Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Plant carotenoid cleavage dioxygenases (CCD) are a family of enzymes that catalyse the oxidative cleavage of carotenoids and/or apocarotenoids. Carotenoids are synthesised in plastids (primarily chloroplasts and chromoplasts), where they are involved in light-harvesting and protecting the photosynthetic apparatus from photo-oxidation. The carotenoid-derived apocarotenoids fulfil a number of roles in plants such as phytohormones, pollinator attractants and flavour and aroma compounds. Due to the floral and fruity characteristics that apocarotenoids contribute to wine, these C13 compounds have received interest in grapevine (Vitis vinifera L.). The CCD gene family in Arabidopsis consists of nine members, all encoding for enzymes that catalyse the cleavage of carotenoids. The enzymes in this family include 9-cis-epoxydioxygenases (NCEDs) and four classes of CCD. NCEDs and CCD7 and CCD8 are involved with plant hormone synthesis, e.g. abscisic acid (ABA) through cleavage by NCED and strigolactone (SL) through the sequential cleavage of carotenoids by CCD7 and CCD8, respectively. SLs are a fairly new class of plant hormone which are involved in several aspects of plant growth and development. The most extensively characterised role of SLs is their involvement in the inhibition of shoot-branching. CCD1 and CCD4 cleave a variety of carotenoids to form pigments and aroma compounds. For example, CCD1 forms β-ionone and β-damascenone, which are important varietal flavours of wine, and CCD4 is involved in synthesis of the pigment and aroma compounds of saffron and annatto. CCD1 enzymes symmetrically cleave the 9,10 (9’,10’) double bonds of multiple carotenoids to produce a C14 dialdehyde and two C13 products. Additional CCD1 cleavage activity at 5,6 (5’,6’) double bonds of lycopene has been reported. Previous studies have shown that CCD1 isolated from V. vinifera (VvCCD1) was able to cleave multiple carotenoid substrates in vitro, namely zeaxanthin, lutein and β-carotene at 9,10 (9’,10’) double bonds and both the 5,6 (5’,6’) and 9,10 (9’,10’) double bonds of lycopene. None of the other VvCCDs, except VvCCD4a have been isolated (but no functionality was illustrated) and characterised yet. CCD4 enzymes also cleave carotenoids at the 9,10 (9’,10’) double bond positions. The presence of plastid-target peptides implies that the CCD4 enzymes have continuous access to carotenoids. Therefore it is suggested that CCD4s are responsible for carotenoid maintenance, where CCD1s contribute towards volatile production. To test this hypothesis VvCCD1, VvCCD4a and VvCCD4b were isolated from V. vinifera (cv Pinotage) cDNA and cloned into a pTWIN1 protein expression vector. Substrate specificity of each VvCCD was tested by co-transforming a carotenoid accumulating E. coli strain with a CCD expression vector. Carotenoids synthesized by the bacteria were identified and quantified by UPLC-analysis, while the concentration of the apocarotenoids, were measured in the headspace of the bacterial cultures using HS-SPME-GC-MS. Several optimisations were done to minimize the natural degradation of the carotenoids; to ensure that the apocarotenoid formation is predominantly due to the enzymatic cleavage by the VvCCDs and not due to oxidation or other non-enzymatic degradation. The HS-SPME-GC-MS analysis indicated that all isoforms cleaved phytoene, lycopene and ε-carotene. Additionally VvCCD1 cleaved a carotenoid involved in photosynthesis, namely β-carotene, while VvCCD4a cleaves neurosporene and VvCCD4b cleaves neurosporene and ζ-carotene, carotenoids not involved in photosynthesis. This study has illustrated that VvCCD1 cleave carotenoids necessary for photosynthesis and VvCCD4s cleave carotenoids which were not present in berry tissue, suggesting their role in carotenoid maintenance. Therefore in planta substrates for CCD1 could possibly be C27 apocarotenoids generated from enzymatic cleavage through CCD4 (role in carotenoid maintenance), CCD7 and/or photo-oxidation, which are then transported from the plastid to the cytosol or possibly C40 carotenoids that are released during senescence or when the plastid membrane is damaged, thus releasing important aroma compounds. Thus the identification of the in vivo substrates has contributed to the understanding the in planta functions of these enzymes / AFRIKAANSE OPSOMMING: Die plant ensiemfamilie van karotenoïedsplitsingdioksigenases (CCDs) kataliseer die oksidatiewe splitsing van karotenoïede en/of apokarotenoïede. Karotenoïede word in plastiede (primêr chloroplaste en chromoplaste) sintetiseer en is betrokke by lig-absorpsie en die beskerming van die fotosintetiese apparaat teen foto-oksidasie. Die apokarotenïede afkomstig van karotenoïede dien onder meer as planthormone, geur- en aromakomponente en om bestuiwers aan te lok. Aangesien apokarotenoïede bydra tot die vrug- en blomgeure van wyn is die C13-verbindings binne wingerd (Vitis vinifera L.) van belang. Al nege lede van die CCD geenfamilie in Arabidopsis kodeer karotenoïedsplitsingsensieme. Die ensiemfamilie sluit 9-sis-epoksidioksigenases (NCEDs), en vier klasse CCD in. NCEDs en CCD7 en 8 is betrokke by die sintese van planthormone, naamlik absissiensuur (ABA) deur NCED en strigolaktone (SL) deur die opeenvolgende aksie van onderskeidelik CCD7 en CCD8. SLe is redelik onlangs as planthormone indentifiseer en is betrokke by ‘n verskeie aspekte van die groei en ontwikkeling van plante. Die rol van SL in inhibisie van vertakking is die beste gekarakteriseerde van hierdie aspekte. CCD1 en CCD4 splits ‘n verskeidenheid karotenoïede om pigmente en aromakomponente te vorm. CCD1 vorm byvoorbeeld β-jonoon en β-damasenoon, beide belangrike kultivar-spesifieke wyngeure. CCD4 vorm weer die pigment en aromakomponente van saffraan en annatto. Die CCD1 ensieme splits die 9,10 (9’,10’) dubbelbindingsetels van verskeie karotenoïede simmetries en vorm een C14-dialdehied en twee C13-produkte. Daar is voorheen melding gemaak van verdere splitsing deur CCD1 by die 5,6 (5’,6’) dubbelbindingsetels van likopeen. Vroeër is getoon dat die CCD1 isovorm wat uit V. vinifera geïsoleer is, naamlik VvCCD1, in vitro seaxantin, luteïen en β-karoteen by die 9,10 (9’,10’) dubbelbindingsetels kon splits, en likopeen by beide die 9,10 (9’,10’) en 5,6 (5’,6’) dubbelbindingsetels. Geen ander VvCCDs is al isoleer en funksioneel gekarakteriseer. VvCCD4a is isoleer, maar geen funksie is bepaal nie. CCD4 ensieme splits ook die 9,10 (9’,10’) dubbelbindingsetels van karotenoïede. Aangesien CCD4 ensieme ‘n plastied-bestemmingspeptied besit behoort dié ensieme konstant toegang tot karotenoïede te hê, wat dui op hul rol in die handhawing van die karotenoïedbalans, terwyl CCD1-ensieme bydra tot die sintese van vlugtige verbindings. Om hierdie hipotese te toets is VvCCD1, VvCCD4a en VvCCD4b uit V. vinifera (kv Pinotage) kDNS isoleer in binne ‘n pTWIN1 proteïenuitdrukkingsvektor kloneer. Die substraatspesifisiteit van elke VvCCD is getoets deur ‘n karotenoïedakkumulerende E. coil stam te transvormeer met ‘n CCD-uitdrukkingsvektor. UPLC-analise is gebruik om karotenoïede wat deur die bakterium sintetiseer is te kwantifiseer en identifiseer, terwyl die apokarotenoïedinhoud en -konsentrasie van die boruimte van die bakteriële kultuur met HS-SPME-GC-MS bepaal is. Verskeie aspekte van die proses is optimaliseer om natuurlike afbreking van karotenoïede te minimeer. Daardeur is verseker dat die apokarotenoïedvorming primêr vanweë die ensiematiese splitsing deur VvCCDs plaasvind en nie deur oksidasie of ander nie-ensiematiese afbreking. Die HS-SPME-GC-MS metings het aangedui dat al drie isovorme fitoëen, likopeen en ε-karoteen kan splits. VvCCD1 kan daarby β-karoteen splits, terwyl VvCCD4a neurosporeen, en VvCCD4b neurosporeen en ζ-karoteen kan splits, beide karotene wat nie betrokke is by fotosintese nie. Dié studie toon dat VvCCD1 die karotenoïede splits wat benodig word vir fotosintese, terwyl beide VvCCD4 isovorme karotenoïede splits wat nie in druiwekorrels gevind word nie. Dit dui op hulle rol in die handhawing van karotenoïedpoele. Die in planta substrate vir CCD1 mag dus die C27-apokarotenoïede wees wat deur CCD4 (as deel van karotenoïedhandhawing), CCD7 en/of foto-oksidasie gevorm word en na die sitosol vervoer word, of moontlik die C40-karotenoïede wat tydens veroudering óf wanner die plastiedmembraan beskadig is in die sitosol vrygestel word. Die identifisering van die in vivo substrate het dus bygedra to die begrip van die in planta funksies van die ensieme.