Many plants accumulate organic osmolytes in response to the imposition of environmental stresses that cause cellular dehydration. Of these, proline is the most extensively studied. Conclusive demonstration that this imino acid acts as a compatible solute which mediates osmotic adjustment has yet to be achieved, although a causal relationship between increased proline synthesis and plant tolerance of hyperosmotic stresses has previously been demonstrated. It is proposed that in many plants, the metabolic implications of the regulated increase in proline synthesis and/or a decline in proline degradation during stress may play a more important role in acclimation to adverse conditions than the simple accumulation of the end-product of these adjustments. In particular, the stress-induced increase in the transfer of reducing equivalents into proline by Δ¹-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) may be a protective mechanism whereby many species ameliorate shifts in cellular redox potential which accompany all biotic and abiotic stresses which cause proline accumulation, including those that do not cause cellular dehydration. The presence of several putative stress-regulated promoter elements in the AtP5CS1, AtP5CS2 and AtP5CR genes of Arabidopsis thaliana strongly implicates an adaptive role for stress-induced increases in proline synthesis in this species. Sequence homologies of several regions within the 5' untranslated regions of these genes to promoter elements which have been shown to participate in redox control of gene expression, the actions of phytochrome and hormones, and tissue-specific regulation of gene expression are also identified. These provide useful indicators both of the mechanisms by which proline synthesis is regulated and how these may relate to its importance in maintaining metabolic homeostasis. In an attempt to resolve the functionality of proline accumulation under stress, chimeric antisense genes comprising 1050 bp and 999 bp fragments of Arabidopsis cDNAs encoding AtP5CS1 and AtP5CR respectively were inserted in the reverse orientation between the CaMV 35S promoter and the GUS gene (encodes β-glucuronidase) in the plant transformation vector pBI121. These constructs were introduced separately into Arabidopsis by cocultivation with Agrobacterium tumefaciens strains carrying the pBI-P5CS1 (AS) and pBI-P5CR(AS) plasmids. Transgenic plants, which were selected on the basis of kanamycin resistance, regenerated at a low frequency in the presence of 1 mM proline. Transformation of 13 pBI-P5CS1(AS) and 7 pBI-P5CR(AS) lines was confirmed by PCR-mediated amplification of gene fragments within the introduced T-DNA. Segregation ratios for kanamycin resistance indicated that most of the lines have multiple T-DNA insertions. Transformants were characterised with respect to their growth rates and free proline content. In at least two pBI-P5CS1(AS) transformants and two pBI-P5CR(AS) transformants, a reduction in root growth rates in the presence of inhibitory concentrations of NaCI correlated with reduced β-glucuronidase activity relative to transgenic lines that were no more sensitive to NaCI than were controls. A reduction in root growth rate both in the absence and presence of hyperosmotic stress was noted in two pBI-P5CS1(AS) transformants, designated A5 and B12. In 14 day-old plants of the T₂ generation of both A5 and B12, free proline levels were significantly lower than in wild-type plants both in the absence of stress and following 24 h incubation in either 250 mM NaCI or 550 mM sorbitol or at 5 °C. In both lines, reduced growth rates in the absence of osmotic stress could be restored by exogenous proline, but not by exogenous glutamate. When used at isosmotic concentrations, sorbitol caused a larger reduction in free proline levels in both A5 and B12 than did NaCI. This observation may relate to an ABM-mediated post-transcriptional effect on AtP5CS1 gene expression which affects NaCI-, but not sorbitol-mediated proline accumulation in Arabidopsis. Post-transcriptional regulation of the expression of the genes involved in proline biosynthesis may account, at least partly, for the absence of dramatic phenotypic effects in any of the pBI-P5CS1(AS) or pBI-P5CR(AS) lines. Under the premise that regulation of shifts in proline metabolism regulate cellular redox potential under conditions of stress may be mirrored by the involvement of proline metabolism in modulating metabolism during normal growth and development, the effects of exogenous proline on Arabidopsis seed germination, seedling growth and in vitro shoot organogenesis were investigated. A dose-dependent inhibition of radicle emergence by millimolar concentrations of proline could be overcome by the artificial oxidants methylene blue and phenazine ethosulphate. Assays of the rate-limiting dehydrogenases of the oxidative pentose phosphate pathway (OPPP), as well as changes in the contributions of ¹⁴C₁ - and ¹⁴C₆ -labelled glucose to respired CO₂ during germination, are consistent with activation of the OPPP during Arabidopsis seed germination. An approximately four-fold increase in free proline, which peaked at the time of radical emergence, was not parallelled by changes in other amino acids and could not be ascribed to degradation of seed storage proteins. Delayed radical emergence in T₂ generation seeds of the pBI-P5CS1(AS) lines A5 and B12 correlated with an approximately 35% reduction in the maximal concentration of proline accumulated during germination. Millimolar concentrations of exogenous proline had a dose-dependent inhibitory effect on Arabidopsis seedling growth both in the light and in darkness. This reduction in growth arises at least in part from a decline in cell elongation. Accordingly, exogenous proline increased total extractable peroxidase activity in Arabidopsis seedlings through the selective induction of peroxidase isoforms. Histochemical analysis of the hypocotyls of plants grown in the presence of exogenous proline suggested that proline increased the levels of lignin and/or the phenolic precursors thereof. A dose-dependent decrease in extractable chlorophyll and damage to chloroplastic and mitochondrial ultrastructure was observed in 21 day-old Arabidopsis seedlings grown in the presence of millimolar concentrations of exogenous proline. In vitro shoot organogenesis from Arabidopsis hypocotyl explants was stimulated by 1 mM proline, and to a lesser extent by 5 mM proline, but inhibited by inclusion of 10 mM proline in the hormonallysupplemented regeneration media. The ability of low concentrations of proline analogues (azetidine-2-carboxylate and thioproline) to overcome the stimulatory effect of 1 mM proline, and a slight increase in the stimulative effects of 1 mM proline by D-proline, are consistent with an important role for the interconversions of proline and its precursors in regulating cell division and differentiation. Together, these data strongly support an important role for the interconversions of proline and its precursors in the regulation of intermediary metabolism under both normal and stressful conditions. These findings draw into question the widely accepted, although poorly investigated, hypothesis that proline is an inert compatible solute that can be accumulated to high levels with minimal effects on cellular metabolism. The novel proposal that stress-induced changes in proline metabolism exert a regulatory effect though an influence on the level of reduction of the cellular NADP pool is discussed in relation to recent evidence that a signal related to proline synthesis and/or degradation selectively increases the expression of stress-induced plant genes. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

y-Aminonbutyric acid (GABA) is a ubiquitous non-protein amino acid found ill many plants and organisms. GABA accumulation in plants has previously been reported as result of stresses such as water deprivation, high salinity and temperature extremes. It is thought that GABA accumulates as a compatible solute in the cytoplasm where it becomes a major constituent of the free amino acid pool. GABA is synthesised from the decarboxylation of glutamate by glutamate decarboxylase (GDC). In some plants, GDC is activated by the lowering of the cytoplasmic pH and the presence of calmodulin and Ca²+ A calmodulin-induced activation of may be due to the physiological factors and environmental stimuli acting in concert leading to the synthesis and accumulation of GABA. The GABA content of Arabidopsis thaliana var. Columbia (L) Heynh leaves was found to increase by over 130% due to water deprivation. NaCl concentrations of up to 100 mM seemed to cause GABA accumulation due to a decrease in osmotic potential. Concentrations of NaCl above 100 mM probably caused GABA accumulation due to combined hyperosmosis and salt toxicity effects. The high levels of GABA in the leaves were maintained throughout a 24 h stress-application period, consistent with its role as compatible solute. The accumulation of GABA followed by its decline in the dark could be attributed to its rapid metabolism because of an active GABA shunt. This is in contrast to the absence of major variations in the amount of GABA in the light confirming its decreased role as a channel for the glutamate carbon and nitrogen under such conditions. A substantial increase in the GABA content was followed by a dramatic decrease in the last 12 h of incubation. This profile of GABA could support its proposed role as a temporary sink for nitrogen and carbon from glutamate during environmental stress. Glutamate decarboxylase appears to be encoded by a single gene in the genome of Arabidopsis. Sequence analysis reveals that the protein possesses what could be a carboxy-terminal, calmodulin- binding domain, which is consistent with other glutamate decarboxylases. The 30-amino acid peptide contains a TrpLysLys motif found in some calmodulin targets. The secondary structure predictions of this peptide suggest a potential to form an a- helix which is also consistent with proteins known calmodulin- binding domains. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.

In Arabidopsis thaliana (L.) Heyhn, the size of the pool of free proline increases up to 27-fold in response to osmotic stress. The magnitude of this accumulation is dependent upon the rate of imposition of the stress. Numerous reports have suggested a role for proline accumulation as a general adaptation to environmental stress. However, controversy surrounds the beneficial effect of proline accumulation in plants under adverse environmental conditions. Stress-induced proline accumulation in plants occurs mainly by de novo synthesis from glutamate. The final and only committed step of proline biosynthesis in plants is catalysed by Δ¹-pyrroline-5-carboxylate reductase (P5CR). The sequence of an incomplete 999 bp cDNA encoding P5CR from A. thaliana was determined. This enabled a preliminary molecular study of the structure and function of both the gene and the corresponding enzyme. The 999 bp cDNA insert in the clone Y AP057 was sequenced on the sense and antisense strands following subcloning of four sub-fragments in appropriate orientations. Comparison with known plant P5CR sequences revealed that Y AP057 does not encode the first 23 N-terminal amino acids of P5CR from Arabidopsis. However, it does encode the remaining 253 amino acid residues of Arabidopsis P5CR The cDNA Y AP057 is complete on the 3' end as indicated by the presence of a poly(A) tail. The nucleotide sequence determined shows complete homology to the corresponding exons of the genomic copy of a bona fide gene encoding P5CR in A. thaliana (Verbruggen et al, 1993). The only difference observed between the sequence of Y AP057 and that of a cDNA sequenced by these workers is that polyadenylation was initiated seven nucleotides earlier in Y AP057 than in the sequence of the published cDNA. Genomic Southern analysis suggests the presence of only a single copy of the gene encoding P5CR in Arabidopsis. Restriction mapping and sequencing the ends of another incomplete Arabidopsis P5CR cDNA clone FAFJ25 (664 bp) indicated that the regions sequenced were completely homologous to the corresponding portions of Y AP057. Analysis of codon usage in the Arabidopsis gene encoding P5CR revealed it to closely resemble the consensus pattern of codon usage in A. thaliana. This suggests that the gene is moderately. expressed. Expression of the gene encoding P5CR in Arabidopsis is not likely to be subject to translational control. Although P5CR from A. thaliana has a fairly high composition of hydrophobic amino acid residues, it does not possess any stretches of hydrophobic amino acids of sufficient length to act as membrane-spanning domains or to anchor the enzyme in a membrane. Neither does it contain an N- terminal leader sequence capable of directing it to either the plastid or mitochondrion. The enzyme therefore appears to be cytosolic. The nucleic acid and deduced amino acid sequences of Arabidopsis P5CR were compared with those from·eleven other organisms for which P5CR sequences are currently available. Except among the three different plants examined, P5CR sequences displayed less identity at the amino acid level than at the nucleotide level. The deduced amino acid sequence of Arabidopsis P5CR exhibits high similarity to the corresponding genes and amino acid sequences of P5CR from soybean and pea. Lower but significant similarity was observed to the amino acid sequences of P5CRs from human, Saccharomyces cerevisiae and the bacteria Escherichia coli, Pseudomonas aeruginosa, Thermus thermophilus, Mycobacterium leprae; Treponema pallidum and Methanobrevibacter smithii. Similarity was also observed to the translational product of a gene from Bacillus subtilis with high homology to the E. coli proC gene. However, construction of a phenogram indicating the relatedness of the various P5CR enzymes suggests that sequence analysis of this enzyme is not a good indicator of evolutionary relatedness of organisms from different biological kingdoms. Multiple alignment of the twelve known P5CR sequences indicated homology between the sequences across their entire lengths. Homology was particularly high in the C-terminal portions of the P5CRs studied. It is speculated that this region may be of importance in binding of the substrate Δ¹-pyrroline-S-carboxylate (P5C). Another region displaying high sequence conservation was found in the central portion of all P5CRs. All P5CRs studied, with the exception of PSCR from T. pallidum contained an N-terminal domain capable of binding a nicotinamide dinucleotide cofactor. Comparison of this region with consensus sequences for NADH and NADPH binding sites in proteins suggests that NADPH is the preferred reductant used by P5CRs from plants and human. In contrast, the N-terrninal domains of P5CRs from S. cerevisiae, M smithii, T. thermophilus and M leprae display greater similarity to a consensus NADH-binding site. The definite preference of plant P5CRs for NADPH in comparison with NADH suggests that P5CR may be involved in regulating the redox potential within plant cells and that this step in proline biosynthesis from glutamate may be of importance in overall metabolic regulation. Three amino acid residues are universally conserved in all P5CRs studied. All are found within blocks of high sequence similarity. These residues are likely to be of importance in the structure or catalytic mechanism of P5CR. A number of other residues are common to several of the enzymes examined. These may also be of importance in subsequent manipulation of Arabidopsis P5CR at the molecular level. Prediction of the putative secondary structures of A. thaliana, soybean, pea, human and E. coli indicated a high degree of similarity between the enzymes. This was particularly evident in the region of the putative P5C-binding domain. Considerable similarity exists in hydrophobicity profiles of P5CRs from these five organisms. Proline levels in reproductive organs of unstressed Arahidopsis plants were considerably higher than those in vegetative tissues. This suggests differential expression of enzymes involved in proline metabolism in these organs. In situ hybridisation studies indicated an increase in levels of mRNA transcripts encoding P5CR in stem tissues in response to water deprivation stress. Regulation of levels of mRNA transcript encoding P5CR in Arabidopsis therefore appears to be an osmotically sensitive process. Furthermore, this accumulation of transcript occurred in a tissue-specific manner. In particular, an increase in levels of transcript encoding P5CR was observed in the cortical parenchyma, phloem, vascular cambium and pith parenchyma in the vicinity of the protoxylem. The significance of these findings in contributing to a better understanding of the role of proline in adaptation to environmental stress is discussed. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

The chloroplast originates from an endosymbiotic event 1.5 billion years ago, when a free living photosynthetic bacteria was engulfed by a eukaryotic host. The chloroplastic genome has through evolution lost many genes to the nuclear genome of the host. To coordinate the gene expression between the two genomes, plants have evolved two types of communication, nucleus-to-plastid (anterograde) and plastid-to-nucleus (retrograde) signalling. This thesis will focus on retrograde communication with emphasis on redox and tetrapyrrole mediated signalling. In this thesis, we establish the tetrapyrrole Mg-ProtoIX as an important retrograde negative regulator of nuclear encoded plastid proteins. We show that Mg-ProtoIX accumulates in both artificial and natural stress conditions, and that the accumulation is tightly correlated to regulation of nuclear gene expression. Using confocal microscopy, we could visualize Mg-ProtoIX in the cytosol during stress conditions. In addition, exogenously applied Mg-ProtoIX stayed in the cytosol and was enough to trigger a signal to the nucleus. The results presented here indicate that Mg-ProtoIX is transported out of the chloroplast to control nuclear gene expression. Mg-ProtoIX mediated repression of the nuclear gene, COR15a, occurs via the transcription factor HY5. HY5 is influenced by both plastid signals and the photoreceptors. Here, we show that photoreceptors are part of Mg-ProtoIX mediated signalling as well as excess light adaptation. We identified the blue light receptor, CRY1, as a light intensity sensor that partly utilizes HY5 in the high light response. To further understand the high light regulation of nuclear genes, we isolated a mutant with redox insensitive (rin) high light response. The rin2 mutant has a mutated plastid protein with unknown function. Characterization of the rin2 mutant revealed that the protein is important in regulating plastid gene expression as well as nuclear gene expression. The rin2 mutant is the first characterized rin mutant and could prove important in elucidating the cross-talk between redox mediated coordination between the plastid and the nuclear genome.

<p>Environmental stress affects plant development and productivity. Sulfur deficiency is a key nutrient deficiency that adversely affects crop yield. The model plant Arabidopsis thaliana has played an informative role in deciphering the mechanisms involved in sulfur assimilation, as well as, the response to limited conditions. Using Arabidopsis thaliana as a model to investigate gene expression in the root, microarray data sets have been generated. These data sets consist of whole root sections for 6 time points across 72 hours, and enriched populations of 5 radial cell-types and 4 sections of 3 developmental zones of the root at 3 hrs on sulfur limited conditions. With these data it was determined which cellular tissues and developmental zones were affected most by sulfur limited conditions. Furthermore, a novel phenotype was characterized that occurs in roots after growth on low sulfur conditions. Cellular inclusions build up within the cytoplasm of mature cortical root cells. These inclusions have been termed "sulfur pox" and their composition remains to be determined.</p> / Dissertation

No description available.

När ett blad gulnar genomgår det både morfologiska och metaboliska förändringar. Denna process benämns senescence och en förbättrad förståelse av dess mekanismer är viktiga både ur ett grundvetenskapligt perspektiv och för potentiella bioteknologiska applikationer. Denna avhandling rapporterar om flera viktiga aspekter relaterade till de cellulära och metaboliska mekanismer som sker under senescencen med tonvikt på mitokondriernas bidrag till denna process. I ett första steg utvecklade vi metoder för att isolera antingen mycket funktionella mitokondrier eller mycket rena mitokondrier från blad av Arabidopsis thaliana. Dessa metoder användes sedan till för att studera mitokondriella bidrag till cellens redox balans och att uppskatta mitokondriernas kapacitet under senescence-processen. Framför allt jämfördes induktionen av senescencen berodende på olika mörkerbehandlingar av Arabidopsis. Jämförelse mellan individuellt mörklagda blad med hela mörklagda växter visade en betydande skillnad i metabolisk strategi mellan de två mörkerbehandlingarna. Genom att integrera data från mätningar av fotosyntes, respiration och konfokal mikroskopi med transcriptomics- och metabolomics-profiler föreslår vi att metabolismen hos blad från helt mörklagda växter antar ett ”stand-by läge” för att kunna bibehålla fotosynteskapaciteten så länge som möjligt. I kontrast till detta visar mitokondrier från individuellt mörklagda blad en hög aktivitet och kan därmed producera energi och kolskelett för degraderingen av cellkomponenter, vilket möjliggör återvinning av näringsämnen. Vi har även studerat dynamiken av det mikrotubulibaserade cytoskelettet under mörkerindicerad senescence. Mitokondriernas rörlighet påverkades av en tidig nedbrytning av mikrotubuli hos individuellt mörklagda blad men inte hos blad där hela växten mörkerbehandlats. Dessutom verkade ett flertal mikrotubuliassocierade proteiner (MAP’s) att vara involverade i buntningen av mikrotubuli runt kloroplasterna. Sammanfattningsvis belyser det arbete som presenteras i denna avhandling ett flertal viktiga steg med avseende på metabolisk anpassning och andra cellulär mekanismer i Arabidopsisblad som utsätts för långvarig mörkerbehendling. Specifikt föreslår vi att mitokondrierna bidrar med speciella och viktiga funktioner under bladens senescence eftersom mitokondriernas roll under långvarig mörkerbehandling av blad verkar bero på den totala statusen av metabolismen hos växten. / When switching from green to yellow, a leaf undergoes both morphological and metabolic changes. This process is known as senescence and improved understanding of its mechanisms is important both from a fundamental scientific perspective but also for biotechnological applications. The present thesis reports on several important aspects regarding the cellular and metabolic mechanisms occurring during leaf senescence with an emphasis on the mitochondrial contribution to this process. As a first step, we developed methods to isolate either highly functional crude mitochondria or highly purified mitochondria from leaves of Arabidopsis thaliana. These methods were further used to study mitochondrial contributions to cellular redox homeostasis and to estimate the mitochondrial capacities in leaves undergoing senescence. In particular, we compared the induction of senescence by different dark treatments in Arabidopsis. The comparison between individually darkened leaves and leaves from whole darkened plants revealed different metabolic strategies in response to darkness. Integrating data from measurements of photosynthesis, respiration and confocal laser microscopy with transcriptomics and metabolomics profiling, we suggested that metabolism in leaves of the whole darkened plants enter a “stand-by mode” with low mitochondrial activity in order to maintain the photosynthetic machinery for as long as possible. In contrast, mitochondria from individually darkened leaves are more active and may provide energy and carbon skeletons for the degradation of cell constituents, facilitating the retrieval of nutrients. We also investigated the dynamic of the microtubular cytoskeleton during dark-induced senescence. Mitochondrial mobility was affected by an early disruption of the microtubules in individually darkened leaves but not in whole darkened plants. In addition, several microtubules associated proteins (MAPs) seemed to be involved in the bundling of the microtubules around the chloroplasts. Altogether, the work presented in this thesis highlights several important steps regarding the metabolic adjustments and the cellular mechanisms in Arabidopsis leaves submitted to prolonged darkness. In particular, we suggest the mitochondria to fulfill specific and important functions during leaf senescence since the role of mitochondria in leaves experiencing prolonged darkness appears very dependant on the whole metabolic status of the plant.

Plant growth and development depends on controlled cell expansion. This, in itself, is determined by the plant cell wall, a structural matrix of polysaccharides encasing the plant cell. One line of investigation that has proven particularly successful in elucidating the components of the plant cell wall machinery has been the forward genetic screens of cell wall mutants. In this study, the molecular and cellular characterisation of sloughy, a cell separation mutant in Arabidopsis thaliana, was commenced. This mutant has a striking phenotype, with files of elongating epidermal cells snaking away from the adjacent epidermal cells and from the underlying cortex, loosing contact from the side walls while remaining attached at the cell ends, in a manner reminiscent of border-like cells in the root cap of arabidopsis. The sloughy mutation was fine mapped to a short region on chromosome I using high resolution melt point analysis. On sequencing all five genes in this region, a single nucleotide mutation, introducing a stop codon, was detected in exon 2 in the previously-described heat shock transcription factor SCHIZORIZA that results in a truncated protein missing several conserved domains essential for activity. SCHIZORIZA acts as a cell fate determinate in the root meristem to promote cortex fate, while suppressing epidermal and root cap fate in the mature ground tissue. Although the literature on schizoriza mutants has focused on the developing root meristem, with little documentation on the cell separation phenotype further up in the roots, the investigation of a collection of schizoriza TILLING mutants revealed that aberrant cell separation was ubiquitous to schizoriza mutants with a severely truncated protein. To investigate cell identity in the mature roots, sloughy was crossed to GAL4-GFP enhancer trap lines that act as cell-specific markers. Epidermal identity lines revealed that sloughy possessed a supernumerary ground tissue layer with epidermal identity. A cortex and endodermal line revealed that these two identities are restricted to the endodermal layer and the next ground tissue layer out. There was no indication of root cap identity in the mature root with any of the root cap lines used, although partial lateral root cap identity has been previously described in the epidermal and subepidermal cell layers in the meristem of schizoriza mutants expressing SOMBRERO-GFP, a lateral root cap-specific transcription factor. Immunolabelling of cell wall epitopes revealed that the JIM13 antibody, which specifically labels arabinogalactan-proteins in wild-type root caps, often labelled the epidermal cells and surrounding mucilage further up the in the roots of sloughy. The aberrant cell separation present in sloughy is thought to be a consequence of epidermal cells possessing partial lateral root cap identity. The data on sloughy/schizoriza is sufficient to generate a model on how a meristem developmental gene can generate a cell separation phenotype in the mature roots. Loss of SCHIZORIZA causes confused cell identity in the root meristem that results in an epidermal and subepidermal layer possessing mixed epidermal and lateral root cap identity. The distinctive properties of border-like cells in the root cap of arabidopsis have been linked to unique cell wall maturation and developmental processes, implicating the cellulases CEL3 and CEL5, the pectin glycosyltransferase QUA1, the pectin methyltransferase QUA2 and other pectolytic enzymes. The ectopic expression of these cell wall enzymes in the epidermal and subepidermal layers of sloughy roots result in reduced adhesion along the sides of the cell, while the ends remain attached, causing the observed cell separation phenotype.

La paroi végétale assure des fonctions biologiques majeures définissant la singularité des plantes ; elle est également à l'origine de multiples applications en tant que ressource agro-alimentaire, source de biomatériaux ou encore pour la production de biocarburants. Malgré cette importance fondamentale et pratique de la paroi végétale, la connaissance de sa biosynthèse apparaît à ce jour toujours très limitée. En effet, la faible abondance des glycosyltransférases (GTs) responsables de sa biosynthèse, l'absence de substrat spécifique et les difficultés à obtenir certains nucléotides-sucres nécessaires aux tests enzymatiques, a souvent rendu difficile les approches de biochimie classiques. Cependant, le séquençage de génomes (Arabidopsis thaliana, Oryza sativa, Poplar populus), la création de banques de mutants d'insertion et la classification des activités glycosyltransférases dans la base de données CAZy (www.cazy.org) sont autant d'outils récents ayant permis des avancées significatives vers la compréhension de la biosynthèse de la paroi des végétaux. Le CERMAV a participé à ce type d'avancée en 2009, en publiant une liste de 24 gènes candidats, nommés " NGT " pour " Nouvelles GlycosylTransférases ", présentant des signatures caractéristiques des glycosyltransférases. Afin de démontrer l'implication des gènes NGT dans les processus d'édification de la paroi végétale, nous avons développé une approche de génomique fonctionnelle, analysant en parallèle des lignées mutantes d'Arabidopsis altérées pour les gènes NGT et testant l'activité GT de ces protéines exprimées en systèmes hétérologues. Durant mes travaux de thèse j'ai pu caractériser 15 lignées mutantes à l'état homozygote pour 7 des 24 gènes NGT. Ces lignées homozygotes ont été criblées afin de rechercher un phénotype d'altération du développement ou de la composition en sucres de leur paroi qui soit corrélé à l'altération des gènes NGT. Ce travail de criblage a conduit à s'intéresser plus particulièrement aux mutants ngt1-1 et ngt1-2 altérés pour le gène NGT1 (At5g28910). La caractérisation des lignées mutantes ngt1-1 et ngt1-2 a permis de quantifier un phénotype de croissance foliaire réduit de 38%, par comparaison au développement des feuilles de la plante sauvage. Par ailleurs, la caractérisation biochimique de la paroi des mutants a révélé des réductions significatives et quantitatives de l'arabinose, du galactose et du rhamnose dans la paroi des mutants, ainsi que des modifications qualitatives marquées principalement des arabinanes. L'altération des arabinanes a d'ailleurs pu être confirmée par microscopie après immuno-marquage de sections d'hypocotyle de mutants à l'aide des anticorps monoclonaux LM6 et LM13 dirigés contre des épitopes α-1,5-arabinanes. Il a pu être montré également que la complémentation des mutants par une construction 35S::NGT1 permet de restaurer un phénotype sauvage à ces mutants. Par ailleurs, de façon à tester l'activité glycosyltransférase de la protéine NGT1, nous avons réalisé son expression en système hétérologue. A ce jour, malgré des résultats préliminaires encourageants, il n'a pas été possible de déterminer des conditions de tests permettant d'observer une activité glycosyltransférase suffisante et reproductible pour la protéine NGT1, que ce soit une activité fucosyltransférase (correspondant à la signature de la séquence du gène) ou bien une activité arabinosyltransférase (correspondant au phénotype biochimique des mutants ngt1).

ABSTRACT: Formation of organ shape is an intriguing yet largely unanswered question in developmental biology. Shapes arise as a result of tightly controlled spatial variation in the rates and directions of tissue expansion over the course of development; therefore, quantifying these growth patterns could provide information about the underlying mechanisms of morphogenesis. Here we present a novel technique and computational tools for quantifying growth and shape changes in developing leaves, with a few unique capabilities. This includes the ability to compute growth from three-dimensional (3D) coordinates, which makes this the first method suitable for studying leaf growth in species or mutants with non-flat leaves, as well as small leaves at early stages of development, and allows us to simultaneously capture 3D shape changes. In the following, we apply these methods to study growth and shape changes in the first rosette leaf of Arabidopsis thaliana. Results reveal clear spatiotemporal patterns in growth rates and directionality, and tissue deformation maps illustrate an intricate balance involved in maintaining a relatively flat leaf surface in wild type leaves. Semi-automated tools presented make a high throughput of data possible with this method, and algorithms for generating mean maps of growth will make it possible to perform standardized comparative analyses of growth patterns between wild type and mutants and/or between species. The methods presented in this thesis will therefore be useful for studying leaf growth and shape, to further investigate the mechanisms of morphogenesis.   RÉSUMÉ: Comment un organe acquiert sa forme particulière au cours du développement est une question intéressante mais largement non résolue. La forme d’un organe résulte de la façon dont les taux et directions de croissance de ses tissues varient dans l’espace et dans le temps. Quantifier les motifs de croissance est donc nécessaire pout élucider les mécanismes sous-jacents de la morphogenèse. Nous présentons ici une nouvelle méthodologie pour quantifier la croissance et les changements de forme dans les feuilles en développement. Cette méthodologie s’appuie sur le développement de nouvelles techniques expérimentales et de programmes informatiques, et présente des avantages uniques : la croissance de la surface des feuilles et le changement de forme peuvent être analysés en trois dimensions (3D), pour une longue période et de large déformations. De plus l’analyse de multiples échantillons permet de générer une cartographie moyenne des motifs de croissance à la surface des feuilles au cours de leur développement, ainsi qu’une description quantitative de la déformation des tissus sous l’effet de leur croissance. Dans cette thèse, nous présentons les résultats de croissance et de changements de forme de la première feuille de rosette d'Arabidopsis thaliana au cours de son développement. Les cartes moyennes de croissance révèlent des motifs spatio-temporels évidents tant pour les taux que pour les directions de croissance. De plus, la description de la déformation des tissus démontre l'équilibre complexe impliqué dans le maintien d'une surface relativement plane dans les feuilles. La méthode proposée et les logiciels associés permettra d’effectuer des analyses comparative de la croissance entre feuilles de type sauvage et feuilles de mutants aux formes altérées, afin d’élucider les mécanismes de la morphogenèse foliaire.