Technical advance: near-infrared femtosecond laser pulses as a novel non-invasive means for dye-permeation and 3D imaging of localised dye-coupling in the Arabidopsis root meristem

We have used near-infrared femtosecond Titanium: Sapphire laser pulses as novel non-invasive means for dye loading into various cell types of the Arabidopsis root meristem, and by 3D imaging have assessed the extent of dye coupling between the meristematic cells. The post-embryonic primary root of Arabidopsis thaliana has an invariant ontogeny and fixed cellular organisation which makes it an attractive model system to study developmental events involving cell fate determination, cellular differentiation and pattern formation. Local intercellular communication and local transmission of positional signals are likely to play a pivotal role in cell proliferation and regulation of differentiation. We have therefore examined the extent to which the constituent cells in the root meristem are symplastically coupled. Following laser-assisted loading of membrane impermeate fluorescent dye propidium iodide (PI) in single cells, we show by time-lapse and 3D imaging that in the root tip all undifferentiated cells are dye-coupled. When PI is permeated into the central cells, it rapidly moved into the adjacent initials of the columella, cortex, pericycle and stele. Interestingly, when only either of the initials were loaded with the dye, it never moved into any of the central cells. Amongst the epidermal cells, the differentiated hair cells are symplastically isolated. Our data provide evidence (1) for differential dye-coupling behaviour between quiescent centre cells and the neighbouring initials; (2) that cells in the root are coupled during stages at which the cell-lineage pattern is formed and that it becomes progressively secluded as they differentiate and the pattern is fixed. Taken together, our NIR-laser mediated approach is highly efficient and has numerous potential applications for non-invasive permeation of dyes in different cell types.