Branching is a highly plastic trait, enabling plants to adapt their growth form in response to environmental stimuli. In flowering plants, shoot branching is regulated through the activity of axillary buds, which grow into branches. Several classes of plant hormones have been shown to play pivotal roles in regulating bud outgrowth. Auxin derived from the primary shoot apex and active branches inhibits bud outgrowth, whereas cytokinin promotes it. Strigolactones also inhibit bud outgrowth, by changing properties of the auxin transport network, increasing the competition between buds. This occurs by modulating access to the polar auxin transport stream (PATS) in the main stem. The PATS provides directional, long distance transport of auxin down the stem, involving basal localisation of the auxin transporter PIN-FORMED1 (PIN1). Buds need to export their auxin across the stem towards the PATS in order to activate, but since PIN1 is mainly expressed in narrow files of cells associated with the stem vasculature, PIN1 itself it is unlikely to facilitate this connectivity. This thesis re-examines the role of auxin transport in the stem, showing that, besides the PIN1-mediated PATS, other auxin transport proteins constitute a more widespread and less polar auxin transport stream, allowing auxin exchange between the PATS and surrounding tissues. Disruption of this transport stream is shown to reduce bud-bud communication and to partially rescue the increased branching observed in strigolactone mutants. Furthermore, it is shown that distinct classes of auxin transport proteins within this stream can differentially affect bud outgrowth mediated by BRANCHED1 (BRC1). BRC1 is a transcription factor proposed to determine bud activation potential. Taken together, the data presented here provide a more comprehensive understanding of the shoot auxin transport network and its role in shoot branching regulation.

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Auxin controls a range of growth related characteristics by a mechanism dependent upon polar auxin transport. We have identified a leaf vein patterning mutant that shows a simple first leaf vein pattern. The veins are often non-meeting and form somewhat parallel to one another. The leaves are narrow and pointed so that the overall leaf phenotype is reminiscent of grass leaves; hence the mutant name grassy (gsy). A range of shoot and root characteristics are also altered in gsy plants. Compared to wild type, gsy plants have shorter primary roots with reduced numbers of lateral roots and increased numbers of longer root hairs. Upon gravitropic stimulation, the root tip bends slightly away from the normal vector. As well, gsy plants produce an inflorescence with altered internode elongation and branching pattern. The intensity of the auxin responsive reporter gene DR5::GUS is unchanged in both roots and developing leaves of gsy, however, it shows subtle differences to the wild type DR5:GUS expression pattern. Finally, gsy leaf and root phenotypes are more sensitive to low doses of the auxin efflux inhibitor NPA and external auxin 2, 4-D. We suggest that this overall pattern is consistent with defects in auxin transport. / xii, 76 leaves : ill. ; 29 cm.

A functional water-conducting system, the tracheary elements of the xylem, is required to sustain plant growth and development. Tracheary element formation is dependent on many biological processes terminated by programmed cell death and cellular autolysis. The final two processes are probably dependent on the activity of hydrolytic enzymes such as XCP1 and XCP2 known to be expressed in tracheary elements during these final two processes. Thus, the transcriptional regulation of XCP1 and the function of XCP2 were investigated. Qualitative and quantitative assessments of GUS activity as directed by various fragments of the XCP1 promoter showed that a 237-bp internal region was able to drive GUS expression in a tracheary element-specific manner in Arabidopsis. A 25-bp deletion at the 3' end of this region abolished GUS expression. The 237-bp region served as bait in a yeast one-hybrid analysis. Screening of yeast colonies retrieved 109 putative positive interactions, which included a potential transcriptional regulator, indole acetic acid-induced protein 8 (IAA8). An auxin responsive element that potentially binds auxin responsive transcription factors was found within the 25-bp deletion. Cis-elements were predicted by Genomatix and Athamap computer programs. The cis-elements form pyrimidine and gibberellic acid responsive elements that can potentially bind Dof and Myb transcription factors, respectively. In an independent effort, attempts to develop a mapping population to isolate upstream regulators of XCP1 expression did not succeed. Functionally, tracheary element-specific expression of XCP2 in Arabidopsis suggested a specialized role for XCP2 in final phases of tracheary element differentiation. The function of XCP2 was assessed using T-DNA insertional mutants, post-transcriptional gene silencing, and through tracheary element-specific expression of the cysteine protease inhibitor, soyacystatin N in Arabidopsis. Our findings revealed that the absence of XCP2 expression due to T-DNA insertional mutagenesis did not affect plant growth and development in the laboratory. Soyacystatin N was an effective in vitro inhibitor of cysteine proteases. Plants expressing 35S-driven cytosolic form of soyacystatin exhibited stunting and reduced apical dominance. Plants expressing pXCP1-driven cytosolic soyacystatin did not differ from wild type plants. Additionally, transgenic plants expressing pXCP1- and 35S-directed XCP2-double-stranded RNA for the silencing of XCP2 showed no unusual phenotypes compared to their wild type counterparts / Ph. D.

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The plant hormone auxin regulates many aspects of plant growth and development. At the cellular level, auxin can stimulate cell division, cell elongation, and cell differentiation. Little is known about the molecular mechanisms that mediate auxin action at this level, although changes in gene expression have been implicated in each of them. An important step in elucidating the signal transduction steps involved in these responses is the identification of intermediates that respond to auxin treatment within a physiologically relevant range, and can be functionally linked to specific response(s). The Aux/IAA family of auxin-regulated genes, which has been identified in several species, may constitute such a group of signalling intermediates. This dissertation reports on the isolation and characterization of eleven tomato genes, LeIAA1 - 11, which are homologous to these Aux/IAA genes. Expression characteristics in response to exogenously-applied auxin were determined in segments of etiolated hypocotyls. The LeIAA genes fell into three kinetic classes and displayed differential responsiveness to the treatment. Comparison of auxin-induced expression in wild-type and the auxin-insensitive diageotropica (dgt) mutant revealed that induction of only a subset of genes was affected by the mutation while the other genes retained wild-type response levels in the mutant. This finding indicates that at least two signal transduction chains lead to auxin-induced gene expression, and the dgt gene product acts on only one of them. The expression pattern of nine LeIAA genes was determined in a wide variety of tissue samples in order to link individual LeIAA genes with specific physiological responses. To that end, a qualitative multiplex RT-PCR method was developed that allowed quick assessment of relative expression levels in a large number of small RNA samples. It was found that LeIAA10 expression was highest in tissues undergoing rapid growth, suggesting a role for this gene in the process of cell expansion. The expression of LeIAA1 1 was also variable during development, while LeIAA2 was strongly down-regulated after light treatment. These experiments demonstrated organ-preferential expression of LeIAA genes under the control of environmental factors and depending on developmental stages. The LeIAA genes may thus act as integrators of several other signals with the auxin stimulus. / Graduation date: 1998

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references. Also available in electronic version. Access restricted to campus users.

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