Hybrid lethality in interspecific F1 hybrid Nicotiana gossei x N. tabacum involves a MAP-kinases signalling cascade

A cultured cell line, GTH4 (Nicotiana gossei Domin x N. tabacum L.), which exhibits hybrid lethality, died at 26 degrees C, but not at 37 degrees C. Pharmacological experiments using inhibitors of protein phosphatases and protein kinases indicated the involvement of a protein kinase signalling pathway in the cell death process. Immunoblot analysis revealed that salicylic acid-induced protein kinase (SIPK) was phosphorylated soon after the shift in temperature from 37 degrees C to 26 degrees C. Cultured cells of the hybrid of N. gossei x transgenic N. tabacum harboring a steroid (dexamethasone; DEX)-inducible NtMEK2 (DD) or NtMEK2 (KR), constitutively active and inactive forms of NtMEK2, respectively, were established. Induction of NtMEK2 (DD) by DEX in the hybrid cells induced the activation of SIPK, the generation of hydrogen peroxide (H (2)O (2)), and cell death at 37 degrees C. The activation of SIPK, generation of H (2)O (2), and cell death at 26 degrees C were compromised by DEX treatment in hybrid cells harbouring NtMEK2 (KR). This study provides evidence for the involvement of MAPK signalling in the regulation of cell death in hybrids.     

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 F₁ hybrid seedlings of Nicotiana gossei Domin and N. tabacum L. The cultured cells started to die at 26°C, but not at 37°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°C, while no such oxidative burst was observed at 37°C. The reactive oxygen intermediates are potentially involved in the death of the hybrid cells.     

Facile induction of apoptosis into plant cells associated with temperature-sensitive lethality shown on interspecific hybrid from the cross Nicotiana suaveolens x N. tabacum

Two lines of suspension culture cells were obtained from a hybrid seedling of Nicotiana suaveolens Lehm. x N. tabacum L. cv. Hicks-2 expressing temperature-sensitive lethality. One of them (LH line) was inducible cell death in accordance with the lethality at 28 degrees C but not under high-temperature conditions (36 degrees C). Another one (SH line) lost the lethality and survived at 28 degrees C. The cells of LH line showed apoptotic changes when they were cultured at 28 degrees C. Fragmentation of nuclei was correlated with the lethality in the cells, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from the cells expressing the lethality revealed a specific ladder pattern suggesting nucleosomal fragmentation that is one of the biochemical characteristics of apoptosis. From these facts, we confirmed that the process of cell death leading to hybrid lethality in the cells is certainly apoptosis. Hybrid cells were used in the experiments to estimate the point of no return in temperature-sensitive lethality and to examine the influence of cation in DNA fragmentation during apoptosis. The utility of hybrid cells as an experimental system for studies of hybrid lethality and apoptosis in plants was confirmed.     

Cell death in seedlings of the interspecific hybrid of <Emphasis Type="Italic">Nicotiana gossei</Emphasis> and <Emphasis Type="Italic">N. tabacum</Emphasis>; 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 ×N. 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°C. However, no such cytological defects were observed in the seedlings grown at 37°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°C before apparent cell death proceeded, but it remained unchanged in the seedlings grown at 37°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.     

Possible involvement of auxin-induced ethylene in an apoptotic cell death during temperature-sensitive lethality expressed by hybrid between Nicotiana glutinosa and N. repanda

Interspecific hybrids of Nicotiana glutinosa L. x N. repanda Willd. express temperature-sensitive lethality induced by apoptotic cell death. Hybrid seedlings cultured at 28 degrees C began to exhibit lethal symptoms during early growth stages, and then they showed a high level of endogenous auxin compared with those of parental seedlings. Meanwhile, the level of auxin in hybrid seedlings cultured at 32 degrees C, which is a condition suppressing the lethality of this cross combination, was equal to or lower than those of parental seedlings. Administration of 2,3,5-triiodobenzoic acid (TIBA) as an auxin transport inhibitor into the hybrid seedlings suppressed lethal symptoms and had a life-extending effect. Additionally, TIBA has an effect to suppress DNA fragmentation, which is one of characteristics of apoptosis and has been detected in the hybrid seedlings expressing the lethality. Administration of aminooxyacetic acid (AOA) as an ethylene synthesis inhibitor, which could inhibit ethylene production, also showed the same effects as TIBA for the lethality. From these results, we suggested that auxin and ethylene were involved in an apoptotic cell death during the lethality, and the abnormal increase of endogenous auxin may lead to the ethylene production in hybrid seedlings during early growth stages.     

Seven of eight species in Nicotiana section Suaveolentes have common factors leading to hybrid lethality in crosses with Nicotiana tabacum

Background and Aims

Reproductive isolation is a mechanism that separates species, and is classified into two types: prezygotic and postzygotic. Inviability of hybrids, or hybrid lethality, is a type of postzygotic isolation and is observed in some plant species, including Nicotiana species. Previous work has shown that the Q chromosome, which belongs to the S subgenome of N. tabacum, encodes one or more genes leading to hybrid lethality in some crosses.

Methods

Interspecific crosses of eight wild species were conducted in section Suaveolentes (which consists of species restricted to Australasia and Africa) with the cultivated species Nicotiana tabacum. Hybrid seedlings were cultivated at 28, 34 or 36 °C, and PCR and chromosome analysis were performed.

Results and Conclusions

Seven of eight wild species produced inviable hybrids after crossing. Hybrid lethality, which was observed in all crosses at 28 °C, was Type II lethality, with the characteristic symptoms of browning of hypocotyl and roots; lethality was suppressed at elevated temperatures (34 or 36 °C). Furthermore, one or more genes on the Q chromosome of N. tabacum were absolutely responsible for hybrid lethality, suggesting that many species of section Suaveolentes share the same factor that triggers hybrid lethality by interaction with the genes on the Q chromosome. Exceptionally, only one wild species, N. fragrans, produced 100 % viable hybrids after crossing with N. tabacum, suggesting that N. fragrans has no factor triggering hybrid lethality.     

Overproduced ethylene causes programmed cell death leading to temperature-sensitive lethality in hybrid seedlings from the cross<Emphasis Type="Italic"> Nicotiana suaveolens</Emphasis> ×<Emphasis Type="Italic"> N. tabacum</Emphasis>

Reproductive isolation mechanisms (RIMs) often become obstacles in crossbreeding. Hybrid lethality is a subtype of RIM but its physiological mechanism remains poorly elucidated. Interspecific hybrids of Nicotiana suaveolens Lehm. x N. tabacum L. cv. Hicks-2 expressed temperature-sensitive lethality. This lethality was induced by programmed cell death (PCD) that was accompanied by the characteristic changes of animal apoptosis in hybrid seedlings at 28 degrees C but not at 36 degrees C. When hybrid seedlings were cultured at 28 degrees C, DNA fragmentation started in the cotyledon, and nuclear fragmentation subsequently progressed with lethal symptoms spreading throughout the seedlings. At 28 degrees C, ethylene production in hybrid seedlings was detectable at a high level compared with the level in parental seedlings. In contrast, the ethylene production rate in hybrid seedlings cultured at 36 degrees C was equal to that in parental seedlings. Treatment with ethylene biosynthetic inhibitors, amino-oxyacetic acid and amino-ethoxyvinyl glycine, suppressed lethal symptoms and apoptotic changes, and also prolonged survival of hybrid seedlings. Thus, the increase in the ethylene production rate correlated closely with expression of lethal symptoms and apoptotic changes in hybrid seedlings. From these observations, we conclude that overproduced ethylene acts as an essential factor mediating PCD and subsequent lethality in hybrid seedlings. Furthermore, the present study has provided the first evidence that ethylene is involved in the phenomenon of hybrid lethality.     

Characterization of a temperature-sensitive mutant of Saccharomyces cerevisiae that undergoes uncontrolled protein synthesis.

A mutant of Saccharomyces cerevisiae, DW137, isolated after treatment of a wild-type strain with ICR-170. The mutant was respiration-deficient and showed abnormal cell division when grown at 30 degrees C. In addition, the mutant was temperature-sensitive and underwent lysis when grown at 37 degrees C. Random spore analysis, induced reversion profiles, and complementation analysis indicated that the abnormal phenotypes were under the control of a single recessive mutation caused by a base-pair substitution in a nuclear gene. Macromolecular analysis of the mutant at permissive and restrictive temperatures showed that at restrictive temperatures the mutant cannot synthesize DNA. Surprisingly, at restrictive temperatures, protein synthesis in the mutant continued at a rate greater than that observed at permissive temperatures. Cell death and lysis of the mutant could be prevented by treatment of cultures with cycloheximide, an inhibitor of protein synthesis. The data suggest that the abnormally high rate of protein synthesis and the inability to synthesize DNA are jointly responsible for death of the cells, and most probably play and integrating role in the incipient cell lysis.     

Hybrid lethality in interspecific hybrids between Nicotiana tabacum and N. suaveolens: evidence that the Q chromosome causes hybrid lethality based on Q-chromosome-specific DNA markers

Hybrid seedlings from the cross Nicotiana tabacum × N. suaveolens express lethality at 28°C. We carried out a cross between monosomic lines of N. tabacum lacking the Q chromosome and N. suaveolens by test-tube pollination and ovule culture at 28°C. To suppress hybrid lethality, hybrid seedlings obtained were transferred to 36°C immediately after germination and cultured. We determined whether Q-chromosome-specific DNA markers were detected among hybrid seedlings. When hybrid seedlings cultured at 36°C were transferred to 28°C, hybrid seedlings in which Q-chromosome-specific DNA markers were detected expressed hybrid lethality, while hybrid seedlings in which Q-chromosome-specific DNA markers were not detected did not express hybrid lethality. From these results, we concluded that the presence of the Q chromosome of N. tabacum is related to hybrid lethality observed in crosses between N. tabacum and N. suaveolens. This is the first report that clearly demonstrates the relationship between a certain chromosome and hybrid lethality in the genus Nicotiana using chromosome-specific DNA markers. Additionally, we confirmed that the Q chromosome belongs to the S subgenome because Q-chromosome-specific DNA markers were detected only in N. sylvestris.     

Identification and characterization of genes involved in hybrid lethality in hybrid tobacco cells (<Emphasis Type="Italic">Nicotiana suaveolens</Emphasis> × <Emphasis Type="Italic">N. tabacum</Emphasis>) using suppression subtractive hybridization

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 × 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. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Temperature-dependent expression of flagella of Listeria monocytogenes studied by electron microscopy, SDS-PAGE and western blotting

Washed cells of Listeria monocytogenes serotype 4b, grown in broth culture at 20 degrees C and at 37 degrees C, were examined by electron microscopy for the presence of flagella. Many flagella were seen in cells grown at 20 degrees C, whereas at 37 degrees C very few were expressed. Flagella sheared from the cell surface were partially purified by differential centrifugation. Using SDS-PAGE and Western blotting two distinct protein bands were seen in this preparation, both with an apparent molecular mass of approximately 29 kDa. Further purification of these proteins was achieved by gel filtration and ion-exchange chromatography. Whole organisms grown at 20 degrees C and 37 degrees C were examined in Western blots using an affinity-purified polyclonal antibody, and a monoclonal antibody, both directed against 29 kDa putative flagellin. Bacteria grown at 20 degrees C expressed abundant flagellin, whereas only trace amounts could be detected in organisms grown at 37 degrees C. It is concluded that organisms grown at 20 degrees C both produce and assemble flagellin at the cell surface, and that flagellin production is a less marked feature of organisms grown at 37 degrees C.     

The morphological and physiological properties of the genetic tumours from a Nicotiana interspecific hybrid

Many plant tumours have been loosely referred to as genetic tumours when no obvious external cause was identifiable. In this study, the morphology of genetic tumours was detected by scanning electron microscopy (SEM) and the influence of exogenous phytohormones on genetic tumour growth was examined. Genetic tumours in Nicotiana hybrid were first observed in the hypocotyl area and later appeared from stems as a cluster complex. In vitro, genetic tumours were produced 5-6 days after explant treatment near each of the cut ends. The morphology of genetic tumours was investigated using SEM. Genetic tumours are mainly composed of epidermal cells surrounded by a well developed extracellular matrix (ECM), that is organized in a network linking surface cells. Globular structures containing several cells were formed from these clusters of epidermal cells. Vigorous tumourous growth from the leaf discs of the Nicotiana hybrid was obtained on medium supplemented with auxin and cytokinin at 0 to 10 muM, but 100 muM of auxin and cytokinin inhibited genetic tumour growth. Leaf discs treated with 10 muM cytokinin or auxin alone produced genetic tumours, but 10 muM cytokinin supplemented with 100 muM auxin stopped growth. Therefore genetic tumours are tissues that are, clearly, disturbed in endogenous hormone balance or have altered sensitivity to hormones and response to exogenous hormones.     

Mutants of Streptococcus pneumoniae that contain a temperature-sensitive autolysin

Two mutants of Streptococcus pneumoniae deficient in autolysin activity produced a protein that showed immunological identity with the N-acetyl-muramyl-L-alanyl-amidase present in the wild-type strain, when tested with antiserum obtained against this enzyme. The protein was produced by the mutant cultures grown either at 37 degrees C or at 30 degrees C, although only the cell extracts obtained at 30 degrees C showed significant cell wall hydrolysing activity. In contrast to the lysis resistance of these bacteria grown at 37 degrees C, mutant cultures grown at 30 degrees C exhibited significant degrees of autolysis when treated with detergent or cell wall inhibitors. Extracts of the mutant cultures contained a cell wall hydrolysing activity that was rapidly inactivated during incubation at 37 degrees C.     

Temperature-sensitive expression of all-Torpedo and Torpedo-rat hybrid AChR in mammalian muscle cells

When the four subunits of the Torpedo californica nicotinic acetylcholine receptor (AChR) are expressed in mammalian fibroblasts, they properly assembly into alpha 2 beta gamma delta pentamers only at temperatures lower than 37 degrees C (Claudio, T., W. N. Green, D. S. Hartman, D. Hayden, H. L. Paulson, F. J. Sigworth, S. M. Sine, and A. Swedlund. 1987. Science (Wash. DC). 238:1688-1694). Experiments here with rat L6 myoblast cell lines indicate that this temperature sensitivity is not specific to fibroblasts, but is intrinsic to Torpedo subunits. A clonal isolate of L6 cells cotransfected with the four Torpedo subunit cDNAs synthesizes the exogenous AChR subunits at 37 degrees and 26 degrees C, but expresses Torpedo AChR complexes only at the lower temperature. When Torpedo alpha alone is expressed in L6 myotubes, hybrid AChRs are formed, again only at temperatures below 37 degrees C. These hybrid AChRs can contain either two Torpedo alpha subunits or one each of rat and Torpedo alpha, proving that the two alpha subunits in an AChR pentamer need not derive from the same polysome. Further analysis of hybrid and all-Torpedo AChR established that there is no internally sequestered pool of AChR at the nonpermissive temperature, and that the AChR, once formed, is thermostable. Two lines of experimentation with alpha subunits expressed in fibroblasts indicate that alpha polypeptides exhibit different conformations at 26 degrees and 37 degrees C, favoring the hypothesis that the temperature-sensitive step occurs before assembly and reflects, at least in part, misfolding of subunits: at 37 degrees C, there is a reduction in the fraction of alpha subunits that (a) bind the AChR antagonist alpha-bungarotoxin with high affinity; and (b) bind a monoclonal antibody that recognizes correctly folded and/or assembled alpha subunit.     

Genomic factors controlling the lethality exhibited in the hybrid between Nicotiana suaveolens Lehm. and N. tabacum L.

Interspecific hybrid plants between Nicotiana suaveolens and N. tabacum exhibit lethal symptoms at the seedling stage and cannot grow to maturity. In this investigation, an attempt was made to clarify the genomic factors responsible for this lethality. N. suaveolens was crossed to N. sylvestris (genomic constitution: SS) and N. tomentosiformis (TT), these latter two species being the progenitors of N. tabacum (SSTT). From the cross N. suaveolens x N. tomentosiformis, many seedlings were obtained through ovule culture, and these subsequently grew to maturity without exhibiting any lethality. In the reciprocal crossing between N. sauvelons and N. sylvestris, only a few hybrid seedlings were obtained through ovlue culture and all died after unfolding their cotyledons when cultured at 28 °C. This lethality could be avoided by culturing the ovules at 36 °C. These features of hybrid lethality resembled those observed in the interspecific hybrid between N. suaveolens and N. tabacum. These findings suggest that the S genome in N. tabacum is responsible for the lethality exhibited in the hybrid between N. suaveolens and N. tabacum.     

Outer membrane protein composition of Yersinia pestis at different growth stages and incubation temperatures.

The protein composition of the outer membrane of Yersinia pestis grown at 26 and at 37 degrees C was examined. The outer membrane was isolated by isopycnic sucrose density centrifugation, and its degree of purity was determined with known inner and outer membrane components. Using two-dimensional gel electrophoresis, we identified a large number of heat-modifiable proteins in the outer membrane of cells grown at either incubation temperature. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heated preparations indicated five proteins in the outer membrane of 37 degrees C-grown cells not evident in 26 degrees C-grown cells. Differences in the protein composition of the outer membrane due to the stage of growth were evident at both 26 degrees C and 37 degrees C, although different changes were found at each temperature. When cell envelopes were examined for the presence of peptidoglycan-associated proteins, no differences were seen as a result of stage of growth. Envelopes from 26 degrees C-grown cells yielded two peptidoglycan-associated proteins, E and J. Cells grown at 37 degrees C, however, also contained an additional protein (F) which was not found in either the bound or free form 26 degrees C. The changes in outer membrane protein composition in response to incubation temperature may relate to known nutritional and antigenic changes which occur under the same conditions.     

Somatic hybrid plants of Nicotiana glauca and Nicotiana tabacum obtained by protoplast fusion

Somatic hybrid plants were produced by fusion of protoplasts from cell cultures of the Nicotiana tabacum L. sulfur mutant Su/Su and from leaf mesophyll of Nicotiana glauca Graham. After fusion the N. glauca protoplasts failed to survive under the selected culture condition. From the hybrid cells light green shoots were produced. The hybrid plants exhibited intermediate characters between parental species with respect to leaf morphology, trichome density, floral structure and flower color. The chromosome number of 25 hybrid plants was 2n = 72 and both N. glauca and N. tabacum chromosomes were identified in the hybrids. Results of isoenzyme analysis showed bands of both parents and a specific (hybrid) band for aspartate amino-transferase. Small subunit fraction-1-protein of somatic hybrids also consisted of the sum of N. glauca and N. tabacum bands. Leaf spot formation associated with the Su locus of N. tabacum was observed in somatic hybrids.     

Apoptotic cell death induces temperature-sensitive lethality in hybrid seedlings and calli derived from the cross of Nicotiana suaveolens×N. tabacum

Hybrid lethality expressed in the interspecific hybrid of Nicotiana suaveolens Lehm. ×N. tabacum L. cv. Hicks-2 is one of the mechanisms for reproductive isolation and it is temperature-sensitive. Apoptotic changes were detected in the cells of hybrid seedlings and calli expressing lethality at 28 °C but not under high-temperature conditions (36 °C), when the lethality is suppressed. Condensation of chromatin, fragmentation of nuclei and cytoplasmic reduction are the cytological changes associated with apoptosis leading to hybrid lethality. Fragmentation of nuclei was correlated with the lethal symptoms in both hybrid seedlings and calli, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from hybrid seedlings and calli showing lethal symptoms revealed a specific ladder pattern suggesting nucleosomal fragmentation which is one of the biochemical changes of apoptosis. In-situ detection using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) showed that this process occurred in distinct stages on each organ of hybrid seedlings and centripetally in hybrid calli. From these results, we confirmed that cell death inducing hybrid lethality was indeed apoptosis. Received: 23 December 1999 / Accepted: 5 April 2000