Italian Alps, where intense metallurgic activities have created strong landscape heterogeneity. Then, we combined genome de novo assembly and genome resequencing of parental genotypes to obtain single-nucleotide polymorphism markers and achieve high-throughput genotyping of the progeny. QTL analysis was performed using growth parameters and photosynthetic yield to assess zinc tolerance levels. One major QTL was identified for photosynthetic yield. It explained about 27% of the phenotypic variance. Functional annotation of the QTL and gene expression analyses highlighted putative candidate genes. Our study represents a successful approach combining evolutionary genetics and advanced molecular tools, helping to better understand how a species can face new selective pressures of anthropogenic origin.

Number of inadvertent RNA targets for morpholino knockdown in Danio rerio is largely underestimated: evidence from the study of Ser/Arg-rich splicing factors.

Although the involvement of Ser/Arg-rich (SR) proteins in RNA metabolism is well documented, their role in vertebrate development remains elusive. We, therefore, elected to take advantage of the zebrafish model organism to study the SR genes' functions using the splicing morpholino (sMO) microinjection and the programmable site-specific nucleases. Consistent with previous research, we revealed discrepancies between the mutant and morphant phenotypes and we show that these inconsistencies may result from a large number of unsuspected inadvertent morpholino RNA targets. While microinjection of MOs directed against srsf5a (sMOsrsf5a) led to developmental defects, the corresponding homozygous mutants did not display any phenotypic traits. Furthermore, microinjection of sMOsrsf5a into srsf5a-/- led to the previously observed morphant phenotype. Similar findings were observed for other SR genes. sMOsrsf5a alternative target genes were identified using deep mRNA sequencing. We uncovered that only 11 consecutive bases complementary to sMOsrsf5a are sufficient for binding and subsequent blocking of splice sites. In addition, we observed that sMOsrsf5a secondary targets can be reduced by increasing embryos growth temperature after microinjection. Our data contribute to the debate about MO specificity, efficacy and the number of unknown targeted sequences.

In vivo chlorophyll fluorescence screening allows the isolation of a Chlamydomonas mutant defective for NDUFAF3, an assembly factor involved in mitochondrial complex I assembly.

The qualitative screening method used to select complex I mutants in the microalga Chlamydomonas, based on reduced growth under heterotrophic conditions, is not suitable for high-throughput screening. In order to develop a fast screening method based on measurements of chlorophyll fluorescence, we first demonstrated that complex I mutants displayed decreased photosystem II efficiency in the genetic background of a photosynthetic mutation leading to reduced formation of the electrochemical proton gradient in the chloroplast (pgrl1 mutation). In contrast, single mutants (complex I and pgrl1 mutants) could not be distinguished from the wild type by their photosystem II efficiency under the conditions tested. We next performed insertional mutagenesis on the pgrl1 mutant. Out of about 3000 hygromycin-resistant insertional transformants, 46 had decreased photosystem II efficiency and three were complex I mutants. One of the mutants was tagged and whole genome sequencing identified the resistance cassette in NDUFAF3, a homolog of the human NDUFAF3 gene, encoding for an assembly factor involved in complex I assembly. Complemented strains showed restored complex I activity and assembly. Overall, we describe here a screening method which is fast and particularly suited for the identification of Chlamydomonas complex I mutants.

Contrasting cadmium resistance strategies in two metallicolous populations of Arabidopsis halleri.

While cadmium (Cd) tolerance is a constitutive trait in the Arabidopsis halleri species, Cd accumulation is highly variable. Recent adaptation to anthropogenic metal stress has occurred independently within the genetic units of A. halleri and the evolution of different mechanisms involved in Cd tolerance and accumulation has been suggested. To gain a better understanding of the mechanisms underlying Cd tolerance and accumulation in A. halleri, ionomic inductively coupled plasma mass spectrometry (ICP-MS), transcriptomic (RNA sequencing) and metabolomic (high-performance liquid chromatography-mass spectrometry) profiles were analysed in two A. halleri metallicolous populations from different genetic units (PL22 from Poland and I16 from Italy). The PL22 and I16 populations were both hypertolerant to Cd, but PL22 hyperaccumulated Cd while I16 behaved as an excluder both in situ and when grown hydroponically. The observed hyperaccumulator vs excluder behaviours were paralleled by large differences in the expression profiles of transporter genes. Flavonoid-related transcripts and metabolites were strikingly more abundant in PL22 than in I16 shoots. The role of novel A. halleri candidate genes possibly involved in Cd hyperaccumulation or exclusion was supported by the study of corresponding A. thaliana knockout mutants. Taken together, our results are suggestive of the evolution of divergent strategies for Cd uptake, transport and detoxification in different genetic units of A. halleri.

Adaptation to high zinc depends on distinct mechanisms in metallicolous populations of Arabidopsis halleri.

Zinc (Zn) hyperaccumulation and hypertolerance are highly variable traits in Arabidopsis halleri. Metallicolous populations have evolved from nearby nonmetallicolous populations in multiple independent adaptation events. To determine whether these events resulted in similar or divergent adaptive strategies to high soil Zn concentrations, we compared two A. halleri metallicolous populations from distant genetic units in Europe (Poland (PL22) and Italy (I16)). The ionomic (Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES)) and transcriptomic (RNA sequencing (RNA-Seq)) responses to growth at 5 and 150 µM Zn were analyzed in root and shoot tissues to examine the contribution of the geographic origin and treatment to variation among populations. These analyses were enabled by the generation of a reference A. halleri transcriptome assembly. The genetic unit accounted for the largest variation in the gene expression profile, whereas the two populations had contrasting Zn accumulation phenotypes and shared little common response to the Zn treatment. The PL22 population displayed an iron deficiency response at high Zn in roots and shoots, which may account for higher Zn accumulation. By contrast, I16, originating from a highly Zn-contaminated soil, strongly responded to control conditions. Our data suggest that distinct mechanisms support adaptation to high Zn in soils among A. halleri metallicolous populations.