Two R2R3-MYB transcription factors from Chinese cedar (Cryptomeria fortunei Hooibrenk) are involved in the regulation of secondary cell wall formation.

As the most abundant renewable energy source, wood comprises the secondary cell wall (SCW). SCW biosynthesis involves lignin and cellulose deposition. Increasing studies have illustrated that R2R3-MYB transcription factors (TFs) play pivotal roles in affecting lignin accumulation and SCW formation. Nevertheless, the regulatory roles of R2R3-MYBs are still unresolved in Cryptomeria fortunei Hooibrenk cambium and wood formation. To dissect the potentials of CfMYBs, we successfully cloned and intensively studied the functions of CfMYB4 and CfMYB5 in SCW formation and abiotic stress response. They both contained the conserved MYB domain capable of forming a special structure that could bind to the core motifs of downstream genes. The phylogenetic tree implied that two CfMYBs clustered into different evolutionary branches. They were predominantly expressed in the stem and were localized to the nucleus. Furthermore, CfMYB4 functioned as an activator to enhance lignin and cellulose accumulation, and increase the SCW thickness by elevating the expression levels of SCW-related genes. By contrast, CfMYB5 negatively regulated lignin and cellulose biosynthesis, and decreased SCW formation by reducing the expression of SCW biosynthetic genes. Our data not only highlight the regulatory functions of CfMYBs in lignin deposition but also provide critical insights into the development of strategies for the genetic improvement of Cryptomeria fortunei wood biomass.

Biological activities of organic extracts and specialized metabolites from different parts of Cryptomeria japonica (Cupressaceae) - A critical review.

Forest operations and wood industry generate large amounts of residues that are discarded in the field and cause environmental pollution. However, these biomass residues are still raw materials to obtain value-added products, such as essential oils, organic/aqueous extracts and resins that are among the great natural sources of bioactive metabolites. Thus, in recent years, the scientific community is giving special attention to their valorization. To date, different uses of biomass residues have been proposed, such as a source of renewable energy, fertilizers, animal feed and bioactive molecules. In this context, Cryptomeria japonica biomass residues (e.g., bark and its exudate, heartwood, sapwood, leaves, cones and roots) represent a source of diverse specialized metabolites (e.g., sesqui-, di-, tri- and sesquarterpenes, flavonoids, lignans and norlignans) with potential application in different fields, particularly in the agrochemical, food, cosmeceutical, pharmaceutical, phytomedicine and esthetic, due to their valuable multi-bioactivities determined over the last decades. Thus, this review provides an overview of the reported biological activities of organic extracts/fractions and their specialized metabolites obtained from different parts of C. japonica, in order to encourage the alternative uses of C. japonica wastes/byproducts, and implement a sustainable and circular bioeconomy.

CfAPX, a cytosolic ascorbate peroxidase gene from Cryptomeria fortunei, confers tolerance to abiotic stress in transgenic Arabidopsis.

Plants subjected to biotic or abiotic stresses produce a large amount of reactive oxygen species (ROS). If ROS cannot be cleared in time, they cause a series of harmful reactions in plants. Ascorbate peroxidase (APX) is a key enzyme that removes ROS from plant cells and plays a vital role in plant stress resistance. However, to date, no studies on APX homologs in Cryptomeria fortunei have been reported. In this study, we isolated complementary DNA (cDNA) encoding APXfrom C. fortunei needles, which is referred to as CfAPX, by rapid amplification of cDNA ends (RACE). The full-length CfAPX sequence was 1226 bp in length and included a 750-bp open reading frame (ORF) encoding a protein of 249 amino acids. Phylogenetic analysis showed that APXs of different plant species have been highly evolutionarily conserved. CfAPX was shown to belong to the cytoplasmic subgroup and was more closely related to GbAPX of the gymnosperm Ginkgo biloba. CfAPX showed no transcriptional activity in yeast cells but was highly expressed in cones. To better handle abiotic stresses, compared with wild-type (WT) Arabidopsis thaliana, 35S::CfAPX transgenic Arabidopsis strongly expressed CfAPX, presented increased antioxidant enzyme activities, ascorbic acid (AsA) contents, chlorophyll levels and fluorescence parameter and reduced malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents. In addition, CfAPX expression in C. fortunei was mostly upregulated under stress. In summary, CfAPX confers abiotic stress responses to plants, which provides a scientific basis for subsequent breeding for increased stress resistance in C. fortunei.

Diversification of terpenoid emissions proposes a geographic structure based on climate and pathogen composition in Japanese cedar.

Biogenic volatile organic compounds emitted from plants are important constituents of atmospheric chemistry and play a major role in the resistance of plants against various environmental stresses. However, little is known about how abiotic and biotic environments on a geographic scale relate to diversifications of the emission. Here, we present variations of terpenes stored in and emitted from leaves of a single species in a common garden, using genetically differentiated local populations of Japanese cedar, the most dominant and widely distributed tree species in Japan. Furthermore, we determined the composition of fungal communities in 50 locations, based on the presence or absence of 158 fungal species inhabiting the cedar. The results showed that terpenoids, especially those that are emitted, were highly diversified and geographically structured among the 12 populations. The total amount of stored terpenes was negatively affected by warm and less-snow climates. On the other hand, variations in some emitted terpenoid species among the populations were correlated to antagonistic fungal species inhabiting the Japanese cedar. We propose that the diversification of composition and amount of stored and emitted terpenoids in the tree species is not only structured by climate, but also antagonistic fungal communities through biological interactions.

Cloning and cold-resistance analyses of CfICE1 gene in Cryptomeria fortunei.

Cryptomeria fortunei is a conifer species that can attain a height of ~70 m and is cultivated for its timber as well as its ornamental value. It is a subtropical plant that prefers a warm and humid environment. Therefore, low temperature (LT) affects its growth, development, productivity and ecological distribution. Inducer of C-repeat binding factor (CBF) expression 1 (ICE1) plays an important role in the response to cold/freezing stress in plants through the CBF regulation pathway. To date, there is no research on homologue of ICE1 in C. fortunei. In this study, we first isolated the CfICE1 transcript from C. fortunei. The CfICE1 coding sequence was 1728 nucleotides encoding a 575-aa protein and contained a serine-rich motif, a basic helix-loop-helix-Zipper (bHLH-Zip), an ACT domain and a nuclear localization signal (NLS), which were conserved in ICE1 homologous genes. Phylogenetic analysis showed that CfICE1 and all dicots ICE1 proteins were clustered together. CfICE1 had transcriptional activity in yeast cells, was predominantly located in the nucleus and highly expressed in tender needles and roots. 35S::CfICE1 transgenic Arabidopsis thaliana could increase antioxidant enzyme activities and photosynthesis and reduce the malondialdehyde content compared to the wild-type to better cope with LT. Under LT, CfICE1 expression was higher; the C. fortunei clone with stronger cold resistance (CR) significantly upregulated the expression of CfICE1 compared to the weaker clone. In conclusion, these results suggest that CfICE1 plays an active role in CR, which provides a theoretical basis for breeding for CR in C. fortunei.

Identification and genetic diversity analysis of a male-sterile gene (MS1) in Japanese cedar (Cryptomeria japonica D. Don).

Identifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.

Surface Structural Transformation of Pre-carbonized Solid Biomass from Japanese Cedar via ATR-FTIR and PCA.

This present research applied the ATR-FTIR technique and principle component analysis (PCA) to investigate molecular surface changes in pre-carbonized solid biomass, called Kindai Bio-coke (BIC) and Japanese cedar. The product is utilized as an alternative to coal coke in the cupola furnace in the steel industry in order to reduce CO2 emissions. The aim is to explore key elements for improving the BIC product applications from the fundamental molecular scale by using PCA to distinguish between changes during the BIC transformation and the differences in BIC samples. Results revealed that transformation occurred at the surface of Japanese cedar raw materials and Japanese cedar BIC. Major changes were observed in the O-H, C-H and C-O stretching regions. The intensity of the IR bands attributed to aliphatic methyl (CH3) and methylene (CH2) stretching modes increased, while a weak O-H stretching intensity associated with BIC hydrophobic characteristic decreased.

Cloning and Functional Analysis of Lignin Biosynthesis Genes Cf4CL and CfCCoAOMT in Cryptomeria fortunei.

Cryptomeria fortunei, also known as the Chinese cedar, is an important timber species in southern China. The primary component of its woody tissues is lignin, mainly present in secondary cell walls. Therefore, continuous lignin synthesis is crucial for wood formation. In this study, we aimed to discover key genes involved in lignin synthesis expressed in the vascular cambium of C. fortunei. Through transcriptome sequencing, we detected expression of two genes, 4CL and CCoAOMT, known to be homologous to enzymes involved in the lignin synthesis pathway. We studied the function of these genes through bioinformatics analysis, cloning, vascular cambium expression analysis, and transgenic cross-species functional validation studies. Our results show that Cf4CL and CfCCoAOMT do indeed function in the pathway of lignin synthesis and likely perform this function in C. fortunei. They are prime candidates for future (gene-editing) studies aimed at optimizing C. fortunei wood production.

Prediction of Lignin Contents from Infrared Spectroscopy: Chemical Digestion and Lignin/Biomass Ratios of Cryptomeria japonica.

A method for the high-throughput analysis of the relative lignin contents of Cryptomeria japonica samples over a wide concentration range (3-73%), independent of the type of chemical pretreatment, was developed by using Fourier transform infrared spectroscopy. First, the assignments of the infrared absorbance related to lignin were reviewed. Then, various chemical treatments, including alkaline, acid, and hydrothermal processes, and a sodium chlorite oxidation treatment, were performed to prepare samples containing a wide range of different lignin contents. Principal component analysis indicated high variability among the chemical treatments in terms of the corresponding lignin contents as well as the resulting changes in the chemical structure of hemicellulose; this conclusion was supported by the loading vectors. The intensity of the key band of lignin at 1508 cm-1 was calculated using the absorbance at 2900 cm-1 as a reference; a reliable calibration curve with an R2 of 0.968 was obtained independent of the chemical treatment performed. This simple and rapid method for determining the lignin content is expected to be widely applicable for optimizing bioethanol production, as well as monitoring biomass degradation processes.

Translocation of 133Cs administered to Cryptomeria japonica wood.

To reveal the in planta behaviour of caesium (Cs), the stable isotope 133Cs was administered into 3-year-old Cryptomeria japonica seedlings by the application of 133CsCl aqueous solution to the bark surface. The administered 133Cs was quantified by ICP-MS measurements, which showed transportation of 133Cs in an ascending direction in the stem. Distribution of 133Cs was visualized using freeze-fixed C. japonica woody stem samples and cryo-time-of-flight secondary ion mass spectrometry/scanning electron microscopy (cryo-TOF-SIMS/SEM) analysis. Cryo-TOF-SIMS/SEM visualization suggested that 133Cs was rapidly transported radially by ray parenchyma cells followed by axial transportation by pith and axial parenchyma cells. Adsorption experiments using powdered C. japonica wood samples and X-ray absorption fine structure (XAFS) analysis suggested that 133Cs was in the hydrated state following its deposition into tracheid cell walls.

Effect of ionic liquid treatment on the ultrastructural and topochemical features of compression wood in Japanese cedar (Cryptomeria japonica).

The morphological and topochemical changes in wood tissues in compression wood of Japanese cedar (Cryptomeria japonica) upon treated with two types of ionic liquids, 1-ethyl-3-methylimidazolium chloride ([C2mim][Cl]) and 1-ethylpyridinium bromide ([EtPy][Br]) were investigated. Compression wood tracheids were swollen by both ionic liquids but their swelling behaviors were different in the types of ionic liquids used. Under the polarized light, we confirmed that crystalline cellulose in compression wood is amorphized by [C2mim][Cl] treatment whereas it changes slightly by [EtPy][Br] treatment. Raman microscopic analyses revealed that [C2mim][Cl] can preferentially liquefy polysaccharides in compression wood whereas [EtPy][Br] liquefy lignin. In addition, the interaction of compression wood with ionic liquids is different for the morphological regions. These results will assist in the use of ionic liquid treatment of woody biomass to produce valuable chemicals, bio-fuels, bio-based composites and other products.

Carbon Sequestration Potential in Stands under the Grain for Green Program in Southwest China.

The Grain for Green Program (GGP) is the largest afforestation and reforestation project in China in the early part of this century. To assess carbon sequestration in stands under the GGP in Southwest China, the carbon stocks and their annual changes in the GGP stands in the region were estimated based on the following information: (1) collected data on the annually planted area of each tree species under the GGP in Southwest China from 1999 to 2010; (2) development of empirical growth curves and corresponding carbon estimation models for each species growing in the GPP stands; and (3) parameters associated with the stands such as wood density, biomass expansion factor, carbon fraction and the change rate of soil organic carbon content. Two forest management scenarios were examined: scenario A, with no harvesting, and scenario B, with logging at the customary rotation followed by replanting. The results showed that by the years 2020, 2030, 2040, 2050 and 2060, the expected carbon storage of the GGP stands in Southwest China is 139.58 TgC, 177.50-207.55 TgC, 196.86-259.65 TgC, 240.45-290.62 TgC and 203.22-310.03 TgC (T = 1012), respectively. For the same years, the expected annual change in carbon stocks is 7.96 TgCyr-1, -7.95-5.95 TgCyr-1, -0.10-4.67 TgCyr-1, 4.31-2.24 TgCyr-1 and -0.02-1.75 TgCyr-1, respectively. This indicates that the stands significantly contribute to forest carbon sinks in this region. In 2060, the estimated carbon stocks in the seven major species of GGP stands in Southwest China are 4.16-13.01 TgC for Pinus armandii, 6.30-15.01 TgC for Pinus massoniana, 11.51-13.44 TgC for Cryptomeria fortunei, 15.94-24.13 TgC for Cunninghamia lanceolata, 28.05 TgC for Cupressus spp., 5.32-15.63 TgC for Populus deltoides and 5.87-14.09 TgC for Eucalyptus spp. The carbon stocks in these seven species account for 36.8%-41.4% of the total carbon stocks in all GGP stands over the next 50 years.

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.

Changes in the distribution of radiocesium in the wood of Japanese cedar trees from 2011 to 2013.

The changes in the distribution of (137)Cs in the wood of Japanese cedar (Cryptomeria japonica) trunks within three years after the Fukushima Dai-ichi Nuclear Power Plant (FDNP) accident in 2011 were investigated. Thirteen trees were felled to collect samples at 6 forests in 2 regions of the Fukushima prefecture. The radial distribution of (137)Cs in the wood was measured at different heights. Profiles of (137)Cs distribution in the wood changed considerably from 2011 to 2013, and the process of (137)Cs distribution change in the wood was clarified. From 2011 to 2012, the active transportation from sapwood to heartwood and the radial diffusion in heartwood proceeded quickly, and the radial (137)Cs distribution differed according to the vertical positon of trees. From 2012 to 2013, the vertical diffusion of (137)Cs from the treetop to the ground, probably caused by the gradient of (137)Cs concentration in the trunk, was observed. Eventually, the radial (137)Cs distributions were nearly identical at any vertical positions in 2013. Our results suggested that the active transportation from sapwood to heartwood and the vertical and radial diffusion in heartwood proceeded according to the vertical position of the tree and (137)Cs distribution in the wood approached the equilibrium state within three years after the accident.

Distribution of coniferin in differentiating normal and compression woods using MALDI mass spectrometric imaging coupled with osmium tetroxide vapor treatment.

Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was employed to detect monolignol glucosides in differentiating normal and compression woods of two Japanese softwoods, Chamaecyparis obtusa and Cryptomeria japonica Comparison of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry collision-induced dissociation fragmentation analysis and structural time-of-flight (MALDI-TOF CID-FAST) spectra between coniferin and differentiating xylem also confirmed the presence of coniferin in differentiating xylem. However, as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF CID-FAST spectra of sucrose were similar to those of coniferin, it was difficult to distinguish the distribution of coniferin and sucrose using MALDI-MSI and collision-induced dissociation measurement only. To solve this problem, osmium tetroxide vapor was applied to sections of differentiating xylem. This vapor treatment caused peak shifts corresponding to the introduction of two hydroxyl groups to the C=C double bond in coniferin. The treatment did not cause a peak shift for sucrose, and therefore was effective in distinguishing coniferin and sucrose. Thus, it was found that MALDI-MSI combined with osmium tetroxide vapor treatment is a useful method to detect coniferin in differentiating xylem.

Radiocarbon concentration in modern tree rings from Fukushima, Japan.

A 30-year-old Japanese cedar (Cryptomeria japonica), collected from Iwaki, Fukushima in 2014, was analyzed for the long-lived radionuclide (14)C. Values of Δ(14)C varied from 211.7‰ in 1984 to 16.9‰ in 2013. The temporal Δ(14)C variation can be described as an exponential decline, indistinguishable from the general Northern Hemisphere Zone 2 (NH Zone 2) values in the atmosphere, until at least 1994. Values of Δ(14)C for 1999 and 2004 are slightly depleted compared with NH Zone 2 values, while from 1999 to 2013 the data suggest a clear depletion with a 2-8 ppmV additional CO2 contribution from a (14)C-free (i.e. fossil carbon) source. This change coincides with local traffic increases since two nearby expressways were opened in the 1990's. In addition, the small but visible (14)C pulse observed in the 2011 tree-ring might be caused by release from the damaged reactors during the Fukushima nuclear accident.

Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in Escherichia coli.

Peptides containing T-cell epitopes from allergens, which are not reactive to allergen-specific IgE, are appropriate candidates as antigens for specific immunotherapy against allergies. To develop a vaccine that can be used in practical application to prevent and treat Japanese cedar pollen allergy, four major T-cell epitopes from the Cry j 1 antigen and six from the Cry j 2 antigen were selected to design cry j 1 epi and cry j 2 epi, DNA constructs encoding artificial polypeptides of the selected epitopes. To apply cholera toxin B subunit (CTB) as an adjuvant, cry j 1 epi and cry j 2 epi were linked and then fused to the CTB gene in tandem to construct a fusion gene, ctb-linker-cry j 1 epi- cry j 2 epi-flag. The fusion gene was introduced into a pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). The expressed recombinant protein was purified by a His-tag affinity column and confirmed by western blot analysis using anti-CTB and anti-FLAG antibodies. The purified recombinant protein also proved to be antigenic against anti-Cry j 1 and anti-Cry j 2 antibodies. Expression of the recombinant protein induced with 1mM IPTG reached a maximum in 3-5h, and recovery of the affinity-purified recombinant protein was approximately 120mg/L of culture medium. The present study indicates that production of sufficient amounts of recombinant protein with antigenic epitopes may be possible by recombinant techniques using E. coli or other bacterial strains for protein expression.

Radiocesium distribution in sugi (Cryptomeria japonica) in eastern Japan: translocation from needles to pollen.

We assessed the radiocesium contamination of sugi (Cryptomeria japonica) forests in eastern Japan from November 2012 to February 2013, including 80 sites in Fukushima and 35 sites in other regions (Tohoku and Kanto-Koshinetsu), by measuring the (137)Cs concentrations in needles of different ages, male flowers, and pollen. Over a wide geographic area, needles that were present at the time of the Fukushima Dai-ichi Nuclear Power Plant accident contained much higher (137)Cs concentrations than needles that emerged after the accident. This result, together with visual analysis of (137)Cs distribution using autoradiography, indicated that some of the (137)Cs derived from direct fallout remained on the surface of the older needles. Since we also detected (137)Cs in younger needles and male flowers, we concluded that (137)Cs was translocated toward the tips of sugi needles. The (137)Cs concentration in male flowers was higher than and positively correlated with that in the currently growing (2012) needles. Also, a positive relationship was observed between the (137)Cs concentration of male flowers and pollen, and they were found to be nearly identical (137)Cs concentration. These results indicate the occurrence of acropetal translocation of (137)Cs from old needles to young needles, male flowers and pollen. However, the results as related to (137)Cs concentration in the needles of three different ages differed from the results of similar studies conducted more than 4 y after the Chernobyl accident. This suggests that, 2 y after the Fukushima Dai-ichi NPP accident, the distribution of (137)Cs in the sugi forests has not yet reached a steady state.

Atmospheric direct uptake and long-term fate of radiocesium in trees after the Fukushima nuclear accident.

Large areas of forests were radioactively contaminated by the Fukushima nuclear accident of 2011, and forest decontamination is now an important problem in Japan. However, whether trees absorb radioactive fallout from soil via the roots or directly from the atmosphere through the bark and leaves is unclear. We measured the uptake of radiocesium by trees in forests heavily contaminated by the Fukushima nuclear accident. The radiocesium concentrations in sapwood of two tree species, the deciduous broadleaved konara (Quercus serrata) and the evergreen coniferous sugi (Cryptomeria japonica), were higher than that in heartwood. The concentration profiles showed anomalous directionality in konara and non-directionality in sugi, indicating that most radiocesium in the tree rings was directly absorbed from the atmosphere via bark and leaves rather than via roots. Numerical modelling shows that the maximum (137)Cs concentration in the xylem of konara will be achieved 28 years after the accident. Conversely, the values for sugi will monotonously decrease because of the small transfer factor in this species. Overall, xylem (137)Cs concentrations will not be affected by root uptake if active root systems occur 10 cm below the soil.

Clock genes and diurnal transcriptome dynamics in summer and winter in the gymnosperm Japanese cedar (Cryptomeria japonica (L.f.) D.Don).

BACKGROUND: The circadian clock and diurnal dynamics of the transcriptome are presumed to play important roles in the regulation of physiological, biological and developmental processes synchronized with diurnal and annual cycles of plant environments. However, little is known about the circadian clock and its regulation in gymnosperms, including conifers. Here we present the diurnal transcriptome dynamics of Japanese cedar (Cryptomeria japonica (L.f.) D.Don) in both active (summer) and dormant (winter) periods. RESULTS: Microarray analysis revealed significant differences in transcripts between summer and winter, and diurnal transcriptome dynamics only in the summer. About 7.7% of unique genes (556 out of 7,254) on the microarray were periodically expressed in summer. Expression patterns of some genes, especially light-related genes, did not show significant oscillation in Japanese cedar, thus differing from those reported in angiosperms. Gene network analysis of the microarray data revealed a network associated with the putative core clock genes (CjLHYa, CjLHYb, CjTOC1, CjGI and CjZTL), which were also isolated, indicating their importance in the diurnal regulation of the transcriptome. CONCLUSION: This study revealed the existence of core clock genes and diurnal rhythms of the transcriptome in summer in Japanese cedar. Dampening of diurnal rhythms in winter indicated seasonal change in the rhythms according to environmental conditions. The data also revealed genes that showed different expression patterns compared to angiosperms, suggesting a unique gene regulatory network in conifers. This study provides fundamental data to understand transcriptional regulatory mechanisms in conifers.