Final and Fatal Step of Tracheary Element Differentiation

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ABSTRACT

The process of terminal differentiation which produces the hollow tracheary element cell corpse requires strict coordination of two developmental events, construction of a rigid, persistent secondary cell wall and programmed cell death. We show that tracheary element programmed cell death involves an influx of Ca2+ into the cell which may play a causative role in executing cell death. Ca2+ influx is triggered by an extracellular signal, and leads to the rapid cessation of cytoplasmic streaming and collapse of the large hydrolytic vacuole. This specific means of effecting cell death is a necessary prerequisite for postmortem developmental events including autolysis and chromatin degradation. A protease (“trigger protease) is secreted during secondary cell wall synthesis which may be the primary trigger of cell death, because specific proteolysis of the extracellular matrix is necessary and sufficient to trigger Ca2+ influx, vacuole collapse, cell death, and chromatin degradation. We propose a model in which secondary cell wall synthesis and cell death are coordinated by the concomitant secretion of the trigger protease with secondary cell wall precursors. Subsequent cell death is triggered upon realization of a critical extracellular activity of protease corresponding with completion of a functional secondary cell wall. Because increased Ca2+ levels are associated with cell death involving what has been termed the mitochondrial pathway in animals, we investigated the possible role of released mitochondrial factors in the death mechanism in tracheary elements. We find that some cytochrome c is released to the cytosol at a time when death occurs and induced by calcium influx. Concomitant with this release are changes in the inner membrane voltage potential and the morphology of the mitochondria. However, cytochrome c release is insufficient to induce death in these cells. This suggests that the events triggered by the extracellular “trigger” protease may set in motion events shared by the mitochondrial pathway for apoptosis in animal cells.

Section snippets

INTRODUCTION

Most terminally differentiated cells fulfill specialized functions until they die, but for some cell types, function does not begin until after death. The developmental programs producing such functional cell corpses involve the coordination of cell differentiation with PCD. The classic example of terminal differentiation in plants is the tracheary element (TE), a functional cell corpse that forms a single unit of the water-conducting vessels of the xylem. We previously used a cell-culture

Plants, cell culture, and chemicals

Seedlings of zinnia (Zinnia elegans L. cv Green Envy; Stokes Seed, Buffalo, NY) were grown in a growth chamber at 25 °C and 60% RH with 14 h of light (110 μmol photons m2 s2) per day. Cells were isolated by the method described by Fukuda and Komamine19 using modifications described by Groover and Jones9.

Protein extraction

Intracellular proteins were isolated by homogenizing cells in extraction buffer (50 mM Tris-HCl, pH 7.5, 2 mM DTT, 250 mM sucrose) at 4 °C, followed by centrifugation at 12,000 X g at 4 °C for 15 min to

Cell Death Is Marked by the Rapid Collapse of the Vacuole and Leads to Autolysis and nDNA Fragmentation

The first morphological manifestation of differentiation occurs approximately 72 h after cell isolation, when nascent TEs synthesize an elaborate secondary cell wall between their primary cell wall and the plasma membrane. Approximately 6 h after the appearance of visible cell wall thickenings, the large central vacuole collapses rapidly and cytoplasmic streaming ceases simultaneously1, marking the irreversible termination of normal metabolism and providing a distinct morphological marker of a

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