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The model angiosperm Arabidopsis thaliana produces viable seed through coordinated growth of three constituents; seed coat, embryo and endosperm. During development the embryo grows to fill the space defined by the seed coat. The growing embryo is surrounded by endosperm, an ephemeral, nutritive structure. The process of programmed cell death (PCD) is essential for endosperm consumption by the embryo however very little is known about developmental PCD in the endosperm. ZOU is a transcription factor expressed specifically in endosperm adjacent to the growing embryo in the Embryo Surrounding Region (ESR) (Yang et al., 2008). zou seed likely have reduced PCD resulting in abnormally persistent endosperm and a small embryo at seed maturity which results in seed shriveling. A second zou phenotype is an impairment of cuticle development in the embryonic leaves (cotyledons), suggesting that ZOU may mediate a signal from endosperm to embryo. The ESR expressed gene ALE1 is down-regulated in zou. When ALE1 is artificially expressed in zou ESR by the construct pSUC5::ALE1 the zou epidermal phenotype is rescued but not the seed shriveling phenotype of zou (Xing et al., 2013). Fixed and resin-embedded sections of zou and pSUC5::ALE1 lines herein confirm that zou-like endosperm is exhibited in pSUC5::ALE1 lines. This confirms that the two phenotypes of zou are genetically separable. The involvement of ZOU in epidermal processes is further confirmed through genetic studies showing that ZOU acts in the same pathway to impart embryonic cuticle as the embryo-expressed Receptor Like Kinases GSO1 and GSO2. In order to quantify PCD in the endosperm of wild-type and zou seed, PCD expression marker and TUNEL analysis were conducted. One PCD marker, pCEP:H2A-YFP is shown to be expressed in wild-type ESR, it is not clear if expression is lost in zou. To identify candidate genes under the control of ZOU active in endosperm PCD the results from several transcriptional profiling experiments were analysed and validated; this detailed gene expression in wild-type, ale1 and zou siliques which allowed for the identification of targets of ZOU but not of ALE1, targets predicted to be PCD effectors. In silico expression and ontology analysis confirmed likely roles for some candidates in endosperm PCD processes (particularly cell wall modification). Selected targets were cloned under pSUC5 and expressed in the ESR of zou seed as part of a molecular screen for the rescue of the zou endosperm phenotype. The ZOU target FRINGE-Like, a Glycosyl Transferase which shows strong endosperm expression is shown to partially rescue the zou phenotype but does not rescue the epidermal phenotype, suggesting that it may mediate PCD processes under ZOU control. The initial discovery that a Glycosyl Transferase may be active in a developmental PCD process in plants is exciting and novel and benefits understanding of developmental PCD and endosperm breakdown, two poorly characterized processes in plants.

Genomic imprinting is the epigenetic modification of loci, primarily by DNA methylation, which results in parent-of-origin-specific monoallelic expression of a small subset of genes. In plants, imprinting occurs during endosperm development and a balance of maternally- and paternally-expressed imprinted genes is essential for normal seed development. Dependence on DNA methylation for imprinting highlights the potential to manipulate seed development, and consequently seed size, by altering DNA methyltransferase activity. DNA METHYLTRANSFERASE 1 (MET1) is the primary plant maintenance DNA methyltransferase and plays a significant role in imprinting. However, no evaluation of the potential role for other MET1 family members in genomic imprinting has been reported. The current model for the control of imprinting in plants suggests that maintenance DNA methyltransferases are required throughout development, yet the tissue-specific requirement of these enzymes is unconfirmed as analysis has relied solely on constitutive DNA methyltransferase mutants. To address these problems and to evaluate the potential to alter seed size, the work reported in this thesis investigated the potential involvement of putative maintenance DNA methyltransferases MET2a, MET2b and MET3 and the tissue-specific role of MET1 in imprinting. Imprinting was not significantly altered in met2a-1, met2b-1 and met3-1 mutants, indicating that MET1 is the sole DNA methyltransferase required for imprinting. Transcriptional analysis suggested MET1 is expressed throughout floral organ development and in the male and female gametophyte generation indicating that MET1 is potentially available to maintain imprinting-dependent methylation in these tissues. Tools to suppress MET1 tissuespecifically were developed to investigate the tissue-specific requirement of MET1 for imprinting. Analysis indicates that such tools could also be used to alter seed size by manipulating imprinting in commercially important species. Further work is needed to validate this approach.

Seed desiccation and germination have great potential for oxidative stress. Glutathione, one of the most abundant antioxidants in plant cells, is a crucial to the plant's defense mechanisms. To better understand glutathione's responses during these two stages, we examined its dynamics in wildtype Arabidopsis seeds and in a transgenic line containing an antisense glutathione reductase2 (anGR2) cDNA insert. Seeds from the two genotypes were compared morphologically. Glutathione levels in maturing and germinating seeds were measured by HPLC, and GR activity by native PAGE. Cytosolic glutathione was measured in situ by confocal laser scanning microscopy. Stress in the form of natural and accelerated ageing, and germination at high and low temperature and at low water potential was applied to both WT and anGR2 seeds to test vigor. Results show similar glutathione levels and GR activity (except during late imbibition) in WT and anGR2. In both genotypes, GSH/GSSG ratio increased and GR activity decreased during seed maturation. During imbibition, the glutathione pool becomes very reduced (<1% GSSG) and in WT seeds, GSH levels increase mostly by GSSG recycling. Cytosolic GSH in embryonic epidermal cells was estimated to be 1.1-1.6 mM. AnGR2 seeds aged faster, and were less tolerant of heat and drought stress than WT. Accumulation of glutathione during maturation indicated that glutathione is a major antioxidant in the seed during storage. Changes in GSH levels during imbibition coincided with ROS production during radicle protrusion. Under stress conditions, anGR2 seeds showed lower vigor, indicating perturbations in the ROS scavenging systems particularly GR2. / Master of Science

Canola (Brassica napus) contributes $19.3 billion dollars to the Canadian economy each year as a result of its oil- and protein-rich seeds. These economically important seed products are produced in highest concentration in the embryo. Embryo development is supported nutritionally and structurally by the maternal subregions, which include the inner (ISC) and outer distal seed coat (OSC), the chalazal seed coat (CZSC), and the chalazal proliferating tissue (CPT). Research on the maternal seed subregions is limited to the SC as a result of its accessibility; the embedded CZSC and CPT subregions have yet to be characterized in canola. Using light and transmission electron microscopy, I found the CZSC and CPT to be anatomically distinct and experience profound changes throughout seed development. To understand these changes at the RNA level, laser microdissection and RNA sequencing were used to profile these subregions spatially and temporally from the ovule to mature green stage of seed development. Employing vigorous bioinformatics analyses, I found that the maternal subregions are transcriptomically distinct and possess unique RNA populations. From here I began to elucidate the biological processes operating within the maternal subregions. As a whole, the maternal subregions appear to have a critical role in transporting nutrients to the filial subregions as well as in coping with oxidative stress produced during these energy-rich processes. Additionally, using CanEnrich, I was able to generate predictive transcriptional circuits regulating the biological processes occurring within the maternal seed. This research has produced the most comprehensive dataset on the canola seed to date and will provide a valuable resource for research on seed development as well as seed improvement. / October 2016

Angiosperm seed development requires co-ordinated development of the embryo and a second zygotic tissue, the endosperm. In Arabidopsis thaliana, the endosperm is ephemeral and is largely consumed by the embryo during seed development. In addition to a role in embryo nutrition, it is also likely that the endosperm may play a more direct role in signalling to the embryo to regulate development. Despite their importance for embryo development, these processes are very poorly understood. The ZHOUPI (ZOU) gene provides an important tool to address these problems. Firstly, ZOU likely regulates endosperm breakdown. Whereas wild-type seed have a single layer of endosperm at maturity, zou seed has a large persistent endosperm and a correspondingly small embryo. The small zou embryo does not fill the seed so that the seed shrivels as it desiccates during maturation. Secondly, zou embryos have defects in their cuticle, so that the endosperm adheres to the embryo throughout seed development. After seed germination, zou cotyledons develop holes in their epidermis as they expand, probably due to the defects in the cuticle. ZHOUPI (ZOU) encodes a bHLH transcription factor and is expressed in the embryo surrounding region (ESR) of endosperm but not in the embryo itself. The role of ZOU in cuticle development is partly mediated by the ABNORMAL LEAF SHAPE1 (ALE1) gene. Thus, ale1 mutants also show defects in embryonic cuticle development and ALE1 is specifically expressed in ESR in a ZOU-dependent fashion. It was unclear whether the effects of ZOU upon embryo development are an indirect consequence of the persistent endosperm mechanically impeding embryo expansion, or rather reflect a more direct role of the ESR in signalling to the embryo. The main aims of this thesis were 1) to provide evidence that ZOU regulates endosperm cell death and 2) to test whether ZOU function in controlling endosperm cell death could be separated from that in embryonic epidermal cuticle development. To achieve this goal, 1) TUNEL assays were performed in the seeds to confirm the zou endosperm cell death phenotype, 2) ALE1 expression in the ESR in zou mutants was rescued using the ZOU-independent AtSUC5 promoter to investigate whether one or both of zou phenotypes were complemented, 3) Candidate ZOU target genes were validated and characterized to determine their functions in endosperm cell death and/or embryonic epidermal cuticle development. The TUNEL assays revealed that zou mutants display less DNA fragmentation in the ESR than that of the wild-type, but that zou did not have defects in cell death outside the seeds suggesting ZOU specifically regulated endosperm cell death. The AtSUC5::ALE1 transgene partially rescued zou defects in epidermal cuticle but not in endosperm cell death. This shows that the defects in the zou cuticle are not caused by the defective endosperm, rather zou has distinct, separable functions. Lastly, I characterised several novel ZOU targets and showed that RGP3 may be a direct ZOU target as it is expressed in ESR in ZOU dependent fashion, whereas RGP4 is likely indirect as it is expressed in the testa and up-regulated in zou mutants. In conclusion, ZOU has independent roles in endosperm cell death and embryonic epidermal cuticle development. Because ALE1, which largely mediates the role in cuticle development, is less widely conserved than is ZOU, the role in promoting endosperm cell death may be the ancestral function of ZOU.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy P. Durrett / Many Euonymus species express an acetyltransferase enzyme in their seeds which catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol (DAG) producing unusual acetyl-1,2-diacyl-sn-glycerols (acetyl-TAG). The presence of the sn-3 acetate group gives acetyl-TAG with unique physical properties over regular triacylglycerol (TAG) found in vegetable oils. The useful characteristics of acetyl-TAG oil offer advantages for its use as emulsifiers, lubricants, and 'drop-in' biofuels. One enzyme, Euonymus alatus diacylglycerol acetyltransferase (EaDAcT), responsible for acetyl-TAG synthesis in nature was previously isolated from the seeds of Euonymus alatus (burning bush) and expressed in the oilseed crop Camelina sativa. Expression of EaDAcT successfully led to production of high levels of acetyl-TAG in camelina seeds. To further increase acetyl-TAG accumulation in transgenic camelina seeds, multiple strategies were examined in this study. Expression of a new acetyltransferase enzyme (EfDAcT) isolated from the seeds of Euonymus fortunei, which was previously shown to possess higher in vitro activity and in vivo acetyl-TAG levels compared to EaDAcT, increased acetyl-TAG accumulation by 20 mol%. Suppression of the endogenous competing enzyme DGAT1 further enhanced acetyl-TAG accumulation to 90 mol% in selected transgenic line. Studying the regulation of EfDAcT transcript, protein, and acetyl-TAG levels during seed development further provided new insights on the factors limiting acetyl-TAG accumulation.