Wataru Marubashi

Suppressive effect of trehalose on apoptotic cell death leading to petal senescence in ethylene-insensitive flowers of gladiolus

Petal senescence is a distinct factor affecting vase-life, which is an important determinant of the quality of cut flowers. Programmed cell death (PCD) is associated with petal senescence, but little is known about the triggering or execution of the process of cell death in petals. In the present study, some features of apoptosis were detected in senescing petals taken from ethylene-insensitive flowers of gladiolus. Agarose gel analysis of DNA extracted from petals revealed that DNA fragmentation, an apoptotic process, occurred in cells of senescing petals. Cytological observation of petal cells suggested that apoptotic changes of nuclei, such as chromatin condensation and nuclear fragmentation, also occurred during cell death leading to petal senescence. Using flow cytometry, we confirmed that nuclear fragmentation correlated with cell death in petals after full flower opening. When the florets were treated with ethylene inhibitors (STS or AOA) or saccharides (sucrose or trehalose), both wilting and nuclear fragmentation of petals were suppressed only by trehalose. From these observations, we conclude that the cell death leading to petal senescence of gladiolus is similar to apoptosis. Furthermore, the present study has provided the first evidence that trehalose has a suppressive effect on apoptotic cell death.

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

Abstract. 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.

Apoptosis detected in hybrids between Nicotiana glutinosa and N. repanda expressing lethality

Abstract. Hybrid lethality is one of the mechanisms for reproductive isolation. Apoptotic features were detected in the cells of hybrid seedlings of Nicotiana glutinosa L. ×N. repanda Willd. Condensation of chromatin and fragmentation of nuclei were observed in the leaf protoplasts isolated from hybrid seedlings expressing this lethality. Fragmentation of nuclei was correlated with the progression of lethal symptoms, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from hybrid leaves showing lethality revealed a specific ladder pattern suggesting nucleosomal fragmentation associated with nuclear fragmentation. In-situ detection of DNA fragmentation using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) showed that this process occurred in all leaf cells. This is the first evidence that apoptosis can induce suicide of hybrid plants, thus leading to reproductive isolation.

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.

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.

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.

Interspecific hybridization among wild Gladiolus species of southern Africa based on randomly amplified polymorphic DNA markers

Abstract We obtained information on the genetic relationship in wild Gladiolus species through randomly amplified polymorphic DNA (RAPD) analysis. Out of the 140 tested primers, 32 amplified a total of 133 RAPD bands in 33 Gladiolus species. The genetic distance was calculated from the data of these RAPDs, and a dendrogram was generated. Interspecific crosses were carried out in seven combinations within or between clusters, and F 1 seedlings were obtained from most combinations. The RAPD analysis showed that these F 1 seedlings were real hybrids. The results suggest that RAPD markers are useful for detecting genetic relationships in Gladiolus species, and for interspecific crosses in breeding programs.

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 F1 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.

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.

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.