http://www.dspace.cam.ac.uk:80/handle/1810/223857 Sat, 25 May 2013 22:37:48 GMT 2013-05-25T22:37:48Z http://www.dspace.cam.ac.uk:80/handle/1810/243636 Title: Forest dynamics at regional scales: predictive models constrained with inventory data Authors: Lines, Emily Abstract: Forest ecosystems store more carbon than the atmosphere and harbour the majority of the world's biodiversity, yet their response to changing climate is uncertain. Forest simulation models make landscape-level predictions of forest dynamics by scaling from key tree-level processes, but models typically have no climate dependency. In this thesis I demonstrate how large-scale national inventories combined with improvements in computational methods mean that models that incorporate the climate dependency of demographic processes may be parameterised at regional scales. In Chapter One I outline historical approaches to modelling forest dynamics and present a discussion of competing methods of parameterisation and model selection. In Chapter Two I present a model of individual tree mortality in the eastern United States which incorporates species, climatic and competitive effects parameterised using Markov Chain Monte Carlo methods. The remainder of the thesis concentrates on modelling Spanish forest dynamics, so in Chapter Three I present a brief introduction to Spanish forest ecology. In Chapter Four I examine how aboveground allometry - the scaling of tree height and crown shape - varies with climate and competition in Spain for 26 species. Hierarchical modelling suggests that scaling theories based on wood properties do not explain differences between species, but climatic factors, and in particular hydraulic limitations, do. In Chapter Five I parameterise a model of recruitment in Spanish forests using Approximate Bayesian Computation, a novel computational method which allows parameterisation of individual-based models without individual-based data, and demonstrate that it produces ecologically reasonable results. Chapter Six presents a forest dynamics model parameterised for the major native species in Spain and tests whether it is able to reproduce observed species-climate distributions. Finally, in Chapter Seven I discuss the main findings of the thesis and avenues for extending this research. Mon, 11 Jun 2012 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/243636 2012-06-11T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/239128 Title: Nannoplankton as indicators of climatic variability in the Upper Pliocene Authors: Chepstow-Lusty, Alexander John Abstract: Discoasters are the remains of an enigmatic group of nannoplankton, whose last representatives disappeared globally at 1.89 Ma. The one million years prior to their extinction has been analysed in high time resolution and a global database has been developed from ten sites in the Atlantic, Indian and Pacific Oceans. It has been shown that the variability in Discoaster abundance at low latitudes cannot be attributed solely to changes in sea-surface temperature; discoasters were demonstrated to be suppressed at upwelling sites. In combination with global satellite images of phytoplankton blooms (Lewis, 1989) and maps of modern sea-surface temperatures (CLIMAP, 1981), Discoaster abundance variations were interpreted in terms of the interplay between productivity pressure and sea-surface temperature. Discoasters thrived in the Indian Ocean at Site 709 (4°S) in warm waters with little productivity pressure, whereas Discoaster abundance was suppressed at Sites 677 (l°N) and 662 (l°S) in the Pacific and Atlantic, which although located in warm waters, were affected by upwelling. In addition to the reduced Discoaster abundances associated with upwelling, sea-surface temperature gradients are a significant factor when comparing high and low latitude sites. A marked drop in Discoaster abundance is apparent between 41°N and 56°N (i.e., between Sites 607 and 552). Relative abundances of species in the Discoaster assemblage revealed clearly their contrasting environmental preferences; D. brouweri was produced under a wide range of conditions, favouring warm, low productivity waters; D. asymmetricus and D. tamalis were produced in higher abundances relative to D. brouweri during cooler, low productivity episodes; D. pentaradiatus showed highest absolute abundance in warm, low productivity waters, although increasing in relative abundance at higher latitudes; D. surculus increased in relative abundance in high productivity regions and in cooler waters. At all sites, though most markedly at higher latitudes, Discoaster abundances declined after 2.4 Ma, when glaciation began in the North Atlantic. Tue, 01 Jan 1991 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/239128 1991-01-01T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/237960 Title: A simple way to identify non-viable cells within living plant tissue using confocal microscopy Authors: Truernit, Elisabeth; Haseloff, Jim Abstract: Abstract Background Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. Results Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy. The dyes were selective for non-viable cells and showed very little background staining in living cells. Simultaneous detection of SYTOX dye and fluorescent protein (e.g. GFP) fluorescence was possible. Conclusion The fluorescent SYTOX dyes are useful for an easy and quick first assay of plant cell viability in living plant samples using fluorescence and confocal laser-scanning microscopy. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are. Sun, 22 Jun 2008 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/237960 2008-06-22T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/237902 Title: Population genetics of the understory fishtail palm Chamaedorea ernesti-augusti in Belize: high genetic connectivity with local differentiation Authors: Cibrian-Jaramillo, Angelica; Bacon, Christine D; Garwood, Nancy C; Bateman, Richard M; Thomas, Meredith M; Russell, Steven R; Bailey, Donovan C; Hahn, William J; Bridgewater, Samuel G M; DeSalle, Rob Abstract: Abstract Background Developing a greater understanding of population genetic structure in lowland tropical plant species is highly relevant to our knowledge of increasingly fragmented forests and to the conservation of threatened species. Specific studies are particularly needed for taxa whose population dynamics are further impacted by human harvesting practices. One such case is the fishtail or xaté palm (Chamaedorea ernesti-augusti) of Central America, whose wild-collected leaves are becoming progressively more important to the global ornamental industry. We use microsatellite markers to describe the population genetics of this species in Belize and test the effects of climate change and deforestation on its recent and historical effective population size. Results We found high levels of inbreeding coupled with moderate or high allelic diversity within populations. Overall high gene flow was observed, with a north and south gradient and ongoing differentiation at smaller spatial scales. Immigration rates among populations were more difficult to discern, with minimal evidence for isolation by distance. We infer a tenfold reduction in effective population size ca. 10,000 years ago, but fail to detect changes attributable to Mayan or contemporary deforestation. Conclusion Populations of C. ernesti-augusti are genetically heterogeneous demes at a local spatial scale, but are widely connected at a regional level in Belize. We suggest that the inferred patterns in population genetic structure are the result of the colonization of this species into Belize following expansion of humid forests in combination with demographic and mating patterns. Within populations, we hypothesize that low aggregated population density over large areas, short distance pollen dispersal via thrips, low adult survival, and low fruiting combined with early flowering may contribute towards local inbreeding via genetic drift. Relatively high levels of regional connectivity are likely the result of animal-mediated long-distance seed dispersal. The greatest present threat to the species is the potential onset of inbreeding depression as the result of increased human harvesting activities. Future genetic studies in understory palms should focus on both fine-scale and landscape-level genetic structure. Thu, 08 Oct 2009 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/237902 2009-10-08T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/237861 Title: TobEA: an atlas of tobacco gene expression from seed to senescence Authors: Edwards, Kieron D; Bombarely, Aureliano; Story, Geraint W; Allen, Fraser; Mueller, Lukas A; Coates, Steve A; Jones, Louise Abstract: Abstract Background Transcriptomics has resulted in the development of large data sets and tools for the progression of functional genomics and systems biology in many model organisms. Currently there is no commercially available microarray to allow such expression studies in Nicotiana tabacum (tobacco). Results A custom designed Affymetrix tobacco expression microarray was generated from a set of over 40k unigenes and used to measure gene expression in 19 different tobacco samples to produce the Tobacco Expression Atlas (TobEA). TobEA provides a snap shot of the transcriptional activity for thousands of tobacco genes in different tissues throughout the lifecycle of the plant and enables the identification of the biological processes occurring in these different tissues. 772 of 2513 transcription factors previously identified in tobacco were mapped to the array, with 87% of them being expressed in at least one tissue in the atlas. Putative transcriptional networks were identified based on the co-expression of these transcription factors. Several interactions in a floral identity transcription factor network were consistent with previous results from other plant species. To broaden access and maximise the benefit of TobEA a set of tools were developed to provide researchers with expression information on their genes of interest via the Solanaceae Genomics Network (SGN) web site. The array has also been made available for public use via the Nottingham Arabidopsis Stock Centre microarray service. Conclusions The generation of a tobacco expression microarray is an important development for research in this model plant. The data provided by TobEA represents a valuable resource for plant functional genomics and systems biology research and can be used to identify gene targets for both fundamental and applied scientific applications in tobacco. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are. Fri, 26 Feb 2010 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/237861 2010-02-26T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/237817 Title: RNA silencing in plants: Flash report! Authors: Mosher, Rebecca A; Lewsey, Mathew G; Shivaprasad, Padubidri V Abstract: Abstract Earlier this year plant scientists met in Santa Fe, New Mexico at the Keystone Symposium "RNA Silencing Mechanisms in Plants". Sessions included small RNA biogenesis and signalling, development and stress responses, small RNA-directed DNA methylation, and interaction with pathogens. This report highlights some of the prominent and recurring themes at the meeting and emerging arenas of future research. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are. Tue, 29 Jun 2010 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/237817 2010-06-29T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/237808 Title: baySeq: Empirical Bayesian Methods For Identifying Differential Expression In Sequence Count Data Authors: Hardcastle, Thomas; Kelly, Krystyna A Abstract: Abstract Background High throughput sequencing has become an important technology for studying expression levels in many types of genomic, and particularly transcriptomic, data. One key way of analysing such data is to look for elements of the data which display particular patterns of differential expression in order to take these forward for further analysis and validation. Results We propose a framework for defining patterns of differential expression and develop a novel algorithm, baySeq, which uses an empirical Bayes approach to detect these patterns of differential expression within a set of sequencing samples. The method assumes a negative binomial distribution for the data and derives an empirically determined prior distribution from the entire dataset. We examine the performance of the method on real and simulated data. Conclusions Our method performs at least as well, and often better, than existing methods for analyses of pairwise differential expression in both real and simulated data. When we compare methods for the analysis of data from experimental designs involving multiple sample groups, our method again shows substantial gains in performance. We believe that this approach thus represents an important step forward for the analysis of count data from sequencing experiments. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are. Mon, 09 Aug 2010 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/237808 2010-08-09T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/237643 Title: Neurological and behavioral abnormalities, ventricular dilatation, altered cellular functions, inflammation, and neuronal injury in brains of mice due to common, persistent, parasitic infection Authors: Hermes, Gretchen; Ajioka, James W; Kelly, Krystyna A; Mui, Ernest; Roberts, Fiona; Kasza, Kristen; Mayr, Thomas; Kirisits, Michael J; Wollman, Robert; Ferguson, David J P; Roberts, Craig W; Hwang, Jong-Hee; Trendler, Toria; Kennan, Richard P; Suzuki, Yasuhiro; Reardon, Catherine; Hickey, Adam William; Chen, Lieping; McLeod, Rima Abstract: Abstract Background Worldwide, approximately two billion people are chronically infected with Toxoplasma gondii with largely unknown consequences. Methods To better understand long-term effects and pathogenesis of this common, persistent brain infection, mice were infected at a time in human years equivalent to early to mid adulthood and studied 5–12 months later. Appearance, behavior, neurologic function and brain MRIs were studied. Additional analyses of pathogenesis included: correlation of brain weight and neurologic findings; histopathology focusing on brain regions; full genome microarrays; immunohistochemistry characterizing inflammatory cells; determination of presence of tachyzoites and bradyzoites; electron microscopy; and study of markers of inflammation in serum. Histopathology in genetically resistant mice and cytokine and NRAMP knockout mice, effects of inoculation of isolated parasites, and treatment with sulfadiazine or αPD1 ligand were studied. Results Twelve months after infection, a time equivalent to middle to early elderly ages, mice had behavioral and neurological deficits, and brain MRIs showed mild to moderate ventricular dilatation. Lower brain weight correlated with greater magnitude of neurologic abnormalities and inflammation. Full genome microarrays of brains reflected inflammation causing neuronal damage (Gfap), effects on host cell protein processing (ubiquitin ligase), synapse remodeling (Complement 1q), and also increased expression of PD-1L (a ligand that allows persistent LCMV brain infection) and CD 36 (a fatty acid translocase and oxidized LDL receptor that mediates innate immune response to beta amyloid which is associated with pro-inflammation in Alzheimer's disease). Immunostaining detected no inflammation around intra-neuronal cysts, practically no free tachyzoites, and only rare bradyzoites. Nonetheless, there were perivascular, leptomeningeal inflammatory cells, particularly contiguous to the aqueduct of Sylvius and hippocampus, CD4+ and CD8+ T cells, and activated microglia in perivascular areas and brain parenchyma. Genetically resistant, chronically infected mice had substantially less inflammation. Conclusion In outbred mice, chronic, adult acquired T. gondii infection causes neurologic and behavioral abnormalities secondary to inflammation and loss of brain parenchyma. Perivascular inflammation is prominent particularly contiguous to the aqueduct of Sylvius and hippocampus. Even resistant mice have perivascular inflammation. This mouse model of chronic T. gondii infection raises questions of whether persistence of this parasite in brain can cause inflammation or neurodegeneration in genetically susceptible hosts. Wed, 22 Oct 2008 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/237643 2008-10-22T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/237555 Title: Welcome to Silence Authors: Baulcombe, David C; Zamore, Philip D Abstract: Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are. Tue, 12 Jan 2010 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/237555 2010-01-12T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/237261 Title: Genetic modification of alternative respiration in Nicotiana benthamiana affects basal and salicylic acid-induced resistance to potato virus X Authors: Lee, Wing-Sham; Fu, Shih-Feng; Verchot-Lubicz, Jeanmarie; Carr, John P Abstract: Abstract Background Salicylic acid (SA) regulates multiple anti-viral mechanisms, including mechanism(s) that may be negatively regulated by the mitochondrial enzyme, alternative oxidase (AOX), the sole component of the alternative respiratory pathway. However, studies of this mechanism can be confounded by SA-mediated induction of RNA-dependent RNA polymerase 1, a component of the antiviral RNA silencing pathway. We made transgenic Nicotiana benthamiana plants in which alternative respiratory pathway capacity was either increased by constitutive expression of AOX, or decreased by expression of a dominant-negative mutant protein (AOX-E). N. benthamiana was used because it is a natural mutant that does not express a functional RNA-dependent RNA polymerase 1. Results Antimycin A (an alternative respiratory pathway inducer and also an inducer of resistance to viruses) and SA triggered resistance to tobacco mosaic virus (TMV). Resistance to TMV induced by antimycin A, but not by SA, was inhibited in Aox transgenic plants while SA-induced resistance to this virus appeared to be stronger in Aox-E transgenic plants. These effects, which were limited to directly inoculated leaves, were not affected by the presence or absence of a transgene constitutively expressing a functional RNA-dependent RNA polymerase (MtRDR1). Unexpectedly, Aox-transgenic plants infected with potato virus X (PVX) showed markedly increased susceptibility to systemic disease induction and virus accumulation in inoculated and systemically infected leaves. SA-induced resistance to PVX was compromised in Aox-transgenic plants but plants expressing AOX-E exhibited enhanced SA-induced resistance to this virus. Conclusions We conclude that AOX-regulated mechanisms not only play a role in SA-induced resistance but also make an important contribution to basal resistance against certain viruses such as PVX. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are. Mon, 28 Feb 2011 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/237261 2011-02-28T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/226624 Title: To concentrate or ventilate? Carbon acquisition, isotope discrimination and physiological ecology of early land plant life forms Authors: Meyer, Moritz; Seibt, Ulli; Griffiths, Howard Abstract: A comparative study has been made of the photosynthetic physiological ecology and carbon isotope discrimination characteristics for modern-day bryophytes and closely related algal groups. Firstly, the extent of bryophyte distribution and diversification as compared with more advanced land plant groups is considered. Secondly, measurements of instantaneous carbon isotope discrimination (D), photosynthetic CO2 assimilation and electron transport rates were compared during the drying cycles. The extent of surface diffusion limitation (when wetted), internal conductance and water use efficiency (WUE) at optimal tissue water content (TWC) were derived for liverworts and a hornwort from contrasting habitats and with differing degrees of thallus ventilation (as intra-thalline cavities and internal airspaces). We also explore how the operation of a biophysical carbon-concentrating mechanism (CCM) tempers isotope discrimination characteristics in two other hornworts, as well as the green algae Coleochaete orbicularis and Chlamydomonas reinhardtii. The magnitude of D was compared for each life form over a drying curve and used to derive the surface liquid-phase conductance (when wetted) and internal conductance (at optimal TWC). The magnitude of external and internal conductances, and WUE, was higher for ventilated, compared with non-ventilated, liverworts and hornworts, but the values were similar within each group, suggesting that both factors have been optimized for each life form. For the hornworts, leakiness of the CCM was highest for Megaceros vincentianus and C. orbicular is (approx. 30%) and, at 5%, lowest in C. reinhardtii grown under ambient CO2 concentrations. Finally, evidence for the operation of a CCM in algae and hornworts is considered in terms of the probable role of the chloroplast pyrenoid, as the origins, structure and function of this enigmatic organelle are explored during the evolution of land plants. Thu, 15 May 2008 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/226624 2008-05-15T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/226623 Title: Functional hybrid Rubisco enzymes with plant small subunits and algal large subunits: engineered RbcS cDNA for expression in Chlamydomonas Authors: Genkov, Todor; Meyer, Moritz; Griffiths, Howard; Spreitzer, Robert J Abstract: There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO2 fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of RbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO2/O2 specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the RbcS gene family. Foreign RbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas RbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO2/O2 specificity but a lower carboxylation Vmax than Chlamydomonas Rubisco, the hybrid enzymes have 3–11% increases in CO2/O2 specificity and retain near normal Vmax values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco.Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO2 is concentrated for optimal photosynthesis. Mon, 26 Apr 2010 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/226623 2010-04-26T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/226324 Title: Role of KNOX genes in the evolution and development of floral nectar spurs Authors: Box, Mathew S Abstract: A key question in biology is how changes in gene function or regulation produce new morphologies during evolution. The nectar spur is an evolutionarily labile structure known to influence speciation in a broad range of angiosperm taxa. Here, the genetic basis of nectar spur development, and the evolution of differences in nectar spur morphology, is investigated in Linaria vulgaris and two closely related species of orchid, the primitively longer-spurred Dactylorhiza fuchsii, and more derived short-spurred D. viridis (Orchidinae, Orchidaceae). Despite considerable morphological and phylogenetic differences, nectar spur ontogeny is fundamentally similar in each of the study species, proceeding from an abaxial bulge formed on the ventral petal relatively late in petal morphogenesis. However, spur development is progenetically curtailed in the short-spurred orchid D. viridis. In each case spur development involves class 1 KNOTTED1-like homeobox (KNOX) proteins. KNOX gene expression is not restricted to the spur-bearing petal, indicating that additional components are required to define nectar spur position, e.g. canonical ABC genes, determinants of floral zygomorphy, and additional (currently unknown) factors. However, constitutive expression of class 1 KNOX proteins in transgenic tobacco produces flowers with ectopic outgrowths on the petals, indicating that KNOX proteins alone are, to some degree, capable of inducing structures similar to nectar spurs in a heterologous host. Interestingly, KNOX gene expression is high in the ovary of all study taxa, suggesting that KNOX proteins may also have been involved in the evolution of this key angiosperm feature. Although principally involved in maintaining indeterminacy in the shoot apical meristem (SAM), members of the KNOX gene family have been co-opted in the evolution and development of compound leaves where they suppress differentiation and extend the morphogenetic potential of the leaf. A similar model is presented here to explain the role of KNOX proteins in nectar spur development. Co-option of KNOX gene expression to the maturing perianth delays cellular differentiation, facilitating the development of the nectar spur but requiring additional, unknown factors, to determine nectar spur fate. As facilitators of nectar spur development, changes in the spatio-temporal patterns of KNOX gene expression may alter the potential for nectar spur development and explain the critical length differences observed between the orchids D. fuchsii and D. viridis (and among other angiosperm taxa). Taken together, the available data indicate that KNOX genes confer a meristematic state upon plant tissues in a variety of morphogenetic contexts, making the gene family a potentially versatile tool to mediate a wide variety of evolutionary transformations. Mon, 07 Jun 2010 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/226324 2010-06-07T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/226160 Title: Physiological and molecular determinants of the Chlamydomonas reinhardtii pyrenoid Authors: Meyer, Moritz Abstract: Aquatic photosynthesis accounts for 50% of the global annual net primary production (NPP), despite frequent low availability and limited diffusion of CO2 in the aquatic milieu, and low affinity for CO2 by the primary carboxylating enzyme, Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Many eukaryotic algae, and a single group of land plants, the hornworts, have an inducible carbon concentrating mechanism (CCM), to overcome these limitations. The efficiency of the CCM is improved when RuBisCO is localised to a subcellular compartment, the pyrenoid, which is hypothesised to act as a diffusion barrier for CO2 . Although the pyrenoid is a major player in global carbon balance (we estimate 10-15% of NPP), it is one of the few remaining prominent cellular features without a precise molecular or physiological definition. Under ambient CO2 , at least 90% of the cellular RuBisCO is packed into a dense matrix, together with the chaperone RuBisCO activase. Thylakoid membranes usually traverse the pyrenoid matrix, and the carboxylating substrate is thought to be delivered to the active sites of the enzyme via a carbonic anhydrase located in the lumen of these thylakoids. The mechanism of aggregation of constituents within the pyrenoid, however, still remains largely unknown. Comprehensive mutant screens have yet to reveal mutants incapable of forming pyrenoids other than those mutants with a defective RuBisCO holoenzyme, whereas DNA microarray studies uncovered little with reference to pyrenoid ultrastructure or aggregation. Taken together, this evidence raises the possibility that the basis of pyrenoid ultrastructure and aggregation lies entirely in sequence variations of RuBisCO itself. This work explored, firstly, the advantages conferred by an active CCM in hornworts and in unicellular algae, compared with the passive CO2 acquisition in most terrestrial plants. A physiological framework to CCM and pyrenoid-based photosynthesis, and isotopic discrimination, was provided by comparing the photosynthetic characteristics of selected bryophytes and algae, differing in chloroplast morphology and degrees of internalisation of gas exchanges. The results showed that on-line, carbon isotope discrimination values were a good indicator of CCM occurrence, as well as liquid-phase diffusion limitation, and biochemical limitations resulting from declining RuBisCO activity and electron transport. The methodology was used to diagnose the presence of an active CCM, and the extent of CO2 leakage. Secondly, the effect of RuBisCO sequence variations on the pyrenoid, and associated CCM, was studied using the model alga Chlamydomonas reinhardtii. The starting premise was the report by Nozaki et al. (2002) that, in some species of the family Chlamydomonaceae, a few amino acid residues within the RuBisCO large subunit (LSU) correlated strongly with pyrenoid formation. The specific roles of seven LSU residues were studied by site-directed mutagenesis. Whilst the mutations reduced the affinity of RuBisCO for CO2 and increased CO2 leakage, compared to wild-type Chlamydomonas, there was no effect on the pyrenoid phenotype. Informed by observations that Chlamydomonas mutants with a hybrid RuBisCO, composed of a native LSU, and higher plant small subunit (SSU), lacked a pyrenoid (Genkov et al., 2010), and that defined SSU alterations were neutral with respect to the pyrenoid (Genkov and Spreitzer, 2006), hitherto unexplored SSU domains were modified. A pyrenoid was successfully restored by replacing jointly the two solvent-exposed α-helices, whereas single α-helix replacements had no effect. However, leakage values indicated that the associated CCM was not fully operative, suggesting important correlates between the RuBisCO SSU and the CCM, besides the conditioning of pyrenoid formation. If the pyrenoid is partly defined by simple sequence variations in the RuBisCO SSU, as suggested by the evidence outlined in this thesis, there is the tantalising possibility that transformation of a biophysical CCM into crop plants could be a tractable approach for the future. Description: Thesis is under embargo due to publisher requirements. Mon, 07 Jun 2010 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/226160 2010-06-07T23:00:00Z