Background: The recent determination of complete chloroplast (cp) genomic sequences of various plant species has enabled numerous comparative analyses as well as advances in plant and genome evolutionary studies. In angiosperms, the complete cp genome sequences of about 70 species have been determined, whereas those of only three gymnosperm species, Cycas taitungensis, Pinus thunbergii, and Pinus koraiensis have been established. The lack of information regarding the gene content and genomic structure of gymnosperm cp genomes may severely hamper further progress of plant and cp genome evolutionary studies. To address this need, we report here the complete nucleotide sequence of the cp genome of Cryptomeria japonica, the first in the Cupressaceae sensu lato of gymnosperms, and provide a comparative analysis of their gene content and genomic structure that illustrates the unique genomic features of gymnosperms.

In response to environmental variation, angiosperm trees bend their stems by forming tension wood, which consists of a cellulose-rich G (gelatinous)-layer in the walls of fiber cells and generates abnormal tensile stress in the secondary xylem. We produced transgenic poplar plants overexpressing several endoglycanases to reduce each specific polysaccharide in the cell wall, as the secondary xylem consists of primary and secondary wall layers. When placed horizontally, the basal regions of stems of transgenic poplars overexpressing xyloglucanase alone could not bend upward due to low strain in the tension side of the xylem. In the wild-type plants, xyloglucan was found in the inner surface of G-layers during multiple layering. In situ xyloglucan endotransglucosylase (XET) activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, began at the inner surface layers S1 and S2 and was retained throughout G-layer development, while the incorporation of xyloglucan heptasaccharide (XXXG) for wall loosening occurred in the primary wall of the expanding zone. We propose that the xyloglucan network is reinforced by XET to form a further connection between wall-bound and secreted xyloglucans in order to withstand the tensile stress created within the cellulose G-layer microfibrils.

To combat SARS-CoV-2 and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report a high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only four days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (IC50 = 0.5 nM). The high-speed in vitro selection of antibody-like proteins would be useful for the rapid development of a detection method and a neutralizing protein against a virus responsible for an ongoing, and possibly a future, pandemic.

SummaryThe genome sizes of 13 species of Taxodiaceae, 19 species of Cupressaceae s.s. and Sciadopitys verticillata were determined by flow cytometry of isolated nuclei stained with propidium iodide, using Hordeum vulgare nuclei as an internal standard. In Taxodiaceae, the genomes of Cunninghamia lanceolata (28.34 pg/2C) and Taiwania species (25.78, 26.80 pg/2C) were larger than those of other genera/species, which ranged from 19.85 to 22.87 pg/2C. In Cupressaceae s.s., genome size ranged from 20.03 to 27.93 pg/2C among 16 species. The Calocedrus species and Thujopsis had a larger genome than most other species. Sciadopitys verticillata had a large genome of 41.60 pg/2C. After comparing the diversity in genome size with previously reported cladograms constructed using nucleotide sequence data, the tendency of changes in genome size with phylogenetic differentiation is discussed.

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A genetic transformation procedure for Cryptomeria japonica was developed after co-cultivation of embryogenic tissues with the disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the visual reporter gene sgfp and two selectable marker genes, hpt and nptII. We were able to generate eight and three independent transgenic lines per gram of embryogenic tissue after selection on hygromycin and kanamycin medium, respectively. Transgenic plants were regenerated through somatic embryogenesis in 4 lines out of these 11 lines. Green fluorescent protein fluorescence was observed under fluorescent microscopy. Integration of the genes into the genome was confirmed by polymerase chain reaction analysis of embryogenic tissues and Southern blot analysis of regenerated plantlets.

Japanese red pine, Pinus densiflora, has 2n=24 chromosomes, of which most carry chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) bands at their centromere-proximal regions. It was proposed that these regions contain highly repetitive DNA. The DNA localized in the proximal fluorescent bands was isolated and characterized. In P. densiflora, centromeric and neighboring segments of the somatic chromosomes were dissected with a manual micromanipulator. The centromeric DNA was amplified from the DNA contained in dissected centromeric segments by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) and a cloned DNA library was constructed. Thirty-one clones carrying highly repetitive DNA were selected by colony hybridization using Cot-1 DNA from this species as a probe, and their chromosomal localization was determined by fluorescent in situ hybridization (FISH). Clone PDCD501 was localized to the proximal CMA band of 20 chromosomes. This clone contained tandem repeats, comprising a 27 bp repeat unit, which was sufficient to provide the proximal FISH signal, with a 52.3% GC content. The repetitive sequence was named PCSR (proximal CMA band-specific repeat). Clone PDCD159 was 1700 bp in length, with a 61.7% AT content, and produced FISH signals at the proximal DAPI band of the remaining four chromosomes. Four clones hybridized strongly to the secondary constriction and gave weak signals at the centromeric region of several chromosomes. Clone PDCD537, one of the four clones, was homologous to the 26S rRNA gene. A PCR experiment using microdissected centromeric regions suggested that the centromeric region contains 18S and 26S rDNA. Another 24 clones hybridized to whole chromosome arms, with varying intensities and might represent dispersed repetitive DNA.

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