FREE ELECTRONIC LIBRARY - Dissertations, online materials

Pages:     | 1 |   ...   | 11 | 12 ||

«by CHIEH-TING WANG (Under the Direction of Jeffrey F. D. Dean) ABSTRACT Laccase and related laccase-like multicopper oxidases (LMCOs) have been ...»

-- [ Page 13 ] --

At2g30210 knockout mutants showed increased sensitivity to salt compared to wild-type plants. To evaluate the impact of salt on At2g3021 gene expression, two-week-old Arabidopsis seedlings grown on MS medium containing NaCl were compared to plants grown on standard medium. QPCR results showed that the At2g30210 gene was slightly, but not significantly, repressed by salt (Figure 4.3), which fits with available microarray data. A contrasting observation was reported for tomato, where a LMCO cDNA that was not detectable under control conditions, was strongly up-regulated in salt-treated or salt/ABA-treated seedlings (Wei et al. 2000). A full-length cDNA is not available for the gene. The partial sequence, when aligned with other plant LMCOs, showed strongest similarity to the Acer LMCO, which does not occur in the same phylogenetic group as the At2g30210 LMCO (McCaig et al. 2005).

These results together support the idea that plant LMCOs play multiple functions in plants. It is not surprising to see functions other than lignin biosynthesis associated with plant LMCOs based on their broad substrate ranges, numerous family members, and varied expression profiles in a single species.

–  –  –

Brouwers GJ (1999) cumA, a gene encoding a multicopper oxidase, is involved in Mn2+ oxidation in Pseudomonas putida GB-1.

Appl Environ Microbiol 65:1762-1768 Brouwers GJ, Vijgenboom E, Corstjens PLAM, De Vrind JPM, De Vrind-De Jong EW (2000) Bacterial Mn2+ oxidizing system and multicopper oxidases: An overview of mechanisms and functions. Geomicrobiol J 17:1-24 Dean JFD, Eriksson K-EL (1994) Laccase and the deposition of lignin in vascular plants. Holzforschung 48:21-33 Herbik A, Haebel S, Buckhout TJ (2002) Is a ferroxidase involved in the high affinity iron uptake in Chlamydomonas reinhardtii? Plant Soil 241:1-9 Hoopes JT, Dean JF (2001) Staining electrophoretic gels for laccase and peroxidase activity using 1,8diaminonaphthalene. Anal Biochem 293:96-101 Hoopes JT, Dean JFD (2004) Ferroxidase activity in a laccaselike multicopper oxidase from Liriodendron tulipifera. Plant Physiol Biochem 42:27-33 Kim C, Lorenz WW, Hoopes T, Dean JFD (2001) Oxidation of siderophores by the multicopper oxidase encoded by the Escherichia coli yacK gene. J Bacteriol 183:4866-4875 Mayer AM, Staples RC, Gilead NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing the hypersensitive response. Phytochemistry 58:33-41 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) 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 Roberts Sue A, Weichsel A, Grass G, Thakali K, Hazzard JT, Tollin G, Rensing C, Montfort WR (2002) Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli. Proc Nat Acad Sci USA 99:2766-2771

–  –  –

Shi X, Stoj C, Romeo A, Kosman D J, Zhu Z (2003) Fre1p Cu2+ reduction and Fet3p Cu1+ oxidation modulate copper toxicity in Saccharomyces cerevisiae. J Biol Chem 278:50309–50315.

Singh SK, Grass G, Rensing C, Montfort WR (2004) Cuprous oxidase activity of CueO from Escherichia coli. J Bacteriol 186:7815– 7817 Sterjiades R, Dean JFD, Gamble G, Himmelsbach DS, Eriksson K-EL (1993) Extracellular laccase and peroxidases from sycamore maple (Acer pseudoplatanus) cell-suspension culture. Planta 190:75-87 Stoj C, Kosman DJ (2003) Cuprous oxidase activity of yeast Fet3p and human cerloplasim: implication for function. FEBS lett 554:422-426 Wei JZ, Tirajoh A, Effendy J, Plant AL (2000) Characterization of salt-induced changes in gene expression in tomato (Lycopersicon esculentum) roots and the role played by abscisic acid. Plant Sci 159:135-48 Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol 136:2621-2632

–  –  –

2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5

–  –  –

Figure 4.1.

Responses of Arabidopsis LMCOs to metal treatment.

Transcript levels of three LMCOs gene were analyzed in samples from plants grown in the presence of excess copper (+Cu), iron deficiency (-Fe), and excess iron (+Fe). IRT, an iron transporter gene, was used as a positive control for the iron response (Vert et al. 2002). Y-axis indicates the fold change in transcription compared to control. Error bars represent standard deviation (S.D.).

144 4 3.5 3 2.5 2 1.5 1 0.5 0 0% 1% 4.5% 1 2 3 Figure 4.2. Responses of Arabidopsis LMCOs to sucrose. At2g30210 (blue) and At2g58910 (green) gene expression levels were analyzed in samples from plants grown in the presence of different sucrose concentrations in MS medium. Y-axis indicates the fold change of transcription compared to control (1% sucrose). Error bars represent S.D.

145 1.4 1.2 1 0.8 0.6 0.4 0.2

–  –  –

Figure 4.3.

Responses of At2g30210 gene expression to salt.

At2g30210 was analyzed from plants grown on different sodium chloride concentrations in MS medium. Y-axis indicates the fold change in transcription compared to control. Error bars represent S.D.

–  –  –

Laccase or laccase-like Multicopper oxidases (LMCOs) are blue copper proteins that catalyze oxidation reactions coupled to the four-electron reduction of molecular oxygen to water. Because of their wide range of potential substrates and difficulty of purification, the physiological functions of the plant LMCOs, in particular, have been difficult to determine. Early studies of plant LMCOs were focused primarily on woody plants due to the abundance of the enzyme in certain species and industrial interests. However, well-founded physiological roles for these LMCOs were difficult to identify because of lack of basic genetic information and tools to manipulate genes in these species. Because of its completed genome sequence and simple techniques for manipulating genes, Arabidopsis provides a good system in which to study the physiological function of LMCOs.

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.

–  –  –

encoded LMCO in a heterologous expression system. The predicted amino acid sequence revealed that the At2g30210 protein possesses the four conserved copper-binding domains that characterize the multicopper oxidase, as well as a glycosyl hydrolase family 1 domain near the C-terminus. Amino acid sequence analyses showed that the At2g30210 protein is closest to another Arabidopsis LMCO, At5g07130, with 73% identity.

Although we were unable to produce active At2g30210 enzyme using E. coli, transgenic tobacco cells allowed us to recover sufficient enzyme for biochemical analyses. Heterologously expressed At2g30210 LMCO showed phenoloxidase activity, but no ferroxidase activity. These results argue against our initial hypothesis, that the At2g30210 gene product might be involved in iron metabolism, which was based on its expression pattern, primarily in roots, and the fact that other LMCOs having ferroxidase activity function in iron homeostasis in a variety of eukaryotes. Further analyses showed that transcript levels from the At2g30210 gene were not regulated by external iron availability, and that mutants exposed to different iron conditions did not show significant phenotypic changes in gene expression. Altogether the results strongly suggest that the At2g30210 gene product is not involved in iron metabolism.

–  –  –

transcripts, a promoter-reporter gene fusion was introduced into wild-type Arabidopsis. Histochemical staining for GUS activity revealed that At2g30210 transcripts were primarily expressed in developing endodermis of the root elongation zone and newly matured roots. The result suggested a new function for Arabidopsis LMCOs in modification of the endodermal walls.

Indeed, endodermal walls of roots are characterized by the Casparian strip, a specialized cell wall structure containing suberin and lignin that functions to seal the perimeter of endodermal cells and, thereby, create an apoplastic transport barrier to water and solutes between the root cortex and stele (Sattlemacher 2001). Changes in structure or composition of the Casparian strip are known to impact hydraulic conductivity and ion flow (Schreiber et al. 2005).

The endodermis is one of the most important adaptations of terrestrial plants. In the absence or malfunction of an endodermis plants would not be able to regulate water uptake by the roots or maintain the water balance of the plant. Soil water enters the root through root hairs and then travels via the symplast (cell-to-cell) and/or apoplast (cell wall and intercellular space). However, the endodermis is impervious to water because of a suberized cell wall matrix called the Casparian strip. Therefore, to enter the stele, apoplastic water must

–  –  –

pass by plasmodesmata into the cells of the stele (Kramer and Boyer 1995). Although the water movement is thought to be driven by transpiration, root pressure also plays a role in the transport of water in the xylem in some plants under certain conditions. Root pressure is created by the osmotic potential of xylem sap, which is, in turn, kept high by dissolved minerals and sugars that have been actively transported into the apoplast of the stele (Patrick 1997, Ma and Peterson 2001). One familiar example of the importance of root pressure is seen in the sugar maple where, in early spring, it hydrolyzes starches stored in the roots to produce simple sugars. The increased levels of sugar in these root tissues draws water through the endodermis and into the xylem ducts where the continuous inflow forces sap to rise up the ducts into the above ground portions of the tree where it can be tapped (harvested) (Kimball 2004).

Plant growth and development requires transport of photoassimilates (e.g. sucrose) from source tissues into developmental sinks. The transport of sugars through the differentiated phloem is explained well by Mϋnch’s pressure flow hypothesis (Ross and Tyree 1980, Bret-Harte and Silk 1994). In the growing root, the phloem transport system ends in roots at the point where differentiation of protophloem sieve tubes is initiated. This is the same region where the most rapid root

–  –  –

pathway for solute transport from the phloem is thought to be apoplastic in mature stem (Hayes et al. 1985) and mature roots (Wyse 1979). Thus, the endodermal cell walls with Casparian strip as apoplastic barriers become critical to insure the delivery of the photoassimilates to root tips for growth. A loss-of-function At2g30210 mutant displayed phenotypic responses to sugar availability and salt levels consistent with functional alteration of the Casparian strip such that sugars destined for the growing root tip diffuse from the stele before reaching the zone of rapid growth at the root tip.

Suberin is a plant polymer containing a core of polyphenolic material resembling lignin. Work done by Kolattukudy (1981) showed that the inclusion of atypical phenolic subunits, such as various hydroxycinnamic acids, distinguishes the “lignin” core of suberin from true lignin (Bernards 2002). Although peroxidases have been implicated most frequently in the polymerization events related to suberization (Quiroga et al.

2000, Bernards and Razem 2001), no one has previously associated LMCOs with suberizing cell walls. Our results are the first to suggest a possible association between an Arabidopsis LMCO and deposition of the lignin-like core associated with suberin.

Combining the results of biochemical analyses showing phenoloxidase activity, cell-specific expression pattern in

–  –  –

mutant analyses, we conclude that the At2g30210 gene is unlikely to be involved with iron-uptake, but is very possibly involved with early lignification of the root endodermis in the process giving rise to the Casparian strip, a structure of some significance to root function.

Further study will focus on uncovering addition details of At2g30210 function. Recognition of the glycosyl hydrolase family 1 domain in the At2g30210 protein C-terminus has stimulated new ideas on LMCO function. Rogers and Campbell (2004) proposed a model in which monolignols are transported from cytoplasm to the cell wall as beta-glucoside intermediates. Once the glucosides have been transported to the cell wall, they must be cleaved, before the resulting aglycones can be polymerized. Perhaps the novel glucosyl hydrolase domain found near the C-terminus of 8 Arabidopsis LMCOs functions to hydrolyze the monolignol glucosides. In addition to phenoloxidase active, such a glucosidase activity would fit well with the previously proposed role for these enzymes in the early stages of lignin polymerization. Further study to demonstrate such glucosidase activity will help to clarify this new puzzle.

–  –  –

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 Bret-Harte MS, Silk WK (1994) Nonvascular, symplasmic diffusion of sucrose cannot satify the carbon demands of growth in the primary root tip of Zea mays L. plant Physiol 105:19-33 Esau K (1965) Vascular differentiation in plants. Holt, Rinehart and Winston, New York Hayes PM, Offerler CE, Patrick JK (1985) Cellular structures, plasma membrane surface areas, and plasmodesmatal frequencies of the stem of Phaseolus vulgaris L. in relation to radial photosynthate transfer. Ann Bot 56:125-138 Kimball JW (2004) Transport of water and minerals in plants.

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/X/Xyle m.html#RootPressure Kolattukudy PE (1981) Structure, biosynthesis, and biodegradation of cutin and suberin. Ann Rev Plant Physiol Plant Mol Biol 32:539-567 Kramer PJ, Boyer JS (1995) Water Relations of Plants and Soils.

Academic Press, Orlando, FL Ma S, Peterson CA (2001) Frequencies of plasmodesmata in Allium cepa L. roots: implications for solute transport pathways. J Exp Bot 52:1051-1061 Patrick JW (1997) PHLOEM UNLOADING: Sieve element unloading and post-sieve element transport. Annu Rev Plant Physiol Plant Mol Biol 48:191-222 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 Rogers LA, Campbell MM (2004) Tansley review: The genetic control of lignin deposition during plant growth and development. New Phytol 164:17-30

–  –  –

Sattelmacher B (2001) Tansley review no. 22 - The apoplast and

its significance for plant mineral nutrition. New Phytol 149:

167-192 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 Wyse R (1979) Sucrose uptake by sugar beet tap root tissue.

Plant Physiol 64:837-841


Pages:     | 1 |   ...   | 11 | 12 ||

Similar works:

«FORM-CRITICISM OF THE SYNOPTIC HEALING NARRATIVES LAURENCE J. McGINLEY, S.J. WOODSTOCK COLLEGE III. NOVELLE AND MIRACLE-STORY I N classifying, for form-critical purposes, the stories of miraculous healings recorded in the synoptic Gospels, Dibelius and Bultmann have employed more or less parallel categories. Just as the paradigm of Dibelius corresponds in general to the apothegm of Bultmann,1 so all the healing narratives which Dibelius names novellen are classified by Bultmann under the...»

«JKAU: Mar. Sci., Vol. 21, No. 2, pp: 89-108 (2010 A.D. / 1431 A.H.) DOI : 10.4197/Mar. 21-2.7 Assessment of Marine Ecosystem Degradation Based on Studying the Geological Significances of Bottom Sediments in Sharm Obhur, North Jeddah, Saudi Arabia Amin M. Gheith and Mohamed S. Hariri* Faculty of Science, Geology Department, Mansoura University, Mansoura, Egypt *Faculty of Marine Science, King Abdullaziz University, Jeddah, Saudi Arabia Abstract. Our investigations focus on the significance of...»

«Tailored Optical Properties on the Macroscale by Template-Assisted Self-Assembly of Plasmonic Nanoparticles Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) an der Bayreuther Graduiertenschule für Mathematik und Naturwissenschaften der Universität Bayreuth vorgelegt von Moritz Jan Tebbe geboren in Henstedt-Ulzburg Juli 2015 Die vorliegende Arbeit wurde im Zeitraum vom April 2012 bis Juli 2015 an der Universität Bayreuth in Bayreuth am...»

«UNIVERSITY OF LIMERICK BRAND SPECIFICATIONS Section Contents Page 1.1 Introduction 3 1.2 Communications Services 4 1.3 Procedure for applying to use the UL Logo 4 2.1 The UL Logo 5 2.2 Standard Logo 6 2.3 Logo Usage – Size 7 2.4 Incorrect Use of Logo 8 3.1 Logo Colours 9 3.2 Patterned or Shaded Backgrounds 10 4.1 Typefaces 12 5.1 UL Colour Palette 13 6.1 Use with Other Logos 14 7.1 Divisional Identifiers 15 8.1 Corporate Affairs Image Library 16 9.1 The UL Heraldic Crest 17 Appendix 1 A Guide...»

«S/2012/171* United Nations Security Council Distr.: General 21 March 2012 Original: English Report of the Secretary-General on children and armed conflict in Colombia Summary The present report has been prepared pursuant to Security Council resolution 1612 (2005). It is the second report on the situation of children and armed conflict in Colombia and follows my first report (S/2009/434) and the subsequent conclusions and recommendations of the Working Group on Children and Armed Conflict...»

«Glossary of Mining Terminology After Damp Gasses resulting from underground combustion, normally carbon monoxide. This is a loose term implying any fatal gas in a mine after an explosion or fire. Air Shaft A vertical opening into a mine for the passage of air. Airway Any passage in a mine along which an air current moves. Some passages are driven solely for air. Other passages, such as a main level, are all purpose, to move air, men, coal, and materials. Anthracite Coal of the highest...»

«(Be)sturen en verbinden in Losser Michael Sijbom en Irma Nadorp Af te halen op gemeentehuis Losser: autostickers tegen Duitse tolplannen. Gemaakt ism met Duitse buurgemeenten. Deze tweet van de burgemeester van Losser leidde in augustus 2014 tot (inter-)nationale persaandacht1. Honderden Lossenaren kwamen op het gemeentehuis de sticker ophalen. Burgemeesters uit buurtgemeenten, grensondernemers uit Duitsland en bestuursorganen zoals de Euregio meldden zich om de krachten te kunnen bundelen. Een...»

«FACULTY OF INFORMATICS AND INFORMATION TECHNOLOGIES www.fiit.stuba.sk ANNUAL REPORT 2010 © 2011, Ľudovít Molnár, Mária Bieliková, Pavel Čičák, Tibor Krajčovič, Margaréta Kotočová, Ladislav Hudec, Pavol Návrat, Ľubica Palatinusová Faculty of Informatics and Information Technologies Slovak University of Technology in Bratislava Ilkovičova 3 842 16 Bratislava, Slovakia Visit FIIT STU on the Web: www.fiit.stuba.sk Executive Editor: Katarína Mršková Copy Editor: Mária Hricová,...»

«ASPO Conference 2014 Conference location MartiniPlaza, Leonard Springerlaan 2. 9727 KB Groningen 1 2 Local organizers Jolien van Breen, Anita Buitenkamp, Kai Epstude, Dianne van der Kolk, Namkje Koudenburg, Angela Ruepert, Martijn van Zomeren Selection committee for conference contributions Erik Bijleveld (RU), Lotte van Dillen (UL), Frenk van Harreveld (UvA), Marlon Mooijman (UL), Yvette van Osch (TiU), Tila Pronk (TiU), Russell Spears (RUG), Elze Ufkes (UTwente) Dissertation award committee...»

«Surface Tension: the art of Euan Macleod 1991 2009... A Tweed River Art Gallery touring exhibition, curated by Gavin Wilson EDUCATION RESOURCE... Prepared by Kent Buchanan This exhibition is supported by Visions of Australia, an Australian Government program supporting touring exhibitions by providing funding assistance for the development and touring of Australian cultural material across Australia. Surface Tension: the art of Euan Macleod 1991 – 2009 1/25 EDUCATION RESOURCE... This...»

«Paediatric Ocular Disease Simon Barnard Paediatric ocular disease Notes to accompany lecture to 2nd Year City University undergraduates Dr Simon Barnard PhD BSc FCOptom FAAO DCLP Table of Contents Trauma The red eye Eyelids Proptosis Leukocoria EOM anomalies Nystagmus Congenital abnormalities and syndromes Systemic disease and the eye Neurological disease Paediatric neurodevelopmental disorders Further reading Multiple Choice Questions MCQ Answers. do not view till you have attempted all...»

«PROFILE OF INTERNAL DISPLACEMENT : KENYA Compilation of the information available in the Global IDP Database of the Norwegian Refugee Council (as of 6 October, 2003) Also available at http://www.idpproject.org Users of this document are welcome to credit the Global IDP Database for the collection of information. The opinions expressed here are those of the sources and are not necessarily shared by the Global IDP Project or NRC Norwegian Refugee Council/Global IDP Project Chemin Moïse Duboule,...»

<<  HOME   |    CONTACTS
2016 www.dissertation.xlibx.info - Dissertations, online materials

Materials of this site are available for review, all rights belong to their respective owners.
If you do not agree with the fact that your material is placed on this site, please, email us, we will within 1-2 business days delete him.