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Title: Regulation of carotenoid metabolism and accumulation in banana fruits: A proteomics approach
Other Titles: Regulatie van het caretenoiden metabolisme in banaan: een proteomics benadering
Authors: Amoako-Andoh, Francis
Issue Date: 7-Sep-2016
Abstract: Abstract
The deficiency of micronutrients such as vitamin A, iron, zinc and iodine is worldwide a major health problem. Biofortification is recommended as a sustainable, targeted and cost-effective approach to reducing micronutrient malnutrition, particularly in poorer communities, where people mostly depend on staple crops for micronutrients. Biofortification is “a process by which the nutritional quality of food crops is improved through agronomic practices, conventional plant breeding or modern biotechnology”.
It is estimated that vitamin A deficiency disorders (VAD) affect over 190 million preschool children, and close to 20 million pregnant women in over 118 countries. VAD are reported to be responsible for over 600,000 deaths annually. Bananas are widely produced and consumed in the regions where VAD are usually highly prevalent. Therefore banana varieties with high provitamin A carotenoids (pVACs) contents have a potential to help alleviate VAD in a sustainable manner. Carotenoid metabolism and accumulation underlie pVACs contents in plants. Very little is presently understood in bananas, although this knowledge is essential in developing the necessary molecular and biochemical tools to support breeding activities aimed at improving fruit pVACs contents. Thus the aim of the research presented in this dissertation was to generate deeper insights into the integrated mechanisms that regulate carotenoid metabolism and accumulation in banana fruits using proteomic data derived from high- and low-pVACs-containing banana varieties.
As a first objective, it was important to establish the most appropriate methodology for fruit proteomic analysis. Plant tissues often contain a range of substances that interfere with the extraction, stability and separation of proteins. Generally, to manage the interfering substances, plant proteins are precipitated from the crude protein extracts and then resolubilized for proteomic analysis. However, each basic step involved in plant proteome sample preparation: extraction, precipitation and resolubilization, has several procedures, which individually or collectively may influence the quality of the results of proteomic analysis. So a systematic evaluation and optimization of a total of 18 method combinations of widely-used protocols for protein extraction and cleanup involving three crude protein extraction procedures (SDS-based, Triton X-100-based, and phenol-based), three protein precipitation methods (ammonium acetate/methanol, TCA/acetone, and acetone) and two protein re-solubilization buffers (the classical Rabilloud and the so-called R2D2) was conducted. The method combinations were evaluated for protein yield, efficiency of extraction, the number of resolved spots as well as 2DE gel quality. The phenol-based extraction with its aqueous buffer component containing a neutral detergent (Triton X-100), followed by acetone precipitation of the extracted proteome and resolubilization in the R2D2 buffer - ‘tPAceR’ method, produced the best results. To provide a wider perspective for plant proteomic analysis, the optimized ‘tPAceR’ methodology was tested on ripe fruits of banana, apple, pear and strawberry as well as apple leafs.
There is significant variation in fruit pVACs contents of banana varieties, and although cultivars with high pVACs contents are relatively few, they are widely distributed across the different Musa genome groups. Therefore it was important that the proteomes of closely- and distantly-related banana cultivars with low and high pVACs contents were compared to identify proteins that are associated with carotenoid metabolism and accumulation and also to generate insights into the potential influence of the Musa genomes on the regulation. Reference banana varieties needed to be chosen to provide a broader representation of the Musa genomes. So as a second objective eight banana varieties representing subgroups of the genome groups: AAA (Gros Michel, Grande Naine and ‘Yangambi Km 5’); AAB (‘Batard’, ‘Iholena Iholena’ and Popoulou Cameroon-PCMR) - Eumusa section, and Fe’i (“Utin lap’ and ‘Karat’) - Australimusa section, were selected based on pVACs contents as well as economic and/or agronomic importance, and evaluated for 2DE profile. The results confirmed the anticipated variability in banana fruit proteomes, even for varieties that belong to the same genome group, and that for the extended study closely-related varieties should be selected from the same subgroup within an genome group. It was further revealed that the 2D-PAGE technique was more suitable for closely-related varieties, whereas the LC-MS/MS technique was appropriate for closely-related as well as distantly-related banana varieties.
As a final objective, comparative 2D-PAGE and LC-MS/MS analyses were conducted on fruit pulp proteomes of closely-related plantain cultivars (AAB plantain subgroup) - ‘Mbouroukou-3’ and ‘Batard’, and the distantly-related Fe’i cultivar - ‘Utin lap’ (Australimusa section), for the identification of proteins possibly associated with carotenoid metabolism and accumulation. The identified proteins indicated that the B1 and B6 vitamins are de novo biosynthesized alongside carotenoids in bananas, and that the pool of 1-deoxy-D-xylulose-5-phosphate (DXP) for the B1 and B6 vitamins biosynthesis and the production of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the plastid-localized methylerythritol 4-phosphate (MEP) pathway represent a metabolic link for carotenoid biosynthesis in banana fruits. Moreover, significant quantitative and structural differences in plastid-lipid-associated proteins (PAPs) were observed between the plantain cultivars (‘Mbouroukou-3’ and ‘Batard’) and ‘Utin lap’, and also for proteins of plastid-localized enzymes involved in fatty acid biosynthesis. Whereas proteins involved in the biosynthesis of short-chained as well as very long-chained, relatively more saturated plastidial fatty acid variants were identified in ‘Utin lap’, proteins responsible for relative less longer, (poly)unsaturated fatty acids predominated in ‘plantain cultivars. The differences suggested substantial variations in the morphology of carotenoid sequestration and storage structures between cultivars the plantain cultivars and ‘Utin lap’, and this may influence the types and quantity of carotenoids accumulating in plastoglobules of the respective banana cultivars. The abundance and plastid-localized lipoxygenase variants identified in the banana cultivars suggested that the loss of carotenoids to co-oxidation is also important in bananas, and that this may be highest in the ‘Utin lap’. For the plantain cultivars, ‘Mbouroukou-3’ is more likely to lose higher amounts of carotenoids compared to ‘Batard’. Moreover, considering that plastoglobule membranes of the plantain cultivars apparently contain significant amounts of (poly)unsaturated fatty acids, these cultivars may be relatively more susceptible to ROS mediated oxidation, with consequential co-oxidation of carotenoids that may lead to higher carotenoid losses relative to ‘Utin lap. Nonetheless, the rate of carotenoid losses may be influenced by activity of lipoxygenase variants expressed in banana cultivars.
This study has generated deeper insights into proteins, and thus genes linked to precursor supply for carotenoid biosynthesis, sequestration and storage as well as the fate of stored carotenoids in bananas. The implications are that factors that limit the precursor supply for carotenoid biosynthesis, the impact of nature of storage structures as well as metabolic activities leading to the degradation of carotenoids need be considered by breeding programs that are aimed at increasing carotenoids contents in bananas. However, further research is needed to validate the findings made about the various regulatory aspects for carotenoid metabolism and accumulation in banana fruits.
Table of Contents: Acknowledgements ....................................................................................................... i
Abstract .........................................................................................................................iv
Samenvatting ...............................................................................................................vii
Abbreviations ...............................................................................................................xi
Contents ...................................................................................................................... xix
Chapter 1 ........................................................................................................................ 1
General introduction and objectives ........................................................................... 1
1.1 Motivation ....................................................................................................................... 1
1.2 Potential of bananas (Musa spp.) to reduce vitamin A deficiency disorders .................... 2
1.3 Problem statement ......................................................................................................... 2
1.4 Objectives of the thesis .................................................................................................. 2
1.5 Outline of the thesis ........................................................................................................ 3
Chapter 2 ........................................................................................................................ 5
Literature review ........................................................................................................... 5
2.1 Provitamin A carotenoid and vitamin A deficiency disorders ........................................... 5
2.1.1 Importance of provitamin A carotenoids to humans ........................................................ 5
2.1.2 Significance of vitamin A deficiency disorders ................................................................ 6
2.1.3 Sources of vitamin A ....................................................................................................... 7
2.2 Potential of Musa spp. as principal source of pVACs ...................................................... 7
2.2.1 Musa species and genetics ............................................................................................ 8
2.2.2 Biofortification challenges ............................................................................................... 9
2.3 Determinants of pVACs content in plant storage organs ................................................. 9
2.4 Biosynthesis and regulation of carotenoids ....................................................................10
2.4.1 Carotenoid biosynthesis ................................................................................................10
2.4.2 Regulation of carotenoid biosynthesis ...........................................................................14
2.5 Carotenoid sequestration and its regulation ...................................................................19
2.5.1 Biogenesis and structure of chromoplasts .....................................................................20
2.5.2 Plastid-lipid-associated proteins ....................................................................................21
2.5.3 Lipid biosynthesis ..........................................................................................................21
2.5.4 The Or gene ..................................................................................................................22
2.6 Dynamics of carotenoids content at storage and the regulation .....................................23
2.6.1 Enzymatic cleavage and conversion of carotenoids.......................................................23
2.6.2 Ripening-associated breakdown of carotenoids .............................................................23
2.7 Other factors affecting carotenoid metabolism ...............................................................24
2.7.1 Co-factors for carotenoid biosynthesis ...........................................................................24
2.7.2 Phytohormones .............................................................................................................24
2.8 Proteomics as an investigative platform to study regulation of carotenoid metabolism and accumulation .............................................................................................................................25
2.8.1 The basic principles of proteomics .................................................................................26
2.8.2 Gel-based proteomics: two-dimensional gel electrophoresis .........................................26
2.8.3 Gel-free-based proteomics ............................................................................................34
2.9 Concluding remarks .......................................................................................................35
Chapter 3 ...................................................................................................................... 37
Materials and methods ................................................................................................37
3.1 Plant materials ...............................................................................................................37
3.1.1 Source of Musa fruits .....................................................................................................37
3.1.2 Sampling .......................................................................................................................37
3.2 pVACs identification and quantification in Musa fruit pulp ..............................................38
3.2.1 Extraction of carotenoids ...............................................................................................38
3.2.2 RP-HPLC analysis .........................................................................................................39
3.3 Gel-based proteomic analysis .......................................................................................40
3.3.1 Protein extraction ..........................................................................................................40
3.3.2 Sample preparation .......................................................................................................41
3.3.3 Protein quantitation........................................................................................................41
3.3.4 1D SDS-PAGE ..............................................................................................................41
3.3.5 Isoelectric focusing ........................................................................................................41
3.3.6 Equilibration ..................................................................................................................42
3.3.7 2D SDS-PAGE ..............................................................................................................42
3.3.8 Protein visualization and image acquisition ...................................................................43
3.3.9 Gel image analysis ........................................................................................................43
3.3.10 Statistical analysis .........................................................................................................43
3.3.11 Spot picking ...................................................................................................................44
3.3.12 Protein digestion ............................................................................................................44
3.3.13 Protein identification by MALDI-TOF MS/MS .................................................................45
3.4 Gel-free-based proteomic analysis ................................................................................46
3.4.1 Sample preparation .......................................................................................................46
3.4.2 Protein digestion and sample clean-up ..........................................................................46
3.4.3 Mass spectrometry analysis for gel-free protein identification ........................................46
3.4.4 Protein quantification .....................................................................................................48
Chapter 4 ...................................................................................................................... 49
Method optimization of fruit tissues for comparative proteomics studies of Musa cultivars .......................................................................................................................49
4.1 Introduction ...................................................................................................................49
4.2 Evaluation of protocols for optimizing banana proteomic studies ...................................50
4.2.1 Literature review ............................................................................................................50
4.2.2 Materials and methods ..................................................................................................51
4.2.3 Estimation of extraction buffer volume ...........................................................................51
4.2.4 Recovery efficiency for method combinations ................................................................54
4.2.5 2DE qualitative and quantitative attributes for protein extraction protocols ....................59
4.2.6 Optimization of the phenol/acetone/R2D2 protocol ........................................................62
4.2.7 Validation of optimized protocol for reproducibility .........................................................66
4.2.8 Evaluation of the ‘tPAceR’ methodology on ripe fruits ....................................................69
4.2.9 Concluding remarks .......................................................................................................71
Chapter 5 ...................................................................................................................... 73
2DE fruit proteome profiling of important banana cultivars from different Musa genome groups ...........................................................................................................73
5.1 Identification of ‘reference’ banana cultivars and appropriate techniques for comparative proteomic analysis ....................................................................................................................73
5.1.1 Introduction ...................................................................................................................73
5.1.2 Materials and methods ..................................................................................................75
5.1.3 Results and discussions ................................................................................................75
5.1.4 Concluding remarks .......................................................................................................80
5.2 Characteristic features of ‘reference’ banana cultivars ...................................................81
5.2.1 Genetics and morphology ..............................................................................................81
5.2.2 Provitamin A carotenoids content in ‘reference’ banana cultivars ..................................82
5.2.3 Conclusions ...................................................................................................................82
Chapter 6 ...................................................................................................................... 83
Proteomic analysis of closely- and distantly-related banana cultivars with low and high fruit pVACs content ............................................................................................83
6.1 Introduction ...................................................................................................................83
6.2 Literature review ............................................................................................................84
6.3 Materials and methods ..................................................................................................87
6.3.1 Materials and experimental design ................................................................................87
6.3.2 Methods ........................................................................................................................87
6.4 Results and discussion ..................................................................................................89
6.4.1 Carotenoid biosynthesis regulation ................................................................................95
6.4.2 Regulation of carotenoid sequestration and storage .................................................... 105
6.4.3 Regulation of carotenoid degradation .......................................................................... 113
6.4.4 Energy generation for carotenoid accumulation activities ............................................ 120
Chapter 7 .................................................................................................................... 135
General discussion, conclusions and perspectives ............................................... 135
7.1 General discussion and conclusions ............................................................................ 135
7.1.1 Introduction ................................................................................................................. 135
7.1.2 Method optimization for proteomic analysis ................................................................. 137
7.1.3 Identification of ‘reference’ banana varieties and appropriate techniques for comparative proteomic analysis .................................................................................................................. 138
7.1.4 Carotenoid metabolism and accumulation ................................................................... 139
7.2 Perspectives ................................................................................................................ 144
References ................................................................................................................. 149
Appendices ................................................................................................................ 169
APPENDIX I ............................................................................................................................ 169
The ‘tPAceR’ protein extraction methodology for proteomic analysis ...................................... 169
Crude protein extraction .......................................................................................................... 169
Protein purification .................................................................................................................. 171
Protein re-solubilization ........................................................................................................... 172
APPENDIX II ........................................................................................................................... 175
APPENDIX III .......................................................................................................................... 176
APPENDIX IV ......................................................................................................................... 177
APPENDIX V .......................................................................................................................... 182
APPENDIX VI ......................................................................................................................... 183
APPENDIX VII ........................................................................................................................ 184
APPENDIX VIII ....................................................................................................................... 185
List of publications .................................................................................................................. 186
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Division of Crop Biotechnics

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