The role of post-transcriptional modulators of metalloproteins in response to metal deficiencies.

Copper and iron proteins have a wide range of functions in living organisms. Metal assembly into metalloproteins is a complex process, where mismetalation is detrimental and energy consuming to cells. Under metal deficiency, metal distribution is expected to reach a metalation ranking, prioritizing essential versus dispensable metalloproteins, while avoiding interference with other metals and protecting metal-sensitive processes. In this review, we propose that post-transcriptional modulators of metalloprotein mRNA (ModMeR) are good candidates in metal prioritization under metal-limited conditions. ModMeR target high quota or redundant metalloproteins and, by adjusting their synthesis, ModMeR act as internal metal distribution valves. Inappropriate metalation of ModMeR targets could compete with metal delivery to essential metalloproteins and interfere with metal-sensitive processes, such as chloroplastic photosynthesis and mitochondrial respiration. Regulation of ModMeR targets could increase or decrease the metal flow through interconnected pathways in cellular metal distribution, helping to achieve adequate differential metal requirements. Here, we describe and compare ModMeR that function in response to copper and iron deficiencies. Specifically, we describe copper-miRNAs from Arabidopsis thaliana and diverse iron ModMeR from yeast, mammals, and bacteria under copper and iron deficiencies, as well as the influence of oxidative stress. Putative functions derived from their role as ModMeR are also discussed.

The Copper-microRNA Pathway Is Integrated with Developmental and Environmental Stress Responses in Arabidopsis thaliana.

As an essential nutrient, copper (Cu) scarcity causes a decrease in agricultural production. Cu deficiency responses include the induction of several microRNAs, known as Cu-miRNAs, which are responsible for degrading mRNAs from abundant and dispensable cuproproteins to economize copper when scarce. Cu-miRNAs, such as miR398 and miR408 are conserved, as well as the signal transduction pathway to induce them under Cu deficiency. The Arabidopsis thaliana SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) family member SPL7 binds to the cis-regulatory motifs present in the promoter regions of genes expressed under Cu deficiency, including Cu-miRNAs. The expression of several other SPL transcription factor family members is regulated by miR156. This regulatory miR156-SPL module plays a crucial role in developmental phase transitions while integrating internal and external cues. Here, we show that Cu deficiency also affects miR156 expression and that SPL3 overexpressing plants, resistant to miR156 regulation, show a severe decrease in SPL7-mediated Cu deficiency responses. These include the expression of Cu-miRNAs and their targets and is probably due to competition between SPL7 and miR156-regulated SPL3 in binding to cis-regulatory elements in Cu-miRNA promoters. Thus, the conserved SPL7-mediated Cu-miRNA pathway could generally be affected by the miR156-SPL module, thereby underscoring the integration of the Cu-miRNA pathway with developmental and environmental stress responses in Arabidopsis thaliana.

Characterization of the Copper Transporters from Lotus spp. and Their Involvement under Flooding Conditions.

Forage legumes are an important livestock nutritional resource, which includes essential metals, such as copper. Particularly, the high prevalence of hypocuprosis causes important economic losses to Argentinian cattle agrosystems. Copper deficiency in cattle is partially due to its low content in forage produced by natural grassland, and is exacerbated by flooding conditions. Previous results indicated that incorporation of Lotus spp. into natural grassland increases forage nutritional quality, including higher copper levels. However, the biological processes and molecular mechanisms involved in copper uptake by Lotus spp. remain poorly understood. Here, we identify four genes that encode putative members of the Lotus copper transporter family, denoted COPT in higher plants. A heterologous functional complementation assay of the Saccharomyces cerevisiae ctr1∆ctr3∆ strain, which lacks the corresponding yeast copper transporters, with the putative Lotus COPT proteins shows a partial rescue of the yeast phenotypes in restrictive media. Under partial submergence conditions, the copper content of L. japonicus plants decreases and the expression of two Lotus COPT genes is induced. These results strongly suggest that the Lotus COPT proteins identified in this work function in copper uptake. In addition, the fact that environmental conditions affect the expression of certain COPT genes supports their involvement in adaptive mechanisms and envisages putative biotechnological strategies to improve cattle copper nutrition.

Copper and ectopic expression of the Arabidopsis transport protein COPT1 alter iron homeostasis in rice (Oryza sativa L.).

KEY MESSAGE: Copper deficiency and excess differentially affect iron homeostasis in rice and overexpression of the Arabidopsis high-affinity copper transporter COPT1 slightly increases endogenous iron concentration in rice grains. Higher plants have developed sophisticated mechanisms to efficiently acquire and use micronutrients such as copper and iron. However, the molecular mechanisms underlying the interaction between both metals remain poorly understood. In the present work, we study the effects produced on iron homeostasis by a wide range of copper concentrations in the growth media and by altered copper transport in Oryza sativa plants. Gene expression profiles in rice seedlings grown under copper excess show an altered expression of genes involved in iron homeostasis compared to standard control conditions. Thus, ferritin OsFER2 and ferredoxin OsFd1 mRNAs are down-regulated whereas the transcriptional iron regulator OsIRO2 and the nicotianamine synthase OsNAS2 mRNAs rise under copper excess. As expected, the expression of OsCOPT1, which encodes a high-affinity copper transport protein, as well as other copper-deficiency markers are down-regulated by copper. Furthermore, we show that Arabidopsis COPT1 overexpression (C1 OE ) in rice causes root shortening in high copper conditions and under iron deficiency. C1 OE rice plants modify the expression of the putative iron-sensing factors OsHRZ1 and OsHRZ2 and enhance the expression of OsIRO2 under copper excess, which suggests a role of copper transport in iron signaling. Importantly, the C1 OE rice plants grown on soil contain higher endogenous iron concentration than wild-type plants in both brown and white grains. Collectively, these results highlight the effects of rice copper status on iron homeostasis, which should be considered to obtain crops with optimized nutrient concentrations in edible parts.

Interaction Between ABA Signaling and Copper Homeostasis in Arabidopsis thaliana.

ABA is involved in plant responses to non-optimal environmental conditions, including nutrient availability. Since copper (Cu) is a very important micronutrient, unraveling how ABA affects Cu uptake and distribution is relevant to ensure adequate Cu nutrition in plants subjected to stress conditions. Inversely, knowledge about how the plant nutritional status can interfere with ABA biosynthesis and signaling mechanisms is necessary to optimize stress tolerance in horticultural crops. Here the reciprocal influence between ABA and Cu content was addressed by using knockout mutants and overexpressing transgenic plants of high affinity plasma membrane Cu transporters (pmCOPT) with altered Cu uptake. Exogenous ABA inhibited pmCOPT expression and drastically modified COPT2-driven localization in roots. ABA regulated SPL7, the main transcription factor responsive for Cu deficiency responses, and subsequently affected expression of its targets. ABA biosynthesis (aba2) and signaling (hab1-1 abi1-2) mutants differentially responded to ABA according to Cu levels. Alteration of Cu homeostasis in the pmCOPT mutants affected ABA biosynthesis, transport and signaling as genes such as NCED3, WRKY40, HY5 and ABI5 were differentially modulated by Cu status, and also in the pmCOPT and ABA mutants. Altered Cu uptake resulted in modified plant sensitivity to salt-mediated increases in endogenous ABA. The overall results provide evidence for reciprocal cross-talk between Cu status and ABA metabolism and signaling.

Modulation of copper deficiency responses by diurnal and circadian rhythms in Arabidopsis thaliana.

Copper homeostasis under deficiency is regulated by the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7) transcription factor. The daily oscillating expression of two SPL7-dependent copper deficiency markers, COPPER TRANSPORTER (COPT2) and IRON SUPEROXIDE DISMUTASE (FSD1), has been followed by quantitative PCR and in promoter:LUCIFERASE transgenic plants. Both genes showed circadian and diurnal regulation. Under copper deficiency, their expression decreased drastically in continuous darkness. Accordingly, total copper content was slightly reduced in etiolated seedlings under copper deficiency. The expression of SPL7 and its targets COPT2 and FSD1 was differently regulated in various light signalling mutants. On the other hand, increased copper levels reduced the amplitude of nuclear circadian clock components, such as GIGANTEA (GI). The alteration of copper homeostasis in the COPT1 overexpression line and spl7 mutants also modified the amplitude of a classical clock output, namely the circadian oscillation of cotyledon movements. In the spl7 mutant, the period of the oscillation remained constant. These results suggest a feedback of copper transport on the circadian clock and the integration of rhythmic copper homeostasis into the central oscillator of plants.

Defective copper transport in the copt5 mutant affects cadmium tolerance.

Cadmium toxicity interferes with essential metal homeostasis, which is a problem for both plant nutrition and the consumption of healthy food by humans. Copper uptake is performed by the members of the Arabidopsis high affinity copper transporter (COPT) family. One of the members, COPT5, is involved in copper recycling from the vacuole toward the cytosolic compartment. We show herein that copt5 mutants are more sensitive to cadmium stress than wild-type plants, as indicated by reduced growth. Exacerbated cadmium toxicity in copt5 mutants is due specifically to altered copper traffic through the COPT5 transporter. Three different processes which have been shown to affect cadmium tolerance are altered in copt5 mutants. First, ethylene biosynthesis diminishes under copper deficiency and, in the presence of cadmium, ethylene production diminishes further. Copper deficiency responses are also attenuated under cadmium treatment. Remarkably, while copt5 roots present higher oxidative stress toxicity symptoms than controls, aerial copt5 parts display lower oxidative stress, as seen by reduced cadmium delivery to shoots. Taken together, these results demonstrate that copper transport plays a key role in cadmium resistance, and suggest that oxidative stress triggers an NADPH oxidase-mediated signaling pathway, which contributes to cadmium translocation and basal plant resistance. The slightly lower cadmium levels that reach aerial parts in the copt5 mutants, irrespective of the copper content in the media, suggest a new biotechnological approach to minimize toxic cadmium entry into food chains.

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

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.

The role of the Arabidopsis tandem zinc-finger C3H15 protein in metal homeostasis.

Living organisms have developed finely regulated homeostatic networks to mitigate the effects of environmental fluctuations in transition metal micronutrients, including iron, zinc, and copper. In Saccharomyces cerevisiae, the tandem zinc-finger protein Cth2 post-transcriptionally regulates gene expression under conditions of iron deficiency by controlling the levels of mRNAs that code for non-essential ferroproteins. The molecular mechanism involves Cth2 binding to AU-rich elements present in the 3' untranslated region of target mRNAs, negatively affecting their stability and translation. Arabidopsis thaliana has two TZF proteins homologous to yeast Cth2, C3H14 and C3H15, which participate in cell wall remodelling. The present work examines the expression of representative metal homeostasis genes with putative AREs in plants with altered levels of C3H14 and C3H15 grown under varying metal availabilities. The results suggest that C3H15 may act as a post-transcriptional plant modulator of metal adequacy, as evidenced by the expression of SPL7, the main transcriptional regulator under copper deficiency, and PETE2, which encodes plastocyanin. In contrast to S. cerevisiae, the plant C3H15 affects copper and zinc homeostasis rather than iron. When grown under copper-deficient conditions, adult C3H15OE plants exhibit lower chlorophyll content and photosynthetic efficiency compared to control plants, suggesting accelerated senescence. Likewise, metal content in C3H15OE plants under copper deficiency shows altered mobilization of copper and zinc to seeds. These data suggest that the C3H15 protein plays a role in modulating both cell wall remodelling and metal homeostasis. The interaction between these processes may be the cause of altered metal translocation.

The Arabidopsis COPT6 transport protein functions in copper distribution under copper-deficient conditions.

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.

Calcium- and potassium-permeable plasma membrane transporters are activated by copper in Arabidopsis root tips: linking copper transport with cytosolic hydroxyl radical production.

Transition metals such as copper can interact with ascorbate or hydrogen peroxide to form highly reactive hydroxyl radicals (OH(•) ), with numerous implications to membrane transport activity and cell metabolism. So far, such interaction was described for extracellular (apoplastic) space but not cytosol. Here, a range of advanced electrophysiological and imaging techniques were applied to Arabidopsis thaliana plants differing in their copper-transport activity: Col-0, high-affinity copper transporter COPT1-overexpressing (C1(OE) ) seedlings, and T-DNA COPT1 insertion mutant (copt1). Low Cu concentrations (10 µm) stimulated a dose-dependent Gd(3+) and verapamil sensitive net Ca(2+) influx in the root apex but not in mature zone. C1(OE) also showed a fivefold higher Cu-induced K(+) efflux at the root tip level compared with Col-0, and a reduction in basal peroxide accumulation at the root tip after copper exposure. Copper caused membrane disruptions of the root apex in C1(OE) seedlings but not in copt1 plants; this damage was prevented by pretreatment with Gd(3+) . Our results suggest that copper transport into cytosol in root apex results in hydroxyl radical generation at the cytosolic side, with a consequent regulation of plasma membrane OH(•) -sensitive Ca(2+) and K(+) transport systems.

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.

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.

Deregulated High Affinity Copper Transport Alters Iron Homeostasis in Arabidopsis.

The present work describes the effects on iron homeostasis when copper transport was deregulated in Arabidopsis thaliana by overexpressing high affinity copper transporters COPT1 and COPT3 (COPTOE ). A genome-wide analysis conducted on COPT1OE plants, highlighted that iron homeostasis gene expression was affected under both copper deficiency and excess. Among the altered genes were those encoding the iron uptake machinery and their transcriptional regulators. Subsequently, COPTOE seedlings contained less iron and were more sensitive than controls to iron deficiency. The deregulation of copper (I) uptake hindered the transcriptional activation of the subgroup Ib of basic helix-loop-helix (bHLH-Ib) factors under copper deficiency. Oppositely, copper excess inhibited the expression of the master regulator FIT but activated bHLH-Ib expression in COPTOE plants, in both cases leading to the lack of an adequate iron uptake response. As copper increased in the media, iron (III) was accumulated in roots, and the ratio iron (III)/iron (II) was increased in COPTOE plants. Thus, iron (III) overloading in COPTOE roots inhibited local iron deficiency responses, aimed to metal uptake from soil, leading to a general lower iron content in the COPTOE seedlings. These results emphasized the importance of appropriate spatiotemporal copper uptake for iron homeostasis under non-optimal copper supply. The understanding of the role of copper uptake in iron metabolism could be applied for increasing crops resistance to iron deficiency.

Copper transporter COPT5 participates in the crosstalk between vacuolar copper and iron pools mobilisation.

Copper (Cu) deficiency affects iron (Fe) homeostasis in several plant processes, including the increased Fe requirements due to cuproprotein substitutions for the corresponding Fe counterpart. Loss-of-function mutants from Arabidopsis thaliana high affinity copper transporter COPT5 and Fe transporters NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 3/4 (NRAMP3 and NRAMP4) were used to study the interaction between metals internal pools. A physiological characterisation showed that the copt5 mutant is sensitive to Fe deficiency, and that nramp3nramp4 mutant growth was severely affected under limiting Cu. By a transcriptomic analysis, we observed that NRAMP4 expression was highly induced in the copt5 mutant under Cu deficiency, while COPT5 was overexpressed in the nramp3nramp4 mutant. As a result, an enhanced mobilisation of the vacuolar Cu or Fe pools, when the other metal export through the tonoplast is impaired in the mutants, has been postulated. However, metals coming from internal pools are not used to accomplish the increased requirements that derive from metalloprotein substitution under metal deficiencies. Instead, the metal concentrations present in aerial parts of the copt5 and nramp3nramp4 mutants conversely show compensated levels of these two metals. Together, our data uncover an interconnection between Cu and Fe vacuolar pools, whose aim is to fulfil interorgan metal translocation.

The Altered Expression of microRNA408 Influences the Arabidopsis Response to Iron Deficiency.

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.

Copper uptake mechanism of Arabidopsis thaliana high-affinity COPT transporters.

Copper (Cu) is an essential plant micronutrient. Under scarcity, Cu2+ is reduced to Cu+ and taken up through specific high-affinity transporters (COPTs). In Arabidopsis, the COPT family consists of six members, either located at the plasma membrane (COPT1, COPT2, and COPT6) or in internal membranes (COPT3 and COPT5). Cu uptake by COPT proteins has been mainly assessed through complementation studies in corresponding yeast mutants, but the mechanism of this transport has not been elucidated. To test whether Cu is incorporated by an electrogenic mechanism, electrophysiological changes induced by Cu addition were studied in Arabidopsis thaliana. Mutant (T-DNA insertion mutants, copt2-1 and copt5-2) and overexpressing lines (COPT1OE and COPT5OE) with altered expression of COPT transporters were compared to wild-type plants. No significant changes of the membrane potential (Em) were detected, regardless of genotype or Cu concentration supplied. In contrast, membrane depolarization was detected in response to iron supply in both wild-type and in mutant or transgenic plants. Similar results were obtained for trans-plant potentials (TPP). GFP fusions of the plasma membrane COPT2 and the internal COPT5 transporters were expressed in Xenopus laevis oocytes to potentiate Cu uptake signals, and the cRNA-injected oocytes were tested for electrical currents upon Cu addition using two-electrode voltage clamp. Results with oocytes confirmed those obtained in plants. Cu accumulation in injected oocytes was measured by ICP-OES, and a significant increase in Cu content with respect to controls occurred in oocytes expressing COPT2:GFP. The possible mechanisms driving this transport are discussed in this manuscript.

Induction of a polyubiquitin gene promoter by dehydration stresses in transformed rice cells.

The expression of the maize polyubiquitin gene promoter UBI1 in rice cells has been used to study the involvement of ubiquitin in cell protection responses to dehydration caused by osmotic, saline or freezing stress. The effect of these stresses on UBI1 activity was investigated by the use of stably transformed rice calli (UBI1:GUS), as well as by transient expression experiments performed with cell lines with high or low tolerance to each type of stress. The theoretical analysis of the UBI1 promoter shows several putative stress-regulated boxes that could account for the stress-related UBI1 induction pattern described in this work. We suggest that the study of the differential UBI1 promoter-driven expression in rice cell lines with different level of tolerance to stress might be useful to elucidate complex signal transduction pathways in response to dehydration stresses in monocots.

Expression of the Intracellular COPT3-Mediated Cu Transport Is Temporally Regulated by the TCP16 Transcription Factor.

Copper is an essential element in plants. When scarce, copper is acquired from extracellular environment or remobilized from intracellular sites, through members of the high affinity copper transporters family COPT located at the plasma membrane and internal membrane, respectively. Here, we show that COPT3 is an intracellular copper transporter, located at a compartment of the secretory pathway, that is mainly expressed in pollen grains and vascular bundles. Contrary to the COPT1 plasma membrane member, the expression of the internal COPT3 membrane transporter was higher at 12 h than at 0 h of a neutral photoperiod day under copper deficiency. The screening of a library of conditionally overexpressed transcription factors implicated members of the TCP family in the COPT3 differential temporal expression pattern. Particularly, in vitro, TCP16 was found to bind to the COPT3 promoter and down-regulated its expression. Accordingly, TCP16 was mainly expressed at 0 h under copper deficiency and induced at 12 h by copper excess. Moreover, TCP16 overexpression resulted in increased sensitivity to copper deficiency, whereas the tcp16 mutant was sensitive to copper excess. Both copper content and the expression of particular copper status markers were altered in plants with modified levels of TCP16. Consistent with TCP16 affecting pollen development, the lack of COPT3 function led to altered pollen morphology. Furthermore, analysis of copt3 and COPT3 overexpressing plants revealed that COPT3 function exerted a negative effect on TCP16 expression. Taken together, these results suggest a differential daily regulation of copper uptake depending on the external and internal copper pools, in which TCP16 inhibits copper remobilization at dawn through repression of intracellular transporters.