Unreduced gamete formation in plants: mechanisms and prospects

Polyploids, organisms with more than two sets of chromosomes, are widespread in flowering plants, including many important crop species. Increases in ploidy level are believed to arise commonly through the production of gametes that have not had their ploidy level reduced during meiosis. Although there have been cytological descriptions of unreduced gamete formation in a number of plants, until recently none of the underlying genes or molecular mechanisms involved in unreduced gamete production have been described. The recent discovery of several genes in which mutations give rise to a high frequency of unreduced gametes in the model plant Arabidopsis thaliana opens the door to the elucidation of this important event and its manipulation in crop species. Here this recent progress is reviewed and the identified genes and the mechanism by which the loss of protein function leads to the formation of unreduced gametes are discussed. The potential to use the knowledge gained from Arabidopsis mutants to design tools and develop techniques to engineer unreduced gamete production in important crop species for use in plant breeding is also discusse

A Plant Germline-Specific Integrator of Sperm Specification and Cell Cycle Progression

The unique double fertilisation mechanism in flowering plants depends upon a pair of functional sperm cells. During male gametogenesis, each haploid microspore undergoes an asymmetric division to produce a large, non-germline vegetative cell and a single germ cell that divides once to produce the sperm cell pair. Despite the importance of sperm cells in plant reproduction, relatively little is known about the molecular mechanisms controlling germ cell proliferation and specification. Here, we investigate the role of the Arabidopsis male germline-specific Myb protein DUO POLLEN1, DUO1, as a positive regulator of male germline development. We show that DUO1 is required for correct male germ cell differentiation including the expression of key genes required for fertilisation. DUO1 is also necessary for male germ cell division, and we show that DUO1 is required for the germline expression of the G2/M regulator AtCycB1;1 and that AtCycB1:1 can partially rescue defective germ cell division in duo1. We further show that the male germline-restricted expression of DUO1 depends upon positive promoter elements and not upon a proposed repressor binding site. Thus, DUO1 is a key regulator in the production of functional sperm cells in flowering plants that has a novel integrative role linking gametic cell specification and cell cycle progression

Arabidopsis DUO POLLEN3 Is a Key Regulator of Male Germline Development and Embryogenesis

Male germline development in angiosperms produces the pair of sperm cells required for double fertilization. A key regulator of this process in Arabidopsis thaliana is the male germline-specific transcription factor DUO POLLEN1 (DUO1) that coordinates germ cell division and gamete specification. Here, we uncover the role of DUO3, a nuclear protein that has a distinct, but overlapping role with DUO1 in male germline development. DUO3 is a conserved protein in land plants and is related to GON-4, a cell lineage regulator of gonadogenesis in Caenorhabditis elegans. Mutant duo3-1 germ cells either fail to divide or show a delay in division, and we show that, unlike DUO1, DUO3 promotes entry into mitosis independent of the G2/M regulator CYCB1;1. We also show that DUO3 is required for the expression of a subset of germline genes under DUO1 control and that like DUO1, DUO3 is essential for sperm cell specification and fertilization. Furthermore, we demonstrate an essential sporophytic role for DUO3 in cell division and embryo patterning. Our findings demonstrate essential developmental roles for DUO3 in cell cycle progression and cell specification in both gametophytic and sporophytic tissues

Imprinting of the Polycomb Group Gene MEDEA Serves as a Ploidy Sensor in Arabidopsis

Balanced maternal and paternal genome contributions are a requirement for successful seed development. Unbalanced contributions often cause seed abortion, a phenomenon that has been termed “triploid block.” Misregulation of imprinted regulatory genes has been proposed to be the underlying cause for abnormalities in growth and structure of the endosperm in seeds with deviating parental contributions. We identified a mutant forming unreduced pollen that enabled us to investigate direct effects of unbalanced parental genome contributions on seed development and to reveal the underlying molecular mechanism of dosage sensitivity. We provide evidence that parent-of-origin–specific expression of the Polycomb group (PcG) gene MEDEA is causally responsible for seed developmental aberrations in Arabidopsis seeds with increased paternal genome contributions. We propose that imprinted expression of PcG genes is an evolutionary conserved mechanism to balance parental genome contributions in embryo nourishing tissues

Analysis of Fluorescent Reporter Activity in the Male Germline During Pollen Development by Confocal Microscopy.

The male germline of flowering plants develops within the vegetative cell of the male gametophyte (pollen). The germline is established by asymmetric division of the microspore to form the generative cell. Mitotic division of the generative cell then produces the two sperm cells required for double fertilization. These differentiate to produce the proteins required for gamete attachment and fusion. An important aspect of understanding germline development is the characterization of germline gene expression. Here, we describe the use of a fluorescent reporter to study germline gene expression in developing pollen to assess the timing and specificity of expression

The R2R3 MYB Transcription Factor DUO1 Activates a Male Germline-Specific Regulon Essential for Sperm Cell Differentiation in Arabidopsis[C][W]

The MYB protein DUO1 is a key determinant of sperm cell development in Arabidopsis. This study identifies a diverse range of downstream genes regulated by DUO1 and provides molecular insight into the regulatory networks associated with the differentiation of precursor germ cells into functional sperm cells

Identification of Cis-Regulatory Modules that Function in the Male Germline of Flowering Plants.

The male germline of flowering plants develops within the vegetative cell of the male gametophyte and displays a distinct transcriptional profile. Key to understanding the development of this unique cell lineage is determining how gene expression is regulated within germline cells. This knowledge impacts upon our understanding of cell specification, differentiation, and plant fertility. Here, we describe methods to identify cis-regulatory modules (CRMs) that act as key regulatory regions in the promoters of germline-expressed genes. We detail the complimentary techniques of phylogenetic footprinting and the use of fluorescent reporters in pollen for the identification and verification of CRMs

Meiocyte size is a determining factor for unreduced gamete formation in Arabidopsis thaliana

Polyploidy, the presence of more than two sets of chromosomes within a cell, is a widespread phenomenon in plants. The main route to polyploidy is considered through the production of unreduced gametes that are formed as a consequence of meiotic defects. Nevertheless, for reasons poorly understood, the frequency of unreduced gamete formation differs substantially among different plant species. The previously identified meiotic mutant jason (jas) in Arabidopsis thaliana forms about 60% diploid (2n) pollen. JAS is required to maintain an organelle band as a physical barrier between the two meiotic spindles, preventing previously separated chromosome groups from uniting into a single cell.In this study, we characterized the jas suppressor mutant telamon (tel) that restored the production of haploid pollen in the jas background.The tel mutant did not restore the organelle band, but enlarged the size of male jas tel meiocytes, suggesting that enlarged meiocytes can bypass the requirement of the organelle band. Consistently, enlarged meiocytes generated by a tetraploid jas mutant formed reduced gametes.The results reveal that meiocyte size impacts chromosome segregation in meiosis II, suggesting an alternative way to maintain the ploidy stability in meiosis during evolution