Overexpression of the cell plate-associated dynamin-like GTPase, phragmoplastin, results in the accumulation of callose at the cell plate and arrest of plant growth

Abstract

Phragmoplastin, a dynamin-like GTPase, is associated with cell plate formation in plants. We expressed a phragmoplastin-GFP construct (Phr G) under the control of a 35S promoter in transgenic tobacco plants. High levels of expression of this chimeric protein in transgenic plants were confirmed by Western blot analysis. T1 seedlings formed morphologically normal cotyledons, but most were unable to develop beyond the first pair of true leaves. Only 5–10% of the seedlings could develop into small plants whose progenies continued to exhibit severe growth defects. The primary root growth was arrested after 7 days and no lateral roots were formed. The mature primary root meristem consisted of disorganized isodiametrically enlarged cells and no recognizable quiescent center or collumela. Adventitious root primordia were initiated in the hypocotyl, but completely arrested at an early stage and contained differentiated treachery elements. The orientation of cell plates was abnormal, suggesting a possible defect in its positioning. However, unlike other cytokinesis mutants, no binucleate or polyploid cells were found. Fluorescence image cytometry studies indicated that Phr G root meristem cells had 2C levels of DNA and appear to be arrested in G1 stage of the cell cycle. Heavy callose deposition was observed at the nascent cell plate and callose accumulation persisted in new cell walls. Electron microscopy revealed unusual electron dense substances in the maturing cell plate and accumulation of multivesicular bodies around the cell plate. Since phragmoplastin interacts with callose synthase complex, its overexpression may affect accumulation of callose arresting plant growth due to perturbation in cell division progression.