Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Soilless production of crops relies on the addition of high concentrations of nutrients with the irrigation water. The drained nutrient solution should be re-used to reduce the risk of pollution and to increase the water- and nutrient use efficiency of the system. Besides the risk of pathogen build-up, one of the main impediments of a wider application of this method is the frequent analysis required to maintain optimum nutrient concentrations and ratios in the rootzone. Yield reductions may be caused by an unbalanced nutrient solution. Alternatively the addition level of nutrients can be calculated through the use of nutrient uptake models that simulate the change in the re-circulated nutrient solution. To simulate crop water and nutrient demand necessary for model based regulation it was necessary to quantify the key factors affecting nutrient uptake by plants. The nutrient solution concentration and ratios between the macro-nutrients affected the uptake of water and nutrients. The total nutrient uptake per root dry weight increased and more specifically the nitrate (NO3 -), phosphate (H2PO4 -), potassium (K+) and sulphate (SO4 2-) uptake increased with an increase in nutrient solution electrical conductivity (EC) from 0.8 to 4.0 mS cm-1 while water uptake decreased. Except for Ca2+ uptake there was no correlation between nutrient and water uptake. Nutrient uptake can thus not be calculated based on water uptake. Instead a mechanistic high-affinity Michaelis-Menten based model can be used to estimate macro-nutrient uptake (Un, mg m-2 hr-1). Water and nutrient uptake was also affected by the solar radiation levels. Since nutrient uptake is related to the growth rate, solar radiation levels can be expected to influence nutrient uptake. The uptake of all ions increased with an increase in the solar radiation levels and for NO3 -, K+ and H2PO4 - the uptake rate was higher at higher nutrient solution concentrations. The Michaelis-Menten based model was adjusted to incorporate the effect of solar radiation levels on nutrient uptake. Water uptake (Wu, L m-2 day-1) was simulated as a function of crop transpiration and crop leaf area using a linear regression model, but since leaf area development was affected by solar radiation levels this was additionally incorporated into the estimation of the leaf area index (LAI). The composition of the nutrient solution also affected the biomass allocation of the crop which can again affect nutrient use as well as the fruit yield. There was also a direct effect of nutrient solution composition on fruit yield and quality with higher EC’s resulting in smaller fruit but an increase in fruit dry matter %, total soluble solids (TSS), titratable acidity (TA) and lycopene content. The results in this thesis make a valuable contribution to our understanding of the effect of nutrient availability (concentration and ratios) and nutrient requirement for growth (solar radiation levels) on nutrient uptake. Incorporating these into nutrient uptake models resulted in the development of a handy tool to simulate changes in composition of re-circulating nutrient solutions ultimately resulting in an improvement of the water and nutrient use efficiency of soilless systems. / AFRIKAANSE OPSOMMING: Die grondlose verbouing van gewasse is afhanklik van toediening van voedingselemente teen hoë peile in die besproeiingswater. Die voedingsoplossing wat dreineer moet hergebruik word om die risiko van besoedeling te verminder en ook om die water en nutriënt verbruik doeltreffendheid van die sisteem te verbeter. ʼn Ongebalanseerde voedingsoplossing kan ʼn verlaging in opbrengste veroorsaak. Benewens die risiko van patogene wat opbou, is die gereelde analises nodig word vir die handhawing van optimale nutriënt konsentrasies en verhouding tussen elemente in die wortelsone een van die hoof faktore wat ʼn meer algemene gebruik van die metode verhoed. Alternatiewelik kan die nutriënt toedieningspeile bereken word deur voedingstof opname modelle en simulasie van die verandering in water en nutriente wat dreineer. Om ʼn model gebaseerde reguleringsmetode daar te stel was dit nodig om die belangrikste faktore wat nutriënt opname beïnvloed te kwantifiseer. Beide die konsentrasie van die voedingsoplossing en die verhouding tussen elemente het ‘n effek gehad op die opname van water en nutriënte. Die totale nutriënt opname per wortel droë massa het toegeneem. Terwyl water opname afgeneem het met ‘n toename in die elektriese geleding (EG) van die voedingsoplossing vanaf 0.8 tot 4.0 mS cm-1 het die nitraat (NO3 -), fosfaat (H2PO4 -), kalium (K+) en sulfaat (SO4 2-) opname verhoog. Behalwe vir Ca2+ opname was daar geen korrelasie tussen water en nutriënt opname nie. Nutriënt opname kan dus nie bepaal word gebaseer op wateropname nie. Alternatiewelik is die gebruik van ʼn meganistiese hoë-affiniteit Michaelis-Menten-gebaseerde model voorgestel om die opname van makro-nutriente (Un, mg m-2 hr-1) te bepaal. Water- en voedingstofopname is beinvloed deur die ligintensiteit vlakke. Voedingsopname word bepaal deur die groei van die plant, daarom is dit verwag dat ligintensiteit vlakke die opname van voedingstowwe sal beïnvloed. Die opname van al die ione het toegeneem met 'n toename in die ligintensiteit vlakke en die tempo van NO3 -, K+ en H2PO4 - opname was hoër by 'n hoër voedingsoplossing konsentrasie. Die Michaelis-Menten gebaseerde model is aangepas om die effek van ligintensiteit vlakke op nutriënt opname te inkorporeer. Opname van water (Wu, L m-2 dag-1) is gesimuleer as 'n funksie van transpirasie en blaaroppervlakte met behulp van 'n lineêre regressiemodel en aangesien die blaaroppervlak ontwikkeling ook deur ligintensiteit vlakke beïnvloed word, is dit opgeneem in die skatting van die blaaroppervlakte-indeks (LAI). Die samestelling van die voedingsoplossing het die biomassa verspreiding beïnvloed. Dit kan nutriënt gebruik en vrug opbrengs beïnvloed. Die voedingsoplossing samestelling het vrug opbrengs en - kwaliteit beinvloed met kleiner vrugte, maar 'n toename in droëmateriaal %, totale oplosbare vastestowwe (TOVS), titreerbare suur (TA) en likopeen inhoud by ʼn hoër EG. Die resultate in hierdie tesis lewer 'n waardevolle bydrae tot ons begrip van die effek van nutriënt beskikbaarheid (konsentrasie en verhoudings) en voedingstof behoefte vir groei (ligintensiteit vlakke) op voedingsopname. Deur die inligting te inkorporeer in voedingsopname modelle het gelei tot die ontwikkeling van 'n handige instrument om die veranderinge in die samestelling van hersirkulerende voedingsoplossings te simuleer. Dit lei gevolglik tot die verbetering van die water en voedingstof gebruik doeltreffendheid van grondlose stelsels.

The work in this thesis aimed to demonstrate the contribution of arbuscular mycorrhizal (AM) fungi to the yield and fruit quality of field-grown processing tomatoes, and the potential to increase the sustainability of tomato production through greater fertiliser use efficiency by inoculating tomato seedlings with beneficial AM fungi. Previously, the conclusion that tomato growth is unresponsive to AM colonisation, particularly in high-P soils, has often been based on only a part of the tomato life-cycle. However, there is increasing evidence that that positive AM yield responses can occur in soils with relatively high plant-available P, and that AM responsiveness of tomato during vegetative growth may be a poor predictor of reproductive growth. A preceding industry study found that AM colonisation of field-grown processing tomatoes was very low, mostly less than 5%. The reason for the low colonisation was unclear since previous studies have shown that tomato can become relatively highly colonised by AM fungi. It was not known if farm practices, such as soil cultivation and chemical sterilisation, which have been shown to decrease AM colonisation of tomato and other crops, could have contributed to the low colonisation. Furthermore, it was unclear what contribution AM fungi were making to the yield and fruit quality of tomato in commercial production, and what their potential contribution might be if greater AM colonisation could be achieved through inoculating seedlings. Yield and fruit quality are important to tomato growers as both are used to calculate payment when the fruits are sold. Large amounts of soluble fertilisers, particularly P, are applied during tomato production with the aim of increasing yield and quality. However, fertiliser use efficiency, particularly P, on tomato farms has been identified as being low, and needing to be improved in order to increase the economic and environmental sustainability of tomato farming. Increasing P, and also other nutrients, such as Zn and Ca, in tomatoes could also help to improve agricultural sustainability by alleviating human malnutrition in developing countries and, in the case of Ca, have the potential to reduce blossom end rot, which can severely reduce marketable yield. There is considerable potential for AM fungi to assist in the supply of these nutrients to field-grown tomatoes. AM fungi are widely accepted to increase plant uptake of P. This has mostly been demonstrated in low-P soils, as increases in plant-available P are generally known to be detrimental to AM colonisation and any subsequent growth effects. However, there is increasing evidence of the ability of AM fungi to increase P uptake and yield even in high P soils. There is also good evidence of increased Zn uptake by mycorrhizal supply to plants. Evidence for increased Ca uptake in mycorrhizal plants is in comparison limited and conflicting, but has been demonstrated in some cases. It is possible that AM fungi could allow applications of these nutrients, particularly P, to be reduced while maintaining or increasing fruit yield and quality. However, the ability of indigenous or inoculated AM fungi to do so in the relatively high-P farm soils used in this project was unknown. In order to address these uncertainties a series of pot studies and a field experiment were conducted using field soils from tomato farms and an adjacent nature reserve for comparison. Data on soil characteristics from five farms, collected during the previous industry study, was analysed in conjunction with data from another farm located nearby with contrasting soil properties. Two farm soils and an unfarmed comparison were selected on the basis of their having contrasting levels of P, Zn and Ca, and pH, with the constraint that they were located within 50 km of each other to minimise travel time in the study area. The two farmed soils had a relatively high concentration of plant-available P (103 and 58 mg/kg Colwell), while plant-available P in the unfarmed soil was probably marginal to that required for healthy tomato growth (27 mg/kg Colwell). Samples of the soils were taken soon after commencement of the work and used in pot studies. Firstly, a bioassay was conducted to establish the ability of tomato to become colonised in the three field soils. AM colonisation of tomato and medic, which is known to be highly susceptible to AM colonisation, was compared between three harvests over an approx. 16 week period. Vegetative growth was also measured. The total colonisation of tomato mostly did not differ from that of medic at each harvest in any soil. Furthermore, despite the large differences in plant-available P between the three soils, colonisation and vegetative growth of tomato did not differ between soils at any harvest. In a subsequent pot experiment, the effect of colonisation by AM fungi in the three field soils on the vegetative and reproductive growth, and nutrient status of tomato was determined using the tomato mutant rmc (reduced mycorrhizal colonisation) and its progenitor 76R. A number of non-destructive vegetative and reproductive growth measurements were repeatedly measured over an approx. 24 week period. Destructive measurements were carried out at two harvests, 39 and 164 days after planting. Tomato 76R was again well colonised in all soils. Tomato rmc remained uncolonised, and was therefore an effective non-mycorrhizal control. AM colonisation had little effect on plant growth or nutrient status in any soil at the first harvest, but significant growth and nutrient responses were recorded at the second harvest. In particular, AM colonisation markedly increased vegetative growth in the unfarmed soil. AM colonisation did not affect vegetative growth in either of the farmed soils. However, AM colonisation increased reproductive growth, particularly yield over time, in all soils. AM colonisation increased shoot P concentration and content, but effects on Zn were mixed and largely inconclusive. Shoot Ca concentration and content were mostly reduced by AM colonisation. Similar patterns were observed in fruit nutrient status. The potential of pre-inoculation with AM fungi to increase AM colonisation and/or AM growth and nutrient effects in the field was considered. A commercial AM fungal inoculum was initially proposed for use, but was found to be unreliable and laboratory cultures of Scutellospora calospora and Glomus mosseae were used instead. Tomato seedlings were inoculated by amending a commercial seed-raising medium with an equal mixture of S. calospora and G. mosseae inocula. Seeds of tomato rmc, 76R and the commercial processing tomato cultivar U941 were sown and raised according to the practices followed by a commercial seedling nursery. After 9 weeks a sub-sample of inoculated seedlings of 76R and U941 had become colonised by both AM fungi, although the total colonisation was relatively low (approx. 10%). There was no difference in the shoot or root dry weights between inoculated and non-inoculated seedlings. The remaining seedlings were then used in the field experiment. Seedlings were transplanted amongst a commercial processing tomato crop on two farms and grown to maturity. A substitute farm with soil of moderate P (66 mg/kg Colwell) was used as tomatoes were no longer being grown on the initial farm with moderate P. Two P treatments, ‘normal’ and ‘reduced’ P fertilisation, were imposed in order to investigate the effect of P fertilisation on colonisation by indigenous and inoculated AM fungi, and growth and nutrient status of tomato in the field. Non-destructive growth measurements and soil core samples to assess mycorrhizal colonisation were taken mid-season (approx. 10 weeks after transplanting). Destructive growth measurements and core samples to assess colonisation were taken at harvest (approx. 19 weeks after transplanting). Colonisation of rmc was insubstantial and it again served as an effective non-mycorrhizal control to 76R. Colonisation was insubstantial in all treatments on the farm where soil had moderate plant-available P. On the other farm, where soil had relatively high plant-available P, colonisation of all plants was low mid-season, but was mostly substantial (>20%) in 76R and U941 at harvest. Low colonisation on both farms was probably the result of farming practices, particularly soil cultivation. However, a combination of inoculation and reduced P fertilisation increased colonisation. Colonisation by indigenous AM fungi had no effect on the growth or nutrient status of field grown tomatoes. In contrast, pre-inoculation with AM fungi increased fruit yield by a mean of approx. 40% in 76R and U941. This was the result of an 18% increase in the fresh weight of individual fruits and, when inoculation was combined with reduced P fertilisation, a 21% increase in the number of fruits on each plant. The increase in the number of fruits on each plant was associated with an increase in the number of flowers at the most advanced growth stage. Inoculation also increased vegetative growth, and fruit P, Zn and Ca contents. A small (4%) decrease in fruit brix was more than offset by increased yield. This study has shown that while AM fungi indigenous to tomato farm soils have the ability to substantially colonise tomato, they appear to have little effect on tomato growth, yield or nutrition in the field. In contrast, inoculation of tomato seedlings with mutualistic AM fungi during nursery production can substantially increase the growth, yield and fruit nutrient contents of field-grown tomatoes under commercial conditions. This increase could also be enhanced by a reduction in P fertilisation. Increased yield and fruit nutrient contents, and decreased P fertilisation neatly address the aims of increased agricultural sustainability. Incorporating pre-inoculation of tomato into existing farming practices has a potential to increase the productivity and sustainability of processing tomato production worldwide. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1292847 / Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2007

Non-mycorrhizal fungal endophytes are able to colonize internally roots without causing visible disease symptoms establishing neutral or mutualistic associations with plants. These fungi known as non-clavicipitaceous endophytes have a broad host range of monocot and eudicot plants and are highly diverse. Some of them promote plant growth and confer increased abiotic-stress tolerance and disease resistance. According to such possible effects on host plants, it was aimed to isolate and to characterize native fungal root endophytes from tomato (Lycopersicon esculentum Mill.) and to analyze their effects on plant development, plant resistance and fruit yield and quality together with the model endophyte Piriformospora indica. Fifty one new fungal strains were isolated from desinfected tomato roots of four different crop sites in Colombia. These isolates were roughly characterized and fourteen potential endophytes were further analyzed concerning their taxonomy, their root colonization capacity and their impact on plant growth. Sequencing of the ITS region from the ribosomal RNA gene cluster and in-depth morphological characterisation revealed that they correspond to different phylogenetic groups among the phylum Ascomycota. Nine different morphotypes were described including six dark septate endophytes (DSE) that did not correspond to the Phialocephala group. Detailed confocal microscopy analysis showed various colonization patterns of the endophytes inside the roots ranging from epidermal penetration to hyphal growth through the cortex. Tomato pot experiments under glass house conditions showed that they differentially affect plant growth depending on colonization time and inoculum concentration. Three new isolates (two unknown fungal endophyte DSE48, DSE49 and one identified as Leptodontidium orchidicola) with neutral or positiv effects were selected and tested in several experiments for their influence on vegetative growth, fruit yield and quality and their ability to diminish the impact of the pathogen Verticillium dahliae on tomato plants. Although plant growth promotion by all three fungi was observed in young plants, vegetative growth parameters were not affected after 22 weeks of cultivation except a reproducible increase of root diameter by the endophyte DSE49. Additionally, L. orchidicola increased biomass and glucose content of tomato fruits, but only at an early date of harvest and at a certain level of root colonization. Concerning bioprotective effects, the endophytes DSE49 and L. orchidicola decreased significantly disease symptoms caused by the pathogen V. dahliae, but only at a low dosis of the pathogen. In order to analyze, if the model root endophytic fungus Piriformospora indica could be suitable for application in production systems, its impact on tomato was evaluated. Similarly to the new fungal isolates, significant differences for vegetative growth parameters were only observable in young plants and, but protection against V. dahliae could be seen in one experiment also at high dosage of the pathogen. As the DSE L. orchidicola, P. indica increased the number and biomass of marketable tomatoes only at the beginning of fruit setting, but this did not lead to a significant higher total yield. If the effects on growth are due to a better nutrition of the plant with mineral element was analyzed in barley in comparison to the arbuscular mycorrhizal fungus Glomus mosseae. While the mycorrhizal fungus increased nitrogen and phosphate uptake of the plant, no such effect was observed for P. indica. In summary this work shows that many different fungal endophytes can be also isolated from roots of crops and, that these isolates can have positive effects on early plant development. This does, however, not lead to an increase in total yield or in improvement of fruit quality of tomatoes under greenhouse conditions. / Endophyten, die nicht zu den Mykorrhizapilzen gehören, können das Innere von Wurzeln ohne sichtbare Krankheitssymptome besiedeln und bilden so mit der Pflanze neutrale oder mutualistische Wechselwirkungen. Diese Pilze, auch als nicht-clavicipetale Endophyten bekannt, haben ein breites Wirtsspektrum von mono- und dikotyledonen Pflanzen und weisen eine hohe Diversität auf. Einige von ihnen fördern Pflanzenwachstum und erhöhen Resistenz und Toleranz gegenüber biotischem und abiotischem Stress. Ausgehenden von diesen möglichen Effekten auf ihre Wirtspflanzen war das Ziel der vorliegenden Arbeit die Isolierung und Charakterisierung neuer pilzlicher Wurzelendophyten der Tomate (Lycopersicon esculentum Mill.) und die Analyse ihres Einflusses auf Pflanzenentwicklung und Pflanzenresistenz, sowie auf Ertrag und Fruchtqualität unter Einbeziehung des Modellendophyten Piriformospora indica. Aus vier verschiedenen Anbaugebieten in Kolumbien konnten 51 neue Pilzstämme von oberflächensterilisierten Tomatenwurzeln isoliert werden. Diese Isolate wurden vorcharakterisiert und 14 potentielle Endophyten bezüglich ihrer Taxonomie, ihrer Besiedlungsmuster und ihres Einfluss auf das Pflanzenwachstum näher untersucht. Sequenzierung der ITS Region des ribosomalen RNA Genclusters und genaue morphologische Charakterisierung zeigten, dass sie zu verschiedenen phylogenetischen Gruppen innerhalb der Ascomycota gehören. Neun Morphotypen ließen sich beschreiben, wobei sechs zu den ‚Dark Septate Endophytes’ (DSEs) gehören, aber nicht mit der bekannten Phialocephala Gruppe verwandt waren. Ausführliche konfokale mikroskopische Untersuchungen ergaben sehr verschiedene Besiedelungsmuster der Wurzelendophyten vom Endringen in die Epidermis bis zum Hyphenwachstum durch den Kortex. Topfexperimente unter Gewächshausbedingungen zeigten dass die Isolate in Abhängigkeit von der Inokulumkonzentration und der Zeit der Besiedlung das Wachstum der Tomaten sehr unterschiedlich beeinflussten. Drei neue Isolate (die beiden unbekannte pilzlichen Endophyten DSE48 und DSE49 und eines identifiziert als Leptodontidium orchidicola) mit neutralen oder positiven Effekten wurden für weitere Versuche ausgewählt. In mehreren Experimenten sollte ihr Einfluss auf das vegetative Wachstum, auf Ertrag und auf Fruchtqualität untersucht werden, sowie ihre Fähigkeit die Auswirkungen des Pathogens Verticillium dahliae auf Tomatenpflanzen zu vermindern. Obwohl wachstumsfördernde Effekte durch alle drei Pilze in jungen Pflanzen beobachtet wurden, waren vegetative Wachstumsparameter nach 22 Wochen der Besiedlung nicht mehr beeinflusst bis auf ein signifikante Erhöhung des Wurzeldurchmessers durch den Endophyten DSE49. L. orchidicola dagegen erhöhte die Biomasse und den Glukosegehalt der Früchte, aber nur zu frühen Ernteterminen und bei einer bestimmten Intensität der Wurzelbesiedelung. Hinsichtlich eines schützenden Effekts, konnten die Endophyten DSE49 und L. orchidicola die Krankheitssymptome, die durch V. dahliae verursacht wurden, vermindern, aber nur bei einem geringen Pathogendruck. Um zu überprüfen, ob der Modellendophyt P. indica in Produktionssytemen eingesetzt werden kann, wurde seine Auswirkungen auf Tomaten untersucht. Ähnlich wie die neuen pilzlichen Isolate, zeigte aber auch er seinen fördernden Einfluss nur auf das frühe vegetative Wachstum. Schützende Effekte gegen V. dahliae konnten ebenfalls nur bei niedrigem Pathogendruck konstant beobachtet werden. Wie L. orchidicola erhöhte P. indica die Biomasse an marktfähigen Tomaten am Anfang des Fruchtansatzes, was nicht zu einem insgesamt höheren Ertrag führte. Ob die beobachteten Effekte auf ein verbesserte Nährstoffversorgung der Pflanze zurückzuführen seien, wurde in Gerste im Vergleich mit dem arbuskulären Mykorrhizapilz Glomus mosseae untersucht. Während der Mykorrhizapilz sowohl Phosphat wie Stickstoffaufnehme der Pflanze erhöhte, konnte dies für P. indica nicht festgestellt werden. Zusammenfassend zeigt diese Arbeit, dass auch aus Wurzeln von Kulturpflanzen viele verschiedene pilzliche Endophyten isoliert werden können, und dass einige von diesen durchaus einen positiven Effekt auf die frühe Pflanzenentwicklung aufweisen. Zumindest für Tomate unter Gewächshausbedingungen führen diese Effekte aber nicht zu einer Erhöhung des Gesamtertrags oder einer nachhaltigen Verbesserung der Fruchtqualität.

Analyse fonctionnelle de la protéine Enhancer of Zeste, SlEZ2, chez la tomate, Solanum lycopersicumLes protéines Polycomb, initialement découvertes chez la drosophile, ont récemment caractérisées chez les plantes où elles remplissent des fonctions essentielles au cours du développement de la plante. Chez la drosophile, les protéines polycomb (PcG) agissent sous forme de trois complexes multi-protéiques : PRC1, PRC2 et PhoRC. Seulement, deux de ces complexes ont été identifiés chez les plantes : un orthologue fonctionnel du complexe PRC1 (PRC1-like) et PRC2. Le complexe PRC2 maintien la chromatine dans un état condensé et intervient dans le contrôle du développement des fleurs, des graines, des fruits et des feuilles. Chez la tomate Solanum lycopersicum, le complexe PRC2 est composé de trois protéines polycomb : SlEMF2 (EMbryotic Flower), SlFIE (Fertilization Independent Endosperm) and SlE(Z) (Enhancer of Zeste). Les protéines SlE(Z) portent l’activité histone méthyl transférase qui permet la mise en place de la marque répressive H3K27me3. Chez la plante modèle, Arabidopsis thaliana, cette marque joue un rôle essentiel au cours du développement de la plante Afin d’étudier le rôle du complexe PRC2 dans le développement du fruit et de la plante de tomate, et plus particulièrement de la protéine SlE(Z), nous avons identifié trois gènes codant les protéines SlE(Z) : SlEZ1, SlEZ2 et SlEZ3. Au laboratoire, il a récemment été montré que la protéine SlEZ1 intervient au cours du développement floral (How Kit et al., 2010). L’objectif de ce travail est de déterminer la fonction de la protéine SlEZ2 au cours du développement du fruit et de la plante de tomate. Pour cela, nous avons analysé des plantes transgéniques sous exprimant le gène SlEZ2, orthologue au gène CURLY LEAF d’A. thaliana, par stratégie RNAi. Ce travail indique que la protéine SlEZ2 est impliquée dans la croissance de la plante de tomate, ainsi que dans le développement des feuilles, des fleurs et des fruits. Les plantes transgéniques présentent des phénotypes pléiotropes tels que des fleurs et des feuilles modifiées, un fort taux d’avortement des fruits, des fruits de texture et de couleur altérées ainsi qu’une réduction de la taille des plantes. De plus, nous avons identifiés quatre gènes ciblés par la protéine SlEZ2 dont l’expression est dérégulée dans les feuilles. Il s’agit de deux gènes à MADS box, TAG1 et TAGL1, ainsi que de deux gènes KNOX, LeT6 et TKN4. / Functional analysis SlEZ2, a tomato Enhancer of zeste proteinPolycomb proteins, first discovered in Drosophila, have been identified in plants and play essential functions in plant development. In Drosophila, polycomb proteins (PcG) acts as a complex and three have been identified: PRC1, PRC2 and PhoRC. However, only two polycomb complexes have been identified in plants: like-PCR1 and PRC2. The PCR2 complex maintain chromatin in a closed state and control flower, seed, fruit and leaf development.In tomato Solanum lycopersicum, PRC2 is composed by three polycomb proteins SlEMF2 (EMbryotic Flower), SlFIE (Fertilization Independent Endosperm) and SlE(Z) (Enhancer of Zeste)(Enhancer of Zeste). SlE(Z) proteins have a methyltransferase activity that puts in place an repressive epigenetic mark a trimethylation of lysine 27 histone 3. In plant model, Arabidopsis thaliana, this mark plays an essential role in plant development but little is known about PRC2 role in plant and fruit development of tomato. In order to unravel the function of the E(z) protein in the control of tomato fruit and plant development, we have characterized three E(z) encoding genes, namely SlEz1, SlEz2 and SlEZ3. In a recent work, we reported that SlEZ1 protein plays a role in flower development (How Kit at al., 2010). The aim of this present study was to determine the function of the SlEZ2 protein in plant and fruit development. We present our results focusing on RNAi transgenic plants which underexpressed SlEZ2 gene, homologue of Curly Leaf Arabidopsis gene. This analysis indicates that SlEZ2 protein is implicated in tomato plant growth and affects also leaf, flower and fruit development. Phenotypes include abnormal flowers and leafs, fruit development abortion, altered fruit colour and texture and plant of reduced size. Moreover, we characterize four target genes of SlEZ2 genes in leaves which present a deregulated expression : TAG1, TAGL1, LeT6 and TKN4.

Au sein de l’unité mixte de recherche 619 de l’Institut National de Recherche Agronomique, le groupe « Organogénèse du Fruit et Endoréduplication » étudie les acteurs moléculaires prenant part au contrôle du cycle cellulaire dans le fruit de tomate. L’objet de la présente thèse est l’étude de l’inhibiteur du cycle cellulaire Kip-Related Protein, et son rôle durant le développement du fruit. Identification de motifs protéiques fonctionnels chez l’Inhibiteur de Kinase Cycline-Dependent SlKRP1 chez Solanum lycopersicum : Leur rôle dans les interactions avec des partenaires du cycle cellulaire Les Kip-related proteins (KRPs) jouent un rôle majeur dans la régulation du cycle cellulaire. Il a été montré qu’ils inhibent les complexes CDK/Cyclin et ainsi bloquent la progression du cycle cellulaire. Malgré leur manque d’homologie avec leurs homologues animaux au delà de leur motif de liaison CDK/Cyclin, localisé à l’extrémité C-terminal de la protéine dans les séquences de plante, des études antérieurs ont montré la présence de motifs conservés spécifiques aux plantes chez certaines KRPs. Nous n’avons cependant que peu d’information concernant leur fonction. Nous montrons ici que les KRPs sont distribués en deux sous groupes phylogénétiques, et que chaque sous-groupe dispose de courts motifs spécifiques conservés. Les KRPs du sous-groupe 1 disposent ainsi de six motifs conservés entre eux. Utilisant SlKRP1, qui appartient au sous-groupe 1, nous avons identifié des motifs responsables de la localisation de la protéine et de ses interactions protéine-protéine. Nous montrons que le motif 2 est responsable de l’interaction avec CSN5, une sous-unité du complexe signalosome, et que le motif 5 a un effet redondant avec le motif 3 pour ce qui est de la localisation sub-cellulaire de la protéine. Nous montrons de plus que SlKRP1 est capable de guider SlCDKA1 et SlCycD3;1 vers le noyau, et ce même en l’absence du motif de liaison CDK/Cycline précédemment référencé. Ce nouveau site d’interaction est probablement localisé dans la partie centrale de la séquence de SlKRP1. Ces résultats apportent de nouveaux indices quant au rôle de la partie encore méconnue de cette protéine. La surexpression de SlKRP1 dans le mésocarpe de tomate détruit la proportionnalité entre endoréduplication et taille cellulaire Le fruit est un organe spécialisé résultant du développement de l’ovaire après pollinisation et fertilisation, et qui offre un environnement adéquat pour la maturation des graines et leur dispersion. De part leur importance en nutrition humaine et leur importance économique, les espèces à fruit charnu ont été le sujet d’étude développementales principalement orientée vers la formation de l’ovaire, la mise à fruit et la maturation du fruit. La phase de croissance du fruit a été beaucoup moins étudiée, bien que la division cellulaire et la croissance cellulaire prenant place durant cette période soient cruciales à la détermination de la taille finale du fruit, ainsi que de sa masse et sa forme. Le développement du mésocarpe du fruit de tomate se déroule par la succession d’une phase de division cellulaire suivie d’une phase d’expansion cellulaire associée à l’endoréduplication, menant à la formation de cellules géantes (jusqu’à 0,5mm) avec des niveaux de ploïdie pouvant atteindre 256C. Bien qu’une relation évidente entre endoréduplication et croissance cellulaire ait été montrée par de nombreux exemples chez les plantes, le rôle exact de l’endoréduplication n’a toujours pas été élucidé, étant donné que la plupart des expériences induisant une modification du niveau d’endoréduplication dans la plante affectaient aussi la division cellulaire. Nous avons étudié la cinétique du dévelopement du mésocarpe de tomate au niveau morphologique et cytologique et avons étudié l’effet de la diminution du niveau d’endoréduplication sur le dévelopement du fruit en sur-exprimant l’inhibiteur du cycle cellulaire Kip-Related Protein 1 (SlKRP1) spécifiquement dans les cellules en croissance du mésocarpe de tomate. Nous montrons une proportionnalité directe entre endoréduplication et taille cellulaire durant le développement normal du fruit, ce qui nous a permis de construire un modèle de développement du mésocarpe définissant l’épaisseur du péricarpe en ne prenant en compte que le nombre de divisions cellulaires et le nombre de tours d’endoréduplication. De façon surprenante, les mésocarpes de tomate affectés dans leur niveau d’endoréduplication par la sur-expression de SlKRP1 ne sont pas affectés au niveau de la taille des cellules ou du fruit, ni dans leur contenu métabolique. Nos résultats démontrent pour la première fois qu’alors que le niveau de ploïdie est étroitement lié avec la taille des cellules et du fruit, l’endoréduplication n’est pas responsable de la croissance cellulaire du mésocarpe de tomate. / Within the Joint Research Unit 619 of the National Institute of Agronomic Research (INRA), the group "Organogenesis of the Fruit and endoreduplication" examines the molecular players involved in cell cycle control in tomato fruit. The purpose of this thesis is the study of the cell cycle inhibitor Kip-Related Protein and its role during fruit development. Identification of protein motifs in the functional inhibitor of Cyclin-Dependent Kinase in Solanum lycopersicum SlKRP1: Their role in interactions with partners in the cell cycle The Kip-related proteins (KRPs) play a major role in the regulation of cell cycle. It has been shown to inhibit the CDK / Cyclin and thus block cell cycle progression. Despite their lack of homology with their counterparts in animals beyond their binding motif CDK / Cyclin, located at the C-terminal protein sequences in the plant, previous studies have shown the presence of conserved motifs plant specific in some KRPs, but there is little information about their function. We show here that the KRPs are distributed into two phylogenetic groups, and that each subgroup has specific short conserved motifs. The KRPs from subgroup 1 have six conserved motifs. Using SlKRP1, which belongs to subgroup 1, we have identified the motifs responsible for the localization of the protein and protein-protein interactions. We demonstrate that the pattern 2 is responsible for the interaction with CSN5, a subunit of the signalosome complex, and that the motif 5 is redundant with motif 3 with respect to the sub-cellular localization of the protein. We also show that SlKRP1 is capable of guiding SlCDKA1 and SlCycD3; 1 to the nucleus, even in the absence of CDK / cyclin binding motif previously referenced. This new site of interaction is probably located in the central part of the sequence of SlKRP1. These results provide new clues about the role of the little-known part of this protein. Overexpression of SlKRP1 in tomato mesocarp disrupts the proportionality between endoreduplication and cell size The fruit is a specialized organ which results from the ovary after pollination and fertilization, and provides a suitable environment for seed maturation and dispersal. Because of their importance in human nutrition and economic importance, fleshy fruit species have been the subject of study mainly focused on the developmental formation of the ovary, fruit set and fruit ripening. The stage of fruit growth has been much less studied, although cell division and cell growth taking place during this period are crucial to determining the final size of the fruit, as well as its mass and shape. The development of tomato fruit mesocarp occurs by the estate of a phase of cell division followed by a phase of cell expansion associated with endoreduplication, leading to the formation of giant cells (up to 0.5 mm) with ploidy levels of up to 256C. Although a clear relationship between endoreduplication and cell growth has been shown by many examples in plants, the exact role of endoreduplication has still not been elucidated, since most of the experiments leading to a change in the level of endoreduplication in plants also affected cell division. We studied the kinetics of the development of tomato mesocarp morphologically and cytologically and studied the effect of the reduced level of endoreduplication in the development of the fruit over-expressing the cell cycle inhibitor Kip-Related Protein 1 (SlKRP1) specifically in the growing cells of the tomato mesocarp. We show a direct proportionality between endoreduplication and cell size during normal development of the fruit, which allowed us to build a model for development of mesocarp defining the thickness of the pericarp by taking into account the number of cell divisions and the number of rounds of endoreduplication. Surprisingly, the tomato mesocarps affected in their level of endoreduplication by over-expression of SlKRP1 are not affected in terms of cell size and fruit, or on their metabolic content. Our results demonstrate for the first time that while the level of ploidy is closely linked with cell size and fruit, endoreduplication is not responsible for the cell growth of tomato mesocarp.

Made available in DSpace on 2014-12-17T14:13:22Z (GMT). No. of bitstreams: 1 SeveroP.pdf: 317890 bytes, checksum: b308c1b0abe5acf0e146092bb45e65ca (MD5) Previous issue date: 2007-08-17 / There are strong interests in the evaluation of the biological effects of natural and synthetic products. Blood constituents labeled with technetium-99m (99mTc) are used in nuclear medicine. The aim of this work was to study the effects of Clove (Caryophyllus aromaticas L.) and OZE (preparation used in the Health Sciences) on the labeling blood constituents with 99mTc and on the morphologic red blood cells (RBC) and the action of an extract of tomato (TO) on the labeling of blood constituents Blood samples were incubated with clove or OZE or TO, stannous chloride and 99mTc. Plasma (P), blood cells (BC), insoluble fractions (IF) of plasma and blood cells were separated. The radioactivity was counted and percentage of radioactivity (%ATI) to each blood fraction was calculated. The shape and morphometric parameter (perimeter/area ratio) were evaluated in the studies with clove and OZE. Clove extract and OZE altered significantly (p<0.05) the %ATI of blood constituents and the shape of red blood cells. However, clove extract not altered the red blood cells perimeter/area ratio. The tomato extract used at the highest concentrations reduced significantly (p<0.05) the %ATI in IF-P, although this extract did not modify the radiolabeling on BC, neither the radioactivity fixation on IFBC. The results indicate that these chemical compounds would have oxidative/chelating actions / Existe um grande interesse na avalia??o dos efeitos biol?gicos de produtos naturais e sint?ticos. Constituintes sang??neos marcados com tecn?cio-99m (99mTc) s?o usados em medicina nuclear. O objetivo desse trabalho foi estudar os efeitos do Cravo da ?ndia (Caryophyllus aromaticus L.) e do ?xido de Zinco Eugenol (OZE) (prepara??o usada em Ci?ncias da Sa?de) na marca??o de constituintes sang??neos com 99mTc e na morfologia de c?lulas vermelhas do sangue (CVS) e a a??o de um extrato de tomate (Solanum lycopersicum) (TO) na marca??o de constituintes sang??neos. Amostras de sangue foram incubadas com cravo da ?ndia ou OZE ou TO, cloreto estanoso e 99mTc. Plasma (P), c?lulas sangu?neas (CS), fra??es insol?veis (IFP) do plasma e das c?lulas sang??neas (IFC) foram separadas. A radioatividade foi contada e o percentual de radioatividade (%ATI) para cada fra??o sang??nea foi calculada. A forma e o par?metro morfom?trico (rela??o per?metro/?rea) foram avaliados nos estudos com o cravo da ?ndia e OZE. Os extratos de cravo da ?ndia e OZE alteraram significativamente (p<0,05) a %ATI dos constituintes sang??neos e a forma das CVS. Entretanto, o cravo da ?ndia n?o foi capaz de modificar a rela??o per?metro/?rea das CVS. O extrato do tomate usado na sua maior concentra??o reduziu significativamente (p<0,05) a %ATI na IF-P, embora esse extrato n?o tenha modificado a radiomarca??o na CS, nem a fixa??o de radioatividade na IF-CS. Os resultados indicam que esses compostos qu?micos poderiam ter a??es oxidativa e/ou quelante. O estudo tem car?ter multidisciplinar com a participa??o das seguintes ?reas do conhecimento: Radiobiologia, Bot?nica,Fitoterapia e Hematologia

Currently looking for alternative approaches to crop production which would be in accord with sustainable development. The present thesis was aimed on testing of organic cultivation of tomato (Solanum lycopersicum L.) and leek (Allium porrum L.) by using amendment with organic maize biomass (Zea mays L.), mycorrhizal fungi and saprotrophic fungi. The effects of different combinations of microbial inoculations on nitrogen uptake, plant growth and yield were investigated in greenhouse conditions. Supplied 15 N-labelled organic matter was separated from the root system by a nylon mesh which permitted only fungal hyphae to pass through but not plant roots. In the first year experiments the treatments differed in the presence or absence of three factors: organic matter, saprotrophic fungus Agrocybe sp. and mycorrhizal fungus Glomus mosseae (Nicolaj & Gerd.) Gerd. & Trappe. Plant inoculation with Agrocybe sp. alone or together with G. mosseae increased plant growth of tomato in the presence of organic matter. Tomato yield were not increased significantly. Shoot dry weight of leek increased when plants were treated with mycorrhizal fungus G. mosseae and organic matter. Microbial inoculation did not influence nitrogen (15 N) uptake from the organic source. In following experiments, all treatments contained...

Some microorganisms are known to form mutualistic symbiosis with plant roots and by their impact they can improve some plant parameters. These symbiotic microorganisms, which are able to improve some plant parameters, include especially mycorrhizal fungi, plant growth promoting bacteria and some saprotrophic mycoparasitical fungi. Mechanisms of changes of these parameters, as influenced by symbiotic microorganisms, are known only in part and nowadays are being actively researched. Aims of this work were to find out if selected microbial treatments influence selected growth, physiological and yield parameters of plants and contents of selected substances in fruits. Within this work were made three pot greenhouse experiments (experiments 1, 2 and 3) and three pilot greenhouse experiments (experiments 4, 5 and 6), performed on tomato (Solanum lycopersicum) and cucumber (Cucumis sativus) plants. Plants were grown in hydroponics using a carrier of rockwool and they were watered by nutrient solution. As microbial treatments for plants in experiments have been used a mixture of arbuscular mycorrhizal fungi (AM), mixture of plant growth promoting bacteria (PGPB), saprotrophic mycoparasitical fungus Trichoderma harzianum (Th) and various mutual combinations of these treatments. There have been observed...