Technical Note: On Uncertainties in Plant Water Isotopic Composition Following Extraction by Cryogenic Vacuum Distillation

Recent studies have challenged the interpretation of plant water isotopes obtained through cryogenic vacuum distillation (CVD) based on observations of a large 2H fractionation. These studies have hypothesized the existence of an H-atom exchange between water and organic tissue during CVD extraction with the magnitude of H exchange related to relative water content of the sample; however, clear evidence is lacking. Here, we systematically tested the uncertainties in the isotopic composition of CVD-extracted water by conducting a series of incubation and rehydration experiments using isotopically depleted water, water at natural isotope abundance, woody materials with exchangeable H, and organic materials without exchangeable H (cellulose triacetate and caffeine). We show that the offsets between hydrogen and oxygen isotope ratios and expected reference values (Δ2H and Δ18O) have inversely proportional relationships with the absolute amount of water being extracted, i.e. the lower the water amount, the higher the Δ2H and Δ18O. However, neither Δ2H nor Δ18O values, were related to sample relative water content. The Δ2H pattern was more pronounced for materials with exchangeable H atoms than with non-exchangeable H atoms. This is caused by the combined effect of H exchange during the incubation of materials in water and isotopic enrichments during evaporation and sublimation that depend on absolute water amount. The H exchange during CVD extraction itself was negligible. Despite these technical issues, we observed that the water amount-dependent patterns were much less pronounced for samples at natural isotope abundance and particularly low when sufficiently high amounts of water were extracted (\u3e600 µL). Our study provides new insights into the mechanisms causing isotope fractionation during CVD extraction of water. The methodological uncertainties can be controlled if large samples of natural isotope abundance are used in ecohydrological studies

Technical note: On uncertainties in plant water isotopic composition following extraction by cryogenic vacuum distillation

Recent studies have challenged the interpretation of plant water isotopes obtained through cryogenic vacuum distillation (CVD) based on observations of a large 2H fractionation. These studies have hypothesized the existence of an H-atom exchange between water and organic tissue during CVD extraction with the magnitude of H exchange related to relative water content of the sample; however, clear evidence is lacking. Here, we systematically tested the uncertainties in the isotopic composition of CVD-extracted water by conducting a series of incubation and rehydration experiments using isotopically depleted water, water at natural isotope abundance, woody materials with exchangeable H, and organic materials without exchangeable H (cellulose triacetate and caffeine). We show that the offsets between hydrogen and oxygen isotope ratios and expected reference values (Δ2H and Δ18O) have inversely proportional relationships with the absolute amount of water being extracted, i.e. the lower the water amount, the higher the Δ2H and Δ18O. However, neither Δ2H nor Δ18O values, were related to sample relative water content. The Δ2H pattern was more pronounced for materials with exchangeable H atoms than with non-exchangeable H atoms. This is caused by the combined effect of H exchange during the incubation of materials in water and isotopic enrichments during evaporation and sublimation that depend on absolute water amount. The H exchange during CVD extraction itself was negligible. Despite these technical issues, we observed that the water amount-dependent patterns were much less pronounced for samples at natural isotope abundance and particularly low when sufficiently high amounts of water were extracted (&gt;600 µL). Our study provides new insights into the mechanisms causing isotope fractionation during CVD extraction of water. The methodological uncertainties can be controlled if large samples of natural isotope abundance are used in ecohydrological studies.</p

Recombinant humanized collagen combined with nicotinamide increases the expression level of rat basement membrane proteins and promotes hair growth

BackgroundHair follicle stem cells (HFSCs) play crucial roles in hair growth and are expected to be potential targets in regenerative medicine and tissue engineering.MethodThis study aims to investigate the positive effect on hair growth by the recombinant human collagen complex (RHC complex), composed of rhCOL III, rhCOL XVII, and rhCOL XXI, along with nicotinamide, both in vitro and in vivo, by HFSCs and rat models. The survival rate, function, and differentiation of HFSCs were investigated.ResultsThe CCK-8 experiment showed that the RHC complex was non-toxic to HFSCs, and the cell survival rate exceeded 80% after 8 and 16 h of treatment. The ELISA method showed that the RHC complex significantly increased the intracellular vascular endothelial growth factor (VEGF) levels. In addition, the increase in the content of trichohyalin (a key structural protein of hair) indicates that the structure and function of hair follicles may be enhanced. The expression levels of β-integrin and p63 were significantly upregulated, which are crucial for cell adhesion, migration, and maintenance of HFSCs homeostasis. In the rat model, significant hair growth was observed after a 7-day treatment period. The period of vigorous hair growth in rats was selected for immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and hematoxylin-eosin (HE) staining analysis. The results showed that the RHC complex could promote the expression of Integrin, Laminin and Perlecan, which were conducive to maintaining the stability of the microenvironment of HFSCs. Additionally it facilitated the migration and differentiation of HFSCs, as evidenced by an increased number of hair follicles in HE-stained skin tissues. In conclusion, the RHC complex exhibited high HFSCs survival rates and enhanced the expression of HFSCs-associated factors and basement membrane proteins. These properties make the RHC complex a promising novel ingredient for promoting hair growth, preventing hair loss, and maintaining hair health

Transposable element-initiated enhancer-like elements generate the subgenome-biased spike specificity of polyploid wheat

Transposable elements (TEs) comprise ~85% of the common wheat genome, which are highly diverse among subgenomes, possibly contribute to polyploid plasticity, but the causality is only assumed. Here, by integrating data from gene expression cap analysis and epigenome profiling via hidden Markov model in common wheat, we detect a large proportion of enhancer-like elements (ELEs) derived from TEs producing nascent noncoding transcripts, namely ELE-RNAs, which are well indicative of the regulatory activity of ELEs. Quantifying ELE-RNA transcriptome across typical developmental stages reveals that TE-initiated ELE-RNAs are mainly from RLG_famc7.3 specifically expanded in subgenome A. Acquisition of spike-specific transcription factor binding likely confers spike-specific expression of RLG_famc7.3-initiated ELE-RNAs. Knockdown of RLG_famc7.3-initiated ELE-RNAs resulted in global downregulation of spike-specific genes and abnormal spike development. These findings link TE expansion to regulatory specificity and polyploid developmental plasticity, highlighting the functional impact of TE-driven regulatory innovation on polyploid evolution

The impact of insect herbivory on biogeochemical cycling in broadleaved forests varies with temperature

Herbivorous insects alter biogeochemical cycling within forests, but the magnitude of these impacts, their global variation, and drivers of this variation remain poorly understood. To address this knowledge gap and help improve biogeochemical models, we established a global network of 74 plots within 40 mature, undisturbed broadleaved forests. We analyzed freshly senesced and green leaves for carbon, nitrogen, phosphorus and silica concentrations, foliar production and herbivory, and stand-level nutrient fluxes. We show more nutrient release by insect herbivores at non-outbreak levels in tropical forests than temperate and boreal forests, that these fluxes increase strongly with mean annual temperature, and that they exceed atmospheric deposition inputs in some localities. Thus, background levels of insect herbivory are sufficiently large to both alter ecosystem element cycling and influence terrestrial carbon cycling. Further, climate can affect interactions between natural populations of plants and herbivores with important consequences for global biogeochemical cycles across broadleaved forests

Environmental Effects on Carbon Isotope Discrimination from Assimilation to Respiration in a Coniferous and Broad-Leaved Mixed Forest of Northeast China

Carbon (C) isotope discrimination during photosynthetic CO2 assimilation has been extensively studied, but the whole process of fractionation from leaf to soil has been less well investigated. In the present study, we investigated the &delta;13C signature along the C transfer pathway from air to soil in a coniferous and broad-leaved mixed forest in northeast China and examined the relationship between &delta;13C of respiratory fluxes (leaf, trunk, soil, and the entire ecosystem) and environmental factors over a full growing season. This study found that the &delta;13C signal of CO2 from canopy air was strongly imprinted in the organic and respiratory pools throughout C transfer due to the effects of discrimination and isotopic mixing on C assimilation, allocation, and respiration processes. A significant difference in isotopic patterns was found between conifer and broadleaf species in terms of seasonal variations in leaf organic matter. This study also found that &delta;13C in trunk respiration, compared with that in leaf and soil respiration, was more sensitive to seasonal variations of environmental factors, especially soil temperature and soil moisture. Variation in the &delta;13C of ecosystem respiration was correlated with air temperature with no time lag and correlated with soil temperature and vapor pressure deficit with a lag time of 10 days, but this correlation was relatively weak, indicating a delayed linkage between above- and belowground processes. The isotopic linkage might be confounded by variations in atmospheric aerodynamic and soil diffusion conditions. These results will help with understanding species differences in isotopic patterns and promoting the incorporation of more influencing factors related to isotopic variation into process-based ecosystem models

Different responses of oxygen and hydrogen isotopes in leaf and tree-ring organic matter to lethal soil drought

The oxygen and hydrogen isotopic composition (δ18O, δ2H) of plant tissues are key tools for the reconstruction of hydrological and plant physiological processes and may therefore be used to disentangle the reasons for tree mortality. However, how both elements respond to soil drought conditions before death has rarely been investigated. To test this, we performed a greenhouse study and determined predisposing fertilization and lethal soil drought effects on δ18O and δ2H values of organic matter in leaves and tree rings of living and dead saplings of five European tree species. For mechanistic insights, we additionally measured isotopic (i.e. δ18O and δ2H values of leaf and twig water), physiological (i.e. leaf water potential and gas-exchange) and metabolic traits (i.e. leaf and stem non-structural carbohydrate concentration, carbon-to-nitrogen ratios). Across all species, lethal soil drought generally caused a homogenous 2H-enrichment in leaf and tree-ring organic matter, but a low and heterogenous δ18O response in the same tissues. Unlike δ18O values, δ2H values of tree-ring organic matter were correlated with those of leaf and twig water and with plant physiological traits across treatments and species. The 2H-enrichment in plant organic matter also went along with a decrease in stem starch concentrations under soil drought compared with well-watered conditions. In contrast, the predisposing fertilization had generally no significant effect on any tested isotopic, physiological and metabolic traits. We propose that the 2H-enrichment in the dead trees is related to (i) the plant water isotopic composition, (ii) metabolic processes shaping leaf non-structural carbohydrates, (iii) the use of carbon reserves for growth and (iv) species-specific physiological adjustments. The homogenous stress imprint on δ2H but not on δ18O suggests that the former could be used as a proxy to reconstruct soil droughts and underlying processes of tree mortality.ISSN:0829-318XISSN:1758-446

Hydrogen isotopes in leaf and tree-ring organic matter as potential indicators of drought-induced tree mortality

&amp;lt;p&amp;gt;Drought-induced tree mortality is occurring more frequently in the world, with both the direct impact of drought (i.e., heat and drought events) and the mortality predisposition (e.g., tree nutrition status) influencing death or survival of trees. The oxygen and hydrogen isotopic compositions (&amp;amp;#948;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O and &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H) of plant water are widely used as hydrological indicators. Both elements in water are tightly correlated and are the key source for the isotopic composition of plant organic matter. Yet, recent studies show that the relationship between &amp;amp;#948;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O and &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H values in organic matter is weaker and more divergent. This is probably caused by physiological and metabolic processes (i.e., assimilation, assimilate allocation, use of reserves) that are integrated into &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H but not into &amp;amp;#948;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O. This let us hypothesize that &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H can function as a useful tool in tree mortality research to study assimilate and storage related reason (i.e., carbon starvation) of tree death, but more knowledge is urgently needed.&amp;lt;/p&amp;gt; &amp;lt;p&amp;gt;To test our hypothesis, we studied the pre-disposal fertilization and drought effects on &amp;amp;#948;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O and &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H values in plant water and organic matter in a greenhouse experiment. We planted three years old saplings of &amp;lt;em&amp;gt;Abies alba&amp;lt;/em&amp;gt;, &amp;lt;em&amp;gt;Acer pseudoplatanus&amp;lt;/em&amp;gt;, &amp;lt;em&amp;gt;Picea abies&amp;lt;/em&amp;gt;, &amp;lt;em&amp;gt;Pinus sylvestris&amp;lt;/em&amp;gt; and &amp;lt;em&amp;gt;Quercus petraea&amp;lt;/em&amp;gt;, half of which were treated with a slow-release formula fertilizer (control (F0) and fertilization (F+)); one year later, half of the F0 and half of the F+ plants were selected for a lethal drought treatment (control (D0) and drought (D+)), i.e., D0 plants were watered to field capacity, while D+ plants received no more water until they died. After 6 weeks of drought, leaf and twig samples were collected for &amp;amp;#948;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O and &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H analyses of plant water. After the D+ plants died (i.e., 9-15 weeks after start of drought), additional leaf and stem material were collected from a same number of D0 and D+ plants. Organic matter of leaf and tree-ring of the recent year were prepared for &amp;amp;#948;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O and &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H analyses. Additional physiological and metabolic factors were measured to examine the treatment effects.&amp;lt;/p&amp;gt; &amp;lt;p&amp;gt;Across all species, we found that the pre-disposal fertilization had no significant effect on &amp;amp;#948;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O and &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H values of plant water and organic matter. On the other hand, the drought treatment significantly increased both &amp;amp;#948;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O and &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H values of leaf and twig water, while it only significantly increased &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H values of leaf and tree-ring organic matter. These results indicate that &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H in leaf and tree-ring organic matter in dying trees can capture drought-induced tree mortality signals. We propose that the &amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H-enrichment in the drought-exposed trees might be related to (i) the imprint of &amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H-enriched signal of plant water; (ii) drought-induced changes in the metabolic processes of sugar biosynthesis; (iii) drought-induced changes in the use of carbon reserves. In summary, our study supports the idea that hydrogen isotopes can function as a potential diagnostic tool in tree mortality studies.&amp;lt;/p&amp;gt;</jats:p

On uncertainties in the plant source water isotopic composition extracted with cryogenic vacuum distillation

&amp;lt;p&amp;gt;Recent studies challenge the use of plant water from cryogenic vacuum distillation (CVD) extraction in accurately representing the hydrogen and oxygen isotopic composition (&amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H and &amp;amp;#948;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O) of plant source water. This is hypothesized to be because the &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H in extracted water depends on tissue relative water content (RWC), which might be explained by the exchange of H-atoms between water and organic material. Secondary hypotheses focus on extraction artefacts related to evaporation and sublimation, but clear evidence is lacking. Here, we hypothesized that the observed &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H and &amp;amp;#948;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O offsets (&amp;amp;#916;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H and &amp;amp;#916;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O) are influenced by (i) an H-exchange effect, (ii) tissue water amount or RWC and (iii) evaporation and sublimation enrichments.&amp;lt;br&amp;gt;The hypotheses were systematically tested by three corresponding experiments. Firstly, we added a range of strongly depleted reference water (&amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H: &amp;lt;em&amp;gt;ca.&amp;lt;/em&amp;gt; -460&amp;amp;#8240;; &amp;amp;#948;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O: &amp;lt;em&amp;gt;ca.&amp;lt;/em&amp;gt; -170&amp;amp;#8240;; 50&amp;amp;#8211;1200 &amp;amp;#956;l) to organic materials (with and without exchangeable H) of constant weight (200 mg), followed by a 24 h incubation. In addition, the same range of pure reference water and tap water without any material were used as controls. Secondly, we incubated dry stem segments (&amp;lt;em&amp;gt;Larix decidua&amp;lt;/em&amp;gt;) of different sizes in excess of reference water for 24 hours, then they were took out for extracting known water contents from samples with known RWC. Accordingly, fresh twig segments from the same species were prepared for extracting water with natural abundance. Thirdly, a range of different amounts of reference water (50&amp;amp;#8211;1200 &amp;amp;#956;l) was added directly into the water collection tubes of the CVD extraction system. In addition, 2 ml glass vials containing the same range of reference water amounts were incubated in a climate chamber at 25 &amp;amp;#176;C and 50 % relative humidity with lids open for 2 hours. All the samples, except the water in the glass vials, were extracted using a standard CVD extraction method for 2 hours.&amp;amp;#160;&amp;lt;br&amp;gt;We found that both &amp;amp;#916;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H and &amp;amp;#916;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O values were not related to changes in RWC. In contrast, we observed an inversely proportional relationships with water amount, i.e., the lower the water amount, the higher the &amp;amp;#916;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H and &amp;amp;#916;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O. For &amp;amp;#916;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H, the pattern was more pronounced for materials with exchangeable H, which reached 150&amp;amp;#8240; at the lowest water amount and decreased to -20&amp;amp;#8240; with increasing water amounts when the depleted reference water was used. However, the pattern was much less pronounced for the samples with natural isotopic abundance, indicating that the magnitude of the pattern is probably dependent on isotope ratios of plant water and water vapour in the laboratory. The evaporation and sublimation tests both showed that the pattern was partly caused by an increasing isotopic enrichment with decreasing water amount.&amp;lt;br&amp;gt;In conclusion, we identified a significant artefact of CVD when water is present in small amounts, particularly when &amp;amp;#948;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H and &amp;amp;#948;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O of the water was below natural isotope abundances. We therefore recommend extracting &amp;gt; 600 &amp;amp;#956;l of water. Moreover, we provide first evidence of a significant H-exchange effect, suggesting that using hydrogen isotopes for estimating plant source water will remain challenging in future.&amp;lt;/p&amp;gt;</jats:p