Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3354. Adviser: Thomas W. Jacobs. Includes bibliographical references (leaves 143-158) Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph. D.)--Syracuse University, 2005. / "Publication number AAT 3176987."

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1987. / Vita. Abstract. Includes bibliographical references (leaves 113-122). Also available via the Internet.

Magnesium (Mg2+) is an essential mineral nutrient for plants and is the most abundant free divalent cation in plant cells. However, our knowledge of the role of this ion in the plant cell is limited, and the mechanisms of homeostasis and transport of the ion are almost completely unknown. A. Tutone (this laboratory) identified an Arabidopsis thaliana gene by the complementation of a Mg2+-uptake yeast mutant (CM66). This gene, referred to as AtMRS2-11, was expressed as cDNA from a strong yeast promoter and allowed the growth of the CM66 yeast strain on standard media. The conceptually translated AtMRS2-11 protein sequence was used in this study to identify nine additional proteins by sequence homology searches using the BLAST algorithm. The corresponding genes have been cloned from cDNA (A. thaliana ecotype Landsberg erecta) and sequenced. Protein sequence similarity suggests that the family forms a sub-section of the CorA super-family of Mg2+ transport proteins. The mutant yeast used to identify the family initially was also used to show that two family members in addition to AtMRS2-11 were able to complement the Mg2+-dependent growth phenotype. When fused to eGFP, these proteins showed a localisation consistent with some of the protein reaching the yeast cell membrane. The other members of the family were also fused to eGFP and showed a range of localisation patterns within the yeast cell. None of the three AtMRS2 proteins previously able to complement the yeast mutant phenotype did so when fused to eGFP. RNA transcripts from the AtMRS2 family were detected by RT-PCR in organ-scale preparations of total RNA from A. thaliana. Most family members were detected in all of the organs tested. Northern analysis of AtMRS2-11 RNA transcript level showed that the gene was more highly expressed in leaf tissue, but was not affected by decreased levels of Mg2+ in the growth media. The levels of steady state AtMRS2-11 mRNA transcript were elevated two-fold in the light during the diurnal cycle, but no change was detected during light-induced greening of etiolated seedlings. A stable transgenic A. thaliana line expressing the gusA gene from the promoter region of AtMRS2-11 was used to localise the promoter's activity to cells containing chloroplasts. The expression appeared highest in younger cells. VI The AtMRS2-11 protein was predicted to contain a chloroplast targeting peptide. Western analysis demonstrated that AtMRS2-11 was enriched in the total proteins of isolated chloroplasts as compared to extracts from whole plants. The AtMRS2-11:eGFP fusion protein was also detected in chloroplasts by fluorescence microscopy. Flame atomic absorption spectroscopy was used in conjunction with isolated chloroplasts to try to determine the effects of the overaccumulation of the AtMRS2-11 protein in a transgenic A. thaliana plant line (constructed by A. Tutone). A rapid uptake or binding of Mg2+ was seen in chloroplasts isolated from both wild type and transgenic lines, but no differences were observed in either the rate of Mg2+ uptake/binding or the final Mg2+ content.

The development of the pollen grains within the anther locule relies upon the nourishing and secretory properties of a tissue layer termed tapetum. The transition of the post-meiotic phase of tapetum development depends on the MALE STERILITY 1. In the ms1 mutant tapetum development is arrested post-meiosis and lacks subsequent biological processes, such as biosynthesis and secretion of pollen wall/coat components and the tissue programmed cell death process. MS1 exhibits a transient expression pattern, which is tightly regulated and critical for tapetum development and viable pollen formation. Therefore, understanding the genetic control of MS1 is key to uncover the regulation of post-meiotic tapetum development. During this project, three regulation levels of MS1 were studied: (i) transcriptional activation, (ii) auto-repression and (iii) post-translational proteolysis. Phylogenetic footprinting analysis and molecular promoter dissection was used to investigate the transcriptional control of expression and a distal upstream sequence (−2900 to −2066 bp) was found to be essential for the activation of MS1. Three evolutionarily conserved non-coding sequences (CNS), enriched with unusually long consensus motifs, and binding site combinations of MS1 upstream transcription factors (TFs) were found within the −2 kb MS1 upstream sequence. These may serve as essential cis-regulatory elements (CREs) for MS1 expression. ChIP experiments were used to investigate MS1 autorepression; the MS1 protein was shown to bind to the second exon of its genomic locus and to repress its own expression. Post-translational proteolysis was investigated using a triple mutant of the MS1 interacting gene that encodes for an E3 ubiquitin ligase LRB1 and its two paralogs LRB2 and LRB3; which exhibits a novel tapetum phenotype that may be induced by altered removal of MS1 protein in the lrb123 tapetum. The MS1 protein possesses a Plant Homeotic Domain (PHD) finger and belongs to a plant-specific C-terminal PHD contained protein (CPCP) family. Although extensive research has been carried out on the tapetum regulation role of MS1, very little is known about the underlying molecular mechanism. A series in-silico comparative analysis of the CPCP sequences in this thesis found that this family originated from green algae. Besides the PHD, two evolutionarily conserved domains, termed MS1/MMD1 Associated Domain 1 (MAD1) and MAD2, were identified in the protein. Molecular modelling of the MS1 PHD domain predicted a histone reader role with high affinity to H3K4me2/3 histone peptides. Super-resolution STED confocal observation showed that subnuclear localisation of the MS1 protein is distinctive with canonical TFs and aggregates at rounded speckles that resemble Polycomb bodies. A meta-data-analysis of MS1 microarrays found that most MS1 immediately responding genes are repressed by MS1, which is on the contrary to the previously proposed activator role of MS1. MS1 may therefore be a unique plant-specific histone reader family protein that participates in gene repression as a co-repressor. MYB99 has previously been hypothesised to be a direct target of MS1, regulating late tapetum development. Comprehensive phenotyping was carried out on two MYB99 null alleles; however, no defects were identified, probably due to high function redundancy among the MYB family TFs. In addition, no evidence of direct activation by MS1 was observed by yeast one-hybrid and ChIP analysis. Interestingly, a PCD indicator gene was down-regulated in the myb99 mutant, suggesting a tapetal PCD regulatory role for MYB99.

Green leaf material is recognized as a good source of many valuable nutrients including vitamins, fatty acids and minerals. Chloroplast-rich-fractions (CRFs) were prepared from green plant species and their nutrient composition compared with the whole leaf materials (WLMs). Digestibility of CRFs from spinach was also compared with the WLM using simulated in vitro oral, stomach and small intestinal conditions. The impact of pancreatic lipase-related protein 2 from guinea pig (gPLRP2) on the hydrolysis of galactolipids compared to neutral lipid, Tributyrin was subsequently determined in vitro. Porcine pancreatic lipase was also used against the same substrates compared to gPLRP2. Furthermore, spinach CRFs and WLMs were fed to zebrafish and the impact of CRFs and WLMs on growth performance and transition of certain compounds into zebrafish body was evaluated. Results indicated that compared with the WLM, the CRFs were enriched in more lipids and fatty acids, and more proteins and amino acids. Spinach CRFs possessed the highest α-tocopherol (62 mg 100 g-1 , dry weight (DW)), β-carotene (336 mg 100 g-1 DW) and lutein (341 mg 100 g-1 DW) contents, whilst grass CRFs had the highest alpha-linolenic acid (ALA) concentration (69.5 mg g-1). Of the minerals, iron was most notably concentrated in CRF. The digestive conditions caused changes in the structure and composition of the material providing some indication of its digestibility and bioaccessibility. Whilst PLRP2 was more active on galactolipids, with moderate reaction against the neutral lipid, pancreatin indicated higher activity on Tributyrin with almost no activity against MGDG. Diets with 10% zebrafish meal reduction improved growth rate with significant reduction in feed conversion ratio (FCR) compared to the control. CRFs diets showed greater ALA content and distinct pigmentation of zebrafish egg and flesh due to the CRF carotenoids. Overall, the results indicated that CRF is a potential digestible source of vital nutrients.

The development of new and novel polymorphic assay methods represents one of the most significant developments in plant and animal biology. The exploitation of these genetic markers is of relevance to both applied and fundamental research. Recombinant DNA technology provides the opportunity to develop phenotypically neutral genetic markers in any organism from which DNA can be extracted. The research described in this thesis has focused on the development, evaluation and exploitation of genetic markers in barley. Both protein and DNA based molecular markers were evaluated as a means of detecting polymorphism in H. vulgare and H. spontaneum. New methods for detecting polymorphisms based on the Polymerase Chain Reaction (PCR) were assessed and successfully applied to barley. The segregation of alleles at morphological, isozyme and RFLP loci were monitored in doubled haploid (DH) populations of barley. In order to detect molecular variability, both clones of known function and anonymous clones were employed. Clones homologous to the- hordein gene complex on chromosome IH were used in conjunction with DHs to intra-chromosomally map various members of this multi-gene family. Allelic variation at the genetic loci segregating in the DH population was evaluated in relation to the expression of quantitative traits. This study established that several isozymes and RFLP loci were significantly associated with quantitative trait loci (QTL) of agronomic importance. Grain isozyme and ribosomal DNA (rDNA) variability was examined in Hordeum spontaneum populations sampled from 27 geographical sites in Israel. Considerable phenotypic variability was observed and isozyme and rDNA variants were identified that were not present in the H. vulgare gene pool. Furthermore, the variability detected was quantified and correlated with a range of ecogeographical factors. The distribution of grain isozyme and rDNA phenotypes was non-random with particular phenotypes being restricted to specific geographical areas of Israel. Information on the spatial distribution of diversity in H. spontaneum is a pre-requisite for the optimization of sampling and germplasm collecting strategies. Opportunities and limitations to the exploitation of genetic markers in barley improvement are considered.

The principal aim of the research presented in this thesis was to investigate factors affecting the "male competition" component of sexual selection in the hermaphroditic species, Senecio vulgaris. As male reproductive function consists of attracting pollinators and the success of pollen in contributing genes to the next generation, sexual selection will act on both the sporophytic and gametophytic stages of the life cycle. The potential for, and consequences of male competition were analysed at both pre- and post-pollination stages. A comparison of the relative attractiveness of the radiate and non-radiate morphs of S.vulgaris to pollinators revealed that in mixed stands, pollinators discriminated in favour of the radiate morph irrespective of the frequency of the two morphs in a population. Measurement of intramorph and intermorph maternal outcrossing rates showed the radiate morph always outcrossed at higher levels than the non-radiate morph. Both morphs exhibited levels of male outcrossing, the radiate morph exhibited higher levels of intramorph paternal outcrossing while non-radiate pollen was more successful than radiate pollen in intermorph crosses. The potential levels of intermorph and intramorph pollen competition experienced by the radiate and non-radiate pollen types suggest radiate pollen was subjected to greater levels of competition for access to ovules than non-radiate pollen. Examination of post-pollination events suggested that radiate pollen germinates faster than non-radiate pollen when applied to stigmas of either morph. However, no consistent evidence of radiate pollen tubes outcompeting non-radiate pollen tubes in the style and consequently fertilising a disproportionate share of available ovules was obtained. The problems associated with measuring pollen competitive ability are discussed. In addition to the research on male competition, a study was also conducted to examine the origin of the radiate morph of S.vulgaris. Morphometric and electrophoretic analyses provided strong evidence that a radiate variant from York possessed more 'squalidus-like' characters than are generally found in radiate S.vulgaris. It is suggested that this radiate form may represent an early stage in the origin of radiate S.vulgaris via introgression of S.squalidus into S.vulgaris.

The approach in this study to discover more about the relation between growth rate and adaptation to particular sites was an examination of the quantitative differences in growth, soluble carbohydrate and amino acid content in the four experimental species: Lolium perenne (S24 strain), Dactylis glomerata (S143 strain), Festuca rubra, and Agrostis tenuis. On the basis of the growth studies, the species cloud be seen to differ and form two groups: I. perenne and D. glomerata in one group, and A. tenuis and F. rubra in the other. When complete nutrient solutions were supplied to these species in treatments of increasing concentration the two groups were seen to differ in the pattern of response of their soluble carbohydrates. When the treatments used in further experiments differed only in nitrate, ammonium, nitrate/ammonium proportions and phosphate at different nitrate concentration, definite treatment effects were found in each species, and in many cases, the response of the species could be seen to be significantly different. Further, in many cases, the differences divided the species into the same two groups observed in the growth experiments. However, when the effects of the separate ions were examined, these species differences appeared to be due mainly to quantitative differences rather than to differences in pattern of response. The examination of amino acid content of the species did not appear to clarify the differences observed between the species, though this examination did explain some of the fluctuations of soluble sugar content which could in some cases be related to the yields and rates of growth in the species.

The involvement of the sucrose-cleaving enzymes, acid and alkaline invertases and sucrose synthase in carbohydrate metabolism, was investigated in three different developing sink organs: 1) the starch-storing tubers from Solanum tuberosum L., 2) the starch- and protein-storing cotyledons from Vicia faba L., and 3) the sucrose-storing taproots from Beta vulgaris L. subsp. altissima. In potato, tuberisation is characterised by a change from an invertase- dominated sucrolytic pathway in stolons to one dominated by sucrose synthase in developing tubers. This pathway continues to be the major route for sucrose breakdown during tuber growth but only in tubers receiving a ready supply of photoassimilate. Sucrose flux to the tuber was shown to regulate sucrose synthase activity, excision of developing tubers from the mother plant resulting in a rapid decrease in sucrose synthase activity and an increase in acid invertase. Acid invertase was by far the major sucrolytic enzyme in stored tubers. In contrast, acid invertase does not play a major role in sucrose cleavage in developing bean cotyledons. Sucrose synthase is the dominant sucrolytic enzyme during the early stages of seed growth but in the later stages of development alkaline invertase predominates. During sugar beet development, high acid invertase activity in very young roots declines rapidly when taproot swelling commences, to be replaced by both sucrose synthase and alkaline invertase. Neither enzyme predominates during taproot growth. No significant increase in the activity of any of the sucrolytic enzymes occurred in taproots stored for 80 d at 8°C. Sucrose synthase was purified to homogeneity from bean cotyledons and characterised. Polydonal antibodies were raised to both native and denatured sucrose synthase protein. Similarly alkaline invertase was purified from bean cotyledons and sugar beet taproots and polyclonal antibodies raised to both denatured proteins. Isoforms of bean and sugar beet alkaline invertases were separated by anion-exchange chromatography but were not immunologically distinct. The antibodies produced were used throughout this study to confirm enzyme levels.