Localization and fuction of plant invertases and fructan metabolizing enzymes

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 . . . 201nrpages: 230status: publishe