The combination of a microbial and a non-microbial biostimulant increases yield in lettuce (Lactuca sativa) under salt stress conditions by up-regulating cytokinin biosynthesis.

Salinization poses a significant challenge in agriculture, exacerbated by anthropogenic global warming. Biostimulants, derived from living microorganisms or natural extracts, have emerged as valuable tools for conventional and organic agriculture. However, our understanding of the molecular mechanisms underlying the effects of biostimulants is very limited, especially in crops under real cultivation conditions. In this study, we adopted an integrative approach to investigate the effectiveness of the combined application of plant growth-promoting bacterium (Bacillus megaterium strain BM08) and a non-microbial biostimulant under control conditions (normal watering) and salt stress. After confirming the yield increase under both conditions, we investigated the molecular mechanisms underlying the observed effect by measuring a number of physiological parameters (i.e., lipid peroxidation, antioxidants, chlorophylls, total phenolics and phytohormone content), as well as RNA sequencing and primary metabolite analyses. Our findings reveal that the combined effect of the microbial and non-microbial biostimulants led to a decrease in the antioxidant response and an up-regulation of genes involved in cytokinin biosynthesis under salt stress conditions. This, in turn, resulted in a higher concentration of the bioactive cytokinin, isopentenyladenosine, in roots and leaves and an increase in γ-aminobutyric acid, a non-proteic amino acid related to abiotic stress responses. In addition, we observed a decrease in malic acid, along with an abscisic acid (ABA)-independent up-regulation of SR-kinases, a family of protein kinases associated with abiotic stress responses. Furthermore, we observed that the single application of the non-microbial biostimulant triggers an ABA-dependent response under salt stress; however, when combined with the microbial biostimulant, it potentiated the mechanisms triggered by the BM08 bacterial strain. This comprehensive investigation shows that the combination of two biostimulants is able to elicit a cytokinin-dependent response that may explain the observed yield increase under salt stress conditions.

Modulation of potassium transport to increase abiotic stress tolerance in plants.

Potassium is the major cation responsible for the maintenance of the ionic environment in plant cells. Stable potassium homeostasis is indispensable for virtually all cellular functions, and, concomitantly, viability. Plants must cope with environmental changes such as salt or drought that can alter ionic homeostasis. Potassium fluxes are required to regulate the essential process of transpiration, so a constraint on potassium transport may also affect the plant's response to heat, cold, or oxidative stress. Sequencing data and functional analyses have defined the potassium channels and transporters present in the genomes of different species, so we know most of the proteins directly participating in potassium homeostasis. The still unanswered questions are how these proteins are regulated and the nature of potential cross-talk with other signaling pathways controlling growth, development, and stress responses. As we gain knowledge regarding the molecular mechanisms underlying regulation of potassium homeostasis in plants, we can take advantage of this information to increase the efficiency of potassium transport and generate plants with enhanced tolerance to abiotic stress through genetic engineering or new breeding techniques. Here, we review current knowledge of how modifying genes related to potassium homeostasis in plants affect abiotic stress tolerance at the whole plant level.

The Biostimulant, Potassium Humate Ameliorates Abiotic Stress in Arabidopsis thaliana by Increasing Starch Availability.

Potassium humate is a widely used biostimulant known for its ability to enhance growth and improve tolerance to abiotic stress. However, the molecular mechanisms explaining its effects remain poorly understood. In this study, we investigated the mechanism of action of potassium humate using the model plant Arabidopsis thaliana. We demonstrated that a formulation of potassium humate effectively increased the fresh weight accumulation of Arabidopsis plants under normal conditions, salt stress (sodium or lithium chloride), and particularly under osmotic stress (mannitol). Interestingly, plants treated with potassium humate exhibited a reduced antioxidant response and lower proline accumulation, while maintaining photosynthetic activity under stress conditions. The observed sodium and osmotic tolerance induced by humate was not accompanied by increased potassium accumulation. Additionally, metabolomic analysis revealed that potassium humate increased maltose levels under control conditions but decreased levels of fructose. However, under stress, both maltose and glucose levels decreased, suggesting changes in starch utilization and an increase in glycolysis. Starch concentration measurements in leaves showed that plants treated with potassium humate accumulated less starch under control conditions, while under stress, they accumulated starch to levels similar to or higher than control plants. Taken together, our findings suggest that the molecular mechanism underlying the abiotic stress tolerance conferred by potassium humate involves its ability to alter starch content under normal growth conditions and under salt or osmotic stress.

Seed coat lignification level is crucial in Capsicum spp seed longevity.

Capsicum (pepper) is known for its poor seed germination, particularly seed longevity is usually much shorter than other Solanaceae. However, the molecular mechanisms involved are mostly unknown in these species. The present study examines the differences in seed longevity among Capsicum species and varietal types. Feral or less domesticated species, such as Capsicum chinense and particularly Capsicum frutescens, showed higher germination rates than the more domesticated Capsicum annuum after accelerated seed aging treatments. In addition, variability was detected in the expression of genes involved in the response to seed deterioration. The differences observed in ASPG1 expression led us to study the seed protein profile in dry and germinating seeds. Seed storage protein mobilization during germination was faster in seed aging-resistant genotypes. Similarly, the transcriptional change observed for the orthologous gene of the trans-species regulator AtHB25 prompted us to study the structure and molecular components of the seed coat in peppers. All the Capsicum pepper accessions analyzed presented very lignified testa and we observed a positive correlation between the amount of lignin and seed viability. Our results provide essential information to explain the poor germination observed in pepper seeds and provide an experimental framework for future improvements in this important character.

Identification of distinctive physiological and molecular responses to salt stress among tolerant and sensitive cultivars of broccoli (Brassica oleracea var. Italica).

BACKGROUND: Salt stress is one of the main constraints determining crop productivity, and therefore one of the main limitations for food production. The aim of this study was to characterize the salt stress response at the physiological and molecular level of different Broccoli (Brassica oleracea L. var. Italica Plenck) cultivars that were previously characterized in field and greenhouse trials as salt sensitive or salt tolerant. This study aimed to identify functional and molecular traits capable of predicting the ability of uncharacterized lines to cope with salt stress. For this purpose, this study measured different physiological parameters, hormones and metabolites under control and salt stress conditions. RESULTS: This study found significant differences among cultivars for stomatal conductance, transpiration, methionine, proline, threonine, abscisic acid, jasmonic acid and indolacetic acid. Salt tolerant cultivars were shown to accumulate less sodium and potassium in leaves and have a lower sodium to potassium ratio under salt stress. Analysis of primary metabolites indicated that salt tolerant cultivars have higher concentrations of several intermediates of the Krebs cycle and the substrates of some anaplerotic reactions. CONCLUSIONS: This study has found that the energetic status of the plant, the sodium extrusion and the proline content are the limiting factors for broccoli tolerance to salt stress. Our results establish physiological and molecular traits useful as distinctive markers to predict salt tolerance in Broccoli or to design novel biotechnological or breeding strategies for improving broccoli tolerance to salt stress.

BCL2-ASSOCIATED ATHANOGENE4 Regulates the KAT1 Potassium Channel and Controls Stomatal Movement.

Potassium (K+) is a key monovalent cation necessary for multiple aspects of cell growth and survival. In plants, this cation also plays a key role in the control of stomatal movement. KAT1 and its homolog KAT2 are the main inward rectifying channels present in guard cells, mediating K+ influx into these cells, resulting in stomatal opening. To gain further insight into the regulation of these channels, we performed a split-ubiquitin protein-protein interaction screen searching for KAT1 interactors in Arabidopsis (Arabidopsis thaliana). We characterized one of these candidates, BCL2-ASSOCIATED ATHANOGENE4 (BAG4), in detail using biochemical and genetic approaches to confirm this interaction and its effect on KAT1 activity. We show that BAG4 improves KAT1-mediated K+ transport in two heterologous systems and provide evidence that in plants, BAG4 interacts with KAT1 and favors the arrival of KAT1 at the plasma membrane. Importantly, lines lacking or overexpressing the BAG4 gene show altered KAT1 plasma membrane accumulation and alterations in stomatal movement. Our data allowed us to identify a KAT1 regulator and define a potential target for the plant BAG family. The identification of physiologically relevant regulators of K+ channels will aid in the design of approaches that may impact drought tolerance and pathogen susceptibility.

Saccharomyces cerevisiae as a Tool to Investigate Plant Potassium and Sodium Transporters.

Sodium and potassium are two alkali cations abundant in the biosphere. Potassium is essential for plants and its concentration must be maintained at approximately 150 mM in the plant cell cytoplasm including under circumstances where its concentration is much lower in soil. On the other hand, sodium must be extruded from the plant or accumulated either in the vacuole or in specific plant structures. Maintaining a high intracellular K+/Na+ ratio under adverse environmental conditions or in the presence of salt is essential to maintain cellular homeostasis and to avoid toxicity. The baker's yeast, Saccharomyces cerevisiae, has been used to identify and characterize participants in potassium and sodium homeostasis in plants for many years. Its utility resides in the fact that the electric gradient across the membrane and the vacuoles is similar to plants. Most plant proteins can be expressed in yeast and are functional in this unicellular model system, which allows for productive structure-function studies for ion transporting proteins. Moreover, yeast can also be used as a high-throughput platform for the identification of genes that confer stress tolerance and for the study of protein-protein interactions. In this review, we summarize advances regarding potassium and sodium transport that have been discovered using the yeast model system, the state-of-the-art of the available techniques and the future directions and opportunities in this field.

Reciprocal Regulation of Target of Rapamycin Complex 1 and Potassium Accumulation.

The proper maintenance of potassium homeostasis is crucial for cell viability. Among the major determinants of potassium uptake in the model organism Saccharomyces cerevisiae are the Trk1 high affinity potassium transporter and the functionally redundant Hal4 (Sat4) and Hal5 protein kinases. These kinases are required for the plasma membrane accumulation of not only Trk1 but also several nutrient permeases. Here, we show that overexpression of the target of rapamycin complex 1 (TORC1) effector NPR1 improves hal4 hal5 growth defects by stabilizing nutrient permeases at the plasma membrane. We subsequently found that internal potassium levels and TORC1 activity are linked. Specifically, growth under limiting potassium alters the activities of Npr1 and another TORC1 effector kinase, Sch9; hal4 hal5 and trk1 trk2 mutants display hypersensitivity to rapamycin, and reciprocally, TORC1 inhibition reduces potassium accumulation. Our results demonstrate that in addition to carbon and nitrogen, TORC1 also responds to and regulates potassium fluxes.

FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology.

Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens-mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for HygR or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3α/pDGB3Ω destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community.

Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 α-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein.

Cell viability requires adaptation to changing environmental conditions. Ubiquitin-mediated endocytosis plays a crucial role in this process, because it provides a mechanism to remove transport proteins from the membrane. Arrestin-related trafficking proteins are important regulators of the endocytic pathway in yeast, facilitating selective ubiquitylation of target proteins by the E3 ubiquitin ligase, Rsp5. Specifically, Rod1 (Art4) has been reported to regulate the endocytosis of both the Hxt1, Hxt3, and Hxt6 glucose transporters and the Jen1 lactate transporter. Also, the AMP kinase homologue, Snf1, and 14-3-3 proteins have been shown to regulate Jen1 via Rod1. Here, we further characterized the role of Rod1, Snf1, and 14-3-3 in the signal transduction route involved in the endocytic regulation of the Hxt6 high affinity glucose transporter by showing that Snf1 interacts specifically with Rod1 and Rog3 (Art7), that the interaction between the Bmh2 and several arrestin-related trafficking proteins may be modulated by carbon source, and that both the 14-3-3 protein Bmh2 and the Snf1 regulatory domain interact with the arrestin-like domain containing the N-terminal half of Rod1 (amino acids 1-395). Finally, using both co-immunoprecipitation and bimolecular fluorescence complementation, we demonstrated the interaction of Rod1 with Hxt6 and showed that the localization of the Rod1-Hxt6 complex at the plasma membrane is affected by carbon source and is reduced upon overexpression of SNF1 and BMH2.

Arabidopsis COGWHEEL1 links light perception and gibberellins with seed tolerance to deterioration.

Light is a major regulator of plant growth and development by antagonizing gibberellins (GA), and we provide evidence for a role of light perception and GA in seed coat formation and seed tolerance to deterioration. We have identified two activation-tagging mutants of Arabidopsis thaliana, cog1-2D and cdf4-1D, with improved seed tolerance to deterioration linked to increased expression of COG1/DOF1.5 and CDF4/DOF2.3, respectively. These encode two homologous DOF transcription factors, with COG1 most highly expressed in seeds. Improved tolerance to seed deterioration was reproduced in transgenic plants overexpressing these genes, and loss of function from RNA interference resulted in opposite phenotypes. Overexpressions of COG1 and CDF4 have been described to attenuate various light responses mediated by phytochromes. Accordingly, we found that phyA and phyB mutants exhibit increased seed tolerance to deterioration. The phenotype of tolerance to deterioration conferred by gain of function of COG1 and by loss of function of phytochromes is of maternal origin, is also observed under natural aging conditions and correlates with a seed coat with increased suberin and reduced permeability. In developing siliques of the cog1-2D mutant the expression of the GA biosynthetic gene GA3OX3 and levels of GA1 are higher than in the wild type. These results explain the antagonism between phytochromes and COG1 in terms of the inhibition and the activation, respectively, of GA action.

Evolution by gene duplication of Medicago truncatula PISTILLATA-like transcription factors.

PISTILLATA (PI) is a member of the B-function MADS-box gene family, which controls the identity of both petals and stamens in Arabidopsis thaliana. In Medicago truncatula (Mt), there are two PI-like paralogs, known as MtPI and MtNGL9. These genes differ in their expression patterns, but it is not known whether their functions have also diverged. Describing the evolution of certain duplicated genes, such as transcription factors, remains a challenge owing to the complex expression patterns and functional divergence between the gene copies. Here, we report a number of functional studies, including analyses of gene expression, protein-protein interactions, and reverse genetic approaches designed to demonstrate the respective contributions of each M. truncatula PI-like paralog to the B-function in this species. Also, we have integrated molecular evolution approaches to determine the mode of evolution of Mt PI-like genes after duplication. Our results demonstrate that MtPI functions as a master regulator of B-function in M. truncatula, maintaining the overall ancestral function, while MtNGL9 does not seem to have a role in this regard, suggesting that the pseudogenization could be the functional evolutionary fate for this gene. However, we provide evidence that purifying selection is the primary evolutionary force acting on this paralog, pinpointing the conservation of its biochemical function and, alternatively, the acquisition of a new role for this gene.

Potassium and Sodium Transport in Yeast.

As the proper maintenance of intracellular potassium and sodium concentrations is vital for cell growth, all living organisms have developed a cohort of strategies to maintain proper monovalent cation homeostasis. In the model yeast Saccharomyces cerevisiae, potassium is accumulated to relatively high concentrations and is required for many aspects of cellular function, whereas high intracellular sodium/potassium ratios are detrimental to cell growth and survival. The fact that S. cerevisiae cells can grow in the presence of a broad range of concentrations of external potassium (10 µM-2.5 M) and sodium (up to 1.5 M) indicates the existence of robust mechanisms that have evolved to maintain intracellular concentrations of these cations within appropriate limits. In this review, current knowledge regarding potassium and sodium transporters and their regulation will be summarized. The cellular responses to high sodium and potassium and potassium starvation will also be discussed, as well as applications of this knowledge to diverse fields, including antifungal treatments, bioethanol production and human disease.

ARABIDOPSIS THALIANA HOMEOBOX25 uncovers a role for Gibberellins in seed longevity.

Seed longevity is crucial for agriculture and plant genetic diversity, but it is limited by cellular damage during storage. Seeds are protected against aging by cellular defenses and by structures such as the seed coat. We have screened an activation-tagging mutant collection of Arabidopsis (Arabidopsis thaliana) and selected four dominant mutants with improved seed longevity (isl1-1D to isl4-1D) under both natural and accelerated aging conditions. In the isl1-1D mutant, characterized in this work, overexpression of the transcription factor ARABIDOPSIS THALIANA HOMEOBOX25 (ATHB25; At5g65410) increases the expression of GIBBERELLIC ACID3-OXIDASE2, encoding a gibberellin (GA) biosynthetic enzyme, and the levels of GA1 and GA4 are higher (3.2- and 1.4-fold, respectively) in the mutant than in the wild type. The morphological and seed longevity phenotypes of the athb25-1D mutant were recapitulated in transgenic plants with moderate (4- to 6-fold) overexpression of ATHB25. Simultaneous knockdown of ATHB25, ATHB22, and ATHB31 expression decreases seed longevity, as does loss of ATHB25 and ATHB22 function in a double mutant line. Seeds from wild-type plants treated with GA and from a quintuple DELLA mutant (with constitutive GA signaling) are more tolerant to aging, providing additional evidence for a role of GA in seed longevity. A correlation was observed in several genotypes between seed longevity and mucilage formation at the seed surface, suggesting that GA may act by reinforcing the seed coat. This mechanism was supported by the observation of a maternal effect in reciprocal crosses between the wild type and the athb25-1D mutant.

A functional Rim101 complex is required for proper accumulation of the Ena1 Na+-ATPase protein in response to salt stress in Saccharomyces cerevisiae.

The maintenance of ionic homeostasis is essential for cell viability, thus the activity of plasma membrane ion transporters must be tightly controlled. Previous studies in Saccharomyces cerevisiae revealed that the proper trafficking of several nutrient permeases requires the E3 ubiquitin ligase Rsp5 and, in many cases, the presence of specific adaptor proteins needed for Rsp5 substrate recognition. Among these adaptor proteins are nine members of the arrestin-related trafficking adaptor (ART) family. We studied the possible role of the ART family in the regulation of monovalent cation transporters. We show here that the salt sensitivity phenotype of the rim8/art9 mutant is due to severe defects in Ena1 protein accumulation, which is not attributable to transcriptional defects. Many components of the Rim pathway are required for correct Ena1 accumulation, but not for the accumulation of other nutrient permeases. Moreover, we observe that strains lacking components of the endosomal sorting complexes required for transport (ESCRT) pathway previously described to play a role in Rim complex formation present similar defects in Ena1 accumulation. Our results show that, in response to salt stress, a functional Rim complex via specific ESCRT interactions is required for the proper accumulation of the Ena1 protein, but not induction of the ENA1 gene.

Functional specialization of duplicated AP3-like genes in Medicago truncatula.

The B-class of MADS box genes has been studied in a wide range of plant species, but has remained largely uncharacterized in legumes. Here we investigate the evolutionary fate of the duplicated AP3-like genes of a legume species. To obtain insight into the extent to which B-class MADS box gene functions are conserved or have diversified in legumes, we isolated and characterized the two members of the AP3 lineage in Medicago truncatula: MtNMH7 and MtTM6 (euAP3 and paleoAP3 genes, respectively). A non-overlapping and complementary expression pattern of both genes was observed in petals and stamens. MtTM6 was expressed predominantly in the outer cell layers of both floral organs, and MtNMH7 in the inner cell layers of petals and stamens. Functional analyses by reverse genetics approaches (RNAi and Tnt1 mutagenesis) showed that the contribution of MtNMH7 to petal identity is more important than that of MtTM6, whereas MtTM6 plays a more important role in stamen identity than its paralog MtNMH7. Our results suggest that the M. truncatula AP3-like genes have undergone a functional specialization process associated with complete partitioning of gene expression patterns of the ancestral gene lineage. We provide information regarding the similarities and differences in petal and stamen development among core eudicots.

PIF transcriptional regulators are required for rhythmic stomatal movements.

Stomata govern the gaseous exchange between the leaf and the external atmosphere, and their function is essential for photosynthesis and the global carbon and oxygen cycles. Rhythmic stomata movements in daily dark/light cycles prevent water loss at night and allow CO2 uptake during the day. How the actors involved are transcriptionally regulated and how this might contribute to rhythmicity is largely unknown. Here, we show that morning stomata opening depends on the previous night period. The transcription factors PHYTOCHROME-INTERACTING FACTORS (PIFs) accumulate at the end of the night and directly induce the guard cell-specific K+ channel KAT1. Remarkably, PIFs and KAT1 are required for blue light-induced stomata opening. Together, our data establish a molecular framework for daily rhythmic stomatal movements under well-watered conditions, whereby PIFs are required for accumulation of KAT1 at night, which upon activation by blue light in the morning leads to the K+ intake driving stomata opening.

Maternal Embryo Effect Arrest 31 (MEE31) is a moonlighting protein involved in GDP-D-mannose biosynthesis and KAT1 potassium channel regulation.

Due to anthropogenic global warming, droughts are expected to increase and water availability to decrease in the coming decades. For this reason, research is increasingly focused on developing plant varieties and crop cultivars with reduced water consumption. Transpiration occurs through stomatal pores, resulting in water loss. Potassium plays a significant role in stomatal regulation. KAT1 is an inward-rectifying potassium channel that contributes to stomatal opening. Using a yeast high-throughput screening of an Arabidopsis cDNA library, MEE31 was found to physically interact with KAT1. MEE31 was initially identified in a screen for mutants with delayed embryonic development. The gene encodes a conserved phosphomannose isomerase (PMI). We report here that MEE31 interacts with and increases KAT1 activity in yeast and this interaction was also confirmed in plants. In addition, MEE31 complements the function of the yeast homologue, whereas the truncated version recovered in the screening does not, thus uncoupling the enzymatic activity from KAT1 regulation. We show that MEE31 overexpression leads to increased stomatal opening in Arabidopsis transgenic lines. Our data suggest that MEE31 is a moonlighting protein involved in both GDP-D-mannose biosynthesis and KAT1 regulation.

Endocytic regulation of alkali metal transport proteins in mammals, yeast and plants.

The relative concentrations of ions and solutes inside cells are actively maintained by several classes of transport proteins, in many cases against their concentration gradient. These transport processes, which consume a large portion of cellular energy, must be constantly regulated. Many structurally distinct families of channels, carriers, and pumps have been characterized in considerable detail during the past decades and defects in the function of some of these proteins have been linked to a growing list of human diseases. The dynamic regulation of the transport proteins present at the cell surface is vital for both normal cellular function and for the successful adaptation to changing environments. The composition of proteins present at the cell surface is controlled on both the transcriptional and post-translational level. Post-translational regulation involves highly conserved mechanisms of phosphorylation- and ubiquitylation-dependent signal transduction routes used to modify the cohort of receptors and transport proteins present under any given circumstances. In this review, we will summarize what is currently known about one facet of this regulatory process: the endocytic regulation of alkali metal transport proteins. The physiological relevance, major contributors, parallels and missing pieces of the puzzle in mammals, yeast and plants will be discussed.

Role of the yeast multidrug transporter Qdr2 in cation homeostasis and the oxidative stress response.

We have identified QDR2 in a screening for genes able to confer tolerance to sodium and/or lithium stress upon overexpression. Qdr2 is a multidrug transporter of the major facilitator superfamily, originally described for its ability to transport the antimalarial drug quinidine and the herbicide barban. To identify its physiological substrate, we have screened for phenotypes dependent on QDR2 and found that Qdr2 is able to transport monovalent and divalent cations with poor selectivity, as shown by growth tests and the determination of internal cation content. Moreover, strains overexpressing or lacking QDR2 also exhibit phenotypes when reactive oxygen species- producing agents, such as hydrogen peroxide or menadione were added to the growth medium. We have also found that the presence of copper and hydrogen peroxide repress the expression of QDR2. In addition, the copper uptake of a qdr2 mutant strain is similar to a wild type, but the extrusion is clearly impaired. Based on our results, we propose that free divalent copper is the main physiological substrate of Qdr2. As copper is a substrate for several redox reactions that occur within the cytoplasm, its function in copper homeostasis explains its role in the oxidative stress response.