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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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2003). Each of the N- and C-terminal regions contains a conserved HXH motif (Figure 1.4). Eight histidine residues within the four blocks provide the conserved ligands for the trinuclear center. The Type-2 copper is coordinated by two of these histidine residues, and each of the Type-3 copper atoms is coordinated by three of remaining histidine residues. The conserved ligands for the Type-1 copper center are located in the two C-terminal regions and consist of one cysteine and two histidine residues. The bond between the Type-1 copper and Scys is highly covalent, and is the source of the strong absorption band around 600 nm that gives these proteins their typical blue color. In some LMCOs the Type-1 copper atoms are also coordinated with an axial ligand provided by a methionine, leucine, or phenylalanine residue. The Type-1 center cysteine residue is flanked by two of the histidines coordinating the trinuclear cluster, which provides a proximal link between the Type-1 center and the trinuclear cluster. Through this Cys-His

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one-electron oxidations to the trinuclear cluster (Solomon et al. 1996, Brouwers et al. 2000). The trinuclear cluster is the site of the four-electron reduction of oxygen to water, probably through a peroxide intermediate (Solomon et al. 1996).

Molecular biology of plant LMCOs As discussed previously, biochemical analyses have not yet provided clear functions for plant LMCOs. Thus, genetic analysis will be critical for demonstrating the function of specific plant LMCOs. The first plant LMCO gene was isolated from sycamore maple (LaFayette et al. 1995). Since then, the number of LMCO genes sequenced in plants has increased considerably.

Using the sycamore maple LMCO DNA sequence (APU12757), a search of the GenBank database revealed LMCO genes in at least 15 plant species, drawn from non-vascular plants, conifers, and flowering plants. However, a significant number of these sequences are only partial stretches of putative LMCO genes that have been found in genome-wide sequencing projects, and have been annotated on the basis of their sequence homology with known LMCOs. The number of LMCO genes for which the corresponding protein products have been experimentally characterized is significantly lower.

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large amounts of LMCO protein for biochemical analysis. E. coli has been used routinely for heterologous gene expression;

however, there have been no reports of successful use of this system to express plant LMCOs. In contrast, suspension-cultured tobacco cells have been used successfully to express plant LMCOs (Dean et al. 1998). LaFayette et al. (1999) demonstrated activity from a yellow-poplar LMCO cDNA heterologously expressed in tobacco cells. Subsequently, ferroxidase activity was demonstrated in a LMCO produced by tobacco cells transformed with another LMCO cDNA cloned from yellow-poplar (Hoopes et al.

2004). These results aside, faster progress could be made in studies of plant LMCOs if an easier system for heterologous expression were available.

To attack the question of LMCO function in plants, Ranocha et al. (2002) generated four populations of transgenic hydrid poplar, each expressing an individual poplar LMCO gene in the antisense (AS) orientation. This was the first in-depth characterization of genetic manipulation of LMCOs in plants, although preliminary data for similar studies of L. tulipifera (Dean et al. 1998) and Arabidopsis (Halpin et al. 1999) LMCOs had been reported. Surprisingly, none of these studies showed reproducible alterations in lignin content or composition. Only one of the populations of poplar (lac3AS) exhibited quantitative

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perturbations in xylem fiber cell wall structure. The authors concluded that the product of the lac3 gene is essential for normal cell wall structure and integrity in xylem fibers, although a direct correlation between LMCO gene expression and lignification could not be assigned (Ranocha et al. 2002).

The analysis of mutants is a very powerful tool for revealing the role of a particular gene in the physiological and developmental processes of plants. For many woody plants, such as trees, it is difficult to isolate or screen mutants due to the long life cycle and a dearth of genetic information. In contrast, Arabidopsis, a model plant, grows quickly and has a wide array of available genetic and genomic resources.

Consequently, it has become a valuable model for study of plantspecific gene functions, as well as fundamental processes common to all higher organisms at the molecular and cellular levels.

The completed Arabidopsis genome sequence provides new ways of addressing biological questions, ranging from molecular genetics to evolution, from an integrated perspective (Bouché and Bouchez 2001). The Arabidopsis genome encodes 17 LMCO genes that exhibit a wide variety of expression patterns in different tissues, and can be separated into 6 distinct phylogenetic groups (McCaig et al. 2005). A preliminary report described a phenotype associated with reduced expression of an Arabidopsis

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characterized by chlorosis and poor growth, but having no detectable changes in lignin quantity or composition (Halpin et al. 1999). Recently, the Arabidopsis TT10 gene, which encodes a putative LMCO (At5g48100), was identified by a mutant phenotype having a pale brown seed coat at harvest. The authors suggested that the TT10 LMCO in Arabidopsis serves as a flavonoid oxidase in developing seed coats (Pourcel et al. 2005).





Recently, well-developed bioinformatics systems have been developed to provide online access to whole genome datasets of gene expression data. With these tools researchers can quickly obtain large amounts of information on the expression patterns of specific gene(s) in which they are interested. For example, Figure 1.5 shows the expression profiles of the 17 Arabidopsis LMCOs at different stages of development.

In addition, several hundred thousand T-DNA and transposon insertion lines generated in multiple laboratories have been made available to the research community (Bouché and Bouchez 2001). Such mutants provide powerful tools for revealing the role of particular genes in physiological and developmental processes. Several groups have initiated programs for the systematic sequencing of the flanking DNA in various insert populations, which has greatly accelerated the process of identifying mutants related to specific genes. Most of the

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accessed through the TAIR website (http://www.arabidopsis.org/links/insertion.jsp).

Iron metabolism in plants In plants, iron is taken up from the soil into the root and then distributed throughout the plant, crossing a variety of cellular and organellar membranes. To compete for limited iron in the surrounding environment, plants have developed several mechanisms to enhance iron acquisition. Under iron-deficiency stress, all plants, except grasses, use a reductive strategy, similar to that of yeast, to acquire iron. This strategy, referred to as Strategy I, starts with reduction of Fe3+ by a plasma membrane-bound Fe3+-chelate reductase. This is followed by transport of Fe2+ across the root epidermal cell membrane via divalent metal transporters. The roots of Strategy I plants also release protons under conditions of iron-deficiency, thereby lowering the rhizosphere pH and increasing the levels of soluble Fe3+.

The FRO2 gene, which encodes the Fe3+-chelate reductase in A.

thaliana, was cloned based on the sequence of its yeast homologues, FRE1 and FRE2 (Robinson et al. 1999). FRO2 is only expressed in roots and its mRNA levels are induced by iron deficiency. Loss-of-function mutants in FRO2 result in decreased

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low-iron conditions. Fe3+-chelate reductase from pea, FRO1, encodes a protein that is 55% identical to Arabidopsis FRO2 (Waters et al. 2002).

The IRT1 gene was the first ferrous ion transporter cloned from plants using functional complementation of a yeast strain defective (fet3fet4 mutant) in iron uptake (Edie et al. 1996).

In addition to iron, IRT1 also displays manganese and zinc uptake activities when expressed in yeast (Korshunova et al.

1999). The IRT1 transcript and protein accumulate in Arabidopsis roots in response to iron deficiency, supporting a role for IRT1 in iron uptake in planta (Eide et al. 1996, Vert et al. 2002, Connolly et al. 2002). IRT1 knockout mutants are chlorotic and have a severe growth defect when grown in soil (Henriques et al.

2002, Varotto et al. 2002, Vert et al. 2002). Another putative iron transporter gene, IRT2, was isolated using the same strategy of expressing Arabidopsis cDNA of IRT1 homologues in the fet3fet4 yeast mutant defective in iron uptake (Vert et al.

2001). Although IRT2 is closely related to IRT1, its function as an iron transportor has not yet been demonstrated.

The NRAMP gene family, which has seven members in Arabidopsis, encodes another class of metal transporters. NRAMP1 and NRAMP2 are respectively up-regulated and down-regulated in roots experiencing iron deficiency (Curie et al. 2003, Thomine et al.

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plants results in increased resistance of the plants to iron toxicity. However, there is no data to indicate whether these transporters play a role in iron uptake.

In contrast to Strategy I plants, Strategy II plants, including all of the grasses, release Fe3+-binding compounds, low-molecular-mass secondary amino acids (mugineic acids, MAs) also known as “phytosiderophores” (PS), into the surrounding soil. These chelators bind iron and are then taken up into the root by active transport (Fett et al. 1998, Mori 1998). The biosynthesis pathway for PS has been extensively studied and has been almost completely deciphered in barley (Mori 1999, Negishi et al. 2002). The maize mutant, yellow stripe 3 (ys3), which shows interveinal chlorosis characteristic of iron deficiency, has been associated with a defect in MAs secretion (Basso et al.

1994). Another maize mutant, yellow stripe 1 (ys1), is deficient in Fe3+-phytosiderophore uptake, leading to the suggestion that YS1 is a Fe3+-phytosiderophore transporter (Curie et al. 2001). Interestingly, a family of eight genes in Arabidopsis that encode proteins sharing ca. 80% similarity with YS1 has been identified. This surprising finding suggests that metal-chelated transporters may actually be found in all plants, and not just grasses (Curie and Brait 2003).

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plants. Plastids contain ferritin, an iron storage protein, and iron-ferritin complexes represent more than 90% of iron found in the pea embryo axis (Marentes et al. 1998). In Arabidopsis, four ferritin genes (AtFer1-4) have been cloned, and all four ferritin proteins are predicted to be targeted to the plastids.

Gene AtFer2 is the only gene of the four to be expressed in seeds, whereas AtFer1, AtFer3, and AtFer4 are expressed in various vegetative organs, but not in seeds. Waldo et al. (1995) showed that ferritin mineralization iron loading occurs after protein localization into the plastid, which indicates that the plastid must have an active system to take up iron for incorporation into ferritin. Iron transport into the plastids is, therefore, of major importance in plant iron metabolism.

However, the mechanism of iron uptake into chloroplasts and/or plastids at the molecular level is unclear. Further information on subcellular localization of high-affinity transporters of iron may play a critical role in addressing iron uptake into subcellular compartments. A summarized model for iron acquisition systems in plants is shown in Figure 1.6. In it, we propose an additional transporter class (3), which uses a highaffinity iron uptake system similar to those found in yeast and animal systems. Such transporters could be located in different

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subcellular compartments.

Aims of the study The main goal of the work described in this dissertation was to extend our understanding of the physiological function(s) of the LMCOs in plants. Arabidopsis was used as a model system and a single LMCO gene was selected for characterization via both biochemical and genetic approaches. The initial hypothesis for the study was that the At2g30210 LMCO might function as a Fet3

ortholog, i.e. working as a ferroxidase in high-affinity ironuptake pumps. Specific objectives were as follows:

(1) Clone a full-length cDNA for the At2g30210 gene;

(2) Heterologous expression, as well as phenoloxidase and ferroixdase activity analysis, of the heterologous expressed LMCO gene product;

(3) Test tissue-specific expression of the gene;

(4) Test subcellular localization of the LMCO gene product;

(5) Investigate LMCO knockouts for clues to the physiological function(s) of At2g30210 gene; and (6) Study the regulation of the LMCO gene in response to various environmental perturbations.

The results of this research are detailed in the following

chapters:

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from Arabidopsis and heterologous expression of the cloned gene.

Phenoloxidase and ferroxidase activities of the gene product were tested and the gene expression pattern was examined in multiple tissues.

Chapter 3 presents the identification and characterization of At2g30210 mutants. An At2g30210 promoter:reporter gene fusion was used to demonstrate tissue-specific patterns of gene expression at the cellular level in transgenic Arabidopsis.

Subcellular localization of the At2g30210 protein using a chimeric At2g30210-EYFP fusion protein was also demonstrated.

Chapter 4 presents studies of the regulation of the At2g30210 gene in response to different metal, sugar, and salt stresses.

An alternative function for the At2g30210 protein, other than as a ferroxidase involved in iron metabolism, is proposed.

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