Copper and iron are essential micronutrients for most living organisms because they participate as cofactors in biological processes, including respiration, photosynthesis, and oxidative stress protection. In many eukaryotic organisms, including yeast (Saccharomyces cerevisiae) and mammals, copper and iron homeostases are highly interconnected; yet, such interdependence is not well established in higher plants. Here, we propose that COPT2, a high-affinity copper transport protein, functions under copper and iron deficiencies in Arabidopsis (Arabidopsis thaliana). COPT2 is a plasma membrane protein that functions in copper acquisition and distribution. Characterization of the COPT2 expression pattern indicates a synergic response to copper and iron limitation in roots. We characterized a knockout of COPT2, copt2-1, that leads to increased resistance to simultaneous copper and iron deficiencies, measured as reduced leaf chlorosis and improved maintenance of the photosynthetic apparatus. We propose that COPT2 could play a dual role under iron deficiency. First, COPT2 participates in the attenuation of copper deficiency responses driven by iron limitation, possibly to minimize further iron consumption. Second, global expression analyses of copt2-1 versus wild-type Arabidopsis plants indicate that low-phosphate responses increase in the mutant. These results open up new biotechnological approaches to fight iron deficiency in crops.

Copper is an essential cofactor for key processes in plants, but it exerts harmful effects when in excess. Previous work has shown that the Arabidopsis (Arabidopsis thaliana) COPT1 high-affinity copper transport protein participates in copper uptake through plant root tips. Here, we show that COPT1 protein localizes to the plasma membrane of Arabidopsis cells and the phenotypic effects of transgenic plants overexpressing either COPT1 or COPT3, the latter being another high-affinity copper transport protein family member. Both transgenic lines exhibit increased endogenous copper levels and are sensitive to the copper in the growth medium. Additional phenotypes include decreased hypocotyl growth in red light and differentially affected flowering times depending on the photoperiod. Furthermore, in the absence of environmental cycles, such as light and temperature, the survival of plants overexpressing COPT1 or COPT3 is compromised. Consistent with altered circadian rhythms, the expression of the nuclear circadian clock genes CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) is substantially reduced in either COPT1-or COPT3-overexpressing plants. Copper affects the amplitude and the phase, but not the period, of the CCA1 and LHY oscillations in wild-type plants. Copper also drives a reduction in the expression of circadian clock output genes. These results reveal that the spatiotemporal control of copper transport is a key aspect of metal homeostasis that is required for Arabidopsis fitness, especially in the absence of environmental cues.

Summary Copper is an essential micronutrient that functions as a redox cofactor in multiple plant processes, including photosynthesis. Arabidopsis thaliana possesses a conserved family of CTR‐like high‐affinity copper transport proteins denoted as COPT1‐5. COPT1, the only family member that is functionally characterized, participates in plant copper acquisition. However, little is known about the function of the other Arabidopsis COPT proteins in the transport and distribution of copper. Here, we show that a functional fusion of COPT5 to the green fluorescent protein localizes in Arabidopsis cells to the prevacuolar compartment. Plants defective in COPT5 do not exhibit any significant phenotype under copper‐sufficient conditions, but their growth is compromised under copper limitation. Under extreme copper deficiency, two independent copt5 knockout mutant lines exhibit severe defects in vegetative growth and root elongation, low chlorophyll content, and impairment in the photosynthetic electron transfer. All these phenotypes are rescued when the wild‐type copy of the COPT5 gene is retransformed into a copt5 knockout line or when copper, but not other metals, are added to the medium. COPT5 is expressed in vascular tissues, with elevated levels in roots. Taken together, these results suggest that COPT5 plays an important role in the plant response to environmental copper scarcity, probably by remobilizing copper from prevacuolar vesicles, which could act as internal stores or recycling vesicles to provide the metal cofactor to key copper‐dependent processes such as photosynthesis.

Copper (Cu), an essential redox active cofactor, participates in fundamental biological processes, but it becomes highly cytotoxic when present in excess. Therefore, living organisms have established suitable mechanisms to balance cellular and systemic Cu levels. An important strategy to maintain Cu homeostasis consists of regulating uptake and mobilization via the conserved family of CTR/COPT Cu transport proteins. In the model plant Arabidopsis thaliana, COPT1 protein mediates root Cu acquisition, whereas COPT5 protein functions in Cu mobilization from intracellular storage organelles. The function of these transporters becomes critical when environmental Cu bioavailability diminishes. However, little is know about the mechanisms that mediate plant Cu distribution. In this report, we present evidence supporting an important role for COPT6 in Arabidopsis Cu distribution. Similarly to COPT1 and COPT2, COPT6 fully complements yeast mutants defective in high-affinity Cu uptake and localizes to the plasma membrane of Arabidopsis cells. Whereas COPT2 mRNA is only up-regulated upon severe Cu deficiency, COPT6 transcript is expressed under Cu excess conditions and displays a more gradual increase in response to decreases in environmental Cu levels. Consistent with COPT6 expression in aerial vascular tissues and reproductive organs, copt6 mutant plants exhibit altered Cu distribution under Cu-deficient conditions, including increased Cu in rosette leaves but reduced Cu levels in seeds. This altered Cu distribution is fully rescued when the wild-type COPT6 gene is reintroduced into the copt6 mutant line. Taken together, these findings highlight the relevance of COPT6 in shoot Cu redistribution when environmental Cu is limited.

Iron is a redox active element that functions as an essential cofactor in multiple metabolic pathways, including respiration, DNA synthesis and translation. While indispensable for eukaryotic life, excess iron can lead to oxidative damage of macromolecules. Therefore, living organisms have developed sophisticated strategies to optimally regulate iron acquisition, storage and utilization in response to fluctuations in environmental iron bioavailability. In the yeast Saccharomyces cerevisiae, transcription factors Aft1/Aft2 and Yap5 regulate iron metabolism in response to low and high iron levels, respectively. In addition to producing and assembling iron cofactors, mitochondrial iron-sulfur (Fe/S) cluster biogenesis has emerged as a central player in iron sensing. A mitochondrial signal derived from Fe/S synthesis is exported and converted into an Fe/S cluster that interacts directly with Aft1/Aft2 and Yap5 proteins to regulate their transcriptional function. Various conserved proteins, such as ABC mitochondrial transporter Atm1 and, for Aft1/Aft2, monothiol glutaredoxins Grx3 and Grx4 are implicated in this iron-signaling pathway. The analysis of a wide range of S. cerevisiae strains of different geographical origins and sources has shown that yeast strains adapted to high iron display growth defects under iron-deficient conditions, and highlighted connections that exist in the response to both opposite conditions. Changes in iron accumulation and gene expression profiles suggest differences in the regulation of iron homeostasis genes.

In response to iron deficiency, the budding yeast Saccharomyces cerevisiae undergoes a metabolic remodeling in order to optimize iron utilization. The tandem zinc finger (TZF)-containing protein Cth2 plays a critical role in this adaptation by binding and promoting the degradation of multiple mRNAs that contain AU-rich elements (AREs). Here, we demonstrate that Cth2 also functions as a translational repressor of its target mRNAs. By complementary approaches, we demonstrate that Cth2 protein inhibits the translation of SDH4, which encodes a subunit of succinate dehydrogenase, and CTH2 mRNAs in response to iron depletion. Both the AREs within SDH4 and CTH2 transcripts, and the Cth2 TZF are essential for translational repression. We show that the role played by Cth2 as a negative translational regulator extends to other mRNA targets such as WTM1, CCP1 and HEM15. A structure-function analysis of Cth2 protein suggests that the Cth2 amino-terminal domain (NTD) is important for both mRNA turnover and translation inhibition, while its carboxy-terminal domain (CTD) only participates in the regulation of translation, but is dispensable for mRNA degradation. Finally, we demonstrate that the Cth2 CTD is physiologically relevant for adaptation to iron deficiency.

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MicroRNAs contribute to the adaptation of plants to varying environmental conditions by affecting systemic mineral nutrient homeostasis. Copper and iron deficiencies antagonistically control the expression of Arabidopsis thaliana microRNA408 ( miR408 ), which post-transcriptionally regulates laccase-like multicopper oxidase family members LAC3 , LAC12 , and LAC13 . In this work, we used miR408 T-DNA insertion mutants (408-KO1 and 408-KO2) and a previously characterized transgenic line overexpressing miR408 (35S:408-14) to explore how miR408 influences copper- and iron-dependent metabolism. We observed that the altered expression of miR408 diminished plant performance and the activation of the iron-regulated genes under iron-deficient conditions. Consistently with the low expression of the miR408 -target laccases, we showed that the vascular bundle lignification of the 35S:408-14 plants diminished. The decrease in the phenoloxidase and ferroxidase activities exhibited by wild-type plants under iron deficiency did not occur in the 408-KO1 plants, probably due to the higher expression of laccases. Finally, we observed that the hydrogen peroxide levels under iron starvation were altered in both the 408-KO1 and 35S:408-14 lines. Taken together, these results suggest that Arabidopsis plants with modified miR408 levels undergo multiple deregulations under iron-deficient conditions.