The promoter of an A9 homolog from the conifer Cryptomeria japonica imparts male strobilus-dominant expression in transgenic trees.

KEY MESSAGE : GUS analysis in Cryptomeria japonica revealed that the CjMALE1 promoter is activated in the male strobilus of C. japonica. Toward the development of male sterile technology for Cryptomeria japonica, a male strobilus-dominant promoter of C. japonica was isolated. The CjMALE1 gene was isolated from a male strobilus-specific suppression subtractive hybridization (SSH) library, and the promoter was isolated by the TAIL-PCR method. To characterize the CjMALE1 promoter, ß-glucuronidase (GUS)-fused genes were constructed and introduced into C. japonica using Agrobacterium tumefaciens. GUS expression from CjMALE1-2.5 K (2,718 bp fragment)::GUS C. japonica and CjMALE1-1 K (1,029 bp fragment)::GUS C. japonica was detected in the tapetum and microspore mother cells. These promoter fragments were comparably active in the pre-meiotic stage of the male strobilus of C. japonica. Our analysis showed that the 1,029 bp promoter had all the cis-elements necessary for male strobilus-dominant expression of CjMALE1. When CjMALE1-1 K::GUS was introduced into Arabidopsis, GUS expression was detected in the same spatiotemporal pattern as in C. japonica. These results suggest that the CjMALE1 promoter is subject to transcriptional regulatory systems consisting of cis- and trans-elements that have been highly conserved during evolution.

A frameshift mutation of the chloroplast matK coding region is associated with chlorophyll deficiency in the Cryptomeria japonica virescent mutant Wogon-Sugi.

Wogon-Sugi has been reported as a cytoplasmically inherited virescent mutant selected from a horticultural variety of Cryptomeria japonica. Although previous studies of plastid structure and inheritance indicated that at least some mutations are encoded by the chloroplast genome, the causative gene responsible for the primary chlorophyll deficiency in Wogon-Sugi, has not been identified. In this study, we identified this gene by genomic sequencing of chloroplast DNA and genetic analysis. Chloroplast DNA sequencing of 16 wild-type and 16 Wogon-Sugi plants showed a 19-bp insertional sequence in the matK coding region in the Wogon-Sugi. This insertion disrupted the matK reading frame. Although an indel mutation in the ycf1 and ycf2 coding region was detected in Wogon-Sugi, sequence variations similar to that of Wogon-Sugi were also detected in several wild-type lines, and they maintained the reading frame. Genetic analysis of the 19 bp insertional mutation in the matK coding region showed that it was found only in the chlorophyll-deficient sector of 125 full-sibling seedlings. Therefore, the 19-bp insertion in the matK coding region is the most likely candidate at present for a mutation underlying the Wogon-Sugi phenotype.

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

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. RESULTS: The 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. CONCLUSION: The 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.

[Investigation of the variation of Cry j 2 concentration in pollen among sugi (Cryptomeria japonica d. Don) trees using a newly established extraction method].

BACKGROUND: Selection of low-allergen trees is under consideration for the reduction of Sugi (Cryptomeria japonica) pollinosis. The extraction method of Cry j 2 was established and then the variation of Cry j 2 concentrations in pollen among Sugi trees was investigated. METHODS: The extraction efficiency of Cry j 2 was compared among buffers with different pH, ionic species and NaCl concentration. The Cry j 2 concentration in the pollen of 140 trees was measured using the most efficient buffer. RESULTS: NaHCO3 (0.125 M) + 0.5 M NaCl buffer showed the highest Cry j 2 extraction efficiency. The mean and standard deviation of Cry j 2 concentration among 140 trees was 404 +/- 189 microg/g pollen, which was equivalent to that of Cry j 1 (435 +/- 169 microg). CONCLUSION: We revealed that the variation of Cry j 2 concentrations was as large as that of Cry j 1 in the pollen among trees using the newly developed extraction method.

Influences of genetic and environmental factors on the concentration of the allergen Cry j 1 in sugi (Cryptomeria japonica) pollen.

Pollen from sugi (Japanese cedar, Cryptomeria japonica D. Don), a forest tree species that is widely grown in Japan, causes serious allergic disease. The major allergens from sugi pollen, Cry j 1 and Cry j 2, have been isolated and characterized. It has been reported that Cry j 1 concentration in pollen varies considerably among trees. If Cry j 1 concentration is genetically controlled, the planting of trees with low Cry j 1 concentrations would reduce pollinosis. We investigated genetic and environmental effects on Cry j 1 concentration in eight clones growing at four sites. Concentrations of Cry j 1 in pollen were measured with a monoclonal antibody-based Enzyme Linked Immunosorbent Assay (ELISA). The Cry j 1 concentrations differed significantly among clones and sites, but the site x clone interaction was not significant, suggesting that the Cry j 1 concentration is controlled primarily by genetic factors. We examined correlations between Cry j 1 concentration and temperature and precipitation from July through February. Temperature was not significantly related to Cry j 1 concentration, whereas cumulative precipitation during the 8 months and mean daily precipitation in September showed significant negative correlations with Cry j 1 concentration.

Xyloglucan for generating tensile stress to bend tree stem.

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.

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.

Tandem repeat DNA localizing on the proximal DAPI bands of chromosomes in Larix, Pinaceae.

Repetitive DNA was cloned from HindIII-digested genomic DNA of Larix leptolepis. The repetitive DNA was about 170 bp long, had an AT content of 67%, and was organized tandemly in the genome. Using fluorescence in situ hybridization and subsequent DAPI banding, the repetitive DNA was localized in DAPI bands at the proximal region of one arm of chromosomes in L. leptolepis and Larix chinensis. Southern blot hybridization to genomic DNA of seven species and five varieties probed with cloned repetitive DNA showed that the repetitive DNA family was present in a tandem organization in genomes of all Larix taxa examined. In addition to the 170-bp sequence, a 220-bp sequence belonging to the same DNA family was also present in 10 taxa. The 220-bp repeat unit was a partial duplication of the 170-bp repeat unit. The 220-bp repeat unit was more abundant in L. chinensis and Larix potaninii var. macrocarpa than in other taxa. The repetitive DNA composed 2.0-3.4% of the genome in most taxa and 0.3 and 0.5% of the genome in L. chinensis and L. potaninii var. macrocarpa, respectively. The unique distribution of the 220-bp repeat unit in Larix indicates the close relationship of these two species. In the family Pinaceae, the LPD (Larix proximal DAPI band specific repeat sequence family) family sequence is widely distributed, but their amount is very small except in the genus Larix. The abundant LPD family in Larix will occur after its speciation.