Post-translational regulation of invertases molecular mechanism and impact on plant development in Arabidopsis thaliana
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2014
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| adam_text | DISSERTATION
SUBMITTED TO THE
COMBINED FACULTIES FOR THE NATURAL SCIENCES AND FOR MATHEMATICS
OF THE RUPERTO-CAROLA UNIVERSITY OF HEIDELBERG, GERMANY
FOR THE DEGREE OF
DOCTOR OF NATURAL SCIENCES
PRESENTED BY
MASTER OF SCIENCE TAO SU
BORN IN: QIQIHAR, HEILONGJIANG,
P.R.CHINA
ORAL EXAMINATION: 25.03.2014
HTTP://D-NB.INFO/105140536X
TABLE OF CONTENTS
1 SUMMARY/ZUSAMMENFASSUNG 1
1.1 SUMMARY 1
1.2 ZUSAMMENFASSUNG 2
2 INTRODUCTION 3
2.1 SUGAR IMPACT: METABOLISM AND SENSING IN PLANT 3
2.1.1 SUGAR ALLOCATION AND SOURCE-SINK RELATIONSHIPS 3
2.1.2 SUGAR TRANSPORTERS 5
2.1.3 SUGAR METABOLISM AND SIGNALING IN PLANTS 11
2.2 INVERTASES AND SUCROSE SYNTHASE (SUSY) 15
2.2.1 CYTOSOLIC INVERTASE (CI) 15
2.2.2 VACUOLAR INVERTASE (VI) 16
2.2.3 EXTRACELLULAR INVERTASE (CWI) 18
2.2.4 SIGNALING ROLE OF INVERTASE AND SUSY 21
2.3 REGULATION OF INVERTASES 22
2.3.1 REGULATION BY ABIOTIC STRESS 23
2.3.2 REGULATION BY HORMONES 24
2.4 INVERTASE INHIBITORS (IFS) 25
2.4.1 THE ROLES OF IFS 25
2.4.2 INTERACTIONS BETWEEN INVERTASES AND IFS 27
3 PROJECT AIMS 29
4 RESULTS 30
4.1 ATCIFI FUNCTIONS AS CELL WALL INVERTASE INHIBITOR 30
4.1.1 CELL COMPARTMENTATION OF ATCIFI 30
4.1.2 ATCIFI MUTANTS SHOW EFFECT ON ACID INVERTASES AT TISSUE LEVEL 32
4.2 ATCIFI PHYSIOLOGICAL ROLE IN VIVO 33
4.2.1 ACID INVERTASE WAS AFFECTED IN SEEDS AND SEEDLINGS OF ATCIF1-1 33
4.2.2 INCREASED VI ACTIVITY CAUSED ELONGATED ROOT LENGTH IN SEEDLING 35
4.2.3 CWI IS POST-TRANSLATIONALLY REGULATED IN EARLY SEEDLING GROWTH 36
4.2.4 OVEREXPRESSION OF ATCIFI IMPAIRS CWI ACTIVITY IN SEEDS 38
4.3 LOSS-OF-FUNCTION OF ATCIFI AFFECTS SUGAR BALANCE 39
4.3.1 ELEVATED CWI ACTIVITY INDUCES GLUCOSE TRANSPORTER EXPRESSION 39
4.3.2 ATCIF1-1 SHOWS MODIFIED SUGAR AND STARCH LEVEL 41
4.3.3 DOWNSTREAM SUGAR METABOLISM ENZYMES ARE NOT AFFECTED 42
4.4 ATCIFI EXPRESSION IN RESPONSE TO HORMONAL AND ENVIRONMENTAL CUES 43
4.4.1 ATCIFI IS INDUCED BY ABA 43
4.4.2 ATCIFI RESPONDS TO OSMOTIC STRESS 45
4.4.3 CYTOKININ CONDITIONALLY REGULATES CWI ACTIVITY 45
4.5 ATCIF2 IN VIVO FUNCTION 46
4.5.1 PHYLOGENETIC ANALYSIS OF C/VIFS 46
4.5.2 AMI LINES OF ATCIF2 SHOW INCREASED CWI ACTIVITY 47
4.5.3 DOUBLE MUTANTS SHOW INCREASED SEED BIOMASS 49
4.6 ATC/VIF3 IS A INVERTASE INHIBITOR IN VITRO 51
4.7 ATCIF2 AND ATCIF3 RESPOND TO VARIOUS FACTORS 52
5 DISCUSSION 54
5.1 ATCIFI PHYSIOLOGICAL ROLE AS CWI INHIBITOR 54
5.1.1 ATCIFI IS LOCALIZED TO THE CELL WALL 54
TABLE OF CONTENTS
5.1.2 ATCIFI IS INVOLVED IN EARLY SEEDLING DEVELOPMENT 55
5.1.3 ATCIFI AFFECTED SEED GERMINATION IS DOSE-DEPENDENT IN VIVO 56
5.2 ABA INDUCED ATCIFI EXPRESSION IS TISSUE DEPENDENT 57
5.2.1 LACK OF ATCIFI DELAYS ABA INDUCED SENESCENCE IN SEEDLING? 57
5.2.2 LESS ABA ACCUMULATION IN SEEDS OF ATCIF1-1? 58
5.2.3 ABI
5
POTENTIALLY REGULATES ATCIFI EXPRESSION UPON ABA? 59
5.3 THE ROLE OF ATCWIS AND ATVIS IN ARABIDOPSIS 60
5.3.1 FUNCTION OF ATCWIS 60
5.3.2 FUNCTION OF ATVIS 61
5.3.3 CWI MAY AFFECT VI BY CHANGING THE CAPACITY OF SUGAR? 62
5.4 OUTLOOK 63
5.4.1 WHAT TARGET MECHANISMS ARE INVOLVED IN ATCA/IFS INHIBITORY
FUNCTION 63
5.4.2 WHAT TRANSCRIPTIONAL FACTORS REGULATE ATCA/IFS? 63
5.4.3 WHAT ARE THE ROLE OF ATCWI3 AND ATCWI6 IN VIVO? 64
5.4.4 TO WHAT EXTENT DOSE THE ATCA/IFS INFLUENCE ON PLANT DEVELOPMENT?
64
6 MATERIAL AND METHODS 65
6.1 PLANT MATERIAL 65
6.1.1 ARABIDOPSIS THALIANA 65
6.1.2 NICOTIANA BENTHAMIANA 65
6.2 MICROBIOLOGICAL TECHNIQUES 65
6.2.1 ESCHERICHIA COLI (E. COLI) STRAINS 65
6.2.2 AGROBACTERIUM STRAINS 66
6.2.3 MEDIA AND ANTIBIOTICS FOR E. COLI GROWTH 66
6.2.4 PREPARATION OF ELECTROCOMPETENT E. COLI AND AGROBACTERIA CELLS 67
6.2.5 TRANSFORMATION OF BACTERIA 67
6.2.6 PREPARATION OF GLYCEROL STOCKS 68
6.3 DNA TECHNIQUES 68
6.3.1 OLIGONUCLEOTIDES 68
6.3.2 ISOLATION OF PLASMID DNA FROM BACTERIAL CULTURE 71
6.3.3 ISOLATION OF GENOMIC DNA 71
6.3.4 DETERMINATION OF DNA CONCENTRATION 71
6.3.5 POLYMERASE CHAIN REACTION (PCR) 71
6.3.6 CLONING USING RESTRICTION DIGESTION 72
6.3.7 LIGATION OF BLUNT-END AND DIGESTED PCR PRODUCTS 72
6.3.8 TA CLONING 72
6.3.9 GATEWAY CLONE 72
6.3.10 DNA GEL ELECTROPHORESIS 73
6.3.11 PURIFICATION OF PCR OR RESTRICTION DIGESTED DNA FRAGMENTS 73
6.3.12 DNA SEQUENCING 73
6.4 RNA TECHNIQUES 73
6.4.1 RNA ISOLATION 73
6.4.2 DETERMINATION OF RNA CONCENTRATION 74
6.4.3 RNA ANALYSIS ON DENATURING AGAROSE GEL ELECTROPHORESIS 74
6.4.4 REVERSE TRANSCRIPTION (RT) 74
6.4.5 QUANTITATIVE REAL TIME PCR (QRT-PCR) 74
6.5 PROTEIN TECHNIQUE 75
6.5.1 PREPARATION OF PROTEIN 75
6.5.2 IMMUNOBLOTTING 75
6.5.3 IMMUNOPRECIPITATION 76
TABLE OF CONTENTS
6.5.4 ENZYME ACTIVITY 77
6.5.5 P-GLUCURONIDASE (GUS) HISTOCHEMICAL STAINING 78
6.5.6 NBT (NITROBLUE TETRAZOLIUM) HISTOCHEMICAL STAINING 78
6.5.7 MICROSCOPY AND IMAGE ANALYSIS 78
6.6 PLANT TRANSFORMATION 79
6.6.1 TRANSIENT TRANSFORMATION IN TOBACCO 79
6.6.2 STABLE TRANSFORMATION IN ARABIDOPSIS 79
6.7 METABOLIC ANALYSIS 79
6.7.1 MEASUREMENT OF LEAF PIGMENTS 79
6.7.2 DETERMINATION OF SUGAR CONTENT BY HPLC 79
6.7.3 DETERMINATION OF STARCH CONTENT 80
6.8 SEQUENCES AND ANNOTATIONS 80
6.9 SUPPLEMENT FIGURES 82
7 LIST OF ABBREVIATIONS AND SYMBOLS 84
8 LITERATURE 86
9 ACKNOWLEDGEMENT 99
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| spellingShingle | Su, Tao Post-translational regulation of invertases molecular mechanism and impact on plant development in Arabidopsis thaliana |
| subject_GND | (DE-588)4113937-9 |
| title | Post-translational regulation of invertases molecular mechanism and impact on plant development in Arabidopsis thaliana |
| title_auth | Post-translational regulation of invertases molecular mechanism and impact on plant development in Arabidopsis thaliana |
| title_exact_search | Post-translational regulation of invertases molecular mechanism and impact on plant development in Arabidopsis thaliana |
| title_full | Post-translational regulation of invertases molecular mechanism and impact on plant development in Arabidopsis thaliana [presented by Tao Su] |
| title_fullStr | Post-translational regulation of invertases molecular mechanism and impact on plant development in Arabidopsis thaliana [presented by Tao Su] |
| title_full_unstemmed | Post-translational regulation of invertases molecular mechanism and impact on plant development in Arabidopsis thaliana [presented by Tao Su] |
| title_short | Post-translational regulation of invertases |
| title_sort | post translational regulation of invertases molecular mechanism and impact on plant development in arabidopsis thaliana |
| title_sub | molecular mechanism and impact on plant development in Arabidopsis thaliana |
| topic_facet | Hochschulschrift |
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