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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease system is a versatile and essential biotechnological tool in the life sciences that allows efficient genome editing. When generating gene-edited trees, T0-generation plants are often used for subsequent analysis because of the time that is required to obtain the desired mutants via crossing. However, T0-generation plants exhibit various unexpected mutations, which emphasizes the need to identify mutants with expected mutation patterns. The two critical checkpoints in this process are to confirm the expected mutation patterns in both alleles and to exclude somatic chimeric plants. In this study, we generated gene-edited Cryptomeria japonica plants and established a method to determine chimerism and mutation patterns using fragment analysis and Oxford Nanopore Technologies (ONT)-based amplicon sequencing. In the first screening, fragment analysis, i.e. indel detection via amplicon analysis, was used to predict indel mutation patterns in both alleles and to discriminate somatic chimeric plants in 188 candidate mutants. In the second screening, we precisely determined the mutation patterns and chimerism in the mutants using ONT-based amplicon sequencing, where confirmation of both alleles can be achieved using allele-specific markers flanking the single guide RNA target site. In the present study, a bioinformatic analysis procedure was developed and provided for the rapid and accurate determination of DNA mutation patterns using ONT-based amplicon sequencing. As ONT amplicon sequencing has a low running cost compared with other long-read analysis methods, such as PacBio, it is a powerful tool in plant genetics and biotechnology to select gene-edited plants with expected indel patterns in the T0-generation. [ABSTRACT FROM AUTHOR]

Introductory biology lessons around the world typically teach that plants absorb water through their roots, but, unfortunately, absorption of water through leaves and subsequent transport and use of this water for biomass formation remains a field limited mostly to specialists. Recent studies have identified foliar water uptake as a significant net water source for terrestrial plants. The growing interest in the development of a new model that includes both foliar water uptake (in liquid form) and root water uptake to explain hydrogen and oxygen isotope ratios in leaf water and tree rings demands a method for distinguishing between these two water sources. Therefore, in this study, I have devised a new labelling method that utilizes two different water sources, one enriched in deuterium (HDO + D2O; δ D  = 7.0 × 10 4‰, δ18O = 4.1‰) and one enriched in oxygen-18 (H218O; δ D  = −85‰, δ18O = 1.1 × 104‰), to simultaneously label both foliar-absorbed and root-absorbed water and quantify their relative contributions to plant biomass. Using this new method, I here present evidence that, in the case of well-watered Cryptomeria japonica D. Don, hydrogen and oxygen incorporated into new leaf cellulose in the rainy season derives mostly from foliar-absorbed water (69% from foliar-absorbed water and 31% from root-absorbed water), while that of new root cellulose derives mostly from root-absorbed water (20% from foliar-absorbed water and 80% from root-absorbed water), and new branch xylem is somewhere in between (55% from foliar-absorbed water and 45% from root-absorbed water). The dual-labelling method first implemented in this study enables separate and simultaneous labelling of foliar-absorbed and root-absorbed water and offers a new tool to study the uptake, transport and assimilation processes of these waters in terrestrial plants. [ABSTRACT FROM AUTHOR]

Forest soils are the largest terrestrial sink of methane (CH4), but CH4 produced in tree trunks by methanogenic archaea and emitted into the atmosphere can significantly offset CH4 oxidation in the soil. However, our mechanistic understanding of CH4 accumulation in tree trunks, in relation to CH4 emission from the trunk surface, is still limited. We characterized temporal variations in the molar fraction of CH4 in the heartwood of trees ([CH4]HW) of four different species in a mountain forest and addressed the relationship between [CH4]HW and emission from the surface of the trunk (${F}_{CH_4}$), in connection with the characteristics of the wood. [CH4]HW measurements were made monthly for 15 months using gas-porous tubes permanently inserted into the trunk. [CH4]HW were above ambient CH4 molar fraction for all trees, lower than 100 p.p.m. for seven trees, higher for the nine other trees and greater than 200,000 p.p.m. (>20%) for two of these nine trees. [CH4]HW varied monthly but were not primarily determined by trunk temperature. Heartwood diffusive resistance for CH4 was variable between trees, not only due to heartwood characteristics but probably also related to source location. ${F}_{CH_4}$were weakly correlated with [CH4]HW measured a few days after. The resulting apparent diffusion coefficient was also variable between trees suggesting variations in the size and location of the CH4 production sites as well as resistance to gas transport within the trunk. Our results highlight the challenges that must be overcome before CH4 emissions can be simulated at the tree level., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Compared with stem water storage, leaf water storage is understudied although it may be important for alleviating water stress by contributing quickly and directly to transpiration demand. To quantify the relative contribution of stem versus leaf water storage to daily water deficit, we measured diurnal changes in transpiration rate, sap-flow velocity and stem radius of 10-year-old Cryptomeria japonica D. Don trees. We assumed that the duration of time lags between transpiration rate and sap-flow velocity reflected stored water in the stem and leaf, and that stem volume change represented water content of elastic tissue. The relationship between fresh mass and water potential of the whole tree indicated that the study trees had capacity to store, on average, 91.4 ml of water per kg fresh mass at turgor loss. Leaves, sapwood and elastic tissue contributed around 51%, 29% and 20% of stored water, respectively. During morning, transpiration rates were higher than sap-flow velocity suggesting depletion of stored water. During the first 2 h after onset of transpiration, stored water contributed more than 100% of whole-tree transpiration. Depletion of leaf water (PLeaf) and sapwood water (PSap) coincided with the onset of transpiration and became maximum around 15:00 h. Depletion of elastic tissue water (PElasticm) lagged behind that of PLeaf and PSap by 1-2 h, indicating that replenishment of stored water occurs late in the day when low leaf water potentials resulting from daytime transpiration drive water uptake. Maximum depletion of PLeaf was about 1-3 times and 5-10 times that of PSap and PElasticm, respectively. The contribution of PLeaf to total daily transpiration was 5-8%, while those of PSap and PElasticm were 3-4% and 0.7-1%, respectively. Our results suggest the importance of leaf water storage in maintaining daily transpiration in young C. japonica trees. [ABSTRACT FROM AUTHOR]

Recent studies in the tallest tree species suggest that physiological and anatomical traits of tree-top leaves are adapted to water-limited conditions. In order to examine water retention mechanism of leaves in a tall tree, infrared (IR) micro-spectroscopy was conducted on mature leaf cross-sections of tall Cryptomeria japonica D. Don from four different heights (51, 43, 31 and 19 m). We measured IR transmission spectra and mainly analyzed OH (3700-3000 cm-1) and C-O (1190-845 cm-1) absorption bands, indicating water molecules and sugar groups, respectively. The changes in IR spectra of leaf sections from different heights were compared with bulk-leaf hydraulics. Both average OH band area of the leaf sections and leaf water content were larger in the upper-crown, while osmotic potential at saturation did not vary with height, suggesting higher dissolved sugar contents of upper-crown leaves. As cell-wall is the main cellular structure of leaves, we inferred that larger average C-O band area of upper-crown leaves reflected higher content of structural polysaccharides such as cellulose, hemicellulose and pectin. Infrared microspectroscopic imaging showed that the OH and C-O band areas are large in the vascular bundle, transfusion tissue and epidermis. Infrared spectra of individual tissue showed that much more water is retained in vascular bundle and transfusion tissue than mesophyll. These results demonstrate that IR micro-spectroscopy is a powerful tool for visualizing detailed, quantitative information on the spatial distribution of chemical substances within plant tissues, which cannot be done using conventional methods like histochemical staining. The OH band could be well reproduced by four Gaussian OH components around 3530 (free water: long H bond), 3410 (pectin-like OH species), 3310 (cellulose-like OH species) and 3210 (bound water: short H bond) cm-1, and all of these OH components were higher in the upper crown while their relative proportions did not vary with height. Based on the spectral analyses, we inferred that polysaccharides play a key role in biomolecular retention of water in leaves of tall C. japonica. [ABSTRACT FROM AUTHOR]

In order to predict the effects of future atmospheric conditions on forest productivity, it is necessary to clarify the physiological responses of major forest tree species to high concentrations of ozone (O3) and carbon dioxide (CO2). Furthermore, intraspecific variation of these responses should also be examined in order to predict productivity gains through tree improvements in the future. We investigated intraspecific variation in growth and photosynthesis of Cryptomeria japonica D. Don, a major silviculture species in Japan, in response to elevated concentrations of O3 (eO3) and CO2 (eCO2), separately and in combination. Cuttings of C. japonica were grown and exposed to two levels of O3 (ambient and twice-ambient levels) in combination with two levels of CO2 (ambient and 550 µmol mol-1 in the daytime) for two growing seasons in a free-air CO2 enrichment experiment. There was no obvious negative effect of eO3 on growth or photosynthetic traits of the C. japonica clones, but a positive effect was observed for annual height increments in the first growing season. Dry mass production and the photosynthetic rate increased under eCO2 conditions, while the maximum carboxylation rate decreased. Significant interaction effects of eO3 and eCO2 on growth and photosynthetic traits were not observed. Clonal effects on growth and photosynthetic traits were significant, but the interactions between clones and O3 and/or CO2 treatments were not. Spearman's rank correlation coefficients between growth traits under ambient conditions and for each treatment were significantly positive, implying that clonal ranking in growth abilities might not be affected by either eO3 or eCO2. The knowledge obtained from this study will be helpful for species selection in afforestation programs, to continue and to improve current programs involving this species, and to accurately predict the CO2 fixation capacity of Japanese forests. [ABSTRACT FROM AUTHOR]

In order to predict the effects of future atmospheric conditions on forest productivity, it is necessary to clarify the physiological responses of major forest tree species to high concentrations of ozone (O3) and carbon dioxide (CO2). Furthermore, intraspecific variation of these responses should also be examined in order to predict productivity gains through tree improvements in the future. We investigated intraspecific variation in growth and photosynthesis of Cryptomeria japonica D. Don, a major silviculture species in Japan, in response to elevated concentrations of O3 (eO3) and CO2 (eCO2), separately and in combination. Cuttings of C. japonica were grown and exposed to two levels of O3 (ambient and twice-ambient levels) in combination with two levels of CO2 (ambient and 550 µmol mol-1 in the daytime) for two growing seasons in a free-air CO2 enrichment experiment. There was no obvious negative effect of eO3 on growth or photosynthetic traits of the C. japonica clones, but a positive effect was observed for annual height increments in the first growing season. Dry mass production and the photosynthetic rate increased under eCO2 conditions, while the maximum carboxylation rate decreased. Significant interaction effects of eO3 and eCO2 on growth and photosynthetic traits were not observed. Clonal effects on growth and photosynthetic traits were significant, but the interactions between clones and O3 and/or CO2 treatments were not. Spearman's rank correlation coefficients between growth traits under ambient conditions and for each treatment were significantly positive, implying that clonal ranking in growth abilities might not be affected by either eO3 or eCO2. The knowledge obtained from this study will be helpful for species selection in afforestation programs, to continue and to improve current programs involving this species, and to accurately predict the CO2 fixation capacity of Japanese forests. [ABSTRACT FROM AUTHOR]

Biomass allocation to fine roots often increases under soil nutrient deficiency, but the fine root biomass does not often increase in old stands, even under nutrient limitation. Therefore, in old stands, the morphology, anatomy, branching architecture and mycorrhization of fine roots may compensate efficiently for nutrient acquisition by the low fine root biomass. In this study, changes in the morphology, anatomy and arbuscular mycorrhizal infection at each branching position of fine root clusters were evaluated in relation to stand age. A chronosequence (6-90 years of age) of stands in a Cryptomeria japonica D. Don plantation was used for these analyses. The fine root size parameters, such as length, weight and tip numbers of fine root clusters, increased with stand age. The specific root tip length (SRTL) decreased with increasing stand age, suggesting that the allocation to root active portions decreased with stand age. From the anatomical observation, the ephemeral root tips increased with stand age, suggesting that root tip turnover within a root cluster was high in old stands. The proportions of proto-xylem groups among branching positions indicated that the life cycles in branching hierarchy should be clearer in old stands than that in younger stands. The increasing in the mycorrhizal infection of root tips in old stands should enhance the root tip absorptive functions. The SRTL was correlated with the wood/needle ratio, suggesting that carbon limitation as the stand ages may result in decline of carbon allocation to maintain active root tips. However, increasing of the ephemeral tips and mycorrhizal infection rates may compensate the declines of tip allocation in old stands. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

To investigate the biological mechanism by which trees control the changes in microfibril (MF) orientation among secondary cell wall layers of conifer tracheids, we studied seasonal variation in the orientation of newly deposited MFs during tracheid cell wall development in Japanese cedar (Cryptomeria japonica D. Don) trees growing in Central Japan (36°36′N, 140°39′E). Sample blocks were repeatedly collected from four 16-year-old clones of different origins during the growing season of 2010 to investigate the hypotheses that changes in cellulose MF orientation between wall layers exhibited seasonal and clonal differences. The progressive change in the orientation of newly deposited MFs on the primary and secondary cell wall layers of tracheids was detected by field-emission-scanning electron microscopy. Tracheid production and differentiation was studied by light microscopy. We observed a decreasing trend in the orientation of deposited MFs from earlywood to latewood in the S2 and S1 layers, where MFs appeared in a Z-helix. In contrast, no seasonal pattern in the orientation of the MFs in the S-helix was observed. Minor clonal variation was observed in the phenology of tracheid production and differentiation. We concluded that a seasonal decreasing trend in the orientation of the MFs in the Z-helix in S1 and S2 was present, whereas the MFs in other layers exhibited minor random variations. Thus, the orientation of the MFs in S2 was affected by seasonal factors, whereas the MFs in other layers were more intrinsically controlled. The within-ring variations in the MF orientation and thus the resulting average MF angle might also be related to genotypic differences in the tracheid production and differentiation rate. However, our results do not exclude other intrinsic and environmental regulations in the change in MF orientation, which remains a topic for future studies. [ABSTRACT FROM AUTHOR]

Understanding radial and azimuthal variation, and tree-to-tree variation, in sap flux density (Fd) as sources of uncertainty is important for estimating transpiration using sap flow techniques. In a Japanese cedar (Cryptomeria japonica D. Don.) forest, Fd was measured at several depths and aspects for 18 trees, using heat dissipation (Granier-type) sensors. We observed considerable azimuthal variation in Fd. The coefficient of variation (CV) calculated from Fd at a depth of 0–20 mm (Fd1) and Fd at a depth of 20–40 mm (Fd2) ranged from 6.7 to 37.6% (mean = 28.3%) and from 19.6 to 62.5% (mean = 34.6%) for the ­azimuthal directions. Fd at the north aspect averaged for nine trees, for which azimuthal measurements were made, was ­obviously smaller than Fd at the other three aspects (i.e., west, south and east) averaged for the nine trees. Fd1 averaged for the nine trees was significantly larger than Fd2 averaged for the nine trees. The error for stand-scale transpiration (E) estimates caused by ignoring the azimuthal variation was larger than that caused by ignoring the radial variation. The error caused by ignoring tree-to-tree variation was larger than that caused by ignoring both radial and azimuthal variations. Thus, tree-to-tree variation in Fd would be more important than both radial and azimuthal variations in Fd for E estimation. However, Fd for each tree should not be measured at a consistent aspect but should be measured at various aspects to make accurate E estimates and to avoid a risk of error caused by the relationship of Fd to aspect. [ABSTRACT FROM PUBLISHER]

Sequence similarity between genes of angiosperms and conifers was analyzed to identify genes specific to conifer lineages. Using the BLAST and FASTA algorithms, we identified 156 putative conifer-specific transcripts among 5253 unique transcripts of Cryptomeria japonica D. Don. For more than half of the 156 conifer-specific transcripts obtained, the regions conserved among conifers corresponded to the predicted open reading frames, suggesting that the obtained transcripts encode functional proteins. Four of the putative conifer-specific transcripts appeared to be tissue-specific; three were specific to reproductive organs and the other was specific to wood-forming tissues. Comparisons of highly conserved nucleotide sequences in conifer lineages indicated that the nucleotide compositions of orthologous genes were similar, although no common features were found among the conifer-specific genes. [ABSTRACT FROM AUTHOR]

We report the isolation and characterization of CjNdly, a homolog in Japanese cedar (Cryptomeria japonica D. Don) of the FLORICAULA/LEAFY (FLO/LFY) genes. We determined the entire nucleotide sequence of CjNdly, including short 5″- and 3″-untranslated regions. The deduced amino acid sequence was similar to those of the products of the FLO/LFY genes from other species. The nucleotide sequence showed the closest homology to that of the NEEDLY gene in Pinus radiata D. Don. Although no proline-rich region has been reported previously in homologous gene products from gymnosperms, we found such a region at the amino-terminal end of the deduced amino acid sequence encoded by CjNdly. We detected the expression of CjNdly in both reproductive and vegetative tissues and organs of C. japonica. Heterologous expression of CjNdly in transgenic tobacco plants induced precocious flowering of regenerating shoots on agar-solidified medium and flowers with an abnormal phenotype, namely, petal-like stamens. Our findings suggest that the CjNdly gene may have important roles in flower development in Japanese cedar, resembling those of its angiosperm homologs. [ABSTRACT FROM AUTHOR]

We determined the amount of information needed to estimate watershed-scale transpiration in a Japanese cedar (Cryptomeria japonica D. Don) forest from sap flow measurements of individual trees. Measurements of tree biometrics (diameter at breast height (DBH) and tree sapwood area (AS_tree)), and tree-to-tree and radial variations in xylem sap flux density (Fd) were made in two stand plots, an upper slope plot (UP) and a lower slope plot (LP), during a growing season characterized by significant variations in environmental factors. We then investigated how mean stand sap flux density (JS) and a tree stem allometric relationship (DBH--AS_tree) varied between the stands. Appropriate sample sizes for estimating representative JS values were determined. Both a unique and a general function allowed description of the allometric relationship along the slope, but the data for its formulation was required for both the UP and LP. Values of JS in the UP and LP were similar during the study period despite differences in tree density and size between the plots, implying that JS measured in a partial stand in a watershed is a reasonable estimate of JS in other stands in the watershed, and that stand sapwood area calculated from AS_tree is a strong determinant of water use in a forest watershed. To estimate JS in both the UP and LP, it was necessary to sample at least 10 trees in each plot. [ABSTRACT FROM AUTHOR]

Secondary metabolites called norlignans are produced in the xylem of Cryptomeria japonica D. Don. Several norlignans have roles in the defense of sapwood against microbial invasion and in the coloration of heartwood. The biosynthetic pathway of norlignans is largely unknown. Norlignans have been reported to accumulate in the sapwood during the drying of C. japonica logs. To search for genes encoding enzymes that catalyze the synthesis of norlignans, we carried out suppression subtractive hybridization using the fresh sapwood of a felled log and the drying sapwood in which a norlignan, agatharesinol, accumulated. A total of 1050 expressed sequence tags were obtained from the subtracted cDNA library, and these were assembled into 146 contigs and 361 singletons. Of these 507 unique sequences, 263 were functionally classified into 12 categories. “Metabolism” was the largest category, with 23% (61) of classified sequences. Twenty-six sequences that encode 16 enzymes were assigned to “secondary metabolism.” Expression analysis of 15 genes related to “secondary metabolism” revealed that 12 of these genes had transcripts that were induced during the sapwood drying process. Of the 12 genes, 10 encoded enzymes that use aromatic compounds as substrates. In addition, 58 sequences representing 22 defense-related proteins were found. Our subtraction library should be a useful source for isolating genes encoding proteins involved in secondary metabolism including norlignan biosynthesis and defense in C. japonica xylem. [ABSTRACT FROM PUBLISHER]

Cryptomeria japonica D. Don is one of the most important forest trees in Japan, but more than 10% of the Japanese population is allergic to its pollen. We constructed a cDNA library derived from pollen grains of C. japonica and performed an analysis of expressed sequence tags (ESTs). We obtained partial sequences from 1929 clones, which represented 1365 unique transcripts. Among the unique transcripts, 984 (72%) encoded proteins that were similar to Arabidopsis proteins with E-values of < 10−5. Analysis of funtional composition of the pollen ESTs revealed the overrepresentation of mRNAs for proteins involved in protein synthesis and post-translational modification. The most abundant transcripts were derived from novel genes (CjMP1-related genes) and encoded proteins that were not homologous to any proteins in current databases. The CjMP1-related genes formed a multi-gene family and were expressed specifically in the pollen grains of C. japonica. An analysis of homologies between ESTs from C. japonica pollen and proteins in the Structural Database of Allergenic Proteins revealed that products of 48 of the clones (2.5%) exhibited significant homology to known plant allergens. Our results provide new information about pollen-specific genes and potential allergens in C. japonica pollen. [ABSTRACT FROM PUBLISHER]

Thaumatin-like proteins (TLPs) are induced by a variety of phytopathogens in many plants and several TLPs are allergenic. Previously, we isolated three TLP-encoding cDNAs (Cry j 3.1, Cry j 3.2 and Cry j 3.3) from a cDNA library derived from the pollen of Cryptomeria japonica D. Don. Here, we describe three new TLP cDNAs (Cry j 3.4, Cry j 3.5 and Cry j 3.6). We compared the sequences, the genetic map location and the expression patterns of the Cry j 3 genes. The amino acid sequence predicted from Cry j 3.5 exhibits only limited similarity to those predicted from the other Cry j 3 genes. Linkage analysis showed that the Cry j 3.1 to Cry j 3.4 genes are located in the same linkage group, but Cry j 3.5 is located in a different group. Organ-specificity and induction by stresses and plant hormones differed among the Cry j 3 mRNAs. In pollen grains, the Cry j 3.5 mRNA expression level was higher than that of the other Cry j 3 genes. Exposure to UV-B and salt stress induced expression of Cry j 3.1. The ethylene-releasing compound ethephon strongly induced expression of Cry j 3.4. Salt stress and salicylic acid also induced expression of Cry j 3.4. Abscisic acid weakly induced expression of Cry j 3.5. Arachidonic acid strongly induced expression of Cry j 3.4 and Cry j 3.6, and weakly induced that of Cry j 3.3, whereas expression of Cry j 3.1 and Cry j 3.5 was unaffected. These results suggest that the roles of TLPs and the cascades that regulate their expression differ among the members of the TLP family in C. japonica. [ABSTRACT FROM PUBLISHER]

In many temperate evergreen plant species, reductions in turgor loss point of leaves (Ψtlp) and leaf osmotic potential at full turgor (πsat) occur from late summer to winter. To test the hypothesis that this seasonal change in leaf water relations is driven by root temperature, we manipulated the temperature of the roots and shoots of Cryptomeria japonica D. Don seedlings separately. Whole-plant warming diminished the seasonal changes in shoot water relations observed in the control plants, whereas shoot warming did not. Compared with the controls, root warming diminished the change in Ψtlp but not in πsat, whereas cooling accelerated the seasonal changes in shoot water relations. These results indicate that: (1) temperature responses of roots are involved in the seasonal changes in Ψtlp from late summer to winter; and (2) root temperature is partly responsible for the simultaneous changes in πsat. Whole-plant cooling caused increased root hydraulic resistance, suggesting that seasonal changes in shoot water relations represent adaptive responses to increased root hydraulic resistance at low root temperatures. [ABSTRACT FROM AUTHOR]

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 × 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. [ABSTRACT FROM AUTHOR]

Cell behavior in the cambium and developing xylem of 3-year-old Japanese cedar (Cryptomeria japonica D. Don.) trees, during and after an 11-day suspension of irrigation, was analyzed. Leaf xylem pressure potential and tangential strain of the stem surface were monitored throughout the experiment. Anatomical features and numbers of developing tracheids and cambial cells were observed in four trees, sampled on Days 0, 4, 8 and 11 after irrigation was suspended. Daytime xylem pressure potential decreased to –1.9 MPa on Day 7 and remained the same until irrigation was resumed on Day 11. The transverse dimensions of the tracheids, which began to form secondary walls, began to decrease on Day 4. The number of cells in the cambial zone and cell expansion zone decreased abruptly on Day 8. Tangentially aligned developing tracheids with collapsed cell walls were observed in samples harvested on Days 8 and 11. Secondary wall formation was recognized in these tracheids. After the resumption of irrigation, xylem pressure potential recovered rapidly to the same value as before the suspension of irrigation. Tangential strain increased within 30 min after the resumption of irrigation, and continued to increase until the onset of light the next day. Eighteen days after the resumption of irrigation, anatomical features of cells in the cambium and cell-expansion zone were similar to those observed before suspension of irrigation. [ABSTRACT FROM AUTHOR]

The distribution of growth stresses in leaning trunks of Cryptomeria japonica (L.f.) D. Don was determined by measuring the stresses released by the kerf method with strain gauges glued at specified positions along the trunks. Effects of both tree height and peripheral positions on the surface of leaning trunks on surface growth stress were determined. The inner residual growth strains in leaning trunks were also measured. We found high compression stresses in the lower side of leaning trunks that differed greatly from the tensile stresses in normal erect trunks. However, transverse compression stress was found around the tree trunk in both normal and compression wood. In leaning trees, the distribution of internal stresses in the bent trunk portion differed from that in the erect trunk portion, being compressive on the outside and tensile on the inside. The resistant moment introduced by compression stress generated in compression wood is released by the bending of the leaning trunk. The bending stresses are then superimposed on the original internal growth stress. We demonstrated that Poisson's effect of longitudinal stresses should be considered when evaluating transverse surface growth stresses. The existence and intensity of compression wood development can be assessed by growth stress measurements. We conclude that the compressing force of compression wood functions physiologically to give an upward righting response in a leaning trunk. [ABSTRACT FROM PUBLISHER]

Responses of cambium to warming were recorded three times (December 14–27, 1990, January 18–February 3 and February 27–March 13, 1991) on 14-year-old Cryptomeria japonica D. Don and four times (December 12–26, 1990, January 18–February 2, February 26–March 12 and March 28–April 13, 1991) on 27-year-old Larix leptolepis Gord., during a period of winter cambial dormancy. Stem surfaces at breast height, mid-tree height and the crown base were warmed to 25–30 °C for 2 weeks. After heat treatment, cambia in the treated regions and in untreated regions 1 m above each treated area were examined by optical and transmission electron microscopy (TEM). In C. japonica, heat treatment often resulted in cambial reactivation in the treated regions, and this response to heat gradually increased as the dormant season passed from winter to spring. Conversely, in L. leptolepis, no cell division was observed in the cambial region of warmed stems until natural resumption of cambial activity, which occurred after bud break. [ABSTRACT FROM PUBLISHER]

In order to predict the effects of future atmospheric conditions on forest productivity, it is necessary to clarify the physiological responses of major forest tree species to high concentrations of ozone (O3) and carbon dioxide (CO2). Furthermore, intraspecific variation of these responses should also be examined in order to predict productivity gains through tree improvements in the future. We investigated intraspecific variation in growth and photosynthesis of Cryptomeria japonica D. Don, a major silviculture species in Japan, in response to elevated concentrations of O3 (eO3) and CO2 (eCO2), separately and in combination. Cuttings of C. japonica were grown and exposed to two levels of O3 (ambient and twice-ambient levels) in combination with two levels of CO2 (ambient and 550 µmol mol-1 in the daytime) for two growing seasons in a free-air CO2 enrichment experiment. There was no obvious negative effect of eO3 on growth or photosynthetic traits of the C. japonica clones, but a positive effect was observed for annual height increments in the first growing season. Dry mass production and the photosynthetic rate increased under eCO2 conditions, while the maximum carboxylation rate decreased. Significant interaction effects of eO3 and eCO2 on growth and photosynthetic traits were not observed. Clonal effects on growth and photosynthetic traits were significant, but the interactions between clones and O3 and/or CO2 treatments were not. Spearman's rank correlation coefficients between growth traits under ambient conditions and for each treatment were significantly positive, implying that clonal ranking in growth abilities might not be affected by either eO3 or eCO2. The knowledge obtained from this study will be helpful for species selection in afforestation programs, to continue and to improve current programs involving this species, and to accurately predict the CO2 fixation capacity of Japanese forests., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Biomass allocation to fine roots often increases under soil nutrient deficiency, but the fine root biomass does not often increase in old stands, even under nutrient limitation. Therefore, in old stands, the morphology, anatomy, branching architecture and mycorrhization of fine roots may compensate efficiently for nutrient acquisition by the low fine root biomass. In this study, changes in the morphology, anatomy and arbuscular mycorrhizal infection at each branching position of fine root clusters were evaluated in relation to stand age. A chronosequence (6–90 years of age) of stands in a Cryptomeria japonica D. Don plantation was used for these analyses. The fine root size parameters, such as length, weight and tip numbers of fine root clusters, increased with stand age. The specific root tip length (SRTL) decreased with increasing stand age, suggesting that the allocation to root active portions decreased with stand age. From the anatomical observation, the ephemeral root tips increased with stand age, suggesting that root tip turnover within a root cluster was high in old stands. The proportions of proto-xylem groups among branching positions indicated that the life cycles in branching hierarchy should be clearer in old stands than that in younger stands. The increasing in the mycorrhizal infection of root tips in old stands should enhance the root tip absorptive functions. The SRTL was correlated with the wood/needle ratio, suggesting that carbon limitation as the stand ages may result in decline of carbon allocation to maintain active root tips. However, increasing of the ephemeral tips and mycorrhizal infection rates may compensate the declines of tip allocation in old stands.

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., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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)., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)