Masayasu Inoue

Unusual core histones specifically expressed in male gametic cells of Lilium longiflorum

Abstract.We have cloned three novel histone genes using antibodies that recognize only nuclei of the male gametic (generative and sperm) cells of Lilium longiflorum. The deduced amino acid sequence of each clone shows only between 40% and 50% identity with the H2A, H2B and H3 somatic core histones of other plant species. Transcripts of these genes were first detected in bicellular pollen soon after microspore mitosis, and their mRNAs, as revealed by in situ hybridization, were observed only in the cytoplasm of the generative cells. As expression of these three genes was specific to generative cells within the bicellular pollen, we designated the clones gH2A, gH2B and gH3. Immunocytochemistry further revealed that the proteins encoded by these genes accumulated in the elongating and condensing generative nucleus during development of bicellular pollen, and were most abundant in the two sperm nuclei within an elongated pollen tube. We therefore propose that these male gamete-specific core histones contribute to chromatin condensation of male gametes or to chromatin remodeling, and result in the repression of gene expression in male gametes.

Residual structure and constituent proteins of the peripheral framework of the cell nucleus in somatic embryos from Daucus carota L.

Nuclei were isolated from somatic embryos of carrot (Daucus carota L.) using a buffer system containing non-ionic detergent. To prepare nuclear matrices, the purified membrane-depleted nuclei were digested with DNase I in combination with RNase A, followed by extraction with 1 M NaCl. The DNA residue in the final insoluble fraction was less than 4% of that in isolated nuclei, and most of the residual nuclei retained their sphericity. Electron microscopy revealed that the nuclear matrix was composed of a distinct peripheral layer, an internal matrix structure and some fibrils; residual nucleoli were observed when exogeneous RNase was not incorporated. The proteins extracted from the nuclei and nuclear subfractions were compared by gel electrophoresis, which showed that the residual fraction contained many minor proteins. To identify proteins showing specific localization at the nuclear periphery, we prepared monoclonal antibodies (MAbs) against an ion-exchange chromatography fraction extracted from carrot nuclear matrices. Immunofluorescence microscopy with one of the MAbs, CML-1, showed exclusive staining of the nuclear periphery. The MAb recognized several spots showing microheterogeneity, with a narrow range of pI and molecular mass upon immunoblotting. A complete set of these spots was shown to be conserved in nuclear matrices. On the other hand, MAb CML-13 appeared to react with the nuclear interior as well as the periphery, recognizing a 96-kDa polypeptide of the nuclear matrix. These proteins were thus demonstrated to lie at the nuclear periphery, and to constitute the nuclear matrices in carrot. The 96-kDa polypeptide is suggested to be similar to the 92-kDa nuclear protein reported by Beven et al. in carrot (Beven et al., 1991, J. Cell Sci. 98, 293–302).

Callus formation and plant regeneration from rice protoplasts purified by density gradient centrifugation

Abstract Protoplasts were isolated from germinating embryo-derived cell suspensions of rice (Oryza sativa L.). The protoplast yield from cultured cells was sufficient, but heterogeneity in size and the degree of vacuolation of freshly isolated protoplasts were commonly observed for all preparations from four varieties tested. The use of density gradient centrifugation with media based on a mixture of sucrose and mannitol was effective for separation of a relatively uniform protoplast preparation from the heterogeneous population. The fraction with a high specific gravity from density gradients contained a high percentage of cytoplasmic-rich protoplasts. The plating efficiency (percentage of protoplasts that formed colonies) was improved markedly by the incorporation of this purification step. The purified fraction could be cultured in a liquid medium and a reproducible plating efficiency of up to 0.7% was obtained depending on the variety and the plating density. Otherwise the eliminated fraction with low specific gravity, which contained mainly transparent protoplasts with large vacuoles, rarely underwent sustained divisions. Protoplast-derived calli of tested rice varieties produced plantlets on regeneration media with a frequency of 10–22%. Regenerated plants were transfered to soil and grown to set seeds in a greenhouse.

A simple procedure for the isolation of pure nuclei from carrot embryos in synchronized cultures

A simple method is presented for the isolation of nuclei from somatic embryos of carrot (Daucus carota L.), which is applicable to small amounts of material in synchronized culture. The method employs buffers containing a high concentration of glycerol to stabilize the structure of the nuclei. Purification was carried out by centrifugation using preformed Percoll gradients. Treatment with cell wall-degrading enzymes prior to homogenization improved the efficiency of isolation and permitted a reproducible yield of nuclei. The pure preparations were obtained with an efficiency of approximately 60%. The isolated nuclei retained their morphological characteristics as demonstrated by phase — contrast and electron microscopy. Nuclear proteins displayed the expected species of histones by two-dimensional gel electrophoresis. The isolated nuclei showed high RNA polymerase activity.

Reduced glutathione promotes callus growth and shoot development in a shoot tip culture of apple root stock M26

Abstract Reduced glutathione (GSH) was applied to prevent browning in shoot tip explants of apple (Malus pumila Mill.). Development was compared between shoot tips treated either by (a) dipping into 0.1 mm GSH solution prior to culture (dip treatment), (b) dipping into the GSH solution and transferring to a medium containing 0.1 mm GSH (dip-and-add treatment), or (c) without dipping or culturing with GSH (control). In the dip treatment, 100% of shoot tips developed into normal shoots after 120 days, while the results with the dip-and-add treatment and control were 50 and 40%, respectively. The results show that application of antioxidant GSH in the initial phase of culture promoted the normal development of shoot tips.

Nucleotide sequences of the coat protein gene of potyviruses infecting Ornithogalum thyrsoides

Summary. Species of three viral genera infecting Ornithogalum thyrsoides plants showing mosaic symptoms were identified using RT-PCR and degenerate universal primers for each viral genus. The DNA fragments obtained encoded the coat protein (CP) gene and were sequenced. The plants were found to be infected with one or other of three potyvirus species, one of them was Ornithogalum mosaic virus (OrMV). The other two viruses were previously unrecorded and were named Ornithogalum virus 2 (OV-2) and 3 (OV-3). Direct comparison and phylogenetic analysis with published OrMV isolates revealed that the CP of the three OrMV-like clones were more similar to Pterostylis virus Y (PtVY) than to OrMV. No carlavirus or potexvirus was isolated.

Molecular characterization of a novel protein disulfide isomerase in carrot.

A protein disulfide isomerase (PDI) coding sequence was cloned from a cDNA library derived from carrot (Daucus carota L.) somatic embryos. The cDNA is 2060 bp in length and encodes for a protein of 581 amino acids and molecular weight of 64.4 kDa. Primary structure analysis of the deduced protein revealed two thioredoxin-like active sites and an endoplasmic reticulum-retention signal at its C-terminus, which is also found in PDIs in plants and animals. Although between the carrot protein and other plant PDIs there is only about 30% identity, the active site regions are almost identical. The corresponding mRNA was found in varying amounts, in all tissues investigated. A recombinant protein expressed from the carrot cDNA clone effectively catalyzed both glutathione-insulin transhydrogenation and the oxidative renaturation of denatured RNase A. These results suggest that the protein coded for by the carrot gene is a novel member of the PDI family in plants. We therefore designated this novel carrot gene PDIL1. The protein expressed by the PDIL1 cDNA sequence had a highly acidic stretch at its N-terminal region (no such domain exists in known plant PDIs), and was located far from known plant PDIs on a maximum likelihood tree. The PDIL1 gene, together with closely-related genes identified in Arabidopsis and tomato, was suggested to belong to a novel subfamily of PDIs.

Male gametic cell-specific histone gH2A gene of Lilium longiflorum: genomic structure and promoter activity in the generative cell.

A genomic clone containing the gH2A gene, a histone variant specifically expressed in male gametic cells within the pollen of Lilium longiflorum, was isolated. Sequence analysis revealed that the coding region of the gene is interrupted by one intron, as is the case with the somatic type of plant histone H2A genes, suggesting derivation from the same ancestral gene containing one intron. In addition, a 2.8-kbp fragment of the 5′ upstream region of gH2A contained TATA and CAAT boxes, but neither a plant histone-specific regulatory DNA element nor vegetative cell-specific cis-elements were found. A histochemical study of stable transformants demonstrated that the 5′ upstream region of the gene can drive gene expression specifically in the generative cell of pollen; no activity was detectable in the vegetative cell or in other reproductive and vegetative tissues of transgenic Nicotiana tabacum. These results strongly suggest that the generative cell can direct specific gene expression, that this expression may be regulated by a putative male gametic factor, and that the gH2A promoter may therefore serve as a useful male gametic cell fate marker in angiosperms.

A Missense Mutation in Tomato mosaic virus L11A-Fukushima Genome Determines Its Symptomless Systemic Infection of Tomato

The genome of an attenuated isolate L11A-Fukushima (L11A-F) of Tomato mosaic virus was cloned, its complete nucleotide sequence was determined, and full-length clone of L11A-F was then assembled. Its transcripts systemically infected tomato without causing any symptom development. When a missense back mutation at the nucleotide correspondent to nucleotide 2350 of the genome was introduced into the clone, its transcripts produced distinct mosaic on the upper leaves of the inoculated tomato, and virus accumulation in the upper leaves increased about five times. The missense mutation in the L11A-F genome was thus confirmed to be sufficient to attenuate its virulence on tomato.

Discrimination between Virulent and Attenuated Isolates of Tomato mosaic virus by Restriction Fragment Length Polymorphism

The three missense mutations on the gene for the 130-K protein of Tomato mosaic virus (ToMV) L11A have been thought to be responsible for the attenuation of its virulence. The Eco47I RFLP detecting the missense mutation at 2349 successfully discriminated L11A and its derivative attenuated isolates from ToMV virulent ones. RFLP analysis and mismatch amplification assay detecting the missense mutations at 1117 and 2754, respectively, could not discriminate some of the attenuated isolates from the virulent ones. These results indicated that, of the three missense mutations, only the one at 2349 was conserved in all the L11A-derivative attenuated isolates.