Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Previous research has shown that carotenoids are precursors of C13-norisoprenoid aroma compounds in wine. C13-norisoprenoids have low threshold values in wine with the most prominent C13-norisoprenoids being β-damascanone and β-ionone which contribute honey and floral like aroma to wine. Chlorophyll and its derivates have also been detected in wine with potential to be precursors to aroma compounds. Apart from the contribution of these pigments to wine aroma and quality they are vital role players in photosynthesis and are widely found in plants and plant products. The main functions of these pigments in plants are light collection and light-protection. Research has shown that environmental conditions, climate, light exposure of bunches and soil water deficit influence the carotenoid content of grape berries. Furthermore the concentration of carotenoids and chlorophylls has also been shown to differ between cultivars. No research in this regard has been done on Merlot grape berries. With this in mind, the aim of this study was to evaluate the effect of vigour and soil water content on the evolution of carotenoids and chlorophylls through ripening of grape berries from the cv. Merlot. However, when looking at methods to analyse carotenoids and chlorophylls in berry tissue, especially lyophilised tissue, there were no readily available methods. Thus, an extraction method to identify and quantify the carotenoid and chlorophyll profile of lyophilised tissue from unripe (green) to ripe (red) Merlot grape berries was needed. In this study the RPHPLC method of Taylor et al. (2006) for carotenoids and the extraction method of Mendes-Pinto et al. (2004) were adapted to analyse both carotenoids and chlorophylls in lyophilised grape tissue. The RP-HPLC method baseline separated all the carotenoids and chlorophylls and their derivatives. Recovery of standards from mock extractions was high, indicating that the extraction procedure was acceptable. However, extraction recovery tested in the matrix of the grape tissue showed less promising results due to the high acid content of grape tissue. Violaxanthin, neoxanthin and the chlorophylls were especially sensitive to low pH conditions which facilitated their degradation. The degradation products of these compounds under acidic conditions were identified as pheophytin a, b, chlorophillide a, pyropheophytin b, cisviolaxanthin, cis-neoxanthin, neochrome, mutatoxanthin and luteoxanthin. There is a possibility that some degradation products were already present in the tissue due to lyophilisation (since the water in the berry was then removed and the acid concentrated). More work is needed to investigate the effect of lyophilisation and storage on the composition of grape tissue of different maturity. The extraction method for grape berry tissue at different ripening stages should also be optimised further to effectively neutralise tissue acidity, without compromising the extraction of carotenoids significantly, in especially green berry tissue. The question as to whether cisisomers and chlorophyll degradation products are naturally present in grape berries or are formed during sampling and processing remains unanswered in the current study. This study confirmed that in general carotenoids and chlorophylls decrease on a per berry (μg/berry) and concentration (μg/g) basis from veraison to harvest. Furthermore, this study was inconclusive in showing that vigour differences have an effect on the rate of synthesis/degradation of carotenoids, chlorophyll and some other ripening parameters, namely malic acid, total glucose and fructose, total tannin and total anthocyanin, from pre-veraison (pea size) to harvest. Additionally, no significant effect of soil water content on carotenoids, chlorophylls and ripeness parameters was found in this study, most likely due the fact that high soil water capacity was found in lower soil layers which may have prevented significant differences in grapevine water status. Experimental plots selected for vigour differences based on normalised difference vegetation index (NDVI) images, pruning mass and soil water measurements by means of a neutron probe, showed significant differences in soil water content in only the first 30 cm of the soil for the ripening seasons studied. Predawn plant water potential measurements, however, indicated that none of the experimental vines experienced severe water stress which was previously shown to effect carotenoid content of grapes. The carotenoid 5,8-epoxy--carotene was quantified for the first time in grapes and represents a significant amount of the total carotenoids present at harvest. All the carotenoids and chlorophylls except -carotene appeared to be sensitive to seasonal variation in climatic conditions. Lutein and β-carotene were found to be the most abundant carotenoids present in Merlot grape berries together with chlorophyll a for both seasons studied. The values of these carotenoids also correlated well with previous research. However, chlorophyll a was found in much larger quantities in Merlot berries compared to reported data. This is possibly because in this study the chlorophyll degradation products were included in the calculation of chlorophyll a. Multivariate analysis showed promising preliminary prediction models (with correlation values of above 0.8 for both seasons analysed) for the prediction of the concentration of ripeness parameters (glucose, fructose, malic acid, total tannins and anthocyanins) with carotenoid and chlorophyll content. This result highlights the opportunity for the development of a rapid non-destructive method to measure carotenoids and chlorophylls in berries which in turn can predict optimal ripeness. Furthermore, since carotenoids are the precursors to C13- norisoprenoid aroma compounds in wine a preview of the potential contribution of these aromas to wine might be evaluated. Further research is necessary to investigate the possibility of building and validating such models. / AFRIKAANSE OPSOMMING: Vorige navorsing het getoon dat karotenoïede die voorlopers is van C13-norisoprenoïed aromaverbindings in wyn. C13-norisoprenoïede het lae drempelwaardes in wyn, met β- damassenoon en β-jonoon as die prominentste C13-norisoprenoïede wat ‘n bydrae tot die heuning en blomagtige aroma van die wyn maak. Chlorofil en sy derivate is ook reeds in wyn bespeur, met die potensiaal om voorlopers van aromaverbindings te wees. Buiten die bydrae van hierdie pigmente tot wynaroma en -kwaliteit is hulle ook belangrike rolspelers in fotosintese en kom hulle wydverspreid in plante en plantprodukte voor. Die vernaamste funksies van hierdie pigmente in plante is om lig te versamel en om as beskerming teen lig op te tree. Navorsing het getoon dat omgewingstoestande, klimaat, ligblootstelling van die trosse en grondwatertekorte die karotenoïedinhoud van druiwekorrels beïnvloed. Verder is ook getoon dat die konsentrasie van karotenoïede en chlorofille tussen kultivars verskil. Geen navorsing is al in hierdie opsig op Merlot-druiwekorrels gedoen nie. Met hierdie aspek in gedagte was die doelwit van hierdie studie om die effek van groeikrag en grondwaterinhoud op die evolusie van karotenoïede en chlorofille tydens die rypwording van druiwekorrels van die cv. Merlot te evalueer. Wanneer mens egter kyk na die metodes waarvolgens die karotenoïede en chlorofille in korrelweefsel geanaliseer word, is daar geen geredelik beskikbare metodes nie. ‘n Ekstraksiemetode om die karotenoïed- en chlorofilprofiel van geliofiliseerde weefsel van onryp (groen) tot ryp (rooi) Merlot-bessies te identifiseer en kwantifiseer was dus nodig. In hierdie studie is die RP-HPLC metode van Taylor et al. (2006) vir karotenoïede en die ekstraksiemetode van Mendes-Pinto et al. (2004) aangepas om beide karotenoïede en chlorofille in geliofiliseerde druiweweefsel te analiseer. Die basislyn van die RP-HPLC metode het all karotenoïede en chlorofille en hul derivate geskei. Herwinning van die standaarde vanaf skynekstraksies was hoog, wat aandui dat die ekstraksieprosedure aanvaarbaar was. Ekstraksieherwinning wat in die matriks van die druiweweefsel getoets is, het egter minder belowende resultate getoon as gevolg van die hoë suurinhoud van die druifweefsel. Violaxantien, neoxantien en die chlorofille was veral sensitief vir toestande van lae pH, wat hulle afbreking gefasiliteer het. Die afbrekingsprodukte van hierdie verbindings onder suurtoestande is geïdentifiseer as feofitien a en b, chlorofillied a, pirofeofitien b, cis-violaxantien, cis-neoxantien, neochroom, mutatoxantien en luteoxantien. Daar is ‘n moontlikheid dat sommige afbreekprodukte reeds in die weefsel teenwoordig was as gevolg van liofilisering (aangesien die water in die korrel reeds verwyder was en die suur gekonsentreerd was). Meer werk is nodig om die effek van liofilisering en berging op die samestelling van druifweefsel van verskillende rypheid te bepaal. Die ekstraksiemetode vir druifkorrelweefsel op verskillende stadia van rypwording moet ook verder geoptimaliseer word om weefselsuurheid doeltreffend te neutraliseer, sonder om die ekstraksie van karotenoïede noemenswaardig te kompromitteer, veral in groen korrelweefsel. Die vraag of cis-isomere en chlorofil afbreekprodukte natuurlik in die druifkorrels teenwoordig is en of hulle tydens monsterneming en prosessering gevorm word, kon nie in hierdie studie beantwoord word nie. Hierdie studie het bevestig dat karotenoïede en chlorofille oor die algemeen op ‘n korrel (μg/korrel) en konsentrasie (μg/g) basis afneem vanaf deurslaan tot oes. Hierdie studie het nie daarin geslaag om te toon dat groeikragverskille vanaf voor-deurslaan (ertjiekorrelgrootte) tot oes ‘n effek het op die tempo van sintese/afbreking van karotenoïede, chlorofil en ander rypwordingsparameters nie, naamlik op appelsuur, totale glukose en fruktose, totale tannien en totale antosianien. Daar is ook in hierdie studie geen noemenswaardige effek van grondwaterinhoud op karotenoïede, chlorofille en rypheidsparameters gevind nie, heel moontlik as gevolg van die feit dat hoë grondwaterkapasiteit in die laer grondlae gevind is, wat betekenisvolle verskille in wingerdwaterstatus kon verhoed het. Eksperimentele persele wat gekies is vir groeikragverskille op grond van genormaliseerde verskil plantegroei indeks (NDVI) beelde, snoeimassa en grondwatermetings met ‘n neutronvogmeter het net in die eerste 30 cm van die grond noemenswaardige verskille in grondwaterinhoud getoon vir die rypwordingseisoene wat bestudeer is. Voor-sonopkoms plantwaterpotensiaalmetings het egter aangedui dat geen van die eksperimentele wingerdstokke ernstige waterstres ervaar het nie. Sulke stres is voorheen aangedui om ‘n effek op die karotenoïedinhoud van druiwe te hê. Die karotenoïed 5,8-epoksi--karoteen is vir die eerste keer in druiwe gekwantifiseer en verteenwoordig ‘n noemenswaardige hoeveelheid van die totale karotenoïede wat met oes teenwoordig is. Al die karotenoïede en chlorofille behalwe -karoteen blyk sensitief vir seisoenale verskille in klimaatstoestande te wees. Luteïen en β-karoteen was die volopste karotenoïede in die Merlot-druifkorrels, tesame met chlorofil a, vir beide seisoene wat bestudeer is. Die waardes van hierdie karotenoïede was ook goed gekorreleer met vorige navorsing. Chlorofil a is egter in baie groter hoeveelhede in Merlot-korrels gevind in vergelyking met dít wat in die data gerapporteer is. Die rede hiervoor is moontlik dat die chlorofil-afbreekprodukte in hierdie studie in die berekening van chlorofil a ingesluit is. Meerveranderlikeontleding het belowende voorlopige voorspellingsmodelle getoon (met korrelasiewaardes van meer as 0.8 vir beide die seisoene wat geanaliseer is) vir die voorspelling van die konsentrasie van rypheidsparameters (glukose, fruktose, appelsuur, totale tanniene en antosianiene) met karotenoïed- en chlorofilinhoud. Hierdie resultaat beklemtoon die geleentheid vir die ontwikkeling van ‘n vinnige, nie-destruktiewe metode om karotenoïede en chlorofille in korrels te meet, wat op sy beurt optimate rypheid kan voorspel. Aangesien karotenoïede die voorlopers van C13-norisoprenoïed aromaverbindings in wyn is, kan ‘n voorskou van die potensiële bydrae van hierdie aromas tot wyn moontlik verder evalueer word. Verdere navorsing is nodig om die moontlikheid van die bou en geldigheidsbepaling van sulke modelle te ondersoek.

Thesis (PhD(Agric)--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The bright colouration of involucral leaves in Leucadendron is unfortunately transient in nature. Undesirable colour changes render this cut flower unmarketable, resulting in a considerable loss of profit. A deeper understanding of the mechanism leading to colour change is needed to form the framework on which future manipulation strategies can be built. Yellow Leucadendron possess the ability to degreen and regreen naturally, a phenomenon linked to the controlled degradation of chlorophyll and the lesser degradation of carotenoids, which then impart the yellow colour. This colour change is directly linked to the development of the inflorescence. Involucral leaves degreen towards anthesis and are entirely yellow at full bloom. They begin to regreen again when the last florets on the cone have wilted. Deconing before flowering completely inhibits the colour change. Deconing at full bloom, results in leaves regreening sooner. Therefore the inflorescence appears to be the origin of the cue for colour change. Any factor that expedites the death of the florets, results in sooner regreening of involucral leaves. Ultra-structurally, the degreening and regreening resulted from a transdifferentiation of mature chloroplasts to gerontoplast-like plastids, which upon regreening completely redifferentiated into fully functional chloroplasts. In the red Leucadendron cultivar Safari Sunset, the photosynthetic pigment degradation pattern is identical to that of yellow cultivars. However, colour expression is complexed by the presence of anthocyanins. Anthocyanin concentration was shown to be directly related to the opening of the flower head rather than to the phenological development of the inflorescence. With opening, the previously shaded inner involucral leaf surfaces are exposed to higher levels of irradiance and respond by turning red, presumably for photoprotection. Similar to yellow cultivars, any factor leading to the death of the florets before flowering, not only prevents the degreening of involucral leaves, but also prevents the opening of the flower head and therefore the associated change in anthocyanin levels. The ecological significance of regreening was also investigated. What does a female Leucadendron plant stand to gain by regreening rather than discarding the involucral leaves? Regreened involucral leaves were shown not to play a significant role in providing photosynthates for the developing cone. Although the presence of regreened involucral leaves were shown to provide protection against high irradiance and radiant heating of the cone, they were not essential to ensure survival of the cone. The small floral bracts were shown to be very capable of adaptation. The most plausible reason for regreening is therefore assumed to be based on a cost-benefit relationship. As most Leucadendron are adapted to grow on very nutrient poor soils, the question should maybe be rephrased. Why waste valuable resources? Sclerophyllous leaves, like the involucral leaves, are costly to make and therefore reusing, rather than discarding them does seem a sensible strategy for survival. / AFRIKAANSE OPSOMMING: Leucadendron snyblomme word gekenmerk deur die helder kleure van hul omwindselblare. Die helder kleure is egter slegs vir 'n kort periode aanwesig waarna die snyblomme onbemarkbaar word, met aansienlike verlies aan potensiele inkomste. Die ontwikkeling van manipulasies ten einde die bemarkbare periode van Leucadendron te verleng, berus op die verkryging van 'n dieper insig in die meganisme van kleurverandering. Die kleurveranderinge van geel Leucadendron omwindselblare is te wyte aan 'n unieke vermoë tot die gereguleerde degradasie en heropbou van chlorofiele en karotenoiede onder direkte beheer van die ontwikkelende bloeiwyse. Met die aanvang van blom, lei groter proporsionele degradasie van chlorofiele tot geleidelike vergeling van omwindselblare. Die hele blomhofie verkry uiteindelik met volblom 'n helder geel kleur. Sodra die laaste blommetjies doodgaan, neem chlorofiel- en karotenoiedsintese weer in aanvang en binnekort is die omwindselblare weer net so groen soos voor die aanvang van blom. Die geel verkleuring kan verhoed word deur die keël voor blom uit te breek. Enige faktor wat die dood van die blommetjies versnel, asook die uitbreek van keël tydens volblom, lei tot die vroeëre aanvang van vergroening. Die degradasie van plastiedpigmente hang nou saam met die differensiasie van volwasse chloroplaste tot gerontoplast-agtige plastiede wat op hul beurt weer tydens vergroening tot volkome funksionele chloroplaste herdifferensieer. Soortgelyk aan geel Leucadendron kultivars, vind die veranderinge in plastiedpigmente ook plaas tydens blom van die rooi kultivar, Safari Sunset. Kleurveranderinge in 'Safari Sunset' is egter meer ingewikkeld vanweë die aanwesigheid van variërende konsentrasies antosianiene. Antosianienkonsentrasies en rooi kleur neem toe tydens blom vanwee die blootstelling van die beskutte adaksiale binnekante van omwindselblare aan hoe irradiasie met die oopvou van die blomhofie. Die akkumulasie van antosianiene het moontlik 'n fotobeskermende funksie. Kleurveranderinge in 'Safari Sunset' kan, soos in geel kultivars, voorkom word deur blom te verhoed. Antosianiensintese word voorkom deurdat die blomhofie geslote bly en is nie direk gekoppel aan blom soos wat met plastiedpigmente die geval is nie. Die belang van vergroening is ondersoek na aanleiding van die vraag oor wat dit 'n vroulike Leucadendron baat om omwindselblare te behou na die afloop van blom? Die bydrae van foto-assimilasie deur omwindselblare tot die ontwikkeling van keels is beperk. Alhoewel omwindselblare wel keels teen hoe irradiasie en stralingsverhitting beskerm, is die blomskutblare in staat om aan te pas by hierdie kondisies. Die mees waarskynlike verklaring vir die behoud van die omwindselblare na blom berus moontlik op 'n koste-voordele verwantskap. Alhoewel nie essensieel nie, is die beperkte bydrae van die omwindselblare na die afloop van blom tot die oorlewing en welstand van die keel waarskynlik genoegsaam om hul behoud te regverdig. Verskeie Leucadendron spesies groei in gronde wat baie arm is aan nutriente. Sklerefiele blare, soos die van Leucadendron, is verder duur om te vervaardig. Dit maak dus sin om hulle vir meer as een funksie te herontplooi eerder as om hulpbronne te belê in meer gespesialiseerde en minder durende blombykomstighede. Dus dui die behoud van omwindselblare dalk op 'n strategie wat gemik is op die behoud en besparing van beperkte hulpbronne.

Η εκτίμηση της παραγωγικότητας των χερσαίων οικοσυστημάτων προϋποθέτει τη δημιουργία αξιόπιστων μοντέλων παραγωγικότητας. Τα μοντέλα παραγωγικότητας τροφοδοτούνται με μια σειρά βιοτικών (φωτοσυνθετική ταχύτητα, συγκέντρωση χλωροφύλλης, ειδική μάζα φύλλου) και αβιοτικών παραμέτρων (θερμοκρασία, ένταση φωτός, βροχόπτωση) των φυτικών ειδών και του περιβάλλοντος. Μία από τις σημαντικότερες βιοτικές παραμέτρους αποτελεί η μέγιστη φωτοσυνθετική ταχύτητα κάτω από κορεσμένες εντάσεις φωτός, σε φυσιολογικές συγκεντρώσεις CO2 και στις επικρατούσες θερμοκρασίες (Asat). Η μέτρηση της Asat στο πεδίο με τις κλασσικές μεθόδους (αναλυτής αερίων) είναι επίπονη και χρονοβόρος διαδικασία. Για το λόγο αυτό νέοι τρόποι υπολογισμού της Asat προτείνονται. Στην παρούσα εργασία διερευνήθηκε η δυνατότητα εκτίμησης της Asat ορισμένων μεσογειακών φυτών μέσω της ανάλυσης της ταχείας φάσης ανόδου του φθορισμού της χλωροφύλλης (JIP-test). Για το σκοπό αυτό χρησιμοποιήθηκαν τα ετήσια είδη Malva sylvestris και Capparis spinosa, το φυλλοβόλο είδος Cercis siliquastrum, το ημιφυλλοβόλο είδος Phlomis fruticosa, και τα σκληρόφυλλα είδη Nerium oleander, Ceratonia siliqua και Ligustrum japonicum. Οι μετρήσεις της Asat πραγματοποιήθηκαν με τη χρήση αναλυτή αερίων ανοικτού τύπου (LI-6400, LICOR), ενώ για τη μέτρηση της ταχείας φάσης ανόδου του φθορισμού χρησιμοποιήθηκε φθορισμόμετρο υψηλής χρονικής ανάλυσης (HandyPEA, Hansatech). Οι μετρήσεις πραγματοποιήθηκαν σε εποχιακή βάση. Σύμφωνα με τα αποτελέσματα των πειραμάτων μας, δεν βρέθηκε κάποια παράμετρος του JIP-test που μπορεί να χρησιμοποιηθεί ως γενικευμένος δείκτης της Asat, αντίθετα η Asat εμφάνισε πολύ καλή συσχέτιση με τη στοματική αγωγιμότητα. Εντούτοις, στα σκληρόφυλλα είδη και το ημιφυλλοβόλο είδος βρέθηκε καλή συσχέτιση μεταξύ της Asat και των δεικτών ψΕ0, δR0 και φR0 για τα χειμερινή ομάδα μετρήσεων. Η δυσμενέστερη περίοδος για τα πολυετή είδη φαίνεται πως είναι το καλοκαίρι. Τέλος, ο δείκτης VK/VJ μπορεί να υποδείξει τις κύριες καταπονητικές περιόδους. / The evaluation of the productivity of terrestrial ecosystems presupposes the creation of reliable productivity models. The productivity models are supplied with a series of biotic (photosynthetic rate, chlorophyll concentration, specific leaf mass) and abiotic parameters (temperature, light intensity, rainfall) of the plant species and of the environment. One of the most important biotic parameters is the maximum photosynthetic rate under saturated light intensities, under ambient CO2 concetration and under the prevailing temperatures (Asat). The measurement of Asat under field conditions using the traditional methods (gas analyzer) is a laborious and time consuming process. For this reason new methods of Asat calculation are suggested. In the present study the possibility of Asat estimation in some Mediterranean species with the use of the fast chlorophyll fluorescence transient (JIP-test) has been examined. For this purpose, the annual species Malva sylvestris and Capparis spinosa, the deciduous species Cercis siliquastrum, the semi-deciduous species Phlomis fruticosa, and the sclerophyllous species Nerium oleander, Ceratonia siliqua και Ligustrum japonicum have been used. The Asat measurements have been carried out with the use of an open gas exchange analyzer (LI-6400, LICOR), while for the measurement of the fast chlorophyll fluorescence transient a high-time resolution fluorometer (HandyPEA, Hansatech) has been used. The measurements were carried out on a seasonal basis. According to the results of our experiments, no JIP-test parameter has shown to act as a generalized index of Asat, while Asat has shown a very good correlation with stomatal conductance. However, at the sclerophyllous and the semi-deciduous species, a good correlation between Asat and ψΕ0, δR0 και φR0 indices has been shown for the winter team. The most adverse period of the year for the perennial species is summer. Finally, VK/VJ index is indicative of the most stressful periods for our species.

The Avon-Heathcote Estuary is of significant value to Christchurch due to its high productivity, biotic diversity, proximity to the city, and its cultural, recreational and aesthetic qualities. Nonetheless, it has been subjected to decades of degradation from sewage wastewater discharges and encroaching urban development. The result was a eutrophied estuary, high in nitrogen, affected by large blooms of nuisance macroalgae and covered by degraded sediments. In March 2010, treated wastewater was diverted from the estuary to a site 3 km offshore. This quickly reduced water nitrogen by 90% within the estuary and, within months, there was reduced production of macroalgae. However, a series of earthquakes beginning in September 2010 brought massive changes: tilting of the estuary, changes in channels and water flow, and a huge influx of liquefied sediments that covered up to 65% of the estuary floor. Water nitrogen increased due to damage to sewage infrastructure and the diversion pipeline being turned off. Together, these drastically altered the estuarine ecosystem. My study involves three laboratory and five in situ experiments that investigate the base of the food chain and responses of benthic microalgae to earthquake-driven sediment and nutrient changes. It was predicted that the new sediments would be coarser and less contaminated with organic matter and nutrients than the old sediments, would have decreased microalgal biomass, and would prevent invertebrate grazing and bioturbation activities. It was believed that microalgal biomass would become similar across new and old sediments types as the unstable new sediments were resuspended and distributed over the old sediments. Contact cores of the sediment were taken at three sites, across a eutrophication gradient, monthly from September 2011 to March 2012. Extracted chlorophyll a pigments showed that microalgal biomass was generally lower on new liquefied sediments compared to old sediments, although there was considerable site to site variation, with the highly eutrophic sites being the most affected by the emergence of the new sediments. Grazer experiments showed that invertebrates had both positive and negative site-specific effects on microalgal biomass depending on their identity. At one site, new sediments facilitated grazing by Amphibola crenata, whereas at another site, new sediments did not alter the direct and indirect effects of invertebrates (Nicon aestuariensis, Macropthalmus hirtipes, and A. crenata) on microalgae. From nutrient addition experiments it was clear that benthic microalgae were able to use nutrients from within both old and new sediments equally. This implied that microalgae were reducing legacy nutrients in both sediments, and that they are an important buffer against eutrophication. Therefore, in tandem with the wastewater diversion, they could underpin much of the recovery of the estuary. Overall, the new sediments were less favourable for benthic microalgal growth and recolonisation, but were less contaminated than old sediments at highly eutrophic sites. Because the new sediments were less contaminated than the old sediments, they could help return the estuary to a noneutrophic state. However, if the new sediments, which are less favourable for microalgal growth, disperse over the old sediments at highly eutrophic sites, they could become contaminated and interfere with estuarine recovery. Therefore, recovery of microalgal communities and the estuary was expected to be generally long, but variable and site-specific, with the least eutrophic sites recovering quickly, and the most eutrophic sites taking years to return to a pre-earthquake and non-eutrophied state. changes in channels and water flow, and a huge influx of liquefied sediments that covered up to 65% of the estuary floor. Water nitrogen increased due to damage to sewage infrastructure and the diversion pipeline being turned off. Together, these drastically altered the estuarine ecosystem. My study involves three laboratory and five in situ experiments that investigate the base of the food chain and responses of benthic microalgae to earthquake-driven sedimen tand nutrient changes. It was predicted that the new sediments would be coarser and less contaminated with organic matter and nutrients than the old sediments, would have decreased microalgal biomass, and would prevent invertebrate grazing and bioturbation activities. It was believed that microalgal biomass would become similar across new and old sediments types as the unstable new sediments were resuspended and distributed over the old sediments. Contact cores of the sediment were taken at three sites, across a eutrophication gradient, monthly from September 2011 to March 2012. Extracted chlorophyll a pigments showed that microalgal biomass was generally lower on new liquefied sediments compared to old sediments, although there was considerable site to site variation, with the highly eutrophic sites being the most affected by the emergence of the new sediments. Grazer experiments showed that invertebrates had both positive and negative site-specific effects on microalgal biomass depending on their identity. At one site, new sediments facilitated grazing by Amphibola crenata, whereas at another site, new sediments did not alter the direct and indirect effects of invertebrates (Nicon aestuariensis, Macropthalmus hirtipes, and A. crenata) on microalgae. From nutrient addition experiments it was clear that benthic microalgae were able to use nutrients from within both old and new sediments equally. This implied that microalgae were reducing legacy nutrients in both sediments, and that they are

Black wattle (Acacia mearnsii) is one of South Africa's leading commercial exotic species comprising nearly seven percent of South African forestry plantations. The planting of black wattle has become increasingly popular, initially for its high quality tannin content and in more recent times, for its wood and wood products. The industry also provides jobs for more than 36 000 people. Despite the commercial value of black wattle, if left unmanaged, it is one of South Africa's top invader species that aggressively colonise and rapidly out-competes indigenous vegetation. Thus, both plant breeders and environmentalists alike are faced with an interesting paradox of balancing the commercial significance of black wattle on the one hand with increasing environmental concern on the other. At the Institute for Commercial Forestry Research (ICFR), black wattle breeding programmes are being designed and implemented in order to reduce invasiveness whilst still maintaining product quality. One way of minimising invasiveness is to decrease fertility through the introduction of semi-sterility; while at the same time leaving product yield and quality unaffected. A method of achieving semi-sterility is by the induction of autopolyploidy that results in unviable gametes. Autopolyploidy, tetraploidy, is induced chemically through doubling of the chromosomes of diploids. These induced tetraploids may then be crossed with diploids to produce triploids. Thus, an effective method to identify polyploids at the seedling stage would greatly facilitate the success of the abovementioned breeding programmes in the black wattle industry. Polyploidy in plants is often associated with physiological and biochemical changes that become apparent as gigantism of organs which include fruits, flowers and leaves. Polyploidy is also associated with an increase in the number of organelles such as the number of stomatal chloroplasts and nucleoli, as well as an increased production of some proteins and pigments such as chlorophyll. These ploidy-related manifestations are often utilised in breeding programmes to increase the size and quality of plant products as well as a tool to discriminate between polyploids and diploids. Two putative diagnostic procedures to differentiate between diploid and tetraploid black wattle were developed in this investigation. The study focused on the discriminating power of stomatal chloroplast numbers and arrangements as well as the chlorophyll content in the two different ploids. A number of associated experiments were initially conducted to establish the optimal conditions for chlorophyll content analyses such as the type of leaf material and storage conditions. Stomatal chloroplast frequencies were determined in diploid and tetraploid black wattle and comprised three lines per ploidy level with five plants per line. A thin epidermal layer from the abaxial surface of a pinnule was stripped, stained, mounted and 15 stomatal guard cells per plant were viewed at 40X magnification. The mean number of chloroplasts per cell in diploids (9.89 ± 0.222) was found to be statistically different (p < 0.001) to that of tetraploids (22.43 ± 0.222) with no overlapping of the mean chloroplast values between the two ploidy levels. The ratio of diploid and tetraploid stomatal chloroplast numbers was roughly 1:2. An analysis of the least significant difference (LSD) was performed and indicated significant differences between plants within lines, between lines of different ploids (LSD =0.6266), as well as between the different ploids (LSD =0.2802). Furthermore, stomatal chloroplasts spatial arrangements were distinctly different in diploids and tetraploids. In diploids, chloroplasts were clustered into two regions, each towards the extreme ends of the kidney shaped stomatal cells. In the tetraploids, no clustering of chloroplasts could be identified, with an even distribution around the convex curvature/perimeter of the cells. There are a number of factors that influence chlorophyll content and degradation, which are either environmental or genetic in nature. Environmental factors that were considered are sample age and sample storage conditions. Genetic factors include genetic composition and, specifically, the number of sets of chromosomes, that is, the ploidy. Chlorophyll content was investigated by chemically extracting chlorophyll from leaf material and obtaining absorbance spectra with a PerkinElmer UV/vis spectrometer for wavelengths from 400 nm to 700 nm. Chlorophyll absorbance spectra were generated in terms of leaves stored prior to chlorophyll extraction, leaves of different ages, trees of different ages and ploidy. The effects of storage of leaves on chlorophyll content were determined in five non-identical two year-old nursery diploid black wattle genotypes. Fifteen leaf samples from each genotype were either oven dried and then stored for one week or one month at room temperature, or frozen for one week or one month at -4 °C, before chlorophyll was extracted and absorbance spectra determined. Chlorophyll absorbance values of chlorophyll extracted from leaf material on the day of collection (day-0) was used as the control. An analysis of variance (ANOVA) revealed that the chlorophyll absorbance values of the different storage treatments were all significantly lower than the chlorophyll absorbance values of the control (p < 0.001). Assessment of the mean chlorophyll absorbance (TĀ), sum of the three peak absorbance values at three wavelengths, namely, 433 nm, 456 nm and 663 nm, revealed significant differences (p < 0.001) from the control (TĀ = 1.275) for all treatments. Dried leaves that were stored for seven days (TĀ = 1.132) resulted in the least amount of chlorophyll degradation followed by 28 day ice storage (TĀ = 1.114), seven day ice storage (TĀ = 1.103) and lastly 28 day dried storage (TĀ = 1.093). An analysis of least significant differences (LSD) revealed that chlorophyll absorbance values within lines and between wavelengths were significantly different (LSD = 0.005). Furthermore, LSD analysis revealed significant differences between all treatments (LSD =0.003) which also supported the ANOVA findings. Chlorophyll absorbance values within dried and frozen treatments were compared with respect to storage time periods of one week and one month. It was noted that whilst all treatments decreased from the control (day-0), dried samples responded differently to storage periods as compared to frozen samples. Chlorophyll absorbance values of dried material decreased steadily over time from control to seven-day storage to one-month storage, whereas, in the case of frozen material, a similar trend could not be identified. A greater decrease from the control to seven day ice storage was recorded than for the decrease from the control to 28 day ice storage. The effects of tree and leaf ages of diploid black wattle on chlorophyll content were determined. Two types of leaf flushes namely, old and new flush, were examined in relation to different tree ages; two, four, six, eight and nine year-old; in order to assess whether the choice of material impacts on chlorophyll absorbance values. Five leaf samples from each tree were collected, bagged and chlorophyll extracted within two hours of collection. These chlorophyll absorbance values were compared to young diploid seedling material as a base-value and as a control value. An analysis of variance (ANOVA), revealed significant differences between tree ages and between leaf ages (p < 0.001). An analysis of least significant differences (LSD) revealed that new flush of all tree age groups were significantly different from the control (LSD = 0.006). This was mostly true for old flush, except that of six year-old old flush which was not significantly different from the control (LSD =0.006). The chlorophyll absorbance values of both old and new flush of different age groups produced spectral graphs for which no specific trends could be ascertained. Therefore, the data from the two flush types were pooled and revealed a marked increase in chlorophyll absorbance as trees became older. Moreover, this increase was more apparent in new flush than in old flush. Interestingly, juvenile characteristics were identified in two year-old black wattle trees, where a marked increase in chlorophyll content was noted. The effects of the number of chromosome sets on chlorophyll content were assessed for diploid and tetraploid black wattle. Seedlings, bagged juveniles as well as two year-old field trees were analysed. Three genetic lines per ploidy level comprising of ten plants per line were used in the analysis. An analysis of variance (ANOVA) revealed significant increases of chlorophyll absorbance values (p < 0.001) for diploid seedlings (TĀ = 1.1086) to bagged trees (TĀ = 1.149) to field trees (TĀ = 1.224). Similar significant increases were recorded for the tetraploid seedlings (TĀ = 1.886) to bagged trees (TĀ = 1.931) to field trees (TĀ = 2.059). There were distinct differences in chlorophyll absorbance between the two levels of ploidy (LSD =0.002). Furthermore, chlorophyll absorbance within lines, between wavelengths were found not to be significant (p = 0.984), which was supported by an analysis of least significant differences (LSD = 0.004). Moreover, the ratio of diploid to tetraploid chlorophyll absorbance was roughly 2:3. Additionally, the increase of chlorophyll content from seedlings to bagged juveniles to field material of both diploid and tetraploid black wattle further supported the findings in the previous age study that there was an increase in chlorophyll content as the tree matures. Stomatal chloroplast frequencies and chlorophyll content have been identified as two methods that are able to effectively, and with ease, discern between diploid and tetraploid black wattle. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

This study investigated the use of optical remote sensing for estimating leaf and canopy scale light use efficiency (LUE) and carbon exchange. In addition, a new leaf level model capable of predicting dynamic changes in apparent reflectance due to chlorophyll fluorescence was developed. A leaf level study was conducted to assess the applicability of passive remote sensing as a tool to measure the reduction, and the subsequent recovery, of photosynthetic efficiency during the weeks following transplantation. Spectral data were collected on newly planted saplings for a period of 8 weeks, as well as gas exchange measurements of LUE and PAM fluorescence measurements. A set of spectral indices, including the Photochemical Reflectance Index (PRI), were calculated from the reflectance measurements. A marked depression in photosynthetic rate occurred in the weeks after outplanting followed by a gradual increase, with recovery occurring in the later stages of the experimental period. As with photosynthetic rate, there was a marked trend in PRI values over the study period but no trend was observed in chlorophyll based indices. The study demonstrated that hyperspectral remote sensing has the potential to be a useful tool in the detection and monitoring of the dynamic effects of transplant shock. Relationships between hyperspectral reflectance indices, airborne carbon exchange measurements and satellite observations of ground cover were then explored across a heterogeneous Arctic landscape. Measurements were collected during August 2008, using the University of Edinburgh’s research aircraft, from an Arctic forest tundra zone in northern Finland as part of the Arctic Biosphere Atmosphere Coupling at Multiple Scales (ABACUS) study. Surface fluxes of CO2 were calculated using the eddy covariance method from airborne data that were collected from the same platform as hyperspectral reflectance measurements. Airborne CO2 fluxes were compared to MODIS vegetation indices. In addition, LUE was estimated from airborne flux data and compared to airborne measurements of PRI. There were no significant relationships between MODIS vegetation indices and airborne flux observations. There were weak to moderate (R2 = 0.4 in both cases) correlations between PRI and LUE and between PRI and incident radiation. A new coupled physiological radiative transfer model that predicts changes in the apparent reflectance of a leaf, due to chlorophyll fluorescence, was developed. The model relates a physically observable quantity, chlorophyll fluorescence, to the sub leaf level processes that cause the emission. An understanding of the dynamics of the processes that control fluorescence emission on multiple timescales should aid in the interpretation of this complex signal. A Markov Chain Monte Carlo (MCMC) algorithm was used to optimise biochemical model parameters by fitting model simulations of transient chlorophyll fluorescence to measured reflectance spectra. The model was then validated against an independent data set. The model was developed as a precursor to a full canopy scheme. To scale to the canopy and to use the model on trans-seasonal time scales, the effects of temperature and photoinhibition on the model biochemistry needs to be taken into account, and a full canopy radiative transfer scheme, such as FluorMOD, must be developed.

Chlorins, as the core structures of chlorophylls, have been extensively studied for harvesting solar energy, fluorescent imaging and photodynamic therapy against cancer. This thesis is concerned with design and synthesis of novel chlorins as antennae for harvesting light and sensitising near infrared lanthanide luminescence. In the first part, a series of chlorin monomers, dimers and polymers were synthesised and their photophysical properties were characterised. The chlorin monomers were substituted with five-membered heterocycles, such as thiophenes and furans. These heterocycles function as auxochromes analogous to the natural ones in chlorophylls, and extend chlorin absorption and emission strongly to the red (up to λem = 680 nm). A borylation method was developed to prepare borylated chlorins, which gave access to directly linked chlorin dimers through Suzuki coupling reaction. Different regioisomers of chlorin dimer were prepared, including β-meso homodimers, meso-meso homodimers and heterodimers. The dimerisation resulted in red-shifted absorption and emission. Chlorin polymerisations were performed both electrochemically and chemically. Bis-thienylchlorins yielded chlorin films and an organic solvent soluble copolymer with hexylthiophene, respectively. These polymers from both polymerisations have red absorptions beyond 700 nm, and might be used as light-harvesting antennae. In the second part, chlorins were used as chromophores to sensitise near infrared lanthanide luminescence. Two types of chlorin-lanthanide dyads were prepared through lanthanide coordination with cyclen derivatives and dipicolinic acids (DPA). The cyclen-based dyads were poorly soluble in water, thus their near infrared emissions were not observed. The other type of complexes was fully soluble in H2O and THF. Both Nd and Yb emission were recorded even upon excitation into the Q bands of chlorins. In the dyads with free base chlorins, the singlet state of chlorins might be involved in the sensitisation of lanthanide luminescence. These DPA-based dyads presented two-color emission based on one chlorin and two-color excitation based on one lanthanide ion. These dyads would enable in theory 4-color imaging. In the last part, a microwave-assisted two-step synthesis was described to prepare dipyrromethanes, which are the key intermediates in the chlorin synthesis. This mild method took advantage of the nucleophilicity of pyrrole and the electrophilicity of N,N-dimethylaminomethyl pyrroles. The usually used acid catalysis is detrimental to many functionalities, thus our methods enable the synthesis of dipyrromethanes with acid sensitive groups or a formyl group.

Cold pressed Hemp (Cannabis sativa L) seed oil, which contains a favorable ratio of omega-6/omega-3 and a considerable amount of vitamin E, is a high nutrition value product with various health benefits. However, the presence of a large amount of chlorophyll makes the oil highly susceptible to photo-oxidation and limits its applications. Chlorophyll and other pigments in edible oils are commonly reduced through oil bleaching process. The objective of this study was to reduce the chlorophyll content in cold pressed hemp seed oil using ultrasonic bleaching and evaluate the impacts on carotene content, peroxide value, conjugated diene, total phenolic content and oxidative stability. The bleaching efficiency increased significantly as the ultrasound power (0 to 60% pressure amplitude) and concentration of bleaching clay (20 to 40 g/kg) increased. At 20% ultrasound power, the chlorophyll reduction (%) of different clays was found to be 99.4% (industrial clay) > 97.8% (activated bentonite) > 82.7% (sepiolite) > 47.1% (non-activated bentonite). Ultrasonic bleaching significantly reduced (p<0.05) the total chlorophyll content, total carotene content and primary oxidation products of hemp seed oil. During accelerated oxidation tests (40 and 60 ℃), the developments of peroxide value and conjugated diene were significantly (p<0.05) slower for hemp seed oil treated with ultrasonic bleaching compared to the control oil. Enhanced oxidative stability was observed in hemp seed oil after ultrasonic bleaching. In addition, another objective of this study was to evaluate the impacts of solvent and heat treatment on phenolic profile and antioxidant activity of cold pressed canola and hemp meals. For canola meal, a solvent-mixture of aqueous methanol (70%) and aqueous acetone (70%) in a ratio of 1:1 (v/v) extracted (p<0.05) slightly higher total phenolic content (11.3 mg SAE/g), while aqueous methanol (70%) extracts exhibited higher DPPH scavenging effect (40.4%). For hemp meal, aqueous acetone (80%) extracts exhibited higher total phenolic content (6.0 mg GAE/g) and DPPH scavenging effect (12.5%). Total phenolic content and antioxidant activity in canola meal extracts were significantly higher (p<0.05) than those in hemp meal extracts. After heat treatments at temperature from 140 to 180 ℃, canola and hemp meal extracts showed equal or lower total phenolic content and DPPH scavenging effect. / May 2017

Woody plant expansion has been documented for decades in many different ecosystems, often due to anthropogenic disturbances to the environment and yielding vast changes in ecosystem functioning. While causes and consequences of woody expansion have been well documented, few studies have investigated functional traits of woody species that promote rapid expansion in range. My objective was to determine if hydraulic efficiency confers enhanced photosynthetic efficiency so that functional traits representing light and water use may be possible mechanisms facilitating woody encroachment into grasslands and forest understories. I quantified leaf-level light environment, photosynthetic activity, and hydraulic characteristics of three sympatric broadleaf evergreens of varying leaf life span (Ilex opaca, Kalmia latifolia, and Myrica cerifera) in a deciduous forest understory to understand seasonal intra- and interspecific ranges of broadleaf evergreen physiology. Additionally, I investigated the effects of age on physiological efficiency of M. cerifera across a chronosequence (i.e., space for time substitution) of shrub thicket development in order to understand possible age-related physiological mechanisms facilitating shrub expansion. Lastly, I determined functional traits and resulting physiology that contribute to rapid expansion and thicket formation of an invasive, deciduous, N-fixing shrub, Elaeagnus umbellata, and a native, evergreen, N-fixing shrub M. cerifera. When compared to co-occurring evergreen species, electron transport rate (ETR) of M. cerifera was nearly double that of I. opaca or K. latifolia in summer. Photosynthetic capacity was positively related to hydraulic capacity among understory evergreens. Furthermore, photosynthetic and hydraulic efficiency of M. cerifera remained consistent despite considerable differences in thicket age and development. Both similar and contrasting functional traits of E. umbellata and M. cerifera allowed for enhanced light capture and water movement, and reductions in subcanopy light penetration. Enhanced hydraulic and photosynthetic efficiency relative to co-occurring species contributes to rapid range expansion and thicket formation by promoting enhanced productivity and limiting successful colonization of other species. My results indicate that there may be suites of functional traits linked to expansive success and thicket-formation, yet differences in functional traits between native and invasive species represent alternative strategies leading to rapid growth and thicketization.

The smallest free living eukaryote known as Ostreococcus tauri became along with some related species great experimental models for iron uptake research in marine picoplankton. The ecological context of such topic is very interesting considering the nature of adaptation to iron limitation and its connection to the copper metabolism. Our experiments has simulated iron and copper limiting conditions of the ocean, as a control we used iron and copper repleted cultivation media. The maximum cell counts were two- to threefold higher in iron-repleted medium compared to iron- depleted one. There was the only exception showing no difference in growth - O. lucimarinus coming from open ocean with high irradiance levels and very low iron concentrations, which all together made it the best adapted species. The reinoculation of cell cultures after a week cultivation into to the same iron/copper containing media led to unmasking the copper effect on growth, which was much weaker than encountered with iron (again except of O. lucimarinus). Iron sufficiency positively affects heme b and chlorophyll a and b content with no significant copper dependency. The circadian timing of heme b and chlorophyll a and b content shows the increasing trend during the day followed by decrease at night. This might be caused by...