The hybrid lethality of interspecific F1 hybrids of Nicotiana: a clue to understanding hybrid inviability-a major obstacle to wide hybridization and introgression breeding of plants.

Reproductive isolation poses a major obstacle to wide hybridization and introgression breeding of plants. Hybrid inviability in the postzygotic isolation barrier inevitably reduces hybrid fitness, consequently causing hindrances in the establishment of novel genotypes from the hybrids among genetically divergent parents. The idea that the plant immune system is involved in the hybrid problem is applicable to the intra- and/or interspecific hybrids of many different taxa. The lethality characteristics and expression profile of genes associated with the hypersensitive response of the hybrids, along with the suppression of causative genes, support the deleterious epistatic interaction of parental NB-LRR protein genes, resulting in aberrant hyper-immunity reactions in the hybrid. Moreover, the cellular, physiological, and biochemical reactions observed in hybrid cells also corroborate this hypothesis. However, the difference in genetic backgrounds of the respective hybrids may contribute to variations in lethality phenotypes among the parental species combinations. The mixed state in parental components of the chaperone complex (HSP90-SGT1-RAR1) in the hybrid may also affect the hybrid inviability. This review article discusses the facts and hypothesis regarding hybrid inviability, alongside the findings of studies on the hybrid lethality of interspecific hybrids of the genus Nicotiana. A possible solution for averting the hybrid problem has also been scrutinized with the aim of improving the wide hybridization and introgression breeding program in plants.

The role of chaperone complex HSP90-SGT1-RAR1 as the associated machinery for hybrid inviability between Nicotiana gossei Domin and N. tabacum L.

Two cultured cell lines (GTH4 and GTH4S) of a Nicotiana interspecific F1 hybrid (N. gossei × N. tabacum) were comparatively analyzed to find genetic factors related to hybrid inviability. Both cell lines proliferated at 37 °C, but after shifting to 26 °C, GTH4 started to die similar to the F1 hybrid seedlings, whereas GTH4S survived. As cell death requires de novo expression of genes and proteins, we compared expressed protein profiles between the two cell lines, and found that NgSGT1, a cochaperone of the chaperone complex (HSP90-SGT1-RAR1), was expressed in GTH4 but not in GTH4S. Agrobacterium-mediated transient expression of NgSGT1, but not NtSGT1, induced cell death in leaves of N. tabacum, suggesting its possible role in hybrid inviability. Cell death in N. tabacum was also induced by transient expression of NgRAR1, but not NtRAR1. In contrast, transient expression of any parental combinations of three components revealed that NgRAR1 promoted cell death, whereas NtRAR1 suppressed it in N. tabacum. A specific inhibitor of HSP90, geldanamycin, inhibited the progression of hypersensitive response-like cell death in GTH4 and leaf tissue after agroinfiltration. The present study suggested that components of the chaperone complex are involved in the inviability of Nicotiana interspecific hybrid.

Cell physiology of mortality and immortality in a Nicotiana interspecific F1 hybrid complies with the quantitative balance between reactive oxygen and nitric oxide.

The cultured cell line, GTH4, of an interspecific F1 hybrid between Nicotiana gossei Domin and N. tabacum L. died after a shift in temperature from 37°C to 26°C. Fluctuations in the cellular amounts of reactive oxygen species (ROS) and nitric oxide (NO) were detected in GTH4 after the temperature shift, but not in the mutant, GTH4S, which did not die at 26°C presumably due to the lack of genetic factors involved in cell death. The removal of ROS or NO suppressed cell death in GTH4, suggesting that ROS and NO both acted as mediators of cell death. However, excess amounts of the superoxide anion (O2-) or NO alleviated cell death. A series of experiments using generators and scavengers of ROS and NO showed that O2- affected the cellular levels of NO, and vice versa, indicating that a quantitative balance between O2- and NO was important for hybrid cell death. The combination of NO and hydrogen peroxide (H2O2) was necessary and sufficient to initiate cell death in GTH4 and GTH4S. Hypoxia, which suppressed cell death in GTH4 at 26°C, reduced the generation of H2O2 and NO, but allowed for the production of O2-, which acted as a suppressor and/or modulator of cell death. The activation of MAPK was involved in the generation of H2O2 in GTG4 cells under normoxic conditions, but promoted O2- generation under hypoxic conditions. More protective cellular conditions against ROS, as estimated by the expression levels of genes for ROS-scavenging enzymes, may be involved in the mechanisms responsible for the low cell death rate of GTH4 under hypoxic conditions.

Cell death in seedlings of the interspecific hybrid of Nicotiana gossei and N. tabacum; possible role of knob-like bodies formed on tonoplast in vacuolar-collapse-mediated cell death.

Vacuolar collapse plays a direct role in the cell death of the interspecific hybrid of Nicotiana gossei Domin xN. tabacum L. which exhibits hybrid lethality at the seedling stage. We have previously reported that cell death in these seedlings began at the base of hypocotyls and spread throughout the plant (Mino et al. 2002). A light microscopic analysis revealed that the process involved disruption of the intra-cellular membranes, plasmolysis, and retraction of the wall of the cell in hypocotyls. A transmission electron microscopic analysis showed that there were several abnormal structures, i.e. knob-like bodies on the tonoplast and small vesicles in the cytoplasm, and the disintegration of the tonoplast, in the cells of seedlings grown at 26 degrees C. However, no such cytological defects were observed in the seedlings grown at 37 degrees C, at which temperature the expression of lethality was suppressed. The activity levels of vacuolar processing enzyme (VPE), which might be involved in the vacuolar collapse of plant cells, temporarily increased in the seedlings grown at 26 degrees C before apparent cell death proceeded, but it remained unchanged in the seedlings grown at 37 degrees C. Applications of acetyl-L: -tyrosyl-L: -valyl-L: -alanyl-L: -aspart-1-aldehyde, an inhibitor for VPE, and cycloheximide to the seedlings suppressed VPE's activities, the formation of knob-like bodies on the tonoplast, and cell death. VPE might be involved in the structural anomalies on the tonoplast which lead to cell death triggered by vacuolar collapse in hybrid seedlings.

Hybrid lethality of cultured cells of an interspecific F1 hybrid of Nicotiana gossei Domin and N. tabacum L.

Cultured cells were established from the hypocotyl of F(1) hybrid seedlings of Nicotiana gossei Domin and N. tabacum L. The cultured cells started to die at 26 degrees C, but not at 37 degrees C, which is similar to what occurred in cells of the original hybrid plants. An increase in the number of cells without cytoplasmic strands and acidification of the cytoplasm followed by decomposition of the mitochondria and chloroplasts indicated that vacuolar collapse plays a central role in the execution of cell death. Oxygen but not light was required for cell death. Cellular levels of the superoxide anion and hydrogen peroxide temporarily increased during the early phase at 26 degrees C, while no such oxidative burst was observed at 37 degrees C. The reactive oxygen intermediates are potentially involved in the death of the hybrid cells.

Reduced glutathione is a novel regulator of vernalization-induced bolting in the rosette plant Eustoma grandiflorum.

The transition from the vegetative rosette stage to the reproductive growth stage (bolting) in the rosette plant Eustoma grandiflorum has a strict requirement for vernalization, a treatment that causes oxidative stress. Since we have shown that reduced glutathione (GSH) and its biosynthesis are associated with bolting in another rosette plant Arabidopsis thaliana, we here investigated whether a similar mechanism governs the vernalization-induced bolting of E. grandiflorum. Addition of GSH or its precursor cysteine, instead of vernalization, induced bolting but other thiols, dithiothreitol and 2-mercaptoethanol, did not. The inductive effect of vernalization on bolting was nullified by addition of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, without decreasing the plant growth rate. BSO-mediated inhibition of bolting was reversed by addition of GSH but not by cysteine. These indicate that vernalization-induced bolting involves GSH biosynthesis and is specifically regulated by GSH. Plant GSH increased during the early vernalization period along with the activity of gamma-glutamylcysteine synthetase that catalyzes the first step of GSH biosynthesis, although there was little change in amounts of GSH precursor thiols, cysteine and gamma-glutamylcysteine. These findings strongly suggest that vernalization stimulates GSH synthesis and synthesized GSH specifically determines the bolting time of E. grandiflorum.

Cell death processes during expression of hybrid lethality in interspecific F1 hybrid between Nicotiana gossei Domin and Nicotiana tabacum.

Hybrid lethality, a type of reproductive isolation, is a genetically controlled event appearing at the seedling stage in interspecific hybrids. We characterized the lethality of F(1) hybrid seedlings from Nicotiana gossei Domin and Nicotiana tabacum cv Bright-Yellow 4 using a number of traits including growth rate, microscopic features of tissues and cells, ion leakage, DNA degradation, reactive oxygen intermediates including superoxide radical (O(2)(-)) and hydrogen peroxide (H(2)O(2)), and expression of stress response marker genes. Lethal symptoms appeared at 4 d after germination in the basal hypocotyl and extended toward both the hypocotyl and root of the plants grown at 26 degrees C. Microscopic analysis revealed a prompt lysis of cell components during cell death. Membrane disruption and DNA degradation were found in the advanced stage of the lethality. The death of mesophyll cells in the cotyledon was initiated by the vascular bundle, suggesting that a putative factor inducing cell death diffused into surrounding cells from the vascular tissue. In contrast, these symptoms were not observed in the plants grown at 37 degrees C. Seedlings grown at 26 degrees C generated larger amounts of reactive oxygen intermediate in the hypocotyl than those grown at 37 degrees C. A number of stress response marker genes were expressed at 26 degrees C but not at 37 degrees C. We proposed that a putative death factor moving systemically through the vascular system induced a prompt and successive lysis of the cytoplasm of cells and that massive cell death eventually led to the loss of the hybrid plant.