Triterpenoids and their derivatives are ubiquitous in sediment samples. Land plants are major sources of nonhopanoid triterpenoids; these terpenoids comprise a vast number of chemotaxonomically distinct biomolecules. Hence, geologically occurring plant-derived triterpenoids (geoterpenoids) potentially record unique characteristics of paleovegetation and sedimentary environments, and serve as source-specific markers for studying paleoenvironments. This review is aimed at explaining the origin of triterpenoids and their use as biomarkers in elucidating paleoenvironments. Herein, application of plant-derived triterpenoids is discussed in terms of: (i) their biosynthetic pathways. These compounds are primarily synthesized via oxidosqualene cyclase (OSCs) and serve as precursors for a variety of membrane sterols and steroid hormones. Studies on OSCs and resulting compounds have helped elucidate the diversity and origin of the parent terpenoids. (ii) their chemotaxonomic significance. Geochemically important classes of triterpenoid skeletons are useful in gathering and substantiating information on botanical origin of these compounds, evolution and diversification of angiosperms, and organic matter preservation during different periods in geological history. (iii) current knowledge on their transformation into geoterpenoids via diagenetic alterations. This knowledge helped in paleoenvironmental reconstructions from the local depositional environments to paleoclimatic variations. This review focuses on triterpenoids as paleoenvironmental biomarkers, and consolidates relevant literature that can form the basis for developing tools and techniques for improved paleoenvironment reconstruction. Future investigations should focus on detecting as yet unknown classes of triterpenoids and their biosynthetic pathways, inclusion of this information in automated databases, and identification of geoterpenoids as potential biomarkers to further our understanding of paleoenvironments and paleoclimate.
Ester-bound compounds (compounds released by saponification) from the resistant macromolecules (RMMs) were analyzed in the extant and Pliocene fossil Liquidambar fruit from the Tokai Group distributed in Gifu Prefecture, central Japan. We compared the distributions of the ester-bound triterpenoids from the fossil with those of free and ester-bound triterpenoids of the extant fruits. The series of triterpenoid acids such as oleanolic and ursolic acids were identified in free compounds but hardly detected in ester-bound compounds from the extant Liquidambar samples. Most of free triterpenoids identified in the extant Liquidambar were also present in the fossil Liquidambar sample as the ester-bound compounds. These results indicate the occurrence of post-depositional incorporation of the free triterpenoids into the macromolecules such as the RMMs via ester bonds. Thus, it is suggested that the ester-bound triterpenoids in RMMs serve as excellent archives of the indigenous assemblage of triterpenoids. The class distributions of the oleanoids and ursanoids in the ester-bound compounds from the fossil sample are considerably different from those in the free polar compounds of the extant samples. Selective preservation of C-28 carboxyl group compared to C-3 oxygenated functions indicates that the triterpenoid acids were incorporated into RMM via ester-bound formed between C-28 carboxyl and hydroxyl groups of the RMM. These results indicate that the ester-bound triterpenoid acids obtained from the fossil sample are selectively preserved by incorporation into the RMM of the fruits. Res. Org. Geochem. 34, 37 − 45 (2018) Ken Sawada, Takaaki Arai, Hideto Nakamura and Minoru Tsukagoshi -38In the present study, we analyzed the ester-bound compounds from the RMMs in the Pliocene plant macrofossils, a fossil fruit of angiosperm Liquidambar (family Altingiaceae), from the Tokai Group distributed in Gifu Prefecture, central Japan. We compared the distributions of the ester-bound triterpenoids from the fossil with those of free and ester-bound triterpenoids of the extant fruits, and examine the preservation processes of the terpenoid biomarkers in the RMMs of plant fossil. 2. Materials and methods 2.1. Samples A fruit fossil of Liquidambar formosana (Fig. 1) was collected from the Pliocene Middle Sand and Mud Member (Loc. NN06), Toki Sand and Gravel Formation (ca. 4 Ma; Todo Collaborative Research Group, 1994; Tsukagoshi and Todo Collaborative Research Group, 1995), the Tokai Group in Ena City, Gifu Prefecture, central Japan. The Toki Sand and Gravel Formation was deposited in a fluvial system and contained excellentpreserved plant macrofossils (Todo Collaborative Research Group, 1994). This fossil has been preserved within a bottle filled with the solution of ethyl alcohol in the Osaka Museum of Natural History, Japan. Therefore, most of the free (solvent-extractable) compounds have been removed from this fossil and probably added the contamination. The fruits of extant L. formosana and Liquidambar styraciflua (Fig. 1) were collected in the Nagai Botanical Garden in Osaka City, Japan in November of 2002. 2.2. Lipid extraction and separation The fruit fossil was treated by hydrolysis (saponification) with KOH / MeOH. Prior to the hydrolysis, the fossil was ‘washed’ by pure water, methanol (MeOH) and dichloromethane (DCM). The residue after this extraction was saponified within a sealed glass tube by 1M KOH in MeOH at 110oC for 3 hours. The non-saponifiable (neutral) lipids were extracted by partitioning with hexane-ethyl acetate (8:1 v / v), and the carboxylic and hydroxy acids were extracted in the same way after acidification to pH 2 with HCl. The acid and neutral fractions were silylated by using bis(trimethylsilyl) trifluoroacetamide (BSTFA, Wako) at 60oC for 1 hour. The extant fruits were washed with distilled water and then cut into fine tips by pruning shears. Organic constituents were extracted from the fine tips of fruit samples by successive treatment with MeOH / DCM (3 / 1 v / v, x2), DCM (x2) for 20 min, and subsequently with MeOH by steeping overnight (for about 12 hours). The lipids were separated by adding pure water to the combined extracts, before the DCM layer was siphoned off and passed through an anhydrous Na2SO4 column for removing labile and polar compounds such as amino acid and sugar. The extract was dried in a rotary evaporator and then re-dissolved in hexane. The hexane extract was passed through a silica gel column (95% activated) as modified in Sawada et al. (2013), and the polar fraction was eluted with ethyl acetate MeOH (1:1 v / v). The hydrolysis of the residue after the extraction was carried out as mentioned above. Also, the residues after extraction of extant fruits were treated by hydrolysis (saponification) with KOH / MeOH with the same methods for the fossil fruit sample as mentioned above. 2.3. Gas chromatography-mass spectrometry (GC-MS) Identification of the lipid was carried out by GC-MS using a Hewlett Packard 6890 attached to a capillary GC (30 m x 0.25 mm i.d. DB-5HT column, J&W Scientific) directly coupled to a Hewlett Packard MSD quadrupole mass spectrometer. (electron voltage, 70 eV; emission current, 350 μA; mass range, m / z 50 – 650 in 2.91 scan / sec.) The GC temperature was programmed as follows: 60oC for 5 min, 60 – 250oC at 10oC / min, 250 – 320oC at 3oC / min and 320oC for 20 min in the polar fraction of free compounds, and 50oC for 4 min, 50 – 300oC at 4oC / min and 300oC for 20 min in the acid fraction. The lipids were quantified with a Hewlett Packard 6890 capillary GC equipped with a flame-ionization Fig.1 Fossil Liquidambar formosana Extant Liquidambar formosana Extant Liquidambar styraciflua Fig. 1. Photographs of a Liquidambar fruit fossil (L. formosana) from the Toki Sand and Gravel Formation, Tokai Group in Ena City (Gifu Prefecture), central Japan, and extant fruits (L. formosana and L. styraciflua). Scale bars = 2.0 cm. Higher plant triterpenoids bound in resistant macromolecules in extant and Pliocene fossil Liquidambar fruits -39detector (FID), the capillary column and temperature program used being the same as those used for GC / MS. Identification of the compounds was made from mass chromatographic responses (mass fragmentation pattern and molecular ion etc.) and relative retention times in comparison with library data (NIST14) and the literature. Quantification of the compounds was made from the peak areas determined by the FID responses, and / or the responses of individual base peaks (e. g., m / z 203 for oleanoids) determined from the authentic standards of oleanolic acid (Wako co.) and betulin (Aldrich co.). 3. Results and Discussion 3.1. Free compounds In the extant L. formosana and L. styraciflua samples, C16-C24 even-carbon number alkanoic acids, C18 alkenoic acids, C9 dicarboxylic acid, lignin phenols such as vanillic and syringic acids, and triterpenoid acids such as oleanolic and ursolic acids were identified as the major free polar compounds (Fig. 2a). The oleanolic and ursolic acids are angiospermous biomarkers, and several peaks in the mass fragmentogram (MF) of m / z 203 are attributed to their isomers and analogues (Table 1 and Fig. 3). Fig. 4 shows mass spectra of TMS derivatives of these compounds. The 3-oxo-oleanolic acid (oleanonic acid) and 3-epi-oleanolic acid can be identified according to the mass spectra of extracts from Liquidambar resins reported by Pastorova et al. (1998). The other triterpenoids were tentatively identified by molecular ions, mass fragment pattern and retention times. Concentrations of the triterpenoid acids in the free polar fractions of the extant L. formosana and L. styraciflua are shown in Table 1 and Fig. 5. It was found that the concentrations of 3-oxo-oleanolic acids are the highest in both extant species (425.98 μg / g in L. formosana and 34.25 μg / g in L. styraciflua). The oleanolic acid and 3-oxo-ursolic acid (ursanonic acid) are also major components in these species. 3.2. Ester-bound compounds Figure 2b shows the total ion chromatograms (TICs) of the compounds obtained by the KOH / MeOH hydrolysis (saponification) in the extant and fossil L. formosana, and extant L. styraciflua samples. In the extant samples, cutin acids, C16 and C18 alkanoic acids, C4, C9, and C16 dicarboxylic acids, and phenolic acids such as vanillic and syringic acids were identified as the major compounds released. On the other hand, C16-C26 even-carbon number alkanoic acids and lignin phenols such as vanillic and syringic acids were mainly identified in the compounds released from the fossil L. formosana. These released compounds can be interpreted to have existed as the acyl moieties in the RMM of fruits, namely, is the ester-bound components. The cutin acids, which are derived from cuticle polyesters, detected in Table 1. Peak assignments and concentrations of triterpenoids labelled in Fig. 3 in the acid fractions from extant and fossil Liquidambar. Concentrations (μg/g) Peak Compound name MW fossil L. formosana extant L. formosana extant L. styraciflua Bound*1 Free*2 Free*2 1 olean-2, 12-dienoic acid 510 1.82 0.59 n.d. 2 ursan-2, 12-dienoic acid 510 1.29 2.28 n.d. 3 unknown triterpenoid 512 0.48 1.40 n.d. 4 3-epi-oleanolic acid 600 0.96 4.69 24.80 5 ursolic acid (isomer) 600 0.85 5.03 8.17 6 3-oxo-oleanolic acid (isomer) 526 1.30 0.80 n.d. 7 3-oxo-oleanolic acid 526 0.60 34.25 425.98 8 3-oxo-lup-20(29)-en-28-oic acid 526 n.d. 21.54 162.40 9 unknown triterpenoid 538 n.d. 4.39 n.d. 10 oleanolic acid 600 0.75 11.17 70.65 11 3-oxo-ursolic acid 526 0.54 17.60 51.10 12 unknown triterpenoid 614 n.d. 8.40 6.55 13 unknown triterpenoid 612 0.35 5.32 n.d. 14 ursolic acid 600 0.82 8.39 36.92 15 unknown triterpenoid *3 n.d. 11.71 n.d. 16 23-oxo-ursolic acid 614 n.d. 11.67 28.40 17 unknown triterpenoid 658 n.d. 16.78 22.72 18 unknown triterpenoid 614 n.d. 11.06 n.d. 19 23 or 24-oxo-triterpenoic acid 614 n.d. n.d. 74.22 20 unknown triterpen
Elevation in the δN value of amino acids (δNAAs) from the diet to its consumer (i.e. ‘inter’-trophic discrimination factor: TDF) has been widely used to illustrate the trophic hierarchy among organisms in ecological food webs. However, there is ‘intra’-trophic discrimination factor (TDF’) within a single organism, which is attributable to the catabolism of storage compounds for adjusting the energy balance between supply and demand, independent of the TDF between two separate organisms. The δNAAs values of the deciduous plant Cerasus lannesiana reveal that the TDF’ is 0.1 ± 1.0‰ (mean ± 1σ) for leaf senescence from spring to autumn, whereas that is gradually decreased from 5.3‰ to 0.9‰ for leaf flush in early spring. These results imply that plants can use sufficient photosynthetically-fixed energy for the leaf senescence, but use a large amount of catabolically-released energy (from deamination of storage amino acids) for the leaf flush under no / less photosynthetic activities. Thus, we predict that the metabolic energy fluxes can be considered in the isotope ecology, as such TDF’ potentially propagates into the δNAAs values in consumers that particularly feed on buds and flush leaves. Res. Org. Geochem. 33, 1 − 6 (2017) Yuko Takizawa and Yoshito Chikaraishi -2βGlu / Phe is derived from the same offset in the δ N value between glutamic acid and phenylalanine in primary producers to that used in the eq. (1). As similar to the flowers, if the energy supply frequently leans from the photosynthesis to the catabolism of amino acids even in plant leaves, the TDF’ can propagate through consumers in food webs. Takizawa et al. (2017) indeed speculated that the TDF’ would be detectable in buds and flush leaves during plant phenology. Identifying specific factors, when / how the TDF’ is substantial large in plant leaves, is thus required to improve accuracy of the TP estimation in the isotope ecology, particularly for studies of green food webs where plant leaves considerably contribute to basal resources. In the present study, we determined the δNAAs values in leaves of the deciduous plant Cerasus lannesiana for leaf senescence (March-October in 2015) and leaf flush (January-March in 2016) periods, to evaluate diversity and variation in the TDF’ with respect to the phenology of plant leaves. Furthermore, we discuss the potential impact whether or not the TDF’ in leaves propagates into the δNAAs values in food webs. 2. Materials and Methods Leaf and flower samples We collected leaves of the deciduous plant C. lannesiana for leaf senescence (March-October in 2015) and flush (January-March in 2016) periods and flowers of the same plant for blooming periods (early spring in both 2015 and 2016), from a house-garden in Yugawara, Japan (35°08 ́N, 139°07E) (Table 1). This plant commonly thrives in the temperate region of Japan. The phenology of this plant is composed of growing seasons and winter dormancy, as flush leaves and flowers in spring, mature leaves in summer, turned leaves in autumn, and no leaves in winter. The bloom of flowers generally starts for about 2-3 weeks prior to the flush of leaves. For leaf-senescence and flowering, approximately ten leaves and ten flowers were collected, respectively, cleaned with distilled water to remove surface contaminants, homogenized to a fine powder using a Tube-Mill (IKA), and freeze-dried. On the other hand, for leaf flush, approximately five small leaves were collected, cleaned with distilled water, and cut into small pieces, and total ~4 mm x 8 mm area of each sample were used. These samples were stored at − 20oC until the isotope analysis. Analysis of the δNAA values These samples were prepared for the δNAAs analysis after HCl hydrolysis and N-pivaloyl / isopropyl (Pv / iPr) derivatization, according to the procedure in Chikaraishi et al. (2009). In brief, the samples were hydrolyzed using 12M HCl at 110oC overnight (> 12 hours). The hydrolysate was washed with n-hexane / dichloromethane (3 / 2, v / v) to remove hydrophobic constituents. The derivatization was performed sequentially with thionyl chloride / 2-propanol (1 / 4, v / v) at 110oC for 2 hours and pivaloyl chloride / dichloromethane (1 / 4, v / v) at 110oC for 2 hours. The δNAAs values were determined by gas chromatography / isotope ratio mass spectrometry (GC / IRMS) using a 6890N GC (Agilent Technologies) instrument coupled to a DeltaXP IRMS instrument through combustion (950oC) and reduction (550oC) furnaces, a countercurrent dryer (Permeable membrane, Table 1. Nitrogen isotopic composition of amino acids in plant leaves and flowers, examined in this study. Collection date (yy/mm/dd) δN (‰) TPGlu / Phe 2 TDF’ 3 Reference Alanine Glycine Valine Leucine Isoleucine Proline Glutamic acid Phenyl alanine
Organisms have complex metabolic networks, and their fluxes could be sensitive depending on various physio logical and environmental conditions. As one of these factors, reproduction (e.g., egg production) is a common and essential process to create new individuals. However , little is known about the effect of reproduction in metabolic fluxes of organisms. In this study, we cultured the calanoid copepod Acartia steueri, and measured the stable nitrogen isotopic composition (δ 15 N) of amino acids in adult males and females and their eggs in order to demonstrate the effect of reproduction in amino acid metabolic fluxes of this species. The δ 15 N values of glutamic acid and phenylalanine and the estimated trophic position were 6.3‰, −2.6‰ and 1.73 for male adults; 7.0‰, −2.6‰ and 1.78 for female adults; and 7.3‰, −2.3‰ and 1.82 for eggs, respectively. These results demonstrate no substantial change in the isotopic signature of amino acids among the examined samples, implying that the balance in the amino acid metabolism between assimilation (i.e., absorbing input and biomass construction) and dissimilation (i.e., amino
Sweet potatoes sometimes sprout the purple color of stems with several small leaves in the house pantry. In the present study, we investigated the trophic hierarchy between a mother sweet potato and its baby sprouts grown without any light in a dark house pantry, based on stable nitrogen isotopic composition (δN, ‰ vs. AIR) of glutamic acid and phenylalanine. The isotope data reveal that glutamic acid has a significant N-enrichment (by 6.9‰) from the mother sweet potato to its baby sprout while phenylalanine has a little N-enrichment (by 0.6‰) between them. Interestingly, the isotopic heterogeneity found within the sweet potato is very similar to the isotopic discrimination generally found in the combination between plants and herbivores during grazing food webs (ca. 8.0‰ for glutamic acid and ca. 0.4‰ for phenylalanine). These results suggest that the proteins in the mother sweet potatoes are major resources for not only proteins in their baby sprouts but also growth energy in the sprouting, when they are grown heterotrophically without any light.
