Functional analysis of tocopherol biosynthesis in plants [Elektronische Ressource] / vorgelegt von Ali-Reza Abbasi

Lars Thalmann

Sujets

• Biologie

Informations

• 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 .........................