Seed-coat dormancy in Grevillea linearifolia: little change in permeability to an apoplastic tracer after treatment with smoke and heat.

BACKGROUND AND AIMS: Seeds of Grevillea linearifolia germinate following fire, and have seed-coat dormancy broken by smoke and heat shock. Smoke breaks seed coat dormancy in Emmenanthe penduliflora by altering the permeability of the seed coat to an internal germination inhibitor, which subsequently escapes. This model was tested for in G. linearifolia by investigating the permeability of the seed coat to diffusion of high-molecular-weight compounds, and whether this changed after exposure to fire cues. METHODS: Germination response of the seeds to heat shock, smoke or heat + smoke was tested. Penetration of Lucifer Yellow dye into intact seeds was examined after 24 and 48 h of exposure, and penetration of the dye from the inside of the seed coat outwards was examined after 24 h. Histochemical staining with Nile Red and Acridine Orange was used to locate cuticles, suberin and lignin. KEY RESULTS: Twenty-three per cent of untreated seeds germinated; heat shock and smoke increased germination additively up to approx. 80 % for both cues combined. Lucifer Yellow did not penetrate fully through the seed coat of untreated seeds, whether diffusing inwards or outwards. Three barriers to diffusion were identified. Treatment with heat or smoke slightly increased penetration of the dye, but did not completely remove the barriers. Suberin was identified in secondary walls of exotestal and mesotestal cells, and was absent from primary cell walls. Movement of Lucifer Yellow occurred through the middle lamella and primary cell wall of suberized cells; movement of the dye was impeded where suberin was absent. CONCLUSIONS: Fire cues did not significantly decrease barriers to diffusion of high-molecular-weight compounds in the seed coat of Grevillea, and must be breaking dormancy by another mechanism.

SYBR Green I-induced fluorescence in cultured immune cells: a comparison with Acridine Orange.

Using fluorescence microscopy, we explored the ability of cultured immune cells to take up aqueous SYBR Green I (SGI). SYBR Green I, a highly sensitive fluorescent nucleic acid stain, which preferentially binds to dsDNA over ssDNA or RNA with little background fluorescence from unbound molecules. A time course study over 2h using final dilutions of SGI of 1:10,000 and 1:100,000 at 22 and 37 degrees C, revealed the dye quickly entered the cells, stained mitochondrial DNA then nuclear DNA, and SGI-induced green fluorescence increased over time. As staining progressed, heterochromatin appeared as more intense green fluorescent lines, patches and circles against the lower fluorescence of the nucleoplasm. The lower fluorescence from the nucleoplasm indicated SGI also bound to areas of euchromatin. Similar progressive uptake experiments were carried out with the permeant DNA dye Acridine Orange (AO) to provide insight into staining patterns and mode of uptake. Statistical analysis of cells prestained with SGI then tested with Trypan Blue for changes in membrane permeability, revealed no significant difference between controls and treatment for each temperature. It appears that SGI does not compromise cells for up to 2 h following initial exposure.

Live Cell Characterization of DNA Aggregation Delivered through Lipofection.

DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation.