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.

Ferrous iron uptake via IRT1/ZIP evolved at least twice in green plants.

Iron (Fe) is essential for virtually all organisms, being irreplaceable because of its electrochemical properties that enable many biochemical processes, including photosynthesis. Besides its abundance, Fe is generally found in the poorly soluble form of ferric iron (Fe3+ ), while most plants uptake the soluble form Fe2+ . The model angiosperm Arabidopsis thaliana, for example, captures Fe through a mechanism that lowers rhizosphere pH through proton pumping that increases Fe3+ solubility, which is then reduced by a membrane-bound reductase and transported into the cell by the zinc-regulated, iron-regulated transporter-like protein (ZIP) family protein AtIRT1. ZIP proteins are transmembrane transporters of divalent metals such as Fe2+ , Zn2+ , Mn2+ , and Cd2+ . In this work, we investigated the evolution of functional homologs of IRON-REGULATED TRANSPORTER 1/ZIP in the supergroup Archaeplastida (Viridiplantae + Rhodophyta + Glaucophyta) using 51 genomes of diverse lineages. Our analyses suggest that Fe is acquired through deeply divergent ZIP proteins in land plants and chlorophyte green algae, indicating that Fe2+ uptake by ZIP proteins evolved independently at least twice throughout green plant evolution. Our results indicate that the archetypical IRON-REGULATED TRANSPORTER (IRT) proteins from angiosperms likely emerged before the origin of land plants during early streptophyte algae terrestrialization, a process that required the evolution of Fe acquisition in terrestrial subaerial settings.

Regulatory module WRKY33-ATL31-IRT1 mediates cadmium tolerance in Arabidopsis.

Cadmium (Cd) is one of the most dangerous environmental pollutants among heavy metals, and threatens food safety and human health by accumulating in plant sink tissues. Here, we report a novel regulatory cascade that profoundly influences Cd tolerance in Arabidopsis. Phenotypic analysis showed that an insertional knockdown mutation at the Arabidopsis Tóxicos en Levadura 31 (ATL31) locus resulted in hypersensitivity to Cd stress, most likely due to a significant increase in Cd accumulation. Consistently, ATL31-overexpressing lines exhibited enhanced Cd stress tolerance and reduced Cd accumulation. Further, IRON-REGULATED TRANSPORTER 1 (IRT1) was identified, and yeast two-hybrid, co-immunoprecipitation and bimolecular fluorescence complementation assays demonstrated its interaction with ATL31. Biochemical, molecular, and genetic analyses showed that IRT1 is targeted by ATL31 for ubiquitin-conjugated degradation in response to Cd stress. Intriguingly, transcription of ATL31 was strongly induced by Cd stress. In addition, transgenic and molecular analyses showed that WRKY33 directly activated the transcription of ATL31 in response to Cd stress and positively regulated Cd tolerance. Genetic analysis indicated that ATL31 acts upstream of IRT1 and downstream of WRKY33 to regulate Cd tolerance. Our study revealed that the WRKY33-ATL31-IRT1 module plays a crucial role in timely blocking Cd absorption to prevent metal toxicity in Arabidopsis.

Establishment of hairy root system of transgenic IRT1 brassica campestris L. and preliminary study of its effect on cadmium enrichment.

Cadmium (Cd) is the main heavy metal pollutant in soil. The combination of genetic engineering technology and Rizobium rhizogenes mediated technology can effectively improve the enrichment efficiency of heavy metals in super accumulators and reduce soil heavy metal pollution. In this study, the transgenic hairy root system containing the IRT1 gene of Cd hyperaccumulator-Brassica campestris L. was successfully constructed by the R. rhizogenes mediated method (IRT1 gene come from Arabidopsis thaliana). The hairy roots of each subculture can grow stably within 6 weeks, and IRT1 gene will not be lost within 50 subcultures., which is detected using PCR method. The results of Cd enrichment experiments showed that after treatment with 100 µmol/L Cd for 14 days, the growth state of transgenic IRT1 hairy roots only showed slight browning. Also, the accumulation value of Cd reached 331.61 µg/g and the enrichment efficiency of transgenic IRT1 hairy roots was 13.8% higher than that of wild-type hairy roots. Western blotting results showed that the expression of IRT1 protein in transgenic hairy roots was significantly higher than that of wild-type hairy roots under Cd stress. The above results indicated that the overexpression of IRT1 gene can help B. campestris L. hairy roots to effectively cope with Cd stress and improve its ability to enrich Cd.

In this study, the transgenic hairy root system containing the IRT1 gene of Cd hyperaccumulator-Brassica campestris L. was successfully constructed by the Rizobium rhizogenes mediated method. At the same time, the growth state and cadmium enrichment efficiency of transgenic hairy roots under different concentrations of Cd stress were studied. Overexpression of IRT1 gene can effectively improve the tolerance of hairy root to Cd. The enrichment efficiency of transgenic IRT1 hairy roots was 13.8% higher than that of wild-type hairy roots. The transgenic IRT1 hairy root system established in this study can be used as a reliable experimental model for the study of Cd adsorption mechanism, and can be further regenerated to obtain transgenic IRT1 B. campestris L. plants for the study of heavy metal Cd pollution remediation.

Birth, life and death of the Arabidopsis IRT1 iron transporter: the role of close friends and foes.

MAIN CONCLUSION: IRT1 intracellular dynamics and function are finely controlled through protein-protein interactions. In plants, iron uptake from the soil is tightly regulated to allow optimal growth and development. Iron acquisition in Arabidopsis root epidermal cells requires the IRT1 transporter, which also mediates the entry of non-iron metals. In this mini-review, we describe how protein-protein interactions regulate IRT1 intracellular dynamics and IRT1-mediated metal uptake to maintain iron homeostasis. Recent interactomic data provided interesting clues on IRT1 secretion and the putative involvement of COPI- and COPII-mediated pathways. Once delivered to the plasma membrane, IRT1 can interact with other components of the iron uptake machinery to form an iron acquisition complex that likely optimizes iron entrance in root epidermal cells. Then, IRT1 may be internalized from the plasma membrane. In the past decade, IRT1 endocytosis emerged as an essential mechanism to control IRT1 subcellular localization and thus to tune iron uptake. Interestingly, IRT1 endocytosis and degradation are regulated by its non-iron metal substrates in an ubiquitin-dependent manner, which requires a set of interacting-proteins including kinases, E3 ubiquitin ligases and ESCRT complex subunits. This mechanism is essential to avoid non-iron metal overload in Arabidopsis when the iron is scarce.

Endocytosis in plants: Peculiarities and roles in the regulated trafficking of plant metal transporters.

The removal of transmembrane proteins from the plasma membrane via endocytosis has emerged as powerful strategy in the regulation of receptor signalling and molecule transport. In the last decade, IRON-REGULATED TRANSPORTER1 (IRT1) has been established as one of the key plant model proteins for studying endomembrane trafficking. The use of IRT1 and additional other metal transporters has uncovered novel factors involved in plant endocytosis and facilitated a better understanding of the role of endocytosis in the fine balancing of plant metal homoeostasis. In this review, we outline the specifics of plant endocytosis compared to what is known in yeast and mammals, and based on several examples, we demonstrate how studying metal transport has contributed to extending our knowledge of endocytic trafficking by shedding light on novel regulatory mechanisms and factors.

Transcriptional effects of cadmium on iron homeostasis differ in calamine accessions of Noccaea caerulescens.

Calamine accessions of the zinc/cadmium/nickel hyperaccumulator, Noccaea caerulescens, exhibit striking variation in foliar cadmium accumulation in nature. The Ganges accession (GA) from Southern France displays foliar cadmium hyperaccumulation (>1000 µg g-1 DW), whereas the accession La Calamine (LC) from Belgium, with similar local soil metal composition, does not (<100 µg g-1 DW). All calamine accessions are cadmium hypertolerant. To find out the differences between LC and GA in their basic adaptation mechanisms, we bypassed the cadmium excluding phenotype of LC by exposing the plants to 50 µm cadmium in hydroponics, achieving equal cadmium accumulation in the shoots. The iron content increased in the roots of both accessions. GA exhibited significant decreases in manganese and zinc contents in the roots and shoots, approaching those in LC. Altogether 702 genes responded differently to cadmium exposure between the accessions, 157 and 545 in the roots and shoots, respectively. Cadmium-exposed LC showed a stress response and had decreased levels of a wide range of photosynthesis-related transcripts. GA showed less changes, mainly exhibiting an iron deficiency-like response. This included increased expression of genes encoding five iron deficiency-regulated bHLH transcription factors, ferric reduction oxidase FRO2, iron transporters IRT1 and OPT3, and nicotianamine synthase NAS1, and decreased expression of genes encoding ferritins and NEET (a NEET family iron-sulfur protein), which is possibly involved in iron transfer, distribution and/or management. The function of the IRT1 gene in the accessions was compared. We conclude that the major difference between the two accessions is in the way they cope with iron under cadmium exposure.

Phospho-mutant activity assays provide evidence for alternative phospho-regulation pathways of the transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR.

The key basic helix-loop-helix (bHLH) transcription factor in iron (Fe) uptake, FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), is controlled by multiple signaling pathways, important to adjust Fe acquisition to growth and environmental constraints. FIT protein exists in active and inactive protein pools, and phosphorylation of serine Ser272 in the C-terminus, a regulatory domain of FIT, provides a trigger for FIT activation. Here, we use phospho-mutant activity assays and study phospho-mimicking and phospho-dead mutations of three additional predicted phosphorylation sites, namely at Ser221 and at tyrosines Tyr238 and Tyr278, besides Ser 272. Phospho-mutations at these sites affect FIT activities in yeast, plant, and mammalian cells. The diverse array of cellular phenotypes is seen at the level of cellular localization, nuclear mobility, homodimerization, and dimerization with the FIT-activating partner bHLH039, promoter transactivation, and protein stability. Phospho-mimicking Tyr mutations of FIT disturb fit mutant plant complementation. Taken together, we provide evidence that FIT is activated through Ser and deactivated through Tyr site phosphorylation. We therefore propose that FIT activity is regulated by alternative phosphorylation pathways.

Comparative RNA-seq analysis of nickel hyperaccumulating and non-accumulating populations of Senecio coronatus (Asteraceae).

Most metal hyperaccumulating plants accumulate nickel, yet the molecular basis of Ni hyperaccumulation is not well understood. We chose Senecio coronatus to investigate this phenomenon as this species displays marked variation in shoot Ni content across ultramafic outcrops in the Barberton Greenstone Belt (South Africa), thus allowing an intraspecific comparative approach to be employed. No correlation between soil and shoot Ni contents was observed, suggesting that this variation has a genetic rather than environmental basis. This was confirmed by our observation that the accumulation phenotype of plants from two hyperaccumulator and two non-accumulator populations was maintained when the plants were grown on a soil mix from these four sites for 12 months. We analysed the genetic variation among 12 serpentine populations of S. coronatus, and used RNA-seq for de novo transcriptome assembly and analysis of gene expression in hyperaccumulator versus non-accumulator populations. Genetic analysis revealed the presence of hyperaccumulators in two well supported evolutionary lineages, indicating that Ni hyperaccumulation may have evolved more than once in this species. RNA-Seq analysis indicated that putative homologues of transporters associated with root iron uptake in plants are expressed at elevated levels in roots and shoots of hyperaccumulating populations of S. coronatus from both evolutionary lineages. We hypothesise that Ni hyperaccumulation in S. coronatus may have evolved through recruitment of these transporters, which play a role in the iron-deficiency response in other plant species.

The bifunctional transporter-receptor IRT1 at the heart of metal sensing and signalling.

Transporters are at the centre of regulatory modules allowing optimal assimilation, distribution or efflux of substrate molecules. The IRT1 root metal transporter represents a textbook example in which detailed regulatory networks have been shown to integrate several endogenous and exogenous cues at various levels to regulate its expression and to fine tune iron uptake. Here, we summarise recent advances in the dissection of the transcriptional and posttranslational control of IRT1 by its various metals substrates and discuss the emerging role of IRT1 in the direct sensing of non-iron metals flowing through IRT1 to drive its degradation. We propose that transporters that also act as receptors are likely to be a common theme in the regulation of nutrient transport by sensing local nutrient concentrations.