Maize defective kernel (dek) mutants identify genes necessary for the successful passage of embryos through the embryogenic and maturation phases of embryo development. The goal of this thesis was to characterize the developmental potential of three dek mutants that appeared to be morphologically blocked prior to the maturation phase of embryogenesis. Descriptive and experimental studies of the mutants and their wild-type counterparts were used to compare their morphological and physiological progression through seed development. Parameters of growth, morphogenesis, maturation and germination were measured throughout their ontogeny. Two mutants, cp*-1311C and cp*-1399A, slowly progress to morphological stages 2 and 3, respectively. Growth and maturation processes remain in synchrony with morphology, as indicated by their size, germination behavior and level of storage reserve accumulation. Not every facet of development is retarded. Both dehydration of the seed and the accumulation of desiccation proteins, maize Lea group 3 (MLG3) and Lea group 2 dehydrin (DHN), are more globally regulated, since their accumulation is precocious with respect to embryo morphology. This suggests that some aspects of the embryonic program are mediated by maternal factors. Genetic and developmental characterization of a third mutant, dks8, indicates that it defines a pattern gene that functions to specify the initiation or maintenance of the embryonic shoot. The dks8 mutant is variable in phenotype; mutants with partial and abnormal shoot development are sometimes found on ears segregating for shootless dks8 embryos. dks8 is not allelic to other shootless dek mutants. The dks8 mutation was isolated from an active Mutator transposon stock. RFLP analyses for cosegregation of various Robertson's Mutator transposable elements with the dks8 allele demonstrate that a Mu8 element and dks8 are closely linked. The dks8 allele is under-represented on segregating ears, which may reflect either epigenetic suppression of the mutant phenotype or a bias against the mutant allele in the formation of the female gametophyte. / Graduation date: 1995

Experiments were performed to assess regulatory factors governing maize embryo maturation and vivipary. Both visual and molecular markers of embryo development were used to examine the roles of the hormones abscisic acid (ABA) and gibberellins (GAs), as well as water stress in governing transit from early embryogeny to maturation-phase development. A differential screen identified cDNAs whose expression is impaired in maize viviparous mutants which fail to undergo maturation and instead precociously germinate. The cDNAs isolated in this screen absolutely required both ABA and the Viviparousl (Vpl) gene product for expression both in vivo and in vitro. Two novel clones were isolated: a maize homologue of the wheat metallothionein gene E[subscript]c and a second clone which may encode a novel seed storage protein of maize. In a separate screen, a maize cDNA encoding a Lea group 3 protein was isolated. Like many maturation-associated genes, maize Lea 3 was shown to ABA-inducible but is also expressed in response to water stress in the absence of ABA or the Vp 1 gene. We examined whether gibberellins might also be a factor modulating precocious germination. Gibberellin inhibitors applied to cultured wildtype embryos suppressed precocious germination and enhanced anthocyanin accumulation in a developmentally specific manner. These behaviors mimicked the effect of ABA and they were reversed by the addition of exogenous GA���. Vivipary in vivo resulting from diminished ABA levels could be suppressed by either chemical or genetic reduction of GA levels in immature kernels and resulted in desiccation-tolerant seed. In contrast, reduction of endogenous gibberellins did not suppress vivipary of the ABA-insensitive mutant vp1. Temporal analysis of gibberellin accumulation in developing kernels revealed the accumulation of two bioactive species (GA��� and GA���) during a developmental window just prior to peak ABA levels. It is suggested that these species stimulate a developmental program leading to vivipary in the absence of sufficient levels of ABA and that reduction of GA levels reestablishes a hormone balance appropriate for suppression of germination and induction of maturation in ABA-deficient kernels. The failure to suppress vivipary via reduction of GA levels in the ABA-insensitive mutant vp1 suggests that the wildtype gene product functions downstream of the sites of GA and ABA action in regulation of maturation versus germination. / Graduation date: 1995

No description available.

Embryogenesis in plants, as in animals, requires the regulated expression of sets of genes involved in developmental processes. To gain insight into the processes regulating gene expression during embryogenesis differential screening was used to identify embryo-specific sequences in a cDNA library constructed from Zea mays embryo RNA. Four embryo-specific sequences and one constitutive sequence were characterized further by RNA blot hybridization and DNA sequence determination. The constitutive sequence and two of the embryo-specific sequences were found to encode parts of the previously-reported chloroplast 23S rRNA, Oleosin KD-18, and RAB-17 genes. Two sequences, named Emb5 and Emb564, were found to encode novel maize homologs of a gene expressed during late embryogenesis in a wide range of seed plants. These 5 genes exhibited differential temporal and spatial accumulation during development. Moreover, analysis of RNA from cultured embryos suggested that 4 of these genes were regulated by abscisic acid. The ABA-responsive genes could be divided into 3 classes, based on their developmental expression, tissue-specificity, and sensitivity to ABA. Antibodies raised against a $ beta$-galactosidase:EMB564 fusion protein were used to analyze the accumulation of the EMB564 and/or EMB5 proteins. These polyclonal antibodies detected one or several polypeptides with a molecular weight less than 14 kD which exhibited patterns of developmental accumulation and regulation similar to Emb5 and Emb564 transcripts.