The Arabidopsis Copper Transporter COPT1 Functions in Root Elongation and Pollen Development
2004; Elsevier BV; Volume: 279; Issue: 15 Linguagem: Inglês
10.1074/jbc.m313321200
ISSN1083-351X
AutoresVicente Sancenón, Sergi Puig, Isabel Mateu‐Andrés, Eavan Dorcey, Dennis J. Thiele, Lola Peñarrubia,
Tópico(s)Aluminum toxicity and tolerance in plants and animals
ResumoCopper plays a dual role in aerobic organisms, as both an essential and a potentially toxic element. To ensure copper availability while avoiding its toxic effects, organisms have developed complex homeostatic networks to control copper uptake, distribution, and utilization. In eukaryotes, including yeasts and mammals, high affinity copper uptake is mediated by the Ctr family of copper transporters. This work is the first report on the physiological function of copper transport in Arabidopsis thaliana. We have studied the expression pattern of COPT1 in transgenic plants expressing a reporter gene under the control of the COPT1 promoter. The reporter gene is highly expressed in embryos, trichomes, stomata, pollen, and root tips. The involvement of COPT1 in copper acquisition was investigated in CaMV35S::COPT1 antisense transgenic plants. Consistent with a decrease in COPT1 expression and the associated copper deprivation, these plants exhibit increased mRNA levels of genes that are down-regulated by copper, decreased rates of 64Cu uptake by seedlings and reduced steady state levels of copper as measured by atomic absorption spectroscopy in mature leaves. Interestingly, COPT1 antisense plants also display dramatically increased root length, which is completely and specifically reversed by copper addition, and an increased sensitivity to growth inhibition by the copper-specific chelator bathocuproine disulfonic acid. Furthermore, COPT1 antisense plants exhibit pollen development defects that are specifically reversed by copper. Taken together, these studies reveal striking plant growth and development roles for copper acquisition by high affinity copper transporters. Copper plays a dual role in aerobic organisms, as both an essential and a potentially toxic element. To ensure copper availability while avoiding its toxic effects, organisms have developed complex homeostatic networks to control copper uptake, distribution, and utilization. In eukaryotes, including yeasts and mammals, high affinity copper uptake is mediated by the Ctr family of copper transporters. This work is the first report on the physiological function of copper transport in Arabidopsis thaliana. We have studied the expression pattern of COPT1 in transgenic plants expressing a reporter gene under the control of the COPT1 promoter. The reporter gene is highly expressed in embryos, trichomes, stomata, pollen, and root tips. The involvement of COPT1 in copper acquisition was investigated in CaMV35S::COPT1 antisense transgenic plants. Consistent with a decrease in COPT1 expression and the associated copper deprivation, these plants exhibit increased mRNA levels of genes that are down-regulated by copper, decreased rates of 64Cu uptake by seedlings and reduced steady state levels of copper as measured by atomic absorption spectroscopy in mature leaves. Interestingly, COPT1 antisense plants also display dramatically increased root length, which is completely and specifically reversed by copper addition, and an increased sensitivity to growth inhibition by the copper-specific chelator bathocuproine disulfonic acid. Furthermore, COPT1 antisense plants exhibit pollen development defects that are specifically reversed by copper. Taken together, these studies reveal striking plant growth and development roles for copper acquisition by high affinity copper transporters. Plants use copper as a cofactor for a wide variety of physiological processes such as photosynthesis, mitochondrial respiration, superoxide scavenging, cell wall metabolism, and ethylene sensing. In the absence of this essential micronutrient, plants display diverse symptoms, most of which affect young leaves and reproductive organs (1Märschner H. Mineral Nutrition of Higher Plants. Academic Press, San Diego1995Google Scholar). Typical symptoms of copper deficiency appear first at the tips of young leaves and then extend downward along the leaf margins. The leaves may also be twisted or malformed and show chlorosis or even necrosis (1Märschner H. Mineral Nutrition of Higher Plants. Academic Press, San Diego1995Google Scholar, 2Dell B. Plant Soil. 1994; 167: 181-187Crossref Scopus (8) Google Scholar). In addition to its essentiality, copper is a potentially toxic agent at supraoptimal levels. Copper reactivity can lead to the generation of harmful reactive oxygen species that cause severe oxidative damage to cells (3Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4392) Google Scholar). To deal with this dual nature of copper, plants, as well as other organisms, have evolved sophisticated homeostatic networks controlling copper uptake, utilization, and detoxification (for reviews see Refs. 4Himelblau E. Amasino R.M. Curr. Opin. Plant Biol. 2000; 3: 205-210Crossref PubMed Google Scholar, 5Clemens S. Planta. 2001; 212: 475-486Crossref PubMed Scopus (1089) Google Scholar, 6Mira H. Sancenón V. Peñarrubia L. Handbook of Copper Pharmacology and Toxicology. Humana Press, Totowa, NJ2002: 543-557Google Scholar). The copper homeostasis network has been primarily described in the yeast Saccharomyces cerevisiae (for reviews see Refs. 7Peña M.M. Lee J. Thiele D.J. J. Nutr. 1999; 129: 1251-1260Crossref PubMed Scopus (599) Google Scholar, 8Harris E.D. Annu. Rev. Nutr. 2000; 20: 291-310Crossref PubMed Scopus (214) Google Scholar, 9Puig S. Thiele D.J. Curr. Opin. Chem. Biol. 2002; 6: 171-180Crossref PubMed Scopus (562) Google Scholar), and its components are widely conserved among eukaryotes. Intracellular copper distribution is performed by metallochaperones, a set of soluble copper-binding proteins that direct the metal to its final destination (for a review see Refs. 10Harrison M.D. Jones C.E. Solioz M. Dameron C.T. Trends Biochem. Sci. 2000; 25: 29-32Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar and 11Huffman D.L. O′Halloran T.V. Annu. Rev. Biochem. 2001; 70: 677-701Crossref PubMed Scopus (411) Google Scholar). The copper chaperone CCH was the first metallochaperone described in plants (12Himelblau E. Mira H. Lin S.-J. Culotta V.C. Peñarrubia L. Amasino R.M. Plant Physiol. 1998; 117: 1227-1234Crossref PubMed Scopus (171) Google Scholar). The amino-terminal half of CCH displays homology to yeast Atx1 (13Lin S.J. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3784-3788Crossref PubMed Scopus (232) Google Scholar), and it possesses a plant-specific carboxyl-terminal extension (14Mira H. Vilar M. Perez-Paya E. Peñarrubia L. Biochem. J. 2001; 15: 545-549Crossref Google Scholar), which has been proposed to participate in intracellular copper transport through the plant symplasmic connections or plasmodesmata (15Mira H. Martínez-García F. Peñarrubia L. Plant J. 2001; 25: 521-528Crossref PubMed Scopus (89) Google Scholar). Similarly to the task performed by homologues in other organisms, CCH is predicted to deliver copper to a P-type copper-transporting ATPase, denoted RAN1, that is putatively localized at a post-Golgi compartment (16Hirayama T. Kieber J.J. Hirayama N. Kogan M. Guzman P. Nourizadeh S. Alonso J.M. Dailey W.P. Dancis A. Ecker J.R. Cell. 1999; 97: 383-393Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). The RAN1 (responsive to antagonist 1) gene from Arabidopsis thaliana codes for a homologue of the yeast Ccc2 and human Menkes (ATP7A) and Wilson (ATP7B) proteins (17Fu D. Beeler T.J. Dunn T.M. Yeast. 1995; 11: 283-292Crossref PubMed Scopus (142) Google Scholar, 18Yuan D.S. Dancis A. Klausner R.D. J. Biol. Chem. 1997; 272: 25787-25793Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 19Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14035Crossref PubMed Scopus (189) Google Scholar, 20Hung I.H. Suzuki M. Yamaguchi Y. Yuan D.S. Klausner R.D. Gitlin J.D. J. Biol. Chem. 1997; 272: 21461-21466Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). This copper homeostasis-related gene was isolated in a screen for plants with an atypical response to the ethylene antagonist trans-cyclooctene, underscoring the critical role of copper in the ethylene-signaling pathway (16Hirayama T. Kieber J.J. Hirayama N. Kogan M. Guzman P. Nourizadeh S. Alonso J.M. Dailey W.P. Dancis A. Ecker J.R. Cell. 1999; 97: 383-393Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 21Woeste K.E. Kieber J.J. Plant Cell. 2000; 12: 443-455Crossref PubMed Scopus (182) Google Scholar). This role is explained by the fact that the ethylene receptor requires copper for high affinity ethylene binding (22Rodriguez F.I. Esch J.J. Hall A.E. Binder B.M. Schaller G.E. Bleecker A.B. Science. 1999; 283: 996-998Crossref PubMed Scopus (489) Google Scholar). A main step in the control of copper homeostasis is its uptake through the plasma membrane. This function is accomplished by the Ctr family of eukaryotic high affinity copper transporters. Ctr family members have been identified in yeasts and mammals (for a review see Ref. 9Puig S. Thiele D.J. Curr. Opin. Chem. Biol. 2002; 6: 171-180Crossref PubMed Scopus (562) Google Scholar). The importance of copper transport into cells has been underscored by recent observations demonstrating that a murine knockout of the gene encoding Ctr1 exhibits severely defective embryonic development and prenatal lethality (23Kuo Y.M. Zhou B. Cosco D. Gitshier J. Proc. Natl. Acad. Sci, U. S. A. 2001; 98: 6836-6841Crossref PubMed Scopus (295) Google Scholar, 24Lee J. Prohaska J.R. Thiele D.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6842-6847Crossref PubMed Scopus (357) Google Scholar). Ctr proteins contain three predicted transmembrane domains, a variable number of putative metal-binding motifs at their extracellular amino-terminal domain, and a conserved and absolutely essential MXXXM motif within the putative second transmembrane domain (25Puig S. Lee J. Lau M. Thiele D.J. J. Biol. Chem. 2002; 277: 26021-26030Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). A cDNA from Arabidopsis encoding a Ctr-related copper transporter (COPT1) was isolated in a functional complementation screen performed in yeast cells defective in high affinity copper uptake (26Kampfenkel K. Kushinr S. Babychuk E. Inzé D. Van Montagu M. J. Biol. Chem. 1995; 270: 28479-28486Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Surprisingly, a role for COPT1 in copper uptake from the soil has not been supported because RNA blotting analysis indicated that the level of COPT1 in roots was undetectable (26Kampfenkel K. Kushinr S. Babychuk E. Inzé D. Van Montagu M. J. Biol. Chem. 1995; 270: 28479-28486Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). In this work, we focus on the function of copper transport in Arabidopsis by investigating both the COPT1 expression pattern and the phenotypes obtained in COPT1 antisense plants. COPT1 tissue-specific expression was monitored using COPT1::GUS transgenic plants, and the phenotype of COPT1 antisense transgenic plants is described on the basis of the main tissues expressing the transporter, which include root apical zones and pollen. Our results demonstrate the important role of COPT1 in copper uptake, root growth, and pollen development. Plant Growth Conditions and Treatments—Seeds of A. thaliana, ecotype Columbia (Col 0), were grown in pots and covered with a clear plastic dome. The covered pots were placed in a cold room at 4 °C for 2 days to synchronize germination and then moved to a growth cabinet at 23 °C with a 16-h photoperiod (65 mmol/m2 of cool white fluorescent light). The plants were watered regularly with standard commercial greenhouse nutritive solution. The ethylene response in seedlings was analyzed as described (27Guzmán P. Ecker J.R. Plant Cell. 1990; 2: 513-523Crossref PubMed Scopus (971) Google Scholar). Briefly, seeds from the indicated lines were germinated in MS medium (28Murashige T. Skoog F. Physiol. Plant. 1962; 15: 437-497Crossref Scopus (52758) Google Scholar) and grown under hydrocarbon-free or ethylene (10 ppm) atmospheres and complete darkness. The triple response was determined after 3 days as a decrease in hypocotyl length and an increase in hook curvature and hypocotyl width. To study the sensitivity of seedlings to copper availability, the seeds were germinated in either standard MS medium plates including 1% sucrose (except for the root length experiments) (28Murashige T. Skoog F. Physiol. Plant. 1962; 15: 437-497Crossref Scopus (52758) Google Scholar) or supplemented with the indicated concentrations of bathocuproine disulfonic acid (BCS), 1The abbreviations used are: BCS, bathocuproine disulfonic acid; WT, wild type; RT, reverse transcription. metal ions or both. The plates were photographed 2 weeks after germination, and seedling fresh weight and root length was determined from five seedlings with the standard deviation. Plasmid Constructs and Plant Transformation—For the promoter studies a genomic library (λGEM-11; Promega) was screened, and a positive clone containing the COPT1 5′ region was isolated. EcoRV/BamHI (-1334 to +102, where +1 indicates the position of the starting codon) and a XhoI/MnlII (-359 to -15) restriction fragments were fused in frame to the GUS gene in the pBI121 plasmid, replacing the CaMV35S promoter. For functional studies, the COPT1 complementary strand was placed under the control of the CaMV35S promoter. The COPT1 cDNA was recovered from previous work (29Sancenón V. Puig S. Mira H. Thiele D.J. Peñarrubia L. Plant Mol. Biol. 2003; 51: 577-587Crossref PubMed Scopus (246) Google Scholar) and cloned into the SacI/SmaI sites of pBI121 vector. The C58 strain of Agrobacterium tumefaciens was used to transform Arabidopsis by following the floral dip protocol (30Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Crossref PubMed Google Scholar). Gene Expression by Semiquantitative RT-PCR—Total RNA was isolated from Arabidopsis tissues as described by Prescott and Martin (31Prescott A. Martin C. Plant Mol. Biol. Rep. 1987; 4: 219-224Crossref Scopus (211) Google Scholar). RNA was quantified by UV spectrophotometry, and its integrity was visually assessed on ethidium bromide-stained agarose gels. Total RNA (1-5 μg) isolated from the indicated tissues was first converted to cDNA by reverse transcription using SuperScript II reverse transcriptase (Invitrogen) and the anchored oligo(dT)15 (Roche Applied Science) and 18 S primer (reverse, 5′-CTGGATCCAATTACCAGACTCAA). To quantify endogenous COPT1 mRNA in the antisense lines, primer COPT1 (reverse, 5′-CCTGAGGGAGGAACATAGTTAG) was added instead of oligo(dT)15. For PCRs, 1.5-10 μl of the diluted (1:5) cDNA products were used as templates. The primers were designed to specifically anneal with COPT1 (forward, 5′-CAATGGATCCATGAACGAAGG; reverse, see above), COPT2 (forward, 5′-ACGTGTCAGTGGCTCAACC; reverse, 5′-GACGGCGGAAGAAGCTCGGCGG) and CCH (forward, 5′-ATGGCTCAGACCGTTGTCCT-3′; reverse, 5′-GCTCAGCTTCAGCCTCCACT-3′). The reactions were run at 94 °C (30 s), 55 °C (30 s), and 72 °C (30 s) for 25 (COPT2), 26 (CCH), or 27 (COPT1) cycles, which was within the linear reaction range. 18 S rRNA was also measured in the same reaction as an internal control with 18 S primers (forward, 5′-TGGGATACCTGCCAGTAGTCAT; reverse, see above) together with the competimers 18 S (having the same sequence as their counterparts, but a 3′-terminal dideoxy nucleotide). To balance the great excess of 18 S rRNA with respect to the message of COPT2 and CCH, a series of PCRs were performed varying the ratio of competimers/(primers + competimers) from 0 to 1. The ratio that yielded the best relation of COPT to 18 S intensities, without saturating the employed detection method (see below), was selected for further analysis. PCR products were run on a 1.8% agarose gel and stained with ethidium bromide. The relative values of COPT message levels in each sample were normalized with respect to 18 S. The band intensities were analyzed and quantified with the applications ImageStore 5000 and GelBlot (Ultra Violet Products Ltd.). GUS Expression Analyses—The assays were performed as described by Jefferson et al. (32Jefferson R.A. Kavanagh T.A. Bavan M.W. EMBO J. 1987; 6: 3901-3907Crossref PubMed Scopus (8126) Google Scholar). Seedlings or organs from adult plants were recovered and vacuum-infiltrated with the substrate solution (50 mm NaPO4, pH 7.2, 0.5 mm K3Fe(CN)6, 0.5 mm K4Fe(CN)6, 0.1% Triton X-100, 0.5 mm 5-bromo-4-chloro-3-indolyl β-d-glucuronide (X-Gluc; Research Organics Inc.)). The reactions were developed at 37 °C for times ranging from 20 min to several hours and stopped with an ethanol series (30-70%). Fresh GUS-stained seedlings or anthers were fixed in 4% formaldehyde for 4 h, followed by several washes in 0.11 m NaH2PO4, pH 7.0. The samples were dehydrated in ethanol series (70-100%), cleared with xylene, and embedded in low melting temperature paraplast (Sigma). Sections of 10 μm width were obtained in a rotating microtome and mounted in glass slides. The preparations were photographed in a Zeiss optical microscope. Copper Uptake and Accumulation Measurements—For the metal transport experiments, 10 mg of A. thaliana seeds ecotype Columbia (Col 0) and COPT1 antisense A 2-1, A 3-4, and A 3-6 transgenic lines were sterilized and incubated at 4 °C for 1-2 days to synchronize germination. The seeds were germinated at 23 °C for 60 h in water with white fluorescent light. One ml of phosphate-buffered saline was added to the seedlings derived from 2 mg of dry seeds. The seedlings were incubated either at 23 °C or on ice for 1 h. 64CuCl2 (Mallinckrodt Institute of Radiology, Washington University, St. Louis, MO), specific activity 15-30 mCi/μg of copper, was added to a final concentration of 5 μm, and the seedlings were incubated at 23 or at 4 °C for 30 min with continuous agitation. The samples were quenched by adding ice-cold EDTA (final concentration, 10 mm), filtered, and washed with quenching buffer (10 mm EDTA in 0.1 m Tris-succinate, pH 6.0). 64Cu was quantified in a γ-counter (Packard Cobra II) by using a standard curve. The values obtained at 0-4 °C were subtracted from 23 °C values and normalized to dry weight. The experiments were independently repeated at least in triplicate, and standard deviation were represented. To determine the copper content in wild type and transgenic plants, leaves from 4-week-old adult plants were dried and frozen in liquid N2, ground in a mortar, and lyophilized. 50 mg of dried tissue was digested in 1 ml of HNO3 for 10 h at 80 °C. After adding 4 ml of H2O, the copper content of each sample was determined by atomic absorption spectrometry (Analytical Technology, Inc., Unicam Solar System 939). Pollen Preparations for Scanning Electron Microscopy—Pollen was mounted on standard stubs and coated with gold-palladium in a Bio-Rad E5600 ion sputter for 3 min prior to observation on a Hitachi S4100 FE scanning electron microscope. Digital images were acquired with the application EMIP. COPT1 belongs to a five-member family (COPT1-5) of putative Arabidopsis copper transporters (26Kampfenkel K. Kushinr S. Babychuk E. Inzé D. Van Montagu M. J. Biol. Chem. 1995; 270: 28479-28486Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 29Sancenón V. Puig S. Mira H. Thiele D.J. Peñarrubia L. Plant Mol. Biol. 2003; 51: 577-587Crossref PubMed Scopus (246) Google Scholar). Consistent with a putative role as plasma membrane copper transporters, two members of the family, COPT1 and COPT2, robustly restore the respiratory deficiency of a yeast mutant defective in high affinity copper transport, and their corresponding mRNAs are down-regulated in response to copper excess (29Sancenón V. Puig S. Mira H. Thiele D.J. Peñarrubia L. Plant Mol. Biol. 2003; 51: 577-587Crossref PubMed Scopus (246) Google Scholar). The present study is focused on the functional characterization of COPT1 and its physiological role in copper transport in Arabidopsis. COPT1 Is Expressed in Root Tips and Pollen Grains—To study the COPT1 spatial expression pattern, a 1.4-kb COPT1 promoter region (-1334 to +102, where +1 indicates the position of the first nucleotide in the COPT1 cDNA) was fused to the uidA gene, which encodes β-glucuronidase (GUS). Stable homozygous transgenic Arabidopsis lines harboring the COPT1::GUS chimeric gene were obtained, and those that exhibited GUS activity were selected for further analysis (results not shown). Transgenic lines expressing GUS under the control of the Cauliflower mosaic virus 35S (CaMV35S) general promoter and a minimal COPT1 promoter (-359 to -15) were used as positive and negative controls for GUS expression, respectively. In Arabidopsis plants transformed with the COPT1::GUS construct, embryos at the heart stage are uniformly GUS-stained except in the suspensor (Fig. 1A). GUS expression was also present in the hydrated seeds of mature Arabidopsis embryos and the cotyledons of young seedlings (not shown). Histological analyses of adult plants showed COPT1 promoter-driven GUS expression specifically restricted to trichomes and stomata guard cells in adult leaves (Fig. 1, B and C). Moreover, although GUS staining is not detected during the early stages of flower development (Fig. 1D), mature pollen grains are intensely stained at the pistil, and they remain so during pollination (Fig. 1E). A mature anther cross-section shows that pollen is uniformly stained (Fig. 1F), indicating that at least the vegetative cell is expressing the COPT1::GUS fusion. In all cases, negative controls with the COPT1 minimal promoter remained unstained, and CaMV35S::GUS positive controls were uniformly stained (not shown). Although a previous analysis of total RNA indicated the absence of COPT1 mRNA in roots (26Kampfenkel K. Kushinr S. Babychuk E. Inzé D. Van Montagu M. J. Biol. Chem. 1995; 270: 28479-28486Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar), we detected COPT1 expression in roots by RT-PCR (29Sancenón V. Puig S. Mira H. Thiele D.J. Peñarrubia L. Plant Mol. Biol. 2003; 51: 577-587Crossref PubMed Scopus (246) Google Scholar) and showed that GUS expression is restricted to a specific area at the root apical zone (Fig. 2). Expression in roots persisted throughout the whole life of the plant and could be observed both at the primary root (Fig. 2A) and at lateral root primordia of adult plants (Fig. 2B). GUS staining was restricted to an annular region immediately after the root tip (Fig. 2C). As a positive control, CaMV35S::GUS transgenic plants showed constitutive and global GUS staining in roots (Fig. 2D). To further address the cellular location of COPT1 expression at root apical zones, histochemical detection of GUS staining was carried out in longitudinal (Fig. 2E) and transverse (Fig. 2F) root apical sections of 2-day-old Arabidopsis seedlings. Labeling was mainly detected at the columella, lateral root cap, epidermis, and cortex layers, whereas expression was only poorly detected at the endodermis and the stele (Fig. 2, E and F). Copper Deficiency Responses Are Up-regulated in COPT1 Antisense Transgenic Plants—The effects of reduced COPT1 expression on plant phenotypes were studied in transgenic lines expressing COPT1 in antisense orientation under the control of the CaMV35S promoter. Independent Arabidopsis transgenic lines expressing CaMV35S::COPT1 antisense were generated and stable homozygous lines were obtained. All of the lines expressed high levels of COPT1 antisense mRNA (not shown). To gain initial insight whether antisense COPT1 expression causes defects in copper acquisition in transgenic lines, we accurately measured the COPT1 mRNA endogenous levels by real time PCR in three transgenic lines. COPT1 mRNA levels quantified by this technique turned out to be approximately the same as those found in wild type plants (not shown). Although the critical issue would be to detect COPT1 protein stationary levels in these lines, unfortunately specific antibodies are not available. For this reason, we measured the transcript levels of CCH and COPT2, two genes that are up-regulated in response to decreased copper concentrations (12Himelblau E. Mira H. Lin S.-J. Culotta V.C. Peñarrubia L. Amasino R.M. Plant Physiol. 1998; 117: 1227-1234Crossref PubMed Scopus (171) Google Scholar, 29Sancenón V. Puig S. Mira H. Thiele D.J. Peñarrubia L. Plant Mol. Biol. 2003; 51: 577-587Crossref PubMed Scopus (246) Google Scholar). COPT1 antisense lines (A 2-1, A 3-4, and A 3-6) displayed CCH, COPT2, or both mRNA levels up to four times higher than wild type controls (Fig. 3). The observation that genes whose expression is induced by copper limitation are induced in these lines is consistent with a reduction in bioavailable copper levels. Therefore, these lines were used for further experiments. Copper Uptake in Arabidopsis COPT1 Antisense Transgenic Plants—We previously demonstrated that heterologous expression of the Arabidopsis COPT1 gene in a yeast mutant defective in high affinity copper uptake specifically stimulates 64Cu uptake, and competition experiments strongly support COPT1 specificity for Cu(I) over other transition metals (29Sancenón V. Puig S. Mira H. Thiele D.J. Peñarrubia L. Plant Mol. Biol. 2003; 51: 577-587Crossref PubMed Scopus (246) Google Scholar). To further evaluate the ability of the COPT1 protein to transport copper into Arabidopsis, 64Cu uptake assays were carried out in wild type seedlings and compared with the COPT1 antisense lines. Arabidopsis seeds were germinated for 2.5 days, and radioactive copper accumulation in seedlings was measured after incubation with 64CuCl2. All three COPT1 antisense Arabidopsis lines (A 2-1, A 3-4, and A 3-6) showed a 40-60% reduction in the short term levels of 64Cu accumulation as compared with the wild type (WT) (Fig. 4A). To evaluate whether the reduced levels of 64Cu resulted in reduced steady state copper accumulation in COPT1 antisense adult plants, copper content in plant leaves from both wild type and COPT1 antisense transgenic lines was determined by atomic absorption spectroscopy. Consistent with the copper transport data in Fig. 4A, the average copper content in 4-week-old Arabidopsis rosette leaves was reduced ∼40-60% in the COPT1 antisense transgenic lines when compared with the control (WT; Fig. 4B). Taken together, these results strongly implicate the COPT1 protein in copper uptake and accumulation in Arabidopsis plants. COPT1 Antisense Transgenic Plants Display Increased Root Length—Despite the reduced copper uptake levels and accumulation, COPT1 antisense plants are apparently as healthy as controls when grown under standard laboratory conditions in growth cabinets. Because of the well established requirement of copper ions for the ethylene receptor (16Hirayama T. Kieber J.J. Hirayama N. Kogan M. Guzman P. Nourizadeh S. Alonso J.M. Dailey W.P. Dancis A. Ecker J.R. Cell. 1999; 97: 383-393Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 22Rodriguez F.I. Esch J.J. Hall A.E. Binder B.M. Schaller G.E. Bleecker A.B. Science. 1999; 283: 996-998Crossref PubMed Scopus (489) Google Scholar), a more detailed study was performed to determine whether the COPT1 antisense RNA-induced copper deficiency impacts ethylene sensing and/or signaling. For this purpose, seeds derived from the COPT1 A 2-1, A 3-4, and A 3-6 transgenic lines were germinated in MS medium plates with 1% sucrose (28Murashige T. Skoog F. Physiol. Plant. 1962; 15: 437-497Crossref Scopus (52758) Google Scholar), grown for 3 days in darkness under hydrocarbon-free or ethylene (10 ppm) atmospheres, and analyzed for their triple response to ethylene (27Guzmán P. Ecker J.R. Plant Cell. 1990; 2: 513-523Crossref PubMed Scopus (971) Google Scholar). Arabidopsis seedlings from both wild type and a mutant (ctr1-1) for an ethylene signal transduction component conferring constitutive triple response (33Kieber J.J. Rothenberg M. Roman G. Feldmann K.A. Ecker J.R. Cell. 1993; 72: 427-441Abstract Full Text PDF PubMed Scopus (1463) Google Scholar) were used as controls in the assays. All of the COPT1 antisense lines tested behaved as the wild type seedlings, indicating that ethylene sensing and signaling are not disturbed in these plants. Moreover, the addition of 30 μm of the copper chelator BCS to the growth medium did not cause any effect on the triple response, showing that ethylene action remains unaltered in these plants under these conditions (not shown). Interestingly, seedlings from COPT1 antisense transgenic plants exhibited roots three to six times longer than controls when grown on the sucrose-depleted medium (Fig. 5A, line A 3-6). The roots returned to wild type length when treated with 30 μm copper sulfate, whereas the addition of the same concentration of transition metals such as iron or zinc failed to restore the phenotype (Fig. 5, A and B). COPT1 antisense roots remained 3.9 and 2.2 times longer than controls in media supplemented with iron or zinc, respectively (Fig. 5B). These results were also observed with the other two COPT1 antisense lines, A 2-1 and A 3-4, and were abolished by 1% sucrose addition (not shown). Interestingly, when wild type seedlings were supplemented with different concentrations of the copper chelator BCS, root length increased in a dose-dependent manner, whereas the COPT1 antisense plants were not significantly affected (Fig. 5C). Therefore, the copper chelator BCS mimics the root elongation phenotype observed in the COPT1 antisense transgenic plants in wild type plants. Taken together, these results strongly suggest that a defect in copper uptake from soil dramatically affects root elongation. Moreover, seedlings from all three COPT1 antisense transgenic lines grown in the presence of BCS showed a reduced rate of growth when compared with wild type; this was observed as both a decrease in c
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