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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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Plant Physiol Biochem 42:27-33 Hofacker IL, Fontana W, Stadler PF, Bonhoeffer S, Tacker M, Schuster P (1994) Fast folding and comparison of RNA secondary structures. Monatsh Chem 125:167-188 Karahara I, Ikeda A, Kondo T, Uetake Y (2004) Development of the Casparian strip in primary roots of maize under salt stress. Planta 219:41-47 Keilin E, Mann T (1939) Laccase, a blue copper-protein oxidase from the latex of Rhus succedanea. Nature 143: 23-24 Kiefer-Meyer M, Gomord VO, Connell A, Halpin C, Faye L (1996) Cloning and sequence analysis of laccase-encoding cDNA clones from tobacco. Gene 178:205-207 Kim C, Lorenze WW, Hoopes JT, Dean JFD (2001) Oxidation of phenolate siderophores by the multicopper oxidase encoded by the Escherichia coli yacK gene. J Bacteriol 183:4866LaFayette PR, Eriksson K-EL, Dean JFD (1999) Characterization and heterologous expression of laccase cDNAs from xylem tissues of yellow-poplar (Liriodendron tulipifera). Plant Mol Biol 40:23-35 LaFayette PR, Eriksson K-EL, Dean JFD (1995) Nucleotide sequence of a cDNA clone encoding an acidic laccase from sycamore maple (Acer pseudoplatanus L.). Plant Physiol 107:667-668 73 Liu L, Tewari RP, Williamson PR (1999) Laccase protects Cryptococcus neoformans from antifungal activity of alveolar macrophages. Infect Immunity 67:6034-6039 Ma F, Peterson CA (2003) Current insights into the development, structure, and chemistry of the endodermis and exodermis of roots. Can J Bot 81:405-421 Mayer AM, Staples RC (2002) Laccase: new functions for an old enzyme. Phytochemistry 60:551-565 McDougall GJ (2000) A comparison of proteins from the developing xylem of compression and non-compression wood of branches of Sitka spruce (Picea sitchensis) reveals a differentially expressed laccase. J Exp Bot 51: 1395-1401 Messerschmidt, A and Huber R (1990) The blue copper oxidases, ascorbate oxidase, laccase and ceruloplasmin: Modeling and structural relationships. Eur J Biochem 187:341-352 Meyers BB, Lee DK, Vu TH, Tej SS, Edberg SB, Matvienko M, Tindell LD (2004) Arabidopsis MPSS: an online resource for quantitative expression analysis. Plant Physiol 135:801-813 Novy R, Don Drott D, Keith Yaeger K, Mierendorf R (2001) Overcoming the codon bias of E. coli for enhanced protein expression. inNovations 12:1-3 O’Mally DM, Whetten R, Bao W, Chen CL, Sederoff RR (1993) The role of laccase in lignification. Plant J 4:751-757 Ranocha P, Chabannes M, Chamayou S, Danoun S, Jauneau A, Boudet AM, Goffner D (2002) Laccase down-regulation causes alterations in phenolic metabolism and cell wall structure in poplar. Plant Physiol 129: 145-155 Ranocha P, McDougall G, Hawkins S, Sterjiades R, Borderies G, Stewart D, Cabanes-Macheteau M, Boudet A-M, Goffner D (1999) Biochemical characterization, molecular cloning and expression of laccases - a divergent gene family- in poplar. Eur J Biochem 259:485-495 Ryden LG, Hunt LT (1993) Evolution of protein complexity: The blue copper-containing oxidases and related proteins. J Mol Evol 36:41-66

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Sterjiades R, Dean JFD, Eriksson K-EL (1992) Laccase from sycamore maple (Acer pseudoplatanus) polymerizes monolignols. Plant Physiol 99:1162-1168 Thurston CF (1994) The structure and function of fungal laccase.

Microbiology 140:19-26 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680– 685 Vuple CD, Kuo YM, Murphy TL, Cowley L, Askwith C, Libian N, Gitschier J, Anderson GJ (1999) Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse. Nat Genet 21:195-199 Wang GD, Li QJ, Luo B, and Chen XY (2004) Ex planta phytoremediation of trichlorophenol and phenolic alleochemicals via an engineered secretory laccase. Nature Biotech 22:893-897 Weigel D and Glazebrook J (2002) ARABIDOPSIS: A laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New Work. PP. 354 Yokoyama M, Karahara I (2001) Radial widening of the Casparian strip follows induced radial expansion of endodermal cells.

Planta 213:474–477 Zeier J, Ruel K, Ryser U, Schreiber L (1999) Chemical analysis and immunolocalisation of lignin and suberin in endodermal and hypodermal/rhizodermal cell walls of developing maize (Zea mays L.) primary roots. Planta 209:1-12 Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol 136: 2621-2632 75 Table 2.1. Different codon usages of Arg and Pro and Leu for E.

coli and the At2g30210 gene. Codon usage is expressed as the fraction of all possible codons for a given amino acid. “All genes” is the fraction represented in all 4,290 coding sequences in the E. coli genome. “Class II” is the fraction represented in 195 genes highly and continuously expressed during exponential growth. Table is modified from Novy et al. (2001).

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76 Figure 2.1. Expression profile of LMCOs in Arabidopsis thaliana.

LMCO gene expression data in selected Arabidopsis tissues obtained using from massively parallel signature sequencing (MPSS) was collected from the Arabidopsis MPSS website (http://mpss.udel.edu/at/). Values represent the pooled number of times the predominant signature tag for each Arabidopsis LMCO gene was seen in MPSS libraries from various Arabidopsis tissues.

77 Figure 2.2. Structure of the Arabidopsis At2g30210 gene. The Genomic DNA sequence of At2g30210 is shown (A). Introns (gray) and the untranslated regions (blue and underlined) are represented in lowercase letters. A putative TATA box is shown in boldface. Intron positions were based on cDNA sequence compared with genomic sequence. Relative locations of the signal peptide (red) and copper-binding domains (yellow) in the At2g30210 gene are shown (B). Exon color indicates featured domains.





78Figure 2.2.A.

1287044 aataaattgt ttaattgcta acgaattcac gaagacctct gttgtttccc gcttagtgtg 1286984 actccttcct tttctccaaa ctcatctcac tataaatacc aaatctccaa cacagagagc

ATGGAGTCTT TTCGGCGATT CTCCTTGCTA TCCTTCATTG

1286924 ttctcacagt gaagcaaaca

1286864 CCCTACTTGC CTACTTCGCT TTCCTCGCTT CTGCTGAACA TCACGTCCAT CAATTCGTGg

1286804 taagctagct actaatgtta cattaaagct ttttggtatg ttcttacgtt tatagaattc 1286744 ccttaattta agtttaaacg acgatgactt tagATCACAC CGACACCAGT GAAGAGGCTG

1286684 TGCAGAACTC ACCAAAGCAT CACTGTGAAT GGTCAGTACC CTGGTCCAAC GCTTGTGGTC

1286624 AGGAACGGTG ACTCTCTCGC AATCACTGTC ATCAACAGAG CCCGTTACAA CATTAGTATT

1286564 CATTGgtaaa atatttgacc atcaaattca acaaacatat gttatgttat gttctctgtt 1286504 ttcatacttt gttttttgct ctgttttttg ggatagGCAT GGAATCAGAC AGCTGCGGAA

1286444 TCCGTGGGCC GATGGTCCAG AGTATATAAC ACAATGTCCG ATCCGTCCAG GACAAACCTA

1286384 CACTTACAGA TTCAAAATCG AGGATCAAGA GGGTACGCTT TGGTGGCACG CTCATAGCCG

1286324 CTGGCTCAGA GCCACGGTCT ATGGTGCTCT CATCATTTAC CCTCGTCTTG GTTCTCCTTA

1286264 TCCCTTCTCT ATGCCCAAAC GTGACATTCC AATTCTTCTT Ggtaaacaac taaacatttt 1286204 gtaacttcta atcacttcaa attatatttt catgatcaca actaataata cttactaaaa 1286144 tagGGGAATG GTGGGATAGA AACCCAATGG ATGTTTTGAA GCAAGCACAA TTTACGGGAG

1286084 CAGCAGCTAA TGTCTCTGAC GCTTACACAA TCAACGGTCA ACCAGGCGAT CTTTACCGCT

1286024 GCTCGCGGGC TGGGACAATC CGTTTTCCAA TTTTCCCCGG GGAGACGGTG CAACTCCGTG

1285964 TCATCAACGC TGGTATGAAC CAAGAGCTCT TCTTCTCAGT CGCCAACCAC CAGTTCACAG

1285904 TTGTAGAAAC TGATTCCGCC TACACGAAAC CATTCACCAC AAATGTCATC ATGATCGGTC

1285844 CTGGCCAAAC CACTAACGTC CTCCTCACGG CAAACCAGAG ACCAGGCCGC TACTACATGG

1285784 CAGCTCGAGC CTACAACAGC GCAAACGCCC CGTTCGACAA CACAACCACT ACTGCTATCT

1285724 TACAATACGT CAACGCTCCA ACAAGACGTG GCCGTGGTCG TGGTCAAATC GCTCCTGTTT

1285664 TCCCAGTCCT CCCCGGGTTC AACGACACCG CAACCGCAAC TGCTTTCACC AACCGTCTCC

1285604 GATACTGGAA ACGAGCTCCA GTACCACAAC AAGTCGACGA GAACCTCTTT TTCACCGTCG

1285544 GATTAGGGCT AATCAACTGT GCCAACCCAA ACAGTCCCCG TTGCCAAGGT CCTAACGGGA

1285484 CCCGATTCGC AGCAAGCATG AACAACATGT CCTTCGTGCT ACCACGAAGT AACTCCGTCA

1285424 TGCAAGCATA TTACCAAGGC ACCCCAGGAA TCTTCACAAC GGATTTTCCG CCCGTTCCAC

1285364 CGGTGCAATT CGATTACACA GGTAACGTTA GCCGCGGGTT ATGGCAGCCC ATAAAAGGAA

1285304 CCAAAGCTTA CAAGCTTAAG TACAAATCTA ATGTTCAGAT TGTGTTACAA GACACTAGCA

1285244 TTGTCACGCC AGAGAATCAT CCCATGCATC TACACGGGTA CCAATTCTAC GTGGTCGGGT

1285184 CAGGTTTCGG TAATTTCAAC CCGAGAACAG ACCCGGCTAG GTTTAACTTA TTTGACCCAC

1285124 CAGAGAGGAA CACCATTGGA ACACCTCCAG GTGGTTGGGT GGCAATTCGG TTCGTCGCTG

1285064 ATAATCCAGg ttagatgatc ttattgactc gaaagcattt gatactttgt aaccataggt 1285004 taaatgatct tattgactca aaaatgaaaa acatattttg atatacttgt ggtgtaatgc

1284944 agGAGCATGG TTTATGCATT GTCACATTGA TTCACATTTG GGATGGGGTT TGGCTATGGT

1284884 TTTCTTGGTA GAGAACGGTC GTGGACAGTT GCAATCGGTG CAGGCTCCAC CATTGGATCT

1284824 TCCAAGATGC TAAtaaaaaa cttctaccgt tggatctaaa gaatttatgg ggtttggttt 1284764 tttgttcttg ttttttaatc aaaaaaagac ttcgtgagtc ttgatatgtt aaagatggac 1284704 aactgctcta ttgaaagtgg ttcaaccttt gttttcgatg tacttttgtt attttcctcg 1284644 taatagagtg agtgttttca tttttccgat taaaaattta ccaaataaaa gctgtgttag ttagttatta acttaatatg 1284584 ttaatcttct ttttctagct gactttccca aacaacttta 1284444 aacccacaag atacttttaa ttacattatc atacataaac ttttcttgca aaaatagcat B.

5’ 3’ 79 Figure 2.3. Possible secondary structure in the At2g30210 LMCO 5’UTR. An optimal secondary structure based on the minimum free energy was predicted using RNAfold. The 5’UTR sequence was directly submitted to the RNAfold program at a web interface server (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) (Hofacker et al. 1994).

80 Figure 2.4. Heterologous expression of At2g30210 in E. coli.

Aliquots (10µl) of crude sample lysates and different purification steps were loaded onto gels for SDS-PAGE and immunoblot analysis, respectively. A. Crude extracts from transformed (Lane 1) and control cells (Lane 2) after four hours of induction were analyzed by 10% SDS-PAGE (left panel) and immunoblot with an antibody against XpressTM tag (right panel).

B. Purification was performed with an affinity purification column under denaturing conditions using 8M urea buffer. Each step of the purification was collected and analyzed by 10% SDSPAGE (left panel) and immunoblot (right panel). Lane 1: crude

extract: Lane 2: flow through of binding buffer, pH 7.8; Lane 3:

flow through of wash buffer, pH 6.0; Lane 4: flow through of wash buffer, pH 5.3; Lane 5~10: a series of fractions eluted using elution buffer, pH 4.0. Samples were analyzed by SDS-PAGE (left). C. Lane 1: molecular weight marker; Lane 2: crude lysate of induced cells; Lane 3: unbound fraction of sample through affinity column; Lane 4: pooled final elution using wash buffer (pH 4.0); Lane 5: sample after dialysis against 10mM Tris, pH 8.0, 0.1% triton X-100 overnight at 4 oC; Lane 6: insoluble pellet after dialysis; Lane 7: 1 µg BSA: Lane 8: 5 µg BSA.

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Figure 2.5.

Enzyme activity analysis of the At2g30210 LMCO expressed in tobacco cells. A. Phenoloxidase activity was demonstrated in three kanamycin-resistant cell lines by testing the ability to oxidize the laccase substrate, ABTS, in liquid assays. B. Activity staining of multicopper oxidases separated using SDS-PAGE. Crude protein extracts from three cell lines (2,3,5) transformed with the AT2g30210 gene and a vector control cell line (C) were stained with 1,8-diaminonaphthalene (DAN) for phenoloxidase activities.

83 Figure 2.6. Testing the At2g30210 protein for ferroxidase activity. A. Partially purified protein (20 µg) from one resistant cell line (#2) was used for phenoloxidase staining with DAN (left panel). For detecting ferroxidase activity (right panel), the amount loaded for the sample was 10x that loaded (200 µg, Lane #2) for phenoloxidase staining and an equivalent amount of ABTS-oxidizing activity from the yacK protein (Lane yacK) was used as a positive control (Kim et al. 2001). Cleared zones representing ferroxidase activity were visualized by applying 3-(2-pyridyl)-5,6-bis (4-phenylsulfonic acid)-1,2,4triazine (ferrozine). B. Solution assay for ferroxidase activity was determined using ferrous sulfate as the electron donor and ferrozine as a specific chelator to bind ferrous iron remaining at the end of the reaction. Aliquots of 3 µg protein from tobacco cells transformed with At2g3210 cDNA (#2) or vector alone (Vec) were added to a total 1 mL reaction. Reaction buffer (Buffer) and a boiled sample (Boiled #2) were also included for negative control. Another set of samples were also analyzed in the same buffer with addition of 1 µM apo-transferrin (+Tf), as a Fe3+ acceptor to promote reactions.

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0.5 0 0 50 100 150 200 250 300

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85 Figure 2.7. Expression profile of the At2g30210 gene in various tissues at different developmental stages. A. Messenger RNA isolated from the identified tissues was reverse transcribed, and diluted cDNA product was used as template for each PCR reaction. QUB10 was included to control for equal cDNA input between tissue samples. fl: flowers, sil: siliques, lf: leaves, px: inflorescence, rt: roots, sdl: two weeks old seedling. B.

Gene expression for the At2g30210 gene in selected Arabidopsis tissues as determined using massively parallel signature sequencing (MPSS), as described in Figure 1. C. Affymetrix expression data for the At2g30210 gene represented on the ATH1 whole-genome chip. Microarray data for selected tissues were collected from the public database (https://www.genevestigator.ethz.ch) (Zimmermann et al. 2004)

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A LACCASE-LIKE MULTICOPPER OXIDASE (LMCO) MAY BE INVOLVED IN

ENDODERMIS FORMATION IN ARABIDOPSIS THALIANA ROOTS1

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

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