Characterization of complex photosynthetic pigment profiles in European deciduous tree leaves by sequential extraction and reversed-phase high-performance liquid chromatography

Leaf pigments, including chlorophylls and carotenoids, are important biochemical indicators of plant photosynthesis and photoprotection. In this study, we developed, optimized, and validated a sequential extraction and liquid chromatography-diode array detection method allowing for the simultaneous quantification of the main photosynthetic pigments, including chlorophyll a, chlorophyll b, β-carotene, lutein, neoxanthin, and the xanthophyll cycle (VAZ), as well as the characterization of plant pigment derivatives. Chromatographic separation was accomplished with the newest generation of core–shell columns revealing numerous pigment derivatives. The sequential extraction allowed for a better recovery of the main pigments (+25 % chlorophyll a, +30 % chlorophyll b, +42 % β-carotene, and 61% xanthophylls), and the characterization of ca. 5.3 times more pigment derivatives (i.e., up to 62 chlorophyll and carotenoid derivatives including isomers) than with a single-step extraction. A broad working range of concentrations (300–2,000 ng.mL$^{−1}$) was achieved for most pigments and their derivatives and the limit of detection was as low as a few nanograms per milliliter. The method also showed adequate trueness (RSD < 1%) and intermediate precision (RSD < 5%). The method was developed and validated with spinach leaves and their extracts. The method was successfully performed on leaf pigment extracts of European deciduous tree species. Within a case study using Fagus sylvatica L. leaves, pigment derivatives revealed a high within-individual tree variability throughout the growing season that could not be detected using the main photosynthetic pigments alone, eventually showing that the method allowed for the monitoring of pigment dynamics at unprecedented detail

Source or decomposition of soil organic matter: what is more important with increasing forest age in a subalpine setting?

Afforestation has been the dominant land-use change in the Swiss Alps during the last decades which has not only the potential to increase soil organic carbon sequestration, but it has also the potential to alter soil organic matter (SOM) dynamics through the vegetation shift and change in organic matter (OM) input into soils. The effects of afforestation on SOM dynamics, however, are still not fully understood as specific sources of OM and modifications of soil processes influencing decomposition and preservation remain largely unknown on alpine to subalpine slopes. Within this study we aimed to identify the potential sources and the decomposition of OM in a subalpine afforestation chrono-sequence (0–130 years) with Norway spruce (Picea abies L.) on a former pasture by using a multi-proxy molecular marker approach. We observed that leaf-derived OM plays an essential role in the pasture areas, while root-derived OM only plays a minor role in pasture and forest areas. Needle-derived OM represents the dominant source of SOM with increasing forest age, while understory shrubs and moss also contribute to the OM input in younger forest stand ages. However, needle litter and buildup of organic layers and subsequently less input of fresh OM from organic horizons to mineral soil can result in increased OM decomposition in mineral soils rather than contributing to additional SOM stabilization in mineral soils. This was most pronounced in the oldest forest stand (130-year-old) in the investigated afforestation sequence, particularly in deeper soil horizons (10–45 cm). Thereby, our study provides new insights into SOM dynamics following afforestation, especially with respect to the long-term SOM sequestration potential of afforestation of subalpine pasture soils

Severe drought-influenced composition and δ ¹³C of plant and soil n- alkanes in model temperate grassland and heathland ecosystems

Drought events are predicted to increase under future climate change. In temperate ecosystems, plants are capable of resisting drought due to their hydrophobic wax layer, in which n-alkanes are important constituents. In soils, plant-derived n-alkanes are comparatively resistant to degradation. To improve understanding of the significance of n-alkanes in plant-soil systems during a severe drought period (104 days), we investigated bulk organic carbon (Corg) concentration, total lipid extract (TLE) concentration, n-alkane molecular ratios such as average chain length (ACL), carbon preference index (CPI) and chain length ratios of different n-alkane compounds, in addition to the compound-specific isotope composition (δ13C) of n-alkanes in model temperate grassland and heathland plant-soil systems. Although plant communities of two (heathland) and four (grassland) species were available, only one representative species per biome was accessible for the current study. Heathland plants and soil revealed significantly higher concentrations of Corg and TLE compared with grassland. TLE and alkane composition responded quickly during the first drought phase (0 – 40 days). This indicates that plants were actively utilizing C and produced more n-alkanes in order to withstand drought, which was confirmed by increased (2 – 3‰) δ13C values for n-alkanes in shoot biomass. However, during later drought phases all the parameters remained constant for plants and soils. This suggests limited change in biosynthesis and cycling of plant lipids such as n-alkanes during intense drought. Surprisingly, during the first drought phase, increased ACL and CPI ratios in soil demonstrated a rapid input of plant-derived long chain n-alkanes to soil, which was not expected due to the decadal residence time of alkanes in soil. The study enabled tracing of plant metabolic response in terms of alkane biosynthesis under different phases of drought and rapid cycling of alkanes in the plant-soil system

Pyrogenic molecular markers: Linking PAH with BPCA analysis

Molecular characterization of pyrogenic organic matter (PyOM) is of great interest to understand the formation and behavior of these increasingly abundant materials in the environment. Two molecular marker methods have often been used to characterize and trace PyOM: polycyclic aromatic hydrocarbon (PAH) and benzenepolycarboxylic acid (BPCA) analysis. Since both methods target pyrogenic polycyclic compounds, we investigated the linkages between the two approaches using chars that were produced under controlled conditions. Rye and maize straws and their analogues charred at 300, 400 and 500 °C, respectively, were thus analyzed with both methods. Moreover, we also measured BPCAs directly on the lipid extracts, on which PAHs were analyzed, and on the respective extraction residues, too. Both methods revealed important features of the chars, in particular the increasing degree of aromatic condensation with increasing highest heating temperature (HTT). The overlap between the two methods was identified in the lipid fraction, where the proportion of benzenetricarboxylic acids (B3CAs) correlated with PAH abundance. The results confirmed the validity and complementarity of the two molecular marker methods, which will likely continue to play a crucial role in PyOM research due to the recent developments of compound-specific PAH and BPCA stable carbon (d¹³C) and radiocarbon (¹⁴C) isotope methods

Late-season biosynthesis of leaf fatty acids and n-alkanes of a mature beech (Fagus sylvatica) tree traced via13CO2 pulse-chase labelling and compound-specific isotope analysis

Leaf cuticular waxes play an important role in reducing evapotranspiration via diffusion. However, the ability of mature trees to regulate the biosynthesis of waxes to changing conditions (e.g., drought, light exposition) remain an open question, especially during the late growing season. This holds also true for one of the most widely distributed trees in Central Europe, the European beech tree (Fagus sylvatica L.). In order to investigate the ongoing formation of wax constituents like alkanes and fatty acids, we conducted a 13CO2 pulse-chase labelling experiment on sun-exposed and shaded branches of a mature beech tree during the late summer 2018. The 13C-label was traced via compound-specific δ13C isotope analysis of n-alkanes and fatty acids to determine the de-novo biosynthesis within these compound classes. We did not observe a significant change in lipid concentrations during the late growing season, but we found higher n-alkane concentrations in sun-exposed compared to shaded leaves in August and September. The n-alkane and fatty acid composition showed ongoing modifications during the late growing season. Together with the uptake and following subsequent decrease of the 13C-label, this suggests ongoing de-novo biosynthesis, especially of fatty acids in European beech leaves. Moreover, there is a high variability in the 13C-label among individual branches and between sun-exposed and shaded leaves. At the same time, sun-exposed leaves invest more of the assimilated C into secondary metabolites such as lipids than shaded leaves. This indicates that the investigated mature beech tree could adjust its lipid production and composition in order to acclimate to changes in microclimates within the tree crown and during the investigated period

High-resolution record of stable isotopes in soil carbonates reveals environmental dynamics in an arid region (central Iran) during the last 32 ka

Although central Iran is pivotal for palaeoclimatic correlations, palaeoenvironmental data for this region is very sparse and a reliable chronology for pedogenic features is lacking. We therefore tried to answer the question how the environmental conditions and, in particular, the climate developed over time by using the isotopic signatures of pedogenic carbonates. We present a chronology of pedogenic carbonates in association with stable carbon and oxygen isotopes in both the matrix and coating carbonates of a relict palаeosol (Baharan palaeosol) in central Iran to understand the dynamics of environmental changes in this region during the late Quaternary. The palаeosol experienced several episodes of leaching during pedogenesis as reflected in its morphology (carbonate coatings under the rock fractions) and geochemical characteristics (Ba/Sr ratios). The δ$^{18}$O values of both the matrix and coating carbonates in the upper 60 cm (especially in the upper 20 cm) of the pedon are enriched (∼4‰) compared to the subsoil and are mainly related to the impact of evaporation. Moreover, the δ$^{13}$C values of the carbonates are in isotopic disequilibrium with the modern vegetation cover (desert shrubs) of the study area and are enriched in different degrees. The carbonates in the top 60 cm are formed by the input of atmospheric CO$_{2}$ and calcareous dust while deeper carbonates formed in an environment exhibiting a higher contribution of C$_{4}$ plants. Based on the radiocarbon chronology of carbonate coatings, it seems that three main stages of palaeoenvironmental changes occurred in the region during the last 32 ka. The first stage lasted ca. 5,000 years (between 31.6 and 26.0 ka) and was accompanied by deep leaching under sub-humid climatic conditions and the expansion of C$_{4}$ plants. Under the dominance of semi-arid conditions during the second stage until the late Holocene, a gradual increase in the δ$^{18}$O values and aridity occurred in the region. The last phase in the late Holocene was characterised by the establishment of an arid and evaporative environment with a sparse vegetation cover. A climatic correlation using the oxygen isotopic composition of secondary carbonates from the Baharan palaeosol, Soreq Cave (the Levant) and Hoti Cave (Oman; both having speleothems records) suggested a climatic connection between central Iran and the eastern Mediterranean during the late Pleistocene and between central Iran and northern Oman during the late Holocene

Soil organic carbon stocks did not change after 130 years of afforestation on a former Swiss Alpine pasture

Soil organic matter (SOM) plays an important role in the global carbon cycle, especially in alpine ecosystems. However, ongoing forest expansion in high-elevation systems potentially alters SOM storage through changes in organic matter (OM) inputs and microclimate. In this study, we investigated the effects of an Picea abies L. afforestation chrono-sequence (0 to 130 years) of a former subalpine pasture in Switzerland on soil organic carbon (SOC) stocks and SOM dynamics. We found that SOC stocks remained constant throughout the chrono-sequence, with comparable SOC stocks in the mineral soils after afforestation and previous pasture (SOC forest40 = 11.6 ± 1.1 kg m−2, SOC forest130 = 11.0 ± 0.3 kg m−2 and SOC pasture = 11.5 ± 0.5 kg m−2). However, including the additional carbon of the organic horizons in the forest, reaching up to 1.7 kg m−2 in the 55-year old forest, resulted in an increase in the overall SOC stocks following afforestation. We found that the soil C:N ratio in the mineral soil increased in the topsoil (0–5 cm) with increasing forest stand age, from 11.9 ± 1.3 in the pasture to 14.3 ± 1.8 in the 130-year old forest. In turn, we observed a decrease in the soil C:N ratio with increasing depth in all forest stand ages. This suggests that litter-derived organic matter (C:N from 35.1 ± 1.9 to 42.4 ± 10.8) is likely to be incorporated and translocated from the organic horizon to the mineral topsoil (0–10 cm) of the profiles. Due to the high root C:N ratio (pasture 63.5 ± 2.8 and forests between 54.7 ± 3.9 and 61.2 ± 2.9), particulate root-derived organic matter seems to have a rather small effect on forest soil C:N ratios, as well as on SOC accumulation in the mineral soil. These results suggest that, although afforestation does not change the SOC stock in the mineral soil, there is an apparent alteration in the SOM dynamics through changes in the litter composition caused by the vegetation shift. We conclude that, at our study site, spruce afforestation on a former subalpine pasture does not change the total SOC stock and that, consequently, there is no additional SOC sequestration on a decadal to centennial scale

Rapid loss of complex polymers and pyrogenic carbon in subsoils under whole-soil warming

Subsoils contain more than half of soil organic carbon (SOC) and are expected to experience rapid warming in the coming decades. Yet our understanding of the stability of this vast carbon pool under global warming is uncertain. In particular, the fate of complex molecular structures (polymers) remains debated. Here we show that 4.5 years of whole-soil warming (+4 °C) resulted in less polymeric SOC (sum of specific polymers contributing to SOC) in the warmed subsoil (20–90 cm) relative to control, with no detectable change in topsoil. Warming stimulated the subsoil loss of lignin phenols (−17 ± 0%) derived from woody plant biomass, hydrolysable lipids cutin and suberin, derived from leaf and woody plant biomass (−28 ± 3%), and pyrogenic carbon (−37 ± 8%) produced during incomplete combustion. Given that these compounds have been proposed for long-term carbon sequestration, it is notable that they were rapidly lost in warmed soils. We conclude that complex polymeric carbon in subsoil is vulnerable to decomposition and propose that molecular structure alone may not protect compounds from degradation under future warming

Comparison of paleobotanical and biomarker records of mountain peatland and forest ecosystem dynamics over the last 2600 years in central Germany

As peatlands are a major terrestrial sink in the global carbon cycle, gaining an understanding of their development and changes throughout time is essential in order to predict their future carbon budget and potentially mitigate the adverse outcomes of climate change. With this aim to understand peat development, many studies have investigated the paleoecological dynamics by analyzing various proxies, including pollen, macrofossil, elemental, and biomarker analyses. However, as each of these proxies is known to have its own benefits and limitations, examining them in parallel allows for a deeper understanding of these paleoecological dynamics at the peatland and a systematic comparison of the power of these individual proxies. In this study, we therefore analyzed peat cores from a peatland in Germany (Beerberg, Thuringia) to (a) characterize the vegetation dynamics over the course of the peatland development during the late Holocene and (b) evaluate to what extent the inclusion of multiple proxies, specifically pollen, plant macrofossils, and biomarkers, contributes to a deeper understanding of those dynamics and interaction among factors. We found that, despite a major shift in the regional forest composition from primarily beech to spruce as well as many indicators of human impact in the region, the local plant population in the Beerberg area remained stable over time following the initial phase of peatland development up until the last couple of centuries. Therefore, little variation could be derived from the paleobotanical data alone. The combination of pollen and macrofossil analyses with the elemental and biomarker analyses enabled further understanding of the site development as these proxies added valuable additional information, including the occurrence of climatic variations, such as the Little Ice Age, and more recent disturbances, such as drainage

Warming and elevated CO2 promote rapid incorporation and degradation of plant-derived organic matter in an ombrotrophic peatland

Rising temperatures have the potential to directly affect carbon cycling in peatlands by enhancing organic matter (OM) decomposition, contributing to the release of CO2 and CH4 to the atmosphere. In turn, increasing atmospheric CO2 concentration may stimulate photosynthesis, potentially increasing plant litter inputs belowground and transferring carbon from the atmosphere into terrestrial ecosystems. Key questions remain about the magnitude and rate of these interacting and opposing environmental change drivers. Here, we assess the incorporation and degradation of plant- and microbe-derived OM in an ombrotrophic peatland after 4 years of whole-ecosystem warming (+0, +2.25, +4.5, +6.75 and +9°C) and two years of elevated CO2 manipulation (500 ppm above ambient). We show that OM molecular composition was substantially altered in the aerobic acrotelm, highlighting the sensitivity of acrotelm carbon to rising temperatures and atmospheric CO2 concentration. While warming accelerated OM decomposition under ambient CO2, new carbon incorporation into peat increased in warming × elevated CO2 treatments for both plant- and microbe-derived OM. Using the isotopic signature of the applied CO2 enrichment as a label for recently photosynthesized OM, our data demonstrate that new plant inputs have been rapidly incorporated into peat carbon. Our results suggest that under current hydrological conditions, rising temperatures and atmospheric CO2 levels will likely offset each other in boreal peatlands