Microtubules and actin filaments co-localize in pollen tubes ofNicotiana tabacum L. andLilium longiflorum Thunb.

Summary Double labeling with fluorescent probes showed that in the cortical cytoplasm of pollen tubes ofNicotiana tabacum andLilium longiflorum, the microtubules and actin filaments co-localize for the most part. They displayed complex net-axial or helical distributions. They structural association of microtubules and actin filaments implies a functional relationship with respect to organelle movement and/or the organization of the cortical cytoplasm and cell surface.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetra-acetic acid - FITC fluoresceine iso-thiocyanate     

Asymmetrical conspecific seed-siring advantage between Silene latifolia and S. dioica

Background and Aims

Silene dioica and S. latifolia experience only limited introgression despite overlapping flowering phenologies, geographical distributions, and some pollinator sharing. Conspecific pollen precedence and other reproductive barriers operating between pollination and seed germination may limit hybridization. This study investigates whether barriers at this stage contribute to reproductive isolation between these species and, if so, which mechanisms are responsible.

Methods

Pollen-tube lengths for pollen of both species in styles of both species were compared. Additionally, both species were pollinated with majority S. latifolia and majority S. dioica pollen mixes; then seed set, seed germination rates and hybridity of the resulting seedlings were determined using species-specific molecular markers.

Key Results

The longest pollen tubes were significantly longer for conspecific than heterospecific pollen in both species, indicating conspecific pollen precedence. Seed set but not seed germination was lower for flowers pollinated with pure heterospecific versus pure conspecific pollen. Mixed-species pollinations resulted in disproportionately high representation of nonhybrid offspring for pollinations of S. latifolia but not S. dioica flowers.

Conclusions

The finding of conspecific pollen precedence for pollen-tube growth but not seed siring in S. dioica flowers may be explained by variation in pollen-tube growth rates, either at different locations in the style or between leading and trailing pollen tubes. Additionally, this study finds a barrier to hybridization operating between pollination and seed germination against S. dioica but not S. latifolia pollen. The results are consistent with the underlying cause of this barrier being attrition of S. dioica pollen tubes or reduced success of heterospecifically fertilized ovules, rather than time-variant mechanisms. Post-pollination, pre-germination barriers to hybridization thus play a partial role in limiting introgression between these species.     

Does anthocyanin affect outcrossing rates in Datura Stramonium (Solanaceae)?

In this paper, I investigate whether the presence or absence of anthocyanin is neutral with respect to reproduction in Datura stramonium. The observations concern the portion of the life cycle spanning pollination to germination. Pollinators do not appear to distinguish between floral morphs, as revealed by nonbiased distribution of fluorescent powder used as a pollen analogue. Pollen-tube growth is also equal for the two morphs. Seed germination is affected by the presence of anthocyanin, but apparently only by the genotype of the mother at the anthocyanin locus, and not by the genotype of the embryo itself. In addition, there was an interaction between maternal morph and the maternal source population, with seeds from high-elevation anthocyanin-producing mothers germinating most rapidly and seeds from low-elevation anthocyanin-producing mothers germinating most slowly in a common garden at low elevation. However, because germination of anthocyanin-producing and anthocyaninless progeny proceed equally quickly, the anthocyanin marker provides unbiased estimates of outcrossing rates. The results overall support the use of anthocyanin as a neutral marker, with the alternate phenotypes unlikely to be differentially impacted by the processes of pollination, pollen-tube growth, and germination. Fluctuating selection on conditions for dormancy release may be partially responsible for the maintenance of the anthocyanin polymorphism in Datura stramonium.     

The effect of varying the number of pollen grains used in fertilization

Summary The number of pollen grains placed upon a stigma influence both the development of pollen tubes and subsequently the progeny which result from fertilizations by gametes from these pollen tubes. The first influence is demonstrated by reduced pollen tube growth rates when pollen grains are few in number. This may indicate direct effects of pollen tubes upon the stylar tissues or perhaps more complex interactions between pollen and style. The second and potentially more important influence of limited pollination is upon the progeny. This was demonstrated with studies on three species. In each case, variation among the resultant plants was greater when pollen was limited than when normal, that is excessive, pollen was used. The mechanism of this phenomenon is not certain, but our data indicate that it is not simply an artefact of variation in seed size.     

Metabolic profiling of Chinese hamster ovary cell cultures at different working volumes and agitation speeds using spin tube reactors

Culture systems based on spin tube reactors have been consolidated in the development of manufacturing processes based on Chinese hamster ovary (CHO) cells. Despite their widespread use, there is little information about the consequences of varying operational setting parameters on the culture performance of recombinant CHO cell lines. Here, we investigated the effect of varying working volumes and agitation speeds on cell growth, protein production, and cell metabolism of two clonally derived CHO cell lines (expressing an IgG1 and a “difficult-to-express” fusion protein). Interestingly, low culture volumes increased recombinant protein production and decreased cell growth, while high culture volumes had the opposite effect. Altering agitation speeds exacerbated or moderated the differences observed due to culture volume changes. Combining low agitation rates with high culture volumes suppressed growth and recombinant protein production in CHO cells. Meanwhile, high agitation rates narrowed the differences in culture performance between low and high working volumes. These differences were also reflected in cell metabolism, where low culture volumes enhanced oxidative metabolism (linked to a productive phenotype) and high culture volume generated a metabolic profile that was predominately glycolytic (linked to a proliferative phenotype). Our findings indicate that the culture volume influence on metabolism modulates the balance between cell growth and protein production, a key feature that may be useful to adjust CHO cells toward a more productive phenotype.     

Cytoskeleton in plant development

The plant cytoskeleton has crucial functions in a number of cellular processes that are essential for cell morphogenesis, organogenesis and development. These functions have been intensively investigated using single cell model systems. With the recent characterization of plant mutants that show aberrant organogenesis resulting from primary defects in cytoskeletal organization, an integrated understanding of the importance of the cytoskeleton for plant development has begun to emerge. Newly established techniques that allow the non-destructive visualization of microtubules or actin filaments in living plant cells and organs will further advance this understanding.     

Single cell and tissue specific methods for evaluation of radiation and microgravity effects

A discussion of different methods to evaluate dose/response and biological effects of ionizing radiation is given. Confocal scanning laser microscopy (CSLM) is presented as a high performing observation method for evaluating different cytological effects. Standard cytochemical techniques can be used to analyse the cell in situ with minimal disturbance of morphology and structure. If a relatively small number of cells are affected by the treatment, the use of confocal microscope observations is fast and has a better resolution than conventional fluorescence microscopy. The optical sectioning capability of the CSLM makes it possible to analyse stacks of cells on detectors up to a depth of 200 micrometer with a resolution of 0.7 micrometer. This is used to analyse single cell electrophoresis results and nuclear track analysis in poly allyl diglycol carbonate (PADC). Consecutive analysis of cells cultivated on PADC, and analysis of nuclear tracks after chemical etched tracks in the PADC, will make it possible to correlate physical dose with direct cellular effects. This is a promising method for single cell analysis and the study of the effects of ionizing radiation at low particle flux density.