Expressing the Diphtheria Toxin A Subunit from the HAP2(GCS1) Promoter Blocks Sperm Maturation and Produces Single Sperm-Like Cells Capable of Fertilization

Department of Molecular Biology, Brown University, Providence, Rhode Island 02912, USA.
Plant physiology (Impact Factor: 6.84). 10/2009; 151(3):1390-400. DOI: 10.1104/pp.109.144204
Source: PubMed


After meiosis, the male germline of flowering plants undergoes two mitoses, producing two sperm that are carried within a pollen tube to an ovule. One sperm fuses with the egg to form the zygote and the other fuses with the central cell to form the primary endosperm. The mechanisms that control male germline development and gene expression, and ensure that sperm properly fuse with female gametes are just beginning to be understood. Expression of the potent translation inhibitor, diphtheria toxin A subunit, from the Arabidopsis (Arabidopsis thaliana) HAP2(GCS1) promoter blocked sperm development before the final cell division, resulting in pollen tubes that carried a single sperm-like cell rather than two sperm. These pollen tubes targeted ovules and fertilized either the egg or the central cell, producing seeds with either endosperm or an embryo, but not both. Endosperm-only seeds significantly outnumbered embryo-only seeds, suggesting that single sperm-like cells preferentially fuse with the central cell. These experiments show that de novo translation is required for completion of sperm development, that the HAP2(GCS1) promoter is very tightly controlled, and that disruption of gene expression can result in male germ cells with a bias for gamete fusion.

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    • "In contradiction to the hypotheses of single sperm-like fertilization and semigamy, Sarkar and Coe (1966) reported that all pollen grains produced by inducer lines are tricellular and, in previous studies (Fischer, 2004; Li et al., 2009), genome fragments from the inducer were found in a few haploid plants. Furthermore, except for some extreme examples such as B chromosomes, it is known that each of the two sperm cells can fertilize either the egg cell or the central cell (Chen et al., 2008; Frank and Johnson, 2009; Aw et al., 2010). Consequently, if the single sperm that carried the sed1 haplotype fused with either the egg cell or the central cell and eventually resulted in a haploid kernel or a defective kernel, respectively, then the endosperm of aborted kernels should not always carry the sed1 haplotype. "
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    ABSTRACT: Production of maternal haploids via a male inducer can greatly accelerate maize breeding and is an interesting biological phenomenon in double fertilization. However, the mechanism behind haploid induction remains elusive. Segregation distortion, which is increasingly recognized as a potentially powerful evolutionary force, has recently been observed during maternal haploid induction in maize. The results present here showed that both male gametophytic and zygotic selection contributed to severe segregation distortion of a locus, named segregation distortion 1 (sed1), during maternal haploid induction in maize. Interestingly, analysis of reciprocal crosses showed that sed1 is expressed in the male gametophyte. A novel mapping strategy based on segregation distortion has been used to fine-map this locus. Strong selection for the presence of the sed1 haplotype from inducers in kernels with haploid formation and defects could be detected in the segregating population. Dual-pollination experiments showed that viable pollen grains from inducers had poor pollen competitive ability against pollen from normal genotypes. Although defective kernels and haploids have different phenotypes, they are most probably caused by the sed1 locus, and possible mechanisms for production of maternal haploids and the associated segregation distortion are discussed. This research also provides new insights into the process of double fertilization.
    Journal of Experimental Botany 01/2013; 64(4). DOI:10.1093/jxb/ers393 · 5.53 Impact Factor
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    • "During pollination, the vegetative cell forms the pollen tube, one sperm cell fertilizes the egg cell to form the zygote, and the other sperm cell fertilizes the central cell to produce the endosperm. Because the pollen generative cell and subsequent sperm cells contain the genetic material that will be transferred to progeny, male gametic cellspecific promoters have the unique ability to express a protein with direct access to the heritable male genome, such as a site-specific recombinase, a nuclease, or a toxin (Frank and Johnson 2009; Viswanathan et al. 2011; Yang et al. 2011). This could be particularly useful for precise expression of a transgene that would be undesirable and/or detrimental in other plant tissues. "
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    ABSTRACT: Expression control elements (i.e., promoters) are crucial components required for the genetic engineering of plants, but relatively few well-characterized organ-specific promoters are available. We have characterized the rice Gamete Expressed 2 (OsGEX2) gene promoter in transgenic rice plants. The OsGEX2 gene (Os09g25650) is homologous to Arabidopsis GEX2, a gene that exhibits expression specifically in the gametic cells of Arabidopsis pollen. The OsGEX2 gene transcript was only detected in rice pollen (or mixed tissues containing pollen) and not found in other organs or tissues. Transgenic rice plants containing an OsGEX2 promoter fused to a GUSPlus reporter gene displayed cell type-specific β-glucuronidase enzyme activity localized within the sperm cells of mature rice pollen. This expression pattern was clearly distinct from the uniform reporter gene activity observed in the pollen vegetative cell of transgenic rice carrying the rice Pollen Specific 1 promoter. The sperm cell reporter gene activity in OsGEX2–GUSPlus transgenic plants correlated well with the native gene transcript levels, suggesting that the cis-regulatory elements necessary for this specificity are present in the 1.9-kb OsGEX2 promoter fragment. The OsGEX2 promoter with its cell type-specific expression will be a useful tool for precisely controlling sperm cell gene expression in rice and potentially other cereal crop plants.
    Plant Molecular Biology Reporter 10/2012; 30(5). DOI:10.1007/s11105-012-0429-3 · 1.66 Impact Factor
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    • "An increasing number of sperm genes have proved to be essential for fertilization and normal embryo establishment , including, for example, HAPLESS2 (HAP2) and SHORT SUSPENSOR (SSP). HAP2 is known to encode a sperm-specific, surface-linked protein required for fertilization and is also implicated in directing pollen tubes to their female targets (von Besser et al., 2006; Frank & Johnson, 2009). SSP is transmitted as a sperm transcript into the egg cell during gamete fusion and encodes the protein SSP, which activates the developmentally critical asymmetrical division of the zygote, producing a polarized proembryo, which has been shown to establish embryogenesis (Bayer et al., 2009). "
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    ABSTRACT: Genomic assay of sperm cell RNA provides insight into functional control, modes of regulation, and contributions of male gametes to double fertilization. Sperm cells of rice (Oryza sativa) were isolated from field-grown, disease-free plants and RNA was processed for use with the full-genome Affymetrix microarray. Comparison with Gene Expression Omnibus (GEO) reference arrays confirmed expressionally distinct gene profiles. A total of 10,732 distinct gene sequences were detected in sperm cells, of which 1668 were not expressed in pollen or seedlings. Pathways enriched in male germ cells included ubiquitin-mediated pathways, pathways involved in chromatin modeling including histones, histone modification and nonhistone epigenetic modification, and pathways related to RNAi and gene silencing. Genome-wide expression patterns in angiosperm sperm cells indicate common and divergent themes in the male germline that appear to be largely self-regulating through highly up-regulated chromatin modification pathways. A core of highly conserved genes appear common to all sperm cells, but evidence is still emerging that another class of genes have diverged in expression between monocots and dicots since their divergence. Sperm cell transcripts present at fusion may be transmitted through plasmogamy during double fertilization to effect immediate post-fertilization expression of early embryo and (or) endosperm development.
    New Phytologist 06/2012; 195(3):560-73. DOI:10.1111/j.1469-8137.2012.04199.x · 7.67 Impact Factor
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