Developmental analyses reveal early arrests of the spore-bearing parts of reproductive organs in unisexual flowers of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

To understand the regulatory mechanisms governing unisexual flower development in cucumber, we conducted a systematic morphogenetic analysis of male and female flower development, examined the dynamic changes in expression of the C-class floral organ identity gene CUM1, and assessed the extent of DNA damage in inappropriate carpels of male flowers. Accordingly, based on the occurrence of distinct morphological events, we divided the floral development into 12 stages ranging from floral meristem initiation to anthesis. As a result of our investigation we found that the arrest of stamen development in female flowers, which occurs just after the differentiation between the anther and filament, is mainly restricted to the primordial anther, and that it is coincident with down-regulation of CUM1 gene expression. In contrast, the arrest of carpel development in the male flowers occurs prior to the differentiation between the stigma and ovary, given that no indication of ovary differentiation was observed even though CUM1 gene expression remained detectable throughout the development of the stigma-like structures. Although the male and female reproductive organs have distinctive characteristics in terms of organ differentiation, there are two common features regarding organ arrest. The first is that the arrest of the inappropriate organ does not affect the entirety of the organ uniformly but occurs only in portions of the organs. The second feature is that all the arrested portions in both reproductive organs are spore-bearing parts.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy - TUNEL TdT-mediated dUTP nick-end labeling     

Ethylene perception is involved in female cucumber flower development

It is well established that ethylene promotes female flower development in cucumber. However, little is known about how the gaseous hormone selectively affects female flowers, and what mechanism it uses. Previously, we found organ‐specific DNA damage in the primordial anther of female cucumber flowers. This finding led to a hypothesis that ethylene might promote female flower development via the organ‐specific induction of DNA damage in primordial anthers. In this study, we tested this hypothesis first by demonstrating ethylene induction of DNA damage via the ethylene signaling pathway using cucumber protoplasts. Then, using representative component genes of the ethylene signaling pathway as probes, we found that one of the ethylene receptors, CsETR1, was temporally and spatially downregulated in the stamens of stage‐6 female cucumber flowers, especially along with the increase of the nodes. Furthermore, by constructing transgenic Arabidopsis plants with organ‐specific expression of antisense CsETR1 under the control of an AP3 promoter to downregulate ETR1 expression in the stamens, we generated Arabidopsis ‘female flowers’, in which the abnormal stamens mimic those of female cucumber flowers. Our data suggest that ethylene perception is involved in the arrest of stamen development in female cucumber flowers through the induction of DNA damage. This opens up a novel perspective and approach to solve the half‐century‐long puzzle of how gaseous ethylene selectively promotes female flowers in the monoecious cucumber plant.     

Participation of <Emphasis Type="Italic">SRM5/CDC28</Emphasis>, <Emphasis Type="Italic">SRM8/NET1</Emphasis>, and <Emphasis Type="Italic">SRM12/HFI1</Emphasis> genes in checkpoint control in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

About twenty genes participating in checkpoint control are known in yeast Saccharomyces cerevisiae. The involvement of SRM genes in the cell cycle arrest under the action of DNA damaging agents was studied in this work. These genes were earlier defined as genes affecting genetic stability and radiosensitivity. It was shown that mutations srm5/cdc28-srm, srm8/net1-srm, and srm12/hfi1-srm fail the cell cycle arrest in the presence of DNA damage and influence the checkpoint arrest in G0/S (srm5, srm8), G1/S (srm5, srm8, srm12), S (srm5, srm12), and G₂/M (srm5). It seems likely that genes SRM5/CDC28, SRM12/HFI1/ADA1, and SRM8/NET1 are involved in a cell response to DNA damage, and in checkpoint regulation in particular.     

Morphological development of anthers induced by the dimorphic smut fungus<Emphasis Type="Italic"> Microbotryum violaceum</Emphasis> in female flowers of the dioecious plant<Emphasis Type="Italic"> Silene latifolia</Emphasis>

When inoculated with the dimorphic smut fungus Microbotryum violaceum (Pers.) G. Deml and Oberwinkler, the female flower of the dioecious plant Silene latifolia (Miller) E.H.L. Krause develops anther-like structures filled with spores instead of pollen grains. Using natural scanning electron microscopy, Nomarski interference microscopy, and fluorescence microscopy, we investigated the morphological modifications of the host plant resulting from this parasitism and the localization of smut hyphae in the flower bud. Flowers of infected plants lasted significantly longer than those of healthy plants, probably because the infection strengthened floral organs, such as the flower base and the anther filaments. Smut hyphae were observed throughout all organs of the young flower buds of infected plants, including sepals, petals, stamens, and pistil primordia. In healthy female flowers, anthers initiated sporogenous cell formation, but lacked parietal cell layers. By contrast, the parietal cell layers of infected female flowers differentiated into tapetal tissue, middle cell layers, and endothecial layers, as in the anthers of healthy male flowers. Smut spore formation in the infected anther was initiated in intercellular regions between the sporogenous cells, resulting in degeneration of premature sporogenous cells, tapetal tissue, and middle cell layers. The development of the endothecial layers and epidermis in the infected anther were morphologically normal.Abbreviations DAPI 4,6-diamidino-2-phenylidole - i infected - PMC pollen mother cell     

Lithium induces cell plate dispersion during cytokinesis inTradescantia

Summary We have found that 10mM LiCl added toTradescantia stamen hair cells prior to early anaphase appears to prevent the vesicle coalescence phase of cell plate formation. In a fashion similar to caffeine inhibition of cell plate formation [Bonsignore and Hepler 1985, Protoplasma 129, 28–35], cell plate vesicle aggregation occurs at its normal time in LiCl, forming an incipient plate which is similar in morphology to that of an untreated cell during the first 10min, but the structure subsequently disperses and the resultant cells are binucleate. The addition of 10–20M myo-inositol was sufficient to reverse the inhibitory effect of Li⁺ in the majority of our experiments while scyllitol, an isomer of myo-inositol, or buffer without myoinositol were usually insufficient to reverse the inhibition. The timing of the addition of myo-inositol was critical for reversal; if the rescue solution was added more than 4 min after the onset of cell plate vesicle aggregation, the cell was usually irreversibly destined to become binucleate. The addition of 100M CaCl₂ within 2min of cell plate vesicle aggregation also overcame Li⁺-induced plate dispersal, but the kinetics of reversal were substantially slower than with myo-inositol. The results show that cell plate formation and in particular, cell plate vesicle coalescence, is sensitive to exogenously applied LiCl.Abbreviations AO Anaphase Onset - CP Cell Plate (completed) - CPD Cell Plate Dispersion - CPVA Cell Plate Vesicle Aggregation - DAG 1,2-diacylglycerol - InsP₃ 1,4,5-inositol trisphosphate - IP inositol-1-phosphate - polyPI polyphosphoinositide     

Temperature conditional cAMP-requiring mutant strains ofChlamydomonas reinhardtii arrest in G1 and are rescued by added cAMP

Summary Three independent isolates ofChlamydomonas, selected for caffeine resistance, were found to arrest in G₁ phase, as determined by quantitative fluorescence measurements of DNA, when grown at a non-permissive temperature. This cell cycle arrest correlated with lowered levels of cAMP and of adenylate cyclase activity. The arrested cells could be rescued by added cAMP but not AMP, hence the defect was not one of general purine metabolism. Back-crosses to wild type revealed that the phenotypes observed result from a combination of three separable mutations. It is clear that the mutations define functions that are more stringently required for cell division than for growth since the mutant strains are able to grow up to fifteen times normal size while blocked at the non-permissive temperature. The possible interaction of cAMP dependent events with division is discussed.Abbreviations AMP adenosine 5-monophosphate - ATP adenosine 5-triphosphate - BSA bovine serum albumin - cAMP adenosine 3,5-cyclicmonophosphate - db-cAMP dibutyryl-cAMP - DNA deoxyribonucleic acid - DTT dithiothreitol - -cAMP 1,N⁶-etheno-cAMP - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid - HPLC high performance liquid chromatography - LSA low sulphur-high salt-acetate medium - LYP LSA media containing yeast extract and proteose peptone - M1, 2, 3 mutants 1, 2, 3 - PDE phosphodiesterase - TAP trisacetate-phosphate medium - TLC thin layer chromatography - TYP TAP medium containing yeast extract and proteose peptone     

Protein markers for anther culturability in barley

Two-dimensional electrophoresis of proteins from a recombinant population of anther culture-derived doubled haploid lines identified 4 loci or linkage groups showing a deviation from an expected 11 segregation. It was hypothesized that these markers are linked to genes involved in the process of haploid plant production and that the deviation was due to a selection for alleles conferring higher anther culture response. To check this hypothesis, the anther culturability of 50 of the doubled haploid lines and their two inbred parents was assessed. It was found that 2 of the loci which had a distortion of segregation showed a significant effect on anther culture response, the most efficient allele being the most frequent in both loci. In addition, 2 more markers associated with anther culturability were found. One of the first mentioned 2 loci and one of the latter 2 were found to be linked to genes involved in both embryoid production and subsequent green plant regeneration. The remaining two were linked to genes involved only in green plant regeneration. Of the 4 favorable alleles 3 were inherited from one parent.     

Suppression of heterotrimeric G-protein β-subunit affects anther shape,pollen development and inflorescence architecture in tobacco

The role of the heterotrimeric G-protein -subunit in plant development was studied in transgenic tobacco (Nicotiana tabacum L.) plants with reduced -subunit levels due to the antisense expression of the -subunit mRNA. The antisense plants had aberrant anther shape and produced non-germinating pollen. The anthers were sporadically transformed to petals, whereas other floral organs were not affected. The pollen grains were smaller than the wild-type pollen and had abnormal cell walls. The architecture of mature antisense plants was altered. The plants had long branched panicles and short stems. These data suggest that the -subunit of the plant heterotrimeric G-proteins is involved in the regulation of the reproductive phase of the tobacco life cycle, particularly in stamen development and pollen maturation.     

Behavior of organelles and their nucleoids in the shoot apical meristem during leaf development in Arabidopsis thaliana L.

The behavior of organelle nucleoids and cell nuclei was studied in the shoot apical meristem and developing first foliage leaves of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4-6-diamidino-2-phenylindole to observe DNA. Fluorimetry was performed using a video-intensified microscope photon-counting system. The DNA content of individual mitochondria was more than 1 Mbp in the shoot apical meristem and the young leaf primordium, and decreased to approximately 170 kbp in the mature foliage leaf. In contrast, the DNA content of individual plastids was low in the shoot apical meristem and increased until day 7 after sowing. Application of 5-bromo-2-deoxyuridine, an analogue of thymidine, was usesd to investigate DNA synthesis in situ. The activities of DNA synthesis in the mitochondria and plastids changed according to the stage of development. Mitochondrial DNA was actively synthesized in the shoot apical meristem and young leaf primordia. This strongly suggests that the amount of mitochondrial DNA per mitochondrion, which has been synthesized in the shoot apical meristem and young leaf primordium, is gradually reduced due to continual divisions of the mitochondria during low levels of mitochondrial DNA synthesis. Synthesis of DNA in the plastid became active in the leaf primordia following DNA synthesis in the mitochondria, and the small plastids were filled with large plastid nucleotids. This enlargement of the plastid nucleoids occurred before the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the development of thylakoids.Abbreviations BrdU 5-bromo-2-deoxyuridine - DAPI 4-6-diamidino-2-phenylindole - DiOC₆a 3,3-dihexyloxacarbocyanine - mtDNA mitochondrial DNA - mt-nucleoid mitochondrial nucleoid - ptDNA plastid DNA - pt-nucleoid plastid nucleoid - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This work was supported by grant No. 2553 to M.F. and Nos. 04454019, 03304005 and 06262204 to T.K. from the Ministry of Education, Science and Culture of Japan, and by a grant for a pioneering research project in biotechnology from the Ministry of Agriculture, Forestry and Fisheries of Japan.     

Variation in structural gender in the hermaphrodite Helleborus foetidus (Ranunculaceae): within- and among-population patterns

In hermaphrodite plants, variations in structural gender (defined as the ratio between male and female gametes) may occur at different levels (among flowers, plants, and populations). In this study, we investigated variation in four traits influencing structural gender (number of carpels, ovules per carpel, stamens, and pollen grains per stamen) within and among six distant populations of the hermaphrodite perennial herb Helleborus foetidus (Ranunculaceae) in the Iberian Peninsula. Our results show that the four traits investigated varied significantly at all levels considered. Traits influencing the female sexual component (number of carpels and ovules per carpel) showed greater variation at the lowermost levels (within flower and plant) than traits influencing the male component, which in turn varied more markedly among populations. Number of carpels per flower and number of pollen grains per anther were the most important traits affecting between-plant variation in structural gender. There was no evidence of significant plant-level trade-offs or correlations between the various male and female traits, which covaried differently across populations. The observed between-population variation in structural gender of Helleborus foetidus can be explained as a consequence of differences in self-pollination levels related to a flowers mating environment.     

Function of poly(ADP-ribose) polymerase in response to DNA damage: Gene-disruption study in mice

To elucidate the biological functions of poly(ADP-ribose) polymerase (PARP, [EC 2.4.2.30]) in DNA damage responses, genetic and biochemical approaches were undertaken. By disrupting exon 1 of the mouse PARP gene by a homologous recombination, PARP-deficient mouse embryonic stem (ES) cell lines and mice could be produced without demonstrating lethality. PARP-/- ES cells showed complete loss of PARP activity and increased sensitivity to -irradiation and an alkylating agents, indicating a physiological role for PARP in the response to DNA damage. p53, a key molecule in cellular DNA damage response, was found to stimulate PARP activity and became poly(ADP-ribosyl)ated in the presence of damaged DNA. However, PARP-/- ES cells showed p21 and Mdm-2 mRNA induction following -irradiation, indicating that PARP activity is not indispensable for p21 and Mdm-2 mRNA induction in the established p53-cascade. On the other hand, in a reconstituted reaction system, purified PARP from human placenta suppressed the pRB-phosphorylation activity in the presence of NAD and damaged DNA. Human PARP expressed in E. coli showed a similar effect on pRB-phosphorylation activity of cdk2. These findings suggest a direct involvement of PARP in the regulation of cdk activity for cell-cycle arrest.     

Inheritance of anther culture derived green plantlet regeneration in wheat (Triticum aestivum L.)

A study was set up to determine the inheritance and combining ability of the factors anther culture response and green plant regeneration. Reciprocal crosses were made between cultivar Ringo Sztar, showing high anther culture response and the cultivars Ciano 067 and Benoist H77022, showing a high level of green plant regeneration. Averaged over all genotypes, 23.0% of the anthers responded and a callus induction frequency of 77.8% was observed. Of all the embryos, 43.0% developed into plantlets, 25.6% of the regenerants being green, the result being that 3.3 green plants per 100 anthers were formed. Genotypic effects accounted for 57.7%, 86.3% and 77.5% of the total variance of anther culture response, callus induction frequency and embryo induction frequency, respectively. Additive and dominant gene action was detected for all characteristics, including green plant regeneration. No reciprocal differences were found for anther culture response, embryo induction frequency and green plant regeneration, indicating no cytoplasmic effects. A small but significant reciprocal difference was found for callus induction frequency. Embryo production was primarily correlated with anther culture response and not with the number of embryos produced per plated anther or per responding anther. Possible mechanisms for the inheritance of green plant regeneration are discussed.Abbreviations CIRA callus induction frequency per responding anther - ERA embryo induction frequency per responding anther - FHB fusarium head blight - MS-medium Murashige & Skoog (1962) medium - REML residual maximum likelihood     

Changes in DNA methylation during somatic embryogenesis in <Emphasis Type="Italic">Cucurbita pepo</Emphasis> L.

Three pumpkin embryogenic lines were initiated on wounded zygotic embryos cultured on medium with or without 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryo development was controlled by the availability of various compounds in the medium: presence/absence of 2,4-D, nitrogen sources. The highest rate of DNA methylation was in the early embryo stages, predominantly on MSC medium with 2,4-D and on auxin-free medium supplemented with 1.0 mM NH₄Cl. DNA methylation was correlated with early embryo development in a manner that was not exclusively dependent on the presence/absence of exogenous auxin. DNA methylation decreased during embryo maturation on auxin-free MSC medium and on auxin-free MSC supplemented with 12.3 M 5-azacytidine (5-azaC). The embryogenic features of the pumpkin tissue were preserved, even after a 2-month treatment with 5-azaC.Abbreviations 5-azaC 5-Azacytidine - CRED-RA Coupled restriction enzyme digestion and random amplification - 2,4-D 2,4-Dichlorophenoxyacetic acid - DNMRT Duncans new multiple range test - IAA Indole-3-acetic acid - 5-mC 5-Methylcytosine     

Radiomimetic Effect of Cisplatin on Cucumber Root Development: the Relationship between Cell Division and Cell Growth

Cisplatin [DDP, cis-dichlorodiammine platinum (II)], a strongcytostatic and antineoplastic agent, was tested on seedlingsof cucumber Cucumis sativus L. for its general effect on rootdevelopment and its particular effects on root cell divisionand cell growth. DDP was characterized as a radiomimetic compoundsince both DDP (1·3 x 10^-5 M) and -irradiation (2·5-10kGy) drastically and irreversibly stopped development of embryoniclateral root primordia (LRPs) in the radicle by inhibiting bothmitotic activity and cell growth. In 20% of the LRPs of DDP-treatedroots, cells did not divide at all. Dividing cells completedno more than two cell cycles. These effects were specific becausewhen DDP was available to the roots only at the onset of celldivision, cell proliferation and cell growth were similar tothat produced by constant incubation. Neither DDP nor -irradiationaffected non-meristematic cell elongation. It was concludedthat cell growth of meristematic cells is closely related tocell division. However, non-meristematic cell growth is independentof DNA damage. This suggests DDP as a tool to reveal these autonomousprocesses in plants development and to detect tissue compartmentsin mature plant embryos which contain potentially non-meristematiccells.Copyright 1993, 1999 Academic Press Cucumis sativus, cucumber, cisplatin, cell growth, cell elongation, cell division, lateral root, root development     

Effects of growth temperature upshift on cell cycle progression in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cell cycle progression of Cryptococcus neoformans was studied for cells grown exponentially at 15°, 24°, and 30°C. Except for speed, cell cycle progression was similar. In particular, budding occurred relatively soon after initiation of DNA synthesis at 15°, 24°, and 30°C. After growth temperature was shifted from 15° to 30°C, cells were transiently arrested before initiation of DNA synthesis. Thus, similar to Saccharomyces erevisiae, Start was the main susceptible cell cycle controlling point in C. neoformans. However, after spontaneous release from arrest as above, cells were further arrested in the unbudded state. Thus, the timing of budding was delayed just before the G₂ phase, or even until after entering the G₂ phase, but it was also transient, and 5h after the shift buds emerged relatively soon after initiation of DNA synthesis. Thus, C. neoformans cells can respond adaptively to mild stress by delaying budding. The existence of the second susceptible cell cycle control point, i.e., budding, appears to endow C. neoformans with a unique characteristic of stronger inhibition of multiplication than growth. A model of the C. neoformans cell cycle is also presented.     

Senescence in the nongreening region of the rice (Oryza sativa) coleoptile

Summary The coleoptile of rice (Oryza sativa L. cv. Nippon-bare) emerges from the imbibed seed on day 2 after sowing and ceases its growth on day 3. In cross section, the cells near the outer epidermis turn into green between days 2 and 3, while those near the inner epidermis remain colorless. In this study, the complete process of the development in the nongreening cells in the coleoptile was examined by fluorescence and electron microscopy. Embryonic morphology on day 0 was rapidly converted into the differentiated greening or nongreening cells between days 1 and 2. Senescence in the inner, nongreening region first appeared on day 4 in the third or fourth cell layer from the inner epidermis and then spread towards both the inner and the outer epidermis, and the inner cells collapsed completely before the outer cells senesced. Cells adjacent to the inner epidermis, which senesced slowly, followed a sequence of events during development: (1) degradation of plastid DNA; (2) dispersal of nuclear chromatin, differentiation of plastids into amyloplasts, degradation of mitochondrial DNA; (3) degradation of the starch in amyloplasts; (4) disorganization of plastids; (5) condensation of the nucleus, shrinkage of mitochondria; (6) complete loss of cellular components, distortion of cell walls. In the interior cells, the early events including degeneration of plastid DNA and mitochondrial DNA occurred in parallel with those in the cells adjacent to the inner epidermis, yet rapid collapse of all the cellular components proceeded between days 3 and 5, and nuclear condensation could not be detected.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole - DiOC₇ 3,3-dihexyloxacarbocyanine - IE inner epidermis - mtDNA mitochondrial DNA - mt-nucleoid mitochondrial nucleoid - OE outer epidermis - ptDNA plastid DNA - pt-nucleoid plastid nucleoid     

Spermidine/spermine N1-acetyltransferase overexpression in kidney epithelial cells disrupts polyamine homeostasis, leads to DNA damage, and causes G2 arrest

Expression of spermidine/spermine N¹-acetyltransferase (SSAT) increases in kidneys subjected to ischemia-reperfusion injury (IRI). Increased expression of SSAT in vitro leads to alterations in cellular polyamine content, depletion of cofactors and precursors of polyamine synthesis, and reduced cell proliferation. In our model system, a >28-fold increase in SSAT levels in HEK-293 cells leads to depletion of polyamines and elevation in the enzymatic activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, suggestive of a compensatory reaction to increased polyamine catabolism. Increased expression of SSAT also led to DNA damage and G₂ arrest. The increased DNA damage was primarily due to the depletion of polyamines. Other factors such as increased production of H₂O₂ due to polyamine oxidase activity may play a secondary role in the induction of DNA lesions. In response to DNA damage the ATM/ATR Chk1/2 DNA repair and cell cycle checkpoint pathways were activated, mediating the G₂ arrest in SSAT-expressing cells. In addition, the activation of ERK1 and ERK2, which play integral roles in the G₂/M transition, is impaired in cells expressing SSAT. These results indicate that the disruption of polyamine homeostasis due to enhanced SSAT activity leads to DNA damage and reduced cell proliferation via activation of DNA repair and cell cycle checkpoint and disruption of Raf MEK ERK pathways. We propose that in kidneys subjected to IRI, one mechanism through which increased expression of SSAT may cause cellular injury and organ damage is through induction of DNA damage and the disruption of cell cycle. ischemia-reperfusion injury; polyamine depletion; cell proliferation; DNA repair; cell cycle arrest     

Evidence of DNA replication in an arbuscular mycorrhizal fungus in the absence of the host plant

Summary This study provides evidence thatGigaspora margarita replicates its nuclear DNA, even in the absence of a host plant. Three experimental approaches were used: (i) static cytofluorimetry to quantify the DNA content, (ii) pulse treatments with bromodeoxyuridine (BrdU), which is an analogue of thymidine, to reveal nuclei undergoing DNA synthesis, and (iii) ultrastructural observations to study changes in chromatin morphology during the fungal cell cycle. A slight second peak of approximately twice the value of a major peak was found by cytofluorimetry, showing that a small number of nuclei had entered in cycle during in vitro development. Nuclei which had incorporated BrdU were observed after pulses of 24 h; nuclei with condensed chromatin were also apparent at this time. The results demonstrate thatG. margarita has all the metabolic pathways needed to replicate its nuclear DNA even in the absence of the host, suggesting that more complex mechanisms inhibit the extended growth in vitro of arbuscular mycorrhizal fungi.Abbreviations AM-fungi arbuscular mycorrhizal fungi - A.U. arbitrary units - BrdU 5-bromo-2-deoxyuridine - DAPI 4,6-diamidino-2-phenylindole - UV ultraviolet light