Nitric oxide signalling in Arabidopsis thaliana [Elektronische Ressource] : redox modification in mitochondria and regulation of transcription / Maria Cristina Palmieri

Juggernalz

Sujets

• Naturwissenschaften

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 63
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Biochemische Pflanzenpathologie




Nitric Oxide Signalling in Arabidopsis thaliana:
Redox Modification in Mitochondria and Regulation of Transcription

Maria Cristina Palmieri


Vollständiger Abdruck von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt der Technischen Universität München zur Erlangung des
akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. C. Schwechheimer


Prüfer der Dissertation: 1. Univ.-Prof. Dr. J. Durner
2. Univ.-Prof. Dr. E. Grill




Die Dissertation wurde am 01.04.2009 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 10.11.2009 angenommen.
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2 Parts of this thesis are already published, or publication is in progress, respectively.

Palmieri MC, Lindermayr C, Bauwe H, Durner J (2009)
Glycine decarboxylase associated plant defence responses and cell death are mediated by S-
nitrosylation and glutathionylation (submitted; corresponds to PART I)

Palmieri MC, Sell S, Huang X, Scherf M, Werner T, Durner J, Lindermayr C (2008)
Nitric oxide-responsive genes and promoters in Arabidopsis thaliana: a bioinformatics
approach. J Exp Bot 59: 177-186 (corresponds to PART II)

Zemojtel T, Frohlich A, Palmieri MC, Kolanczyk M, Mikula I, Wyrwicz LS, Wanker EE, Mundlos
S, Vingron M, Martasek P, Durner J (2006)
Plant nitric oxide synthase: a never-ending story? Trends Plant Sci 11: 524-525; author
reply 526-528 (not in this thesis)

















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Contents

List of Figures and Tables........................................................................... 9
Abbreviations ........................................................................................... 11
Abstract.................................................................................................... 13
1 Introduction....................................................................................... 15
1.1 NITRIC OXIDE SIGNALLING ........................................................................15
1.1.1 NO Chemistry .......................................................................................15
1.1.2 NO Biosynthesis ....................................................................................16
1.1.3 NO Function in Plants .............................................................................18
1.1.4 NO Perception and Signal Transduction.....................................................20
1.2 MITOCHONDRIA IN REDOX SIGNALLING.......................................................25
1.2.1 Mitochondrial ROS .................................................................................25
1.2.2 Mitochondria and NO..............................................................................26
1.2.3 Function of Mitochondria during Plant – Pathogen Interactions .....................27
1.2.4 Photorespiration: Role in Stress Protection ................................................28
Aim of the Study and Research Strategy ............................................. 29

PART I: MODIFICATION OF MITOCHONDRIAL PROTEINS BY
S-NITROSYLATION
2 Results............................................................................................... 33
2.1 NITRIC OXYDE PRODUCTION IN ARABIDOPSIS MITOCHONDRIA ......................33
2.2 PREPARATION OF MITOCHONDRIAL FRACTION..............................................33
2.3 DETECTION OF S-NITROSYLATED PROTEINS IN ARABIDOPSIS LEAVES
MITOCHONDRIA........................................................................................38
2.4 THE GLYCINE DEHYDROGENASE P PROTEIN..................................................41
2.4.1 Production of recombinant P protein .........................................................41
2.4.2 Purification of the P Subunit of GDC..........................................................41
5 2.4.3 Activity of the Partially Purified P Protein...................................................43
2.5 S-NITROSYLATION / S-GLUTATHIONYLATION OF THE P PROTEIN.....................44
2.5.1 Biotin Switch Method on Enriched P Protein ...............................................45
2.5.2 MS Analysis of GSNO Treated P Protein.....................................................45
2.5.3 S-Nitrosylation or S-Glutathionylation?46
2.6 INHIBITION OF GDC BY NO ........................................................................46
2.6.1 Modulation of GDC Activity by GSNO in Mitochondria ..................................46
2.6.2 Modulation ofivity by GSNO in Arabidopsis Leaves..........................47
2.7 ROLE OF GDC IN PLANT RESPONSE TO HARPIN .............................................49
2.7.1 Effect of Harpin on GDC in Arabidopsis Leaves ...........................................49
2.7.2 Effepin on Photorespiratory Rate ..................................................50
2.7.3 Effect of Harpin on Glycine and Serine Content ..........................................52
2.8 MITOCHONDRIAL AND CELLULAR RESPONSE TO GDC INHIBITION ...................52
2.8.1 Generation of Mitochondrial ROS..............................................................53
2.8.2 Mitochondrial Transmembrane Potential....................................................55
2.8.3 Induction of cell death............................................................................56
3 Discussion.......................................................................................... 59
3.1 MITOCHONDRIA AND NO59
3.2 S-NITROSYLATION OF MITOCHONDRIAL PROTEINS........................................61
3.3 EFFECT OF NO ON THE GLYCINE DECARBOXYLASE.........................................63
3.4 RESPONSE TO GDC INHIBITION ..................................................................66
3.5 CONCLUSION ...........................................................................................68
4 Materials and Methods....................................................................... 69
4.1 PLANT MATERIAL and TREATMENTS .............................................................69
4.1.1 Arabidopsis thaliana Suspension Culture ...................................................69
4.1.2 Arabidopsis thaliana Plants......................................................................69
4.2 ISOLATION OF MITOCHONDRIA...................................................................70
4.2.1 Crude Mitochondria Extract .....................................................................70
4.2.2 Pure Mitochondria: Free Flow Electrophoresis.............................................70
4.2.3 Treatments of Arabidopsis Mitochondria ....................................................71
4.3 ANALYSIS OF MITOCHONDRIA PURITY .........................................................71
4.3.1 Cytochrome c Oxidase Assay71
4.3.2 Catalase Assay......................................................................................72
4.3.3 Determination of Cellular Respiration........................................................73
4.3.4 2D Gel Electrophoresis ...........................................................................74
6 4.4 HARPIN PREPARATION and CHEMICALS........................................................74
4.4.1 Preparation of Harpin Protein...................................................................74
4.4.2 Chemicals.............................................................................................75
4.5 S-NITROSYLATION ....................................................................................75
4.5.1 S-Nitrosylated Proteins Detection and Purification.......................................75
4.5.2 Polyacrylamide Gel Electrophoresis...........................................................77
4.5.3 Staining of Proteins with Sypro® Ruby Protein Gel Stain .............................77
4.5.4 Staining of Proteins with Colloidal Comassie (Blue Silver) ............................78
4.5.5 Protein Transfer and Immunoblotting........................................................78
4.5.6 Maldi-Tof Mass Spectrometry for Peptide Fingerprint Analysis and
NanoLC/MS/MS .....................................................................................79
2/34.5.7 Nano-HPLC-MS and Data Analysis .........................................................79
4.6 PURIFICATION OF P-PROTEIN FROM MITOCHONDRIA EXTRACT .......................80
4.6.1 Solubilization of Glycine-Cleavage System.................................................81
4.6.2 Size Exclus