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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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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. This study analyzed in greater detail the product of the At2g30210 gene, a member of the LMCO family that is predominately expressed in young root tissues of Arabidopsis thaliana. To determine the spatial expression pattern of At2g30210 transcripts, a promoter-reporter gene fusion was introduced into Arabidopsis. Histochemical analyses of transgenic plants expressing GUS activity revealed that At2g30210 transcripts were preferentially expressed in developing endodermis of the root elongation zone and newly matured roots. Low levels of expression were also detected in hypocotyls, as well as major veins of some cotyledons under some conditions. A chimeric fusion protein comprised of the At2g30210 coding sequence and a modified green-fluorescent protein (YFP) was used to examine subcellular localization of the protein in transgenic plants. Confocal microscopy indicated that the At2g30210-YFP fusion product was localized to the cell periphery, likely extracellularly and possibly to the cell wall.

A genetrap insertion (GT7855) mutant of Atg30210 was confirmed by sequence analysis and PCR to have a transposon in the second exon of the gene. RT-PCR of seedling cDNA confirmed loss of

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mutant were grown under various culture conditions for comparison with wild-type plants. On solid MS medium containing 1% sucrose, the GT7855 mutant showed more lateral root production than wild-type plants. When grown in soil or MS media without sucrose, growth of the mutant was severely repressed. The At2g30210 knockout mutants were also much more sensitive to salt stress than wild-type plants. The results suggest involvement of the At2g30210 LMCO gene product in endodermal cell function, possibly in wall development events leading to formation of the Casparian strip.

Introduction Laccase is a member of the multicopper oxidase (MCO) family that also includes yeast FET3 class ferroxidases (De Silva et al. 1995), bacterial metal oxidases (Brouwers et al. 1999; Grass and Rensing 2001), mammalian and avian ceruloplasmin (Musci 2001), and plant ascorbate oxidases (Messerschmidt 1993).

Laccases, or more correctly Laccase-like Multicopper Oxidases (LMCOs), have been founded in a wide range of organisms, including bacteria, fungi, insects, and plants, yet their physiological functions remain unclear. Wound healing through oxidative polymerization of alkylcatechols in the sap of Rhus species (true laccase) had been the only strongly supported

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staples 2002; McCaig et al. 2005), yet even the exact mechanism in that function remains unclear. Plant LMCOs have also been proposed to assist in lignin biosynthesis in previous reports (Dean and Eriksson 1994; Dean et al. 1998; Mayer and Staples 2002). Early studies suggested that laccase and laccase-like oxidase activities were associated with lignification in plants based on their spatial and temporal correlation with lignification (O'Malley et al. 1993; Dean and Eriksson 1994, Ranocha et al. 1999), their capability to oxidize monolignols to produce dehydrogenation polymers (DHPs)(Sterjiades et al. 1992, Bao et al. 1993; Ranocha et al. 1999), and the impact on xylem tissue formation in transgenic plants expressing sense and antisense LMCO genes (Dean et al. 1998; Ranocha et al. 2002).

Other putative functions attributed to plant LMCOs derive from their specific enzyme activities. Recent demonstration of ferroxidase activity in a LMCO from yellow-poplar (Hoopes and Dean 2004) was taken to suggest that some plant LMCOs might take part in iron metabolism, similar to the function provided by FET3 in the high-affinity iron uptake system of yeast. Also, LMCO is one of several oxidative enzymes with demonstrated capacity to degrade chemical compounds in their immediate vicinity (Boyajian and Carreira 1997). That transgenic Arabidopsis over-expressing a secretory LMCO from cotton

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phenolic allelochemicals and 2,4,6-trichlorophenol suggests plant LMCOs could also play a role in detoxification of environmental compounds (Wang et al. 2004). However, in most cases, definitive demonstration of physiological functions for plant LMCOs has been difficult because the enzymes are typically able to oxidize a wide variety of potential phenolic and inorganic substrates. In addition, multiple LMCO gene products may be expressed simultaneously in the same tissues.

The completed Arabidopsis genome sequence is expected to provide new ways of addressing biological questions from an integrated perspective, ranging from molecular genetics to evolution (Bouché and Bouchez 2001). Previous study showed that like other plants, the Arabidopsis genome encodes multiple (17) LMCOs. The seventeen LMCO genes exhibit a wide variety of expression patterns in different tissues and can be separated into six phylogenetic groups (McCaig et al. 2005). We have taken a combined genetic and biochemical approach to dissecting the physiological functions of the LMCO gene family in Arabidopsis using promoter-reporter gene constructs, gene knockouts, and chimeric fusion proteins. Our studies of the At2g30210 LMCO gene point to an extracellular role for the enzyme in development of cell walls in the root endodermis.

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Identification of At2g30210 insertion mutants Arabidopsis thaliana mutants were identified by searching the insertion line databases listed on the TAIR server (http:/www.Arabidopsis.org). The Salk_031901 T-DNA insertion line was identified in a collection at the Salk Institute (http://signal.salk.edu/cgi-bin/tdnaexpress) (Alonso et al.

2003). Annotation information was obtained from the website. PCR analysis was used to confirm the insertion and screen for homozygous transgenic plants using a combination of genespecific primers (Table 3.1) from the 3’ (031901RP) and 5’(031901LP) ends of the At2g30210 gene, as well as the T-DNA left border (Lba1) and right border (RB) primers, respectively.

GT3416 and GT7855 were identified from among the gene-trap lines generated using Ac/Ds transposons at Cold Spring Harbor Laboratory (http://genetrap.cshl.org/). Annotation information was obtained from the website. Ds-specific primers (Ds3-4 and Ds5-4) together with At2g30210 primers (1F and 1016R) were used to confirm the insertions, as well as screen for homozygous individuals. All oligonucleotides used in this study were synthesized by Integrated DNA Technologies, Inc (Corvalville, IA) and used without further purification.

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A. thaliana plants were cultivated on soil mixes in growth chambers at 22°C and a 16h light/8h dark illumination cycle. For some experiments, plants were grown in petri dishes on solid MS media (Invitrogen, Carlsbad, CA) supplemented with 1% sucrose and 0.8% phytoagar. For root growth analysis, seedlings were grown on plates set vertically. Tobacco cells were maintained on agar plates as described by Lafayette et al. (1999).

Promoter expression analysis For promoter activity analysis, 1 kb of genomic DNA upstream of the translational start site and including the first 2 codons was amplified by PCR using Pwo high-fidelity DNA polymerase (Boehringer Mannheim, Indianapolis, IN) with the PMT_F and PMT_R primers. The resultant amplimer was digested with SpeI/SptII and ligated in frame to the 5’ end of the GUS gene contained in the pUPC5-GUS vector (from Dr. C Joseph Nairn, University of Georgia, USA). The resultant At2g30210 promoter:GUS fusion was subcloned into the pZP-NPTII binary vector developed by Dr.

Nairn, and introduced into wild-type plants by Agrobacterium tumefaciens-mediated transformation using the floral dip method (Clough and Bent 1998). Transgenic seedlings were selected on MS medium containing 50µg/mL of kanamycin. Kanamycin-resistant lines were tested for the construct using GUS-specific primers.

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Arabidopsis tissues and stages using 5-bromo-4-chloro-3-indolylβ-D-glucuronide as a substrate (Jefferson et al. 1987). The tissues were stained with the substrate, fixed in FAA (50% ethanol, 10% glacial, and 5% formaldehyde) solution, and then cleared in 70% ethanol before observations. Selected GUS-stained roots were embedded in LRR white resin after ethanol dehydration. Cross sections obtained using a vibratome (TPI, Inc., St. Louis, MO) were observed under a light microscope.

At2g30120 protein localization For subcellular localization analysis, the coding region of the At2g30210 cDNA was amplified by PCR using Pwo DNA polymerase (Boehringer Mannheim) with primers At2g30210-YFPC-F and At2g30210-YFPC-R. Care was taken to omit the stop codon (TAA) of At2g30210 and fuse the coding sequence in-frame with the EYFP sequence. The At2g30210 PCR product was digested and cloned into the SpeI/SptII sites of the p35S-EYFPC vector provided by Dr.

Nairn (University of Georgia, USA). The resultant construct was confirmed by DNA sequencing and restriction digest analysis.

The AT2g30210-EYFP fusion construct, under 35S promoter control, was directly transformed into suspension-cultured tobacco cells (BY2) using particle bombardment as described in Chapter 2.

After over-night incubation in the dark, cells were observed using fluorescence microscopy with UV illumination (ARC lamp)

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nm barrier filter).

To generate stable transgenic plants, the construct was subcloned into the pZP-NPTII binary vector and introducted into Arabidopsis as described for the promoter:reporter gene construct. Kanamycin-resistant lines were confirmed for transgene presence using At2g30210 gene-specific primers (1F and 1016R) that could distinguish the transgene from the endogenous gene. T2-generation progeny were used for EYFP localization. EYFP fluorescence from roots of 5-day-old transgenic seedlings was observed using a Leica TCS SP2 confocal microscope (Leica Microsystems, Heidelberg, Germany).

Quantitative PCR (qPCR) and RT-PCR experiments Total RNA was extracted from two-week old seedlings grown in liquid medium. Template cDNA for all samples was made using 5µg of Rnase-free Dnase-treated total RNA and Superscript III (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The first strand RT reaction was diluted 10 times with water and 10uL of diluted cDNA product was used for each qPCR reaction. Primers specific to At2g30210 (44F and 147R) were used to amplify a 140 bp region upstream of the mapped insertions. The internal control gene, G3PDH, was amplified using the specific primers, G3PDH-F and G3PDH-R. Primer concentration was 300nM for the reactions. DyNAmo SYBR Green

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experiments following the manufacturer’s protocol. Samples were replicated three times for the At2g30210 gene and twice for the internal control (G3PDH). A standard curve based on known quantities of plasmid containing the At2g30210 cDNA was determined for each experiment. All experiments were run using an iCycler thermal cycler (Bio-Rad, Hercules, CA) under the following cycling conditions: 95oC for 10 min followed by 40 cycles of 95 oC for 1 sec and 55oC for 1 min. The instrument was set to measure dye florescence at the end of each cycle. Melting curve analysis was performed at the end of reaction to ascertain the quality of the PCR products. The Cycle Threshold (cT) line was determined manually as the point where the R2 value for the standard curve was highest.

The same cDNA pools were used for RT-PCR analyses to confirm gene disruption in the insertion lines. To test the Salk_031901 line, 1F and 1694R and 326F and 1694R primer pairs were used in different experiments. For GT7855, the 1F and 1016R pair was used. The PCR products were analyzed on 1% agarose gels stained with ethidium bromide.

Metal ion content The metal ion concentration in Arabidopsis seeds was measured from soil-grown plants. Seeds from three individual plants each of Salk-031901, GT7855, wild-type, and At2g30120-EYFP transgenic

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desiccator under vacuum, and 0.2 g of dry seeds were digested in 70% HNO3 using a microwave-assisted method (U.S. EPA, Method 3051). Trace elements were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS) at the Environmental Analysis Lab (University of Georgia, USA). Before analysis, solutions were diluted by a factor of ~200.

Results Isolation and molecular characterization of the At2g30210 mutants The analysis of mutants is a powerful tool for revealing the role of a particular gene in physiological and developmental processes in plants. To date, several hundred thousand T-DNA and transposon insertion lines have been generated in various laboratories that make them available to the research community.

Recently, several groups have initiated programs for the systematic sequencing of insertion sites in various populations (Bouché and Bouchez 2001), and the data are made available through online resources, such as TAIR (http://www.arabidopsis.org/links/insertion.jsp). Using this system, we identified three potential knockout insertion lines from different populations.

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