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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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ANALYSIS OF PHYSIOLOGICAL FUNCTION(S) OF

A LACCSKE-LIKE MULTICOPPER OXIDASE IN ARABIDOPSIS THALIANA

by

CHIEH-TING WANG

(Under the Direction of Jeffrey F. D. Dean)

ABSTRACT

Laccase and related laccase-like multicopper oxidases

(LMCOs) have been studied in plants for more than a century, yet our understanding of their physiological function(s) remains limited. The work described in this dissertation was directed toward understanding of the physiological function of one particular Arabidopsis LMCO gene, At2g30210, by measuring its enzyme activities, patterns of tissue-specific expression, subcellular localization, effects of loss-of-function mutations, and regulation by external factors. Heterologously expressed At2g30210 LMCO showed phenoloxidase active, but no ferroxidase activity. RT-PCR and transcriptional profiling showed that the At2g30210 LMCO gene is expressed primarily in root tissues.

Histochemical analyses of transgenic, GUS-expressing plants revealed that At2g30210 transcripts were preferentially expressed in developing endodermis of the root elongation zone.

Analyses of subcellular localization indicated that the At2g30210-YFP fusion product was localized to the cell periphery. A loss-of-function At2g30210 mutant displayed phenotypic responses to sugar availability and salt levels consistent with alterations in endodermal function. The At2g30210 LMCO gene was not regulated by changes in iron levels.

However, the gene was up-regulated in seedlings grown on MS medium lacking sucrose, a response that was consistent with a phenotype observed in knockout mutants of this gene. These results altogether suggest a possible physiological function for the At2g30210 LMCO gene product in endodermal cell development, possibly in formation of the lignin-like phenolic polymers observed in the specialized cell walls that constitute the Casparian strip in endodermal cells.

INDEX WORDS: laccase, laccase-like multicopper oxidase, LMCO, phenoloxidase, ferroxidase, Casparian strip, GUS, knockout mutant, sugar, salt stress, qPCR, gene expression, regulation, transformation, heterologous expression, suberin, lignin, Arabidopsis thaliana, endodermis

ANALYSIS OF PHYSIOLOGICAL FUNCTION(S) OF

A LACCSKE-LIKE MULTICOPPER OXIDASE IN ARABIDOPSIS THALIANA

by

CHIEH-TING WANG

B.S., National ChungHsing University, Taiwan, 1993 M.S., National Taiwan University, Taiwan, 1995 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA

–  –  –

Chieh-Ting Wang All Rights Reserved

ANALYSIS OF PHYSIOLOGICAL FUNCTION(S) OF

A LACCSKE-LIKE MULTICOPPER OXIDASE IN ARABIDOPSIS THALIANA

–  –  –

Electronic Version Approved:

Maureen Grasso Dean of the Graduate School The University of Georgia August 2005 To Shu-Chen, Erin, and Ethan, my love and future

–  –  –

Thank you all that help me with this document and my academic career. I have to say a special Thank you to Dr.

Jeffrey Dean for all of his guidance and help that he has provided along the way. I would like to thank Dr Bonnie McCaig who serves as a mentor from the beginning of my project. I also want to thank you my committee: Dr. Joe Nairn, Dr. Sarah Covert, Dr. Scott Merkle, Dr. Zheng-Hua Ye for their valuable comments and critical review.

Finally, I have to say thank you to my love, Shu-Chen, who has been with me all way long. Without her I would have not accomplished this goal. I also want to say thank you to my lovely daughter and son who always entertain me.

–  –  –

ACKNOWLEDGEMENTS

LIST OF TABLES

LIST OF FIGURES

CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW

2 CLONING, CHARACTERIZATION, AND EXPRESSION ANALYSIS OF A

LACCASE-LIKE MULTICOPPER OXIDASE GENE FROM ARABIDOPSIS

THALIANA

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

IN ENDODERMIS FORMATION IN ARABIDOPSIS THALIANA ROOTS

4 ANALYSIS OF A LACCASE-LIKE MULTICOPPER OXIDASE (LMCO)

IN RESPONSE TO ABIOTIC CONDITIONS

5 SUMMARY AND CONCLUSIONS

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Table 2.1: Different codon usages of Arg and Pro and Leu for E.

coli and the At2g30210 gene

Table 3.1: Oligonucleotide primers used in this study.

........117

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Figure 1.1: A model for copper and iron uptake systems in yeast

Figure 1.2: Neighbor-joining phylogeny depicting relatedness of plant LMCOs

Figure 1.3: Three-dimensional structure of the LMCO from Melanocarpus albomyces





Figure 1.4: An overview of the conserved domains identified by multiple sequence alignment for LMCOs.

.................42 Figure 1.5: An expression profile for Arabidopsis LMCOs......44 Figure 1.6: Iron acquisition systems in plants................45 Figure 2.1: Expression profile of LMCOs in Arabidopsis thaliana

Figure 2.2: Structure of the Arabidopsis At2g30210 gene.

.......78 Figure 2.3: Possible secondary structure in the At2g30210 LMCO 5’UTR

Figure 2.4: Heterologous expression of At2g30210 in E.

coli..81 Figure 2.5: Enzyme activity analysis of the At2g30210 LMCO expressed in tobacco cells

Figure 2.6: Testing the At2g30210 protein for ferroxidase activity

Figure 2.7: Expression profile of the At2g30210 gene in various tissues at different developmental stages.

.............86 Figure 3.1: Insertion position in the At2g30210 mutants.....118 Figure 3.2: Genetic analyses of the At2g30210 insertion mutants

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Figure 3.4: Transcription of At2g30210 in wild-type and insertion mutants of Arabidopsis

Figure 3.5: T-DNA insertion mutants showing reduced growth in the absence of sugar

Figure 3.6: Growth alterations in the GT7855 mutant.

..........124 Figure 3.7: GT7855 insertion line grown on MS media without sucrose

Figure 3.8: Exogenous sugar enables growth of the GT7855 insertion mutant in soil

Figure 3.9: Salt sensitivity in the gene-trap insertional mutant

Figure 3.10: At2g30210 promoter expression patterns.

........128 Figure 3.11: Developmental regulation of the At2g30210 promoter

Figure 3.12: Subcellular localization of the At2g30210-EYFP chimeric protein in BY2 tobacco cells and transgenic Arabidopsis

Figure 4.1: Responses of Arabidopsis LMCOs to metal treatment

Figure 4.2: Responses of Arabidopsis LMCOs to sucrose.

.......145 Figure 4.3: Responses of At2g30210 gene expression to salt...146

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Multicopper oxidases Multicopper oxidases (MCOs) typically contain four copper atoms per protein molecule and catalyze oxidation reactions in which electrons are removed from reducing substrates and transferred to oxygen to form water. In general, MCOs possess three spectroscopically distinct copper centers. These centers are called Type-1 (or blue), Type-2 (or normal) and Type-3 (or coupled binuclear), although in some MCOs Type-2 and Type-3 centers may be organized into a trinuclear cluster (Solomon et al. 1996). Type-1 copper (blue copper) centers exhibit intense spectral absorption at about 600 nm, owing to a charge-transfer reaction between Cu2+ and a coordinated cysteine residue. Type-2 copper centers show little or no visible light absorption, and function as a one-electron acceptor. Type-3 copper centers contain two copper atoms that absorb at 330 nm, and function as a two-electron acceptor (Solomon et al. 1996).

Well-known MCOs include laccases in fungi and plants, ascorbate oxidase in plants, ceruloplasmin in mammals, and the Fet3 ferroxidase in yeast. In addition to these enzymes, there are a number of other proteins, which are similar to the multicopper oxidases, including the copper-resistance protein A

–  –  –

dihydrogeodin oxidase, and tyrosinase enzymes from various fungi; and the phenoxazinone synthase enzyme found in several Streptomyces species (Messerschmidt 1997, Brouwers et al. 2000).

All these enzymes are involved in the oxidation of organic compounds (Solomon et al. 1996; Brouwers et al. 2000).

Ceruloplasmin is unique among the multicopper oxidases in that it contains two additional Type-1 centers (Solomon et al.

1996). Although isolated ceruloplasmin is able to oxidize aromatic substrates, it oxidizes ferrous iron with much higher affinity (Harris et al. 1995). Ceruloplasmin has been implicated in iron metabolism because of its catalytic oxidation of Fe2+ to Fe3+ (ferroxidase activity) (Harris et al. 1995, Mukhopadhyay et al. 1998). Its main function is thought to be in mobilization of cellular iron. Mutations leading to a loss of ceruloplasim activity disrupt mammalian iron homoeostasis (Harris et al.

1995, 1999, 2004). Hephaestin, a ceruloplasmin homologue involved in releasing iron from intestinal enterocytes into the circulatory system (Vulpe et al. 1999), can oxidize ferrous iron as well as p-phenylenediamine, a typical laccase substrate (Chen et al. 2004).

Fet3p is a MCO required for a high-affinity iron uptake system in yeast (Askwith et al. 1994, Stearman et al. 1996). A model for this high-affinity (Km = 2µM) iron uptake system is

–  –  –

ferrireductases is converted into Fe3+ by the Fet3p, and is subsequently transferred to an Fe-binding site on the associated permease protein, Ftr1p (Eide 1998, Shi et al. 2003).

Interaction between Fet3p and Ftr1p is required for exact protein targeting, and in the absence of any Ftr1p, Fet3p fails to properly localize to the plasma membrane in yeast (Shi et al.

2003). The Fet5p/Fth1p complex, a homologue of the Fet3p/Ftr1p complex, plays a role in releasing iron from the yeast vacuole to the cytoplasm (Urbanowski and Piper 1999).

The discovery that the Fet3 protein plays a key role in yeast iron metabolism has had a great impact on our understanding of iron metabolism across a spectrum of eukaryotes, in particular supporting the function of the plasma MCOs, ceruloplasmin and hephaestin, in vertebrates (Askwith and Kaplan 1998, Harris et al. 1999, Attieh et al. 1999, Vulpe et al. 1999). Recently, Harris et al. (2004) showed that injection of yeast Fet3p could restore iron homeostasis in phlebotomized mice having a deletion of their endogenous ceruloplasmin genes. This analysis strongly supports the conservation of function for copper-containing proteins in eukaryotic iron metabolism.

Ascorbate oxidase, which catalyzes the oxidation of ascorbate to dehydroascorbate, is found mainly in higher plants, and is especially abundant in members of the Cucurbitaceae (e.g.,

–  –  –

discovered by Szent-Gyorgyi (1928), this enzyme has undergone extensive biochemical study, and numerous genes encoding ascorbate oxidases have been isolated and sequenced (Messerschmidt 1997). Ascorbate oxidase is one of the few multicopper oxidases that have been well characterized by X-ray crystallography (Messerschmidt et al. 1992). Immunohistochemical localization in green zucchini revealed that ascorbate oxidase is distributed ubiquitously over vegetative and reproductive organs (Chichiricco et al. 1989). Although ascorbate oxidase has been characterized in detail with regard to enzyme structure and expression, its in vivo role in plants remains unclear. The enzyme has been suggested to play a role in cell expansion (Lin and Varner 1991), in regulation of the cell cycle (Arrigoni 1994, Diallinas et al. 1997), and in plant oxidative defense systems (Foryer et al. 1994).

Laccases and laccase-like multicopper oxidases (LMCOs) are common enzymes in nature, and they are widespread in plants and fungi, as well as in some bacteria and insects. They are structurally homologous to ceruloplasmin and ascorbate oxidase, and are interesting as models for understanding multicopper oxidases (Messerschmidt 1997). Laccase is a polyphenol oxidase (p-diphenol:oxygen oxidoreductases, EC 1.10.3.2), first reported in Japanese lacquer tree (Rhus vernicifera) sap by Yoshida

–  –  –

Bertrand (1895). Although some enzymes in the MCO family oxidize specific, identified substrates, and have been named accordingly, most of the better known MCOs, including the “laccases,” are capable of oxidizing multiple potential substrates, and this has created significant confusion in enzyme nomenclature (Mayer and Harel 1979). Indeed, the name “laccase” should be reserved for those members of the MCO family that are found in plant saps containing the unsaturated alkylcatechols (“laccol,” also known as “urushiol”) that are their natural substrates (Du et al. 1984). However, the name has frequently been used to identify any monomeric MCO that contains four copper atoms in a specific arrangement of Type-1, Type-2 and Type-3 electromagnetic centers and displays some level of phenoloxidase activity (Messerschmidt 1997). Recently, many more “laccases” have been identified from genomic sequence data by sequence similarity with known laccase genes, rather than biochemical characterization. Due to the multiplicity of functions likely performed by the numerous MCOs appearing in plants, “laccase-like multicopper oxidases” or “LMCOs” has been proposed as a more appropriate for these newly discovered fourcopper MCOs (Hoopes and Dean 2004; McCaig et al. 2005).

–  –  –

Only a few bacterial LMCOs have been described so far. The first bacterial LMCO was detected in the plant root-associated bacterium, Azospirillum lipoferum (Givaudan et al. 1993), where it was shown to be involved in melanin formation (Faure et al.



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