This study focused on characterizing and determining the inheritance pattern of very high tillering and dwarf traits of a rice mutant. To characterize the new mutant, field phenotyping studies, and response of two mutant lines (M-13662 & M-13684) to three levels of nitrogen (179, 202, 224 kg ha-1) and five planting densities (1, 2, 3, 4, 5 plants hill-1) in greenhouse conditions were conducted. A separate study was carried out to determine the response of the two mutant lines to gibberellic acid (GA) application. The mutants were 50-55 cm tall and produced 89-121 tillers plant-1 at harvest. Dwarfness of the mutants was due to average shortening of the top four internodes as well as compression of 2-3 basal internodes. The first tiller emerged at the 4th leaf stage whereas no tiller was observed in semi-dwarf rice cultivar, Cocodrie. Results showed that the production of high tiller numbers was the result of the release of axillary buds from a dormant stage rather than the initiation of additional axillary buds. The mutants were late maturing than controls (Cocodrie & Zhe733). The panicles were very short (10-12 cm) and had 25-30 small grains. The majority of tillers of the mutants followed the dn-type dwarf pattern based on Takeda’s classification, but a few plants had a different dwarfing pattern not included in the classification. Both mutant lines were found to have similar agronomic traits but were significantly different from controls. The tillering ability of the mutants was affected by the five different planting densities as well as the three nitrogen levels. Mutants produced more tillers, both productive and non-productive, at the lowest plant density. The longest and shortest panicles were observed at 202 kg ha-1 and 179 kg ha-1, respectively. Variations in other agronomic traits were found not significant. The response of the mutant to GA application was similar to Cocodrie, and thus was considered GA responsive. Preliminary DNA data using SSR markers supported the presumed origin of the mutants and the genetic analysis indicated that one recessive gene controlled both the dwarfing and very high tillering traits.

<i>Arabidopsis thaliana</i> trichomes exhibit strong birefringence under polarized light, a characteristic of cell walls containing large amounts of highly ordered cellulose microfibrils. The <i>tbr1</i> mutant of <i>Arabidopsis</i> lacks trichome birefringence and is deficient in secondary cell wall cellulose synthesis (Potikha and Delmer, 1995). The <I>TBR</i> gene was identified by recombinational mapping, candidate gene sequencing and molecular complementation using genomic cosmid clones, as well as a p35S:<I>TBR</i> genomic DNA construct, fully rescuing the mutant phenotype in both cases. The only mutant allele available (<i>tbr-1</i>) carries a substitution (G to E) in a conserved aminoacid domain of the protein. <br><br> <I>TBR</i> gene structure was proved to have a longer size than the one found to be annotated at the time of identification in the data-base. A full cDNA clone containing the full transcript was available and also complementation experiments using different gene fragments (annotated and suggested) leaded to the result that TBR gene is indeed, longer. <I>TBR</i> encodes a novel plant-specific protein with predicted plasma membrane localization, therefore being consistent with idea that is required for-, or is a novel component of a functional cellulose synthase complex. <I>TBR</i> is part of an Arabidopsis gene/protein family, (TBL-trichome birefringence like) which, depending on homology, comprises up to 20 members, none of which has a biological or biochemical function attributed.<br><br> T-DNA insertion lines in <I>TBR</i> gene and two close homologues have been screened by PCR, but no homozygous were found and no trichomes phenotype was identified. Promoter-GUS lines were produced for <I>TBR</i>, as well as for its two closest homologues (one being a segmentally duplicated gene on chromosome III), using 1.6-2 kb of promoter sequence upstream of the annotated start codons. The <I>TBR</i> promoter was the only one of the three that yielded trichome expression, this probably explaining the phenotype of the <i>TBR</i> mutant. Moreover, <I>TBR</i> is expressed in leaves, in growing lateral roots, and in vascular tissues of young <i>Arabidopsis</i> seedlings and plantlets. Later on, the expression appears in inflorescens, stems, flowers and green siliques. This expression pattern is largely overlapping with those of the two analyzed homologues and it corresponds with data of RT-PCR expression profiling performed for <I>TBR</i> and the two analyzed homologues in different tissues, at different developmental stages. Biochemical analysis of cell wall (leaves and trichomes), as GC and MALDI-TOF, were performed, but revealed no major differences between tbr1 and wild type plants. Scanning electron microscopy analysis and cell wall polysaccharides antibody labeling showed a clear difference in the trichomes cell wall structure between mutant plant and wild type. / Genetische Kartierung und molekulare Charakterisierung der tbr-Mutante in <i>Arabidopsis thaliana</i>. <br><br> Arabidopsis Trichome zeigen eine starke Doppelbrechung unter polarisiertem Licht, ein charakteristisches Merkmal der Sekundärzellwand, die aus großen Mengen parallel angeordneten Zellulose-Mikrofibrillen besteht. In der Arabidopsis tbr-Mutante ist die Synthese der Sekundärzellwand fehlerhaft. Die Trichome der tbr-Mutante zeigen unter polarisiertem Licht den Doppelbrechungseffekt nicht (Potikha and Delmer, 1995). <br><br> TBR ist ein pflanzenspezifisches Protein, das in der Plasmamembran lokalisiert ist. Diese Tatsache lässt vermuten, dass TBR eine Rolle bei der Zellulose-Synthese spielt oder Teil des Zellulose-Synthese Komplexes ist. Die TBR Genfamilie besteht aus 20 Genen, deren biologische oder biochemische Funktion nicht bekannt ist.<br><br> Das TBR Gen wurde durch „recombinational mapping“ und Sequenzierung identifiziert. Die molekulare Komplementation erfolgte unter Verwendung von Cosmidklonen und einem p35S:TBR genomischen DNA-Konstrukt und konnte den Wildtypphänotyp wieder vollständig herstellen. Die tbr-1 Mutante trägt einen Basenaustausch (G nach A) in einer hochkonservierten Aminosäuresequenz im Protein. In einer Arabidopsis T-DNA Linie mit Insertion in einem Exon konnten in einem PCR-Screen nur heterozygote Pflanzen identifiziert werden. Diese zeigten alle im polarisierten Licht den Doppelbrechungseffekt nicht. Ein möglicher Hinweis für den Phänotyp der tbr-Mutante könnte die Expressionsanalyse durch ein Promotor-GUS Konstrukt sein. Hierfür wurde eine 1,6 kb Sequenz, die upstream des TBR Startcodons liegt verwendet. Es zeigte sich, dass die Expression in den sowohl in den Trichomen zu finden war, als auch in Blättern, der Seitenwurzel und in vaskulären Gewebe junger Arabidopsis Keimlinge. In späteren Entwicklungsstadien ist eine Expression in den Stämmen, Blüten und grünen Schoten sichtbar. Diese Ergebnisse konnten durch RT-PCR Expressionsprofile und anhand der AtGenExpress Daten verifiziert werden. Zellwandanalysen durch GC und MALDI-TOF der Blätter und Trichome zeigten keine signifikanten Unterschiede zwischen der tbr-Mutante und Wildtyppflanzen. In den Pektinen der primären Zellwand konnten Unterschiede durch Immunolabeln der Zellwände festgestellt werden. <br><br> Lokalisationsanalysen wurden mit Hilfe von C- und N-terminalen GFP-Konstrukten durchgeführt. Es konnte kein Signal detektiert werden. Mit Hilfe der Konstrukte konnte jedoch der Wildtyp Phänotyp wieder hergestellt werden. <br> Es konnte also gezeigt werden, dass durch die genetische Kartierung und molekulare Charakterisierung des TBR Gens, ein an der Synthese der Zellulose beteiligtes Gen gefunden wurde.

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An <i>Escherichia coli</i> strain carrying the <i>phs</i> mutation was isolated by other workers who were interested in determining the role of the Na /H antiport in pH homeostasis. The mutation was observed to cause impaired growth on L-glutamate amd melibiose and was also reported to cause a sensitivity to alkaline conditions due to an impairment in pH homeostasis. Since uptake of both glutamate and melibiose is energised by a Na⁺ gradient, these observations were interpreted as evidence that the <i>phs</i> mutation impairs Na⁺ /H⁺ antiport activity, and so by implication, that the Na⁺ /H⁺ antiport is involved in pH homeostasis in <i>E. coli</i>. In work for this thesis, evidence was accumulated that the <i>phs</i> mutation does not affect Na⁺ /H⁺ antiport activity. Proline uptake, which is energised by a Na⁺ gradient, was found to be normal in a mutant strain. Also, arabinose uptake via two different transport systems, neither of which is energised by a Na⁺ gradient, was found to be impaired by the mutation. K⁺ uptake by Na⁺ loaded cells, a phenomenon which is thought to give a measure of Na⁺ /H⁺ antiport activity, was not affected by the mutation. Studies on growth under alkaline conditions indicated that growth is not impaired by the mutation. Evidence was obtained however, that the <i>phs</i> mutation causes filamentation under these conditions. There is a possibility that this may be due to an effect on the penicillin-binding protein content of the cell envelope. Investigations of the effect of the mutation on pH homeostasis suggested that it affects either pH homeostasis, or the validity of the techniques used for determining the cytoplasmic pH. In view of the mapping of the mutation to the gene coding for the α-subunit of RNA polymerase (Rowland <i>et al</i>, 1985), the effects of the mutation on transcription were studied. It was found that the mutation dramatically impairs transcription of genes under the positive regulation of the <i>AraC</i> gene product. An examination of the effect of the mutation on transcription from a variety of different promotors showed that the transcription defect is highly selective. Some evidence was obtained that the mutation impairs the process of positive regulation. To test this, the effect of the mutation on transcription from AraC independent derivatives of an AraC dependent promoter was determined. In the absence of AraC, the mutation had no effect. In the presence of AraC, complex effects were observed. It was concluded that the mutation affects the process of positive regulation or CRP-cAMP mediated derepression of this promoter.

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Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.