A functional genomics approach to the plant soluble pyrophosphatase family [Elektronische Ressource] / presented by Zahide Neslihan Ergen

Neslihan Asutay-Effenberger

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2006
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

DISSERTATION

Submitted to the

Combined Faculties
for the NATURAL SCIENCES and for MATHEMATICS
of the RUPERTO-CAROLA UNIVERSITY OF HEIDELBERG, GERMANY

for the degree of
DOCTOR OF NATURAL SCIENCES







Presented by
Zahide Neslihan ERGEN
Ankara, Turkey

8 June 2006


A FUNCTIONAL GENOMICS APPROACH TO
THE PLANT SOLUBLE PYROPHOSPHATASE FAMILY













Referees: Prof. Dr. Thomas RAUSCH
Prof. Dr. Michael WINK

TABLE OF CONTENTS

1 SUMMARY ........................................................................................................................................... 1
ZUSAMMENFASSUNG...................................................................................................................... 3
2 INTRODUCTION................................................................................................................................. 5
2.1 INORGANIC PYROPHOSPHATE........................................................................................................... 5

2.1.1 INORGANIC PYROPHOSPHATE AS AN ENERGY SOURCE ....................................................................6

2.1.2 INORGANIC PYROPHOSPHATE IN PLANT CARBOHYDRATE METABOLISM ........................................7
2.1.2.1 Carbohydrate Metabolism in Plants ..................................................................................................7
2.1.2.1.1 Sucrose Metabolism .......................................................................................................................7
2.1.2.1.2 Starch Metabolism....................................................................................................10
2.1.2.2 Function of Inorganic Pyrophosphate in Carbohydrate Metabolism...............................................10

2.2 INORGANIC PYROPHOSPHATASES 12

2.2.1 TYPES OF INORGANIC PYROPHOSPHATASES ...................................................................................12

2.2.2 SOLUBLE PYROPHOSPHATASES.......................................................................................................13
2.2.2.1 Plastidial Plant Soluble Pyrophosphatases15
2.2.2.2 Mitochondrial Plant Soluble Pyrophosphatases...........................................................................16
2.2.2.3 Cytosolic Plant Solu ..................................................................................17
2.2.2.4 Effect of Soluble Pyrophosphatase Overexpression in Plant Metabolism...................................18

2.2.3 MEMBRANE BOUND INORGANIC PYROPHOSPHATASES ..................................................................21
2.2.3.1 Vacuolar Membrane Bound Pyrophosphatases22
2.2.3.2 Golgi Membrane Bound Pyrophosphatases.................................................................................25
2.2.3.3 Plasma Membrane and Endoplasmic Reticulum Associated Pyrophosphatases..........................26
2.2.3.4 Effect of Overexpression of Vacuolar Membrane Bound Pyrophosphatases ..............................26

2.3 MODEL ORGANISMS USED............................................................................................................... 28

2.3.1 ARABIDOPSIS THALIANA....................................................................................................................28
2.3.2 BETA VULGARIS L.............................................................................................................................28

2.4 AIM OF THE PROJECT ...................................................................................................................... 29

3 RESULTS ............................................................................................................................................31
3.1 THE ARABIDOPSIS THALIANA SOLUBLE PYROPHOSPHATASE FAMILY ......................................... 31

3.1.1 SUBCELLULAR LOCALIZATION OF A. THALIANA SOLUBLE PYROPHOSPHATASES............................34
3.1.1.1 In Silico Analysis of Subcellular Localization of A. thaliana sPPases ........................................34
3.1.1.2 Subcellular Localization of Arabidopsis thaliana Soluble Pyrophosphatases in vivo .................35

3.1.2 DIFFERENTIAL EXPRESSION OF A. THALIANA SOLUBLE PYROPHOSPHATASES DURING PLANT
DEVELOPMENT ...............................................................................................................................37
3.1.2.1 Analysis of Plants with A. thaliana sPPase Promoter Driven GUS Expression ..........................38
3.1.2.2 Quantitative Expression Analysis of ASP Isoforms by Real Time PCR .....................................50

3.1.3 RESPONSE OF A. THALIANA SPPASES TO DIFFERENT STRESSES.......................................................53
3.1.3.1 Transcriptional Factor Binding Motifs in Promoter Regions of ASPases ...................................53
3.1.3.2 Response of A. thaliana sPPase Expression to Etiolation............................................................54
3.1.3.3 Expression of ASP Isoforms in Response to Different Sugars Analyzed in A. thaliana Cell
Culture.............. ...........................................................................................................................55
3.1.3.4 Expression of ASP Isoforms in Response to Different Sugars in planta .....................................57
3.1.3.5 Response of A. thaliana sPPase Expression to ABA and to Different Environmental Stresses..59

3.2 STUDIES USING BETA VULGARIS SOLUBLE AND VACUOLAR PYROPHOSPHATASE ISOFORMS.....70

3.2.1 POST-TRANSLATIONAL REGULATION OF SOLUBLE PYROPHOSPHATASES STUDIED WITH
RECOMBINANT BETA VULGARIS SPPASE ISOFORM 1.......................................................................71
3.2.1.1 Redox Regulation of Recombinant B. vulgaris Soluble Pyrophospatase Isoform 1....................72
3.2.1.2 Phosphorylation of Recombinant Bsp1 with Protein Kinase C ...................................................73

3.2.2 EFFECT OF HOMOLOGOUS OVEREXPRESSION OF BSP1 OR BVP1 ON WOUND RESPONSE AND
SUCROSE LOADING IN BETA VULGARIS ...........................................................................................75
3.2.2.1 Post-transcriptional Regulation of Bsp1 and Bvp1 Observed upon Wounding...........................75
3.2.2.2 Changes in Sucrose Loading and Hexoses in B. vulgaris Taproot Tissue upon Overexpression
of Homologous Bvp1 or Bsp1 ....................................................................................................78

3.2.3 EFFECT OF ECTOPIC EXPRESSION OF BSP1 AND BVP1 ON SALT RESISTANCE OF ARABIDOPSIS
THALIANA.........................................................................................................................................81

4 DISCUSSION ...................................................................................................................................... 84
4.1 A. THALIANA SPPASES SHARE A HIGH HOMOLOGY, EXCEPT FOR THE PLASTIDIAL ISOFORM..84

4.2 A. THALIANA SPPASES LOCALIZE IN THE CYTOPLASM, NUCLEUS AND PLASTIDS, BUT NOT IN
MITOCHONDRIA85

4.3 A. THALIANA SPPASES ARE DIFFERENTIALLY REGULATED .......................................................... 87

4.3.1 PROMOTER ACTIVITIES OF A. THALIANA SPPASES IN DIFFERENT TISSUES INDICATE ISOFORM
SPECIFICITY AND DEVELOPMENTAL STAGE DEPENDENCY............................................................ 88
4.3.2 REAL TIME PCR ANALYSIS CONFIRMS GENE EXPRESSION OF A. THALIANA SPPASES IN SEVERAL
PLANT TISSUES...............................................................................................................................91

4.4 SUGAR-MEDIATED AND STARVATION INDUCED RESPONSES OF A. THALIANA SPPASES .............92

4.4.1 ASP2B IS INDUCED BY SUGAR STARVATION ................................................................................. 93
4.4.2 LIGHT AND PHOTOSYNTHESIS REGULATION OF THE PLASTIDIAL ISOFORM................................... 94
4.4.3 SUGAR-MEDIATED CHANGES IN GENE EXPRESSIONS OF A. THALIANA SPPASE ISOFORMS; ONLY
ASP3 EXPRESSION IS REGULATED BY SUCROSE............................................................................ 95


4.5 CHANGES IN THE EXPRESSIONS OF A. THALIANA SPPASES UPON DIFFERENT ENVIRONMENTAL
CONDITIONS CONFIRM THEIR IMPORTANCE IN STRESS RESPONSES .......................................... 96

4.5.1 ABA, SALT AND COLD SPECIFICALLY INDUCE SOME ASP ISOFORMS, WHILE REPRESSING THE
OTHERS...........................................................................................................................................97
4.5.2 RESPONSES OF ASP ISOFORMS TO HEAT STRESS ARE TIME DEPENDENT .................................... 100
4.5.3 PHOSPHATE STARVATION REPRESSES THE EXPRESSION OF A. THALIANA SPPASES......................101
4.5.4 DIFFERENTIAL GENE EXPRESSION OF SOLUBLE AND VACUOLAR PYROPHOSPHATASES TO
MECHANICAL WOUNDING............................................................................................................ 103
4.5.4.1 Indirect Evidence on the Transient Induction of Bsp1 and Bvp1 up