Manabu Kurita

Complete nucleotide sequence of the Cryptomeria japonica D. Don. chloroplast genome and comparative chloroplast genomics: diversified genomic structure of coniferous species

BackgroundThe 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.ResultsThe C. japonica cp genome is 131,810 bp in length, with 112 single copy genes and two duplicated (trnI-CAU, trnQ-UUG) genes that give a total of 116 genes. Compared to other land plant cp genomes, the C. japonica cp has lost one of the relevant large inverted repeats (IRs) found in angiosperms, fern, liverwort, and gymnosperms, such as Cycas and Gingko, and additionally has completely lost its trnR-CCG, partially lost its trnT-GGU, and shows diversification of accD. The genomic structure of the C. japonica cp genome also differs significantly from those of other plant species. For example, we estimate that a minimum of 15 inversions would be required to transform the gene organization of the Pinus thunbergii cp genome into that of C. japonica. In the C. japonica cp genome, direct repeat and inverted repeat sequences are observed at the inversion and translocation endpoints, and these sequences may be associated with the genomic rearrangements.ConclusionThe observed differences in genomic structure between C. japonica and other land plants, including pines, strongly support the theory that the large IRs stabilize the cp genome. Furthermore, the deleted large IR and the numerous genomic rearrangements that have occurred in the C. japonica cp genome provide new insights into both the evolutionary lineage of coniferous species in gymnosperm and the evolution of the cp genome.

Regeneration of transgenic Cryptomeria japonica D. Don after Agrobacterium tumefaciens-mediated transformation of embryogenic tissue

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.

Evolutionary Relationship and Structural Characterization of the EPF/EPFL Gene Family

EPF1-EPF2 and EPFL9/Stomagen act antagonistically in regulating leaf stomatal density. The aim of this study was to elucidate the evolutionary functional divergence of EPF/EPFL family genes. Phylogenetic analyses showed that AtEPFL9/Stomagen-like genes are conserved only in vascular plants and are closely related to AtEPF1/EPF2-like genes. Modeling showed that EPF/EPFL peptides share a common 3D structure that is constituted of a scaffold and loop. Molecular dynamics simulation suggested that AtEPF1/EPF2-like peptides form an additional disulfide bond in their loop regions and show greater flexibility in these regions than AtEPFL9/Stomagen-like peptides. This study uncovered the evolutionary relationship and the conformational divergence of proteins encoded by the EPF/EPFL family genes.

Biosafety assessment of transgenic poplars overexpressing xyloglucanase (AaXEG2) prior to field trials

We performed biosafety assessments of transgenic poplars prior to field trials. Constitutive expression of the Aspergillus aculeatus xyloglucanase in Populus alba increased the cellulose content and specific gravity of its stem, the leaves of which were visibly greener, thicker, and smaller than those of the wild-type plant. Although the young transgenic poplars grew faster than the wild type in a growth chamber, there was no distinguishable difference in growth between the poplars when they were placed in a special screened greenhouse. Allelopathic tests showed that the transgenic poplars do not produce harmful substances. Based on all the biosafety assessments and the scientific literature on poplar species, we came to the conclusion that transgenic poplars probably do not disturb the biological diversity of the surrounding environment, even when they are submitted to field trials.

Enlargement of individual cellulose microfibrils in transgenic poplars overexpressing xyloglucanase

Holocellulose samples prepared from transgenic poplars overexpressing xyloglucanase had crystal widths of 3.2–3.5 nm as a result of the (2 0 0) plane, based on their X-ray diffraction patterns, and crystal widths were greater than those of the wild type (3.0 nm). Cellulose microfibril widths in the holocellulose samples were further determined from transmission electron microscopic (TEM) images of individualized fibrils prepared by 2,2,6,6-tetramethylpiperidine-1-oxy radical-mediated oxidation of the holocelluloses and the successive disintegration of the oxidized products in water. The TEM images also supported the finding that cellulose microfibril widths of transgenic poplars were larger than those of the wild type. The cellulose microfibril widths of transgenic poplars were approximately 6 nm, whereas those of the wild type were about 5 nm. However, such enlargement of cellulose microfibril widths could not be explained by the increased cellulose contents of the transgenic poplars alone.

Analyses of leaves from open field-grown transgenic poplars overexpressing xyloglucanase

The transgenic expression of Aspergillus xyloglucanase cDNA (AaXEG2) with 35S promoter in the leaves of open field-grown poplars was studied. The level of xyloglucan in the transgenic poplars was decreased to 15–16% in the non-fertile soil (forest-field soil) and to 21–22% in the fertile soil (farming-field soil) compared with that of the wild-type poplars. The leaves exhibited a smaller surface area with more rounded teeth than those of the wild-type plants, similar to the sun leaf variety that was grown in the incubation room and subsequently greenhoused. The majority of total veins with water-conducting vascular bundles were shorter in the leaves of the transgenic poplars than those of the wild type. This decrease in vein length may result from a decrease in xyloglucan during leaf development, from which large numbers of proteins were markedly downregulated in the leaves of the transgenic plants via proteomic analysis. It seems likely that the leaves of the transgenic poplars came to relax the edges of their tooth rather than extend their veins as a result of the loosening of the xyloglucan cellulose networks in the leaves.

Determination of male strobilus developmental stages by cytological and gene expression analyses in Japanese cedar (Cryptomeria japonica)

The molecular mechanisms that control male strobilus development in conifers are largely unknown because the developmental stages and related genes have not yet been characterized. The determination of male strobilus developmental stages will contribute to genetic research and reproductive biology in conifers. Our objectives in this study were to determine the developmental stages of male strobili by cytological and transcriptome analysis, and to determine the stages at which aberrant morphology is observed in a male-sterile mutant of Cryptomeria japonica D. Don to better understand the molecular mechanisms that control male strobilus and pollen development. Male strobilus development was observed for 8 months, from initiation to pollen dispersal. A set of 19,209 expressed sequence tags (ESTs) collected from a male reproductive library and a pollen library was used for microarray analysis. We divided male strobilus development into 10 stages by cytological and transcriptome analysis. Eight clusters (7324 ESTs) exhibited major changes in transcriptome profiles during male strobili and pollen development in C. japonica Two clusters showed a gradual increase and decline in transcript abundance, respectively, while the other six clusters exhibited stage-specific changes. The stages at which the male sterility trait of Sosyun was expressed were identified using information on male strobilus and pollen developmental stages and gene expression profiles. Aberrant morphology was observed cytologically at Stage 6 (microspore stage), and differences in expression patterns compared with wild type were observed at Stage 4 (tetrad stage).

Tissue culture of two medicinal trees native to Japan

Wadatsuminoki (Nothapodytes amamianus) is an endangered tree species observed in only Amami Oshima Island located in southern part of Japan. According to the Red list published by Ministry of Environment, it is classified as 1A (Critically endangered) and naturally remaining number is only 20. It contains camptotesin which is used for anti-cancer drugs. Kagikazura (Uncaria rhynchophylla) is an medicinal tree species observed widely in Japan and China. It contains alkaloids (rhynchophylline, iso-rhynchophylline, hirstine and so on) which are good for remedy of high blood pressure and dementia. For the purpose of micropropagation and development of basis of useful substance production by cell culture as well as conservation of endangered species, tissue culture procedure was developed for those two species. Excised shoots of 2 years old seedling of Wadatsuminoki rooted in the 1/2DCR medium containing 3 g/l activated charcoal powder. Newly shoots were induced from in vitro root segments subcultured to 1/2MS medium containing 2uM BAP. This cycle can be used for micropropagation of Wadatsuminoki. We have succeeded in micropropagation by tissue culture of Wadatsuminoki (Figure 1). Callus proliferation from stem or root segments was observed on the 1/2LP medium containing 0.5uM BAP and 1 uM 2,4-D, this subculture cell line may be used for the possible production of secondary metabolites in vitro. Shoots were induced from stem spine (thorn) of Kagikazura in the 1/2MS medium containing BAP or Zeatin. Regenerated plants were obtained by rooting of these shoots on 1/2MS medium containing 1 uM IBA. Callus induced around the stem segments were continuously subcultured in the fresh 1/2LP medium containing 0.5 uM BAP and 1 uM 2, 4-D. These cell lines can be used for the possible secondary metabolite production and for breeding by somaclonal variation or genetic engineering.

Potential of Genome-Wide Studies in Unrelated Plus Trees of a Coniferous Species, Cryptomeria japonica (Japanese Cedar)

A genome-wide association study (GWAS) was conducted on more than 30,000 single nucleotide polymorphisms (SNPs) in unrelated first-generation plus tree genotypes from three populations of Japanese cedar Cryptomeria japonica D. Don with genomic prediction for traits of growth, wood properties and male fecundity. Among the assessed populations, genetic characteristics including the extent of linkage disequilibrium (LD) and genetic structure differed and these differences are considered to be due to differences in genetic background. Through population-independent GWAS, several significant SNPs found close to the regions associated with each of these traits and shared in common across the populations were identified. The accuracies of genomic predictions were dependent on the traits and populations and reflected the genetic architecture of traits and genetic characteristics. Prediction accuracies using SNPs selected based on GWAS results were similar to those using all SNPs for several combinations of traits and populations. We discussed the application of genome-wide studies for C. japonica improvement.