Cell Death and Cell Cycle Arrest of Silene latifolia Stamens and Pistils After Microbotryum lychnidis-dioicae Infection.

Mechanisms of suppression of pistil primordia in male flowers and of stamen primordia in female flowers differ in diclinous plants. In this study, we investigated how cell death and cell cycle arrest are related to flower organ formation in Silene latifolia. Using in situ hybridization and a TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, we detected both cell cycle arrest and cell death in suppressed stamens of female flowers and suppressed pistils of male flowers in S. latifolia. In female flowers infected with Microbotryum lychnidis-dioicae, developmental suppression of stamens is released, and cell cycle arrest and cell death do not occur. Smut spores are formed in S. latifolia anthers infected with M. lychnidis-dioicae, followed by cell death in the endothelium, middle layer, tapetal cells and pollen mother cells. Cell death is difficult to detect using a fluorescein isothiocyanate-labeled TUNEL assay due to strong autofluorescence in the anther. We therefore combined a TUNEL assay in an infrared region with transmission electron microscopy to detect cell death in anthers. We show that following infection by M. lychnidis-dioicae, a TUNEL signal was not detected in the endothelium, middle layer or pollen mother cells, and cell death with outflow of cell contents, including the nucleoplast, was observed in tapetal cells.

ADAM family protein Mde10 is essential for development of spore envelopes in the fission yeast Schizosaccharomyces pombe.

We report the identification of Schizosaccharomyces pombe mde10+ as a gene possessing a FLEX element, which forms a binding site for the meiosis-specific transcription factor Mei4. In fact, mde10+ is transcribed only in diploid cells that are induced to meiosis in a Mei4-dependent manner. Western blot analysis indicated that the epitope-tagged Mde10 protein accumulates transiently during meiosis and then rapidly decreases. Mde10 is a multidomain protein containing a metalloprotease catalytic domain, a disintegrin domain, a cysteine-rich domain, and membrane-spanning regions, all of which are shared by members of the mammalian ADAM family. A fusion protein of Mde10 and green fluorescent protein localized to the endoplasmic reticulum during meiosis and was located at the peripheral region of spores at the end of meiosis. An mde10Delta deletion mutant showed no apparent defects in meiosis, sporulation, or spore germination. However, the mutant spores exhibited an aberrant surface appearance, in which the ragged outer spore wall was lost to a large extent. Furthermore, mde10Delta spores were found to be less tolerant to ethanol and diethyl ether than were wild-type spores. The mutagenic replacement of the conserved glutamic acid in the putative protease active site with an alanine residue did not affect the surface morphology or the resistance of spores to environmental stress. Our observations indicate that Mde10 is important in the development of the spore envelope, although this function of Mde10 seems to be independent of its metalloprotease activity.

DNA chain unwinding and annealing reactions of lipocortin (annexin) I heterotetramer: regulation by Ca(2+) and Mg(2+).

Lipocortin I-S100 (calcyclin) heterotetramer exhibited ATPase activity in the presence of dsDNA but not ssDNA. To demonstrate its helicase activity, an 80-mer polynucleotide complementary to the replication origin of M13mp18 was synthesized, and the oligonucleotide, (dC)(20), was ligated to either its 5'- or 3'- end for binding to lipocortin. Lipocortin I heterotetramer displaced chains of the partially Y-shaped duplexes with a dC-tail at either the 5'- or 3'- end. The chain displacement required ATP and Mg(2+). Nonhydrolyzable ATP analogues were not effective. Lipocortin I heterotetramer also catalyzed annealing of the polynucleotides to M13mp18. Ca(2+) and phospholipids but not ATP and Mg(2+) were essential for this reaction. Since the chain displacing and annealing reactions were inhibited by monospecific anti-lipocortin I or anti-S100 antibodies, the present observations suggest that the lipocortin I heterotetramer regulates unwinding and annealing of DNA by Mg(2+) (plus ATP) and Ca(2+) (and phospholipids), respectively.