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.

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.

Contact Dermatitis From Gum Mastic (Pistacia lentiscus) and Gum Storax (Liquidambar styraciflua) in Mastisol-Allergic Patients.

BACKGROUND: Mastisol Liquid Adhesive is widely used on the skin, especially after surgical procedures. It contains gum mastic, gum storax, methyl salicylate, and ethanol. OBJECTIVE: The aims of the study were to review our experience patch testing patients allergic to Mastisol and to assess coreacting substances. METHODS: We identified 18 patients who were allergic to Mastisol. Most of these had a history of postoperative or cardiac electrode dermatitis and underwent patch testing with multiple surgically related substances, including ingredients of Mastisol, compound tincture of benzoin, and fragrance-related ingredients and botanicals. RESULTS AND CONCLUSIONS: Among Mastisol-allergic patients, 13 (72%) of 18 were allergic to gum mastic, whereas 7 (44%) of 16 were allergic to gum storax. There was frequent coreactivity with various fragrance-related materials, including Majantol, Styrax benzoin, Myroxylon balsamum, Myroxylon pereirae, propolis, and others. Two gum mastic-allergic patients had positive patch tests with hydroperoxides of linalool and several other linalool-containing essential oils. As gum mastic contains linalool, it may explain some gum mastic reactions. Among patients without a history of postoperative contact dermatitis, 1 (0.4%) of 250 was patch test positive for gum mastic. This patient had allergic contact dermatitis from fragrances, so the gum mastic reaction was likely a true-positive relevant reaction.

Lfo-miR164b and LfNAC1 as autumn leaf senescence regulators in Formosan sweet gum (Liquidambar formosana Hance).

In this study, a microRNA microarray was used to investigate the microRNA profiles from young green leaves, and senescent red leaves and yellow leaves of Formosan sweet gum (Liquidambar formosana Hance). The conserved microRNA miR164 was highly expressed in green leaves compared to senescent leaves. The pri-microRNA of miR164 was identified and named lfo-miR164b based on its secondary structure. In Agrobacterium-mediated transient expression experiment, lfo-miR164b was confirmed to regulate the leaf senescence-associated gene LfNAC1 and LfNAC100. Transient overexpression of LfNAC1 induced the expression of leaf senescence genes in Nicotiana benthamiana. In addition, LfNAC1 activated the expression of proLfSGR::YFP, suggesting the regulatory role of LfNAC1 in leaf senescence. In summary, miR164 inhibits the expression of LfNAC1 in spring and summer, later on LfNAC1 actives leaf senescence-associated genes to cause leaf senescence following a gradual decline of miR164 as the seasons change. The "miR164-NAC" regulatory mechanism was confirmed in Formosan sweet gum autumn leaf senescence.

Identification, Functional Characterization, and Seasonal Expression Patterns of Five Sesquiterpene Synthases in Liquidambar formosana.

Terpenoids are a large group of important secondary metabolites that are involved in a variety of physiological mechanisms, and many are used commercially in the cosmetics and pharmaceutical industries. During the past decade, the topic of seasonal variation in terpenoid biosynthesis has garnered increasing attention. Formosan sweet gum ( Liquidambar formosana Hance) is a deciduous tree species. The expression of terpene synthase and accumulation of terpenoids in leaves may vary in different seasons. Here, four sesquiterpene synthases (i.e., LfTPS01, LfTPS02, LfTPS03, and LfTPS04) and a bifunctional mono/sesquiterpene synthase ( LfTPS05) were identified from Formosan sweet gum. The gene expression of LfTPS01, LfTPS02, and LfTPS03 showed seasonal diversification, and, in addition, expression of LfTPS04 and LfTPS05 was induced by methyl jasmonate treatment. The major products LfTPS01, LfTPS02, LfTPS04, and LfTPS05 are hedycaryol, α-selinene, trans-ß-caryophyllene, α-copaene/δ-cadinene, and nerolidol/linalool, respectively. The data indicated that the sesquiterpenoid content in the essential oil of Formosan sweet gum leaves shows seasonal differences that were correlated to the sesquiterpene synthase gene expression.

A R2R3-MYB Gene LfMYB113 is Responsible for Autumn Leaf Coloration in Formosan sweet gum (Liquidambar formosana Hance).

The regulation of autumn leaf coloration in deciduous trees has long been an enigma. Due to the fact that different coloration phenotypes may be considered when planting, more understanding of the regulation mechanism is needed. In this study, a R2R3-MYB transcription factor gene LfMYB113 was identified from a subtropical deciduous tree species Formosan sweet gum (Liquidambar formosana Hance). The expression patterns of LfMYB113 in four selected phenotypes were different and were positively correlated with leaf anthocyanin content. In a 35S::LfMYB113 transgenic Nicotiana tabacum plant, both the early and late genes in the anthocyanin biosynthetic pathway were shown to be up-regulated. It was also shown that LfMYB113 can activate the promoter sequence of LfDFR1 and LfDFR2. Transient overexpression of LfMYB113 in Nicotiana benthamiana showed strong anthocyanin accumulation and pre-senescence; the latter was confirmed by up-regulation of senescence-associated genes. In addition, the activation of proLfSGR::YFP by LfMYB113 in transient experiments indicated that LfMYB113 may have a role in regulation of Chl degradation. To our knowledge, this is the first time a R2R3-MYB transcription factor has been functionally identified as one of the key regulators of autumn leaf coloration and autumn leaf senescence.

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.

Molecular evidence for natural intergeneric hybridization between Liquidambar and Altingia.

Since its establishment, a hybrid origin for Semiliquidambar has been proposed based on morphological intermediacy and sympatric distribution with Altingia and Liquidambar. This hypothesis, however, has lacked convincing molecular evidence. In this study, two nuclear genes, pin2 and cab4, and a chloroplast gene, matK, from Semiliquidambar cathayensis and its putative parental species Liquidambar and Altingia in Jianfengling, Hainan, and Heishiding and Nanling, Guangdong, China, were sequenced to test this hypothesis. Our results showed that L. formosana and L. acalycina were closely related and constituted an inseparable clade in the phylogenetic trees of both pin2 and cab4 genes. Phylogenetic analyses revealed two types of sequences for S. cathayensis, which were clustered with its putative parents, L. formosana-L. acalycina and A. obovata in Jianfengling, and with L. formosana-L. acalycina and A. chinensis in Heishiding and Nanling. The partial chloroplast matK gene sequences showed four nucleotide substitutions between L. formosana and A. obovata in Jianfengling; the sequences of the two individuals of S. cathayensis were identical with those of A. obovata. No diagnostic chloroplast markers including matK and three other chloroplast genes were found to distinguish L. formosana and A. chinensis in Heishiding and Nanling. Molecular data clearly demonstrated that S. cathayensis is of intergeneric hybrid origin between L. formosana-L. acalycina and A. obovata or A. chinensis and that A. obovata functions as the maternal parent in the hybridization event in Jianfengling, Hainan.

[Effects of phosphorus stress on the growth and nitrogen and phosphorus absorption of different Formosan sweet gum provenances].

Aiming at the ecological value of Formosan sweet gum (Liquidambar formosana) as a pioneer species and the status of red soil phosphorus (P) deficiency, a sand culture experiment of split design was conducted to study the responses of three-leaf stage seedlings of seven Formosan sweet gum provenances from Yixing of Jiangsu, Jingxian of Anhui, Yongkang of Zhejiang, Nanchang of Jiangxi, Shaowu of Fujian, Yanping of Fujian, and Nandan of Guangxi to four levels of P (P0, P1/2, P1, P2). With increasing P stress, the biomass and the N and P absorption of test provenances decreased, whereas the utilization efficiency increased. In higher P treatments, the provenances from Nanchang and Yixing had higher biomass and higher N and P absorption but lower utilization efficiency, while the provenance from Nandan had lower N and P absorption but higher utilization efficiency. In lower P treatments, the biomass and the P absorption and utilization efficiency of the provenances from Nanchang and Nandan were all higher. All the results illustrated that the provenances with high biomass had high P absorption at high P level, and had both high P absorption and high utilization efficiency at low P level. The provenance from Nanchang could be considered to be an excellent P stress-resistant provenance, followed by that from Nandan. Phosphorus was not a limiting nutritional factor of Formosan sweet gum, biomass, leaf delta (N/P) ratio and P efficiency could be used as the indicators of P stress-tolerance of Formosan sweet gum provenances.

Phylogeographical structure and temporal complexity in American sweetgum (Liquidambar styraciflua; Altingiaceae).

Eastern North American plant biogeography has traditionally focused on two primary issues: (i) the location of temperate Pleistocene refugia and their proximity to the southern margin of the ice sheet during the last glacial maximum, and (ii) the origin of the temperate element of northern Latin America. While numerous population genetic and phylogeographical studies have focused on the first issue, few (if any) have considered the second. We addressed these issues by surveying 117 individuals from 24 populations of Liquidambar styraciflua (American sweetgum; Altingiaceae) across the southeastern USA, eastern Mexico, and Guatemala, using more than 2200 bp of chloroplast DNA sequence data. To specifically address the issue of timing, we estimated intraspecific divergence times on the basis of multiple fossil-based calibration points, using taxa from Altingiaceae (Liquidambar and Altingia) and Hammamelidaceae (Hamamelis) as outgroups. More than half of the sampled localities exhibited multiple haplotypes. Remarkably, the greatest variation was observed within the USA, with Mexico and Guatemala sharing widespread haplotypes with Texas, Mississippi, Kentucky, Ohio, and northern Virginia. This lack of differentiation suggests shared ancestral polymorphisms, and that the genetic signal we observed is older than the disjunction itself. Our data provide support for previously proposed hypotheses of Pleistocene refugia in peninsular Florida and along the eastern Atlantic, but also for deeper divergences (approximately 8 million years ago) within the USA. These patterns reflect a dynamic biogeographical history for eastern North American trees, and emphasize the importance of the inclusion of a temporal component in any phylogeographical study.

Phylogeny and biogeography of Altingiaceae: evidence from combined analysis of five non-coding chloroplast regions.

The Altingiaceae consist of approximately 15 species that are disjunctly distributed in Asia and North America. The genus Liquidambar has been employed as a biogeographic model for studying the Northern Hemisphere intercontinental disjunctions. Parsimony and Bayesian analyses based on five non-coding chloroplast regions support that (1) Liquidambar is paraphyletic; (2) the temperate Liquidambar acalycina and Liquidambar formosana are nested within a large tropical to subtropical Asian clade; (3) Semiliquidambar is scattered in the eastern Asian clade and is of hybrid origin involving at least two maternal species: L. formosana and L. acalycina; and (4) the eastern North American Liquidambar styraciflua groups with the western Asian Liquidambar orientalis, but is highly distinct from other lineages. Biogeographically, our results demonstrate the complexity of biogeographic migrations throughout the history of Altingiaceae since the Cretaceous, with migration across both the Bering and the North Atlantic land bridges.

Genotypic effects of fertilization on seedling sweetgum biomass allocation, N uptake, and N use efficiency.

Screening and selecting tree genotypes that are responsive to N additions and that have high nutrient use efficiencies can provide better genetic material for short-rotation plantation establishment. A pot experiment was conducted to test the hypotheses that (1) sweetgum ( Liquidambar styraciflua L.) families have different patterns in biomass production and allocation, N uptake, and N use efficiency (NUE), because of their differences in growth strategies, and (2) sweetgum families that are more responsive to N additions will also have greater nutrient use efficiencies. Seedlings from two half-sib families (F10022 and F10023) that were known to have contrasting responses to fertility and other stress treatments were used for an experiment with two levels of N (0 vs. 100 kg N/ha equivalent) and two levels of P (0 vs. 50 kg P/ha equivalent) in a split-plot design. Sweetgum seedlings responded to N and P treatments rapidly, with increases in both size and biomass production, and those responses were greater with F10023 than with F10022. Growth response to N application was particularly strong. N and P application increased the proportional allocation of biomass to leaves. Under increased N supply, P application increased foliar N concentration and content, as well as total N uptake by the seedlings. However, NUE was decreased by N addition and was higher in F10023 than in F10022 when P was not limiting. A better understanding of genotype by fertility interactions is important in selecting genotypes for specific site conditions and for optimizing nutrient use in forestry production.