Sergi Puig

Molecular mechanisms of copper uptake and distribution.

In the past few years, exciting advances have been made toward understanding how copper is transported into and distributed to cupro-proteins within cells. Recent work has identified high-affinity copper transporters at the plasma membrane in a number of organisms. The elucidation of the three-dimensional structure of copper chaperones and target cupro-proteins has shown that highly specific interactions between homologous domains foster copper transfer between conserved copper ligands, and facilitate a detailed understanding of vectorial copper-transfer reactions. Furthermore, the recent generation of mouse-knockout models, deficient in a high-affinity copper transporter, or in copper chaperones, has demonstrated the importance of copper uptake and targeted distribution in both predicted and fascinating unanticipated ways in growth and development.

Copper and iron homeostasis in Arabidopsis: responses to metal deficiencies, interactions and biotechnological applications

Plants have developed sophisticated mechanisms to tightly control the acquisition and distribution of copper and iron in response to environmental fluctuations. Recent studies with Arabidopsis thaliana are allowing the characterization of the diverse families and components involved in metal uptake, such as metal-chelate reductases and plasma membrane transporters. In parallel, emerging data on both intra- and intercellular metal distribution, as well as on long-distance transport, are contributing to the understanding of metal homeostatic networks in plants. Furthermore, gene expression analyses are deciphering coordinated mechanisms of regulation and response to copper and iron limitation. Prioritizing the use of metals in essential versus dispensable processes, and substituting specific metalloproteins by other metal counterparts, are examples of plant strategies to optimize copper and iron utilization. The metabolic links between copper and iron homeostasis are well documented in yeast, algae and mammals. In contrast, interactions between both metals in vascular plants remain controversial, mainly owing to the absence of copper-dependent iron acquisition. This review describes putative interactions between both metals at different levels in plants. The characterization of plant copper and iron homeostasis should lead to biotechnological applications aimed at the alleviation of iron deficiency and copper contamination and, thus, have a beneficial impact on agricultural and human health problems.

Placing metal micronutrients in context: transport and distribution in plants.

Plants have developed finely tuned mechanisms to efficiently acquire and balance the concentrations of essential metal micronutrients including iron, zinc, copper, and manganese, both at the cellular and systemic levels. The application of new emerging technologies to the study of Arabidopsis thaliana is providing a novel spatiotemporal view of plant metal homeostasis. These advances are uncovering unexpected links of metal homeostasis to central cellular processes, such as compartmentalization, daily redox oscillations, or transcriptional regulation. The intracellular compartmentalization of metals seems essential for optimizing the use of micronutrients during development and in response to deficiencies. Furthermore, recent discoveries indicate that protein metallation is highly sensitive to surrounding conditions, including metal redox state and concentration. Thus, some steps in metal delivery occur during protein folding at specific intracellular compartments. Finally, the daily nature in redox oscillations should be taken into account for a comprehensive understanding of global plant metal homeostasis.

Cooperation of Two mRNA-Binding Proteins Drives Metabolic Adaptation to Iron Deficiency

Iron (Fe) is an essential cofactor for a wide range of cellular processes. We have previously demonstrated in yeast that Cth2 is expressed during Fe deficiency and promotes degradation of a battery of mRNAs leading to reprogramming of Fe-dependent metabolism and Fe storage. We report here that the Cth2-homologous protein Cth1 is transiently expressed during Fe deprivation and participates in the response to Fe deficiency through the degradation of mRNAs primarily involved in mitochondrially localized activities including respiration and amino acid biosynthesis. In parallel, wild-type cells, but not cth1Deltacth2Delta cells, accumulate mRNAs encoding proteins that function in glucose import and storage and store high levels of glycogen. In addition, Fe deficiency leads to phosphorylation of Snf1, an AMP-activated protein kinase family member required for the cellular response to glucose starvation. These studies demonstrate a metabolic reprogramming as a consequence of Fe starvation that is dependent on the coordinated activities of two mRNA-binding proteins.

Mitotic Recombination and Genetic Changes in Saccharomyces cerevisiae during Wine Fermentation

ABSTRACT Natural strains of Saccharomyces cerevisiae are prototrophic homothallic yeasts that sporulate poorly, are often heterozygous, and may be aneuploid. This genomic constitution may confer selective advantages in some environments. Different mechanisms of recombination, such as meiosis or mitotic rearrangement of chromosomes, have been proposed for wine strains. We studied the stability of the URA3 locus of a URA3/ura3 wine yeast in consecutive grape must fermentations. ura3/ura3homozygotes were detected at a rate of 1 × 10−5 to 3 × 10−5 per generation, and mitotic rearrangements for chromosomes VIII and XII appeared after 30 mitotic divisions. We used the karyotype as a meiotic marker and determined that sporulation was not involved in this process. Thus, we propose a hypothesis for the genome changes in wine yeasts during vinification. This putative mechanism involves mitotic recombination between homologous sequences and does not necessarily imply meiosis.

Stress response and expression patterns in wine fermentations of yeast genes induced at the diauxic shift.

During wine fermentation yeasts quickly reach a stationary phase, where cells are metabolically active by consuming sugars present in grape must. It is, consequently, of great interest at this stage to identify suitable gene promoters that may be used to induce the expression of genes with enological applications. With this aim, we have studied a group of genes showing an induction peak at the diauxic shift, and possessing stress response elements (STRE) at their promoters. We have determined their induction levels under individualized stress conditions, such as carbon source starvation or high salt concentrations. In all the cases studied, the activation and/or basal transcription are dependent on the transcriptional factors Msn2p and Msn4p. We have analysed the expression patterns and mRNA levels during wine fermentation, and have found that they are all activated at the stationary phase. Finally, we have identified SPI1, a new highly expressed yeast gene which is specifically induced at the stationary phase of both microvinification and laboratory growth conditions. Copyright

Deregulated copper transport affects Arabidopsis development especially in the absence of environmental cycles.

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.

Regulation of copper transport in Arabidopsis thaliana : a biochemical oscillator?

Plants are among the most versatile higher eukaryotes in accommodating environmental copper availability to largely variable demands. In particular, copper deficiency in soils is a threat for plant survival since it mostly affects reproductive structures. One of the strategies that plant cells use to overcome this situation is to increase copper levels by expressing high-affinity copper transporters delivering the metal to the cytosol. In this minireview, we discuss recent advances in the structure, function, and regulation of the CTR/COPT family of copper transporters, and pay special attention to the Arabidopsis thaliana counterparts. These are constituted by transmembrane polypeptides, containing several copper-binding sequences of functional and/or regulatory value, and assembling as trimers. Copper deficiency activates the expression of some members of the COPT family via the interaction of the SPL7 transcription factor with reiterative GTAC motifs present in their promoters. Interestingly, the regulation of the synthesis of these transporters by copper itself constitutes a negative-feedback loop that could cause a sustained oscillation in the cytosolic copper levels. We analyze the theoretical conditions required for this hypothetical copper oscillation and the potential advantages of synchronization with other cycles. Diverse data in other organisms point to the relationship between copper homeostasis and circadian cycles.

The intracellular Arabidopsis COPT5 transport protein is required for photosynthetic electron transport under severe copper deficiency

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.

Arabidopsis copper transport protein COPT2 participates in the cross talk between iron deficiency responses and low-phosphate signaling.

A copper transport protein affects copper, iron, and phosphate deficiency responses. 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.