RESUMO
Maize mutants of the centromeric histone H3 (CENP-A/CENH3) gene can form haploids that inherit only chromosomes of the pollinating parent but the cytoplasm from the female parent. We developed CENH3 haploid inducers carrying a dominant anthocyanin colour marker for efficient haploid identification and harbouring cytoplasmic male sterile cytoplasm, a type of cytoplasm that results in male sterility useful for efficient hybrid seed production. The resulting cytoplasmic male sterility cyto-swapping method provides a faster and cheaper way to convert commercial lines to cytoplasmic male sterile compared to conventional trait introgression.
Assuntos
Haploidia , Zea mays , Zea mays/genética , Zea mays/fisiologia , Infertilidade das Plantas/genética , Mutação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Centrômero/genética , Histonas/metabolismo , Histonas/genética , Melhoramento Vegetal/métodosRESUMO
Maize, Zea mays, the second-most-widely-grown crop, yields 20 % of all consumed calories worldwide.1 Despite its agronomic importance, research progress is limited by costly transformation. We recently described the Trojan horse method as a useful tool to study maize proteins in situ that circumvents time- and space-consuming whole plant transformation. The Trojan horse approach uses the protein-folding and secretory properties of the corn smut fungus Ustilago maydis to secrete maize proteins from fungal cells into the maize apoplast. Here, we discuss the timing and location of U. maydis during infection and the protein secretion site in relation to anther anatomy. This spatiotemporal analysis enables the study of apoplastic anther proteins in various premeiotic anther developmental stages, and could be adapted for larger screens.
RESUMO
Small proteins are crucial signals during development, host defense, and physiology. The highly spatiotemporal restricted functions of signaling proteins remain challenging to study in planta. The several month span required to assess transgene expression, particularly in flowers, combined with the uncertainties from transgene position effects and ubiquitous or overexpression, makes monitoring of spatiotemporally restricted signaling proteins lengthy and difficult. This situation could be rectified with a transient assay in which protein deployment is tightly controlled spatially and temporally in planta to assess protein functions, timing, and cellular targets as well as to facilitate rapid mutagenesis to define functional protein domains. In maize (Zea mays), secreted ZmMAC1 (MULTIPLE ARCHESPORIAL CELLS1) was proposed to trigger somatic niche formation during anther development by participating in a ligand-receptor module. Inspired by Homer's Trojan horse myth, we engineered a protein delivery system that exploits the secretory capabilities of the maize smut fungus Ustilago maydis, to allow protein delivery to individual cells in certain cell layers at precise time points. Pathogen-supplied ZmMAC1 cell-autonomously corrected both somatic cell division and differentiation defects in mutant Zmmac1-1 anthers. These results suggest that exploiting host-pathogen interactions may become a generally useful method for targeting host proteins to cell and tissue types to clarify cellular autonomy and to analyze steps in cell responses.
Assuntos
Zea mays/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Interações Hospedeiro-Patógeno , Zea mays/genética , Zea mays/microbiologiaRESUMO
In seed plants, anthers are critical for sexual reproduction, because they foster both meiosis and subsequent pollen development of male germinal cells. Male-sterile mutants are analyzed to define steps in anther development. Historically the major topics in these studies are meiotic arrest and post-meiotic gametophyte failure, while relatively few studies focus on pre-meiotic defects of anther somatic cells. Utilizing morphometric analysis we demonstrate that pre-meiotic mutants can be impaired in anticlinal or periclinal cell division patterns and that final cell number in the pre-meiotic anther lobe is independent of cell number changes of individual differentiated somatic cell types. Data derived from microarrays and from cell wall NMR analyses allow us to further refine our understanding of the onset of phenotypes. Collectively the data highlight that even minor deviations from the correct spatiotemporal pattern of somatic cell proliferation can result in male sterility in Zea mays.
Assuntos
Flores/citologia , Meiose , Infertilidade das Plantas/genética , Zea mays/genética , Contagem de Células , Divisão Celular , Linhagem da Célula , Genes de Plantas , Microscopia Confocal , Ressonância Magnética Nuclear Biomolecular , Fenótipo , Pólen , RNA de Plantas/análise , RNA de Plantas/genética , Análise Serial de Tecidos , Zea mays/citologiaRESUMO
Research into anther ontogeny has been an active and developing field, transitioning from a strictly lineage-based view of cellular differentiation events to a more complex understanding of cell fate specification. Here we describe the modern interpretation of pre-meiotic anther development, from the earliest cell specifications within the anther lobes through SPL/NZZ-, MSP1-, and MEL1-dependent pathways as well as the initial setup of the abaxial and adaxial axes and outgrowth of the anther lobes. We then continue with a look at the known information regarding further differentiation of the somatic layers of the anther (the epidermis, endothecium, middle layer, and tapetum), with an emphasis on male-sterile mutants identified as defective in somatic cell specification. We also describe the differences in developmental stages among species and use this information to discuss molecular studies that have analyzed transcriptome, proteome, and small-RNA information in the anther.
Assuntos
Diferenciação Celular , Flores/fisiologia , Organogênese Vegetal , Células Vegetais/fisiologia , Plantas , Perfilação da Expressão Gênica , Meiose , Plantas/genética , Proteômica , RNA Interferente PequenoRESUMO
PREMISE OF THE STUDY: Although anthers of Zea mays, Oryza sativa, and Arabidopsis thaliana have been studied intensively using genetic and biochemical analyses in the past 20 years, few updates to anther anatomical and ultrastructural descriptions have been reported. For example, no transmission electron microscopy (TEM) images of the premeiotic maize anther have been published. Here we report the presence of chloroplasts in maize anthers. METHODS: TEM imaging, electron acceptor photosynthesis assay, in planta photon detection, microarray analysis, and light and fluorescence microscopy were used to investigate the presence of chloroplasts in the maize anther. KEY RESULTS: Most cells of the maize subepidermal endothecium have starch-containing chloroplasts that do not conduct measurable photosynthesis in vitro. CONCLUSIONS: The maize anther contains chloroplasts in most subepidermal, endothecial cells. Although maize anthers receive sufficient light to photosynthesize in vivo and the maize anther transcribes >96% of photosynthesis-associated genes found in the maize leaf, no photosynthetic light reaction activity was detected in vitro. The endothecial cell layer should no longer be defined as a complete circle viewed transversely in anther lobes, because chloroplasts are observed only in cells directly beneath the epidermis and not those adjacent to the connective tissue. We propose that chloroplasts be a defining characteristic of differentiated endothecial cells and that nonsubepidermal endothecial cells that lack chloroplasts be defined as a separate cell type, the interendothecium.
Assuntos
Cloroplastos/ultraestrutura , Flores/ultraestrutura , Zea mays/ultraestrutura , Cloroplastos/genética , Cloroplastos/fisiologia , Flores/genética , Flores/fisiologia , Perfilação da Expressão Gênica , Microscopia Eletrônica de Transmissão , Análise de Sequência com Séries de Oligonucleotídeos , Fotossíntese , Amido/metabolismo , Zea mays/genética , Zea mays/fisiologiaRESUMO
Compared to the diversity of other floral organs, the steps in anther ontogeny, final cell types, and overall organ shape are remarkably conserved among Angiosperms. Defects in pre-meiotic anthers that alter cellular composition or function typically result in male-sterility. Given the ease of identifying male-sterile mutants, dozens of genes with key roles in early anther development have been identified and cloned in model species, ordered by time of action and spatiotemporal expression, and used to propose explanatory models for critical steps in cell fate specification. Despite rapid progress, fundamental issues in anther development remain unresolved, and it is unclear if insights from one species can be applied to others. Here we construct a comparison of Arabidopsis, rice, and maize immature anthers to pinpoint distinctions in developmental pace. We analyze the mechanisms by which archesporial (pre-meiotic) cells are specified distinct from the soma, discuss what constitutes meiotic preparation, and review what is known about the secondary parietal layer and its terminal periclinal division that generates the tapetal and middle layers. Finally, roles for small RNAs are examined, focusing on the grass-specific phasiRNAs.
RESUMO
Plants lack a germ line; consequently, during reproduction adult somatic cells within flowers must switch from mitotic proliferation to meiosis. In maize (Zea mays L.) anthers, hypoxic conditions in the developing tassel trigger pre-meiotic competence in the column of pluripotent progenitor cells in the center of anther lobes, and within 24 hr these newly specified germinal cells have patterned their surrounding neighbors to differentiate as the first somatic niche cells. Transcriptomes were analyzed by microarray hybridization in carefully staged whole anthers during initial specification events, after the separation of germinal and somatic lineages, during the subsequent rapid mitotic proliferation phase, and during final pre-meiotic germinal and somatic cell differentiation. Maize anthers exhibit a highly complex transcriptome constituting nearly three-quarters of annotated maize genes, and expression patterns are dynamic. Laser microdissection was applied to begin assigning transcripts to tissue and cell types and for comparison to transcriptomes of mutants defective in cell fate specification. Whole anther proteomes were analyzed at three developmental stages by mass spectrometric peptide sequencing using size-fractionated proteins to evaluate the timing of protein accumulation relative to transcript abundance. New insights include early and sustained expression of meiosis-associated genes (77.5% of well-annotated meiosis genes are constitutively active in 0.15 mm anthers), an extremely large change in transcript abundances and types a few days before meiosis (including a class of 1340 transcripts absent specifically at 0.4 mm), and the relative disparity between transcript abundance and protein abundance at any one developmental stage (based on 1303 protein-to-transcript comparisons).
Assuntos
Regulação da Expressão Gênica de Plantas , Meiose/genética , Proteoma , Transcriptoma , Zea mays/genética , Zea mays/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes e Vias Metabólicas , Família Multigênica , Especificidade de Órgãos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Zea mays/crescimento & desenvolvimentoRESUMO
During maize anther development, somatic locular cells differentiate to support meiosis in the pollen mother cells. Meiosis is an important event during anther growth and is essential for plant fertility as pollen contains the haploid sperm. A subset of maize male sterile mutants exhibit meiotic failure, including ms8 (male sterile 8) in which meiocytes arrest as dyads and the locular somatic cells exhibit multiple defects. Systematic proteomic profiles were analysed in biological triplicates plus technical triplicates comparing ms8 anthers with fertile sibling samples at both the premeiotic and meiotic stages; proteins from 3.5 to 20 kDa were fractionated by 1-D PAGE, cleaved with Lys-C and then sequenced using a LTQ Orbitrap Velos MS paradigm. Three hundred and 59 proteins were identified with two or more assigned peptides in which each of those peptides were counted at least two or more times (0.4% peptide false discovery rate (FDR) and 0.2% protein FDR); 2761 proteins were identified with one or more assigned peptides (0.4% peptide FDR and 7.6% protein FDR). Stage-specific protein expression provides candidate stage markers for early anther development, and proteins specifically expressed in fertile compared to sterile anthers provide important clues about the regulation of meiosis. 49% of the proteins detected by this study are new to an independent whole anther proteome, and many small proteins missed by automated maize genome annotation were validated; these outcomes indicate the value of focusing on low molecular weight proteins. The roles of distinctive expressed proteins and methods for mass spectrometry of low molecular weight proteins are discussed.
Assuntos
Flores/química , Flores/crescimento & desenvolvimento , Infertilidade das Plantas/fisiologia , Proteínas de Plantas/análise , Pólen/fisiologia , Proteoma/química , Zea mays/genética , Produtos Agrícolas/fisiologia , Perfilação da Expressão Gênica , Variação Genética , Genótipo , Peso Molecular , Infertilidade das Plantas/genética , Proteínas de Plantas/química , Pólen/genética , Reprodutibilidade dos Testes , Zea mays/fisiologiaRESUMO
To ensure fertility, complex somatic and germinal cell proliferation and differentiation programs must be executed in flowers. Loss-of-function of the maize multiple archesporial cells 1 (mac1) gene increases the meiotically competent population and ablates specification of somatic wall layers in anthers. We report the cloning of mac1, which is the ortholog of rice TDL1A. Contrary to prior studies in rice and Arabidopsis in which mac1-like genes were inferred to act late to suppress trans-differentiation of somatic tapetal cells into meiocytes, we find that mac1 anthers contain excess archesporial (AR) cells that proliferate at least twofold more rapidly than normal prior to tapetal specification, suggesting that MAC1 regulates cell proliferation. mac1 transcript is abundant in immature anthers and roots. By immunolocalization, MAC1 protein accumulates preferentially in AR cells with a declining radial gradient that could result from diffusion. By transient expression in onion epidermis, we demonstrate experimentally that MAC1 is secreted, confirming that the predicted signal peptide domain in MAC1 leads to secretion. Insights from cytology and double-mutant studies with ameiotic1 and absence of first division1 mutants confirm that MAC1 does not affect meiotic cell fate; it also operates independently of an epidermal, Ocl4-dependent pathway that regulates proliferation of subepidermal cells. MAC1 both suppresses excess AR proliferation and is responsible for triggering periclinal division of subepidermal cells. We discuss how MAC1 can coordinate the temporal and spatial pattern of cell proliferation in maize anthers.