published_or_final_version / abstract / Botany / Master / Master of Philosophy

Sze, Wing Ho Angel. / "December 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 90-112). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review / Chapter 2.1. --- Carotenoids in plants --- p.5 / Chapter 2.2 --- Carotenoid biosynthesis in plants --- p.7 / Chapter 2.3. --- Carotenoids in animals --- p.11 / Chapter 2.4. --- Vitamin A deficiency (VAD) --- p.13 / Chapter 2.5. --- Recommended requirement of vitamin A --- p.15 / Chapter 2.6. --- Bioavailability and bioconversion of dietary carotenoids --- p.17 / Chapter 2.7. --- Efforts to improve carotenoid contents in food crops --- p.19 / Chapter 2.8. --- Carotenoid catabolism --- p.20 / Chapter 2.9. --- Carotenoid cleavage dioxygenase (CCD) --- p.21 / Chapter 2.10. --- Carotenoid-derived phytohormones --- p.24 / Chapter 2.11. --- "CCD and carotenoid-derived colors, aromas and flavors" --- p.27 / Chapter Chapter 3. --- Hypothesis and Objectives --- p.35 / Chapter Chapter 4. --- Materials and methods / Chapter 4.1. --- General cloning and sequencing --- p.36 / Chapter 4.2. --- Extraction of RNA and DNase treatment --- p.36 / Chapter 4.3. --- Reverse transcription --- p.37 / Chapter 4.4. --- Real-time quantitative RT-PCR --- p.39 / Chapter 4.5. --- Cloning of OsCCD1 cDNA --- p.40 / Chapter 4.6. --- Bacterial in vivo assay of OsCCD 1 activity --- p.41 / Chapter 4.7. --- Construction of OsCCD1 RNAi constructs --- p.42 / Chapter 4.8. --- "Construction of ""Super-Golden"" rice constructs" --- p.46 / Chapter 4.8.1. --- "Construction of ""GluC-Y1-Nos"" cassette" --- p.46 / Chapter 4.8.2. --- "Construction of ""Gt1-TCN"" cassette" --- p.46 / Chapter 4.8.3. --- "Construction of""pGT-PCC""" --- p.47 / Chapter 4.8.4. --- "Construction of ""pGYGC""" --- p.47 / Chapter 4.9. --- Rice transformation --- p.54 / Chapter 4.10. --- Detection of transgene --- p.57 / Chapter 4.10.1. --- Southern blot --- p.57 / Chapter 4.10.2. --- HPLC analysis of carotenoids in seeds --- p.59 / Chapter Chapter 5. --- Results / Chapter 5.1. --- Expression profiles of carotenogenic genes in rice endosperms --- p.62 / Chapter 5.2. --- Expression of CCDs in developing rice seeds --- p.64 / Chapter 5.3. --- Features of OsCCD1 --- p.68 / Chapter 5.4. --- Characterization of OsCCD1-knock down transgenic rice --- p.72 / Chapter 5.5. --- "Construction of ""Super-Golden"" rice" --- p.78 / Chapter 5.6. --- Phenotypic characterization of PCC transgenic rice --- p.79 / Chapter 5.7. --- HPLC analysis on seed carotenoid content --- p.80 / Chapter Chapter 6. --- Discussion --- p.82 / Chapter Chapter 7. --- Conclusion --- p.89 / References --- p.90

Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2007. / Plants are constantly exposed to adverse environmental conditions including variations in light intensity and the availability of water resources. These abiotic factors are expected to worsen as the changing global climate places additional daily and seasonal demands on plant growth and productivity. As plants are incapable of avoiding stress they have developed a number of mechanisms to manage and adapt to the unfavourable conditions. Carotenoids represent one of these mechanisms; with the xanthophylls (oxygenated carotenes) playing an essential role in photoprotection following exposure to excess light energy. They are also precursors to the plant hormone abscisic acid (ABA) which plays a known role in stomatal regulation and thus drought tolerance. Carotenoids have been identified as potential targets for genetic manipulation to meet the existing nutritional demands (particularly vitamin A) and to enable plants to survive the climatic variations predicted. Thorough investigations into the regulation and functioning of each carotenoid biosynthetic gene in vivo as well as the roles of their encoded proteins are prerequisite. Within the Grapevine Biotechnology Programme, a number of isoprenoid biosynthetic genes have been isolated from Vitis vinifera L. cv. Pinotage. From this vast resource two genes were chosen; namely a lycopene b-cyclase (b-LCY) and 9-cis epoxycarotenoid dioxygenase (NCED) for detailed in planta analyses to address knowledge gaps in our current understanding of carotenoid biosynthesis in general, its regulation and the roles of the two target genes in these processes. Currently, the role of b-LCY within the chloroplasts is not well known. Although the relationship between NCED overexpression, ABA levels, reduced stomatal conductance and increased tolerance to water stress has been well-established, comprehensive physiological analysis of the resulting mutants during conditions of both water availability and shortage is not well documented. To assess their in planta role, functional copies of both genes were isolated from Vitis vinifera (cv. Pinotage), characterised and independently transformed into the genome of the model plant, Arabidopsis thaliana, in the sense orientation under a constitutive promoter. In order to investigate these pertinent scientific questions and thus to evaluate the physiological role of each gene in vivo, a number of technologies were developed and/or adopted. These included a high-performance liquid chromatography method for profiling the major plant pigments in leaf tissue, a combination vapour phase extraction and electron impact-gas chromatography/mass spectrometry method for the phytohormone profiling as well as various physiological analyses including the use of chlorophyll a fluorescence to assess the photosynthetic and non-photochemical quenching (NPQ) capacities of the plants. Overexpression of grapevine b-LCY (Vvb-LCY) decreased lutein levels due to preferential partitioning of lycopene into the b-branch. This decrease was not met by an increase in either b-carotene or the xanthophyll cycle pigments implying that Vvb-LCY is not able to regulate the flow of carbon through the pathway and provides additional evidence to the fluidity of this pathway whereby pigment levels are continually balanced. The decreased lutein levels observed under low light (LL) did not compromise the plants’ ability to induce and maintain NPQ over a wide actinic light range. Vvb-LCY transgenics also had lower neoxanthin levels (and specifically the cis-isomer) under both LL and following exposure to high light (HL), which could be correlated to an increase in malondialdehyde. Although not corroborated, a novel and unexpected finding was an essential role for neoxanthin, and potentially lutein, in preventing or at least reducing lipid peroxidation under HL stress. The lower neoxanthin amounts may be due to silencing of the Arabidopsis b-LCY by the Vvb-LCY, as the former may function as a NSY paralog as NSY is not encoded for in the Arabidopsis genome. Clearly, this study has confirmed that Vvb-LCY partitions the carbon flux between the a- and b-branches, however, the catalytic action of this enzyme is dependent on the amount of substrate available and is thus not a regulatory step directing the flux within the pathway. Enzyme kinetic and detailed transcriptional analyses would confirm the above findings. Overexpression of grapevine NCED1 (VvNCED1) increased ABA concentrations, delayed seed germination and resulted in a slight to severe reduction in the overall plant growth rate. NCED cleaves the 9-cis xanthophylls regulating ABA synthesis. However, contrary to expectations, constitutive levels of this regulatory enzyme did not deplete the total and individual chlorophylls and carotenoids in well-watered plants. Instead the VvNCED1 transgenics simply exhibited a lower chloroplastic pigment complement with no concomitant effects on their photosynthetic capacity. Of particular interest, well-watered plants overexpressing the VvNCED1 gene had an increased NPQ capacity of which the thermal energy dissipation component (qE) was the most significant. It has been speculated that this NPQ is associated with the phenotype conferred by VvNCED1 overexpression and occurs independently of the xanthophyll cycle, and specifically zeaxanthin. This study confirmed that VvNCED1 functions during drought tolerance via ABA regulation of stomatal conductance. A detailed study was done to understand the plants’ response during water deficit. Typically, decreases in total and individual carotenoids and the maximum efficiency of photochemistry (Fv/Fm) as well as the relative water and soil moisture content were recorded. No changes were recorded in salicylic acid (SA) levels, while indole acetic acid (IAA) was positively correlated to ABA or vice versa. In contrast, the physiology of VvNCED1 overexpressing lines was largely unaffected, indicating that a reduced stomatal conductance protects the plants against water stress. This study has resulted in the isolation and characterisation of a carotenoid biosynthetic gene (b-LCY) and an abscisic acid synthesising gene (NCED). Significant advancements in our existing knowledge of the in planta role of both genes have been achieved. We have also reaffirmed that strict regulatory control and fluidity exists within the carotenoid biosynthetic pathway whereby individual pigment levels are constantly brought back into balance despite constitutive expression of one of the pathway gene members. These analyses provide valuable baseline information about individual genes which can be extended upon with other omic technologies in order to comprehend the full complexity involved in carotenogenesis.

Thesis (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Plants cannot avoid stress and must therefore be capable of rapidly responding to extreme environmental changes. An inability to control and regulate the photosynthetic process during stress conditions will lead to the formation of highly reactive oxygen species that concomitantly causes photo-oxidative damage to the pigments and proteins of the photosynthetic apparatus. Since light is the primary source of energy for the photosynthetic process, it is clear that plants are continuously required to balance the light energy absorbed for the photochemical reactions against photoprotection in a dynamic way in order to survive. Carotenoids are precursors of abscisic acid, but more importantly structural components of the photosynthetic apparatus. During photosynthesis carotenoids function as accessory light-harvesting pigments, and also fulfil a photoprotective function by quenching the reactive molecules formed during conditions that saturate the photosynthetic process. Due to the importance of carotenoids to plant fitness and human health (as Vitamin A precursors) this study has attempted to isolate and characterise genes that are directly, or indirectly involved in carotenoid biosynthesis in Vitis vinifera. In total eleven full-Iength- and eight partial genes have been isolated, cloned and sequenced. These genes can be grouped into the following pathways: (i) the 1- deoxy-D-xylulose 5-phosphate (DOXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (i.e. the plastidic isopentenyl diphosphate biosynthetic pathway); (ii) the mevalonate pathway (i.e. the cytosolic/mitochondrial IPP biosynthetic pathway); (iii) the carotenoid biosynthetic pathway; (iv) the abscisic acid biosynthetic pathway (as a degradation product of carotenoids); and general isoprenoid biosynthetic pathways (as precursors of carotenoids). The full-length genes (i.e. from the putative ATG to the STOP codon) of DOXP synthase (DXS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), IPP isomerase (IPI), 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), phytoene synthase (PSY), Iycopene ~-cyclase (LBCY), ~-carotene hydroxylase (BCH), zeaxanthin epoxidase (lEP), 9-cis-epoxy carotenoid dioxygenase (NCED), farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) have been isolated from cDNA. In addition, the full-length genomic copy and putative promoters of DXS, PSY, LBCY, BCH, NCED and lEP have also been isolated from genomic DNA by the construction and screening of sub-genomic libraries. Alignments of the genomic copies of these genes to the corresponding cDNA sequences have provided useful information regarding the genomic organisation of these genes, including the intron-exon junction sites in V. vinifera. The copy number of the DXS, PSY, LBCY, BCH, NCED and lEP encoding genes in the Vitis genome have been determined. DXS, PSY, BCH and lEP are single copy genes, whereas LBCY and NCED have two and three copies, respectively. The transcriptional activity of the putative promoters of six of the isolated genes (i.e. DXS, PSY, LBCY, BCH, lEP and NCED) were tested with a transient reporter gene assay. None of the putative promoters tested showed any transcriptional activity of the reporter gene. The transcription of these genes, has however been shown using northern blot analysis and/or RT-PCR. Preliminary expression profiles for PSY, LBCY, BCH, and lEP were determined in different plant organs and the expression of these genes was generally higher in photosynthetically active tissues. The expression of these genes following different treatments (abscisic acid, NaCI and wounding) was also assayed. The functionality of five of the isolated full-length genes (IPI, GGPS, PSY, LBCY and BCH) has been shown in a bacterial colour complementation assay. In silica analysis of the predicted protein sequences of all eleven isolated genes revealed that they are conserved and share a high degree of homology to the corresponding proteins in other plant species. The sequences were further analysed for conserved domains in the protein sequences, and these proteins typically demonstrated similar domain profiles to homologues in other species (plant, bacteria and algae). The predicted protein sequences were further analysed for transit peptides, the presence of which would provide evidence for the sub-cellular localisation of the mature peptides. Since these genes are involved in biosynthetic pathways that are active in discrete organelles, the sub-cellular localisation of most of these proteins is known. The carotenoid biosynthetic genes (PSY, LBCY, BCH and ZEP), the abscisic acid biosynthetic gene, NCED, as well as the DOXP/MEP pathway genes (DXS, lytB and IPI) were all localised to the chloroplast. The mevalonate pathway gene, HMGS, was localised to both the cytosol and the mitochondria, and the general isoprenoid precursor genes, FPS and GGPS, were localised to the cytosol and the chloroplast, respectively. All these results are in agreement with the localisation of the respective pathways. In order to increase our understanding of carotenoid biosynthesis and functions in plants, we constitutively overexpressed one of the isolated genes (BCH) in the model plant, Nicotiana tabacum. Plants expressing the BCH gene in the sense orientation maintained a healthy photosynthetic rate under stress conditions that typically caused photoinhibition and photodamage in the untransformed control plants. This result was inferred using chlorophyll fluorescence and confirmed using CO2 assimilation rates and stomatal conductance. Chlorophyll fluorescence measurements indicated that the photo protective non-photochemical quenching ability of the BCH-expressing plants increased, enabling the plants to maintain photosynthesis under conditions that elicited a stress response in the untransformed control plants. An integral photosynthetic protein component, the D1 protein, was specifically protected by the additional zeaxanthin in the BCH sense plants. Plants expressing an antisense BCH proved the converse, i.e. lower levels of BCH resulted in decreased zeaxanthin levels and made the transgenic plants more susceptible to high-light induced stress. These results have shown the crucial role of carotenoids (specifically the xanthophylls) in the photoprotective mechanism in plants. The increased photoprotection provided by the BCH expressing plants suggests that the scenario in plants is not optimal and can be improved. Any improvement in the photoprotective ability of a plant will affect both the fitness and productivity of the plant as a whole and will therefore find application in a number of crop plants on a global scale. This study has resulted in the successful isolation and characterisation of genes involved in the direct, or indirect, carotenoid biosynthetic pathways. The further study and manipulation of these genes in model plants will provide useful insights into the physiological role of specific carotenoids in photosynthesis and in plants as a whole. / AFRIKAANSE OPSOMMING: Plante het nie die vermoë om stres te ontwyk nie en moet dus vinnig op veranderinge in hulomgewingstoestande kan reageer. Indien hulle nie die fotosinteseproses kan kontroleer en reguleer tydens streskondisies nie, sal dit tot die vorming van hoogs reaktiewe suurstofspesies lei, wat beide die pigmente en proteiene van die fotosintetiese apparaat sal beskadig. Lig is die primêre energiebron vir fotosintese en daarom is dit noodsaaklik dat plante deurgaans 'n dinamiese balans tussen fotosintese en fotobeskerming moet handhaaf. Karotenoiëde is voorlopers vir die vorming van absisiensuur, maar meer belangrik vir die plant, ook integrale komponente van die fotosintetiese apparaat. Tydens fotosintese word karotenoiëde vir die opneem van lig benodig, terwyl dit ook die fotosintetiese apparaat beskerm wanneer lig 'n versadigingspunt bereik vir fotosintese. Weens die belang van karotenoiëde vir plant- en menslike gesondheid (as Vitamiene A voorlopers), het hierdie studie beoog om gene te isoleer en karakteriseer wat direk of indirek 'n rol in karoteenbiosintese in Vitis vinifera speel. Elf vollengte- en agt gedeeltelike gene is geïsoleer, gekloneer, en gekarakteriseer. Hierdie gene kan in die volgende biosintetiese paaie gegroepeer word: (i) die 1- deoksi-D-xilulose 5-fosfaat (DOXP)/2-C-metiel-D-eritritol-4-fosfaat (MEP) pad (d.w.s. die plastiediese isopenteniel difosfaat biosintetiese pad); (ii) die mevalonaat pad (d.w.s. the sitosoliese/mitokondriale IPP biosintetiese pad); (iii) die karotenoiëd biosintetiese pad; (iv) die absisiensuur biosintetiese pad (as 'n afbraak produk van karotenoiëde) en die algemene isoprenoïed bisintetiese paaie (as voorlopers van karotenoiëde ). Die vollengte gene (d.w.s. vanaf die geskatte ATG tot die STOP kodon) van DOXP-sintase (DXS), 4-hidroksi-3-metielbut-2-eniel difosfaatreduktase (lytB), IPPisomerase (IPI), 3-hidroksi-3-metielglutariel koensiem A sintase (HMGS), fitoeën sintase (PSY), likopeen p-siklase (LBCY), p-karoteen hidroksilase (BCH), zeaxantien oksidase (ZEP), 9-cis-epoksi karotenoiëd dioksigenase (NCED), farnesiel difosfaat sintase (FPS)en geranielgeraniel difosfaat sintase (GGPS) is met behulp van. RTPKR vanaf eDNA geïsoleer. Die vollengte genomiese kopieë en die verwagte promotors van die DXS, PSY, LBCY, BCH, NCED and ZEP gene is ook geïsoleer d.m.v. die opstel en sifting van subgenomiese biblioteke. Vergelykende analises van die genoom- en eDNA kopieë het insiggewende data oor die genomiese rangskikking van die gene, insluitende die intron-ekson setels in V. vinifera gelewer. Die kopiegetalle van DXS, PSY, LBCY, BCH, NCED en ZEP is bepaal. DXS, PSY, BCH en ZEP is in die Vitis-genoom as enkel kopieë teenwoordig, terwyl LBCYen NCED twee en drie kopieë, repektiewelik, beslaan. Die transkipsionele aktiwiteit van die verwagte promotors van ses van die geïsoleerde gene (naamlik DXS, PSY, LBCY, BCH, ZEP en NCED) is d.m.v. 'n tydelike verklikkergeentoets ondersoek. Geeneen van die promotors het die transkripsie van die verklikkergeen bemiddel nie. Die transkripsie van die gene is egter wel bewys deur van northernhibridisasies en/of RT-PKR gebruik te maak. Die promotors van hierdie gene kan dus as transkipsioneel aktief beskou word. Voorlopige uitdrukkingsprofiele van PSY, LBCY, BCH, en ZEP is in verskillende plantorgane bepaal; die profiele was deurgaans hoër in fotosinteties aktiewe weefsels. Die uitdrukkingsprofiele van die gene is verder ook in reaksie op verskillende induktiewe behandelings (absisiensuur, NaCI en beskadiging) bepaal. Vyf van die vollengte gene (IPI, GGPS, PSY, LBCYen BCH) is funksioneel bewys in 'n bakteriese funksionele kleurkomplementasiesisteem. In silico analises van die afgeleide proteïene van al elf geïsoleerde gene het 'n hoë vlak van homologie met ooreenstemende proteiene van ander plantspesies getoon. Gekonserveerde domeine is ook in die proteïensekwense van die geïsoleerde gene teenwoordig. Hierdie proteïene het deurgaans dieselfde domeinprofiele vertoontoon as homoloë in ander spesies (bakterieë, alge en plante). Die sub-sellulêre teikening van die gene kon voorspel word deur die seinpeptiede in die proteiensekwense te eien. Aangesien hierdie gene betrokke is by biosintetiese paaie wat in diskrete kompartemente plaasvind; is die sub-selluiêre lokalisering van hierdie proteïene voorspelbaar. Die karotenoïed biosintetiese gene (PSY, LBCY, BCH en ZEP), die absisiensuur biosintetiese geen, NCED, sowel as die DOXP/MEP pad se gene (DXS, lytB en IPI) kom almal in die chloroplast voor. Die mevalonaatpadgeen, HMGS, word na beide die sitosol en die mitokondria geteiken, terwyl die algemene isoprenoïed voorlopergene, FPS en GGPS, onderskeidelik na die sitosol en die chloroplast geteiken word. Die verkreë voorspellings stem met die lokalisering van die biosintetiese paaie in die selooreen. Om ons kennis rakende karotenoïed biosintese en veral hulle funksie(s) in plante te verbreed, het ons een van die geïsoleerde gene, BCH, in die model plant, Nicotiana tabacum, konstitutief ooruitgedruk. Plante wat die BCH geen in die "sense" orientasie uitgedruk het, kon normale fotosintetiese aktiwiteit handhaaf onder kondisies wat foto-inhibisie en foto-osidatiewe skade in die ongetransformeerde kontrole plante veroorsaak het. Hierdie resultaat is met chlorofil fluoresensie analises aangetoon terwyl dit met CO2 assimilasie- en huidmondjie geleidingseksperimente bevestig is. Chlorofil fluoresensie metings het aangetoon dat die beskermingsvermoë van die transgeniese plante verhoog is, en dit dan die plante in staat stelom fotosintetese te handhaaf onder streskondisies van hoë lig. Proteïen analises het aangetoon dat 'n integrale fotosintetiese proteien, die 01 proteïen, word veral deur die verhoogde zeaxantien vlakke in die BCH transgeniese plante beskerm. Plante wat verminderde zeaxantien vlakke gehad het, weens die konstitutiewe ooruitdrukking van die BCH geen in die anti-"sense" orientasie, het die teenoorgestelde bewys. Met ander woorde. laer BCH vlakke (en dus laer zeaxantien vlakke) het tot plante wat meer vatbaar was vir hoë lig geïnduseerde stress gelei. Hierdie resultate het die essensiële beskermende rol wat karotenoiede tydens fotosintese speel, uitgelig. Die vermoë om hierdie beskermende meganisme te manipuleer in transgenies plante het aangetoon dat die sisteem in plante, alhoewel effektief, nie optimaal is nie. Enige verbetering in 'n plant se inherente vermoë om streskondisies te weerstaan sal die plant se algemene gesondheid en dus produktiwiteit beïnvloed. As sulks sal hierdie in meeste gewasspesies toepassing vind. Hierdie studie beskryf die isolering en karakterisering van gene wat direk, of indirek, by karotenoïedbiosintese betrokke is. Verdere studies, en veral die manipulering van hierdie gene in model plante, sal die fisiologiese rol van spesifieke karotenoïeede in fotosintese, en die plant as 'n geheel, ontrafel.