Masanobu Mino

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 F1hybrid seedlings from Nicotiana gosseiDomin 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 (O2 −) and hydrogen peroxide (H2O2), 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°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°C. Seedlings grown at 26°C generated larger amounts of reactive oxygen intermediate in the hypocotyl than those grown at 37°C. A number of stress response marker genes were expressed at 26°C but not at 37°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.

Elevated sensitivity to gibberellin by vernalization in the vegetative rosette plants of Eustoma grandiflorum and Arabidopsis thaliana

Changes in the sensitivity to gibberellin (GA) after vernalization were studied in the vegetative rosette of Eustoma grandiflorum and late flowering Arabidopsis thaliana mutant, fca-1. The sensitivity to GA after vernalization was monitored using the bolting rate of plants that were grown on a medium containing GA(3) or ancymidol. The bolting rates were higher in vernalized plants than non-vernalized plants when the same GA(3) concentration was used. There was a positive relationship between the duration of vernalization and the bolting rate in E. grandiflorum. In contrast, a negative relationship between the duration of treatment and bolting rate was found in the non-vernalized plants. In fca-1, the flowering time of vernalized plants was significantly reduced compared with the non-vernalized plants under various concentrations of GA(3) treatment. To elucidate whether this elevated sensitivity relates to the efficiency of GA signal transduction, we measured the transcript amounts of Expansin (Exp), which is up-regulated by GA, and GA20-oxidase (GA20ox) and GA3 beta-hydroxylase (GA3betahy), which are down-regulated by GA. The transcript amounts were estimated using the Taq-Man PCR system based on combinations of primers and probes that specifically detect the genes, and normalized by the transcript amount of ubiquitin gene measured as an internal standard. For each concentration of GA treatments examined, Expansin of both E. grandiflorum and A. thaliana was induced at a higher rate in the vernalized plants than in the non-vernalized plants. The expression of GA20ox and GA3betahy of E. grandiflorum decreased faster in the vernalized plants than the non-vernalized plants. We conclude that vernalization is a critical environmental cue not only for initiating GA biosyntheses in vegetative rosette, but also for elevating the GA sensitivity of the plants via a GA signal transduction pathway.

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.

RNA and protein synthesis during germination process of F1 hybrid and its parental inbred lines of maize

Abstract The emergence of coleorhiza and radicle were faster in an F1 hybrid of maize than in the parental inbreds. During the germination period, larger amounts of RNA and protein, and higher ratios of the amounts of RNA and protein to that of DNA were found in F1 kernels. Furthermore, the incorporation of [3H] uridine into RNA and [3H] leucine into protein were significantly greater in the embryonic cells of the F1 hybrid as compared to those of the parental lines. It is suggested that the faster germination of the F1 hybrid is due to the more vigorous activity of cellular metabolism following the imbibition.

Assessment of ribozyme-mediated gene suppression using a luciferase reporter gene containing the sequence of Chrysanthemum Stunt Viroid as a target

Transgenic tobacco plants expressing hammerhead ribozyme (Rz), which specifically cleaves Chrysanthemum Stunt Viroid (CSVd) RNA, were produced via Agrobacterium-mediated transformation. To analyze the effects of Rz in cells, fire-fly luciferase (Luc) reporter genes, driven by the Cauliflower Mosaic Virus 35S promoter, which have the entire sense or anti-sense cDNA of the CSVd genome in between the Luc ORF and nopalin synthase terminator were constructed. Using particle bombardment, the Luc reporter constructs were delivered into the leaves of the transgenic tobacco plants. Levels of Luc mRNA and Luc enzyme activity were lower in the transgenic plants than in control wild-type plants, suggesting that the transcript of the Luc reporter gene was made unstable in the presence of Rz. On the other hand, mutations at the cleavage sites of the CSVd sequence in the reporter constructs suppressed the reduction of Luc enzyme activity in transgenic plants. A possibility of post-transcriptional gene silencing was ruled out. A direct evidence of the cleavage activity in cells by the experiment of rapid amplification of 5′ cDNA end could not obtained, but the present results suggest that the levels of Luc mRNA and Luc enzyme activity was affected by the ribozymes.

The siRNAs targeting the left or right terminal region of chrysanthemum stunt viroid (CSVd) sequence suppress the development of disease symptoms caused by CSVd infection of chrysanthemum, but do not suppress viroid propagation

ABSTRACT Transgenic chrysanthemum plants harbouring the genes producing small interfering RNAs (siRNAs) whose sequence was designed based on the chrysanthemum stunt viroid (CSVd) genome were established. The siRNAs are expected to target and decompose CSVd RNA through a silencing mechanism. The CSVd-infected plants were grafted as scions onto the stocks of either non-transgenic or transgenic plants, and axial shoots that elongated from the stocks were rooted and grown to investigate if the plants are resistant to CSVd under greenhouse conditions. The non-transgenic plants infected with CSVd showed severe disease symptoms, i.e. stunted growth and spotty chlorotic lesions on leaves, but no such symptoms were observed in the transgenic plants. The real-time quantitative reverse transcription PCR for monitoring the amounts of CSVd indicated that CSVd equally accumulated in both non-transgenic and transgenic plants. The present results suggest that the resistant feature of the transgenic plants to CSVd was due to interference with the pathway of symptom development, and not the inhibition of CSVd replication.

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.