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«by CHIEH-TING WANG (Under the Direction of Jeffrey F. D. Dean) ABSTRACT Laccase and related laccase-like multicopper oxidases (LMCOs) have been ...»

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Eur J Biochem 259:485-495 Roberts Sue A, Weichsel A, Grass G, Thakali K, Hazzard JT, Tollin G, Rensing C, Montfort WR (2002) Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli. Proc Natl Acad Sci USA 99:2766-2771 Robinson NJ, Procter CM, Connolly EL, and Guerinot ML (1999) A ferric-chelate reductase for iron uptake from soils. Nature 397:694-697 Salas SD, Bennett JE, Kwon-Chung KJ, Perfect JR, Williamson PR (1996) Effect of the laccase gene CNLAC1, on virulence of Crytococcus neoformans. J Exp Med 184:377-386 Sato Y, Bao W, Sederoff R, Whetten R (2001) Molecular cloning and expression of eight cDNAs in loblolly pine (Pinus taeda).

J Plant Res 114:147–155 Schlosser D, Hofer C (2002) Laccase-catalyzed oxidation of Mn2+ in the presence of natural Mn3+ chelators as a novel source of extracellular H2O2 production and its impact on manganese peroxidase. Appl Environ Microbiol 68:3514-3521 Shi X, Stoj C, Romeo A, Kosman D J, Zhu Z (2003) Fre1p Cu2+ reduction and Fet3p Cu1+ oxidation modulate copper toxicity in Saccharomyces cerevisiae. J Biol Chem 278:50309–50315 Singh SK, Grass G, Rensing C, Montfort WR (2004) Cuprous oxidase activity of CueO from Escherichia coli. J Bacteriol 186:7815– 7817 Solano F, Lucas-Elio P, Fernandez E, Sanchez-Amat A (2000)

Marinomonas mediterranea MMB-1 transposon mutagenesis:

isolation of a multipotent polyphenol oxidase mutant. J Bacteriol 182:3754-3760

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Stearman R, Yuan DS, Yamaguchi-Iwai Y, Klausner RD, Dancis A (1996) A permease-oxidase complex involved in high-affinity iron uptake in yeast. Science 271: 1552-1557 Sterjiades R, Dean JFD, Eriksson K-EL (1992) Laccase from sycamore maple (Acer pseudoplatanus) polymerizes monolignols.

Plant Physiol 99:1162-1168 Sterjiades R, Dean JFD, Gamble G, Himmelsbach DS, Eriksson K-EL (1993) Extracellular laccase and peroxidases from sycamore maple (Acer pseudoplatanus) cell-suspension culture. Planta 190:75-87 Thomine S, Wang R, Ward JM, Crawford NM, Schroeder JI (2000) Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes. Proc Natl Acad Sci USA 97:4991–96 Thurston CF (1994) The structure and function of fungal laccase.

Microbiology 140:19-26 Urbanowski J, Piper RC (1999) The iron transporter fth1p forms a complex with the fet5 iron oxidase and resides on the vacuolar membrane. J Biol Chem 274:38061-38070 Van Waasbergen LG, Hildebrand M, Tebo BM (1996) Identification and characterization of a gene cluster involved in maganese oxidation by spores of a marine Bacillus sp. strain SG-1. J Bacteriol 178:3517-3530 Varotto C, Maiwald D, Pesaresi P, Jahns P, Salamini F, Leister D (2002) The metal ion transporter IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana. Plant J 31:589–99 Vert G, Grotz N, Dedaldechamp F, Gaymard F, Guerinot ML (2002) IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth. Plant Cell 14:1223–33 Vulpe CD, Kuo YM, Murphy TL, Cowley L, Askwith C, Libina N, Gitschier J, Anderson GJ (1999) Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the SLA mouse. Nature Genet 21:195-199

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Wang GD, Li QJ, Luo B, Chen XY (2004) Ex planta phytoremediation of trichlorophenol and phenolic allelochemicals via an engineered secretory laccase. Nature Biotech 22:893–897 Worral JJ, Chet I, Hüttermann A (1986) Association of rhizomorph formation with laccase activity in Armillaria spp. J Gen Microbiol 132:2527-2533 Yoshida H (1883) Chemistry of lacquer (Urushi) part 1. J Chem Soc 43:472–486 Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol 136:2621-2632 37 Figure 1.1. A model for copper and iron uptake systems in yeast.

(picture taken from Department for molecular genetics, University Medical Center Utrecht, Netherlands http://genomics.med.uu.nl/~harm/research ) 38 Figure 1.2 Neighbor-joining phylogeny depicting relatedness of plant LMCOs. Arabidopsis genes are shown in boxes. Note that Arabidopsis genes are spread across all groups and there are corresponding homolog genes on chromosome 2 and chromosome 5 from 4 of the phylogenetic groups (1,2,3,and 4). (from McCaig et al. 2005).

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40 Figure 1.3. Three-dimensional structure of the LMCO from Melanocarpus albomyces. Domains A, B,and C, are colored red, green, and blue, respectively. The four copper atoms are shown as yellow balls and carbohydrates as grey sticks. (modified from Hakulinen et al. 2002).

41 Figure 1.4. An overview of the conserved domains identified by multiple sequence alignment for LMCOs. Consensus sequence obtained after multiple sequence alignment of (A) 64 fungal LMCO sequences; (B) 40 plant LMCO sequences; (C) consensus sequence from (A) and (B). The conserved regions are shaded if the length of the sub-sequence is greater than 7 residues. The corresponding regions, which are conserved both in plant and fungal laccases, are shaded in the same pattern. Sequence logos are shown for selected regions, L1–L4, which represent the frequency of occurrence of each amino acid at a particular position in the ungapped region of the aligned protein sequences. Height of a letter in the logo represents the information content at that location. (Picture adapted from Kumar et al. 2003) 42 43 Figure 1.5. An expression profile for Arabidopsis LMCOs. Data were collected from online microarray studies using the MetaAnalyzer on the GENEVESTIGATOR web server (https://www.genevestigator.ethz.ch) (Zimmermann et al. 2004).

The blue/white color indicates the averaged signal intensities.

Similar profiles were clustered together.

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Figure 1.6.

Iron acquisition systems in plants. In order to acquire sufficient iron, plants utilize multiple iron

acquisition and transport systems. Chl: chloroplast, Mt:

mitochondria, Vac: vacuole.

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1 Wang, C. –T. and J.F.D. Dean. To be submitted to Plant molecular biology Today.

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Laccases or laccase-like multicopper oxidases (LMCOs) have been found in a wide range of living organisms, yet in many cases, their physiological functions remain unclear. Because of its completed genome sequence, Arabidopsis provides a good system in which to study the physiological function of LMCOs.

Here we report the cloning, characterization, and expression analysis of one particular LCMO gene from Arabidopsis thaliana.

Analysis of the gene structure and predicted amino acid sequence revealed that the At2g30210 LMCO possesses the conserved copper binding domains that characterize multicopper oxidases.

Heterologously expressed LMCO showed phenoloxidase activity, but no ferroxidase activity. RT-PCR and transcriptional profiling showed that the At2g30210 LMCO gene is expressed primarily in root tissues. Putative physiological functions for this specific LMCO are discussed in the context of these results.

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Laccases, or p-diphenol:oxygen oxidoreductases (EC, belong to an enzyme superfamily called the multicopper oxidase (MCO), which also includes ascorbate oxidase (E.C. No. from plants, the yeast FET3 protein, and ceruloplasmin (E.C. No. from vertebrate animals (Messerschmidt and Huber, 1990; Ryden and Hunt, 1993). Laccases or laccase-like multicopper oxidases (LMCOs) have been found in a wide range of living organisms, including bacteria, fungi, insects, and plants. Fungal LMCOs are the most extensively documented group of enzymes in this superfamily, and their functions have been shown to be associated with a variety of physiological processes, including lignin degradation, morphogenesis, and pathogenicity (Thurson, 1994; Mayer and Staples 2002). In plants, LMCOs encoded by multigene families were observed in Arabidopsis thaliana, rice (Oryza sativa), tobacco (Nicotiana tabacum)(Kiefer-Meyer et al. 1996), poplar (Populus trichocarpa)(Rancha et al. 1999), yellow-poplar (Liriodendron tulipifera)(Lafayette et al. 1999), Sitka spruce (Picea sitchensis)(McDougall 2000), and loblolly pine (Pinus taeda)(Bao et al. 1993; Sato et al. 2001), yet their physiological functions remain unclear.

Although early studies suggested that laccase and laccaselike oxidase activities were associated with lignification in

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tightly coordinated with lignin deposition in vascular tissues (O'Malley et al. 1993; Dean and Eriksson 1994), definitive evidence to support a role for LMCOs in this process has not yet been demonstrated.

Previous studies of plant LMCOs have focused either on enzymological or molecular genetic aspects independently, and confirmed links between the corresponding gene products has been limited. The first complete DNA sequence reported for a plant LMCO was for a cDNA isolated from sycamore maple (Acer pseudoplatanus) (LaFayette et al. 1995). The capabilities of LMCOs from this species to polymerize lignin precursors and localize to the cell wall of lignifying vascular tissues in Acer stems were demonstrated earlier (Sterjiades et al. 1992, Driouich et al. 1992). Ranocha et al. (1999) were the first to report in a single study the cloning of several distinct LMCO cDNAs from poplar together with the biochemical characterization of one of the corresponding gene products. Recently, the same group was able to demonstrate a linkage to vascular tissue development by genetic modification of 3 independent LMCO genes.

Although the results did not clearly point to a role for an LMCO in lignification, they did suggest that the product of at least one LMCO gene is essential for structural integrity of xylem fiber cell walls (Ranocha et al. 2002).

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some LMCOs. For instance, FET3 encodes a multicopper oxidase required for a high-affinity iron uptake system in yeast (Askwith et al, 1994). Recent identification of a ferroxidase activity in a LMCO from yellow-poplar (Hoopes and Dean 2004) suggests that at least some LMCOs in plants have the capacity to take part in iron metabolism systems similar to the FET3 system found in yeast.

Because of its completed genome sequence, Arabidopsis provides a good system in which to study the physiological function of plant LMCOs. Previous work from our lab (McCaig et al. 2005) and data available from public databases showed that the 17 LMCO genes in Arabidopsis exhibit a wide variety of expression patterns in different tissues (Figure 2.1). One particular LMCO gene (At2g30210) showed strong expression in young roots, a tissue not thought to be highly lignified. To develop a better understanding of the physiological functions of plant LMCOs, we cloned, characterized, and measured expression of the At2g30210 LCMO gene from root tissues of Arabidopsis thaliana. Its likely physiological function(s) are discussed on the basis of these results.

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RNA isolation and cDNA synthesis Wild-type A. thaliana (var. Columbia) seeds were sterilized in 50% EtOH for 1 minute, transferred to 50% bleach and 0.1% Tween for 10 minutes, and washed three times in sterile water.

Surface sterilized seeds were germinated and grown for two weeks in liquid Murashige and Skoog medium (Invitrogen, Carlsbad, CA) containing sucrose (20g/L), with shaking at 22oC under constant illumination. Roots were harvested from 2 week-old seedlings, immediately frozen in liquid nitrogen, and stored at –80oC until used. For tissue-specific analyses, different tissues were collected from 8-week old plants grown in soil.

Total RNA was extracted by the method of Durbin et al.

(2000), or using Trizol reagent according to manufacture’s instructions (Invitrogen). For cDNA synthesis, 5µg of total RNA was reverse transcribed using 200 units of Superscript II reverse transcriptase (Invitrogen) with an adapter primer (AP) in a 50µl 3’ RACE (Rapid Amplification of cDNA Ends) reaction, according to the manufacturer’s instructions (Invitrogen). For tissue-specific analysis, cDNA pools from different tissues were first diluted 10-fold before amplification with a pair of PCR primers (1F, 5’-ATGGAGTCTTTTCGGCGATT-3’ and 1016R, 5’TCGGAGACGGTTGGTGAAAG-3’). The ubiquitin, UBQ10, gene was used as an internal control to show that the same amount of cDNA was

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UBQ10 gene was a 483 bp fragment obtained using the UBQ10


5’-CGACTTGTCATTAGAAAGAAAGAGATAACAGG-3’)(Weigel and Glazebrook 2002).

Cloning of the At2g30210 cDNA The coding region of the At2g30210 gene was amplified using the forward primer, 1F, and the reverse primer 5’TTAGCATCTTGGAAGATCCA-3’ (1694R). A different forward primer, 5’- GAACATCACGTCCATCAATTC-3’ (76F), along with the reverse primer (1694R) was used to amplify the coding region lacking the signal peptide. The PCR reaction mixtures in a total volume of 50 µl, consisted of 1µl cDNA template from a 20µl cDNA synthesis reaction using 5µg of total RNA, 5µl reaction buffer (500 mM KCl, 100 mM Tris-HCl pH 7.0, and 1.0% Triton® X-100), 2 mM MgCl2, 0.2 mM dNTP, 0.2 µM each of forward and reverse primer, and 2.5 units proof-reading Taq DNA polymerase (Promega, Madison, WI). The reaction mixtures underwent an initial 3-minute denaturation at 94oC, then 30 cycles at 94oC (1 minute), 55oC (1 minute) and 72oC (2 minutes 30 seconds), and a final extension time of 10 minutes at 72oC. The PCR products were resolved on 0.8% agarose gels and stained with ethidium bromide. Aliquots (4µl) of the RT-PCR products were used for cloning into the cloning vector (PCR2.1)

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(Invitrogen). Putative positive clones were first analyzed by digestion with EcoRI restriction enzyme, and then sequenced using an automated DNA sequencer (ABI 310 or Licor 4200).

Sequences were matched exactly to the confirmed genomic sequence in GenBank.

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