Thesis (MScAgric)--University of Stellenbosch / ENGLISH ABSTRACT: An understanding of the infection pathways of Botrytis cinerea in grape bunches will help to combat this devastating pathogen of grape. Many studies have been done to determine the possible infection pathways of B. cinerea. Most of these studies made use of artificial inoculations that deposit groups of conidia on the plant surface. The deposition of clusters of conidia is not a common phenomenon in nature. The aim of this study was to investigate the infection pathways of (i) naturally- as well as (ii) artificially inoculated B. cinerea conidia during all the phenological stages of three wine grape cultivars, and to compare the (iii) pathogenicity and virulence, on grape and nectarine fruit, of isolates obtained from different host plants. In the natural infection study the occurrence of Botrytis cinerea and subsequent disease expression at different positions in bunches of wine grapes (cultivars Chenin Blanc, Shiraz and Chardonnay) was determined from 1999 to 2001. Different techniques were used to detect viable inoculum at different positions (rachises, laterals, pedicels, and the peicel end, cheek and style end of berries) in bunches. Isolations were made on Kerssies' B. cinerea selective medium, or bunches were used untreated, or treated with paraquat. Paraquat was used to terminate host resistance and to promote the development of the pathogen from the tissues. The material was used untreated to detect the pathogen on the surface, or were surface-sterilized to detect mycelia (latent infection) in the tissue. In the artificial inoculation study, bunches of wine grapes (cultivars Chenin Blanc, Chardonnay and Shiraz) at pea size, bunch closure, and harvest were dusted with dry conidia of Botrytis cinerea in a settling tower and incubated for 24 h at high relative humidity (±93%). Following incubation, the bunches were divided in two groups. The one group was surface-sterilised in 70% ethanol for 5 s, the other group was left untreated. Bunches of the sterile group, and from the untreated group were used for isolation. From each bunch rachis segments, laterals, pedicels and berry skin segments (from the pedicel-end and cheek) were removed. The sections were placed in Petri dishes on Kerssies' B. cinerea selective medium and on a water agar medium supplemented with paraquat, and incubated at 22°C under diurnal light. Occupation by the pathogen was positively identified by the formation of sporulating colonies of B. cinerea on the different tissues. Lastly, in the virulence and pathogenicity experiment on grape and nectarine fruit Botrytis cinerea isolates, which were obtained from different host plants, were compared by simulating natural infection. Cold-stored fruit, considered highly susceptible to B. cinerea were therefore inoculated with single, airborne conidia of the pathogen. Different tests were conducted to assess surface penetration and lesion formation. Isolations were made from fruit skins on Kerssies' B. cinerea selective medium. Nectarine fruit were treated with paraquat, and grape berries were frozen for 1 h at -12°C. Paraquat and freezing were used to terminate host resistance and to promote the development of the pathogen from the tissues. In the natural infection studies B. cinerea occurred in a consistent pattern in bunches of the three cultivars. B. cinerea consistently developed from the tissue of the rachis, laterals, pedicel and pedicel-end, but not from the berry cheek. The rachis, lateral and pedicel contained much higher levels of B. cinerea than any position on the berry. Furthermore, the pathogen consistenly occurred at relatively high levels on the rachises throughout the season. Collectively, the data showed that in the Western Cape province, B. cinerea occured more regularly in wine grape bunches during the early part of the season, than later in the season. The data of the artificial studies confirmed the findings made with the natural infection studies. In these experiments the pathogen resided more often on the structural bunch parts than on the berries. Overall, the isolation studies revealed that conidia occurred predominantly on the rachis. The incidence of B. cinerea was furthermore constantly high in the inner bunch after each inoculation, and in bunches of different maturities. The data therefore indicated that, when available, conidia penetrated loose and tight clustered bunches in a similar way. Finally, in the virulence and pathogenicity experiments the results showed clearly that no host specialisation exists in the B. cinerea isolates used in this study. From these studies it is clear that in the Western Cape province B. cinerea occurs more readily in the inner structural parts of the bunches and more so during the earlier parts of the season. These findings should be considered when planning and implementing disease control programmes. / AFRIKAANSE OPSOMMING: INFEKSIEWEË VAN BOTRYTIS CINEREA OP GESELEKTEERDE WYNDRUIF KULTIVARS Indiepte kennis van die infeksieweë van Botrytis cinerea op druiwetrosse word benodig vir die beheer van dié vernietigende patogeen van druiwe. Vele studies is al gedoen om die moontlike infeksieweë van die swam op druiwe trosse te ondersoek. Die meeste van die studies het gebruik gemaak van kunsmatige inokulasie tegnieke waar die konidia van die swam in groepe op die korreloppervlak gedeponeer is. In die natuur is dit 'n rare verskynsel dat konidia in groepe op die korreloppervlak land. Die doel van die studie was om die infeksieweë van B. cinerea op drie wyndruif kultivars te ondersoek wat (i) natuurlik- en (ii) kunsmatig geïnokuleer is met konidia gedurende al die fenologiese stadia, en om die (iii) virulensie en patogenisisteit van isolate wat van verskillende gashere verkry is, op druiwe en nektariens te vergelyk. In die natuurlik-geïnokuleerde druiwe is die voorkoms van B. cinerea en die gevolglike siektevoorkoms op verkillende posisies in trosse van wyndruiwe (Chenin Blanc, Chardonnay, Shiraz) gedurende 1999 tot 2001 bepaal. Verskillende tegnieke is gebruik om lewensvatbare inokulum by verskillende posisies (ragis, lateraal, pedisel en pedisel-end van die korrel) in die tros waar te neem. Isolasies is op Kerssies' B. cinerea selektiewe medium gemaak, of trosse is onbehandeld gebruik, of behandel met paraquat. Paraquat is gebruik om die gasheer se natuurlike weerstand te verlaag en om die ontwikkeling van die patogeen te bevorder. Die plantmateriaal is onbehandeld gelaat om die patogeen op die oppervlak waar te neem, of die oppervlak is gesteriliseer om die latente myselium in die weefsel waar te neem. In die kunsmatige inokulasiestudies is trosse, van wyndruiwe (Chenin Blanc, Chardonnay, Shiraz), geïnokuleer met droë spore, van B. cinerea, in 'n inokulasietoring en die plantmateriaal is dan geinkubeer vir 24 h by 'n hoë relatiewe humiditeit (93%). Na die inkubasie proses is die trosse in twee groepe verdeel. Die een groep druiwe het oppervlak sterilisasie ondergaan in 70% etanol vir 5 s, en die ander groep was onbehandeld gelaat. Trosse van die onbehandelde en gesteriliseerde groep druiwe is gebruik vir isolasies. Vanuit elke tros is daar segmente van die ragis, laterale, pediselle en korrels (van die pedisel-end en wang gedeeltes) geïsoleer. Die segmente is in Petri bakkies met Kerssies' B. cinerea selektiewe medium en op water agar medium, wat paraquat bevat het, geïsoleer en geïnkubeer onder 'n 12 h dagligperiode teen 22°C. Die patogeen is positief geïdentifiseer deur sporuierende kolonies op die onderskeie weefseltipes. Laastens, in die virulensie- en patogenisiteitsproewe op druiwe en nektariens is verskillende isolate van B. cinerea, verkry vanaf verskillende gasheerplante, vergelyk deur natuurlike inokulasie toestande na te boots. Koue opgebergde vrugte, wat beskou word as hoogs vatbaar vir die infeksie van B. cinerea, is geïnokuleer met droë, enkel luggedraagde spore van die patogeen. Verskillende toetse is gedoen om die oppervlak penetrerende en letselvormende vermoëns van die onderskeie isolate te toets. Isolasies is van die skille van die vrugte gemaak en op Kerssies' B. cinerea selektiewe medium geplaas. Die nektarienvrugte is met paraquat behandel en die druifkorrels is gevries vir 1 h teen -12°C. Paraquat en bevriesing is gebruik om die gasheer se weerstand te verlaag en om die ontwikkeling van die patogeen te bevorder. In die natuurlik-geïnokuleerde studies het B. cinerea 'n konstante patroon getoon in die trosse van die drie verskillende wyndruif kultivars. B. cinerea het konstant ontwikkel uit die ragis, laterale, pedisel en pedisel-end, maar selde uit die korrelwang. Die ragis, lateral en pedisel dele het baie hoër vlakke van van die swam bevat as enige deel op die korrel. Die patogeen het ook konstant volop deur die hele seisoen op die ragis voorgekom. Gesamentlik wys die data dat, B. cinerea in wyndruiwe, in die Wes Kaap provinsie, meer geredelik vroeër in die seisoen voorkom, eerder as later. Data van die kunsmatige inokulasiestudies het die bevindinge van die natuurlike inokulasiestudies tot 'n groot mate bevestig. In dié studies het die patogeen meer geredelik die strukturele dele van die tros, eerder as op die korrels, bewoon. Oor die algemeen het die isolasieproewe gewys dat die konidia meer op die ragis voorkom as op enige ander deel. Die voorkoms van B. cinerea was ook oor die algemeen baie hoër in die strukturele dele van die tros, as op die korrel self. Die verskynsel het onder trosse van verskillende ontwikkelingsvlakke voorgekom. Die data het dus ook gewys dat konidia, wanner dit beskikbaar is, minder- sowel as meer kompakte trosse op 'n soortgelyke manier penetreer. Laastens, in die virulensie en patogenisiteitseksperimente het die resultate duidelik gewys dat daar geen gasheer spesifieke gedrag onder B. cinerea isolate is nie. In die studies het dit duidelik na vore gekom dat, B. cinerea meer geredelik in die strukturele binne dele van die wyndruif tros, in die Wes Kaap provinsie voorkom. En so ook eerder aan die begin van die seisoen, as later in die seisoen. Dié kennis moet in aanmerking geneem word by die beplanning en implementering van siektebeheerprogramme.

Thesis (MScAgric)--Stellenbosch University, 1956. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (PhD) -- Stellenbosch University, 1991. / ENGLISH ABSTRACT: The effect of partial defoliation as canopy management practice on metabolism and grape composition of the grapevine, Vitis vinifera L cv. Cabernet Sauvignon, was investigated. The establishment of physiologically sound principles on how to overcome deleterious effects associated with vigorous and dense-canopy vines is emphasized. Experimental vines were defoliated 33 % and 66 % evenly over the whole canopy from different developmental stages in a field study. Effects on canopy microclimate, photosynthesis, photosynthate translocation, vegetative growth, reproductive growth, root development and distribution, as well as grape and wine quality, were determined. A method for the simultaneous extraction of sugars and organic acids from freeze-dried berries at different developmental stages is described. Partial defoliation of vines improved canopy microclimate and photosynthetic efficiency of remaining leaves. Normal translocation and distribution patterns of photosynthates were apparently unaffected by partial defoliation. Translocation to and accumulation of photosynthetic products in the leaves and bunches of partially defoliated vines were, however, improved. Remaining leaves of partially defoliated vines were in comparison photosynthetically more active. Apart from a less favourable canopy microclimate, it seemed that the sink capacity of non-defoliated vines did not comply to the source capacity, inducing a reduced rate of photosynthesis. Normal sigmoidal growth patterns of vines were not affected by partial defoliation as applied in this study. This is important for the longevity, healthiness and productivity of vines. Vegetative growth was differentially affected by partial defoliation. No compensatory leaf growth occurred in reaction to partial defoliation from different developmental stages. Main shoot length, however, decreased slightly. Lateral shoot length and number of laterals increased, whereas cane mass decreased when vines were partially defoliated, particularly the earlier and more severe the defoliation. Reproductive growth in terms of yield was deleteriously affected by 33 % defoliation prior to pea size and 66 % defoliation prior to veraison. Budding percentage was, however, improved by 33 % and 66 % defoliation, whereas bud fertility was only improved by 33 % defoliation. Partial defoliation changed the canopy microclimate to conditions favourable for pest and disease control and higher grape quality. Subterranean growth was favourably affected by partial defoliation, particularly when applied from pea size stage. These changes included higher root densities, development of higher numbers of fine and medium diameter roots and occurrence of higher total root numbers in all soil layers. Generally, defoliations from pea size and veraison were more efficient regarding root development than defoliations from just after bud break and from berry set. Partially defoliated vines reacted by forming new roots, creating a more efficient nutrient absorption capacity and utilization of soil and available water and that, together with higher photosynthetic activities of leaves, provided an efficient mechanism for continued high performance. Grape quality was not affected markedly by partial defoliation. Total soluble solids in berries of defoliated vines were comparable to and even significantly higher than those of non-defoliated vines in some cases, in spite of much lower leaf areas. Generally, total titratable acidity of musts was also slightly higher for partially defoliated vines. Glucose and fructose concentrations in berries were unaffected by partial defoliation, while tartaric acid concentrations were slightly increased and malic acid concentrations slightly decreased. Partial defoliation generally increased the anthocyanin concentration of berry skins. These changes in grape composition suggest higher grape quality and seemed to result from improved light conditions in the canopy interior. Berry volume decreased with partial defoliation, which lowered the pulp:skin ratio. These berries are more desirable for quality wines. Regardless of severity or the developmental stage defoliation was commenced, wine cultivar character and overall wine quality were significantly improved. Partial defoliation changed the general metabolism of vines, mainly in terms of more favourable source:sink ratios, resulting in more efficient photosynthesis, subterranean performance and canopy microclimate. In general, the results suggest that an even removal of 33 % of leaves opposite and below bunches during the period from flowering or berry set to pea size stage may be applied. lt is further suggested that existing vigorous and dense-canopy vines be 33 % defoliated evenly on the lower half of the shoot (canopy) from pea size or veraison. This hypothesis proved effective in improving canopy microclimate, photosynthetic activity and yield, while vegetative growth was inhibited. Grape and wine quality were higher. On the whole, partial defoliation as applied in this study, is recommended as canopy management practice in order to facilitate the abolishment of deleterious effects of excessive vegetative growth and canopy density on balanced metabolic activity, fruit and wine quantity and quality, as well as longevity and healthiness of grapevines. / AFRIKAANSE OPSOMMING: Die invloed van blaarverwydering as loofbestuurspraktyk op die metabolisme en druifsamestelling van die wingerdstok, Vitis vinifera L cv. Cabernet Sauvignon, is ondersoek. Die vestiging van fisiologies-betroubare beginsels oor hoe nadelige effekte geassosieerd met geil en lower-verdigte wingerde uitgeskakel kan word, word beklemtoon. Proefstokke is in 'n veldondersoek 33 % en 66 % eweredig oor die hele lower vanaf verskillende ontwikkelingstadiums ontblaar. Effekte op lowermikroklimaat, fotosintese, translokasie van fotosintetiese produkte, vegetatiewe groei, reproduktiewe groei, wortelontwikkeling en verspreiding asook druif- en wynkwaliteit is bepaal. 'n Metode vir die gelyktydige ekstraksie van suikers en organiese sure uit gevriesdroogde korrels op verskillende groeistadiums is ontwikkel. Blaarverwydering het lowermikroklimaat verbeter en fotosintetiese doeltreffendheid van oorblywende blare op die stok verhoog. Normale translokasie en verspreidingspatrone van produkte van fotosintese is skynbaar nie deur blaarverwydering befiwloed nie. Translokasie na, en akkumulering van fotosintetiese produkte in die blare en druiwe van gedeeltelik ontblaarde stokke, is egter verbeter. Oorblywende blare van gedeeltelik ontblaarde stokke was fotosinteties meer aktief. Afgesien van 'n ongunstiger lowermikroklimaat, het die sinkkapasiteit van nie-ontblaarde stokke skynbaar ook nie teen die bronkapasiteit opgeweeg nie en is 'n verlaagde tempo van fotosintese verkry. Normale sigmofdale groeipatrone van die stokke is nie deur blaarverwydering befrivloed nie. Dit is belangrik vir langlewendheid, gesondheid en produktiwiteit van stokke. Vegetatiewe groei is differensieel deur blaarverwydering befrivloed. Geen kompenserende blaargroei het in reaksie op blaarverwydering vanaf verskillende ontwikkelingstadiums voorgekom nie. Hooflootlengte was egter effens korter. Sylootlengte en aantal sylote het toegeneem, terwyl lootmassa afgeneem het met blaarverwydering, veral hoe vroeer en strawwer dit toegepas is. Reproduktiewe groei in terme van opbrengs is nadelig befrivloed deur 33 % ontblaring voor ertjiekorrelstadium en 66 % ontblaring voor die deurslaanstadium. Botpersentasie is egter deur blaarverwydering verbeter, terwyl oogvrugbaarheid slegs deur 33 % ontblaring verbeter is. Blaarverwydering het 'n gunstige lowermikroklimaat vir die beheer van plae en siektes en hoer druifkwaliteit geskep. Ondergrondse groei is gunstig deur blaarverwydering befrivloed, veral wanneer toegepas vanaf ertjiekorrelstadium. Hierdie veranderinge het ingesluit hoer worteldigtheid, ontwikkeling van groter hoeveelhede wortels met 'n fyn en medium deursnit en die voorkoms van 'n groter aantal totale wortels in alle grondlae. Blaarverwydering vanaf ertjiekorrel- en deurslaanstadia was in die algemeen meer doeltreffend ten opsigte van wortelontwikkeling as blaarverwydering vanaf net na bot en vanaf korrelset. Gedeeltelik ontblaarde stokke het gereageer deur nuwe wortels te vorm, waardeur 'n meer doeltreffende vermoe tot voedingstofopname en benutting van grand an beskikbare water verkry is. Tesame met hoer fotosintetiese aktiwiteite van die blare is 'n doeltreffende meganisme vir aanhoudende hoe prestasie verkry. Druifkwaliteit is nie aanmerklik deur blaarverwydering beirwloed nie. Totale oplosbare stowwe in druiwe van gedeeltelik ontblaarde stokke was vergelykbaar en selfs betekenisvol hoer as die van nie-ontblaarde stokke in sekere gevalle, ten spyte van die baie laer blaaroppervlakte. In die algemeen was die totale titreerbare suur in die mos van gedeeltelik ontblaarde stokke oak effens hoer. Glukose- en fruktosekonsentrasies in die druiwe is nie deur blaarverwydering beinvloed nie. Die konsentrasie wynsteensuur is egter effens verhoog en die appelsuurkonsentrasie effens verlaag. Antosianienkonsentrasie van korreldoppe is in die algemeen deur blaarverwydering verhoog. Hierdie veranderinge in druifsamestelling dui op hoer druifkwaliteit en is skynbaar die resultaat van verbeterde ligtoestande in die binnekant van die lower. Korrelvolume het afgeneem met blaarverwydering. Dit het die pulp:dop verhouding verlaag. Sulke korrels is meer gewens vir bereiding van kwaliteitswyne. Wyn cultivarkarakter en totale wynkwaliteit is betekenisvol deur blaarverwydering verhoog, onafhanklik van die strafheid of ontwikkelingstadium waarvandaan blare verwyder is. Blaarverwydering het die algemene metabolisme van die wingerdstok verander, hoofsaaklik ten opsigte van meer gunstige bron:sink-verhoudings en derhalwe meer doeltreffende fotosintese, ondergrondse groei en lowermikroklimaat. Die resultate dui in die algemeen daarop dat 'n eweredige blaarverwydering van 33 % regoor en onderkant die trosse tydens blomvorming of korrelset tot ertjiekorrelstadium toegepas kan word. Die resultate dui verder daarop dat 33 %van bestaande geil en lower-verdigte stokke se blare eweredig verwyder kan word op die onderste helfte van die loot (lower) vanaf ertjiekorrel- of deurslaanstadium. Hierdie hipotese was suksesvol in die verbetering van lowermikroklimaat, fotosintetiese aktiwiteit en oesmassa. Vegetatiewe groei is gestrem. Druif- en wynkwaliteit was hoer. In die geheel kan blaarverwydering soos toegepas in hierdie ondersoek aanbeveel word as loofbestuurspraktyk ten einde die uitskakeling van nadelige effekte van oormatige vegetatiewe groei en lowerdigtheid op gebalanseerde metaboliese aktiwiteit, druif- en wynkwantiteit en kwaliteit, asook langlewendheid en gesondheid van wingerdstokke te bevorder.

Thesis (MScAgric)--Stellenbosch University, 1973. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (MSc)--Stellenbosch University, 1981. / ENGLISH ABSTRACT: No abstract available / AFRIKAANSE OPSOMMING: Die aard, omvang en beheer van voelskade by wyndruiwe is ondersoek in die streek Stellenbosch - Simonsberg. Die Mossie (Passer melanurus, Willer) en die Europese spreeu (Sturnus vulgaris, Linnaeus) veroorsaak die meeste voelskade aan.wyndruiwe in die streek. Die kultivars gebruik in die ondersoek Was Chenin blanc, Pint noir, Riesling, Cinsaut, Clairette blanche en Cabernet Sauvignon. Die laagste suiker- en tic.ogsbasuurgehaltes waarby voelskade voorgehom het was onderskeidelik 11,2° Balling en 25,9g per l,- terwyl vreetskade by gemiddelde 12,4°. Balling en 19,1 g per l totale suur begin het. Die belangrikste enkele faktor wat t rol gespeel het by beskadiging was die suikerkonsentrasie. Die patroon van vreetskade oor t wingerdry is bepaal en het getoon dat die eerste 30 stokke aan weerskante van die wingerd die meeste beskadig word. 'n Metode is vasgestel cm die hoeveelheid skade aangerig te bepaal met behulp van tellings van beskadigde korrels in verteenwoordigende wingerdrye. Die skade in die streek is op 2,5% van die totale opbrengs per hektaar bepaal. Die bespuiting van wingerdblokke en alternatiewe buffer-kantrye met die afskrikmiddel metiokarb (Mesurol) teen 'n konsentrasie van 0,1% or genoemde kultivars, asook Muskadel, het onvoldoende beheer gebied. Bespuitings teen hoer konsentrasies word aanbeveel.

Thesis MSc(Agric)--Stellenbosch University, 1973. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

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.

Climate is one of the most important factors influencing where wine grapes can be grown and the quality of wine produced from those grapes. A plants habitat has a profound influence on its growth and development. The surrounding climatic conditions at both the macro- and meso-scales influence the plant-climate miro-scale interactions. The main study site is the McKenzie Vineyard that is owned by Torlesse Wines. The climatic conditions of the surrounding Waipara region was also studied using climate data from the following vineyards; Canterbury House, River Terrace and Waipara West. The overall aim of this research is to improve understanding of the influence of the climatic environment on grapevine development at the meso- to micro-scale. The main findings of the research were firstly, that the most important climatic factor influencing grapevine development and growth is temperature and secondly that there is variability in the temperature across the Waipara Basin. Future research should be conducted for the entire growth season to gain a better understanding of how temperature influences the development of grapevine over the growing season as a whole.

Field and greenhouse experiments to determine the response of grapes to saline irrigation water were conducted. The goal of this research were: (1) to study the effect of salinity on grape and wine quantity and quality and (2) to evaluate the degree of salt tolerance of some of the grape rootstocks. For the greenhouse study, seven grape rootstocks were grown in the soil columns irrigated with three levels of salinity, EC of 0.45, 2.5, and 5 mmhos/cm. The later two waters were prepared by adding MgSO₄ and CaC1₂ salts to tap water with EC of 0.45 mmhos/cm. Shoot growth, pruning weight, leaf area, and trunk diameter were significantly reduced by salinity. Reduction in shoot growth and pruning weight were more pronounced than leaf area and trunk diameter. Maximum ECₑ values (1007 reduction in growth) varied from 8.81 mmhos/cm for 41B rootstock to 16.43 mmhos/cm for Ramsey rootstock. Maximum ECₑ for Barbera (Vitis vinifera) was 11.04 mmhos/cm. Based on percent reduction in growth, the relative tolerance of grapes could be arranged as follows: Ramsey > 5BB > SO4 > 1613 > Barbera > 99R > 41B. The field study included two sources of water and six grape rootstocks which were grafted to Barbera. Two sources of irrigation water were city and well water with EC of 0.42 and 2.6 mmhos/cm, respectively. The response of grapes to salinity was evaluated by fruit yield and pruning weight. Well water application significantly reduced fruit yield and pruning weight. The average fruit yield and pruning weight of Barbera grapes with all the rootstocks decreased by 49.5 7e and 26.7 7e with the well water compared to the city water, respectively. Must and wine analysis indicated that salt treated grape had higher total acidity and lower pH. Alcohol of the wines was not affected uniformly by treatment. Except for 99R rootstocks, the color of the wines were darker in city water than well water. Quality of wine from 3309 rootstock was lowered considerably by well water. With well water, only Barbera wine from 5BB rootstock appeared to be commercially acceptable. The six rootstocks differed from each other in their ability to growth in saline condition. Barbera grape grafted on 5BB and Ramsey rootstocks showed higher tolerance to salinity than Barbera on 99R, 3309, Harmony, and 41B rootstocks.