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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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Increased salt sensitivity in the At2g30210 mutants is also consistant with impaired Casparian strip function. Arabidopsis seed germination is greatly impaired at salt concentrations above 75mM, but low concentrations of NaCl( 50mM) may actually have a slight stimulating effect on fresh weight increase in seedlings grown in culture medium (Xiong and Zhu 2002). Our data showed that seedlings grown on medium containing 80mM NaCl only developed a very short radical, with mutant and wild-type plants affected to an equivalent degree. However, in medium containing 40mM NaCl, while both wild-type and mutant seedlings showed repressed growth, the mutant was severely chlorotic and eventually died. The ability to restrict Na+ transport from the root to aerial tissues is one of several important determinants of salt tolerance in plants (Xiong and Zhu 2002). Amongst several steps that control the long distance transport of Na+ are the radial transport of Na+ across the root cortical cells and the loading of Na+ into the xylem. The Casparian bands are impermeable to small ions because of their lignin and suberin

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site retarding passive movement of specific ions and solutes between the cortex and the stele. Thus, it plays a critical role in establishing an apoplastic transport barrier in roots (Ma and Peterson 2003). If the At2g30210 mutant is impaired in depositing suberin and/or lignin in endodermal cell walls, the resulting defective Casparian strips could quite conceivably allow solutes to freely pass through to the stele.

The Salk insertion mutant showed a subtler phenotype compared to wild-type and the GT7855 mutant. There are two possibilities to explain this phenomenon. First, the mutation may be leaky because the intron location allows the insert to be spliced out at some low frequency during post-transcriptional processing.

This hypothesis is supported by the presence of low levels of mature mRNA as detected using RT-PCR. The other possibility is that the insert results in an enzyme that retains partial function. Stop codons in the insert would result in loss 44 amino acids (the last exon) from the C-terminus. These amino acids encode part of the type-1 copper-binding domain, but the resulting enzyme could still retain partial oxidase activity.

The SKU5 gene in A. thaliana, a LMCO homologue lacking the type I copper-binding domain, has been shown to be localized to the plasma membrane and the cell wall, and is involved in directed root tip growth (Sedbrook et al. 2002). Similarly, Chen et al.

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of ceruloplasmin belonging to the MCO family) expressed in sexlinked anemia (sla) mice still has measurable, but decreased, oxidase activity. Our analyses of the Salk insert line do not eliminate either possibility. Thus, it may not be surprising that only a subtle phenotype was detected in this mutant.

Recent work on Arabidopsis is beginning to shed light on the genetic and molecular mechanisms of endodermal differentiation.

Two genes are known to be required for the proper differentiation of root cortex tissues; they are SCARECROW (SCR) and SHORTROOT (SHR) (Ma and Peterson 2003). In the shr mutant, the endodermis is missing (Benfey et al. 1993). In the scr mutant, the mutant cell layer has the conformation of cortical parenchyma, but Casparian bands remian present (Di Laurenzio et al. 1996). Plants that over-express SHR result in a multiplelayered endodermis (Helariutta et al. 2000). It is unknown whether the At2g30210 gene is regulated by SHR, the principal transcription factor controlling differentiation of endodermis.

Although our data showed fluorescent-tagged At2g30210 protein localized to cell periphery in both BY2 tobacco cells and transgenic Arabidopsis, we did not verify the actual localization of the endogenous At2g30120 LMCO protein to Casparian bands. Our GUS expression data showed that the promoter activity is expressed predominantly in endodermal

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chimeric EYFP fusion protein driven from the endogenous At2g30210 promoter may be required to see specific localization to the Casparian bands. Even then, it may turn out that the EYFP domain will disrupt proper localization of the protein.

Suberin is a plant polymer, containing both aromatic and polyester moieties, which functions as a barrier in below ground tissues, wounded surfaces, and a variety of internal organs.

Suberin is also a dominant material in Casparian strips. Work by Kolattukudy (1981) demonstrated that suberin contains a core of polyphenolic material resembling lignin, but the inclusion of

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Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, AguilarHenonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653-657 Bao W, O'Malley DM, Whetten R, Sederoff RR (1993) A laccase associated with lignification in loblolly pine xylem. Science 260:672-674 Benfey PN, Linstead PJ, Roberts K, Schiefelbein JW, Hauser MT, Aeschbacher RA (1993) Root development in Arabidopsis: four mutants with dramatically altered root morphogenesis.

Development 119:53-70 Bernards MA (2002) Demystifying suberin. Can J Bot 80:227-240 Bernards MA, Razem FA (2001) The poly(phenolic) domain of potato suberin: a non-lignin cell wall bio-polymer. Phytochemistry 57:1115-1122 Bouché N, Bouchez D (2001) Arabidopsis gene knockout: phenotypes wanted. Curr Opin Plant Sci 4:111-117 Boyajian GE, Carreira LH (1997) Phytoremediation: a clean transition from laboratory to marketplace? Nat Biotechnol 15:127−128 Brouwers GJ, de Vrind JPM, Corstjens PLAM, Cornelis P, Baysse C, de Jong EWDV (1999) cumA, a gene encoding a multicopper oxidase, is involved in Mn2+ oxidation in Pseudomonas putida GB-1. Appl Environ Microb 65:1762-1768 Brundertt MC, Enstone DE, Peterson CA (1988) A berberin-aniline blue fluorescent staining procedure for suberin, lignin, and callose in plant tissue. Protoplasma 146:133-142 Chen H, Attieh ZK, Su T, Syed BA, Gao H, Alaeddine RM, Fox TC, Usta J, Naylor CE, Evans RW, McKie AT, Anderson GJ, Vulpe CD (2004) Hephaestin is a ferroxidase that maintains partial activity in sex-linked anemia mice. Blood 103:3933-3939

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David KM, Perrot-Rechenmann C (2001) Characterization of a tobacco bright yellow 2 cell line expressing the tetracycline repressor at a high level for strict regulation of transgene expression. Plant Physiol 125: 1548-1553 De Silva DM, Askwith CC, Eide D, Kaplan J (1995) The FET3 gene product required for high affinity iron transport in yeast is a cell surface ferroxidase. J Biol Chem 270:1098-1101 Dean JFD, Eriksson K-EL (1994) Laccase and the deposition of lignin in vascular plants. Holzforschung 48:21-33 Dean JFD, LaFayette PR, Rugh C, Tristram AH, Hoopes JT, Merkle SA, Eriksson K-EL (1998) Laccases associated with lignifying tissues. In “Lignin and Lignan Biosynthesis”, Lewis NG and Sarkanen S (eds.), ACS Symp Ser 697:96-108 Deeken R, Geiger D, Fromm J, Koroleva O, Ache P, LangenfeldHeyser R. Sauer N, May ST, Hedrich R (2002) Loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis. Planta 216:334-344 Di Laurenzio L, Wysocka-Diller J, Malamy JE, Pysh L, Helariutta Y, Freshour G, Hahn MG, Feldmann KA, Benfey PN (1996) The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell 86:423-433 Gibson SI (2000) Plant sugar-response pathways. Part of a complex regulatory web. Plant Physiol 124:1532-1539.

Grass G, Rensing C (2001) Genes involved in copper homeostasis in Escherichia coli. J Bacteriol 183:2145-7 Helariutta Y, Fukaki H, Wysocka-Diller J, Nakajima K, Jung J, Sena G, Hauser MT, Benfey PN (2000) The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling. Cell 101:555-567 Hoopes JT, Dean JFD (2004) Ferroxidase activity in a laccaselike multicopper oxidase from Liriodendron tulipifera. Plant Physiol Biochem 42:27-33

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Ma FS, Peterson CA (2003) Current insights into the development, structure, and chemistry of the endodermis and exodermis of roots. Can J Bot 81:405–421 Martienssen and Springer 1998 Enhancer and Gene trap transposon mutagenesis in Arabidopsis. In "Insertional Mutagenesis: a practical approach" Oxford University Press (G. Coupland, ed.) McCaig BM, Meagher RB, Dean JFD (2005) Gene structure and molecular analysis of the laccase-type multicopper oxidase gene family in Arabidopsis thaliana. Planta (in press) Mayer AM, Staples RC (2002) Laccase: new functions for an old enzyme. Phytochemistry 60:551-565 Messerschmidt A (1997) Multi-Copper Oxidases. World Scientific, Singapore Musci G (2001) Ceruloplasmin, the unique multi-copper oxidase of vertebrates. Prot Pep Lett 8:159-169 O’Mally DM, Whetten R, Bao W, Chen CL, Sederoff RR (1993) The role of laccase in lignification. Plant J 4:751-757 Quiroga M, Guerrero C, Botella MA, Barcelo A, Amaya I, Medina MI, Alonso FJ, de Forchetti SM, Tigier H, Valpuesta V (2000) A tomato peroxidase involved in the synthesis of lignin and suberin. Plant Physiol 122:1119-1127 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 Sattelmacher B (2001) Tansley review no. 22 - The apoplast and

its significance for plant mineral nutrition. New Phytol 149:

167-192 115 Schreiber L, Franke R, Hartmann KD, Ranathunge K, Steudle E (2005) The chemical composition of suberin in apoplastic barriers affects radial hydraulic conductivity differently in the roots of rice (Oryza sativa L. cv. IR64) and corn (Zea mays L. cv. Helix). J Exp Bot 56:1427-1436 Sedbrook JC, Carroll KL, Hung KF, Masson PH, Somerville CR (2002) The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth. Plant cell 14:1635-1648 Sterjiades R, Dean JFD, Eriksson K-EL (1992) Laccase from sycamore maple (Acer pseudoplatanus) polymerizes monolignols.

Plant Physiol 99:1162-1168 US Environmental Protection Agency (1994) Microwave assited acid digestion of sediments, sludges, soils, and oils.

Environmental Monitoring System Lab, Cincinnati, Ohio Wang GD, Li QJ, Luo B, Chen XY (2004) Ex planta phytoremediation of trichlorophenol and phenolic allelochemicals via an engineered secretory laccase. Nat Biotech 22:893–897 Xiong L, Zhu JK (2002) Salt tolerance. In “The Arabidopsis Book” eds. Somerville CR and Meyerowitz EM, American Society of Plant Biologists, Rockville, MD, doi/10.1199/tab.0009, http://www.aspb.org/publications/arabidopsis/.

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Figure 3.1.

Insertion position in the At2g30210 mutants. A.

thaliana mutants were identified by a search of the insertion line database accessible through TAIR (http:/www.Arabidopsis.org). Arrows show the relative position of primers used to confirm the insertions.

118 Figure 3.2. Genetic analyses of the At2g30210 insertion mutants. A. Three homozygous lines (3,38,41) were isolated from the Salk031901 mutant using gene-specific and T-DNA specific primers. RP and LP: At2g30210 gene-specific primers that spanned the insertion site. LBa1: T-DNA left border-specific primer. B.

RT-PCR analysis of Salk-031901 mutant with a pair of genespecific primers (76F and 1694R). C. Several homozygous (Ho) lines were isolated from the GT7855 insertion mutant. 76F and 1016R: At2g30210 gene-specific primers that spanned the insertion site. Ds5-4: Ds transposon-specific primer. D. RT-PCR analysis of GT7855 mutant with a pair of gene-specific primers (1Fand 1016R). UBQ10 was used as the control to show comparable cDNA loading. Wt: wild type. He: heterozygous. Ho: homozygous.

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Figure 3.3.

GUS expression in tissues from the GT3416 insertion line. Whole mount GUS staining showed that the highest activity was observed in roots (A, F, and G), flowers (c), the abscission zones of siliques (c and D), and developing seed (H), while activity was weak in the node (E).

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Figure 3.4.

Transcription of At2g30210 in wild-type and insertion mutants of Arabidopsis. A. qPCR analysis showed slightly reduced transcription in both the Salk-031901 and GT7855 lines. Y-axis displays the fold change compared to wildtype plants and error bars represent one standard deviation (S.D.). B. RT-PCR analyses using primers spanning the insertion sites showed that the insertions disrupted levels of normal

At2g30210 transcriptss. Wt: wild type, gt: GT7855 line, sk:

Salk-031901 line, oe: At2g3021-EYFP transgenic plant.

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41 38 41 38 Figure 3.5. T-DNA insertion mutants showing reduced growth in the absence of sugar. Three homozygous Salk insertion lines (3,38, and 41) and wild-type (wt) seedlings were grown on water agar (panel A) or MS medium containing 1% sucrose (panel B) for a week. Note that several seedlings of line 38 grown on the water agar plate (panel A) developed extended cotyledons, which were also seen in wild-type seedlings. Similar sporadic growth was not seen amongst the line 3 or 41 plants.

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Figure 3.6.

Growth alterations in the GT7855 mutant. The mutant showed altered root morphology and severe repression in soil, but not on MS medium containing sucrose. A. 14-day-old wild-type (Wt) and GT7855 mutant (GT7855) seedlings were vertically grown on MS media supplemented with 1% sucrose. B. 14-day-old wildtype (Wt), GT7855 mutant (GT), and Salk-031910 mutant (Salk) seedlings were grown on MS medium supplemented with 1% sucrose (left panel) or in soil (right panel).

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