[A new cinnamic acid ester derivative from Liquidambaris Resina].

Twelve compounds were isolated from Liquidambaris Resina by silica gel column chromatography and thin layer chromatography. Their structures were identified on the basis of spectral data, electron capture detector data, and physicochemical properties as(2'R, 3'R)-2',3'-dihydroxy-hydrocinnamyl-(E)-cinnamate(1),(E)-cinnamyl-(E)-cinnamate(2), cinnamic acid(3), 28-norlup-20(29)-en-3-one-17ß-hydroperoxide(4), erythrodiol(5), 13ß,28-epoxy-30-hydroxyolean-1-en-3-one(6),(3ß)-olean-12-ene-3,23-diol(7), 2α,3α-dihydroxy-olean-12-en-28-oic acid(8), 28-hydroxyolean-12-en-3-one(9), 3-epi-oleanolic acid(10), 3-oxo-oleanolic acid(11), and hederagenin(12). Compound 1 was a new cinnamic acid ester derivative and compounds 2-4,6-8, and 12 were isolated from Liquidambaris Resina for the first time. Compounds 4, 5, 10, and 12 exerted inhibitory effects on the proliferation of human umbilical vein endothelial cells(HUVEC) with the IC_(50) values of(17.43±2.17),(35.32±0.61),(27.50±0.80), and(46.30±0.30) µmol·L~(-1), respectively.

First report of Neopestalotiopsis clavispora causing leaf spot on Liquidambar formosana in China.

Liquidambar formosana Hance, a deciduous tree, is widely cultivated in China for its ornamental and afforestation value (Yin et al. 2021). In July 2019, leaf spot symptoms were observed with 20 to 30% disease incidence in Li shan forest farm (27°19'27.2″N, 115°32'51.08″E) in Ji'an city, Jiangxi province, China. Initial disease symptoms were small spots, which enlarged and circular to irregular, gray in the center, and dark brown to black circular on the lesion margin. Leaf pieces (5 × 5 mm) from the lesion borders were surfaced and sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water (Si et al. 2022). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and the representative isolates, FX-2, FX-5, and FX-9 were used for morphological studies and phylogenetic analyses. The colonies of three isolates on PDA grew fast, covering the entire plate with white cottony mycelia with black acervuli after 8 to 10 days. Conidia were 5-celled, clavate to fusiform, smooth, 19.6-24.2 × 6.2-8.5 µm (n = 100). The 3 median cells were dark brown to olivaceous, central cell was darker than other 2 cells, and the basal and apical cells were hyaline. All conidia developed one basal appendage (3.5-8.2 µm long; n = 100), and 2-3 apical appendages (18-31 µm long; n = 100), filiform. Morphological features were similar to Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) regions, ß-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, T1/Bt-2b, EF1-728F/EF-2 (Maharachchikumbura et al. 2014), respectively. All sequences were deposited into GenBank (ITS, ON622512- ON622514; TUB2, ON676532 - ON676534; TEF1-α, ON676529 - ON676531). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed FX-2, FX-5, and FX-9 in the clade of N. clavispora. Based on the multi-locus phylogeny and morphology, three isolates were identified as N. clavispora. To confirm pathogenicity, 10 healthy 2-year-old seedlings, and 5 leaves per seedling were wounded with a sterile needle (Φ=0.5 mm) and inoculated with 200 µL conidial suspension per leaf(106 conidia/mL). Ten control plants were inoculated with ddH2O. All the inoculated leaves were covered with plastic bags and kept in a greenhouse at 26 ± 2 °C and RH 70%. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 8 days. N. clavispora was reisolated from the lesions, whereas no fungus was isolated from control leaves. N. clavispora can cuase leaf diseases in a variety of hosts, including × Taxodiomeria peizhongii (Zhang et al. 2022), Macadamia integrifolia (Qiu et al. 2020), Dendrobium officinale (Cao et al. 2022). N. cocoes, N. chrysea, Pestalotiopsis neglecta and P. neolitseae were also reported to infect L. formosana (Fan et al. 2021). However, this is the first report of N. clavispora infecting L. formosana in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

Volatile Profiling and Transcriptome Sequencing Provide Insights into the Biosynthesis of α-Pinene and ß-Pinene in Liquidambar formosana Hance Leaves.

Liquidambar formosana Hance is a pinene-rich deciduous plant species in the Altingiaceae family that is used as a medicinal plant in China. However, the regulatory mechanisms underlying α-pinene and ß-pinene biosynthesis in L. formosana leaves remain unknown. Here, a joint analysis of the volatile compounds and transcriptomes of L. formosana leaves was performed to comprehensively explore the terpene synthase (TPS) that may participate in α-pinene and ß-pinene biosynthesis. Headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) jointly detected volatile L. formosana leaves. Trees with high and low levels of both α-pinene and ß-pinene were defined as the H group and L group, respectively. RNA sequencing data revealed that DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase), HDS [(E)-4-hydroxy-3-methylbut-2-eny-l-diphosphate synthase], and TPS may be the major regulators of monoterpenoid biosynthesis. We identified three TPSs (LfTPS1, LfTPS2, and LfTPS3), which are highly homologous to α-pinene and ß-pinene synthases of other species in phylogenetic analysis. Four TPS genes (LfTPS1, LfTPS2, LfTPS4, LfTPS5) may be critically involved in the biosynthesis and regulation of α-pinene and ß-pinene in L. formosana. Bioinformatic and transcriptomic results were verified using quantitative real-time PCR. We identified LfTPS1, LfTPS2 as candidate genes for α-pinene and ß-pinene biosynthesis that significantly improve the yield of beneficial terpenoids.

Liquidambarines A - C, three new abietane diterpenoids from Liquidambar formosana Hance and their anti-inflammatory activities.

Liquidambarines A - C (1-3), three new abietane-type diterpenoids, together with five known compounds (4-8) were isolated from the resin of Liquidambar formosana Hance. Their structures were elucidated by the combination of spectroscopic and computational methods. We explored their anti-inflammatory potential by analyzing the protein expression of iNOS and COX-2. Compounds 1 and 3 exhibit significant anti-inflammatory activities without cytotoxicity. These experimental studies suggest these new abietane-type diterpenoids have the potential to be candidates for inflammation-associated diseases.

A new cinnamic acid ester derivative from Liquidambaris Resina / 中国中药杂志

Twelve compounds were isolated from Liquidambaris Resina by silica gel column chromatography and thin layer chromatography. Their structures were identified on the basis of spectral data, electron capture detector data, and physicochemical properties as(2'R, 3'R)-2',3'-dihydroxy-hydrocinnamyl-(E)-cinnamate(1),(E)-cinnamyl-(E)-cinnamate(2), cinnamic acid(3), 28-norlup-20(29)-en-3-one-17β-hydroperoxide(4), erythrodiol(5), 13β,28-epoxy-30-hydroxyolean-1-en-3-one(6),(3β)-olean-12-ene-3,23-diol(7), 2α,3α-dihydroxy-olean-12-en-28-oic acid(8), 28-hydroxyolean-12-en-3-one(9), 3-epi-oleanolic acid(10), 3-oxo-oleanolic acid(11), and hederagenin(12). Compound 1 was a new cinnamic acid ester derivative and compounds 2-4,6-8, and 12 were isolated from Liquidambaris Resina for the first time. Compounds 4, 5, 10, and 12 exerted inhibitory effects on the proliferation of human umbilical vein endothelial cells(HUVEC) with the IC_(50) values of(17.43±2.17),(35.32±0.61),(27.50±0.80), and(46.30±0.30) μmol·L~(-1), respectively.

Evaluation of Ecological Service Function of Liquidambar formosana Plantations.

A Liquidambar formosana plantation is a kind of fast-grown forest in the subtropical region, providing a variety of ecosystem services such as superior wood, carbon fixation and oxygen release, and biodiversity maintenance. However, the ecological service function value of Liquidambar formosana plantations is not clear. To gain insights into the characteristics and importance of its ecological and economic benefits, the Liquidambar formosana plantation in the Tianjiling Forest Farm of Changsha City was taken as the specific research object in this paper. The ecological service function evaluation index system for Liquidambar formosana plantations was established based on the relevant research worldwide and the actual situation. The market value method, shadow engineering method, carbon tax method, and other environmental economics methods were used to estimate the value of seven ecological service functions (including organic matter production, carbon fixation and oxygen release, water conservation, soil conservation, soil improvement, air purification, and biodiversity maintenance) of the forest of Liquidambar formosana. The results indicated that the total economic value of ecological service function provided by the Liquidambar formosana plantation of Changsha was 103,277.82 RMB/(hm2·a), and the indirect economic value was 8.47 times that of the direct economic value. Among the seven ecological service functions, the value of carbon fixation and oxygen release was the highest (36,703.33 RMB·hm-2·a-1), thus suggesting that the Liquidambar formosana plantation had strong photosynthesis and significant carbon fixation. This study directly reflects the value of forest ecological service function in the form of currency, which is beneficial to provide more insights into forest ecological service function so as to provide basic data and a scientific basis for the protection, construction, and promotion of the sustainable utilization and development of urban forest resources.

Transcriptomic Analyses Reveal Key Genes Involved in Pigment Biosynthesis Related to Leaf Color Change of Liquidambar formosana Hance.

Liquidambar formosana Hance has a highly ornamental value as an important urban greening tree species with bright and beautiful leaf color. To gain insights into the physiological and molecular mechanisms of L. formosana leaf color change, the leaves of three different clones were sampled every ten days from October 13, 2019, five times in total, which are S1, S2, S3, S4 and S5. Transcriptome sequencing was performed at S1 and S4. The chlorophyll content of the three clones decreased significantly, while the anthocyanins content of the three clones increased significantly in the coloring stage. The anthocyanins content of clone 2 was far more than that of the other two clones throughout the period of leaf color change. The transcriptome analysis showed that six DEGs related to anthocyanins biosynthesis, including CHS (chalcone synthase), CHI (chalcone isomerase), F3'H (flavonoid 3'-hydroxylase), DFR (dihydroflavonol 4-reductase), ANS (anthocyanidin synthase) and FLS (flavonol synthase), were found in three clones. Clone 2 has another three DEGs related to anthocyanins biosynthesis, including PAL (Phenylalanine ammonia-lyase), F3'5'H (flavonoid 3',5'-hydroxylase) and UFGT (flavonoid 3-O-glucosyltransferase). We lay a foundation for understanding the molecular regulation mechanism of the formation of leaf color by exploring valuable genes, which is helpful for L. formosana breeding.

Accumulation of Anthocyanidins Determines Leaf Color of Liquidambar Formosana as Revealed by Transcriptome Sequencing and Metabolism Analysis.

Liquidambar formosana is important for its ornamental value in China; it is increasingly used for landscaping and gardening trees due to its diverse leaf colors and seasonal changes. Varieties including either a fixed leaf color, the purplish 'Fuluzifeng' (ZF), or seasonal changes in leaf color, the reddish 'Nanlinhong' (NLH) have been bred and registered as new plant varieties under the International Union for the Protection of New Plant Varieties (UPOV) system. To gain practical insights into the anthocyanin biosynthetic process, transcriptome sequencing (Illumina) was performed to clarify the metabolic pathways present in the three seasonal changes in leaf colors in NLH and in the springtime purple-red color of ZF. qRT-PCR was used to verify the speculation. Based on the differentially expressed genes and flavonoids analyses, the spring, summer, and autumn leaves of NLH were compared to study the seasonal differences. NLH and ZF were compared to study the formation mechanism of the two leaf colors in spring. Transcriptome sequencing produced a total of 121,216 unigenes from all samples, where 48 unigenes were differentially expressed and associated with the anthocyanidin pathway. The expression levels of LfDFR and LfANS genes corresponded to the accumulation of concentrations of cyanidins in spring (NLHC) and autumn leaves (NLHQ), respectively, with different shades of red. Moreover, the LfF3'5'H gene corresponded to the accumulation of flavonols and delphinidins in purple-red leaves (ZFC). Cyanidin and peonidin were the key pigments in red and dark-red leaves, and purple-red leaves were co-pigmented by cyanidins, pelargonidins, and delphinidins.

CO2 -responsiveness of leaf isoprene emission: Why do species differ?

Leaf isoprene emission rate, I, decreases with increasing atmospheric CO2 concentration with major implications for global change. There is a significant interspecific variability in [CO2 ]-responsiveness of I, but the extent of this variation is unknown and its reasons are not understood. We hypothesized that the magnitude of emission reduction reflects the size and changeability of precursor pools responsible for isoprene emission (dimethylallyl diphosphate, DMADP and 2-methyl-erythritol 2,4-cyclodiphosphate, MEcDP). Changes in I and intermediate pool sizes upon increase of [CO2 ] from 400 to 1500 µmol/mol were studied in nine woody species spanning boreal to tropical ecosystems. I varied 10-fold, total substrate pool size 37-fold and the ratio of DMADP/MEcDP pool sizes 57-fold. At higher [CO2 ], I was reduced on average by 65%, but [CO2 ]-responsiveness varied an order of magnitude across species. The increase in [CO2 ] resulted in concomitant reductions in both substrate pools. The variation in [CO2 ]-responsiveness across species scaled with the reduction in pool sizes, the substrate pool size supported and the share of DMADP in total substrate pool. This study highlights a major interspecific variation in [CO2 ]-responsiveness of isoprene emission and conclusively links this variation to interspecific variability in [CO2 ] effects on substrate availability and intermediate pool size.

Pentacyclic Triterpenes from the resin of Liquidambar formosana have anti-angiogenic properties.

Phytochemical investigation of the resin of Liquidambar formosana Hanc led to the separation and identification of five undescribed pentacyclic triterpenoids, including two lupane type, one taraxerane type, and two oleanane type triterpenoids, in addition to ten known analogues. Structures and relative or absolute configurations were elucidated by intensive spectroscopic methods, and single-crystal X-ray diffraction analysis. All isolated compounds were evaluated for their anti-angiogenic effects in vitro against VEGF-induced endothelial cell proliferation and migration in HUVECs. Among them, (5R, 8R, 9R, 10R, 13S, 14R, 17R, 18R, 19S)-17,18-epoxy-17,18-seco-28-norlupa-17- hydroxy-20 (29) -ene-3-one, (5R, 8R, 9R, 10R, 13S, 14R, 17S, 18S, 19S, 20S)-17, 20-peroxy-28- norlupa -29 -hydroxy- 3-one, 11α,12α:13ß,28-diepoxyoleanane- 3-one, 28-norlup-20 (29)-ene- 3ß,17ß-diol, liquidambaric lactone and 13,28-epoxy-11- oleanene- 3-one significantly inhibited VEGF-induced HUVECs proliferation with IC50 values ranging from 1.64 ± 0.36 to 7.06 ± 0.28 µM. In addition, they also effectively decreased VEGF-induced cell migration with IC50 values ranging from 1.57 ± 0.60 to 4.77 ± 0.62 µM. The structure-activity relationship of these compounds is discussed. The anti-angiogenic property of (5R, 8R, 9R, 10R, 13S, 14R, 17R, 18R, 19S)-17,18-epoxy-17,18-seco-28-norlupa-17- hydroxy-20 (29) -ene-3-one is mediated by the VEGFR2 - AKT signaling pathway.

Intra-annual Dynamics of Xylem Formation in Liquidambar formosana Subjected to Canopy and Understory N Addition.

Increasing N deposition caused by intensive anthropogenic activities is expected to affect forest growth. However, the effects of N deposition on trees are still controversial due to the wide variability in results and experimental methods used. We conducted an experiment involving both canopy and understory N addition to investigate the effects of N-addition on intra-annual xylem formation of Chinese sweetgum (Liquidambar formosana) in a warm-temperate forest of Central China. Since 2013, 50 kg N ha-1 year-1 (2.5 times the current natural N deposition) was applied monthly from April to December. In 2014 and 2015, the timing and dynamics of xylem formation were monitored weekly during March-December by microcoring the stems of control and treated trees. Similar dynamics of wood formation were observed between canopy and understory N addition. Xylem formation of all the experimental trees started in March and lasted for 119-292 days. Compared to the control, no change was observed in the timing and dynamics of wood formation in N-treated trees. Tree ring-width ranged between 1701 and 4774 µm, with a rate of xylem production of 10.52-26.64 µm day-1. The radial growth of trees was not modified by the treatments. Our findings suggest that short-term N addition is unable to affect the dynamics of xylem formation in Chinese sweetgum in Central China. The effects of N on tree growth observed in previous studies might be related to the duration of the experiment or the imbalance between the amount of natural deposition and N added during treatments.

Facile and fast removal of oil through porous carbon spheres derived from the fruit of Liquidambar formosana.

Porous carbon spheres with a diameter of 1-2 cm were prepared via a simple carbonization of the fruit of Liquidambar formosana. After carbonization, the spherical structure and inner finger-like pores were maintained with high resistance to impact. Due to the porous structure and the hydrophobic nature, the carbonized fruit of Liquidambar formosana can float on the water surface and show a super-fast oil or organic solvent sorption ability (sorption saturation can be achieved within 1-2 min). Moreover, about 99% of adsorbed oil can be easily removed from spheres via organic solvent such as ethanol or hexane, which shows good recyclability of samples. In general, considering the low-cost and abundance of raw material collected from nature and the facile synthetic process (only by carbonization), the centimeter-sized porous spheres via the carbonization of fruit of Liquidambar formosana are very promising to be used for the application of oil or organic solvent spill cleanup.

Moderate Genetic Diversity and Genetic Differentiation in the Relict Tree Liquidambar formosana Hance Revealed by Genic Simple Sequence Repeat Markers.

Chinese sweetgum (Liquidambar formosana) is a relatively fast-growing ecological pioneer species. It is widely used for multiple purposes. To assess the genetic diversity and genetic differentiation of the species, genic SSR markers were mined from transcriptome data for subsequent analysis of the genetic diversity and population structure of natural populations. A total of 10645 potential genic SSR loci were identified in 80482 unigenes. The average frequency was one SSR per 5.12 kb, and the dinucleotide unit was the most abundant motif. A total of 67 alleles were found, with a mean of 6.091 alleles per locus and a mean polymorphism information content of 0.390. Moreover, the species exhibited a relatively moderate level of genetic diversity (He = 0.399), with the highest was found in population XY (He = 0.469). At the regional level, the southwestern region displayed the highest genetic diversity (He = 0.435) and the largest number of private alleles (n = 5), which indicated that the Southwestern region may be the diversity hot spot of L. formosana. The AMOVA results showed that variation within populations (94.02%) was significantly higher than among populations (5.98%), which was in agreement with the coefficient of genetic differentiation (Fst = 0.076). According to the UPGMA analysis and principal coordinate analysis and confirmed by the assignment test, 25 populations could be divided into three groups, and there were different degrees of introgression among populations. No correlation was found between genetic distance and geographic distance (P > 0.05). These results provided further evidence that geographic isolation was not the primary factor leading to the moderate genetic differentiation of L. formosana. As most of the genetic diversity of L. formosana exists among individuals within a population, individual plant selection would be an effective way to use natural variation in genetic improvement programs. This would be helpful to not only protect the genetic resources but also attain effective management and exploit genetic resources.

Comparative Proteomic Analysis Reveals the Effects of Exogenous Calcium against Acid Rain Stress in Liquidambar formosana Hance Leaves.

Acid rain (AR) impacts forest health by leaching calcium (Ca) away from soils and plants. Ca is an essential element and participates in various plant physiological responses. In the present study, the protective role of exogenous Ca in alleviating AR stress in Liquidambar formosana Hance at the physiological and proteomic levels was examined. Our results showed that low Ca condition resulted in the chlorophyll content and photosynthesis decreasing significantly in L. formosana leaves; however, these effects could be reversed by high Ca supplementation. Further proteomic analyses successfully identified 81 differentially expressed proteins in AR-treated L. formosana under different Ca levels. In particular, some of the proteins are involved in primary metabolism, photosynthesis, energy production, antioxidant defense, transcription, and translation. Moreover, quantitative real time polymerase chain reaction (qRT-PCR) results indicated that low Ca significantly increased the expression level of the investigated Ca-related genes, which can be reversed by high Ca supplementation under AR stress. Further, Western blotting analysis revealed that exogenous Ca supply reduced AR damage by elevating the expression of proteins involved in the Calvin cycle, reactive oxygen species (ROS) scavenging system. These findings allowed us to better understand how woody plants respond to AR stress at various Ca levels and the protective role of exogenous Ca against AR stress in forest tree species.

Transcriptome analysis of a subtropical deciduous tree: autumn leaf senescence gene expression profile of Formosan gum.

Autumn leaf senescence is a spectacular natural phenomenon; however, the regulation networks controlling autumnal colors and the leaf senescence program remain largely unelucidated. Whether regulation of leaf senescence is similar in subtropical deciduous plants and temperate deciduous plants is also unknown. In this study, the gene expression of a subtropical deciduous tree, Formosan gum (Liquidambar formosana Hance), was profiled. The transcriptomes of April leaves (green leaves, 'G') and December leaves (red leaves, 'R') were investigated by next-generation gene sequencing. Out of 58,402 de novo assembled contigs, 32,637 were annotated as putative genes. Furthermore, the L. formosana-specific microarray designed based on total contigs was used to extend the observation period throughout the growing seasons of 2011-2013. Network analysis from the gene expression profile focused on the genes up-regulated when autumn leaf senescence occurred. LfWRKY70, LfWRKY75, LfWRKY65, LfNAC1, LfSPL14, LfNAC100 and LfMYB113 were shown to be key regulators of leaf senescnece, and the genes regulated by LfWRKY75, LfNAC1 and LfMYB113 are candidates to link chlorophyll degradation and anthocyanin biosynthesis to senescence. In summary, the gene expression profiles over the entire year of the developing leaf from subtropical deciduous trees were used for in silico analysis and the putative gene regulation in autumn coloration and leaf senescence is discussed in this study.

A new triterpenoid isolated from fruits of Liquidambaris Fructus / 中草药

Objective: To study the chemical constituents from Liquidambaris Fructus (fruits of Liquidambar formosana). Methods: The constituents were isolated and purified by preparative TLC and silica gel, Sephadex LH-20, and ODS column chromatography, and their structures were elucidated on the basis of their physicochemical properties and spectral data. Results: Seven compounds were obtained from 70% ethanol extract of the fruits of Liquidambaris Fructus and identified as 11α-methoxyl-28-nor-β-amyrenone (1), 28-nor-β-amyrenone (2), 3-oxo-12α-hydroxy-oleanan-28, 13β-olide (3), 3α-acetoxyl-25-hydroxy-olean-12-en-28-oic acid (4), erythodiol (5), betulonic acid (6), and styracin (7), respectively. Conclusion: Compound 1 is a new triterpenoid named liquidaformone A, and compounds 5 and 7 are isolated from the plants of Liquidambar Linn. for the first time.