William F. Sheridan

Isolation and Characterization of 51 embryo-specific Mutations of Maize.

A plant embryo consists of an embryonic axis, which eventually grows into the adult body, and one or two nutritive structures, the cotyledons. In the grasses embryo morphogenesis can be divided into three periods: during the first the embryo is regionalized into an embryo proper and suspensor, during the second the embryonic axis is established, and during the third vegetative structures are elaborated. Maize, with its well-characterized embryo-genesis, powerful genetics, and transposon tagging stocks, offers an attractive system for mutational analysis of these events. We have isolated 51 embryo-specific (emb) mutations from active Robertsons Mutator maize stocks. These are single-gene recessive lethals that represent at least 45 independent mutation events. Each of the 25 mutations was located to a chromosome arm using a B-A translocation set that uncovers approximately 40% of the genome; the same test failed to locate 20 others. The embryo phenotype of 27 mutations was characterized by examining mature mutant embryos in fresh dissection: the various emb mutations differ in phenotype and each is consistent in its expression. All 27 mutations result in retarded embryos that are morphologically abnormal. Nine mutants are blocked during the first period; 10 mutants are blocked during the second period; and eight mutants are blocked during the third period. Based on both the genetic and developmental data, it is likely that there are many loci that can mutate to give the emb phenotype and that these genes are crucial to the morphogenesis of the embryo.

Female Gametophyte Development in Maize: Microtubular Organization and Embryo Sac Polarity.

The developmental stages of the maize embryo sac were correlated with the corresponding silk lengths of ear florets in the female inflorescence. The development of embryo sacs in the ovules of spikes occurs in a gradient pattern with the initiation of the embryo sac beginning at the base of the ear and progressing to the top. At the beginning of meiosis, the presence of conspicuous cortical microtubules coincides with the extensive elongation of the megasporocyte. The spindles at metaphase I and II align along the long axis of the megasporocyte leading to the linear alignment of the dyad and tetrad of megaspores. During megagametogenesis, micropylar and chalazal nuclei of the embryo sac undergo synchronized divisions and migration at the second and third mitosis. Radiate perinuclear microtubules are present during the interphase of the second and third mitosis, and inter-sister nuclear microtubules occur at the late four-nucleate embryo sac. The configuration and orientation of the spindles, phragmoplasts, and pairs of nuclei result in precise positioning of the nuclei. The fusion of the polar nuclei and the formation of a microtubule organizing center-like structure in the filiform apparatus occur right after the first division of the antipodal cells. The different patterns of organization of microtubules in the cells of the mature embryo sac reflect their structural adaptations for their future function.

Embryo Sac Development in the Maize indeterminate gametophyte1 Mutant: Abnormal Nuclear Behavior and Defective Microtubule Organization.

The indeterminate gametophyte1 mutation in maize has been known to disrupt development of the female gametophyte. Mutant embryo sacs have abnormal numbers and behavior of micropylar and central cell nuclei, which result in polyembryony and elevated ploidy levels in the endosperm of developing kernels. In this study, we confirm abnormal nuclear behavior and present novel findings. In contrast to the normal form, there is no obvious polarity in two-nucleate embryo sacs or in the micropylar cells of eight-nucleate embryo sacs. We show that the second and third mitoses are not fully synchronized and that additional mitoses can occur in all of the nuclei of the mutant embryo sac or in just the micropylar or central regions. After cellularization, individual micropylar cells can undergo mitosis. Abnormal microtubular behavior results in irregular positioning of the nuclei, asynchronous microtubular patterns in different pairs of nuclei, and abnormal phragmoplasts after the third mitotic division. These results indicate that in addition to acting primarily in controlling nuclear divisions, the indeterminate gametophyte1 gene acts secondarily in regulating microtubule behavior. This cytoskeletal activity most likely controls the polarization and nuclear migration underlying the formation and fate of the cells of the normal embryo sac.

Maize AMEIOTIC1 is essential for multiple early meiotic processes and likely required for the initiation of meiosis.

Molecular mechanisms that initiate meiosis have been studied in fungi and mammals, but little is known about the mechanisms directing the meiosis transition in other organisms. To elucidate meiosis initiation in plants, we characterized and cloned the ameiotic1 (am1) gene, which affects the transition to meiosis and progression through the early stages of meiotic prophase in maize. We demonstrate that all meiotic processes require am1, including expression of meiosis-specific genes, establishment of the meiotic chromosome structure, meiosis-specific telomere behavior, meiotic recombination, pairing, synapsis, and installation of the meiosis-specific cytoskeleton. As a result, in most am1 mutants premeiotic cells enter mitosis instead of meiosis. Unlike the genes involved in initiating meiosis in yeast and mouse, am1 also has a second downstream function, whereby it regulates the transition through a novel leptotene–zygotene checkpoint, a key step in early meiotic prophase. The am1 gene encodes a plant-specific protein with an unknown biochemical function. The AM1 protein is diffuse in the nucleus during the initiation of meiosis and then binds to chromatin in early meiotic prophase I when it regulates the leptotene–zygotene progression.

Maize embryogeny: a promising experimental system

Abstract Maize embryos are an excellent experimental system for examining the role of genes in embryogeny. The study of mutants that are defective in embryo and endosperm development has revealed several loci that control different developmental processes essential for normal morphogenesis of the embryo and several other loci essential for germination. Techniques are now available for the detailed genetic and molecular analysis of these developmentally significant mutants.

Novel phenotypes and developmental arrest in early embryo specific mutants of maize

Abstract.Embryo specific (emb) mutants exhibit aberrant embryo development without deleterious effects on endosperm development. We have analyzed five emb mutants of maize, which, based on their developmental profiles can be divided into two groups: mutants arrested at early stages and mutants with novel phenotypes. The members of the first group resemble wild-type proembryos and never reach other developmental stages. In the second group the tube-shaped mutants emb*-8522 and emb*-8535 completely lack apical-basal differentiation, while in mutant emb*-8516 a second embryo-like structure arises from the suspensor. The five emb mutations analyzed are non-allelic and two of the mutations are very likely caused by insertion of the transposon mutator, opening the door for their molecular analysis.

Analysis of four embryo-specific mutants in Zea mays reveals that incomplete radial organization of the proembryo interferes with subsequent development.

Abstract Using confocal laser scanning microscopy we have characterized early and intermediate stages of maize wild-type embryogenesis and compared to mutant development of four different embryo-specific mutations, emb*-8518, emb*-8521, emb*-8537, and emb*-8542. Confocal laser scanning microscopy is well suited to study embryo development in maize in a nondisruptive manner from shortly after fertilization to late stages in embryogenesis. The analysis of the mutant morphology indicated that two of the recessive mutations, emb*-8518 and emb*-8521,cause an early developmental arrest in the proembryo/early transition stage: mutant embryos are unable to enter the morphogenetic phase of embryogenesis. In contrast, homozygous emb*-8537, and emb*-8542 embryos progress at least to the coleoptilar stage and sometimes establish a functional shoot meristem that can determine leaf primordia. The morphological characterization of mutants was confirmed by analysis of the expression pattern of three different marker genes: Lipid transfer protein 2, Zea mays Outer Cell Layer 1, and Knotted 1. Our data indicate that both emb*-8518 and emb*-8521 mutant embryos are impaired in restriction of ZmOCL1 transcripts to the embryonic protoderm and therefore fail to establish a normal radial organization. In contrast, emb*-8537 and emb*-8542 embryos exhibit the wild-type pattern and proceed in development to the formation of a shoot apical meristem and the establishment of bilateral symmetry.

The maize desynaptic1 mutation disrupts meiotic chromosome synapsis

In most eukaryotes, homologous chromosomes undergo synapsis during the first meiotic prophase. A consequence of mutations that interfere with the fidelity or completeness of synapsis can be failure in the formation or maintenance of bivalents, resulting in univalent formation at diakinesis and production of unbalanced spores or gametes. Such mutations, termed desynaptic mutations, can result in complete or partial sterility. We have examined the effect of the maize desynaptic1-9101 mutation on synapsis, using the nuclear spread technique and electron microscopy to examine microsporocytes ranging from early pachytene until the diplotene stage of prophase I. Throughout the pachytene stage, there was an average of about 10 sites of lateral element divergence (indicating nonhomologous synapsis), and during middle and late pachytene, an average of two and three sites of foldback (intrachromosomal) synapsis, per mutant nucleus, respectively. By the diplotene stage, the number of sites of lateral element divergence had decreased to seven, and there was an average of one foldback synapsis site per nucleus. Lateral element divergence and foldback synapsis were not found in spread pachytene nuclei from normal plants. These results imply that the normal expression of the dsy1 gene is essential for the restriction of chromosome synapsis to homologues. The abundance of nonhomologous synapsis and the persistence of extended stretches of unsynapsed axial elements throughout the pachytene stage of dsy1–9101 meiocytes suggests that this mutation disrupts both the fidelity of homology search and the forward course of the synaptic process. This mutation may identify a maize mismatch repair gene. Dev. Genet. 21:146–159, 1997.

Fertilization and Embryogeny in Maize

The processes of fertilization and embryogeny in maize and other flowering plants have been examined extensively. For a general review, the classic book by Maheshwari (1950) is especially valuable for its scope and clarity. In addition, the works of Wardlaw (1955) and Johansen (1950) provide a broad historical perspective. More recent reviews include Johri (1984), Raghavan (1986), and Meinke (1991). For an introduction to maize and other grasses the work of Arber (1934) is recommended. The events of double fertilization in maize were first described by Guignard (1901); more accessible descriptions are those of Miller (1919) and Weatherwax (1919). For an understanding of the events of maize embryogeny the reader is referred to the studies of Randolph (1936), Kiesselbach (1949), and Abbe and Stein (1954). More recent works of interest are those of Van Lammeren (1986, 1987), Neuffer et al (1986), Sheridan and Clark (1987, 1993), and Clark and Sheridan (1991).

Plant Chromosomal Deletions, Insertions, and Rearrangements

With the exception of a small subset found within mitochondria and chloroplasts, the genes of plants are arranged along an essential set of chromosomes that are found in the nucleus. Within a species, the placement of genes along the chromosomes is expected to be the same in all individuals. This chapter is a primer on several major aberrations of gene order. These aberrations have consequences not only to the individual that harbors them but also to the population at large in terms of genome evolution. Here, we limit our discussion mainly to the effects on the individual. We are particularly interested in the use of these aberrations as experimental tools and include some discussions to that effect.