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Publié par | friedrich-alexander-universitat_erlangen-nurnberg |
Publié le | 01 janvier 2007 |
Nombre de lectures | 33 |
Langue | Deutsch |
Poids de l'ouvrage | 6 Mo |
Extrait
Functional analysis of tocopherol biosynthesis in plants
Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangen des Doktorgrades
vorgelegt von
Ali-Reza Abbasi
Aus Tehran, Islamische Republik des Iran
Als Dissertation genehmigt
von den Naturwissenschaftlichen Fakultäten
der Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung: 23. April 2007
Vorsitzender der
Promotionskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter: Prof. Dr. Uwe Sonnewald
Zweitberichterstatter: Prof. Dr.Norbert Sauer
Content - I -
Content
1. Summary / Zusammenfassung .......................................................................1
1.1.y.............................................................................................................1
1.2. Zusammenfassung .............................................................................................3
2. Introduction.......................................................................................................5
2.1. Vitamin E.............................................................................................................5
2.2. Discovery of vitamin E ........................................................................................5
2.3. Chemical structure of Vitamin E .........................................................................6
2.4. Lipid peroxidation................................................................................................7
2.5. Biological function of Vitamin E ..........................................................................9
2.5.1. Antioxidant function of vitamin E ........................................................................9
2.5.2. Pro-oxidant function............................................................................................11
2.5.3. Non antioxidant function .....................................................................................12
2.6. Proposed function of vitamin E in plants ............................................................12
2.6.1. Protection of the photosynthetic apparatus from photo-oxidative damage........13
2.6.2. Protection of the chloroplast membrane from lipid peroxidation........................16
2.6.3. Proposed function of tocopherol in stress signalling..........................................17
2.7. Occurrence and subcellular localization of vitamin E.........................................18
2.8. The vitamin E biosynthetic pathway ...................................................................20
2.9. Genes, function and mutants..............................................................................22
2.9.1. Homogentisate phytyl transferase (HPT) ...........................................................23
2.9.2. Tocopherol cyclase (TC).....................................................................................25
2.9.3. Gamma- tocopherol methyltransferase ( γTMT) .................................................26
2.9.4. 2-Methyl-6-phythylbenzoquinone methyltransferase (MPBQ MT, MT1)............26
2.10. Scientific aims of the work ..................................................................................27
3. Results ...............................................................................................................29
3.1. Approaches to identify tocopherol cyclase interacting proteins .........................29
3.1.1. Creation of Arabidopsis transgenic plants to investigate the localization of
tocopherol cyclase ..............................................................................................30
3.1.1.1. Construction of tocopherol cyclase (TC) fused to green fluorescence protein
(GFP) ..................................................................................................................31
3.1.1.2. Transformation of Arabidopsis vte1 mutant and pre-screening of the
transgenic plants.................................................................................................32
Content - II -
3.1.1.3. Arabidopsis tocopherol cyclase is located into the chloroplast..........................33
3.1.2. Creation of Arabidopsis transgenic plants to study protein-protein interaction
of tocopherol cyclase ..........................................................................................35
3.1.2.1. Construction of tocopherol cyclase (TC) fused to tandem affinity purification
tag protein (TAP-Tag) expression vector ...........................................................35
3.1.2.2. Arabidopsis vte1 mutant was complemented by tocopherol cyclase fused to
TAP-Tag (pBin: TC: TAP-Tag) ...........................................................................37
3.1.3. Protein complex purification from transformed TC-TAP-Tag plants ..................42
3.1.4. Visualization of the protein complex using one or two dimensional gel
electrophoresis....................................................................................................45
3.2. Study the biological function of tocopherol in tobacco plants under optimal
growth conditions................................................................................................49
3.2.1. Tissue specific distribution of tocopherol derivatives in tobacco plants.............49
3.2.2. Generation of transgenic plants with altered content and composition of
tocopherol using dsRNAi ....................................................................................52
3.2.2.1. Generation of tocopherol deficient plants with constitutively silenced
homogentisate phytyl transferase (HPT) using dsRNAi.....................................52
3.2.2.1.1. Creation of HPT: RNAi construct........................................................................52
3.2.2.1.2. Plant transformation and pre-screening the transgenic plants...........................53
3.2.2.1.3. Screening of the transgenic plants .....................................................................53
3.2.2.1.4. Tocopherol deficiency is inherited in selected HPT: RNAi tobacco transgenic
lines.....................................................................................................................55
3.2.2.1.5. Biochemical and physiological characterization of HPT: RNAi tobacco plants
under ambient growth condition .........................................................................56
3.2.2.1.5.1. Silencing of HPT resulted in severe tocopherol deficiency in transgenic plants56
3.2.2.1.5.2. Silencing of HPT by dsRNAi leads to phenotypic alteration in source leaves
of transgenic plants.............................................................................................58
3.2.2.1.5.3. Growth response of tocopherol deficient tobacco plants ...................................58
3.2.2.1.5.4. Inhibition of HPT leads to seed yield reduction of transgenic tobacco plants....60
3.2.2.1.5.5. Plastoquinone analysis.......................................................................................61
Tocopherol deficiency leads to soluble sugar accumulation in transgenic 3.2.2.1.5.6.
tobacco plants.....................................................................................................63
3.2.2.1.5.7. The amino acid content and composition changed in lower source leaves of
the tocopherol deficient tobacco plants ..............................................................66
3.2.2.1.5.8. Analysis of chlorophyll and carotenoids in transgenic plants.............................68
Content - III -
3.2.2.1.5.9. Silencing of tobacco HPT gene decreased photosynthetic capacity in
transgenic plants.................................................................................................70
3.2.2.1.5.10. Severe tocopherol deficiency is paralleled by increased lipid peroxidation in
source leaves of transgenic tobacco plants .......................................................73
3.2.2.1.5.11. The effect of tocopherol deficiency on ascorbate and glutathione content in
transgenic HPT:RNAi tobacco plants .........................