Bromus tectorum, an invasive winter annual, has displaced native vegetation throughout the western United States. Bromus tectorum litter influences nutrient cycling near the soil surface as well as plant establishment. Failed seed production of B. tectorum occasionally occurs in the field, with plants exhibiting weak flowering culms that turn straw-colored in spring when normal plants are green or purple in color. Because annual grasses transport most soluble carbon (C) and nitrogen (N) to reproductive organs, seed production failure results in significantly different fates for these nutrients compared with normal plants. As part of larger efforts to understand events leading to large-scale seedling emergence failure (termed die-offs), occasionally observed in near mono-cultures of B. tectorum, we here test the hypothesis that prematurely senesced litter associated with seed production failure has higher soluble C and N than normal litter. C and N concentrations of aboveground biomass were compared for normal and prematurely senesced B. tectorum plants. Two methods were used to cause premature senescence: fungal pathogen infection with Clarireedia capillus-albis and glyphosate herbicide application. In a related experiment, field sampling of normal and prematurely senesced plants under natural conditions was conducted to compare C and N levels in-situ. Herbicide-induced senescence resulted in 1.5 to 2 times greater soluble C concentrations, but fungal infection had no effect on soluble C under experiment conditions. Prematurely senesced litter had increased total N concentrations, resulting in lower C:N ratios. The C:N ratio for prematurely senesced plants (averaged across all studies) was 68:1, whereas mature normal plants averaged 243:1. These findings illustrate failed seed production associated with premature senescence results in B. tectorum litter with significantly higher N concentrations and can result in increased soluble C concentrations. Altered nutrient status may contribute to changes in soil microbial activity, including activity of soilborne pathogens found in die-offs.

The objective of this research was to isolate genes encoding isoforms of the enzyme D-myo-inositol 3-phosphate synthase (MIPS, E.C. 5.5.1.4) from soybean and to characterize their expression, especially with respect to their involvement in phytic acid biosynthesis. A MIPS-homologous cDNA, designated GmMIPS1, was isolated via PCR using total RNA from developing seeds. Southern blot analysis and examination of MIPS-homologous soybean EST sequences suggested that GmMIPS1 is part of a multigene family of at least four similar members. The sequences of promoter and genomic regions of GmMIPS1 and GmMIPS2 revealed a high degree of sequence conservation. Northern and western blot analyses showed that MIPS transcript and protein are abundantly expressed early in seed development. Immunolocalization of MIPS protein in developing seeds confirmed expression of MIPS early in seed development and correlated MIPS protein accumulation in soybean seed tissue with tissues in which phytic acid is known to accumulate. The promoter region of GmMIPS1 was isolated and analyzed for possible seed-specificity using promoter:GUS fusions. Two GmMIPS1 promoter fragments were capable of conferring GUS expression when bombarded directly into developing soybean seeds. However, preliminary bombardment experiments into soybean cell suspension culture indicated that both promoter fragments drove expression of GUS in undifferentiated tissue, indicating a potential lack of seed-specificity. / Master of Science

Soybean is a dominant oilseed cultivated worldwide for its use in multiple sectors such as food and feed industries, animal husbandry, cosmetics and pharmaceutical sectors, and more recently, in production of biodiesel. Increasing demand of soybean, changing environmental conditions, and evolution of pathogens pose challenges to soybean production in limited acreage. Genetic research is the key to ensure the continued growth in soybean production, with enhanced yield and quality, while reducing the losses due to diseases and pests. This research is focused on the understanding of transcriptional regulation of two economically important agronomic traits of soybean: low seed phytic acid and resistance to Soybean mosaic virus (SMV), using the 'transcriptomics' and 'genomics' approaches. The low phytic acid (lpa) soybean is more desirable than conventional soybean, as phytic acid is an anti-nutritional component of seed and is associated with phosphorus pollution. Despite the eco-friendly nature of the lpa soybean, it shows poor emergence, which reduces soybean yield. This research is mainly focused on addressing the impact of lpa-causing mutations on seed development, which is suspected to cause low emergence in lpa soybeans. The differences in transcriptome profiles of developing seeds in lpa and normal phytic acid soybean are revealed and the biological pathways that may potentially be involved in regulation of seed development are suggested. The second research project is focused on Rsv3-type resistance, which is effective against most virulent strains of Soybean mosaic virus. The Rsv3 locus, which maps on to soybean chromosome 14, contains 10 genes including a cluster of coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) protein-encoding genes. This dissertation employed a comparative sequencing approach to narrow down the list of Rsv3 gene candidates to the most promising CC-NB-LRR gene. The evidence provided in this study clearly indicates a single CC-NB-LRR gene as the most promising candidate to deliver Rsv3-type resistance. / Ph. D.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Physic nut (Jatropha curcas L.) is an important crop due to its ability of storing high content of oil in the seeds, which can serve as raw material for biodiesel production. Because of the presence of toxic constituents like phorbol esters (PEs) and curcins, the seed cake produced as a result of oil extraction cannot be utilize for animal feed. Development of the genotypes better suited for the industrial applications and biodiesel production as well as with lower level of toxic constituents is being hampered by a lack of understanding about the a) proteins related to the biosynthesis and degradation of fatty acids (FAs) and triacylglycerides (TAGs), b) role of proteins deposited during seed development and c) proteins related to the synthesis and storage of toxic compounds during seed development. Agreeing with this, we have performed the anatomical analysis of the developing seeds of J. curcas, followed by the proteome analysis of the endosperm isolated from the seeds of J. curcas at five different developmental stages, which resulted into the identification of the 1517, 1256, 1033, 752 and 307 proteins, from Stage 6, 7, 8, 9 and 10, respectively, summing up to a total of 1760 proteins. Proteins with similar expression pattern were grouped into five different clusters and protein quantification based on spectral counts was determined. Besides identification of the proteins involved in the biosynthesis and degradation of the FAs and TAGs, we also identified a large number of proteins involved in the metabolism of the carbohydrates, which are important for supplying energy and carbon source for the synthesis of TAGs in heterotrophic seeds. Among the members of different classes of seed storage proteins (SSPs), we have identified four SSPs named as nutrients reservoir, which in contrast to the other SSPs showed decreasing deposition pattern during seeds development and revealed to have special role during seed development. In addition, peptidases belong to different mechanistic classes were identified, which have a range of functions, highlighting the role in reserve mobilization during germination. Isoforms of curcin were also identified in this proteome analysis which were absent in our previous proteome analysis of the other tissues from these seeds, suggesting that the deposition of these toxic proteins only occur in the endosperm. Similarly, several enzymes involved in the biosynthesis of diterpenoid precursors were identified in this proteome analysis but, like in our previous proteome analysis of the other tissues from J. curcas seeds,we were unable to identify any terpene synthase/cyclase, enzymes responsible for the synthesis of PEs, which collectively suggesting that the synthesis of PEs may not occur in seeds of this plant. In conclusion, the strategy used here enabled us to provide a first in depth proteome analysis of the endosperm from J. curcas developing seeds, which along with providing information regarding important aspects of the seed development, also set the foundation of a proteomic approach to study biotechnologically important plant species. / PinhÃo manso (Jatropha curcas L.) à uma cultura importante devido à sua habilidade em armazenar alto conteÃdo de Ãleo nas sementes, as quais podem servir como matÃria-prima para a produÃÃo de biodiesel. Devido à presenÃa de constituintes tÃxicos como Ãsteres de forbol e curcina, a torta da semente produzida como resultado da extraÃÃo do Ãleo nÃo pode ser utilizada na alimentaÃÃo animal. O desenvolvimento de genÃtipos mais adequados a aplicaÃÃes industriais e à produÃÃo de biodiesel assim como apresentando baixos nÃveis de constituintes tÃxicos està sendo prejudicado pela falta de entendimento sobre a) proteÃnas relacionadas a biossÃntese e degradaÃÃo de Ãcidos graxos e triacilglicerÃis, b) o papel de proteÃnas depositadas durante o desenvolvimento da semente e c) proteÃnas relacionadas à sÃntese e reserva de compostos tÃxicos durante o desenvolvimento da semente. Diante disso, nÃs realizamos uma anÃlise anatÃmica de sementes em desenvolvimento de J. curcas, seguido por uma anÃlise proteÃmica do endosperma isolado de sementes dessa espÃcie em cinco diferentes estÃgios de desenvolvimento, o que resultou na identificaÃÃo de 1517, 1256, 1033, 752 e 307 proteÃnas, dos estÃgios 6, 7, 8, 9 e 10, respectivamente, somando um total de 1760 proteÃnas. ProteÃnas com padrÃo de expressÃo similar foram agrupadas em cinco grupos diferentes e a quantificaÃÃo das proteÃnas baseada na contagem dos espectros foi determinada. AlÃm da identificaÃÃo das proteÃnas envolvidas na biossÃntese e degradaÃÃo de FAs e TAGs, nÃs identificamos um grande nÃmero de proteÃnas envolvidas no metabolismo de carboidratos, as quais sÃo importantes para o fornecimento de energia e fontes de carbono para a sÃntese de TAGs em sementes heterotrÃficas. Entre os membros de diferentes classes de proteÃnas de reservas de sementes (SSPs), nÃs identificamos quatro SSPs denominadas reservatÃrios de sementes, que em contraste as outras SSPs mostraram decrÃscimo no padrÃo de deposiÃÃo e revelaram ter um papel especial durante o desenvolvimento da semente. Em adiÃÃo, peptidases pertencentes a diferentes classes mecanÃsticas foram identificadas destacando o papel da mobilizaÃÃo de reservas durante a germinaÃÃo. Isoformas da curcina ausentes em nossas anÃlises proteÃmicas prÃvias de outros tecidos da semente foram identificadas sugerindo que a deposiÃÃo dessas proteÃnas tÃxicas sà ocorre no endosperma. Similarmente, vÃrias enzimas envolvidas na biosÃntese de precursores de diterpenÃides foram identificadas nessa anÃlise proteÃmica, mas como em nossas prÃvias anÃlises proteÃmicas de outros tecidos de sementes de J. curcas, nÃs nÃo fomos capazes de identificar sintases/ciclases de terpenos, enzimas responsÃveis pela sÃntese de PEs, o que coletivamente sugere que a sÃntese desses compostos pode nÃo ocorrer nas sementes dessa planta. Em conclusÃo, a estratÃgia utilizada nos fornece a primeira anÃlise proteÃmica profunda do endosperma de sementes em desenvolvimento de J. curcas, o que alÃm de fornecer informaÃÃes sobre aspectos importantes do desenvolvimento da semente, tambÃm estabelece a base para uma pesquisa proteÃmica com o objetivo de estudar espÃcies vegetais importantes biotecnologicamente.

Dans un contexte actuel qui tend vers une réduction d’intrants dans les systèmes de culture et une relance de la production de protéines végétales pour réduire la dépendance alimentaire de la France, la culture des légumineuses constitue une alternative. Les légumineuses à graines offrent une source riche en protéines pour l’alimentation animale et humaine. Au sein de l'UMR1347 Agroécologie, le pôle "déterminismes Génétiques et Environnementaux de l’Adaptation des Plantes à des Systèmes de culture Innovants" (GEAPSI) étudie via une approche multidisciplinaire (génétique, écophysiologie, physiologie moléculaire) l’adaptation des espèces végétales, et notamment des légumineuses aux contraintes environnementales. Ces travaux de thèse ont été réalisés au sein de l'équipe "Étude des Mécanismes Moléculaires" qui s'intéresse plus particulièrement à des critères de qualité de la graine (teneur en protéine, taille de graine) et au déterminisme génétique de ces caractères chez la plante modèle Medicago truncatula en vue d'un transfert de connaissances vers l'espèce cible Pisum sativum. Chez les légumineuses, la taille de la graine est déterminée par la capacité de l’embryon à se diviser lors de l’embryogenèse et à accumuler des réserves lors du remplissage. Aux stades précoces du développement, l’assimilation de nutriments est réalisée majoritairement par les tissus qui entourent l’embryon: l’albumen et le tégument. Les recherches menées visent à évaluer la contribution de l’albumen dans le développement de la graine chez M. truncatula. Nous avons révélé plusieurs gènes DOF (DNA-binding with One zinc Finger) comme étant exprimé dans ce tissu. Ils appartiennent à une large famille de facteurs de transcription impliqués dans de nombreux processus biologiques mais dont les rôles restent à préciser. Un de ces gènes, nommé DASH pour Dof Affecting Seed embryogenesis and Hormone metabolism, est exprimé préférentiellement dans l’albumen lors de l’embryogenèse. Des mutants TILLING et Tnt-1 isolés pour ce facteur de transcription sont affectés dans le développement de la graine (avortement à environ 10 jap). La cytologie du développement aux stades précoces (6 à 10 jap) a révélé que l’expression de ce gène dans l’albumen est requise pour un développement normal de l’embryon, démontrant le rôle de l’albumen dans le contrôle de l’embryogenèse chez les légumineuses. Une étude comparative du transcriptome des gousses de dash vs sauvage a permis d’émettre des hypothèses quant à la fonction du gène DASH. Une dérégulation du métabolisme hormonal, en particulier de l’auxine, a été mise en évidence et plusieurs gènes cibles potentiels de ce facteur de transcription ont été sélectionnés. Une comparaison du transcriptome des trois tissus de la graine à 12 jap a été réalisée chez la lignée sauvage de référence (Jemalong, A17). Elle a permis de préciser la localisation tissulaire de ces gènes cibles putatifs, de mettre en évidence des voies métaboliques plus spécifiques de l’albumen et de proposer des hypothèses quant à la fonction de ce tissu dans le développement de la graine. / In the current context, which necessitates a reduction in inputs in crop systems and boosting of production of plant proteins to reduce France’s dependency on feed imports,, growing legumes represents an alternative. Grain legumes are major sources of proteins for animal and human nutrition. In the UMR1347 Agroécologie, the objectives of the study group "déterminismes Génétiques et Environnementaux de l’Adaptation des Plantes à des Systèmes de culture Innovants" (GEAPSI) are to promote legume cultivation and adaptation to environmental stresses, via multidisciplinary approaches (genetics, ecophysiology, molecular physiology). This thesis project was carried out in the "Étude des Mécanismes Moléculaires" team, particularly interested in seed quality traits such as protein content or seed size and identification of genes implicated in variations of these trait s. Experiments were performed using Medicago truncatula as a model species for legumes with a view to transferring the information to the target crop species Pisum sativum.Legume seed size is determined by the embryo’s capacity to divide during embryogenesis and to accumulate reserves during seed filling. At early developmental stages, nutrient assimilation occurs predominantly in embryo-surrounding tissues: the endosperm and seed coat. This thesis project aims to evaluate the endosperm contribution to seed development in M. truncatula. We have shown several DOF (DNA-binding One Zinc Finger) genes to be expressed in this tissue. They belong to a large family of transcription factors implicated in numerous biological processes, but whose role remain to be elucidated. One of these genes, termed DASH for Dof Affecting Seed embryogenesis and Hormone metabolism, is expressed preferentially in the endosperm during embryogenesis. TILLING and TnT1 mutants isolated for this transcription factor are affected in seed development (abortion at 10 DAP). The cytology of development at early stages (6 to 10 DAP) revealed that the expression of this gene in the endosperm is required for the normal development of the embryo, demonstrating the role of the endosperm in the control of embryogenesis in legumes. A comparative transcriptomics study of dash vs wild-type pods allowed us to suggest hypothesis about the function of the DASH gene. Evidence for a deregulation of hormone metabolism, in particular for auxin, was obtained, and several potential target genes of this transcription factor were selected. A comparison of the transcriptome of the three tissues of the seed at 12 DAP was carried out for the reference wild-type line (Jemalong A17). This allowed the tissue localization of the target genes, to reveal metabolic pathways preferentially expressed in the endosperm, and to propose hypotheses about the role of this tissue during seed development.

Abstract The study gathered information about polyamine metabolism throughout the Scots pine (Pinus sylvestris L.) zygotic embryogenesis and about physiological events occurring simultaneously in the megagametophyte tissue. Additionally, novel sequence data of the Scots pine polyamine genes were used for studying the evolution of polyamine genes in plants. Phylogenetic analyses revealed that the eukaryotic ornithine decarboxylase (ODC) might have evolved from a multifunctional bacterial progenitor. In conifers, the alternative arginine decarboxylase (ADC) pathway is preferred in putrescine biosynthesis, which may have caused the relaxed purifying selection in the ODC genes. The phylogenetic analysis of spermidine synthase (SPDS), spermine synthase (SPMS) and thermospermine synthase (ACL5) sequences supported the view that eukaryotic SPDS genes are derived from a common ancestor, whereas SPMS genes have evolved several times from SPDS genes. The identified Scots pine sequence was defined as a putative thermospermine synthase (TSPMS) encoding gene and named PsACL5. The phylogenetic analysis of polyamine oxidase (PAO) sequences supported the view that plants possess several different PAOs, which may have different catalytic properties. The consistency of the polyamine concentration profiles during Scots pine zygotic embryogenesis suggested that polyamines have an important role in the embryo development and that individual polyamines may have different roles at different developmental stages. Generally, the polyamine concentrations increased at the early stages but decreased at the late stages of embryo development. Only the free putrescine fraction remained stable throughout the embryo development. Putrescine was almost solely produced via the ADC pathway and the ADC enzyme was at least partially transcriptionally regulated. Both ADC mRNA transcripts and ADC protein localized in dividing cells of embryos, which implicated the essential role of ADC in the mitosis of plant cells. The megagametophyte was viable from the early phases of embryo development until the early germination of mature seeds. However, the megagametophyte cells in the narrow embryo surrounding region (ESR) died via morphologically necrotic cell death. In the dying cells, extensive nucleic acid fragmentation caused the unspecific hybridization of probes in an in situ mRNA hybridization assay. The occurrence of necrotic cell death in Scots pine embryogenesis indicated that developmentally and physiologically regulated necrotic cell death is evolutionarily conserved and exists also in plants. / Tiivistelmä Työssä tutkittiin polyamiiniaineenvaihduntaa ja megagametofyyttisolukossa tapahtuvia fysiologisia muutoksia metsämännyn (Pinus sylvestris L.) alkionkehityksen aikana. Polyamiineja (putreskiini, spermidiini ja spermiini) syntetisoivia ja hajottavia entsyymejä koodaavien geenien emäsjärjestys selvitettiin metsämännystä. Sekvenssejä käytettiin kasvien polyamiinigeenien evoluution tutkimiseen. Tutkimuksessa todettiin, että eukaryooteissa putreskiinin biosynteesistä vastaava entsyymi, ornitiinidekarboksylaasi (ODC), on voinut kehittyä bakteerien lysiinikarboksylaasista (LDC), joka dekarboksyloi sekä ornitiinia että lysiiniä. Kasveissa putreskiinia voidaan tuottaa myös arginiinidekarboksylaasin (ADC) kautta, mikä on johtanut ODC-geeneihin kohdistuvan puhdistavan valinnan heikentymiseen. Aminopropyyli-ryhmiä liittävien entsyymien osalta tutkimus tukee käsitystä, jonka mukaan eukaryoottiset spermidiinisyntaasit (SPDS) ovat kehittyneet yhteisestä kantamuodosta, kun taas spermiinisyntaasi (SPMS) on syntynyt useita kertoja SPDS-geenin kahdentumisen kautta. Metsämännystä tunnistettiin termospermiinisyntaasia (TSPMS) koodaava geeni, jolle annettiin nimeksi PsACL5. Fylogeneettisen analyysin perusteella kasveissa on useita erilaisia polyamiinien hajotuksesta vastaavia polyamiinioksidaaseja (PAO), joiden katalyyttiset ominaisuudet voivat poiketa toisistaan. Metsämännyllä polyamiinipitoisuudet vaihtelivat alkionkehitysvaiheen mukaan yhdenmukaisesti eri vuosina, mikä viittaa polyamiinien tärkeään rooliin alkionkehityksessä. Polyamiinipitoisuudet kasvoivat varhaisen ja pienenivät myöhäisen alkionkehityksen aikana lukuun ottamatta vapaan putreskiinin pitoisuutta, joka pysyi samana koko alkionkehityksen ajan. Putreskiinia tuotettiin alkioissa lähes pelkästään ADC-reitin kautta, ja ADC-entsyymin säätelyn todettiin tapahtuvan ainakin osittain transkription tasolla. Koska sekä ADC-geenin lähetti-RNA että ADC-entsyymi löytyivät alkion jakautuvista soluista, on ilmeistä, että ADC-entsyymillä on tärkeä tehtävä kasvisolujen mitoosissa. Megagametofyytti säilyi elossa koko alkionkehityksen ajan lukuun ottamatta alkio-onteloa reunustavia soluja, jotka olivat morfologialtaan nekroottisia. Nukleiinihappojen voimakas pilkkoutuminen aiheutti soluissa koettimien epäspesifisen sitoutumisen, kun geenien ilmenemistä paikannettiin lähetti-RNA:han in situ hybridisaatio-menetelmällä. Tutkimuksessa löydetty männyn alkiokehitykseen liittyvä nekroottinen solukuolema osoitti ensimmäistä kertaa, että fysiologista ja kehityksellistä nekroottista solukuolemaa esiintyy myös kasveissa.

Strategies to improve stem yield and juice quality in sweet sorghum were investigated in this study. Seed quality of sixty five accessions (landraces) from Botswana was investigated. Standard germination tests revealed that only 66% of the accessions had germination percentages in excess of 85%. The Accelerated Ageing test showed that only 50%of the 26 accessions had germination percentages above 80%. The results indicated that Botswana sweet sorghum seed quality is generally poor. Seed development and maturity observations demonstrated that maximum seed quality occurred 14 to 17 days after mass maturity (physiological maturity) and this coincided with maximum seed germination. These results suggest that harvesting sweet sorghum seed prior to mass maturity can lower seed quality. Farmers should, therefore be advised to select plants intended for seed harvesting and allow them to mature properly before the seeds are harvested. Differences in seed colour, shape and compactness of the inflorescences were observed amongst the 65 landraces collected from farmers in Botswana. Ten landraces were characterised and from the results it was evident that there was a range of genetic diversity which can be utilized in the improvement of the crop. Large panicles were characteristic of most sweet sorghum landraces, the effect of tiller, panicle and floret removal on juice quality was consequently studied. Removal of panicles and florets significantly improved juice quality whilst removal of tillers did not. Selection and breeding of genotypes with small panicles and male sterile varieties may improve juice quality and should be investigated. Effect of planting date, spacing and nitrogen were investigated. Early planting (October) resulted in increased stem yields but reduced juice quality. A 30 cm intra-row spacing resulted in high stem yields per plant and good juice quality. Nitrogen fertilisation increased stem yield and improved juice quality. On the bases of the results obtained from this study, early planting (October), application of 60 kg N ha-¹, and 30 cm intra-row spacing could be recommended for sweet sorghum production in pure stands. In pure stands yields of more than 37 000 stems (per hectare) of good quality can be attained. These could be sold at an estimated price of P2.00 (R2.25) per stem indicating the potential of sweet sorghum as a cash crop. However, its economic viability depends on the price elasticity in the supply - demand function. / Dissertation (PHD)--University of Pretoria, 2003. / Plant Production and Soil Science / unrestricted

Les légumineuses sont une source riche pour l’alimentation humaine comme celle du bétail mais elles sont aussi nécessaires à une agriculture durable. Cependant, les fractions majeures des protéines de réserve dans la graine sont pauvres en acides aminés soufrés et peuvent être accompagné de facteurs antinutritionnels, ce qui affecte leur valeur nutritive. Dans ce cadre, Medicago truncatula est une espèce modèle pour l’étude du développement de la graine des légumineuses, et en particulier concernant la phase d’accumulation des protéines de réserve. Vu la complexité des graines de légumineuses, une connaissance approfondie de leur morphogenèse ainsi que la caractérisation des mécanismes sous-jacents au développement de l’embryon et au remplissage de la graine sont essentielles. Une étude de mutagenèse a permis d’identifier le facteur de transcription DOF1147 (DNA-binding with One Finger) appartenant à la famille Zn-finger, qui s’exprime dans l’albumen pendant la transition entre les phases d’embryogenèse et de remplissage de la graine. Lors de mon travail de thèse, il a été possible de générer plusieurs constructions pour l’analyse de l’expression de DOF1147 ainsi que de la protéine DOF1147. Un protocole efficace pour la transformation génétique stable de M. truncatula a été établi et des études de localisation subcellulaire ont montré que DOF1147 est une protéine nucléaire. Un arbre phylogénétique a révélé différents groupes de facteurs de transcription DOF avec des domaines conservés dans leur séquence protéique. L’analyse du promoteur in silico chez plusieurs gènes-cible potentiels de DOF1147 a identifié les éléments cis-régulateurs de divers facteurs de transcription ainsi que des éléments répondant aux auxines (AuxREs), ce qui suggère un rôle possible de l’auxine pendant le développement de la graine. Une étude in vitro du développement de la graine avec divers régimes hormonaux, a montré l’effet positif de l’auxine sur la cinétique du développement de la graine, que ce soit en terme de gain de masse ou de taille, plus fort avec l’ANA que l’AIB. Grâce à une approche cytomique de ces graines en développement nous avons, en plus, démontré l’effet de l’auxine sur la mise en place de l’endoreduplication. En effet, celle-ci est l’empreinte cytogénétique de la transition entre les phases de division cellulaire et d’accumulation de substances de réserve lors du développement de la graine. Dans son ensemble, ce travail a démontré que l’auxine module la transition entre le cycle mitotique et les endocycles chez les graines en développement de M. truncatula en favorisant la continuité des divisions cellulaires tout en prolongeant simultanément l’endoreduplication. / Legumes are not only indispensible for sustainable agriculture but are also a rich source of protein in food and feed for humans and animals, respectively. However, major proteins stored in legume seeds are poor in sulfur-containing amino acids, and may be accompanied by anti-nutritional factors causing low protein digestibility problems. In this regard, Medicago truncatula serves as a model legume to study legume seed development especially the phase of seed storage protein accumulation. As developing legume seeds are complex structures, a thorough knowledge of the morphogenesis of the seed and the characterization of regulatory mechanisms underlying the embryo development and seed filling of legumes is essential. Mutant studies have identified a DOF1147 (DNA-binding with One Finger) transcription factor belonging to the Zn-Finger family which was expressed in the endosperm at the transition period between embryogenesis and seed filling phase. During my PhD work, a number of transgene constructs were successfully generated for expression analysis of DOF1147 gene as well as the DOF1147 protein. A successful transformation protocol was also established for stable genetic transformation of M. truncatula. Subcellular localization studies have demonstrated that DOF1147 is a nuclear protein. A phylogenetic tree revealed different groups of DOF transcription factors with conserved domains in their protein sequence. In silico promoter analysis of putative target genes of DOF1147 identified cis-regulatory elements of various transcription factors along with auxin responsive elements (AuxREs) suggesting a possible role of auxin during seed development. A study of in vitro seed development under different hormone regimes has demonstrated the positive effect of auxin on kinetics of seed development in terms of gain in seed fresh weight and size, with NAA having a stronger effect than IBA. Using the cytomic approach, we further demonstrated the effect of auxin on the onset of endoreduplication in such seeds, which is the cytogenetic imprint of the transition between the cell division phase and the accumulation of storage products phase during seed development. As a whole, this work highlighted that the auxin treatments modulate the transition between mitotic cycles and endocycles in M. truncatula developing seeds by favouring sustained cell divisions while simultaneously prolonging endoreduplication.

Chez les eucaryotes, le rétromère est un complexe protéique composé d’un sous complexe SNX (Sorting Nexin) et d’une sous unité VPS (Vacuolar Protein Sorting) également appelé « core » rétromère. Le rétromère a été décrit comme un complexe régulant le transport des protéines membranaires au niveau de l’endosome. Chez Arabidopsis thaliana, les travaux de notre équipe ont démontré que ce complexe est impliqué dans différents processus développementaux tels que le développement de l’embryon, la maturation des protéines de réserves de la graine et l’initiation des racines secondaires. Dans ce travail, nous avons caractérisé la fonction du rétromère dans le développement des graines et des jeunes plantules d’Arabidopsis thaliana. D’une part, nous avons montré que VPS29 est nécessaire à la mise en place des réserves lipidiques de la graine. Nous avons identifié un nouveau « cargo » du complexe rétromère ; LTP6 (Lipid Transfer Protein 6) dont la perte de fonction engendre des phénotypes liés au métabolisme lipidique similaires à ceux du mutant vps29. Compte tenu de la localisation de LTP6 au niveau d’une structure intracellulaire spongieuse caractéristique du réticulum endoplasmique, le site de synthèse des corps lipidiques, nous supposons que le rétromère participe à la biogenèse des réserves lipidiques via sa fonction dans le trafic de ce nouveau « cargo ». D’autre part, nous avons mis en évidence que le « core » rétromère indépendamment de la sous-unité SNX est impliqué dans la mobilisation des réserves lipidiques, une fonction indispensable pour le développement des jeunes plantules. Nous avons montré que VPS29 est nécessaire à la translocalisation de la triacylglycérol lipase SDP1 (Sugar-Dependent 1) du peroxysome aux corps lipidiques, le compartiment de stockage des réserves lipidiques. Ces résultats nous ont permis d’envisager que le « core » rétromère pourrait emprunter de nouvelles voies de trafics intracellulaires entre des compartiments autre que l’endosome. / In eukaryotes, the retromer is a complex composed of the SNX (Sorting Nexin) subcomplex and the VPS (Vacuolar Protein Sorting) subcomplex also called the core retromer. To date, the retromer is described as a key regulator of proteins trafficking around endosomal compartment. In Arabidopsis thaliana, our group has previously demonstrated that this complex is involved in several developmental pathways, as embryo development, seed storage protein maturation and lateral root emergence. In this work, we characterised the function of the retromer in seeds and seedling development in Arabidopsis thaliana. Firstly, we found that VPS29 is required for the formation of seeds storage lipid. We identified a new cargo of this complex; Lipid Transfer Protein 6 (LTP6). LTP6 lost of function induces similar phenotype than vps29 linked to lipid metabolism. Based on LTP6 localisation on an intracellular structure characteristic of endoplasmic reticulum, the site of OBs formation, we supposed that the retromer may act on oil bodies biogenesis by its function on LTP6 trafficking. Secondly, we demonstrated that the core retromer have a SNX-independent function in lipid reserves breakdown, which is essential for seedling establishment. We showed that VPS29 is required for translocation of the triacylglycerol lipase SDP1 (Sugar-Dependent-1) from the peroxisome to oil bodies, the lipid storage compartment. Altogether, these results allowed us to propose new intracellular route trafficking for VPS sub-complex between compartments other than the endosome.