Expression of fission yeast cdc25 driven by the wheat ADP-glucose pyrophosphorylase large subunit promoter reduces pollen viability and prevents transmission of the transgene in wheat.

Cell number was to be measured in wheat (Triticum aestivum) endosperm expressing Spcdc25 (a fission yeast cell-cycle regulator) controlled by a supposedly endosperm-specific promoter, AGP2 (from the large subunit of ADP glucose pyrophosphorylase). Wheat was transformed by biolistics either with AGP2::GUS or AGP2::Spcdc25. PCR and RT-PCR checked integration and expression of the transgene, respectively. In cv. Chinese Spring, AGP2::GUS was unexpectedly expressed in carpels and pollen, as well as endosperm. In cv. Cadenza, three AGP2::Spcdc25 plants, AGP2::Spcdc25.1, .2 and .3, were generated. Spcdc25 expression was detected in mature leaves of AGP2::Spcdc25.1/.3 which exhibited abnormal spikes, 50% pollen viability and low seed set per plant; both were small compared with the nonexpressing and normal AGP2::Spcdc25.2. Spcdc25 was not transmitted to the T(1) in AGP2::Spcdc25.1 or .3, which developed normally. Spcdc25 was PCR-positive in AGP2::Spcdc25.2, using primers for a central portion, but not with primers for the 5' end, of the ORF, indicating a rearrangement; Spcdc25 was not expressed in either T(0) or T(1). The AGP2 promoter is not tissue-specific and Spcdc25 expression disrupted reproduction.

The Arabidopsis CDC25 induces a short cell length when overexpressed in fission yeast: evidence for cell cycle function.

The putative mitotic inducer gene, Arath;CDC25 cloned in Arabidopsis thaliana, was screened for cell cycle function by overexpressing it in Schizosaccharomyces pombe (fission yeast). The expression pattern of Arath;CDC25 was also examined in different tissues of A. thaliana. Fission yeast was transformed with plasmids pREP1 and pREP81 with the Arath;CDC25 gene under the control of the thiamine-repressible nmt promoter. Using reverse transcription-polymerase chain reaction (RT-PCR), the expression of Arath;CDC25 was examined in seedlings, flower buds, mature leaves and stems of A. thaliana; actin (ACT2) was used as a control. In three independent transformants of fission yeast, cultured in the absence of thiamine (T), pREP1::Arath;CDC25 induced a highly significant reduction in mitotic cell length compared with wild type, pREP::Arath;CDC25 +T, and empty vector (pREP1 +/- T). The extent of cell shortening was greater using the stronger pREP1 compared with the weaker pREP81. However, Arath;CDC25 was expressed at low levels in all tissues examined. The data indicate that Arath;CDC25 can function as a mitotic accelerator in fission yeast. However, unlike other plant cell cycle genes, expression of Arath;CDC25 was not enhanced in rapidly dividing compared with non-proliferative Arabidopsis tissues.