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Key Message: PCD with features of vacuolar cell death including autophagy-related features were detected in hybrid tobacco cells, and detailed time course of features of vacuolar cell death were established. A type of interspecific Nicotiana hybrid, Nicotiana suaveolens × N. tabacum exhibits temperature-sensitive lethality. This lethality results from programmed cell death (PCD) in hybrid seedlings, but this PCD occurs only in seedlings and suspension-cultured cells grown at 28 °C, not those grown at 36 °C. Plant PCD can be classified as vacuolar cell death or necrotic cell death. Induction of autophagy, vacuolar membrane collapse and actin disorganization are each known features of vacuolar cell death, but observed cases of PCD showing all these features simultaneously are rare. In this study, these features of vacuolar cell death were evident in hybrid tobacco cells expressing hybrid lethality. Ion leakage, plasma membrane disruption, increased activity of vacuolar processing enzyme, vacuolar membrane collapse, and formation of punctate F-actin foci were each evident in these cells. Transmission electron microscopy revealed that macroautophagic structures formed and tonoplasts ruptured in these cells. The number of cells that contained monodansylcadaverine (MDC)-stained structures and the abundance of nine autophagy-related gene transcripts increased just before cell death at 28 °C; these features were not evident at 36 °C. We assessed whether an autophagic inhibitor, wortmannin (WM), influenced lethality in hybrid cells. After the hybrid cell began to die, WM suppressed increases in ion leakage and cell deaths, and it decreased the number of cells containing MDC-stained structures. These results showed that several features indicative of autophagy and vacuolar cell death were evident in the hybrid tobacco cells subject to lethality. In addition, we documented a detailed time course of these vacuolar cell death features.

Hybrid lethality is an important problem for cross-breeding; however, its molecular mechanism is not clear. The purpose of the present study was to identify the genes expressed during hybrid lethality in the hybrid cells (Nicotiana suaveolens x N. tabacum). In order to identify these genes, we employed suppression subtractive hybridization (SSH) between RNA isolated from cells expressing lethality (lethal hybrid line; LH line) and cells overcoming lethality fortuitously (a surviving hybrid line; SH line). Four populations of cDNA were created from the time points corresponding to before and during induction, and at and after the point of no return (PNR) during the process of programmed cell death (PCD) that occurs during hybrid lethality. By SSH and following dot-blot macroarray analysis, 99 genes out of 138 isolated clones were identified as hybrid lethality-related (HLR) genes. Quantitative real-time PCR analysis data indicated that ten clones were expressed specifically in LH line cells. The HLR genes in these clones show homology to genes involved in disease resistance, ethylene-induced reactions, phosphorylation, ubiquitination, jasmonic acid-related reactions, calcium signaling and self-incompatibility. These data suggested that at least some parts of the mechanism of hybrid lethality are shared with those of the putative functions of the HLR gene-related pathways.