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.

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.

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.

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.