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.

Multilayered regulation of iron homeostasis in Arabidopsis.

Iron (Fe) is an essential micronutrient for plant growth and development due to its role in crucial processes such as photosynthesis and modulation of the redox state as an electron donor. While Fe is one of the five most abundant metals in the Earth's crust, it is poorly accessible to plants in alkaline soils due to the formation of insoluble complexes. To limit Fe deficiency symptoms, plant have developed a highly sophisticated regulation network including Fe sensing, transcriptional regulation of Fe-deficiency responsive genes, and post-translational modifications of Fe transporters. In this mini-review, we detail how plants perceive intracellular Fe status and how they regulate transporters involved in Fe uptake through a complex cascade of transcription factors. We also describe the current knowledge about intracellular trafficking, including secretion to the plasma membrane, endocytosis, recycling, and degradation of the two main Fe transporters, IRON-REGULATED TRANSPORTER 1 (IRT1) and NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN 1 (NRAMP1). Regulation of these transporters by their non-Fe substrates is discussed in relation to their functional role to avoid accumulation of these toxic metals during Fe limitation.

Imaging and Quantifying the Endocytosis of IRON-REGULATED TRANSPORTER1 from Arabidopsis.

Iron plays an essential role in plant metabolism and the regulation of its transport is essential for the plant. In Arabidopsis thaliana, iron uptake in root epidermal cells is mediated by the IRT1 (IRON-REGULATED TRANSPORTER 1) broad-spectrum transporter. The regulation of the IRT1 protein is controlled by sophisticated mechanisms that allow it to fine-tune the amount of transporter found at the plasma membrane and to modulate the uptake of iron and divalent metals transported by IRT1. IRT1 shows low selectivity and transports different metals such as manganese, zinc, cobalt, and cadmium. An excess of these non-iron metal substrates of IRT1 is toxic for the plant. The ability of plants to adapt to non-iron metal stress is based on the sensing of their excess, leading to the internalization and degradation of IRT1. IRT1 acts as a bifunctional transporter/receptor directly sensing metal non-iron excess and then undergoes a series of post-translational modifications of the protein culminating in its endocytosis and vacuolar degradation. To monitor the intracellular dynamics of IRT1, we describe in this chapter a live cell imaging approach to follow and quantify IRT1-mCitrine trafficking from the plasma membrane to the vacuole.

Differential metal sensing and metal-dependent degradation of the broad spectrum root metal transporter IRT1.

Iron is an essential micronutrient for plant growth and development. Under low iron conditions, Arabidopsis plants take up soil iron using the root iron transporter IRT1. In addition to iron, IRT1 also transports others divalent metals, including cadmium, which consequently accumulates into plant tissues and enters the food chain. IRT1 expression was shown to be regulated at the transcriptional and post-translational levels by its essential metal substrates to maximize iron uptake while limiting the accumulation of zinc, manganese, or cobalt. Here, we characterized the regulation of IRT1 by cadmium. A short-term exposure to cadmium decreased the cell surface levels of IRT1 through endocytosis and degradation, but with a lower efficiency than observed for other IRT1 metal substrates. We demonstrated that IRT1 endocytosis in response to cadmium is mediated through the direct binding of cadmium to histidine residues within the regulatory loop of IRT1. However, we revealed that the affinity of the metal sensing motif is much lower for cadmium compared to other metal substrates of IRT1. Finally, we proved that cadmium-induced IRT1 degradation takes place through ubiquitin-mediated endocytosis driven by the UBC35/36 E2 ubiquitin-conjugating enzymes and the IDF1 E3 ubiquitin ligase. Altogether, this work sheds light on the mechanisms of cadmium-mediated downregulation of IRT1 and provides an additional molecular basis for cadmium accumulation and toxicity in plants.

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.

The many facets of protein ubiquitination and degradation in plant root iron-deficiency responses.

Organisms need to deal with the absolute requirement for metals and also their possible toxicity. This is achieved through an intricate network of signaling pathways that are integrated to ultimately fine-tune iron uptake and metabolism. The mechanisms by which plants cope with iron limitation and the associated genomic responses are well characterized. On top of this transcriptional cascade is another level of regulation involving the post-translational protein modification and degradation. The ubiquitination and/or degradation of several transcription factors in the iron-deficiency signaling pathways and metal transporters has recently come to light. In this review we discuss the mechanisms and possible roles of protein modification and turnover in the regulation of root iron-deficiency responses. We also highlight the tight coupling between metal sensing by E3 ubiquitin ligases or bifunctional transporters and protein degradation.

Dynamic Control of the High-Affinity Iron Uptake Complex in Root Epidermal Cells.

In plants, iron uptake from the soil is tightly regulated to ensure optimal growth and development. Iron absorption in Arabidopsis root epidermal cells requires the IRT1 transporter that also allows the entry of certain non-iron metals, such as Zn, Mn, and Co. Recent work demonstrated that IRT1 endocytosis and degradation are controlled by IRT1 non-iron metal substrates in a ubiquitin-dependent manner. To better understand how metal uptake is regulated, we identified IRT1-interacting proteins in Arabidopsis roots by mass spectrometry and established an interactome of IRT1. Interestingly, the AHA2 proton pump and the FRO2 reductase, both of which work in concert with IRT1 in the acidification-reduction-transport strategy of iron uptake, were part of this interactome. We confirmed that IRT1, FRO2, and AHA2 associate through co-immunopurification and split-ubiquitin analyses, and uncovered that they form tripartite direct interactions. We characterized the dynamics of the iron uptake complex and showed that FRO2 and AHA2 ubiquitination is independent of the non-iron metal substrates transported by IRT1. In addition, FRO2 and AHA2 are not largely endocytosed in response to non-iron metal excess, unlike IRT1. Indeed, we provide evidence that the phosphorylation of IRT1 in response to high levels of non-iron metals likely triggers dissociation of the complex. Overall, we propose that a dedicated iron-acquisition protein complex exists at the cell surface of Arabidopsis root epidermal cells to optimize iron uptake.

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.

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.

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.