Photosynthesis captures light from the sun and converts it into carbohydrates, which are utilised by almost all living organisms. The conversion between the different forms of carbohydrates is the basis to form almost all biological molecules. The main intention of this thesis has been to study the role of UDP-glucose in carbohydrate synthesis and metabolism, and in particular the genes that encode UDP-glucose pyrophosphorylase (UGPase) and UDP-glucose dehydrogenase (UGDH) in plants and their regulation. UGPase converts glucose-1-phosphate to UDP-glucose, which can be utilised for sucrose synthesis, or cell wall polysaccharides among others. UGDH converts UDP-glucose to UDP-glucuronate, which is a precursor for hemicellulose and pectin. As model species I have been working with both Arabidopsis thaliana and poplar. Sequences for two full-length EST clones of Ugp were obtained from both Arabidopsis and poplar, the cDNAs in Arabidopsis correlate with two genes in the Arabidopsis genomic database. The derived protein sequences are 90-93% identical within each plants species and 80-83% identical between the two species. Studies on Ugp showed that the expression is up-regulated by Pi-deficiency, sucrose-feeding and by light exposure in Arabidopsis. Studies with Arabidopsis plants with mutations in sugar/ starch- and Pi-content suggested that the Ugp expression is modulated by an interaction of signals derived from Pi-deficiency, sugar content and light/ dark conditions, where the signals act independently or inhibiting each other, depending on conditions. Okadaic acid, a known inhibitor of certain classes of protein phosphatases, prevented the up-regulation of Ugp by Pi-deficiency and sucrose-feeding. In poplar, sucrose also up-regulated the expression of Ugp. When poplar and Arabidopsis were exposed to cold, an increase of Ugp transcript content was detected as well as an increase in UGPase protein and activity. In poplar, Ugp was found to be expressed in all tissues that were examined (differentiating xylem, phloem, apical leaves and young and mature leaves). By using antisense strategy, Arabidopsis plants that had a decrease in UGPase activity of up to 30% were obtained. In the antisense plants, the soluble carbohydrate content was reduced in the leaves by at least 50%; in addition the starch content decreased. Despite the changes in carbohydrate content, the growth rate of the antisense plants was not changed compared to wild type plants under normal growth conditions. However, in the antisense lines the UGPase activity and protein content in sliliques and roots increased, perhaps reflecting compensatory up-regulation of second Ugp gene. This correlates with a slightly larger molecular mass of UGPase protein in roots and siliques when compared to that in leaves. Maximal photosynthesis rates were similar for both wild type and antisense plants, but the latter had up to 40% lower dark respiration and slightly lower quantum yield than wild type plants. Two Ugdh cDNAs from poplar and one from Arabidopsis were sequenced. The highest Ugdh expression was found in xylem and younger leaves. Expression data from sugar and osmoticum feeding experiment in poplar suggested that the Ugdh expression is regulated via an osmoticumdependent pathway.

Cellulose in particular and wood in general are valuable biomaterials for humanity, and cellulose is now also in the spotlight as a starting material for the production of biofuel. Understanding the processes of wood formation and cellulose biosynthesis could therefore be rewarding, and genomics and proteomics approaches have been initiated to learn more about wood biology. For example, the genome of the tree Populus trichocarpa has been completed during 2006. A single-gene approach then has to follow, to elucidate specific patterns and enzymatic details. This thesis depicts how a gene encoding a membrane-anchored cellulase was isolated from Populus tremula x tremuloides Mich, how the corresponding protein was expressed in heterologous hosts, purified and characterized by substrate analysis using different techniques. The in vivo function and modularity of the membrane-anchored cellulase was also addressed using overexpression and complementation analysis in Arabidopsis thaliana. Among 9 genes found in the Populus EST database, encoding enzymes from glycosyl hydrolase family 9, two were expressed in the cambial tissue, and the membrane-anchored cellulase, PttCel9A1, was the most abundant transcript. PttCel9A1 was expressed in Pichia pastoris, and purified by affinity chromatography and ion exchange chromatography. The low yield of recombinant protein from shake flask experiments was improved by scaling up in the fermentor. PttCel9A1 was however highly heterogenous, both mannosylated and phosphorylated, which made the protein unsuitable for crystallization experiments and 3D X-ray structure determination. Instead, a homology model using a well-characterized, homologous bacterial enzyme was built. From the homology model, interesting point mutations in the active site cleft that would highlight the functional differences of the two proteins could be identified. The real-time cleavage patterns of cello-oligosaccharides by mutant bacterial enzymes, the wildtype bacterial enzyme and PttCel9A1 were studied by 1H NMR spectroscopy, and compared with results from HPAEC-PAD analysis. The inverting stereochemistry for the hydrolysis reaction of the membrane-anchored poplar cellulase was also determined by 1H NMR spectroscopy, and it was concluded that transglycosylation in vivo is not a possible scenario. The preferred in vitro polymeric substrates for PttCel9A1 were shown to be long, low-substituted cellulose derivatives, and the endo-1,4--glucanase activity was not extended to branched or mixed linkage substrates to detectable levels. This result indicates an in vivo function in the hydrolysis of “amorphous” regions of cellulose, either during polymerization or crystallization of cellulose. In addition, overexpressing PttCel9A1 in A. thaliana, demonstrated a correlation with decreased crystallinity of cellulose. The significance of the different putative modules of PttCel9A1 was investigated by the construction of hybrid proteins, that were introduced into a knock-out mutant of A. thaliana, and the potential complementation of the phenotype was examined. A type B plant cellulase catalytic domain could not substitute for a type A plant cellulase catalytic domain, although localization and interaction motifs were added to the N- and C-terminus. / QC 20100802

Protein phosphorylation is an important post-translational modification of proteins, which can either activate or inhibit the function of a given protein. The enzymes, protein kinases and protein phosphatases catalyze the phosphorylation and dephosphorylation of target proteins, respectively. Protein kinases catalyze the transfer of γ-phosphate from ATP to serine, threonine or tyrosine residues in target proteins. They are traditionally classified as protein serine/threonine kinases and protein tyrosine kinases based on the amino acid to which they transfer the phosphate group. Protein tyrosine kinases play vital roles in numerous pathways that regulate growth, development and oncogenesis in animals. However, no protein tyrosine kinase has been cloned so far from plants. The sequence motif, CW(X)6RPXF of sub-domain XI is well conserved among biochemically characterized protein tyrosine kinases from human, rat, mice, worm, fruitfly and Dictyostelium. To seek plant genes encoding tyrosine kinase, we have performed extensive genome-wide analysis of Arabidopsis thaliana using the delineated tyrosine kinase from animal systems. Repetitive database mining with CW(X)6RPXF sequence motif revealed the presence of 57 different protein kinases that have tyrosine kinase motifs. Myosin light chain protein kinase was identified as false positive with this motif. Multiple sequence alignment of all the 57 kinases indicated the presence of twelve conserved sub-domains in their kinase catalytic domain. Out of the 12 sub-domains present in protein kinases, sub-domain VIb confers serine/threonine kinase Specificity and sub-domains VIII and XI confer tyrosine kinase specificity. All the 57 kinases were Verified to contain CW(X) 6RPXF in sub-domain XI. However, the catalytic domain of all the catalogued kinases contain KXXN motif in sub-domain VIb, which is indicative of serine/threonine Kinase specificity. None of the kinases had the tyrosine kinase consensus motif RAA or ARR in sub-domain VIb. Thus, the catalytic domains of all the identified Arabidopsis protein kinases have motifs for serine/threonine specificity in sub-domain VIb and tyrosine kinase motif in sub-domain XI. These results indicate that perhaps all the kinases belong to the dual-specificity kinase family. Hence, we have tentatively named these protein sequences as STY (serine/threonine/tyrosine) protein kinases. To examine the relationships of Arabidopsis STY protein kinases, a topographic cladogram was constructed. Casein kinase 1 was used as an outgroup to perceive the true class of STY protein kinase family. Neighbor joining tree was constructed with the full-length protein sequences following the alignments. Dendrogram of STY protein kinases suggested that the kinases are mainly clustered into four groups. Group I includes kinases related to ATN1-like kinases, peanut STY related kinases, soybean GmPK6-like kinases and ATMRK1-like kinases. Group II consists of MAP3K-like kinases, CTR1 and EDR1 related kinases. Group III includes protein kinases that harbor ankyrin domain repeat motifs. These kinases are related to Medicago sativa ankyrin kinase, MsAPK1. Group IV consists of light sensory kinases that are related to Ceratodon purpureus phytochrome kinase. C. purpureus light sensory kinase has both phytochrome and protein kinase domains. However, the protein kinases of group IV do not have a phytochrome domain. BLAST analysis was performed using CW(X)6RPXF motif against all the available plant sequences in the database. We retrieved 11 rice protein kinases and 14 Dictyostelium kinases. In addition, we obtained STY protein kinases from wheat, barley, soybean, tomato, beech and alfalfa. Dendrogram analysis indicated that the plant STY protein kinases are clustered in similar manner as observed for Arabidopsis. Large number of sequences were retrieved when the tyrosine kinase motif CW(X)6RPXF was used to perform BLAST analysis against all the known sequences from animals. As large numbers of protein tyrosine kinases are available in animals, we have used representative kinases of each family towards the construction of phylogenetic tree. The main difference between the animal and plant tyrosine kinases is in the consensus motif conferring the tyrosine and serine/threonine specificity in the sub-domain VIb. Animal tyrosine kinases have consensus ARR/RAA in sub-domain VIb and plant kinases have KXXN which is indicative of serine/threonine specificity. Expression analysis of Arabidopsis STY protein kinases was performed using Genevestigator online search tool Meta-Analyzer. Genes were grouped based on their relative expression levels during a specific growth stage, in a particular organ or following different environmental stresses. Various kinases are highly expressed in stamens and seeds while some kinases are expressed ubiquitously. A number of biotic and abiotic factors upregulated plant STY protein kinases. Gene expression data suggests the importance of STY protein kinases in plant growth and development. Genome-wide analysis is supported by phosphoproteomics in Arabidopsis seedlings. Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, phosphoamino acid analysis and peptide mass fingerprinting. Alkaline treatment detected two proteins corresponding to 46 and 37.5 kD. Phosphoamino acids analysis confirmed their dual-specificity nature. MALDI mass spectrometry and peptide mass fingerprinting analysis identified these two proteins as ATN1 and peanut serine/threonine/tyrosine protein kinase like protein from Arabidopsis. To further support the in silico approach, we have overexpressed one of the identified Arabidopsis thaliana serine/threonine/tyrosine protein kinases (AtSTYPK) in E. coli. The recombinant kinase was induced with IPTG and purified by using nickel-nitrilotriacetic acid affinity chromatography. AtSTYPK exhibited a strong preference for manganese over magnesium for kinase activity. The autophosphorylation activity of AtSTYPK was inhibited by the addition of calcium to reaction buffer containing manganese. The rate of autophosphorylation reaction was linear with increasing time and protein concentration. The AtSTYPK phosphorylated histone H1 (type III-S), and myelin basic protein (MBP) in substrate phosphorylation reaction and it did not phosphorylate casein or enolase. To see whether calcium or magnesium inhibits phosphorylation of MBP, we have performed the reaction in the presence of combination of different metal ions. The MBP phosphorylation reaction is more efficient in the presence of Mg2++ Mn2+ than Ca2++ Mn2+ under the same conditions. The recombinant kinase autophosphorylated on serine, threonine and tyrosine residues and phosphorylated myelin basic protein on threonine and tyrosine residues. The AtSTYPK harbors a manganese-dependent serine/threonine kinase domain, COG3642. H248 and S265 on COG3642 are conserved in AtSTYPK and the site-directed mutation of H248 to alanine resulted in loss of serine/threonine kinase activity, but the mutation of S265 to alanine showed a slight increase in its kinase activity. The protein kinase activity is regulated by various mechanisms that include autophosphorylation, protein phosphorylation by other kinases and by the action of regulatory domains or subunits. The role of tyrosine residues in the regulation of peanut dual-specificity kinase activity is well documented, but the importance of serine and threonine residues in the regulation of dual-specificity protein kinase is not studied so far. The kinase activity is generally regulated by phosphorylation of one or more residues within the kinase activation loop. The role of threonine residues in the kinase activation loop and the TEY motif of AtSTYPK were investigated in the present study. Four threonine residues in the activation loop and a T208 in the TEY sequence motif were converted to alanine to study their role in the regulation of kinase activity. The protein kinase activity was abolished when T208 and T293 of the activation loop were converted to alanine. Interestingly, the conversion of T284 in the activation loop to alanine resulted in an increase in both auto- and substrate phosphorylations. The mutation of T288 and T291 to alanine had no effect on kinase activity. There are eight serine residues in the kinase catalytic domain of AtSTYPK and surprisingly there is no serine residue in the kinase activation loop. So it is worthwhile to see how phosphorylation of serine residues regulates the dual-specificity protein kinase activity. The role of each serine residue was studied individually by substituting them with alanine. Serines at positions 215, 259, 269 and 315 regulate the kinase activity both in terms of autophosphorylation and substrate phosphorylation of myelin basic protein. The mutation of serine 265 to alanine resulted in slight increase in auto- and substrate phosphorylations, suggesting that it could be autoinhibitory in function. The other serine residues at positions 165, 181 and 360 did not show any change in the phosphorylation status when compared to wild-type AtSTYPK. In conclusion, this data suggests the importance of serine and threonine residues in the regulation of dual-specificity protein kinase activity and emphasizes the complexity of regulation of dual-specificity protein kinases in plants. To summarise, this study suggests that tyrosine phosphorylation in plants could be brought about only by dual-specificity protein kinases that phosphorylate on serine, threonine and tyrosine residues. This raises an interesting possibility that plants lack classical tyrosine kinases. The results provide a first report of manganese-dependent dual-specificity kinase from plant systems. This data also suggests that plant dual-specificity kinases undergo phosphorylation at multiple amino acid residues and their activity is regulated by various mechanisms, suggesting that they could be regulated by different mechanisms at different stages of plant growth and development. However, the role of dual-specificity kinases in planta has to be understood by mutant analysis in order to assign the physiological roles to these kinases. Further studies are needed to identify the upstream kinase(s) and downstream targets. Determination of physiological functions for dual-specificity protein kinases raises an important challenge in future in the area of plant signal transduction.

My dissertation research investigates the genetic architecture and evolutionary significance of physiological variation in two wildflower species, Ipomopsis aggregata and Arabidopsis thaliana. In particular, my work focuses on water use efficiency (WUE), a critical physiological trait that dictates plant growth and performance in resource-limited environments. I used a combination of quantitative trait loci (QTL) mapping, field selection experiments, and classic quantitative genetics to investigate 1) the genetic architecture of water use efficiency and flowering time, 2) patterns of natural selection on water use efficiency, flowering time, and other ecological traits in I. aggregata, and 3) additive genetic variation, genetic correlations, and selection on water use efficiency, flowering time, and plasticity to drought in Arabidopsis thaliana. Using an Ipomopsis aggregata genetic mapping population, I identified four QTL underlying WUE, three QTL-QTL epistatic interactions, and evidence for a possible QTL x cytoplasmic interaction affecting WUE. I found a similar genetic architecture underlying flowering time, with four main effect QTLs that all adjacently localized to the same linkage groups as WUE, and three QTL-QTL epistatic interactions, which occur between the same chromosome pairs as the WUE interactions. The combined main and interactive effects explain 35% and 40% of the phenotypic variation in WUE and flowering time, respectively. The adjacent localization suggests a possible role for the evolution of co-inheritance or, if the true QTL positions actually overlap, a possible role for pleiotropy underlying the phenotypic correlation between WUE and flowering time. Additionally, these results suggest epistasis is a significant factor affecting phenotypic variation in nature. In a reciprocal transplant and water addition experiment, I demonstrated variable natural selection on WUE, flowering time, and nectar production in I. aggregata across elevation/habitat and differential water availability. At low elevation in the water addition treatment, natural selection favors early flowering and greater nectar sugar concentration, while dry conditions favor high WUE and early flowering time. At high elevation, where the growing season is shorter and drier, selection favors early flowering regardless of water addition. These results suggest natural selection on ecophysiological and floral traits varies with resource availability (e.g. water availability and pollinator visitation). Using data from a glasshouse experiment involving a global panel of accessions of Arabidopsis thaliana, I demonstrated strong positive genetic correlation between WUE and flowering time, as well as selection for low WUE and early flowering under experimental season-ending drought. Finally, I found significant genetic variation in plasticity as well as selection favoring greater WUE plasticity under drought, indicating plasticity to drought is adaptive in A. thaliana. / text

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DHS) catalyzes the first step of the shikimate pathway - a pathway involved in Tyrosine (Tyr), Tryptophan (Trp) and Phenylalanine (Phe) biosynthesis - by condensation of phosphoenolpyruvate and erythrose-4-phosphate to DAHP. Our lab previously demonstrated that Arabidopsis thaliana shikimate pathway flux is regulated by Tyr and Trp. This project suggests that A. thaliana DHS1 overexpressor lines have increased Trp accumulation with Tyr treatment, and that an A. thaliana DHS2 overexpressor line treated with Tyr has unchanged Trp accumulation, indicating that AtDHS2 is Tyr-sensitive. Confocal microscopy of all 3 AtDHS isoforms fused to yellow fluorescent protein demonstrates chloroplast localization. Bimolecular fluorescence complementation indicates that protein-protein interactions occur in the cytoplasm, and not in the chloroplast, for AtDHS1 and AtDHS2 with the metabolic regulator At14-3-3ω. These findings suggest that protein-protein interactions could regulate accumulation of AtDHS2 in the chloroplast, and are perhaps modulated by Tyr.

La stabilité génomique des organelles de plantes suscite un grand intérêt dans le domaine de la biologie végétale. En effet, plusieurs études récentes suggèrent que ce type d’instabilité génomique pourrait mener à l’isolation de traits intéressants en l’agronomie. Plusieurs protéines sont d’ailleurs déjà été identifiés comme étant impliqués dans le maintien de la stabilité de ces génomes, tels que MSH1, la famille des POLI, OSB1, les protéines Whirly et les Recombinases A (RECA). Le génome nucléaire d’Arabidopsis thaliana encode trois protéines s’apparentant à la Recombinase A bactérienne et qui sont ciblées à la mitochondrie et/ou au chloroplaste, soit RECA1, RECA2 et RECA3. Globalement, ces gènes partagent une similarité de séquence de 61% avec leur homologue bactérien chez Escherichia coli. Chez les bactéries ces protéines jouent un rôle essentiel dans la recombinaison homologue et sont impliquées dans la réparation de l’ADN. Chez Arabidopsis, il a été démontré que RECA2 et RECA3 sont nécessaires au maintien de l’intégrité du génome mitochondriale. Toutefois leur contribution à la stabilité du génome chloroplastique ainsi que le rôle de RECA1 restent obscures. Le but de ce projet est donc de déterminer la contribution éventuelle des protéines RECA d’Arabidopsis dans la réparation de l’ADN chloroplastique et plus précisément le rôle du gène RECA1. Nous énonçons l’hypothèse que les RECA de plantes se comportent effectivement comme leurs orthologues bactériens en étant impliqués dans la recombinaison homologue. Dans le cadre de ce projet, nous avons tenté d’isoler des lignées mutantes pour chacun des gènes RECA d’Arabidopsis. En somme, nous avons pu obtenir des lignées convenables pour notre étude que dans le cas du gène RECA1. Ces lignées ont été utilisées pour évaluer la contribution de ce gène à la stabilité du génome du chloroplaste. Ensuite, pour étudier la relation épistatique des gènes RECA1, WHY1 et WHY3, un croisement des différentes lignées mutantes pour ces gènes a été réalisé. Nous avons ensuite étudié la sensibilité de toutes ces lignées mutantes à la ciprofloxacine, un agent causant des bris double brin exclusivement dans les organelles de plantes. Finalement, iii nous avons testé la présence de réarrangements dans le génome du chloroplaste en condition normal ou en présence de stress génotoxique. Nos résultats démontrent que les protéines Whirly et RECA1 sont impliquées dans deux voies de réparation de l’ADN différentes et que les Whirly sont suffisantes pour s’occuper des bris d’ADN double brin en l’absence de RECA1. Nous démontrons également que l’absence de Whirly et RECA1 entraine une forte augmentation de la quantité de réarrangements dans le génome du chloroplaste. De plus nous proposons que la polymérase POLIB est impliquée dans la même voie de réparation que RECA1. Finalement nous proposons un modèle pour expliquer nos résultats et impliquons RECA1 dans un mécanisme de réparation d’ADN et aussi un rôle potentiel dans la réplication. / The stability of plant organelles genomes elicits a great interest in the domain of plant biology. In fact, numerous studies suggest that genomic instability can lead to the isolation of interesting traits in the field of agronomy. Some factors such as MSH1, the POLI family, OSB1, the Whirly proteins and the Recombinase A (RECA), have already been identified has being implicated in the maintenance of genome stability. The nuclear genome of Arabidopsis thaliana encodes three proteins, RECA1, RECA2 and RECA3, that shares a high resemblance with bacterial Recombinase A. They are targeted to the mitochondria and/or to the chloroplast. Globally, these genes share a similarity of sequence of 61% with their bacterial homologue in Escherichia coli. In bacteria these proteins play an essential part in homologous recombination and are implicated in DNA repair. In Arabidopsis, RECA2 and RECA3 have been shown as being essential to maintain the integrity of the mitochondrial genome but their contribution to the stability of the chloroplast as well as the role of RECA1 remains obscure. The goal of this project is to establish the eventual contribution of the Arabidopsis RECA proteins in the repair of chloroplast DNA and more precisely the role of the RECA1 gene. We propose the hypothesis that plants RECA act in the same fashion as their bacterial orthologues by being implicated in homologous recombination. Within the framework of this project, we have attempted to isolate mutant lines for each RECA gene of Arabidopsis. In the end, we were able to obtain appropriate lines for our study only for the RECA1 gene. These lines were then used to evaluate the contribution of the gene to chloroplast genome stability. Afterwards, in order to study the epistatic relationship between the RECA1, WHY1 and WHY3 genes, a cross between different mutant lines of these genes was realised. We then studied the sensitivity of all of those mutant lines to ciprofloxacine, an agent causing double stranded breaks exclusively in plant organelles. Finally, we evaluated the presence of rearrangements in the chloroplast genome under normal conditions and under the presence of a genotoxic stress. Our v results show that the Whirly and RECA1 proteins are implicated in two separate pathways of DNA reparation and that the Whirly proteins are sufficient to take in charge DNA double strand breaks generated by the absence of RECA1. We also demonstrate that the absence of Whirly and RECA1 causes an increasein the quantity of rearrangements in the chloroplast genome. Furthermore, we propose that the polymerase POLIB is implicated in the same repair pathway as RECA1. Finally we propose a model to explain our results and implicate RECA1 in a DNA repair mechanism and propose a role for RECA1 in DNA replication.

Municipal treated wastewater entering rivers contain biologically active pharmaceuticals capable of inducing effects in aquatic life. Phytoremediation of three of these pharmaceuticals and an herbicide was investigated using Sandbar willow (Salix exigua) and Arabidopsis thaliana. Both plants were effective at removing compounds from solution, with removal of 86% of the synthetic estrogen, 17α-ethynylestradiol, 65% of the anti-hypertensive, diltiazem, 60% of the anti-convulsant, diazepam (Valium®), and 51% of the herbicide atrazine, in 24 hours. Distribution of compounds within roots and shoots, in soluble and bound forms, differed among compounds. Uptake and distribution of pharmaceuticals within the study plants confirmed pharmaceutical behaviour can be predicted based on a physiochemical property, their octanol-water partitioning coefficients. An effective method for detection of 17α-ethynylestradiol within surface water using solid phase extraction and gas chromatography-mass spectrometry was developed. Previously unreported breakdown of 17α-ethynylestradiol into another common estrogen, estrone, during preparative steps and gas chromatography was resolved. / xv, 216 leaves ; 29 cm.

Le chloroplaste est un organite spécifique de la cellule végétale. Il est délimité par une double membrane ou enveloppe qui renferme de nombreux systèmes de transports d'ions et de métabolites essentiels au fonctionnement du chloroplaste et de la cellule végétale. A ce jour, seuls quelques transporteurs de métaux associés à l'enveloppe des plastes ont été identifiés : un transporteur de fer, un transporteur de magnésium ainsi que deux ATPases à cuivre de type P1B : HMA1 et PAA1. PAA1 représenterait la voie principale d'import du cuivre dans le chloroplaste, notamment pour alimenter la photosynthèse. HMA1 constituerait une voie additionnelle d'import du cuivre, voie essentielle pour répondre à un stress oxydatif qui apparaît en particulier lorsque la plante est cultivée en lumière forte. Afin de mieux comprendre les rôles respectifs de ces deux ATPases dans la régulation de l'homéostasie du cuivre chloroplastique, deux approches complémentaires ont été développées : - une approche in planta visant à produire de nouvelles lignées affectées dans l'expression de l'une (mutant paa1 surexprimant HMA1) ou de ces deux ATPases (double mutant hma1/paa1), puis à identifier des conditions (stress lumineux, stress salin, excès de métaux) révélant le rôle essentiel de ces ATPases ou induisant une réponse transcriptionnelle différente des gènes codant ces ATPases ou d'autres acteurs liés à l'homéostasie du cuivre... Les résultats obtenus montrent que la fonction de HMA1, connue pour être essentielle lors d'un stress lumineux, est aussi requise lorsque la plante subit un stress salin. Ces résultats confortent le rôle de HMA1 dans la délivrance du cuivre à la superoxyde dismutase (cuivre/zinc) du chloroplaste. D'autre part, nous avons montré qu'en condition de culture photoautotrophe, le cuivre permet de supprimer partiellement la photosensibilité du mutant hma1, validant ainsi l'implication de HMA1 dans l'homéostasie du cuivre. Nous avons aussi démontré que les fonctions de HMA1 et PAA1 ne sont pas redondantes. Le cuivre importé par ces deux ATPases est probablement délivré à des protéines cibles par des voies différentes. Enfin, nous avons montré qu'il existe une troisième voie d'import de cuivre dans le chloroplaste, voie encore non caractérisée. - une approche in vitro visant à produire ces deux ATPases HMA1 et PAA1 dans le système hétérologue Lactococcus lactis afin de déterminer leurs spécificités ioniques et leurs caractéristiques biochimiques. Le système d'expression procaryote L. lactis a été mis en place au laboratoire et s'avère parfaitement adapté à la production de protéines membranaires de plantes. Ce système a permis de produire plusieurs protéines membranaires d'Arabidopsis, dont HMA1 et PAA1, en quantités compatibles avec des analyses biochimiques. Nous avons déterminé les conditions de solubilisation et de purification de ces deux protéines. Nous n'avons pas pu mesurer d'activité ATPase associée à ces protéines. En revanche, nos données indiquent que ces deux protéines recombinantes peuvent lier l'un de leur substrat ; l'ATP. Nous avons aussi pu démontrer que PAA1 peut lier du cuivre sous forme 1+ et sous forme 2+. Au bilan, ces données suggèrent que le contrôle de l'homéostasie du cuivre chloroplastique requiert plusieurs systèmes de transport indépendants et une régulation fine de ces voies d'import de cuivre afin d'alimenter les besoins liés à la photosynthèse et les besoins liés aux mécanismes de résistance aux stress oxydatifs.

The ratio of auxin to cytokinin determines de novo organogenesis in plants. Relatively little is known about the effect of genetically altered auxin signaling on in vitro organogenesis. Here, callusogenesis, shoot, and root formation were studied in loss- (LOF) and gain-of-function (GOF) alleles in two phylogenetically related Auxin Response Factors (ARFs), MONOPTEROS (MP/ARF5) and NON-PHOTOTROPHIC HYPOCOTYL 4 (NPH4/ARF7). Reduced MP activity greatly diminished shoot regeneration, and partially diminished callusogenesis and root formation. LOF in NPH4 strongly decreased callusogenesis, and mildly decreased shoot and root regeneration in particular categories of explants. By contrast, organogenesis responses were strongly increased in aerial explants carrying the GOF transgene dMP. Thus, both MP and NPH4 seem to act as positive regulators of certain organogenesis processes and the GOF dMP transgene may be of interest for stimulating organogenesis in plant species with poor regeneration properties. Also, organogenesis in vitro may reveal unknown developmental ARF functions.

The ratio of auxin to cytokinin determines de novo organogenesis in plants. Relatively little is known about the effect of genetically altered auxin signaling on in vitro organogenesis. Here, callusogenesis, shoot, and root formation were studied in loss- (LOF) and gain-of-function (GOF) alleles in two phylogenetically related Auxin Response Factors (ARFs), MONOPTEROS (MP/ARF5) and NON-PHOTOTROPHIC HYPOCOTYL 4 (NPH4/ARF7). Reduced MP activity greatly diminished shoot regeneration, and partially diminished callusogenesis and root formation. LOF in NPH4 strongly decreased callusogenesis, and mildly decreased shoot and root regeneration in particular categories of explants. By contrast, organogenesis responses were strongly increased in aerial explants carrying the GOF transgene dMP. Thus, both MP and NPH4 seem to act as positive regulators of certain organogenesis processes and the GOF dMP transgene may be of interest for stimulating organogenesis in plant species with poor regeneration properties. Also, organogenesis in vitro may reveal unknown developmental ARF functions.