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).

Agatharesinol biosynthesis-related changes of ray parenchyma in sapwood sticks of Cryptomeria japonica during cell death.

MAIN CONCLUSION: The work demonstrates a relationship between the biosynthesis of the secondary metabolite, agatharesinol, and cytological changes that occur in ray parenchyma during cell death in sapwood sticks of Cryptomeria japonica under humidity-regulated conditions. To characterize the death of ray parenchyma cells that accompanies the biosynthesis of secondary metabolites, we examined cell death in sapwood sticks of Cryptomeria japonica under humidity-regulated conditions. We monitored features of ray parenchyma cells, such as viability, the morphology of nuclei and vacuoles, and the amount of starch grains. In addition, we analyzed levels of agatharesinol, a heartwood norlignan, by gas chromatography-mass spectrometry in the same sapwood sticks. Dramatic changes in the amount of starch grains and in the level of agatharesinol occurred simultaneously. Therefore, the biosynthesis of agatharesinol appeared to originate from the breakdown of starch. Furthermore, we observed the expansion of vacuoles in ray parenchyma cells prior to other cytological changes at the final stage of cell death. In our experimental system, we were able to follow the process of cell death and to demonstrate relationships between cytological changes and the biosynthesis of a secondary metabolite during the death of ray parenchyma cells.

Topochemical and morphological characterization of wood cell wall treated with the ionic liquid, 1-ethylpyridinium bromide.

MAIN CONCLUSION : [EtPy][Br] is more reactive toward lignin than toward the PSs in wood cell walls, and [EtPy][Br] treatment results in inhomogenous changes to the cell wall's ultrastructural and chemical components. The effects of the ionic liquid 1-ethylpyridinium bromide ([EtPy][Br]), which prefers to react with lignin rather than cellulose on the wood cell walls of Japanese cedar (Cryptomeria japonica), were investigated from a morphology and topochemistry point of view. The [EtPy][Br] treatment induced cell wall swelling, the elimination of warts, and the formation of countless pores in the tracheids. However, many of the pit membranes and the cellulose crystalline structure remained unchanged. Raman microscopic analyses revealed that chemical changes in the cell walls were different for different layers and that the lignin in the compound middle lamella and the cell corner resists interaction with [EtPy][Br]. Additionally, the interaction of [EtPy][Br] with the wood cell wall is different to that of other types of ionic liquid.

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.

The function of intercellular spaces along the ray parenchyma in sapwood, intermediate wood, and heartwood of Cryptomeria japonica (Cupressaceae).

PREMISE OF THE STUDY: Intercellular spaces along ray parenchyma (ISRP) are common in many conifer xylems, but their function is uncertain because the in-situ structural network among ISRP, ray parenchyma, and tracheids has not been evaluated. Analysis of water distribution in ISRP from sapwood to heartwood is needed to elucidate the function of ISRP in sapwood, intermediate wood, and heartwood. METHODS: We used cryo-scanning electron microscopy, x-ray photography, and water content measurement in xylem to analyze the presence of liquids in ISRP, ray parenchyma, and tracheids from sapwood to heartwood in Cryptomeria japonica (Cupressaceae). KEY RESULTS: In sapwood, almost all ISRP were empty. "Cingulate-cavitated regions", which lose water along the tangential direction within one annual ring, formed in the earlywood tracheids, and their frequency increased toward the inner annual rings, whereas ray parenchyma cells were alive and not involved in the partial cavitation. In intermediate wood, almost all ISRP and earlywood tracheids and many of the ray cells were empty, and only some latewood tracheids retained liquid in their lumina. The ISRP were connected with tracheids via gas-filled ray parenchyma cells. CONCLUSIONS: The ISRP work as a pathway of gas for aspiration of ray parenchyma cells in sapwood. On the other hand, the occurrence of a gas network between ISRP, ray parenchyma, and tracheids facilitates cavitation of tracheids, resulting in the generation of low-moisture, intermediate wood.

A rapid decrease in temperature induces latewood formation in artificially reactivated cambium of conifer stems.

BACKGROUND AND AIMS: Latewood formation in conifers occurs during the later part of the growing season, when the cell division activity of the cambium declines. Changes in temperature might be important for wood formation in trees. Therefore, the effects of a rapid decrease in temperature on cellular morphology of tracheids were investigated in localized heating-induced cambial reactivation in Cryptomeria japonica trees and in Abies firma seedlings. METHODS: Electric heating tape and heating ribbon were wrapped on the stems of C. japonica trees and A. firma seedlings. Heating was discontinued when 11 or 12 and eight or nine radial files of differentiating and differentiated tracheids had been produced in C. japonica and A. firma stems, respectively. Tracheid diameter, cell wall thickness, percentage of cell wall area and percentage of lumen area were determined by image analysis of transverse sections and scanning electron microscopy. KEY RESULTS: Localized heating induced earlier cambial reactivation and xylem differentiation in stems of C. japonica and A. firma as compared with non-heated stems. One week after cessation of heating, there were no obvious changes in the dimensions of the differentiating tracheids in the samples from adult C. japonica. In contrast, tracheids with a smaller diameter were observed in A. firma seedlings after 1 week of cessation of heating. Two or three weeks after cessation of heating, tracheids with reduced diameters and thickened cell walls were found. The results showed that the rapid decrease in temperature produced slender tracheids with obvious thickening of cell walls that resembled latewood cells. CONCLUSIONS: The results suggest that a localized decrease in temperature of stems induces changes in the diameter and cell wall thickness of differentiating tracheids, indicating that cambium and its derivatives can respond directly to changes in temperature.

Temporal and spatial diversities of the immunolabeling of mannan and xylan polysaccharides in differentiating earlywood ray cells and pits of Cryptomeria japonica.

Wood is composed of various types of cells and each type of cell has different structural and functional properties. However, the temporal and spatial diversities of cell wall components in the cell wall between different cell types are rarely understood. To extend our understanding of distributional diversities of cell wall components among cells, we investigated the immunolabeling of mannans (O-acetyl-galactoglucomannans, GGMs) and xylans (arabino-4-O-methylglucuronoxylans, AGXs) in ray cells and pits. The labeling of GGMs and AGXs was temporally different in ray cells. GGM labeling began to be detected in ray cells at early stages of S(1) formation in tracheids, whereas AGX labeling began to be detected in ray cells at the S(2) formation stage in tracheids. The occurrence of GGM and AGX labeling in ray cells was also temporally different from that of tracheids. AGX labeling began to be detected much later in ray cells than in tracheids. GGM labeling also began to be detected in ray cells either slightly earlier or later than in tracheids. In pits, GGM labeling was detected in bordered and cross-field pit membranes at early stages of pit formation, but not observed in mature pits, indicating that enzymes capable of GGM degradation may be involved in pit membrane formation. In contrast to GGMs, AGXs were not detected in pit membranes during the entire developmental process of bordered and cross-field pits. AGXs showed structural and depositional variations in pit borders depending on the developmental stage of bordered and cross-field pits.

Occurrence of xylan and mannan polysaccharides and their spatial relationship with other cell wall components in differentiating compression wood tracheids of Cryptomeria japonica.

Compression wood (CW) tracheids have different cell wall components than normal wood (NW) tracheids. However, temporal and spatial information on cell wall components in CW tracheids is poorly understood. We investigated the distribution of arabino-4-O-methylglucuronoxylans (AGXs) and O-acetyl-galactoglucomannans (GGMs) in differentiating CW tracheids. AGX labeling began to be detected in the corner of the S(1) layer at the early S(1) formation stage. Subsequently, the cell corner middle lamella (ccML) showed strong AGX labeling when intercellular spaces were not fully formed. AGX labeling was uniformly distributed in the S(1) layer, but showed uneven distribution in the S(2) layer. AGX labeling was mainly detected in the inner S(2) layer after the beginning of the helical cavity formation. The outer S(2) layer showed almost no labeling of low substituted AGXs. Only a very small amount of high substituted AGXs was distributed in the outer S(2) layer. These patterns of AGX labeling in the S(2) layer opposed the lignin and ß-1-4-galactan distribution in CW tracheids. GGM labeling patterns were almost identical to AGX labeling in the early stages of CW tracheids, and GGM labeling was detected in the entire S(2) layer from the early S(2) formation stage of CW tracheids with some spatial differences in labeling density depending on developmental stage. Compared with NW tracheids, CW tracheids showed significantly different AGX distributions in the secondary cell wall but similar GGM labeling patterns. No significant differences were observed in labeling after delignification of CW tracheids.

Immunolocalization and structural variations of xylan in differentiating earlywood tracheid cell walls of Cryptomeria japonica.

We investigated the spatial and temporal distribution of xylans in the cell walls of differentiating earlywood tracheids of Cryptomeria japonica using two different types of monoclonal antibodies (LM10 and LM11) combined with immunomicroscopy. Xylans were first deposited in the corner of the S(1) layer in the early stages of S(1) formation in tracheids. Cell corner middle lamella also showed strong xylan labeling from the early stage of cell wall formation. During secondary cell wall formation, the innermost layer and the boundary between the S(1) and S(2) layers (S(1)/S(2) region) showed weaker labeling than other parts of the cell wall. However, mature tracheids had an almost uniform distribution of xylans throughout the entire cell wall. Xylan localization labeled with LM10 antibody was stronger in the outer S(2) layer than in the inner layer, whereas xylans labeled with LM11 antibody were almost uniformly distributed in the S(2) layer. In addition, the LM10 antibody showed almost no xylan labeling in the S(1)/S(2) region, whereas the LM11 antibody revealed strong xylan labeling in the S(1)/S(2) region. These findings suggest that structurally different types of xylans may be deposited in the tracheid cell wall depending on the developmental stage of, or location in, the cell wall. Our study also indicates that deposition of xylans in the early stages of tracheid cell wall formation may be spatially consistent with the early stage of lignin deposition in the tracheid cell wall.

A heartwood norlignan, (E)-hinokiresinol, is formed from 4-coumaryl 4-coumarate by a Cryptomeria japonica enzyme preparation.

An enzyme preparation from the cultured cells of Cryptomeria japonica catalyses the formation of a heartwood norlignan, (E)-hinokiresinol, from two distinct phenylpropanoid monomers: 4-coumaroyl CoA and 4-coumaryl alcohol, and from a dimer: 4-coumaryl 4-coumarate.

Immunohistochemical localization of agatharesinol, a heartwood norlignan, in Cryptomeria japonica.

Localization of a heartwood norlignan, agatharesinol, in Sugi (Japanese cedar, Cryptomeria japonica D. Don, Taxodiaceae) was investigated by immunohistochemistry. Immuno light microscopy showed that the contents of ray parenchyma cells were immunostained in heartwood but not in sapwood. The staining of the heartwood tissue was competitively inhibited by agatharesinol but not by other Sugi heartwood extractives, and was, furthermore, markedly reduced by pre-extraction of the tissue with MeOH. These results indicated that the staining can be ascribed to the immunolabeling of agatharesinol in situ. The accumulations over the inner surface of some tracheid cell walls adjacent to the ray parenchyma cells were also immunolabeled, while the contents in axial parenchyma cells were not. In conclusion, agatharesinol was localized in the ray parenchyma cells in Sugi heartwood, and differences between the chemical structure of the contents of ray and axial parenchyma cells were also suggested.