title: Timing of the G1/S transition in tobacco pollen vegetative cells as a primary step towards androgenesis in vitro.
abstract: Mid-bicellular pollen vegetative cells in tobacco escape from G1 arrest and proceed to the G1/S transition towards androgenesis within 1 day under glutamine starvation conditions in vitro. In the Nicotiana tabacum pollen culture system, immature pollen grains at the mid-bicellular stage can mature in the presence of glutamine; however, if glutamine is absent, they deviate from their native cell fate in a few days. The glutamine-starved pollen grains cannot undergo maturation, even when supplied with glutamine later. Instead, they undergo cell division towards androgenesis slowly within 10 days in a medium containing appropriate nutrients. During the culture period, they ought to escape from G1 arrest to proceed into S phase as the primary step towards androgenesis. However, this event has not been experimentally confirmed. Here, we demonstrated that the pollen vegetative cells proceeded to the G1/S transition within approximately 15-36 h after the start of culture. These results were obtained by analyzing transgenic pollen possessing a fusion gene encoding nuclear-localizing GFP under the control of an E2F motif-containing promoter isolated from a gene encoding one of DNA replication licensing factors. Observations using a 5-ethynyl-2'-deoxyuridine DNA labeling and detection technique uncovered that the G1/S transition was soon followed by S phase. These hallmarks of vegetative cells undergoing dedifferentiation give us new insights into upstream events causing the G1/S transition and also provide a novel strategy to increase the frequency of the androgenic response in tobacco and other species, including recalcitrants.
authors.affiliation: Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa, 761-0795, Japan, kyo@ag.kagawa-u.ac.jp.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Kyo
authors.forename: Ai
authors.initials: A
authors.lastname: Nagano
authors.forename: Naoki
authors.initials: N
authors.lastname: Yamaji
authors.forename: Yuhki
authors.initials: Y
authors.lastname: Hashimoto
mesh_terms: D020816:Amino Acid Motifs; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D054337:Cell Dedifferentiation; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D016193:G1 Phase; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D005973:Glutamine / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011058:Pollen / Q000166:cytology / Q000196:embryology / Q000254:growth & development*; D011401:Promoter Regions, Genetic / Q000235:genetics; D016196:S Phase; D014026:Nicotiana / Q000166:cytology / Q000196:embryology / Q000254:growth & development*
chemical_list: D018797:Cell Cycle Proteins; D017931:DNA Primers; D010940:Plant Proteins; D005973:Glutamine