published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

No description available.

No description available.

This study is concerned with the analysis of the AtEMl0l promoter trap line of Arabidopsis thaliana. AtEMl0l seedlings show GUS expression in the tips of both primary and lateral roots, and more weakly in the hypocotyl and cotyledons. GUS activity in mature plants is found variably in both rosette and cauline leaves, stem nodes and also siliques but not other floral organs. Active auxins rapidly upregulate whilst cytokinins downregulate GUS transcript levels. AtEMl0l roots are shorter than those of the wild-type, a phenotype which is putatively linked to elevated ethylene levels. AtEMl0l roots were also found to be hypersensitive to exogenous cytokinins. Root patterning is not affected, but cells distal to the elongation zone are shorter in the AtEMl0l line than the wild-type. The T-DNA in line AtEMl0l was found to have inserted in a small, low abundance gene named POLARIS, which encodes a putative 36 amino acid polypeptide, which does not share homology to any known genes. POLARIS shows unusual genome organisation, with its 5' end overlapping with the 3' end of an upstream gene. Upstream sequence, embedded within the upstream gene, when fused to GUS were able to direct expression in root tips whilst a longer fragment mimics the GUS expression of the AtEMl0l line. Retransformation of the AtEMl0l line with a wild-type allele of POLARIS was able to complement the mutant phenotype indicating that the T-DNA insertion into POLARIS is responsible for the AtEMlOl phenotype. Overexpression of POLRIS resulted in transgenic plants with reduced sensitivity to both cytokinins and ACC. The structure of the POLARIS locus and the potential role of POLARIS in regulating cytokinin-induced ethylene levels, with regards to the control of root growth, are discussed.

Twenty years ago, N‐acylethanolamines (NAEs) were considered by many lipid chemists to be biological ‘artifacts’ of tissue damage, and were, at best, thought to be minor lipohilic constituents of various organisms. However, that changed dramatically in 1993, when anandamide, an NAE of arachidonic acid (N‐arachidonylethanolamine), was shown to bind to the human cannabinoid receptor (CB1) and activate intracellular signal cascades in mammalian neurons. Now NAEs of various types have been identified in diverse multicellular organisms, in which they display profound biological effects. Although targets of NAEs are still being uncovered, and probably vary among eukaryotic species, there appears to be remarkable conservation of the machinery that metabolizes these bioactive fatty acid conjugates of ethanolamine. This review focuses on the metabolism and functions of NAEs in higher plants, with specific reference to the formation, hydrolysis and oxidation of these potent lipid mediators. The discussion centers mostly on early seedling growth and development, for which NAE metabolism has received the most attention, but also considers other areas of plant development in which NAE metabolism has been implicated. Where appropriate, we indicate cross‐kingdom conservation in NAE metabolic pathways and metabolites, and suggest areas where opportunities for further investigation appear most pressing.

No description available.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

In the following study, I have investigated aberrant integration events of the maize Activator/Dissociation ( Ac/Ds) family of transposable elements (TEs) in Arabidopsis. The purpose of the study was twofold: (i) to identify sequence modifications associated with aberrant transposition that are informative regarding the mechanism of Ac/Ds transposition; and (ii) to extend our understanding of the mechanisms by which class II TEs can influence genome structure. This work focuses on a large inversion identified on chromosome II. A lone Ds element comprises one breakpoint of the inversion and the second breakpoint is composed solely of Arabidopsis sequences. The analysis of the sequence modifications present at both breakpoints indicates that the event was precipitated by the abortive transposition of Ds. This is the first event of its kind identified for an Ac/Ds and the event defines a novel mechanism by which these TEs can induce change within a genome. Further, the presence of deletions at both termini of the implicated Ds suggests that the transposition of Ac/Ds involves fully excised intermediates. To obtain further support for this model, a population of Arabidopsis individuals harboring Ds excision events was screened for reintegrated elements. Several integrations were analyzed at the sequence level and compared to wild-type integration sites. While no genome rearrangements were detected, a number of integrations display small deletions within both the Ds termini and the DNA flanking the elements. These results are consistent with the presence of fully excised Ac/Ds intermediates. Further, the results suggest that dissolution of the transposase active complex at different points in the transposition process will result in the formation of distinct aberrant transposition products. During the characterization of the inversion, a novel Arabidopsis TE family, FARE, was identified. The FARE elements are foldback transposons, a heterogeneous and poorly characteri

Telomeres form a nucleoprotein cap at the end of eukaryotic chromosomes. The telomere protein constituents repress the DNA damage response (DDR) and facilitate maintenance of terminal sequences by a specialized ribonucleoprotein complex called telomerase. In turn, factors involved in the DDR guarantee telomerase acts only in telomere homeostasis, and not at double-strand breaks (DSBs). Thus, the three pathways surrounding telomeres display incredible overlap and are immensely complex. Here, I report a novel regulatory pathway that limits telomerase action during DNA damage. Duplication of the telomerase RNA subunit (TER) in Arabidopsis has given rise to a TER that is not required for telomere homeostasis. Indeed, this TER, termed TER2, is a competitive inhibitor of TER1 RNP complexes. Exposure to genotoxic agents results in TER2 upregulation and a subsequent inhibition of telomerase activity. Using data from the 1,001 Arabidopsis genomes project, I determine that the TER duplication and inhibitory nature of TER2 is likely derived from a transposon-like element within TER2. This element is found throughout Brassicaceae, with at least 32 members in Arabidopsis lyrata. These findings highlight the complex and diverse mechanisms by which an organism will regulate telomerase action. Here I characterize two members of the A. thaliana POT1 gene family. Contrary to POT1a, these proteins appear to have derived unique ways to perform their roles in chromosome-end protection. POT1b may protect telomeres as part of a TER2 telomerase RNP complex, as telomere defects only appear in the absence of both POT1b and TER2. POT1c is also appears to provide for chromosome end protection and appears to compete with POT1a to regulate telomerase access to the G-overhang. Together, these proteins represent part of a critical telomere capping complex distinct from CST. Additionally, I describe a means for elucidating factors that regulate telomere addition at DSBs. This incredibly detrimental process, termed de novo telomere formation (DNTF), is toxic, and thus this work describes the first in depth characterization of DNTF in multicellular eukaryotes. In summary, my work describes several novel regulatory and protective mechanisms for keeping telomeres and DSBs distinct.