A low molecular weight proteome comparison of fertile and male sterile 8 anthers of Zea mays

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 59proteins 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.     

Comparative development of aseptate and septate anthers of Annonaceae

We compared anther development in 13 genera and 15 species of Annonaceae to document the nature and development of anther septa. In aseptate anthers, all sporogenous initials proceed to sporogenesis and meiosis. In septate anthers, a small number of sporogenous initials, in a discontinuous distribution pattern, differentiate into sporogenous cells; the remaining initials become sterile and form cellular septa that partition each anther lobe into multiple sporangial chambers. In species where the septum is 1-2 cell layers thick, the entire septum becomes tapetal (T-type septa) and breaks down before anther dehiscence. In species in which the septum is three or more cell layers thick, only the layer in direct contact with the sporogenous cells becomes tapetal, and the remaining cells become parenchymatous (P-type septa). These thicker P-type septa are sometimes visible in dehisced anthers. Both types are homologous in ontogeny and are highly associated with the production of compound pollen. We propose that the evolution of anther septation in Annonaceae was mainly driven by the requirement for highly efficient nutrient and physical support to the development of large, compound pollen units.     

Maize csmd1 exhibits pre-meiotic somatic and post-meiotic microspore and somatic defects but sustains anther growth

Maize male reproductive development is complex and lengthy, and anther formation and pollen maturation are precisely and spatiotemporally regulated. Here, we document that callose, somatic, and microspore defect 1 (csmd1), a new male-sterile mutant, has both pre-meiotic somatic and post-meiotic gametophyte and somatic defects. Chromosome behavior and cell developmental events were monitored by nuclear staining viewed by bright field microscopy; cell dimensions were charted by Volocity analysis of confocal microscopy images. Aniline blue staining and quantitative assays were performed to record callose deposition, and expression of three callose synthase genes was measured by qRT-PCR. Despite numerous defects and unlike other maize male-sterile mutants that show growth arrest coincident with locular defects, csmd1 anther elongation is nearly normal. Pre-meiotically and during prophase I, there is excess callose surrounding the meiocytes. Post-meiotically csmd1 epidermal cells have impaired elongation but excess longitudinal divisions, and uninucleate microspores cease growth; the microspore nucleoli degrade followed by cytoplasmic vacuolization and haploid cell collapse. The single vascular bundle within csmd1 anthers senesces precociously, coordinate with microspore death. Although csmd1 anther locules contain only epidermal and endothecial cells at maturity, locules are oval rather than collapsed, indicating that these two cell types suffice to maintain an open channel within each locule. Our data indicate that csmd1 encodes a crucial factor important for normal anther development in both somatic and haploid cells, that excess callose deposition does not cause meiotic arrest, and that developing pollen is not required for continued maize anther growth.     

Emergence and patterning of the five cell types of the Zea mays anther locule

One fundamental difference between plants and animals is the existence of a germ-line in animals and its absence in plants. In flowering plants, the sexual organs (stamens and carpels) are composed almost entirely of somatic cells, a small subset of which switch to meiosis; however, the mechanism of meiotic cell fate acquisition is a long-standing botanical mystery. In the maize (Zea mays) anther microsporangium, the somatic tissues consist of four concentric cell layers that surround and support reproductive cells as they progress through meiosis and pollen maturation. Male sterility, defined as the absence of viable pollen, is a common phenotype in flowering plants, and many male sterile mutants have defects in somatic and reproductive cell fate acquisition. However, without a robust model of anther cell fate acquisition based on careful observation of wild-type anther ontogeny, interpretation of cell fate mutants is limited. To address this, the pattern of cell proliferation, expansion, and differentiation was tracked in three dimensions over 30 days of wild-type (W23) anther development, using anthers stained with propidium iodide (PI) and/or 5-ethynyl-2′-deoxyuridine (EdU) (S-phase label) and imaged by confocal microscopy. The pervading lineage model of anther development claims that new cell layers are generated by coordinated, oriented cell divisions in transient precursor cell types. In reconstructing anther cell division patterns, however, we can only confirm this for the origin of the middle layer (ml) and tapetum, while young anther development appears more complex. We find that each anther cell type undergoes a burst of cell division after specification with a characteristic pattern of both cell expansion and division. Comparisons between two inbreds lines and between ab- and adaxial anther florets indicated near identity: anther development is highly canalized and synchronized. Three classical models of plant organ development are tested and ruled out; however, local clustering of developmental events was identified for several processes, including the first evidence for a direct relationship between the development of ml and tapetal cells. We speculate that small groups of ml and tapetum cells function as a developmental unit dedicated to the development of a single pollen grain.     

Misexpression of cyclin D1 in embryonic germ cells promotes testicular teratoma initiation

Testicular teratomas result from anomalies in embryonic germ cell development. In the 129 family of inbred mouse strains, teratomas arise during the same developmental period that male germ cells normally enter G1/G0 mitotic arrest and female germ cells initiate meiosis (the mitotic:meiotic switch). Dysregulation of this switch associates with teratoma susceptibility and involves three germ cell developmental abnormalities seemingly critical for tumor initiation: delayed G1/G0 mitotic arrest, retention of pluripotency, and misexpression of genes normally restricted to embryonic female and adult male germ cells. One misexpressed gene, cyclin D1 (Ccnd1), is a known regulator of cell cycle progression and an oncogene in many tissues. Here, we investigated whether Ccnd1 misexpression in embryonic germ cells is a determinant of teratoma susceptibility in mice. We found that CCND1 localizes to teratoma-susceptible germ cells that fail to enter G1/G0 arrest during the mitotic:meiotic switch and is the only D-type cyclin misexpressed during this critical developmental time frame. We discovered that Ccnd1 deficiency in teratoma-susceptible mice significantly reduced teratoma incidence and suppressed the germ cell proliferation and pluripotency abnormalities associated with tumor initiation. Importantly, Ccnd1 expression was dispensable for somatic cell development and male germ cell specification and maturation in tumor-susceptible mice, implying that the mechanisms by which Ccnd1 deficiency reduced teratoma incidence were germ cell autonomous and specific to tumorigenesis. We conclude that misexpression of Ccnd1 in male germ cells is a key component of a larger pro-proliferative program that disrupts the mitotic:meiotic switch and predisposes 129 inbred mice to testicular teratocarcinogenesis.     

Cyclin E and CDK-2 regulate proliferative cell fate and cell cycle progression in the C. elegans germline

The C. elegans germline provides an excellent model for analyzing the regulation of stem cell activity and the decision to differentiate and undergo meiotic development. The distal end of the adult hermaphrodite germline contains the proliferative zone, which includes a population of mitotically cycling cells and cells in meiotic S phase, followed by entry into meiotic prophase. The proliferative fate is specified by somatic distal tip cell (DTC) niche-germline GLP-1 Notch signaling through repression of the redundant GLD-1 and GLD-2 pathways that promote entry into meiosis. Here, we describe characteristics of the proliferative zone, including cell cycle kinetics and population dynamics, as well as the role of specific cell cycle factors in both cell cycle progression and the decision between the proliferative and meiotic cell fate. Mitotic cell cycle progression occurs rapidly, continuously, with little or no time spent in G1, and with cyclin E (CYE-1) levels and activity high throughout the cell cycle. In addition to driving mitotic cell cycle progression, CYE-1 and CDK-2 also play an important role in proliferative fate specification. Genetic analysis indicates that CYE-1/CDK-2 promotes the proliferative fate downstream or in parallel to the GLD-1 and GLD-2 pathways, and is important under conditions of reduced GLP-1 signaling, possibly corresponding to mitotically cycling proliferative zone cells that are displaced from the DTC niche. Furthermore, we find that GLP-1 signaling regulates a third pathway, in addition to the GLD-1 and GLD-2 pathways and also independent of CYE-1/CDK-2, to promote the proliferative fate/inhibit meiotic entry.     

Microarray Analysis of Gene Expression Involved in Anther Development in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In flowering plants, anthers bear male gametophytes whose development is regulated by the elaborate coordination of many genes. In addition, both gibberellic acid (GA₃) and jasmonic acid (JA) play important roles in anther development and pollen fertility. To facilitate the analysis of anther development genes and how GA₃ and JA regulate anther development, we performed microarray experiments using a 10-K cDNA microarray with probes derived from seedlings, meiotic anthers, mature anthers and GA₃- or JA-treated suspension cells of rice. The expression level change of 2155 genes was significantly (by 2-fold or greater) detected in anthers compared with seedlings. Forty-seven genes, representing genes with potential function in cell cycle and cell structure regulation, hormone response, photosynthesis, stress resistance and metabolism, were differentially expressed in meiotic and mature anthers. Moreover, 314 genes responded to either GA₃ or JA treatment, and 24 GA₃- and 82 JA-responsive genes showed significant changes in expression between meiosis and the mature anther stages. RT-PCR demonstrated that gene y656d05 was not only highly expressed in meiotic anthers but also induced by GA₃. Strong RNA signals of y656d05 were detected in pollen mother cells and tapetum in in situ hybridization. Further characterization of these candidate genes can contribute to the understanding of the molecular mechanism of anther development and the involvement of JA and GA₃ signals in the control of anther development in rice.     

Xenotransplantation exposes the etiology of azoospermia factor (AZF) induced male sterility

Ramathal et al. have employed an elegant xenotransplantation technique to study the fate of human induced pluripotent stem cells (hiPSCs) from fertile males and from males carrying Y chromosome deletions of the azoospermia factor (AZF) region. When placed in a mouse testis niche, hiPSCs from fertile males differentiate into germ cell‐like cells (GCLCs). Highlighting the crucial role of cell autonomous factors in male sterility, hiPSCs derived from azoospermic males prove to be less successful under similar circumstances. Their studies argue that the agametic “Sertoli cell only” phenotype of two of the AZF deletions likely arises from a defect in the maintenance of germline stem cells (GSCs) rather than from a defect in their specification. These observations underscore the importance of the dialogue between the somatic niche and its inhabitant stem cells, and open up interesting questions concerning the functioning of the somatic niche and how it communicates to the GSCs.     

Cytogenetic mapping with centromeric bacterial artificial chromosomes contigs shows that this recombination‐poor region comprises more than half of barley chromosome 3H

Genetic maps are based on the frequency of recombination and often show different positions of molecular markers in comparison to physical maps, particularly in the centromere that is generally poor in meiotic recombinations. To decipher the position and order of DNA sequences genetically mapped to the centromere of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization with mitotic metaphase and meiotic pachytene chromosomes was performed with 70 genomic single‐copy probes derived from 65 fingerprinted bacterial artificial chromosomes (BAC) contigs genetically assigned to this recombination cold spot. The total physical distribution of the centromeric 5.5 cM bin of 3H comprises 58% of the mitotic metaphase chromosome length. Mitotic and meiotic chromatin of this recombination‐poor region is preferentially marked by a heterochromatin‐typical histone mark (H3K9me2), while recombination enriched subterminal chromosome regions are enriched in euchromatin‐typical histone marks (H3K4me2, H3K4me3, H3K27me3) suggesting that the meiotic recombination rate could be influenced by the chromatin landscape.