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  • Max-Planck-Institut für Molekulare Pflanzenphysiologie, Golm Department of Metabolic Networks

    Research-group: Storage Carbohydrate Metabolism

    Redox-regulation of starch and lipid

    synthesis in leaves

    Disseration zur Erlangung des naturwissenschaftlichen Grades

    Doktor rerum naturalium

    Eingereicht an der mathematisch-naturwissenschaftlichen

    Fakultät der Universität Potsdam

    Anna Kolbe

    June 2005

  • This Ph.D. thesis is the account of work done between April 2002 and June 2005 in

    the department of Prof. Dr. Mark Stitt in the Max-Planck-Institut für Molekulare

    Pflanzenphysiologie, Golm, Germany. It is result of my own work and has not been

    submitted for any degree or Ph. D. at any other university.

    Eidesstattliche Erklärung

    Die Disseration ist ein Ergebnis praktischer Arbeit, welche von April 2002 bis Juni

    2005 durgeführt wurde im Departament von Prof. Dr. Mark Stitt im Max-Planck-

    Institut für Molekulare Pflanzenphysiologie, Golm. Ich erkläre, daß ich vorliegende

    Arbeit selbstständig und ohne unerlaubte Hilfe angefertigt habe. Es wurden keine

    anderen als die angegebenen Quellen und Hilfsmittel benutzt, und die den benutzten

    Quellen wörtlichen und inhaltlichen Stellen sind als solche kenntlich gemacht.

    Golm, Juni 2005

    Anna Kolbe

  • Abstract

    Post-translational redox-regulation is a well-known mechanism to regulate enzymes

    of the Calvin cycle, oxidative pentose phosphate cycle, NADPH export and ATP

    synthesis in response to light. The aim of the present thesis was to investigate

    whether a similar mechanism is also regulating carbon storage in leaves.

    Previous studies have shown that the key-regulatory enzyme of starch synthesis,

    ADPglucose pyrophosphorylase (AGPase) is inactivated by formation of an

    intermolecular disulfide bridge between the two catalytic subunits (AGPB) of the

    heterotetrameric holoenzyme in potato tubers, but the relevance of this mechanism to

    regulate starch synthesis in leaves was not investigated. The work presented in this

    thesis shows that AGPase is subject to post-translational redox-regulation in leaves

    of pea, potato and Arabidopsis in response to day night changes. Light was shown to

    trigger posttranslational redox-regulation of AGPase. AGPB was rapidly converted

    from a dimer to a monomer when isolated pea chloroplasts were illuminated and from

    a monomer to a dimer when preilluminated leaves were darkened. Conversion of

    AGPB from dimer to monomer was accompanied by an increase in activity due to

    changes in the kinetik properties of the enzyme. Studies with pea chloroplast extracts

    showed that AGPase redox-activation is mediated by thioredoxins f and m from

    spinach in-vitro. In a further set of experiments it was shown that sugars provide a

    second input leading to AGPase redox activation and increased starch synthesis and

    that they can act as a signal which is independent from light. External feeding of

    sugars such as sucrose or trehalose to Arabidopsis leaves in the dark led to

    conversion of AGPB from dimer to monomer and to an increase in the rate of starch

    synthesis, while there were no significant changes in the level of 3PGA, an allosteric

    activator of the enyzme, and in the NADPH/NADP+ ratio. Experiments with

    transgenic Arabidopsis plants with altered levels of trehalose 6-phosphate (T6P), the

    precursor of trehalose synthesis, provided genetic evidence that T6P rather than

    trehalose is leading to AGPase redox-activation. Compared to Wt, leaves expressing

    E.coli trehalose-phosphate synthase (TPS) in the cytosol showed increased

  • activation of AGPase and higher starch level during the day, while trehalose-

    phosphate phosphatase (TPP) overexpressing leaves showed the opposite. These

    changes occurred independently of changes in sugar and sugar-phosphate levels

    and NADPH/NADP+ ratio. External supply of sucrose to Wt and TPS-overexpressing

    leaves led to monomerisation of AGPB, while this response was attenuated in TPP

    expressing leaves, indicating that T6P is involved in the sucrose-dependent redox-

    activation of AGPase. To provide biochemical evidence that T6P promotes redox-

    activation of AGPase independently of cytosolic elements, T6P was fed to intact

    isolated chloroplasts for 15 min. incubation with concentrations down to 100 µM of

    T6P, but not with sucrose 6-phosphate, sucrose, trehalose or Pi as controls,

    significantly and specifically increased AGPB monomerisation and AGPase activity

    within 15 minutes, implying T6P as a signal reporting the cytosolic sugar status to the

    chloroplast. The response to T6P did not involve changes in the NADPH/NADP+ ratio

    consistent with T6P modulating redox-transfer to AGPase independently of changes

    in plastidial redox-state.

    Acetyl-CoA carboxylase (ACCase) is known as key-regulatory enzyme of fatty acid

    and lipid synthesis in plants. At the start of the present thesis there was mainly in

    vitro evidence in the literature showing redox-regulation of ACCase by DTT, and

    thioredoxins f and m. In the present thesis the in-vivo relevance of this mechanism to

    regulate lipid synthesis in leaves was investigated. ACCase activity measurement in

    leaf tissue collected at the end of the day and night in Arabidopsis leaves revealed a

    3-fold higher activation state of the enzyme in the light than in the dark. Redox-

    activation was accompanied by change in kinetic properties of ACCase, leading to an

    increase affinity to its substrate acetyl-CoA . In further experiments, DTT as well as

    sucrose were fed to leaves, and both treatments led to a stimulation in the rate of

    lipid synthesis accompanied by redox-activation of ACCase and decrease in acetyl-

    CoA content.

    In a final approach, comparison of metabolic and transcript profiling after DTT feeding

    and after sucrose feeding to leaves provided evidence that redox-modification is an

    important regulatory mechanism in central metabolic pathways such as TCA cycle

    and amino acid synthesis, which acts independently of transcript levels.

  • TABLE OF CONTENTS

    Abstract

    Table of contents

    1. INTRODUCTION 9

    1.1. Importance of Carbon Metabolism in Plants 9

    1.2. Photosynthesis and its regulation in leaves 10

    1.3. The pathway of starch synthesis in leaves and its regulation 13 1.3.1. Starch synthesis pathway 13 1.3.2. Regulation of starch synthesis 15

    1.4. The pathway of sucrose synthesis in leaves and its regulation 18

    1.5. Pathway and Regulation of Fatty Acid Synthesis in Leaves 19 1.5.1. The pathway of fatty acid synthesis 19 1.5.2. Regulation of fatty acid synthesis 22

    1.6. Aims and objectives of the present work 24

    2. MATERIALS AND METHODS 27

    2.1. Enzymes and chemicals 27

    2.2. Plant material and growth 27

    2.3. Harvesting procedure, sample storage 28

    2.4. Homogenisation of frozen leaf tissue 28

    2.5. Incubation of Leaves with Sugars in the Darkness 28

    2.6. Incubation of leaf discs with DTT 28

    2.7. Trichloroacetic acid (TCA) extraction 29

    2.8. Extraction of AGPase for western blot and procedures for gels 29

    2.9. Extraction and assay of AGPase 30

    2.10. HPLC (measurement of ADP-glucose) 30

    2.11. Extraction and assay of ACCAse 31

    2.12. Chloroplast preparation 31

    2.13. Incubations of chloroplasts and photosynthesis measurement 32

  • 2.14. Desalting of proteins from isolated chloroplasts 32

    2.15. Thioredoxin reduction 33

    2.16. Extraction and assay of FBPase and NADP malate dehydrogenase 33

    2.17. Ethanolic extraction 34

    2.18. Assay of sucrose, reducing sugars, starch 34

    2.19. Assay of hexose-phosphates 34 2.19.1. Glucose 6-phosphate 34 2.19.2. Glucose 1-phosphate 35 2.19.3. Fructose 6-phosphate 35

    2.20. Pyruvate and phosphoenolpyruvate 36

    2.21. 3PGA 36

    2.22. Glycerol-3-phosphate 36

    2.23. Acetyl-coenzyme A 36

    2.24. NADPH/NADP+ ratio measurement 37

    2.25. Labeling experiments and label separation 37

    2.26. Extraction and derivatisation for GC/MS (gas chromatography/mass spectrometry) 38

    2.27. GC/MS analysis 38

    2.28. Determination of relative metabolite levels 39

    2.29. RNA isolation 39

    2.30. Transcript levels measurement and analysis 40

    3. RESULTS 41

    3.1. AGPase is subject to post-translational redox-activation in leaves in response to light signals 41

    3.1.1. Establishing of a new protocol to measure changes in the reduction state of AGPase in leaves 41

    3.1.2. Changes in AGPB dimerisation and activity in response to day/night changes in leaves 42

    3.1.3. Rapid Changes in AGPB Dimerisation as a Response to Light/Dark Transitions 46

    3.1.4. Light leads to monomerisation of AGPB in isolated pea chloroplasts 47 3.1.5. AGPase activation in chloroplast extracts incubated with thioredoxins 49 3.1.6. Conclusions 52

  • 3.2. AGPase is activated by posttranslational redox-modification in response to sugars in leaves 52