Postmortem lignification of xylem tracheary elements (TEs) has been debated for decades. Here, we provide evidence in Zinnia elegans TE cell cultures, using pharmacological inhibitors and in intact Z. elegans plants using Fourier transform infrared microspectroscopy, that TE lignification occurs postmortem (i.e., after TE programmed cell death). In situ RT-PCR verified expression of the lignin monomer biosynthetic cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase in not only the lignifying TEs but also in the unlignified non-TE cells of Z. elegans TE cell cultures and in living, parenchymatic xylem cells that surround TEs in stems. These cells were also shown to have the capacity to synthesize and transport lignin monomers and reactive oxygen species to the cell walls of dead TEs. Differential gene expression analysis in Z. elegans TE cell cultures and concomitant functional analysis in Arabidopsis thaliana resulted in identification of several genes that were expressed in the non-TE cells and that affected lignin chemistry on the basis of pyrolysis-gas chromatography/mass spectrometry analysis. These data suggest that living, parenchymatic xylem cells contribute to TE lignification in a non-cellautonomous manner, thus enabling the postmortem lignification of TEs.

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.