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.

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.

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.

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.

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.

di-Cysteine motifs in the C-terminus of plant HMA4 proteins confer nanomolar affinity for zinc and are essential for HMA4 function in vivo.

The PIB ATPase heavy metal ATPase 4 (HMA4) has a central role in the zinc homeostasis network of Arabidopsis thaliana. This membrane protein loads metal from the pericycle cells into the xylem in roots, thereby allowing root to shoot metal translocation. Moreover, HMA4 is key for zinc hyperaccumulation as well as zinc and cadmium hypertolerance in the pseudometallophyte Arabidopsis halleri. The plant-specific cytosolic C-terminal extension of HMA4 is rich in putative metal-binding residues and has substantially diverged between A. thaliana and A. halleri. To clarify the function of the domain in both species, protein variants with truncated C-terminal extension, as well as with mutated di-Cys motifs and/or a His-stretch, were functionally characterized. We show that di-Cys motifs, but not the His-stretch, contribute to high affinity zinc binding and function in planta. We suggest that the HMA4 C-terminal extension is at least partly responsible for protein targeting to the plasma membrane. Finally, we reveal that the C-terminal extensions of both A. thaliana and A. halleri HMA4 proteins share similar function, despite marginally different zinc-binding capacity.

Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.

PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases.

Zinc triggers a complex transcriptional and post-transcriptional regulation of the metal homeostasis gene FRD3 in Arabidopsis relatives.

In Arabidopsis thaliana, FRD3 (FERRIC CHELATE REDUCTASE DEFECTIVE 3) plays a central role in metal homeostasis. FRD3 is among a set of metal homeostasis genes that are constitutively highly expressed in roots and shoots of Arabidopsis halleri, a zinc hyperaccumulating and hypertolerant species. Here, we examined the regulation of FRD3 by zinc in both species to shed light on the evolutionary processes underlying the evolution of hyperaccumulation in A. halleri. We combined gene expression studies with the use of ß-glucuronidase and green fluorescent protein reporter constructs to compare the expression profile and transcriptional and post-transcriptional regulation of FRD3 in both species. The AtFRD3 and AhFRD3 genes displayed a conserved expression profile. In A. thaliana, alternative transcription initiation sites from two promoters determined transcript variants that were differentially regulated by zinc supply in roots and shoots to favour the most highly translated variant under zinc-excess conditions. In A. halleri, a single transcript variant with higher transcript stability and enhanced translation has been maintained. The FRD3 gene thus undergoes complex transcriptional and post-transcriptional regulation in Arabidopsis relatives. Our study reveals that a diverse set of mechanisms underlie increased gene dosage in the A. halleri lineage and illustrates how an environmental challenge can alter gene regulation.

Functional analysis of the three HMA4 copies of the metal hyperaccumulator Arabidopsis halleri.

In Arabidopsis halleri, the HMA4 gene has an essential function in Zn/Cd hypertolerance and hyperaccumulation by mediating root-to-shoot translocation of metals. Constitutive high expression of AhHMA4 results from a tandem triplication and cis-activation of the promoter of all three copies. The three AhHMA4 copies possess divergent promoter sequences, but highly conserved coding sequences, and display identical expression profiles in the root and shoot vascular system. Here, an AhHMA4::GFP fusion was expressed under the control of each of the three A. halleri HMA4 promoters in a hma2hma4 double mutant of A. thaliana to individually examine the function of each AhHMA4 copy. The protein showed non-polar localization at the plasma membrane of the root pericycle cells of both A. thaliana and A. halleri. The expression of each AhHMA4::GFP copy complemented the severe Zn-deficiency phenotype of the hma2hma4 mutant by restoring root-to-shoot translocation of Zn. However, each copy had a different impact on metal homeostasis in the A. thaliana genetic background: AhHMA4 copies 2 and 3 were more highly expressed and provided higher Zn tolerance in roots and accumulation in shoots than copy 1, and AhHMA4 copy 3 also increased Cd tolerance in roots. These data suggest a certain extent of functional differentiation among the three A. halleri HMA4 copies, stemming from differences in expression levels rather than in expression profile. HMA4 is a key node of the Zn homeostasis network and small changes in expression level can have a major impact on Zn allocation to root or shoot tissues.