Thesis (Ph. D.)--University of Wisconsin--Madison, 1980. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 148-157).

Thesis--Ohio State University. / Includes bibliographical references. Available online via OhioLINK's ETD Center

The food supply to a growing world population is based on grain crops, which are harvested for their seeds. Therefore, understanding the process of seed development and its regulation has been important to increase production. This has become even more relevant and important as grain production reaches a plateau post green revolution and food security for an ever-increasing population becomes more pressing. This study focuses on altering seed size via manipulation of the endosperm, an important component of the seed that not only nourishes the developing embryo and provides the majority of our food. A classic study done by crossing different ploidies of Arabidopsis thaliana (A. thaliana) results in changes to endosperm-led growth as a consequence of altered parental genome ratios in the endosperm specifically. Increased paternal contribution results in an enlarged endosperm, and a heavier seed, whereas increased maternal contribution has the opposite effect. Whole genome transcript profiling using microarray analysis of siliques generated by interploidy crosses identified A. thaliana genes namely: PHE1, PHE2, AGL40, AGL62, AGL28, AGL45, CKX2, MAPK10, E2L2, GA1, CYCD4;1, CYCD4;2, GA20OX5, GA-regulated and AT5G46950, that are positively associated with endosperm overgrowth. In order to verify the role of these genes in endosperm proliferation, knock in (KI) which causes gene over-expression and knock out (KO) which results in gene inactivation, strategies were used. Constitutive over-expression of CKX2, MAPK10, and E2L2 showed abnormal vegetative and floral phenotypes, whereas targeted endosperm-specific over-expression of PHE2 and GA1 showed an increase in seed size and/or fertility. KI plants of AGL40, AGL62, AGL45, CYCD4;1, CYCD4;2, GA20OX5, GA-regulated and AT5G46950 did not result in any obvious phenotypic effects under normal growth conditions. KO mutant plants namely: phe1, phe2, agl40, agl62, agl28 and agl45, as single individual mutants were also indistinguishable to wild type plants in non-seed phenotypes. Embryo sac area, individual seed weight and total seed yield data obtained from a phe1 line showed a smaller embryo sac area, lighter seed and reduced total seed yield. Loss of function of agl62 showed precocious endosperm cellularisation and seed lethality. It is nearly impossible to generate phe1/phe2 double mutants due to a very tight linkage between these genes (on the chromosome physically the two genes lie next to each other) and hence doubles, triples and quadruple mutants where appropriate were made with either phe1 or phe2 in combination with other mutant lines of the MADS family of transcription factors. None of the double mutant combination tested had obvious developmental defects. However, the triple mutant phe1/phe1::agl40/agl40 ::agl45/agl45 and the quadruple mutant phe2/phe2::agl40/agl40::agl45/agl45 ::agl28/agl28 showed abnormal embryo and endosperm development which resulted to seed death. This indicates a functional redundancy among these MADS box genes. However, it is unclear how PHE1, PHE2, AGL40, AGL45 and AGL28 act to affect embryo and endosperm development. This result confirms that these genes are positively associated with endosperm over-proliferation as growth promoters and function in some cases as singly or as participants in a complex to control seed development. Thus manipulating expression of genes involved in endosperm development in A. thaliana and the potential use of this knowledge in crop plants provides us with a route to improvement of crop yields.

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Background:In angiosperms, the endosperm plays a crucial placenta-like role in that not only is it necessary for nurturing the embryo, but also regulating embryogenesis through complicated genetic and epigenetic interactions with other seed compartments and is the primary tissue in which genomic imprinting occurs.Results:We observed a gradual increase of paternal siRNA expression in the early stages of kernels and an expected 2:1 maternal to paternal ratio in 7-DAP endosperm via sequencing of small interfering RNA (siRNA) transcriptomes in developing kernels (0, 3 and 5 days after pollination (DAP)) and endosperms (7, 10 and 15 DAP) from the maize B73 and Mo17 reciprocal crosses. Additionally, 460 imprinted siRNA loci were identified in the endosperm, with the majority (456/460, 99.1%) being maternally expressed at 10 DAP. Moreover, 13 out of 29 imprinted genes harbored imprinted siRNA loci within their 2-kb flanking regions, a significant higher frequency than expected based on simulation analysis. Additionally, gene ontology terms of "response to auxin stimulus", "response to brassinosteroid stimulus" and "regulation of gene expression" were enriched with genes harboring 10-DAP specific siRNAs, whereas those of "nutrient reservoir activity", "protein localization to vacuole" and "secondary metabolite biosynthetic process" were enriched with genes harboring 15-DAP specific siRNAs.Conclusions:A subset of siRNAs subjected to imprinted expression pattern in maize developing endosperm, and they are likely correlated with certain imprinted gene expression. Additionally, siRNAs might influence nutrient uptake and allocation processes during maize endosperm development.

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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.

The ‘epigenome’ refers to a difference in the transcriptional behaviour of a given allele that cannot be explained by differences in the genetic code, or ‘mutation’ at that locus. The epigenome is associated with certain biochemical marks, and generally exerts a silencing effect upon the transcription of genes under its influence. In Angiosperms such as Arabidopsis thaliana, fertilisation of both the egg and central cell– referred to as ‘double fertilisation’ – giving rise to the embryo and endosperm respectively, the latter of which is one of the most important tissues in the human food chain. Upon double fertilisation the gametes of each parent are known to contribute differing epigenetic ‘imprints’, where one gamete contributes a copy of a given allele in a transcriptionally unavailable state, while the other parent’s copy is in an available state. When a gene resides in such a region, the result is that only one parent’s copy is hence transcribed; such a gene is said to be ‘imprinted.’ Imprinting is known to affect the development of the placenta as well as some of the adult tissues in mammalian models, and in plants is most extensively found in the endosperm, where without the imprint of both parents the resulting seed exhibits reduced viability and defective endosperm development. A relative dearth in the number of known imprinted loci in the model angiosperm, A. thaliana makes is difficult to make reliable assessments of its role and regulation. This thesis initially aimed to extend the count of known imprinted genes using a model that proved insufficient to identify novel imprinted genes, and presents a meta-analysis showing that the reliable attribution of imprinted status to a gene is difficult using high-throughput methods as well. In addition, the further characterisation of a novel imprinted gene identified previously by this lab, MPC, with a view to acquiring a more detailed understanding of its role using mutants carrying point mutations in the MPC protein showed only a subtle phenotype to discern them from wild-type plants. There has also been recent speculation of a role for repeat elements in imprinting. This thesis presents findings suggesting that the apparent association of repeat elements with imprinted genes is an artefact rather of an association of endosperm-expressed genes with transposable elements, rather than genes that are specifically imprinted.