Isotope Distribution Analysis in H218O Pulse-Labeled Trees Frozen with Liquid Nitrogen.

Tracer injection has long been recognized as a valuable tool for delineating tree hydraulics and assessing water transport pathways. Recently, isotope tracers have emerged as innovative instruments for investigating tree hydraulics, providing new insights into tree water dynamics. Nevertheless, there is a critical need for further research to comprehensively grasp water movement and distribution within trees. A previously introduced technique for analyzing the isotopic ratio of water in wet tissues, offering millimeter-scale resolution for visualizing tracer movement, faces challenges due to its underdeveloped sample preparation techniques. In this study, we introduced an H2 18O tracer into S. gracilistyla samples, exclusively comprising indeterminate roots, stems, and leaves, cultivated through hydroponics and grown within the current year. Our objective was to assess the axial distribution of the tracer in the xylem. Additionally, we devised a novel method for preparing frozen wet tissue samples, enhancing the repeatability and success rate of experiments. The results demonstrated that all frozen wet tissue samples exhibited an average water loss rate of less than 0.6%. Isotopic analysis of these samples unveiled a consistent decline in tracer concentration with increasing height in all Salix specimens, with three out of five samples revealing a significant isotope gradient. Our findings affirm the efficacy and practicality of combining isotopic labeling with freezing, stabilization, and preparation techniques. Looking ahead, our isotopic labeling and analysis methods are poised to transcend woody plants, finding extensive applications in plant physiology and ecohydrology.

The difference in the functional water flow network between the stem and current-year root cross-sectional surfaces in Salix gracilistyla stem xylem.

The dye injection method has been applied to many species to analyze the xylem water transport pathway in trees. However, traditional dye injection methods introduced dye tracers from the surface of cut stems, including several annual rings. Furthermore, the traditional dye injection method did not evaluate radial water movement from the outermost annual rings to the inner annual rings. In this study, we assessed the difference in radial water movement visualized by an injected dye, between stem base cut and current-year root cut samples of Salix gracilistyla Miq., with current-year roots grown hydroponically. The results showed that the number of stained annual rings in the root cut samples was smaller than that in the stem cut samples, and the percentage of stained vessels in the root cut samples was significantly smaller than that in the stem base cut samples in the second and third annual rings. In the current-year root cut samples, water transport mainly occurred in the outermost rings from the current-year roots to leaves. In addition, the theoretical hydraulic conductivity of stained vessels in the stem cut samples was higher in the current-year root cut samples in the second and third annual rings. These findings indicate that the previously reported dye injection method using stem cut samples overestimated the water transport pathway in the inner part of the stems. Moreover, previous hydraulic conductivity measurement methods might not have considered the effects of radial resistance through the annual ring boundary, and they might have overestimated the hydraulic conductivity in the inner annual rings.

Decadal trends in 137Cs concentrations in the bark and wood of trees contaminated by the Fukushima nuclear accident.

Understanding the actual situation of radiocesium (137Cs) contamination of trees caused by the Fukushima nuclear accident is essential for predicting the future contamination of wood. Particularly important is determining whether the 137Cs dynamics within forests and trees have reached apparent steady state. We conducted a monitoring survey of four major tree species (Japanese cedar, Japanese cypress, konara oak, and Japanese red pine) at multiple sites. Using a dynamic linear model, we analyzed the temporal trends in 137Cs activity concentrations in the bark (whole), outer bark, inner bark, wood (whole), sapwood, and heartwood during the 2011-2020 period. The activity concentrations were decay-corrected to September 1, 2020, to exclude the decrease due to the radioactive decay. The 137Cs concentrations in the whole and outer bark samples showed an exponential decrease in most plots but a flat trend in one plot, where 137Cs root uptake is considered to be high. The 137Cs concentration ratio (CR) of inner bark/sapwood showed a flat trend but the CR of heartwood/sapwood increased in many plots, indicating that the 137Cs dynamics reached apparent steady state within one year in the biologically active parts (inner bark and sapwood) and after several to more than 10 years in the inactive part (heartwood). The 137Cs concentration in the whole wood showed an increasing trend in six plots. In four of these plots, the increasing trend shifted to a flat or decreasing trend. Overall, the results show that the 137Cs dynamics within forests and trees have reached apparent steady state in many plots, although the amount of 137Cs root uptake in some plots is possibly still increasing 10 years after the accident. Clarifying the mechanisms and key factors determining the amount of 137Cs root uptake will be crucial for predicting wood contamination.

Foliar water uptake as a source of hydrogen and oxygen in plant biomass.

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; δD = 7.0 × 10 4‰, δ18O = 4.1‰) and one enriched in oxygen-18 (H218O; δD = -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.

Seasonal changes of δD and δ18O in tree-ring cellulose of Quercus crispula suggest a change in post-photosynthetic processes during earlywood growth.

Leaf photosynthetic and post-photosynthetic processes modulate the isotope ratios of tree-ring cellulose. Post-photosynthetic processes, such as the remobilization of stored starch in early spring, are important to understanding the mechanisms of xylem formation in tree stems; however, untangling the isotope ratio signals of photosynthetic and post-photosynthetic processes imprinted on tree rings is difficult. Portions of carbon-bound hydrogen and oxygen atoms are exchanged with medium water during post-photosynthetic processes. We investigated the δD and δ18O values of tree-ring cellulose using Quercus crispula Blume trees in two different habitats to evaluate seasonal changes in the exchange rate (f-value) of hydrogen or oxygen with medium water, and examined the associations of the post-photosynthetic processes. Theoretically, if the f-value is constant, δD and δ18O would be positively correlated due to meteorological factors, while variation in the f-value will create a discrepancy and weak correlation between δD and δ18O due to the exchange of carbon-bound hydrogen and oxygen with medium water. The values of δD decreased drastically from earlywood to latewood, while those of δ18O increased to a peak and then decreased toward the latewood. The estimated seasonal f-value was high at the beginning of earlywood and decreased toward the latewood. The post-photosynthetic processes associated with changes in the f-value were the remobilization of stored starch and triose cycling during cellulose synthesis because of the shortage of photo-assimilates in early spring. Although we did not evaluate relevant physiological parameters, the seasonal pattern of δD and δ18O in tree-ring cellulose of Q. crispula was clear, suggesting that the dual isotope (δD and δ18O) approach can be used to reveal the resource allocation mechanisms underlying seasonal xylem formation.

Temporal trends in 137Cs concentrations in the bark, sapwood, heartwood, and whole wood of four tree species in Japanese forests from 2011 to 2016.

To understand the changes in radiocesium (137Cs) concentrations in stem woods after the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, we investigated 137Cs concentrations in the bark, sapwood, heartwood, and whole wood of four major tree species at multiple sites with different levels of radiocesium deposition from the FDNPP accident since 2011 (since 2012 at some sites): Japanese cedar at four sites, hinoki cypress and Japanese konara oak at two sites, and Japanese red pine at one site. Our previous report on 137Cs concentrations in bark and whole wood samples collected from 2011 to 2015 suggested that temporal variations were different among sites even within the same species. In the present study, we provided data on bark and whole wood samples in 2016 and separately measured 137Cs concentrations in sapwood and heartwood samples from 2011 to 2016; we further discussed temporal trends in 137Cs concentrations in each part of tree stems, particularly those in 137Cs distributions between sapwood and heartwood, in relation to their species and site dependencies. Temporal trends in bark and whole wood samples collected from 2011 to 2016 were consistent with those reported in samples collected from 2011 to 2015. Temporal variations in 137Cs concentrations in barks showed either a decreasing trend or no clear trend, implying that 137Cs deposition in barks is inhomogeneous and that decontamination is relatively slow in some cases. Temporal trends in 137Cs concentrations in sapwood, heartwood, and whole wood were different among species and also among sites within the same species. Relatively common trends within the same species, which were increasing, were observed in cedar heartwood, and in oak sapwood and whole wood. On the other hand, the ratio of 137Cs concentration in heartwood to that in sapwood (fresh weight basis) was commonly increased to more than 2 in cedar, although distinct temporal trends were not found in the other species, for which the ratio was around 1 in cypress and pine and below 0.5 in oak, suggesting that 137Cs transfer from sapwood to heartwood shows species dependency. Consequently, the species dependency of 137Cs transfer within the tree appears easily, while that from the environment to the trees can be masked by various factors. Thus, prediction of 137Cs concentrations in stem wood should be carried out carefully as it still requires investigations at multiple sites with a larger sample size and an understanding of the species-specific 137Cs transfer mechanism.

Reproduction-related variation in carbon allocation to woody tissues in Fagus crenata using a natural 13C approach.

The contribution of new photo-assimilates and stored carbon (C) to plant growth remains poorly understood, especially during reproduction. In order to elucidate how mast seeding affects C allocation to both reproductive and vegetative tissues, we measured biomass increase in each tissue, branch starch concentration and stable C isotope composition (δ13C) in bulk leaves, current-year shoots, 3-year branches and tree rings in fruiting and non-fruiting trees for 2 years, as well as in fruits. We isolated the effect of reproduction on C allocation to vegetative growth by comparing 13C enrichment in woody tissues in fruiting and non-fruiting specimens. Compared with 2‰ 13C enrichment in shoots relative to leaves from non-fruiting trees, fruiting reduced the enrichment to 1‰ and this reduction disappeared in the following year with no fruiting, indicating that new photo-assimilates are preferentially used for woody tissues even with fruiting burden. In contrast, fruits had up to 2.5‰ 13C enrichment at mid-summer, which dropped thereafter, indicating that fruit production relies on C storage early in the growing season then shifts to current photo-assimilates. At this tipping point, growth of shoots and cupules had almost finished and nuts had a second rapid growth period thereafter. Together with shorter shoots but higher biomass increment per length in fruiting trees than non-fruiting trees, these results indicate that the C limitation due to fruit burden is minimized by fine-tuning of allocation of old C stores and new photo-assimilates, along with the growth pattern in various tissues. Furthermore, fruiting had no significant effect on starch concentration in 3-year-old branches, which became fully depleted during leaf and flower flushing but were quickly replenished. These results indicate that reproduction affects C allocation to branches but not its source or storage. These reproduction-related variations in the fate of C have implications for evaluating forest ecosystem C cycles during climate change.

Seasonal variations in the stable oxygen isotope ratio of wood cellulose reveal annual rings of trees in a Central Amazon terra firme forest.

In Amazonian non-flooded forests with a moderate dry season, many trees do not form anatomically definite annual rings. Alternative indicators of annual rings, such as the oxygen (δ(18)Owc) and carbon stable isotope ratios of wood cellulose (δ(13)Cwc), have been proposed; however, their applicability in Amazonian forests remains unclear. We examined seasonal variations in the δ(18)Owc and δ(13)Cwc of three common species (Eschweilera coriacea, Iryanthera coriacea, and Protium hebetatum) in Manaus, Brazil (Central Amazon). E. coriacea was also sampled in two other regions to determine the synchronicity of the isotopic signals among different regions. The annual cyclicity of δ(18)Owc variation was cross-checked by (14)C dating. The δ(18)Owc showed distinct seasonal variations that matched the amplitude observed in the δ(18)O of precipitation, whereas seasonal δ(13)Cwc variations were less distinct in most cases. The δ(18)Owc variation patterns were similar within and between some individual trees in Manaus. However, the δ(18)Owc patterns of E. coriacea differed by region. The ages of some samples estimated from the δ(18)Owc cycles were offset from the ages estimated by (14)C dating. In the case of E. coriacea, this phenomenon suggested that missing or wedging rings may occur frequently even in well-grown individuals. Successful cross-dating may be facilitated by establishing δ(18)Owc master chronologies at both seasonal and inter-annual scales for tree species with distinct annual rings in each region.

Radiocesium concentrations in the bark, sapwood and heartwood of three tree species collected at Fukushima forests half a year after the Fukushima Dai-ichi nuclear accident.

Radiocesium ((134)Cs and (137)Cs) distribution in tree stems of Japanese cedar (aged 40-56 y), red pine (42 y), and oak (42 y) grown in Fukushima Prefecture were investigated approximately half a year after the Fukushima Dai-ichi nuclear accident. Japanese cedar, red pine, and oak were selected from five sites, one site, and one site, respectively. Three trees at each site were felled, and bark, sapwood (the outer layer of wood in the stem), and heartwood (the inner layer of wood in the stem) separately collected to study radiocesium concentrations measured by gamma-ray spectrometry. The radiocesium deposition densities at the five sites were within the range of 16-1020 kBq m(-2). The radiocesium was distributed in bark, sapwood, and heartwood in three tree species, indicating that very rapid translocation of radiocesium into the wood. The concentration of radiocesium in oak (deciduous angiosperm) bark was higher than that in the bark of Japanese cedar and red pine (evergreen gymnosperms). Both sapwood and heartwood contained radiocesium, and the values were much lower than that in the bark samples. The results suggest that radiocesium contamination half a year after the accident was mainly attributable to the direct radioactive deposition. The radiocesium concentrations in the Japanese cedar samples taken from five sites rose with the density of radiocesium accumulation on the ground surface. To predict the future dynamics of radiocesium in tree stems, the present results taken half a year after the accident are important, and continuous study of radiocesium in tree stems is necessary.

Mycoheterotrophy evolved from mixotrophic ancestors: evidence in Cymbidium (Orchidaceae).

BACKGROUND AND AIMS: Nutritional changes associated with the evolution of achlorophyllous, mycoheterotrophic plants have not previously been inferred with robust phylogenetic hypotheses. Variations in heterotrophy in accordance with the evolution of leaflessness were examined using a chlorophyllous-achlorophyllous species pair in Cymbidium (Orchidaceae), within a well studied phylogenetic background. METHODS: To estimate the level of mycoheterotrophy in chlorophyllous and achlorophyllous Cymbidium, natural (13)C and (15)N contents (a proxy for the level of heterotrophy) were measured in four Cymbidium species and co-existing autotrophic and mycoheterotrophic plants and ectomycorrhizal fungi from two Japanese sites. KEY RESULTS: δ(13)C and δ(15)N values of the achlorophyllous C. macrorhizon and C. aberrans indicated that they are full mycoheterotrophs. δ(13)C and δ(15)N values of the chlorophyllous C. lancifolium and C. goeringii were intermediate between those of reference autotrophic and mycoheterotrophic plants; thus, they probably gain 30-50 % of their carbon resources from fungi. These data suggest that some chlorophyllous Cymbidium exhibit partial mycoheterotrophy (= mixotrophy). CONCLUSIONS: It is demonstrated for the first time that mycoheterotrophy evolved after the establishment of mixotrophy rather than through direct shifts from autotrophy to mycoheterotrophy. This may be one of the principal patterns in the evolution of mycoheterotrophy. The results also suggest that the establishment of symbiosis with ectomycorrhizal fungi in the lineage leading to mixotrophic Cymbidium served as pre-adaptation to the evolution of the mycoheterotrophic species. Similar processes of nutritional innovations probably occurred in several independent orchid groups, allowing niche expansion and radiation in Orchidaceae, probably the largest plant family.

13CO2 pulse-labelling of photoassimilates reveals carbon allocation within and between tree rings.

Post-photosynthetic fractionation processes during translocation, storage and remobilization of photoassimilate are closely related to intra-annual sigma13C of tree rings, and understanding how these processes affect tree-ring sigma13C is therefore indispensable for improving the quality of climate reconstruction. Our first objective was to study the relationship between translocation path and phloem grain. We pulse-labelled a branch of Larix gmelinii (Rupr.) Rupr. and later analysed the sigma13C distribution in the stem. A 13C spiral translocation path closely related to the spiral grain was observed. Our second objective was to study the use of remobilized storage material for earlywood formation in spring, which is a suspected cause of the autocorrelation (correlation of ring parameters to the climate in the previous year) observed in (isotope) dendroclimatology. We pulse-labelled whole trees to study how spring, summer and autumn photoassimilate is later used for both earlywood and latewood formation. Analysis of intra-annual sigma13C of the tree rings formed after the labelling revealed that earlywood contained photoassimilate from the previous summer and autumn as well as from the current spring. Latewood was mainly composed of photoassimilate from the current year's summer/autumn, although it also relied on stored material in some cases. These results emphasize the need for separating earlywood and latewood for climate reconstruction work with narrow boreal tree rings.

Seasonal course of translocation, storage and remobilization of 13C pulse-labeled photoassimilate in naturally growing Larix gmelinii saplings.

Autocorrelation--correlation of tree-ring parameters such as ring width, density and isotope ratios to the environmental conditions of the previous year(s)--is associated with the use of previous photoassimilate for current year's tree ring formation. To clarify the seasonal course of carbon allocation patterns among needles, branches, stem and roots, we pulse-labeled 10 Larix gmelinii growing in a continuous permafrost zone with 13CO2. Photoassimilate incorporated in June was allocated mainly to above-ground parts, indicating active above-ground growth in spring. Very little was allocated to below-ground parts (2.6-7.9%), probably because root growth is inhibited by low soil temperatures in spring. Conversely, a higher proportion of July and August photoassimilate was allocated to below-ground parts (32-44 and 12-24%, respectively). About half the carbon in new needles was derived from stored material. The starch pool in non-needle parts, which can be used for xylem formation, drew approx. 43% of its carbon from the previous year's photoassimilate, indicating that carbon storage is a key mechanism behind autocorrelation in (isotope) dendroclimatology.

Chronological trends in trace metals recorded by a tree bark pocket in Yakushima Island, Japan.

Bark included within the trunk of a 200-year-old Japanese cedar tree harvested in Yakushima Island, Japan, a World Natural Heritage Site located 150 km south of mainland Japan and 800 km east of Shanghai, China, was analysed for trace metals by ICP-MS providing a chronology of atmospheric pollution. The concentration of V, As and Pb in decadal sections of the bark pocket increased 30 to 50 fold from 1900-09 to 1960-69, indicating increased atmospheric deposition of these metals. The trend coincided with the establishment and expansion of heavy industries in Kyushu, Japan, resulting in locally high levels of air pollution. V, As and Pb subsequently declined, reflecting lower industrial emissions following air pollution control legislation from the late 1960's and decline in heavy industries. Ni, Cu and Zn showed a relatively small, 7 to 10 fold increase over time. Lead isotope ratios in the bark pockets shifted from about 0.84 to 0.86 for 207Pb/206Pb and from 2.04 to 2.10 for 208Pb/206Pb, showing that the origin of atmospheric lead changed over time from coal to more diverse sources.

Temporal water deficit and wood formation in Cryptomeria japonica.

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.

Tree-ring strontium-90 and cesium-137 as potential indicators of radioactive pollution.

To examine whether tree rings can be used to detect or assess local historical 90Sr or 137Cs fallout, such as that resulting from the Hiroshima atomic bomb, radial distribution of 90Sr and 137Cs in trees was examined. We studied a gymnosperm [Japanese cedar, Cryptomeria japonica (L. f.) D. Don] and an angiosperm (Japanese persimmon, Diospyros kaki Thunb.) tree species from the vicinity of the atomic bomb hypocenter, and from other locations in Japan. A significant amount of 137Cs was detected in tree rings formed before 1945, indicating lateral migration of Cs. In contrast, the specific activity of 90Sr in the Hiroshima Japanese cedar showed the highest level in 1945, due to relatively immobile characteristics of Sr compared with Cs. Strontium-90 and Sr analyses in tree rings helped identify and distinguish between residual 90Sr activity from the Hiroshima atomic bomb and the atmospheric nuclear testing. This indicates the possibility of detecting or assessing previous local 90Sr pollution through with treering analysis.