Summary Maturation of the xylem elements involves extensive deposition of secondary cell‐wall material and autolytic processes resulting in cell death. We describe here a unique type of cell‐death program in xylem fibers of hybrid aspen (Populus tremula x P. tremuloides) stems, including gradual degradative processes in both the nucleus and cytoplasm concurrently with the phase of active cell‐wall deposition. Nuclear DNA integrity, as determined by TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling) and Comet (single‐cell gel electrophoresis) assays, was compromised early during fiber maturation. In addition, degradation of the cytoplasmic contents, as detected by electron microscopy of samples fixed by high‐pressure freezing/freeze substitution (HPF‐FS), was gradual and resulted in complete loss of the cytoplasmic contents well before the loss of vacuolar integrity, which is considered to be the moment of death. This type of cell death differs significantly from that seen in xylem vessels. The loss of vacuolar integrity, which is thought to initiate cell degradative processes in the xylem vessels, is one of the last processes to occur before the final autolysis of the remaining cell contents in xylem fibers. High‐resolution microarray analysis in the vascular tissues of Populus stem, combined with in silico analysis of publicly available data repositories, suggests the involvement of several previously uncharacterized transcription factors, ethylene, sphingolipids and light signaling as well as autophagy in the control of fiber cell death.

Evolutionary, as well as genetic, evidence suggests that vascular development evolved originally as a cell death programme that allowed enhanced movement of water in the extinct protracheophytes, and that secondary wall formation in the water-conducting cells evolved afterwards, providing mechanical support for effective long-distance transport of water. The extant vascular plants possess a common regulatory network to coordinate the different phases of xylem maturation, including secondary wall formation, cell death, and finally autolysis of the cell contents, by the action of recently identified NAC domain transcription factors. Consequently, xylem cell death is an inseparable part of the xylem maturation programme, making it difficult to uncouple cell death mechanistically from secondary wall formation, and thus identify the key factors specifically involved in regulation of cell death. Current knowledge suggests that the necessary components for xylem cell death are produced early during xylem differentiation, and cell death is prevented through the action of inhibitors and storage of hydrolytic enzymes in inactive forms in compartments such as the vacuole. Bursting of the central vacuole triggers autolytic hydrolysis of the cell contents, which ultimately leads to cell death. This cascade of events varies between the different xylem cell types. The water-transporting tracheary elements rely on a rapid cell death programme, with hydrolysis of cell contents taking place for the most part, if not entirely, after vacuolar bursting, while the xylem fibres disintegrate cellular contents at a slower pace, well before cell death. This review includes a detailed description of cell morphology, function of plant growth regulators, such as ethylene and thermospermine, and the action of hydrolytic nucleases and proteases during cell death of the different xylem cell types.

Summary Cell death of xylem elements is manifested by rupture of the tonoplast and subsequent autolysis of the cellular contents. Metacaspases have been implicated in various forms of plant cell death but regulation and execution of xylem cell death by metacaspases remains unknown. Analysis of the type II metacaspase gene family in Arabidopsis thaliana supported the function of METACASPASE 9 (AtMC9) in xylem cell death. Progression of xylem cell death was analysed in protoxylem vessel elements of 3‐d‐old atmc9 mutant roots using reporter gene analysis and electron microscopy. Protoxylem cell death was normally initiated in atmc9 mutant lines, but detailed electron microscopic analyses revealed a role for AtMC9 in clearance of the cell contents post mortem, that is after tonoplast rupture. Subcellular localization of fluorescent AtMC9 reporter fusions supported a post mortem role for AtMC9. Further, probe‐based activity profiling suggested a function of AtMC9 on activities of papain‐like cysteine proteases. Our data demonstrate that the function of AtMC9 in xylem cell death is to degrade vessel cell contents after vacuolar rupture. We further provide evidence on a proteolytic cascade in post mortem autolysis of xylem vessel elements and suggest that AtMC9 is part of this cascade.