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Mitochondria are ubiquitous eukaryotic organelles which carry out a range of essential functions, most notably the production of ATP through the process of oxidative phosphorylation. While the main biochemical function of mitochondria was established over 50 years ago, the processes which control mitochondrial morphology are, at present, poorly understood. The thesis aims to add to our knowledge of the factors that control mitochondrial morphology and dynamics in the model plant species, Arabidopsis thaliana. The phenotypic characteristics of two novel mitochondrial morphology mutants, motley mitochondria I (mmtl) and network mitochondria (nmt), were examined and quantified. mmtl has an increased heterogeneity of mitochondrial plan area relative to wild-type, which is matched by a similar chloroplast phenotype. nmt exhibits a reticular mitochondrial morphology, similar to the mitochondria found in yeast and animals. Genetic mapping of the two mutant loci has established that mmtl resides on a short region of chromosome 11w, hile nmt was mapped to a small area of chromosome V. This thesis describes the identification and functional analysis of two novel orthologs of yeast and animal mitochondrial division genes. Using TDNA reverse genetics, it is shown that disruption of the dynamin-like DRP3A or BIGYIN (an Arabidopsis orthologue of yeast FISI) led to an increase in mitochondrial plan area, which is coupled with a decrease in the number of physically discrete mitochondria per cell. Finally, the morphology and behaviour of Arabiclopsis mitochondria is investigated upon the induction of cell death. Abiotic stress treatments that induce cell death led to fast and irreversible changes in mitochondrial morphology. The role of these changes, as possible early indicators of cell death, are discussed.

published_or_final_version / abstract / Botany / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Arabidopsis thaliana Acyl-CoA-Binding Protein 3, one of six acyl-CoA-binding proteins, is unique by the C-terminal location of its acyl-CoA-binding (ACB) domain. It promotes autophagy (ATG)-mediated leaf senescence and confers resistance to Pseudomonas syringae pv. tomato DC3000. To understand the regulation of ACBP3, a 1.7 kb 5’-flanking region of ACBP3 and its deletion derivatives were characterized using β-glucuronidase (GUS) reporter gene fusions. A 374 bp minimal fragment (-151/+223) could drive GUS expression while a 1698 bp fragment (-1475/+223) conferred maximal activity. Further, histochemical GUS staining analysis on transgenic Arabidopsis harboring the largest (1698 bp) ACBP3pro::GUS fusion displayed ubiquitous expression in floral organs and vascular bundles of leaves and stems, consistent with previous results that extracellularly localized ACBP3 functions in plant defense. A 160 bp region (-434/-274) induced GUS expression in extended darkness and conferred down-regulation in extended light. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay showed that the DNA binding with one finger box (Dof-box, -341/-338) interacted specifically with leaf nuclear proteins from dark-treated Arabidopsis while GT-1 (-406/-401) binds both dark- and light-treated Arabidopsis, suggesting that Dof and GT-1 motifs are required to mediate circadian regulation of ACBP3. Moreover, GUS staining and fluorometric measurements revealed that a 109 bp region (-543/-434) was responsive to phytohormones and pathogens. Within this 109 bp region, an S-box of AT-rich sequence (-516/-512) was identified to bind nuclear proteins from pathogen-infected Arabidopsis leaves, providing the basis for pathogen-inducible regulation of ACBP3 expression. Hence, three cis-responsive elements (Dof, GT-1 and S-box) in the 5’-flanking region of ACBP3 were demonstrated to participate in the regulation of ACBP3. The regulation of ACBP3 by circadian control is not surprising given that defense genes are now known to be circadian-regulated; infection being anticipated at dawn coinciding with pathogen activity in spore dispersal during the light period. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

As sessile organisms, coordination of development and reproduction in a dynamic, and often stressful, environment presents a particular challenge for plants. Rapid processing of internal and external cues by complex signal transduction pathways leads to stimulus-appropriate physiological responses on an organismal scale. In plants, myriad signaling pathways are mediated by calcium (Ca2+) signals, and it is thought that different stimuli elicit unique patterns of Ca2+ influx into cells (termed Ca2+ ‘signatures’) that encode information important for proper physiological responses. Encoding of information in the form of Ca2+ signatures requires that decoding elements be present in cells to direct downstream cellular processes. This role is filled by Ca2+-binding proteins that serve as Ca2+ sensors. Interestingly, plant genomes encode multiple expanded families of Ca2+ sensors not found in animal genomes. Among these, the calmodulin (CaM)-like proteins (CMLs) are represented by a 50 member family in Arabidopsis. On the basis of structural homology, CMLs are predicted to function like conserved CaM, however, little work has been done to address this question. Biochemical characterization of CML39 indicates that it possesses structural properties consistent with function as a Ca2+ sensor. Analysis of transgenic CML39 loss-of-function (cml39) mutants revealed that CML39 is important for proper seedling establishment in the absence of exogenous metabolisable carbon as cml39 seedlings entered a state of developmental arrest shortly after germination. cml39 mutants also exhibited a conditional ‘de-etiolated’ phenotype when grown in complete darkness and exaggerated hypocotyl elongation under a short-day light regime. Genetic data suggest that CML39 functions in signaling pathways downstream of light perception, and this idea is supported by the observation that CML39 iii is expressed in light-sensing tissues, and that subunit 5 of the COP9 signalosome, a protein critical for photomorphogenesis, was identified as a putative target of CML39. Collectively, results show that CML39 is Ca2+ sensor that serves a critical regulatory role during seedling establishment when sucrose is limited, and importantly, further underscore the pervasiveness of Ca2+ signaling in plant growth and development. / Thesis (Ph.D, Biology) -- Queen's University, 2013-05-29 18:16:25.769

In transgenic research the precise control of transgene expression is crucial in order to obtain transformed organisms with expected desirable traits. A broad range of transgenic plants use the constitutive cauliflower mosaic virus (CaMV) 35S promoter to drive expression of selectable marker genes. Due to its strong enhancer function, this promoter can disturb the specificity of nearby eukaryotic promoters. When inserted immediately downstream of the 35S promoter in transformation vectors, special DNA sequences called insulators can prevent the influence of the CaMV35S promoter/enhancer on adjacent tissue-specific promoters for the transgene. Insulators occur naturally in organisms such as yeasts and animals but few insulators have been found in plants. Therefore, the goal of this study is to identify DNA sequences with insulator activity in Arabidopsis thaliana. A random oligonucleotide library was designed as an initial step to obtain potential insulators capable of blocking enhancer-promoter interactions in transgenic plants. Fragments from this library with insulator activity were identified and re-cloned into pB31, in order to confirm their activity. To date, one insulator sequence (CLO I-3) has been identified as likely possessing enhancer-blocking activity. Also, two other oligonucleotide sequences (CLO II-10 and CLO III-78) may possess insulator activity but more sampling is needed to confirm their activity. Further studies are needed to validate the function of plant insulator(s) and characterize their associated proteins.

Discrete changes in the amplitude, frequency, and cellular localisation of calcium ion (Ca2+) transients encode information about numerous stimuli and function to mediate stimulus-specific responses. Cytoplasmic Ca2+ (Ca2+cyt) undergoes circadian oscillations in concentration that appear to be under the control of the same endogenous oscillator that regulates expression of genes in the photoperiodic-flowering pathway. It is currently not known whether these circadian [Ca2+cyt] oscillations are biochemical artefacts or are decoded and function to transduce clock dependent responses. Calmodulin (CaM) is a primary node in Ca2+ signalling in plants and as such is a promising target for investigating the role of Ca2+ in clock-controlled processes. In this study, Arabidopsis thaliana were treated with experimentally validated concentrations of pharmacological CaM inhibitors. Under inductive photoperiods (16 h light : 8 h dark), CaM inhibition was found to increase developmental flowering time, whilst under non-inductive photoperiods no such changes were evident. Inhibition of CaM led to changes in expression of the key clock gene TIMING OF CAB EXPRESSION 1 and flowering time genes, CONSTANS and FLOWERING LOCUS T and removed repression of flowering in darkness. These observations are consistent with CaM modulating the activity of the putative clock component GIGANTEA and the proteasomal targeting protein SUPPRESSOR OF PHYA-105. Due to the unwanted side effects often generated by chemical CaM inhibitors, a peptide inhibitor of CaM comprising a green fluorescent protein / calspermin fusion and labelled smGN was developed. Surface plasmon resonance analysis and affinity chromatography showed smGN to have extremely high selectivity for, and affinity to, CaM and to function as a powerful inhibitor of CaM in vitro. Further work on the methodology used to deploy smGN as a recombinant alternative to chemical CaM inhibitors in planta is also described.

Long-distance signaling is an important process in the development of Arabidopsis thaliana. A leucine-rich repeat receptor-like kinase (LRR-RLK), XYLEM INTERMIXED WITH PHLOEM1 a.k.a. C-TERMINALLY ENCODED PEPTIDE RECEPTOR 1 (XIP1/CEPR1), functions in vascular development and has recently been implicated in nitrogen sensing and response. Previous results indicate that XIP1/CEPR1 also interacts with multiple proteins involved in sugar metabolism and transport as well as other metabolic proteins, which indicates a possible role for XIP1/CEPR1 in mediating sugar transport. xip1-1 seeds, which grow slowly in the cold in comparison to Columbia wild-type plants, were previously EMS mutagenized and screened for suppressors of the cold-sensitive phenotype. One of these suppressors, 9-12, maps to the lower region of chromosome V and several possible causative EMS-like mutations have been identified that may link XIP1/CEPR1 to a more general vascular transport role.