Genetic Cause of Hybrid Lethality Observed in Reciprocal Interspecific Crosses between Nicotiana simulans and N. tabacum.

Hybrid lethality, a type of postzygotic reproductive isolation, is an obstacle to wide hybridization breeding. Here, we report the hybrid lethality that was observed in crosses between the cultivated tobacco, Nicotiana tabacum (section Nicotiana), and the wild tobacco species, Nicotiana simulans (section Suaveolentes). Reciprocal hybrid seedlings were inviable at 28 °C, and the lethality was characterized by browning of the hypocotyl and roots, suggesting that hybrid lethality is due to the interaction of nuclear genomes derived from each parental species, and not to a cytoplasmic effect. Hybrid lethality was temperature-sensitive and suppressed at 36 °C. However, when hybrid seedlings cultured at 36 °C were transferred to 28 °C, all of them showed hybrid lethality. After crossing between an N. tabacum monosomic line missing one copy of the Q chromosome and N. simulans, hybrid seedlings with or without the Q chromosome were inviable and viable, respectively. These results indicated that gene(s) on the Q chromosome are responsible for hybrid lethality and also suggested that N. simulans has the same allele at the Hybrid Lethality A1 (HLA1) locus responsible for hybrid lethality as other species in the section Suaveolentes. Haplotype analysis around the HLA1 locus suggested that there are at least six and two haplotypes containing Hla1-1 and hla1-2 alleles, respectively, in the section Suaveolentes.

Analysis of the possible cytogenetic mechanism for overcoming hybrid lethality in an interspecific cross between Nicotiana suaveolens and Nicotiana tabacum.

Hybrid lethality is a type of reproductive isolation in which hybrids die before maturation, due to the interaction between the two causative genes derived from each of the hybrid parents. The interspecific hybrid of Nicotiana suaveolens × Nicotiana tabacum is a model plant used in studies on hybrid lethality. While most of the progeny produced from such a cross die, some individuals grow normally and mature. Separately, a technique for producing mature hybrids by artificial culture has been developed. However, the mechanism by which hybrids overcome lethality, either spontaneously or by artificial culture, remains unclear. In the present study, we found that some hybrids that overcome lethality, either spontaneously or by artificial culture, lack the distal part of the Q chromosome, a region that includes the gene responsible for lethality. Quantitative polymerase chain reaction results suggested that the distal deletion of the Q chromosome, detected in some hybrid seedlings that overcome lethality, is caused by reciprocal translocations between homoeologous chromosomes. The results showed that chromosomal instability during meiosis in amphidiploid N. tabacum as well as during artificial culturing of hybrid seedlings is involved in overcoming hybrid lethality in interspecific crosses of the genus Nicotiana.

Phenylalanine ammonia-lyase and phenolic compounds are related to hybrid lethality in the cross Nicotiana suaveolens×N. tabacum.

Hybrid lethality observed in hybrid seedlings between Nicotiana suaveolens and N. tabacum is characterized by browning, initially of the hypocotyls and eventually of entire seedlings. We investigated the mechanism underlying this browning of tissues. A phenylalanine ammonia-lyase (PAL) gene codes an enzyme involved in a pathway producing phenolic compounds related to the browning of plant tissues. The expression of PAL rapidly increased with the induction of hybrid lethality. Phenolic compounds were observed to be accumulated in whole parts of hybrid seedlings. Treatment of hybrid seedlings with L-2-aminooxy-3-phenylpropionic acid (AOPP), an inhibitor for PAL, suppressed browning and decreased the phenolic content of hybrid seedlings. Although programmed cell death (PCD) was involved in hybrid lethality, AOPP treatment also suppressed cell death and enhanced the growth of hybrid seedlings. These results indicated that PAL is involved in hybrid lethality, and phenolic compounds could be the cause of hybrid lethality-associated tissue browning.

Accumulation of protein aggregates induces autolytic programmed cell death in hybrid tobacco cells expressing hybrid lethality.

Hybrid cells of Nicotiana suaveolens x N. tabacum grow normally at 36 °C, but immediately express lethality due to probable autoimmune response when transferred from 36 to 28 °C. Our recent study showed that the temperature-sensitive lethality of these hybrid cells occurs through autolytic programmed cell death (PCD). However, what happens in hybrid cells following the induction of autoimmune response to autolytic PCD is unclear. We hypothesized that accumulation of protein aggregates in hybrid cells induces autolytic PCD and examined detergent-insoluble protein (protein aggregates) isolated from hybrid cells expressing lethality. The amount of insoluble proteins increased in hybrid cells. Sodium 4-phenylbutyrate, a chemical chaperone, inhibited both the accumulation of insoluble proteins and irreversible progression of cell death. In contrast, E-64, a cysteine protease inhibitor, accelerated both the accumulation of insoluble proteins and cell death. Moreover, proteome analysis revealed that proteasome-component proteins were accumulated specifically in cells treated with E-64, and proteasome activity of hybrid cells decreased after induction of lethality. These findings demonstrate that accumulation of protein aggregates, including proteasome subunits, eventually cause autolytic PCD in hybrid cells. This suggests a novel process inducing plant PCD by loss of protein homeostasis and provides clues to future approaches for elucidating the whole process.

Tumorigenesis inheritance from the putative progenitor species of Nicotiana rustica.

Hybrid seedlings from crosses of Nicotiana rustica×N. langsdorffii and N. rustica×N. alata show tumors including teratomas and vitrification. In the present study, we attempted to elucidate the genetic background leading to tumorigenesis and vitrification from the viewpoint of the amphidiploidy of N. rustica. The species N. undulata, N. paniculata, and N. knightiana have been suggested to be the progenitors of N. rustica or closely related to its progenitors. We tested tumorigenesis in interspecific hybrids between these putative progenitors of N. rustica and N. langsdorffii or N. alata, which are the species in section Alatae. The hybrid seedlings were cultured in test tubes and their morphological characteristics were observed. According to previous reports, most of the hybrid seedlings from the crosses N. rustica×N. langsdorffii and N. rustica×N. alata formed tumors and showed vitrification. In crosses with every putative progenitor of N. rustica, a portion of hybrid seedlings formed tumors and showed vitrification. These observations suggested that N. rustica inherited the factors leading to expression of abnormal symptoms from its putative progenitors. We also observed the influence of high temperature on the expression of abnormal symptoms of hybrid seedlings from the cross N. rustica×N. alata. While these hybrids developed teratomas and other tumors at 28°C, when cultured at 34°C, they did not show any abnormalities. This is the first report to show that phenotypic abnormalities in hybrid seedlings of N. rustica×N. alata are temperature sensitive.

Time course of programmed cell death, which included autophagic features, in hybrid tobacco cells expressing hybrid lethality.

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.

Species origin of genomic factors in Nicotiana nudicaulis Watson controlling hybrid lethality in interspecific hybrids between N. nudicaulis Watson and N. tabacum L.

Hybrid lethality is expressed at 28°C in the cross Nicotiana nudicaulis × N. tabacum. The S subgenome of N. tabacum has been identified as controlling this hybrid lethality. To clarify the responsible genomic factor(s) of N. nudicaulis, we crossed N. trigonophylla (paternal progenitor of N. nudicaulis) with N. tabacum, because hybrids between N. sylvestris (maternal progenitor of N. nudicaulis) and N. tabacum are viable when grown in a greenhouse. In the cross N. trigonophylla×N. tabacum, approximately 50% of hybrids were vitrified, 20% were viable, and 20% were nonviable at 28°C. To reveal which subgenome of N. tabacum was responsible for these phenotypes, we crossed N. trigonophylla with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT). In the cross N. sylvestris × N. trigonophylla, we confirmed that over half of hybrids of N. sylvestris × N. trigonophylla were vitrified, and none of the hybrids of N. trigonophylla × N. tomentosiformis were. The results imply that the S subgenome, encoding a gene or genes inducing hybrid lethality in the cross between N. nudicaulis and N. tabacum, has one or more genomic factors that induce vitrification. Furthermore, in vitrified hybrids of N. trigonophylla × N. tabacum and N. sylvestris × N. trigonophylla, we found that nuclear fragmentation, which progresses during expression of hybrid lethality, was accompanied by vitrification. This observation suggests that vitrification has a relationship to hybrid lethality. Based on these results, we speculate that when N. nudicaulis was formed approximately 5 million years ago, several causative genomic factors determining phenotypes of hybrid seedlings were inherited from N. trigonophylla. Subsequently, genome downsizing and various recombination-based processes took place. Some of the causative genomic factors were lost and some became genomic factor(s) controlling hybrid lethality in extant N. nudicaulis.

Identification of Nicotiana tabacum linkage group corresponding to the Q chromosome gene(s) involved in hybrid lethality.

BACKGROUND: A linkage map consisting of 24 linkage groups has been constructed using simple sequence repeat (SSR) markers in Nicotiana tabacum. However, chromosomal assignments of all linkage groups have not yet been made. The Q chromosome in N. tabacum encodes a gene or genes triggering hybrid lethality, a phenomenon that causes death of hybrids derived from some crosses. METHODOLOGY/PRINCIPAL FINDINGS: We identified a linkage group corresponding to the Q chromosome using an interspecific cross between an N. tabacum monosomic line lacking the Q chromosome and N. africana. N. ingulba yielded inviable hybrids after crossing with N. tabacum. SSR markers on the identified linkage group were used to analyze hybrid lethality in this cross. The results implied that one or more genes on the Q chromosome are responsible for hybrid lethality in this cross. Furthermore, the gene(s) responsible for hybrid lethality in the cross N. tabacum × N. africana appear to be on the region of the Q chromosome to which SSR markers PT30342 and PT30365 map. CONCLUSIONS/SIGNIFICANCE: Linkage group 11 corresponded to the Q chromosome. We propose a new method to correlate linkage groups with chromosomes in N. tabacum.

Genes in S and T subgenomes are responsible for hybrid lethality in interspecific hybrids between Nicotiana tabacum and Nicotiana occidentalis.

BACKGROUND: Many species of Nicotiana section Suaveolentes produce inviable F(1) hybrids after crossing with Nicotiana tabacum (genome constitution SSTT), a phenomenon that is often called hybrid lethality. Through crosses with monosomic lines of N. tabacum lacking a Q chromosome, we previously determined that hybrid lethality is caused by interaction between gene(s) on the Q chromosome belonging to the S subgenome of N. tabacum and gene(s) in Suaveolentes species. Here, we examined if hybrid seedlings from the cross N. occidentalis (section Suaveolentes)×N. tabacum are inviable despite a lack of the Q chromosome. METHODOLOGY/PRINCIPAL FINDINGS: Hybrid lethality in the cross of N. occidentalis×N. tabacum was characterized by shoots with fading color. This symptom differed from what has been previously observed in lethal crosses between many species in section Suaveolentes and N. tabacum. In crosses of monosomic N. tabacum plants lacking the Q chromosome with N. occidentalis, hybrid lethality was observed in hybrid seedlings either lacking or possessing the Q chromosome. N. occidentalis was then crossed with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT), to reveal which subgenome of N. tabacum contains gene(s) responsible for hybrid lethality. Hybrid seedlings from the crosses N. occidentalis×N. tomentosiformis and N. occidentalis×N. sylvestris were inviable. CONCLUSIONS/SIGNIFICANCE: Although the specific symptoms of hybrid lethality in the cross N. occidentalis×N. tabacum were similar to those appearing in hybrids from the cross N. occidentalis×N. tomentosiformis, genes in both the S and T subgenomes of N. tabacum appear responsible for hybrid lethality in crosses with N. occidentalis.

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 degrees 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 degrees C, was Type II lethality, with the characteristic symptoms of browning of hypocotyl and roots; lethality was suppressed at elevated temperatures (34 or 36 degrees 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.

Possible involvement of genes on the Q chromosome of Nicotiana tabacum in expression of hybrid lethality and programmed cell death during interspecific hybridization to Nicotiana debneyi.

Hybrid seedlings from the cross between Nicotiana tabacum, an allotetraploid composed of S and T subgenomes, and N. debneyi die at the cotyledonary stage. This lethality involves programmed cell death (PCD). We carried out reciprocal crosses between the two progenitors of N. tabacum, N. sylvestris and N. tomentosiformis, and N. debneyi to reveal whether only the S subgenome in N. tabacum is related to hybrid lethality. Hybrid seedlings from reciprocal crosses between N. sylvestris and N. debneyi showed lethal characteristics identical to those from the cross between N. tabacum and N. debneyi. Conversely, hybrid seedlings from reciprocal crosses between N. tomentosiformis and N. debneyi were viable. Furthermore, hallmarks of PCD were observed in hybrid seedlings from the cross N. debneyi x N. sylvestris, but not in hybrid seedlings from the cross N. debneyi x N. tomentosiformis. We also carried out crosses between monosomic lines of N. tabacum lacking the Q chromosome and N. debneyi. Using Q-chromosome-specific DNA markers, hybrid seedlings were divided into two groups, hybrids possessing the Q chromosome and hybrids lacking the Q chromosome. Hybrids possessing the Q chromosome died with characteristics of PCD. However, hybrids lacking the Q chromosome were viable and PCD did not occur. From these results, we concluded that the Q chromosome belonging to the S subgenome of N. tabacum encodes gene(s) leading to hybrid lethality in the cross N. tabacum x N. debneyi.

Identification and characterization of genes involved in hybrid lethality in hybrid tobacco cells (Nicotiana suaveolens x N. tabacum) 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 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.

Fine mapping and allelic dosage effect of Hwc1, a complementary hybrid weakness gene in rice.

Hybrid weakness is a reproductive barrier that is found in many plant species. In rice, the hybrid weakness caused by two complementary genes, Hwc1 and Hwc2, has been surveyed intensively. However, their gene products and the molecular mechanism that causes hybrid weakness have remained unknown. We performed linkage analyses of Hwc1, narrowed down the area of interest to 60 kb, and identified eight candidate genes. In the F(2) population, in which both Hwc1 and Hwc2 genes were segregated, plants were separable into four classes according to their respective phenotypes: severe type, semi-severe type, F(1) type, and normal type. Severe type plants show such severe symptoms that they could produce only tiny shoot-like structures; they were unable to generate roots. Genetic analyses using closely linked DNA markers of the two genes showed that the symptoms of the F(2) plants were explainable by the genotypes of Hwc1 and Hwc2. Weakness was observed in plants that have both Hwc1 and Hwc2. In Hwc1 homozygote, the symptoms worsened and severe type or semi-severe type plants appeared. Consequently, Hwc1 should have a gene dosage effect and be a semi-dominant gene. The dosage effect of Hwc2 was recognizable, but it was not so severe as that in Hwc1. These results are useful to elucidate the mechanism that causes the hybrid weakness phenomenon and the role of each causal gene in hybrid weakness.

A homolog of the defender against apoptotic death gene (DAD1) in senescing gladiolus petals is down-regulated prior to the onset of programmed cell death.

We isolated a homolog of the potential anti-apoptotic gene, defender against apoptotic death (DAD1) from gladiolus petals as full-length cDNA (GlDAD1), and investigated the relationship between its expression and the execution processes of programmed cell death (PCD) in senescing petals. RNA gel blotting showed that GlDAD1 expression in petals was drastically reduced, considerably before the first visible senescence symptom (petal wilting). A few days after down-regulation GlDAD1 expression, DNA and nuclear fragmentation were observed, both specific for the execution phase of PCD.

Development of Q-chromosome-specific DNA markers in tobacco and their use for identification of a tobacco monosomic line.

We developed seven Q-chromosome-specific DNA markers in Nicotiana tabacum by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis using two hybrid lines, and we were able to identify tobacco monosomic plants among F1 progeny derived from the cross N. tabacum Haplo-QxN. tabacum cv. Samsun NN using Q-chromosome-specific DNA markers. Based on the results, we discuss the roles of the Q chromosome in embryo sac development and embryogenesis. Here, we propose a new method for identifying DNA markers for a particular chromosome in the genus Nicotiana.

Time course analysis of apoptotic cell death during expression of hybrid lethality in hybrid tobacco cells (Nicotiana suaveolens x N. tabacum).

Hybrid cells from the cross Nicotiana suaveolens x N. tabacum expressed hybrid lethality at 28 degrees C in a thin layer cell culture system. Features characteristic of apoptosis, such as DNA fragmentation, chromatin condensation and nuclear fragmentation, were detected during expression of hybrid lethality. Actinomycin D (ActD) or cycloheximide (CHX) added to the medium suppressed apoptotic cell death during hybrid lethality. This indicates that hybrid lethality requires de novo transcription and translation, and is thus under genetic control. To estimate the time course of apoptotic cell death during the expression of hybrid lethality, we determined when factors controlling hybrid lethality were expressed by observing the point of no return. When cells were exposed to 28 degrees C for 2 h or less in inhibitor-free medium before addition of ActD or CHX, the percentage of dead cells did not increase. However, when cells were exposed to 28 degrees C for 4 h before the addition of inhibitor, the percentage of dead cells increased. When cells were exposed to 28 degrees C for 3 h before the addition of inhibitor, the percentage of dead cells varied from experiment to experiment. These data indicate that the factors controlling hybrid lethality are expressed 3 h after induction of hybrid lethality. In addition, we found a time difference between the expression of cell death and nuclear fragmentation. This suggests that the factor controlling cell death is different from the one controlling nuclear fragmentation.

Overproduced ethylene causes programmed cell death leading to temperature-sensitive lethality in hybrid seedlings from the cross Nicotiana suaveolens x N. tabacum.

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.