The secretory pathway of plant cells consists of several organelles that are connected by vesicle and tubular transport. Every compartment has a distinct function and the specificity of vesicle fusion is essential to maintain the organelles identity. N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in the secretory pathway driving specific vesicle fusions. A vesicle SNARE (v-SNARE) on a vesicle specifically interacts with two or three target SNAREs (t-SNAREs) on the target compartment. This event leads to vesicle membrane fusion with the membrane of the target compartment and the release of cargo molecules into the organelle lumen.<p> The aim of this work was the characterization of two <i>Arabidopsis thaliana</i> SNAREs. The first one is a v-SNARE, Bet11 that is the Arabidopsis ortholog of the yeast and mammal ER-Golgi v-SNARE, Bet1. In these organisms, Bet1 is involved in trafficking between the ER and Golgi apparatus. The second protein studied is a putative SNARE called Bet12 that shares high sequence identity with Bet11. In particular, I was interested in studying the sorting of these two proteins and their role in the secretory pathway of plant cells. By confocal laser microscopy, I demonstrated that these two proteins have different intracellular localization: Bet11 was mainly localized on the ER, Golgi stacks and punctate structures that I have identified as endosomes. Bet12 was localized only on the Golgi stacks. The identification of signal(s) involved in targeting of Bet11 and Bet12 were studied. To reach this aim I generated different mutant chimeras of Bet11 and Bet12. The co-expression of these chimeras with specific protein markers suggested that the distribution of these proteins was the result of a combined influence of multiple domains.<p> A serine in the Bet11 sequence was identified as a putative phosphorylation site and appeared important for proper Bet11 intracellular distribution.<p> The different intracellular distributions of Bet11 and Bet12 suggest different biological roles for the two proteins. To functionally characterize these two proteins homozygous knock-down mutants of Bet11 were screened. These plants had no evident phenotype, suggesting a possible genetic redundancy in this SNARE family.

Plant form and structure is remarkably diverse encompassing a myriad of shapes and sizes, and is a result of coordinated instances of cell growth and division. This varied form and structure represents adaptive strategies enabling plants to exploit and endure their environment maximizing fitness. Common to both cell growth and division processes, is the requirement of growth material and effectors via the secretory system. The secretory system is a dynamic pathway of organelles connected via vesicular traffic, responsible for protein modification and delivery. Challenges in studying secretion are related to its' dynamic flow between organelles, and the prevalence of redundancy and lethality, which cause difficulties in interpretation and genetic modulation. To further our understanding of the secretory process, we employed a chemical genetic approach to identify small molecule probes of secretion by first screening for inhibitors of cell expansion, followed by a microscopy based screen of GFP marker lines for perturbagens of subcellular structures. Small molecules offer the advantages of temporal and spatial application, have been shown to overcome redundancy and can be varied in concentration to control the severity of effects. We identified twenty-five small molecule probes of secretion and pursued two chemicals with striking properties, polarazine and eroonazole. Polarazine was identified as a cell division inhibitor, affecting phragmoplast structure and function, while eroonazole remodels the endoplasmic reticulum into small vesicles and is implicated in auxin signaling. This approach shows the utility of small molecule probes in furthering our understanding of secretory processes.

La embriogénesis es uno de los procesos más cruciales en el desarrollo vegetal. La secuenciación de ESTs (Expressed Sequences Tags) de la genoteca ATISLA, de semillas de 2 a 6 DDA (Días Después de Antesis), permitió identificar categorías funcionales típicas de desarrollo temprano de semilla como ciclo de división celular, reserva de nutrientes y traducción. A través del análisis in silico, considerando las ESTs de semilla inmadura depositadas en las bases de datos en una primera etapa y aquellos genes cuya expresión era elevada en semilla utilizando el programa Meta analyzer de Genevestigator en una segunda, se lograron identificar 49 genes cuya expresión es específica de semilla inmadura, resultados que fueron confirmados por RT-PCR e hibridación in situ. Al comparar estos 49 genes con el genoma completo de Arabidopsis, se encontraron diferencias estadísticas significativas para las categorías funcionales de reserva de reserva de nutrientes, metabolismo de carbohidratos, respuesta a estrés abiótico y tráfico subcelular y transporte.Las repeticiones anquirina son motivos de 33 aminoácidos repetidos en tándem cuya principal función descrita en animales es de interacción proteína-proteína. Participan en diversas funciones tanto en animales como en vegetales. Mediante análisis in silico fue posible identificar 509 repeticiones anquirina, que son codificadas por 105 genes. Se obtuvo una secuencia consenso de repetición anquirina de Arabidopsis que es similar a la de animales, por lo que es posible que las repeticiones anquirina en plantas cumplan la misma función que en animales. Los 105 genes codifican proteínas que se dividieron en 16 clases diferentes en función de los dominios o motivos que acompañaban a las repeticiones anquirina. El grupo más numeroso corresponde a las proteínas con dominios de repeticiones anquirina y transmembrana (37, proteínas ATANKTM). Mediante análisis neighbor joining de la región de dominios de repeticiones anquirina, estas proteínas se clasificaron en 6 familias distintas. Los genes AtAnkTm se encuentran repartidos en los cinco cromosomas de Arabidopsis y existen eventos de duplicación tanto a nivel inter- como intracromosómico, además de la presencia de genes duplicados en tándem (con 1 grupo de hasta 7 miembros). Mediante RT-PCR en diferentes órganos y bajo diferentes condiciones de estrés, fue posible observar que los genes AtAnkTm poseen patrones de expresión muy diferentes incluso dentro de una misma familia, por lo tanto, es posible que cumplan funciones muy variadas. Estos resultados fueron contrastados con análisis de bases de datos de ESTs y micromatrices utilizando el programa de visualización de expresión de genes de AtGenExpress. Se identificó un gen (AtAnkTm28) cuya expresión es en silicua inmadura y específicamente de semilla, mediante RT-PCR. Por hibridación in situ, se detectó expresión en embrión, suspensor y endospermo nuclear libre. La fusión de la región transmembrana de la proteína ATANKTM28 a GFP se localiza en algunos puntos específicos de la membrana citoplasmática. La mutación en los genes AtAnkTm2 y AtAnkTm9 produce una reducción en la viabilidad de los granos de polen. La mutación de AtAnktm2 también produce cambios en la estructura de la exina. / Embryogenesis is one of the most important steps in plant development. We have built a cDNA library (ATISLA) from isolated Arabidopsis seeds 2-6 DAA (Days After Anthesis) and used it for sequencing ESTs (Expressed Sequences Tags). The most abundant functional categories of the genes sequenced corresponded to cell division, nutrient reservoir and translation. We have used a two-step in silico subtraction analysis in order to identify genes specifically expressed in immature seeds. The first subtraction step was based on ESTs abundance in databases. The second subtraction step was based on microarray hybridization data obtained from the database Genevestigator. We identified 49 genes with specific expression in immature seed and we used RT-PCR and in situ hybridization analysis in order to verify the goodness of our selection method. Comparison of the functional categories of the 49 selected genes with the genes in the whole genome we identified statistically differences in some functional categories as for example nutrient reservoir, carbohydrate metabolism, abiotic stress response and sub cellular traffic and transport.Ankyrin repeats are 33-amino-acid domains that occur in tandemly arrayed sets of two or more, and are believed to function as protein-protein interaction domains. Several proteins having ankyrin repeats have been reported in animals and plants and they appear to be involved in many processes. We used in silico analysis to identify all Arabidopsis genes coding for proteins with ankyrin repeats. A total of 509 ankyrin repeats codified by 105 genes were identified. An ankyrin-repeat consensus sequence for Arabidopsis was determined, being very similar to the animal consensus, suggesting that ankyrin-repeats in plants play a similar role as in animals in protein-protein interactions. Arabidopsis ankyrin coding genes were classified in 16 groups according to the protein structures. The most abundant group contains 37 genes coding for proteins with ankyrin repeats and transmembrane domains (ATANKTM proteins). According to sequence similarity, they were classified in 6 families. The AtAnkTm genes are distributed through the five Arabidopsis chromosomes, including duplicated genes (intra- and inter- chromosomally) and groups of tandemly arrays genes. We used semi quantitative RT-PCR in order to determine the patterns of expression of these genes and we found that they were very variable, even inside a single family. These results were compared with ESTs databases and microarrays analysis using the tool Gene Expression Visualization of AtGeneExpress. One of the genes (AtAnkTm28) is expressed specifically in seeds at very early stages of development. In situ hybridization determined that this gene is expressed in the embryo proper, suspensor and free nuclear endosperm. Fusions of the transmembrane region of ATANKTM28 with GFP seem to localize at few specifics points of the plasma membrane. Mutations of two other AtAnkTm genes (AtAnkTm2 and AtAnkTm9) produce a reduction in pollen viability. Mutation in AtAnkTm2 also generates changes in the structure of the pollen surface (exin).

Les lipocalines sont des petites protéines de structure tertiaire simple qui ont l'habileté de lier de petites molécules généralement hydrophobes. De récentes études ont montré que les lipocalines animales jouent un rôle important dans la régulation du développement et sont impliquées dans la tolérance au stress oxydatif. Les plantes possèdent aussi des types variés de lipocalines. Les analyses bioinformatiques ont prédit que certains membres pourraient être localisés dans le chloroplaste, suggérant une fonction dans la protection de l'appareil photosynthétique lors de conditions de stress oxydatif. Ce travail présente la caractérisation fonctionnelle de la lipocaline chloroplastique AtCHL chez Arabidopsis thaliana. Un fractionnement cellulaire a montré qu'AtCHL est une protéine du lumen des thylacoïdes. La sécheresse, les hautes intensités lumineuses, et le traitement au paraquat et à l'acide abscissique induisent l'accumulation du messager et de la protéine AtCHL. Sous des conditions normales de croissance, les lignées de type sauvage, de sous-(KO) et sur-(OEX) expression ne montrent pas de différence au niveau phénotypique même si elles montrent des niveaux d'accumulation d'AtCHL dramatiquement différents. Lors d'un stress de sécheresse ou un stress oxydatif, l'absence d'AtCHL dans les plantes KO mène à plus de dommages alors qu'un haut niveau d'AtCHL permet aux plantes OEX de mieux surmonter les stress. Le niveau d'accumulation d'AtCHL est inversement proportionnel au niveau de lipides peroxydés. Ces résultats montrent qu'AtCHL est vraisemblablement impliquée dans la protection des lipides, localisés du côté luménal des thylacoïdes, contre les espèces réactives d'oxygène générées sous les stress de sécheresse et oxydatif. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Chloroplaste, Peroxydation des lipides, Lipocalines, Tolérance aux stress, Stress oxydatif.

La tesi doctoral s'emmarca dins de l'àrea de la biologia molecular de plantes, incloent algunes consideracions de tipus fisiològic, i descriu la identificació i caracterització de 5 gens Arabidopsis que codifiquen proteïnes amb similitud estructural amb els transportadors de coure eucariotes de la família Ctr. Els resultats del treball s'han dividit en tres capítols. En el primer d'ells s'analitza a nivell teòric les seqüències dels polipèptids de la família COPT d'Arabidopsis, s'investiga la seua funcionalitat en un sistema d'expressió heteròleg i es caracteritza de forma global el perfil d'expressió dels gens de la família. En aquest apartat es mostra que COPT1 i COPT2 restableixen amb gran eficiència el creixement en medi de selecció d'una soca de llevat deficient en el transport de coure d'alta afinitat i s'estableix que l'expressió dels dos transportadors es reprimeix en resposta a un tractament amb dosis tòxiques de coure. Per un altre costat, COPT3 i COPT5 complementen amb baixa eficiència al mutant de llevat i no responen al tractament amb coure. Les conclusions d'aquest primer apartat permeten proposar un model sobre la localització i funció cel·lular dels transportadors de la família. Els altres dos capítols de la tesi es centren específicament en l'estudi del gen COPT1. En el segon capítol, es descriu l'obtenció de plantes transgèniques que expressen un gen quimèric format per la fusió del promotor de COPT1 al gen GUS. L'anàlisi histoquímic d'aquestes plantes permet dilucidar que el gen COPT1 s'expressa en embrions i plàntules d'Arabidopsis. A més, en plantes adultes, l'expressió de COPT1 es restringeix als àpex radiculars, als tricomes, a les cèl·lules guarda i al pol·len. En l'últim capítol de la tesi s'exposa l'anàlisi fenotípic de plantes transgèniques que sobreexpressen i silencien COPT1, focalitzant l'estudi en els teixits descrits en el capítol anterior. Així, es mostra que la sobreexpressió de COPT1 causa hipersensibilitat al coure i l'activació dels sistemes de segrest cel·lulars, representats per la metal·lotioneïna MT1a. D'altra banda, el silenciament postranscripcional de COPT1 causa una reducció en l'absorció de coure, hipersensibilitat al dèficit de coure, un allargament de l'arrel primari i irregularitats en la formació de la coberta d'exina. Globalment, els resultats de la tesi descriuen per primera vegada l'expressió d'un transportador de coure als arrels d'una planta superior i assenyalen la participació de COPT1 en l'absorció de coure i en altres processos fisiològics i del desenvolupament vegetatiu i reproductiu d'Arabidopsis thaliana. / The work presented in this thesis describes the identification and characterization of 5 genes (COPT1-5) encoding putative Ctr-like Arabidopsis copper transporters. The first chapter encompasses a theoretical comparison of the COPT protein sequences, a functional study in a heterologous system and a preliminary expression and copper-regulation pattern analysis. COPT1 and COPT2 restore with high efficiency the growth of a yeast strain deficient in high affinity copper uptake and both genes are downregulated by exposure of leaves to copper treatment. COPT3 and COPT5 poorly substitute for their yeast counterparts and do not respond to copper treatment. Based on these results, a model for the cellular function of the COPT family members is proposed. The second chapter describes the generation and analysis of transgenic plants expressing a GUS reporter gene under the control of the COPT1 promoter. GUS specific staining is specifically detected in embryos, cotyledons, root tips, tricomes, guard cells and pollen grains. The last chapter describes the generation and phenotypical analysis of Arabidopsis transgenic plants overexpressing and silencing the COPT1 gene. Overexpression of COPT1 causes enhanced copper sensitivity and constitutive activation of the metallothionein gene MT1a. Overexpressing the complementary sequence of COPT1 produces a reduction in copper absorption, enhanced sensitivity to copper deficiency, increased root length and abnormalities in the pollen exine layer. Overall, the results presented here describe for the first time the expression of a copper transporter in the roots of a higher plant and indicate the participation of COPT1 in copper uptake and other physiological and developmental processes of Arabidopsis thaliana

In sexually reproducing individuals, intraorganismal genetic heterogeneity (IGH) or mosaicism is thought to occur infrequently while genetic homogeneity is presumed the norm. In organisms that undergo modular development, such as long-lived plants, IGH has been substantially documented. In Arabidopsis thaliana we have shown that non-parental DNA that is inherited at low but detectable rates can also manifest on single plants as genotypically distinct somatic sectors suggesting that even short-lived annual plants show IGH. The underlying mechanism responsible for generating this type of IGH remains unknown. In order to better understand this phenomenon I have tested the hypothesis that among genome changes that occur in response to stress, these putative triggers also up-regulate IGH. Metabolic stress, cold stress, mechanical damage and ROS exposure were examined. To test for IGH, transgene markers and polymorphic molecular markers were used. Also, presented in this thesis is work investigating the effect of in vitro propagation through tissue culture on IGH frequencies. Regenerated plants as well as undifferentiated callus tissue were genotyped and assayed for sequence reversions. Molecular genotyping revealed an outcome contrary to that predicted by the initial hypothesis showing instead that a high frequency of restoration occurred in the progeny of un-treated control plants. With the exception of samples passed through tissue culture, molecular marker changes, including single and double reversions of alleles, were detected in every line at some low level Furthermore, many of the revertants were found to be genetic mosaics. DNA sequence analyses revealed that sequences flanking three molecular markers that had undergone reversion were near identical to the great-grandparent of the sequenced individual. These results suggest that stress is perhaps an inhibitor of restoration. Although there may be other explanations for the results described in this thesis, the evidence implicates genome restoration as a mechanism for generating IGH.

The Arabidopsis TGA family of basic leucine zipper transcription factors regulate the expression of pathogenesis-related genes and are required for resistance to disease. Members of the family possess diverse properties in respect to their ability to transactivate and interact with NPR1, the central regulator of systemic acquired resistance in Arabidopsis. Two TGA factors, TGA1 and TGA2, have 83 % amino acid similarity but possess differing properties. TGA1 does not interact with NPR1 but is able to transactivate, while TGA2 interacts with NPR1 but is unable to transactivate. This study uses these two TGA factors to identify amino acids that are responsible for their function. <p>Four cysteines residues within TGA1 were targeted for study by site-directed mutagenesis and the resulting mutants were tested for interaction with NPR1 in yeast. The construct containing a mutation of cysteine 260 (Cys-260) interacted well with NPR1, while those with mutations at Cys-172 or Cys-266 interacted poorly. The Cys-260 mutant also displayed the greatest decrease in transactivation potential in yeast, while mutation of Cys-172 or Cys-266 resulted in smaller decreases. Mutation of Cys-287 had no effect on NPR1 interaction or transactivation. Combining various point mutations in a single protein did not increase NPR1 interaction or transactivation levels, indicating that Cys-260 is crucial for regulating TGA1 properties. Cysteines possess the unique ability of forming reversible disulfide bonds which have been shown to regulate several mammalian cellular processes. The observation that mutation of a single TGA1 cysteine (Cys-260) greatly alters the proteins properties provides a convincing argument that oxidoreduction of this residue is important for its regulation, possibly through the formation of a disulfide bond with either Cys-172 or Cys-266. <p>To test whether other members of the TGA family could be regulated by oxidoreduction, several TGA2 constructs were created that introduced Cys at positions corresponding to those found in TGA1. When tested in yeast none were able to transactivate but continued to interact with NPR1.

The secretory pathway of plant cells consists of several organelles that are connected by vesicle and tubular transport. Every compartment has a distinct function and the specificity of vesicle fusion is essential to maintain the organelles identity. N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in the secretory pathway driving specific vesicle fusions. A vesicle SNARE (v-SNARE) on a vesicle specifically interacts with two or three target SNAREs (t-SNAREs) on the target compartment. This event leads to vesicle membrane fusion with the membrane of the target compartment and the release of cargo molecules into the organelle lumen.<p> The aim of this work was the characterization of two <i>Arabidopsis thaliana</i> SNAREs. The first one is a v-SNARE, Bet11 that is the Arabidopsis ortholog of the yeast and mammal ER-Golgi v-SNARE, Bet1. In these organisms, Bet1 is involved in trafficking between the ER and Golgi apparatus. The second protein studied is a putative SNARE called Bet12 that shares high sequence identity with Bet11. In particular, I was interested in studying the sorting of these two proteins and their role in the secretory pathway of plant cells. By confocal laser microscopy, I demonstrated that these two proteins have different intracellular localization: Bet11 was mainly localized on the ER, Golgi stacks and punctate structures that I have identified as endosomes. Bet12 was localized only on the Golgi stacks. The identification of signal(s) involved in targeting of Bet11 and Bet12 were studied. To reach this aim I generated different mutant chimeras of Bet11 and Bet12. The co-expression of these chimeras with specific protein markers suggested that the distribution of these proteins was the result of a combined influence of multiple domains.<p> A serine in the Bet11 sequence was identified as a putative phosphorylation site and appeared important for proper Bet11 intracellular distribution.<p> The different intracellular distributions of Bet11 and Bet12 suggest different biological roles for the two proteins. To functionally characterize these two proteins homozygous knock-down mutants of Bet11 were screened. These plants had no evident phenotype, suggesting a possible genetic redundancy in this SNARE family.

The transport of phosphate (Pi) between subcellular compartments is central to metabolic regulation. Although some of the transporters involved in controlling the intracellular distribution of Pi have been identified in plants, others are predicted from genetic and biochemical studies. The Arabidopsis thaliana genome encodes a family of six proteins that share similarity with SLC17/type I Pi transporters, a diverse group of animal proteins involved in the transport of Pi, organic anions and chloride. Heterologous expression in yeast, and gene expression and localization studies in plants were used to characterize all six members of this Arabidopsis family, which we have named PHT4. All of the PHT4 proteins mediate Pi transport in yeast with high specificity. Bioinformatic analysis and localization of PHT4-GFP fusion proteins indicate that five of the proteins are targeted to the plastid inner envelope membrane, and the sixth resides in the Golgi apparatus. PHT4 genes are expressed in both roots and leaves although two of the genes are expressed predominantly in leaves and one mostly in roots. These expression patterns, together with Pi transport activities and subcellular locations, suggest roles for PHT4 proteins in the transport of Pi between the cytosol and chloroplasts, heterotrophic plastids and the Golgi apparatus.

In addition to AtCPL1-4, the genome of Arabidopsis thaliana encodes a large number of putative acid phosphatases. The predicted Arabidopsis SCP1-like small phosphatases (SSP) are highly homologous to the catalytic domain of eukaryotic RNA polymerase II carboxyl terminal domain (pol II CTD) phosphatases. Among the family members, SSP4, SSP4b and SSP5 form a unique group characterized by long N-terminal extensions. These three SSPs showed similar and ubiquitous gene expression. SSP4 and SSP4b were localized exclusively in the nuclei, while SSP5 accumulated both in the nucleus and cytoplasm. In vitro observation revealed that SSP4 and SSP4b dephosphorylated the pol II CTD-PO4 at both Ser2 and Ser5 in the conserved heptad repeats; however, SSP5 dephosphorylated only Ser5 of CTD-PO4. These results indicate that Arabidopsis SSP family encodes active CTD phosphatases similarly to animal SCP1 family proteins and plant CPLs family proteins, but with distinct substrate specificities. ssp mutants did not exhibit phenotypic abnormalities under normal growth conditions. However, ssp5 single mutants and ssp4 ssp4b ssp5 triple mutants showed enhanced sensitivity to ABA and glucose during seed germination. Yet, increased ABA-inducible gene expressions were not distinguishable in triple mutants compared to wild type plants upon ABA treatment. Unlike the ssp mutations, the cpl1 mutation strongly induced RD29A expression in response to cold, ABA and NaCl treatments. Thus, the cpl1 mutant is an ideal genetic background for an inducible gene expression system, in which the detrimental effect to host plants caused by a conventional constitutive expression could be avoided. Production of flavonoid such as anthocyanins in Arabidopsis is relatively easy to monitor and is regulated by transcription factors such as PAP1. PAP1 activates the expression of multiple enzymes in the anthocyanin biosynthesis pathway; however, high level of flavonoid production could cause vegetative growth retardation. To optimize flavonoid accumulation, a three-component system was designed consisting of a cold inducible RD29A-PAP1 expression cassette, a feedforward effector RD29A-CBF3, and a mutation in host repressor CPL1. Transgenic cpl1 plants containing both homozygous PAP1 and CBF3 transgenes produced 30-fold higher level of total anthocyanins than control plants upon cold treatment. LC/MS/MS analysis showed the flavonoid profile in cold-induced transgenic plants resembled that of previously reported pap1-D plants but were enriched for kaempferol derivatives. Furthermore, PAP1 and environmental signals synergistically regulate flavonoid pathway to produce a flavonoid blend that has not been produced by PAP1 overexpression or cold treatment alone. These results delineate the usability of the three-component inducible system in plant metabolic engineering.