Flow cytometric analysis of tracheary element differentiation in Zinnia elegans cells.
BACKGROUND
Tracheary element (TE) differentiation in single cells in culture isolated from Zinnia elegans leaves involves programmed cell death (PCD) co-ordinated with key morphological developments. We have used flow cytometry to analyze physiological and nuclear changes in the differentiating cells. Flow cytometry allows the identification of subpopulations, thereby removing the obscuring effect of population heterogeneity that occurs with the use of other techniques.


METHODS
Cell viability, plasma membrane integrity, oxidative activity, intracellular calcium and pH, cell wall thickening, the possible role of microtubule rearrangement, chromatin condensation, and DNA breakdown were followed by flow cytometry from the first stages of TE induction.


RESULTS
TE differentiation could be enhanced and made more synchronous by a centrifugation step at 72 h after cell isolation. Size and shape changes were the first changes identified in differentiating cells, and these properties could be used to isolate differentiating populations by back-gating. Chromatin condensation and nDNA breakdown followed patterns characteristic of programmed cell death.


CONCLUSIONS
We have used flow cytometry to characterize the morphological and physiological changes that occur during TE differentiation, and our findings indicate that this process is a form of autophagic PCD in which microtubule rearrangement appears to play a role.
Weir
Iona E
IE
Horticulture and Food Research Institute of New Zealand, Auckland, New Zealand. iweir@biodiscovery.co.nz
Maddumage
Ratnasiri
R
Allan
Andrew C
AC
Ferguson
Ian B
IB
D019659:Asteraceae / Q000166:cytology* / Q000502:physiology; D001343:Autophagy; D002118:Calcium / Q000032:analysis; D002454:Cell Differentiation; D002462:Cell Membrane / Q000502:physiology; D048430:Cell Shape; D048429:Cell Size; D002470:Cell Survival; D002478:Cells, Cultured; D002843:Chromatin / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008870:Microtubules / Q000502:physiology; D018384:Oxidative Stress; D018515:Plant Leaves / Q000737:chemistry / Q000166:cytology* / Q000502:physiology
D002843:Chromatin; D005456:Fluorescent Dyes; D002118:Calcium