Phosphorus is an essential element for plant growth and development. Due to its low availability, solubility and mobility, phosphate is often the limiting macronutrient for crops and other plants. Plants have evolved several responses to phosphate deficiency. However, very little is known about the molecular basis of these responses. Here, I study the expression of PAP12, its role in the phosphate starvation response and the interaction of its promoter with nuclear factors. Analysis of a PAP12 T-DNA insertion line (pap12-1) revealed PAP12 is responsible for the majority of the acid phosphatase activity detected by the standard in-gel assay. RNA gel blots showed that PAP12 was induced only by Pi deficiency, and not by general nutrient stress. PAP12 expression, at the RNA and protein level, reflected endogenous phosphate levels in two mutants with altered phosphate accumulation. In the pho1 mutant, PAP12 expression and activity were up-regulated with respect to wild-type plants, and in the pho2 mutant, PAP12 expression and activity were reduced. Analysis of the PAP12 promoter using promoter-GUS fusions revealed expression in leaves, roots, flowers, hydathodes, root tips, and pollen grains. This broad pattern of expression suggests that PAP12 functions throughout the plant in response to low phosphate concentrations. The results showed PAP12 does not play a major role in phosphate remobilization, acquisition or in helping plants cope with low phosphate environments. Instead, the major phenotype associated with PAP12 deficiency was a significant delay in flowering in the low-phosphate pho1 background and a slight acceleration of flowering in the high-phosphate pho2 background over-expressing PAP12. These results suggest that PAP12 may have a role in linking phosphate status with the transition to flowering. Finally, I used promoter deletion and DNA-protein interaction assay to understand PAP12 expression upon phosphate starvation. A 35-bp region of the PAP12 promoter was identified as an important phosphate regulatory cis-element required for induction during phosphate starvation. We isolated a 23.5 kDa nuclear factor, which binds to this 35-bp region of the PAP12 promoter in a phosphate-dependent manner. The work presented here will add to our knowledge about the molecular processes that regulate phosphate nutrition.

Phosphorus is an essential element for plant growth and development. Due to its low availability, solubility and mobility, phosphate is often the limiting macronutrient for crops and other plants. Plants have evolved several responses to phosphate deficiency. However, very little is known about the molecular basis of these responses. Here, I study the expression of PAP12, its role in the phosphate starvation response and the interaction of its promoter with nuclear factors. Analysis of a PAP12 T-DNA insertion line (pap12-1) revealed PAP12 is responsible for the majority of the acid phosphatase activity detected by the standard in-gel assay. RNA gel blots showed that PAP12 was induced only by Pi deficiency, and not by general nutrient stress. PAP12 expression, at the RNA and protein level, reflected endogenous phosphate levels in two mutants with altered phosphate accumulation. In the pho1 mutant, PAP12 expression and activity were up-regulated with respect to wild-type plants, and in the pho2 mutant, PAP12 expression and activity were reduced. Analysis of the PAP12 promoter using promoter-GUS fusions revealed expression in leaves, roots, flowers, hydathodes, root tips, and pollen grains. This broad pattern of expression suggests that PAP12 functions throughout the plant in response to low phosphate concentrations. The results showed PAP12 does not play a major role in phosphate remobilization, acquisition or in helping plants cope with low phosphate environments. Instead, the major phenotype associated with PAP12 deficiency was a significant delay in flowering in the low-phosphate pho1 background and a slight acceleration of flowering in the high-phosphate pho2 background over-expressing PAP12. These results suggest that PAP12 may have a role in linking phosphate status with the transition to flowering. Finally, I used promoter deletion and DNA-protein interaction assay to understand PAP12 expression upon phosphate starvation. A 35-bp region of the PAP12 promoter was identified as an important phosphate regulatory cis-element required for induction during phosphate starvation. We isolated a 23.5 kDa nuclear factor, which binds to this 35-bp region of the PAP12 promoter in a phosphate-dependent manner. The work presented here will add to our knowledge about the molecular processes that regulate phosphate nutrition.

published_or_final_version / abstract / Physiology / Master / Master of Philosophy

Acid phosphatases (APases; E.C. 3.1.3.2) catalyze the hydrolysis of phosphate (Pi) from Pi monoesters and anhydrides within the acidic pH range. Induction of intracellular and secreted purple acid phosphatases (PAPs) is a widespread plant response to nutritional Pi-deficiency. The probable function of intracellular APases is to recycle Pi from expendable intracellular organophosphate pools, whereas secreted APases likely scavenge Pi from the organically bound Pi that is prevalent in most soils. Although the catalytic function and regulation of plant PAPs have been described, their physiological function in plants has not been fully established. Recent biochemical and proteomic studies indicated that AtPAP26 is the predominant intracellular (vacuolar) and a major secreted purple APase isozyme upregulated by Pi-starved (-Pi) Arabidopsis thaliana. The in planta function of AtPAP26 was assessed by molecular, biochemical, and phenotypic characterization of a homozygous Salk T-DNA insertion mutant. Loss of AtPAP26 expression resulted in the elimination of AtPAP26 transcripts and 55-kDa immunoreactive AtPAP26 polypeptides, correlated with a 9- and 5-fold decrease in extractable shoot and root APase activity, respectively, as well as a 40% reduction in secreted APase activity of –Pi seedlings. The results corroborate previous findings implying that AtPAP26 is: (i) the principal contributor to Pi starvation inducible APase activity in Arabidopsis, and (ii) controlled post-transcriptionally mainly at the level of protein accumulation. Total shoot free Pi level was about 40% lower in –Pi atpap26 mutants relative to wild-type controls, but unaffected under Pi-sufficient conditions. Moreover, shoot, root, inflorescence, and silique development of the atpap26 mutant was impaired during Pi deprivation, but unaffected under Pi-replete conditions, or during nitrogen or potassium-limited growth, or oxidative stress. The results suggest that the hydrolysis of Pi from organic-phosphate esters by AtPAP26 makes an important contribution to Pi-recycling and scavenging in –Pi Arabidopsis. / Thesis (Master, Biology) -- Queen's University, 2009-09-01 09:46:39.302

The experimental work described in this thesis was conducted in the surgical research laboratories of Dr Francis D. Moore in the Peter Bent Brigham Hospital (now the Brigham & Women's Hospital) Boston, Massachusetts, USA, between 1978-1979. It formed part of an ongoing programme of research into protein metabolism in man; specifically to measure total body nitrogen turnover and hence protein synthesis and breakdown, initially in normal volunteers receiving various intravenous feeding regimens, and subsequently in patients. The previous year, 1977, had seen the publication of 'Substrate Interaction in Intravenous Feeding' by Bruce Wolfe et al., from the same laboratories. This was an extensive piece of work incorporating many studies and compared nitrogen balance data together with biochemical, hormonal and plasma amino acid data in normal men fed intravenously with a variety of regimens. Shortly afterwards a series of protein turnover studies was embarked upon, using the uN glycine method, and in collaboration with Dr Vernon Young of the Massachusetts Institute of Technology. The first experiments were essentially a repeat of the studies described by Wolfe et al. (vide supra) but in addition nitrogen turnover, protein synthesis and breakdown were estimated using a continuous 60 hour infusion of uN glycine and measuring enrichment of urinary urea with uN when a plateau was reached. Normal volunteers were studied firstly on normal oral diet and then on a iv succession of intravenous regimens:- amino acids alone (AA), amino acids plus 'high dose' glucose (AA + HOG), amino acids plus fat emulsion (AA + FE), amino acids plus 'low dose' glucose (AA + LOG), amino acids, fat emulsion and low dose glucose (AA + LDG+ FE), and finally 'low dose' glucose alone (LOG). The studies on normal diet, AA and AA+HOG were conducted by Andrew Sim (a Glasgow/Harvard exchange fellow) and Bruce Wolfe. The author took no practical role in these experiments, but was responsible for analysis of the data and the protein metabolism calculations, and was a co-author when the work was published in 1979 (Sim et al., Glucose Promotes Whole-Body Protein Synthesis from Infused Aminoacids in Fasting Man, Lancet i, 68-71). Subsequently, the author did the experiments using AA + LOG + FE, AA + FE, and AA + LOG and LOG. The results on these four regimens were incorporated in a paper presented in 1979 at the Tripartite Meeting of the Surgical Research Society at Oxford under the title 'Isotope Studies of substrate interaction in parenteral nutrition', and also at the 2nd European Congress on Parenteral and Enteral Nutrition at Newcastle upon Tyne in 1980, and later published as 'The Effect of Fat Infusion on Protein Metabolism' (Acta. Chir. Scand., Suppl. 507, 475-484, 1981). When the studies on the various intravenous feeding regimens were completed, attention was turned to the possible distorting effects of variables such as exercise and diet v on the behaviour of the isotope equilibrium curve and plateau. Such effects, if present, might have significance when studies were carried out on patients at a later stage in the research programme. Because each study lasted 48-60 hours, changes might occur either unintentionally or as a result of the needs of clinical management, and if they affected the plateau would alter the resultant calculations of turnover, synthesis and breakdown. Such a potential source of error clearly needed investigation. A pilot study was done in two subjects, later repeated on each, to observe any effects on the curve and plateau of both doubling protein intake and bicycle exercise. Subsequently, more extensive studies were done varying the protein and energy intakes, both orally and intravenously, allowing a detailed analysis of curve perturbation, and introducing the concept of basal catabolic rate. Finally, protein turnover, synthesis and breakdown were estimated seven times in four seriously ill patients. All of the studies mentioned above form the basis of the thesis. Unfortunately, the gestation period of this thesis has been long. There are two main reasons for this. Firstly, the work done was part of a five-year programme of research, with the intention of publishing a paper in a scientific journal at the completion of each stage. This was done vi with the first three regimens (normal diet, AA and AA + HDG) but not with the last four (AA + FE, AA + LOO+ FE, AA + LOG, LDG), although the results were presented at two scientific meetings. Shortly after returning to the United Kingdom the author was appointed a consultant surgeon and this career move assumed priority. Secondly, although it was intended to publish the perturbation studies, it proved impossible to reduce the size of the text to a manageable level suitable for publication in the form of a scientific paper. However, despite the long interval since the experiments were done, no similar work has been published. In particular, virtually no attention has been paid to intentional perturbation. Also, whereas there was a spate of interest in protein turnover studies in the late 1970s and early 1980s, virtually no publications have appeared since 1985. It seems that the potential applications of the method are considered exhausted, and interest has been lost, rather as it was in the 1950s following a short flurry of activity exploring the first cumbersome technique. Hence, it seemed all the more pertinent, even at this late stage, to publish the work in the form of a thesis which could describe in chronological order the continuum of studies as briefly mentioned above. In order to preserve such a progression, the following Introduction contains, with few exceptions, only references up to the time that the experimental studies were commenced, 1978, but the subsequent Discussion(s) in the various sections will attempt to include the relevant literature up to the present time.

Post-transcriptional gene silencing (PTGS) is a powerful mechanism that can be adapted to genetically modify crop plants. PTGS operates in many plant signaling pathways including those mediating stress responses. Given the small number of miRNAs known, research on the characterization of stress-related micro-RNA (miRNA) and their targets could provide the basis for engineering stress tolerant traits in crops. Indeed, several examples of miRNA mediated crop tolerance have been reported. In the research presented here, we aimed to analyze the role of small non-coding RNA (smRNA) pathways involved in plant stress. In particular, we focused on miRNA-mediated PTGS in phosphate (Pi) starvation. The analysis was split into two research projects. First, to identify potential miRNA targets we began by analyzing the response and recovery of coding and long non-coding RNAs (lncRNA) to Pi starvation in shoot and root. The results obtained were the first genome-wide description of the root’s Pi starvation response and recovery. We found that the root's response involved a widely different set of genes than that of the shoot. In the second research project, the results of the first project were correlated with the responses of miRNA and trans-acting small-interfering RNA (tasiRNA) during Pi starvation. Many miRNA circuits have been predicted before, however, tasiRNA circuits are not as well defined. Therefore, we made use of the double-stranded RNA-binding protein 4 (DRB4) smRNA libraries to enhance our prediction of tasiRNAs. Altogether, we provided evidence to support the following miRNA-mRNA pairs that may function in Pi starvation: IPS1:miR399:PHO2; miR399:RS4; miR399:NF-YA10; miR398:CSD1/2; miR2111:TPS11; miR164:NAC6; miR157:TMO7; miR157:PSB28; RPS2:miR169:IPS2; miR397:LAC2; TAS4:PAP1; NR1:PAP1; and Chr3_1967672:TMO7. In general, we found that non-miR399 related circuits were active only during the root’s recovery from Pi starvation. The functional roles of the genes targeted by these PTGS circuits suggested that the local root response to Pi starvation was influenced by the plant's systemic response pathways via PHR1-mediated PTGS. Finally, since many PTGS targets function to modulate concentrations of reactive oxygen species and sucrose, we hypothesized that the candidate PTGS circuits found in our research mediate a general stress recovery process by modulating metabolites involved in signaling pathways.

International humanitarian organizations played a prominent role in the Nigeria-Biafra War, but scholars have paid little or no attention to the humanitarian crisis in the war and the global humanitarian intervention that followed it. This thesis aims to fill a gap in the historiography of international humanitarian aid in the Nigeria-Biafra War by focusing on the Joint Church Aid (JCA), a consortium of Catholic and Protestant Churches that provided relief aid for the starving civilians in Biafra. This study of the JCA is broken down into three parts: the humanitarian impulse in the Nigeria-Biafra conflict, the formation of the JCA and its relief organization, and the challenges of relief operation in Biafra. The research provides a window into understanding the complex nature of international humanitarian aid in political conflicts. This dissertation argues that the JCA’s humanitarian operation, though relatively successful, had unintended consequences. While the JCA aimed to provide relief for the starving Biafran population, it was interpreted by the Nigerian government as political support for a “rebellion.” Convinced that the humanitarian organizations engaged in arms dealings with the Biafran government, the Nigerian government intensified military counter-action against the relief operation. The Nigerian government refused to separate international humanitarian aid from the political objectives of the war hence starvation came to be seen as a legitimate instrument of warfare. On the Biafran side, however, there was an effort to separate international humanitarian aid from the politics and hostilities of the war. Consequently, humanitarianism became a deeply contested issue that brought the humanitarian agencies into direct conflict with the Nigerian government. This study contributes to the scholarship on international humanitarianism and the internationalization of armed conflicts in postcolonial Africa. / Thesis / Doctor of Philosophy (PhD)

Improving the efficiency by which crops use nutrients is critical for maintaining high crop productivity while reducing fertility management costs and eutrophication related to fertilizer runoff. The native crucifer and halophyte, Yukon Eutrema salsugineum, was used in this study. Yukon E. salsugineum is closely related to important Brassica crops and thrives in its native habitat on soil that is low in available phosphate (Pi) and high in sulfur (S). To determine how Yukon E. salsugineum copes with low Pi, leaf transcriptomes were prepared from four week-old plants grown in controlled environment chambers using soil lacking or supplemented with Pi and/or S. This thesis focused on using bioinformatic approaches to assemble, analyze and compare the transcriptome profiles produced by the Yukon E. salsugineum plants undergoing four nutrient combinations of high and/or low Pi and S. The objective of the study was to identify traits associated with altered S and/or Pi with the prediction based on other species that low Pi, in particular, would pose the greatest stress and hence elicit the greatest transcriptional reprogramming. Transcriptome libraries were generated from four treatment groups with three biological replicates each. Reads in each library were mapped to 23,578 genes in the E. salsugineum transcriptome with an average unique read mapping ratio of 99.52%. Surprisingly, pairwise comparisons of the transcriptomes showed little evidence of Pi-responsive reprogramming whereas treatments differing in soil S content showed a clear S-responsive transcriptome profile. Principal Component Analysis revealed that the low variance quaternary Principal Component distinguished the transcriptomes of plants undergoing low versus high Pi treatments with differential gene expression analysis only finding 11 Pi-responsive genes. This outcome suggests that leaf transcriptomes of Yukon E. salsugineum plants under low Pi are largely undifferentiated from plants provided with Pi and is consistent with Yukon E. salsugineum maintaining Pi homeostasis through fine-tuning the expression of protein-coding and non-coding RNA rather than large-scale transcriptomic reprogramming. Previous research has shown Yukon E. salsugineum to be very efficient in its use of Pi and this work suggests that the altered expression of relatively few genes may be needed to develop Pi-efficient crops to sustain the crop demand of a growing population. / Thesis / Master of Science (MSc)

Vibrio vulnificus is a model environmental organism exhibiting a classical starvation response during nutrient limitation as well as a non-culturable state when exposed to low temperatures. In addition to these classic global responses, this organism is an opportunistic pathogen that exhibits numerous virulence factors. This organism was chosen as the model organism for the identification of regulators of the viable but nonculturable response (VBNC) and the starvation-induced maintenance of culturability (SIMC) that occurs when cells are starved prior to low temperature incubation. In order to accomplish this, three indirect approaches were used; proteomics, investigation of intercellular signalling pathways and genetic analysis of regulators involved in these responses. Two-dimensional gel electrophoresis was used to identify proteins expressed under conditions that induced SIMC. It was determined that carbon and long-term phosphorus starvation were important in the SIMC response. V. vulnificus was shown to possess genes, luxS and smcR, that are homologues of genes involved in signalling system system 2 in Vibrio harveyi. Signal molecules were produced upon starvation and the entry to stationary phase in V. vulnificus. Furthermore, a null mutation in smcR, a transcriptional regulator was shown to have pleiotropic effects in V. vulnificus, including up-regulation of numerous virulence factors and a defect in starvation survival and development of the SIMC response. We propose that V. vulnificus possesses a signalling system analogous to that of system 2 in V. harveyi, and that this system is involved in the regulation of stationary phase and starvation adaptation in this organism.

BACKGROUND:Honey bees (Apis mellifera) contribute substantially to the worldwide economy and ecosystem health as pollinators. Pollen is essential to the bee's diet, providing protein, lipids, and micronutrients. The dramatic shifts in physiology, anatomy, and behavior that accompany normal worker development are highly plastic and recent work demonstrates that development, particularly the transition from nurse to foraging roles, is greatly impacted by diet. However, the role that diet plays in the developmental transition of newly eclosed bees to nurse workers is poorly understood. To further understand honey bee nutrition and the role of diet in nurse development, we used a high-throughput screen of the transcriptome of 3day and 8day old worker bees fed either honey and stored pollen (rich diet) or honey alone (poor diet) within the hive. We employed a three factor (age, diet, age x diet) analysis of the transcriptome to determine whether diet affected nurse worker physiology and whether poor diet altered the developmental processes normally associated with aging.RESULTS:Substantial changes in gene expression occurred due to starvation. Diet-induced changes in gene transcription occurring in younger bees were largely a subset of those occurring in older bees, but certain signatures of starvation were only evident 8day old workers. Of the 18,542 annotated transcripts in the A. mellifera genome, 150 transcripts exhibited differential expression due to poor diet at 3d of age compared with 17,226 transcripts that differed due to poor diet at 8d of age, and poor diet caused more frequent down-regulation of gene expression in younger bees compared to older bees. In addition, the age-related physiological changes that accompanied early adult development differed due to the diet these young adult bees were fed. More frequent down-regulation of gene expression was observed in developing bees fed a poor diet compared to those fed an adequate diet. Functional analyses also suggest that the physiological and developmental processes occurring in well-fed bees are vastly different than those occurring in pollen deprived bees. Our data support the hypothesis that poor diet causes normal age-related development to go awry.CONCLUSION:Poor nutrition has major consequences for the expression of genes underlying the physiology and age-related development of nurse worker bees. More work is certainly needed to fully understand the consequences of starvation and the complex biology of nutrition and development in this system, but the genes identified in the present study provide a starting point for understanding the consequences of poor diet and for mitigating the economic costs of colony starvation.