The chlorophyll degradation pathway is central to a number of plant processes including senescence and fruit ripening. However, the regulation of the chlorophyll degradation pathway enzymes is not well understood. The aim of this thesis was to elucidate the genetic mechanisms that control changes in pigment composition leading to fruit flesh yellowing in kiwifruit. Actinidia deliciosa and A. chinensis fruit, which are green and yellow, respectively, provide an opportunity to study the regulation of chlorophyll levels. The expression of genes that code for enzymes of the chlorophyll and cytokinin metabolic pathways was measured using qRT-PCR. Candidates for chlorophyll degradation regulatory points were then characterised for functionality by transient transformation in N. benthamiana. The endogenous cytokinin levels were measured in kiwifruit and transient activation assays were carried out with the promoters of key candidate genes. Overall, expression of the chlorophyll degradation genes was elevated in yellow fruit and expression of biosynthetic genes was higher in green fruit. The chlorophyll degradation-associated protein, STAY-GREEN2 (SGR2), was more highly expressed in yellow fruit, and transient over-expression of SGR was sufficient to drive chlorophyll degradation. Expression of isopentenyl transferase (IPT), the rate-limiting step for cytokinin biosynthesis, showed an increase towards maturity in green fruit, but not in yellow fruit. However, both fruit had similar high levels of cytokinin nucleotides and free bases. A gene coding for O-glucosylation was also highly expressed in green fruit. Green fruit contained higher levels of cytokinin O-glucosides and ribosides towards maturity, suggesting differences in cytokinin signalling, which could lead to regulation of chlorophyll levels via activation of the SGR promoter by transcription factors. It is likely that the chlorophyll degradation pathway and cytokinin metabolism are linked. The differential expression of cytokinin response regulators could lead to differential regulation of cytokinin levels in the fruit of the two species, and possibly differential regulation of the chlorophyll degradation pathway. Progress towards elucidation of the control of chlorophyll levels provides knowledge of this key process in kiwifruit and potentially gene-based markers for breeding new kiwifruit cultivars.

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.

Made available in DSpace on 2015-03-26T13:39:34Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1636884 bytes, checksum: c966934c707a7e6bf40bfe5aee85e5a1 (MD5) Previous issue date: 2009-09-25 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The objective of this work was to promote the degreening, without losing internal fruit quality, of Poncã tangerine from two regions of Minas Gerais. Two experiments were carried out. In the first, the fruits were harvested from a commercial orchard in the northern region, and the second experiment used fruits from Zona da Mata. Fruits were harvested when showing minimum attributes of 35% juice, 9 ° Brix and soluble solids/tritable acidity ratio of 9.5. Then the fruits were packed in airtight boxes, which were exposed for 24, 48 and 96 h to different concentrations of ethylene gas: 0, 5, 10, 20 and 40 μL.L-1, at 20 + 1 °C and 90 + 5% relative humidity, respectively. Every 24 h, the boxes were opened for renewal of internal atmosphere, by reapplying the gas until treatment end. A post-treatment phase of 96 h duration took place at the end of each exposure time, when the fruits were kept in ethylene-free atmosphere, in cold chamber, at the same temperature and relative humidity used during the exposure to ethylene. During this phase, the fruits were evaluated every 24 h, and the difference in skin color, Hue angle of the peel, total chlorophyll and carotenoid content, soluble solids content, tritable acidity and SS/TA ratio of juice, solute leakage, CO2 production and loss of fruit fresh mass. In the first experiment, the fruits showed stronger degreening with increasing time of exposure to ethylene, most markedly for 96 h. After this exposure time, the Hue angle increased from 96.1o, in the first assessment days, to 82.3o, at the end of 96 h, which was lower than values observed in naturally degreened fruits. Fruits exposed for 24 h showed evolution of the yellow peel color similar to the untreated ones. It was observed that fruits left to ripe on the plant showed non-homogeneous peel degreening. The fruits exposed to ethylene for 96 h showed no visual signs of loss of commercial quality, although there was increased solute leakage from the peel during the treatment. Soluble solids content, tritable acidity and SS/TA ratio remained unchanged throughout the assessment period. The synthesis of total carotenoids in the peel was low, from 6.0 μg/cm2, in control fruits at the end of evaluations, to a maximum of 8.2 μg/cm2, in fruits exposed to ethylene for 96 h. This synthesis was insufficient to reach the orange color, therefore, the degreening of fruit peel was due, almost exclusively, to chlorophyll degradation. In the second experiment, the fruits showed yellowing of the peel stronger than fruits of the first experiment. The exposure time of 96 h proved to be the most efficient in promoting the fruit degreening in comparison with other times. Color evolution was quite similar between the control and fruits treated for 24 and 48 h. Fruits of all treatments had very low synthesis of total carotenoids in the peel, changing from 7.3 μg/cm2 in control fruits to 9.2 μg/cm2, maximum value observed in fruits exposed to ethylene for 96 h. Even in fruits showing the strongest yellowing, there was little participation of new synthesized carotenoids in the composition of the final color. Fruits fully developed in the plant reached the maximum degreening, becoming orange, the typical color of the species. Thus, for fruits produced in the Zona da Mata region, the post-harvest degreening with ethylene is only justified to anticipate the harvest, since the region has favorable climate to natural degreening. However, for fruits produced in northern Minas Gerais, where the high temperatures prevailing throughout the year impair the natural degreening, the postharvest use of ethylene may serve to obtain a more uniform peel color. / The objective of this work was to promote the degreening, without losing internal fruit quality, of Poncã tangerine from two regions of Minas Gerais. Two experiments were carried out. In the first, the fruits were harvested from a commercial orchard in the northern region, and the second experiment used fruits from Zona da Mata. Fruits were harvested when showing minimum attributes of 35% juice, 9° Brix and soluble solids/tritable acidity ratio of 9.5. Then the fruits were packed in airtight boxes, which were exposed for 24, 48 and 96 h to different concentrations of ethylene gas: 0, 5, 10, 20 and 40 μL.L-1, at 20 + 1 °C and 90 + 5% relative humidity, respectively. Every 24 h, the boxes were opened for renewal of internal atmosphere, by reapplying the gas until treatment end. A post-treatment phase of 96 h duration took place at the end of each exposure time, when the fruits were kept in ethylene-free atmosphere, in cold chamber, at the same temperature and relative humidity used during the exposure to ethylene. During this phase, the fruits were evaluated every 24 h, and the difference in skin color, Hue angle of the peel, total chlorophyll and carotenoid content, soluble solids content, tritable acidity and SS/TA ratio of juice, solute leakage, CO2 production and loss of fruit fresh mass. In the first experiment, the fruits showed stronger degreening with increasing time of exposure to ethylene, most markedly for 96 h. After this exposure time, the Hue angle increased from 96.1o, in the first assessment days, to 82.3o, at the end of 96 h, which was lower than values observed in naturally degreened fruits. Fruits exposed for 24 h showed evolution of the yellow peel color similar to the untreated ones. It was observed that fruits left to ripe on the plant showed non-homogeneous peel degreening. The fruits exposed to ethylene for 96 h showed no visual signs of loss of commercial quality, although there was increased solute leakage from the peel during the treatment. Soluble solids content, tritable acidity and SS/TA ratio remained unchanged throughout the assessment period. The synthesis of total carotenoids in the peel was low, from 6.0 μg/cm2, in control fruits at the end of evaluations, to a maximum of 8.2 μg/cm2, in fruits exposed to ethylene for 96 h. This synthesis was insufficient to reach the orange color, therefore, the degreening of fruit peel was due, almost exclusively, to chlorophyll degradation. In the second experiment, the fruits showed yellowing of the peel stronger than fruits of the first experiment. The exposure time of 96 h proved to be the most efficient in promoting the fruit degreening in comparison with other times. Color evolution was quite similar between the control and fruits treated for 24 and 48 h. Fruits of all treatments had very low synthesis of total carotenoids in the peel, changing from 7.3 μg/cm2 in control fruits to 9.2 μg/cm2, maximum value observed in fruits exposed to ethylene for 96 h. Even in fruits showing the strongest yellowing, there was little participation of new synthesized carotenoids in the composition of the final color. Fruits fully developed in the plant reached the maximum degreening, becoming orange, the typical color of the species. Thus, for fruits produced in the Zona da Mata region, the post-harvest degreening with ethylene is only justified to anticipate the harvest, since the region has favorable climate to natural degreening. However, for fruits produced in northern Minas Gerais, where the high temperatures prevailing throughout the year impair the natural degreening, the postharvest use of ethylene may serve to obtain a more uniform peel color.

Der Abbau des Chlorophyll (Chl) ist ein Prozess, der typischerweise während der Blattseneszenz und der Reifung von Früchten und Samen stattfindet. Eine Störung dieses koordinierten Prozesses unter Frostbedingungen verzögert den Chl-Abbau und ist ein grosses Hindernis bei der Herstellung von hochwertigem Rapsöl. Der Abbau von Chl zu farblosen Kataboliten erfolgt in einer Serie von enzymatischen Schritten und wird durch die Chlorophyllase begonnen (Chlase). Es wurde vorgeschlagen, dass ein wasserlösliches Chl Protein (WSCP) den Transport des Chl von der Thylakoidmembran zum Wirkort der Chlase übernimmt. Weiterhin wurde angenommen, dass die Steigerungen der Genexpressionen dieser frühen Schritte den Prozess des Chl-Abbaus beschleunigen. In der vorliegenden Arbeit werden die Auswirkungen der Überexpression der Chlase aus Citrus clementii (CcCHLASE) und von WSCP aus Blumenkohl (Cau-WSCP) in transgenen Tabakpflanzen analysiert. Dazu wurde die cDNA Sequenz der CcCHLASE in E. coli exprimiert und mittels in vitro Experimenten die Hydrolysierung von Chl durch die Chlase bestätigt. Anschließend wurden CcCHLASE exprimierende Tabakmutanten generiert und drei T1-Linien wurden unter verschiedenen Stress- und Seneszenzbedingungen untersucht. Die Chlase überexprimierenden Linien zeigten unter allen getesteten Bedingungen einen im Vergleich zum Wildtyp erhöhten Chlide a Gehalt. Trotzdem unterschied sich die Menge an Endkataboliten in diesen Mutanten nicht vom Wildtyp. Andererseits zeigten WSCP überexprimierende Linien zwar keine erhöhten Chlide a Gehalte jedoch erhöhte Protochlorophyllid-(Pchlide)-Level. Das deutet auf eine Rolle des WSCP als Speichermolekül für Chlorophyllvorstufen hin. Die photoprotektive Funktion des WSCP wurde zusätzlich in WSCP überexprimierenden Linien bestätigt. Diese zeigen im Vergleich zu Wildtyp-Tabakpflanzen auch bei hohen Lichtintensitäten von 700 – 900 µmol Photonen m-2 s-1 verringerte Gehalte an Zeaxanthin und reduzierte Peroxidaseaktivitäten. / Chlorophyll (Chl) catabolism is characteristically seen during leaf senescence, fruit ripening and seed maturation. Disruption of this coordinated process under frost conditions delays Chl breakdown and is a great concern in rapeseed oil production. The present work addresses this problem by studying the effect of enhanced Chl catabolism in genetically modified tobacco plants. Chl is catabolised to colourless catabolites through a series of enzymatic reactions initiated by Chlorophyllase (Chlase). A water soluble chlorophyll protein (WSCP) has been proposed to transport Chl from thylakoid membranes to the site of action of Chlase. It was assumed that enhancing the gene expression of these early events in Chl catabolism would increase the Chl breakdown process. The present work analysed the overexpression of Chlase from Citrus clementii (CcCHLASE) and WSCP gene from cauliflower (Cau-WSCP) in modified tobacco plants. Initially, the cDNA sequence of CcCHLASE was expressed in E. coli and in vitro tests confirmed the hydrolytic activity of Chlase on Chl. Subsequently, tobacco plants overexpressing CcCHLASE were generated and three T1 lines were analysed at various stress and senescence conditions. The in vivo production of Chlorophyllide (Chlide) indicated the extent of increased Chl breakdown. The Chlase overexpressor lines showed higher Chlide a steady state levels under all tested conditions in comparison to the WT tobacco plants. However, the end catabolites did not show much difference from WT plants. On the other hand, WSCP overexpressor lines did not show any increase in Chlide a levels, but demonstrated an increased protochlorophyllide (Pchlide) levels. This suggested the role of WSCP as a storage molecule of Chl precursors. Additionally, photoprotective function of WSCP was confirmed in WSCP overexpressors, by lower zeaxanthin levels and peroxidase activity even at high light intensities of 700 – 900 µmol photons m-2 s-1 in comparison to the WT tobacco plants.