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Title: Localization and fuction of plant invertases and fructan metabolizing enzymes
Other Titles: Lokalisatie en functie van plant invertasen en fructan metaboliserende enzymen
Authors: Xiang, Li; S0197922
Issue Date: 10-May-2012
Table of Contents: Abstract . . . vii
Research Objectives . . . xi
List of Abbreviations . . . xv
List of Figures . . . xvii
List of Tables . . . xxi

Chapter 1 Sucrose and fructan metabolism . . . 1
1.1 Sucrose metabolism and signaling . . . 1
1.1.1 Introduction . . . 1
1.1.2 Sucrose biosynthesis . . . 2
1.1.3 Sucrose degradation . . . 4
1.1.4 Sugar sensing and signaling . . . 12
1.1.5 Sugar transporters . . . 14
1.1.6 Starch and RFOs in plants . . . 18
1.2 Fructans and fructan metabolism . . . 19
1.2.1 Fructans: occurrence, structure, and physiological function . . . 19
1.2.2 Enzymes involved in fructan metabolism . . . 22

Chapter 2 Protein sorting . . . 27
2.1 Introduction . . . 27
2.2 Protein sorting to the vacuole . . . 28
2.2.1 Introducing the plant vacuole . . . 28
2.2.2 Vesicular trafficking . . . 31
2.2.3 Sorting of plant vacuolar proteins initiates in the ER . . . 32
2.2.4 Intra-Golgi and post-Golgi transport . . . 35
2.2.5 Trafficking from Golgi to the vacuole . . . 40
2.3 Protein sorting to other organelles . . . 42
2.3.1 Protein import into mitochondria . . . 42
2.3.2 Protein import into the chloroplast . . . 44
2.3.3 Protein sorting to the nucleus . . . 45

Chapter 3 A/N-Invs and antioxidant defence . . . 47
3.1 Introduction . . . 47
3.2 Results . . . 48
3.2.1 Phylogenetic relationships within the A/N-Inv gene family . . . 48
3.2.2 At-A/N-InvA is located at mitochondria . . . 50
3.2.3 Enzymatic activities of recombinant At-A/N-InvA and At-A/N-InvG, and inhibition by TRIS . . . 52
3.2.4 Growth phenotype and total A/N-Inv activities of Atinva and Atinvg knockout plants . . . 52
3.2.5 Expression of genes involved in oxidative stress defence . . . 56
3.3 Discussion . . . 56

Chapter 4 Sorting mechanism of AtVI2 . . . 63
4.1 Introduction . . . 63
4.2 Results . . . 64
4.2.1 AtVI2 is targeted to the vacuole by a complex N-terminal propeptide . . . 64
4.2.2 AtVI2 reaches the vacuole through a membrane anchor secretion pathway (MASP) . . . 66
4.2.3 AtVI2 reaches the lytic vacuole (LV) but not the protein storage vacuole (PSV) . . . 67
4.2.4 Sorting of AtVI2 in bp80 and AP3 loss-of-function protoplasts . . . 67
4.2.5 A dileucine motif contributes to MASP sorting . . . 69
4.2.6 MASP requires a basic region . . . 71
4.2.7 Timing of vacuolar delivery: the role of a glutamate stretch . . . 74
4.3 Discussion . . . 77

Chapter 5 Localization and sorting of FTs . . . 85
5.1 Introduction . . . 85
5.2 Phylogenetic relationship and sequence analysis of FTs . . . 86
5.3 Subcellular localization and trafficking of Ci1-SST and Ci1-FFT . . . 91
5.3.1 Subcellular localization of Ci1-SST and Ci1-FFT . . . 91
5.3.2 Ci1-SST and Ci1-FFT are likely transported through MASP . . . 92
5.3.3 Ci1-FFT reaches the central vacuole (CV) through a ER-->Golgi-->LV pathway . . . 93
5.4 Discussion . . . 94

Chapter 6 FEH localization and sorting . . . 99
6.1 Introduction . . . 99
6.2 Results . . . 100
6.2.1 Gene expression of defective invertases (FEHs) in Arabidopsis . . . 100
6.2.2 At6&1FEH: subcellular localization and promoter-GUS/
GFP expression analysis in seedlings . . . 101
6.2.3 Subcellular localization of other candidate FEHs in Arabidopsis . . . 103
6.2.4 Ci1-FEHIIa is sorted to the central vacuole . . . 105
6.2.5 Ci1-FEHIIa is sorted in the BP80/VSR1-mediated trafficking pathway . . . 107
6.2.6 Motifs in the Ci1-FEHIIa NTPP for vacuolar targeting . . . 111
6.3 Discussion . . . 113
6.3.1 Subcellular localization of defective invertases in plants and their possible function . . . 113
6.3.2 Ci1-FEHIIa is sorted through a BP80-dependent pathway . . . 115

Chapter 7 Materials and methods . . . 119
7.1 Growth media and growth conditions . . . 119
7.2 Molecular biology techniques . . . 120
7.3 Construction of transgenic plants . . . 124
7.4 Transient expression in Arabidopsis mesophyll protoplasts . . . 126
7.5 Protein expression and extraction . . . 130

Chapter 8 General conclusions and perspectives . . . 135

Bibliography . . . 141
List of Publications . . . 201
ISBN: 978-90-8649-515-3
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Molecular Physiology of Plants and Micro-organisms Section - miscellaneous
Laboratory for Molecular Plant Physiology (-)

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