Identification and variation analysis of the composition and content of essential oil and fragrance compounds in Phoebe zhennan wood at different tree ages.

Wood essential oil and wood products with special fragrances are high value-added forest products. Despite the availability of essential oil and volatile organic compounds (VOCs) from Phoebe zhennan wood, their variation and dependence on tree age have not been examined. After essential oil extraction and wood processing, the yields and compositions of essential oils and VOCs in wood from P. zhennan trees of different ages (10a, 30a, and 80a) were determined. The yield of essential oil from 30a wood was significantly greater than that from 10a and 80a wood. Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) revealed 672 and 41 volatile compounds, respectively, in the essential oil and wood, the majority of which exhibited large fluctuations in relative content and composition depending on tree age. Sesquiterpenoids, fatty acids and conjugates may greatly contribute to the main components of essential oil from wood. Almost all major sesquiterpenoid compounds, such as caryophyllene α-oxide, eudesmo, and cubebene, were identified in the essential oils from the 30a and 80a wood, and their relative contents were much greater than those in the 10a wood. The main components of the wood fragrance were sesquiterpenoids. The types and relative contents of sesquiterpenoids from wood increased with tree age. These results suggest that choosing wood from trees of a suitable age will significantly improve the efficiency of wood utilization.

Optimization-based adaptive trajectory tracking controller design of self-balanced vehicle with asymptotic prescribed performance.

To handle with the nonlinear external disturbances and unmodeled dynamics of self-balanced vehicle (SBV), a novel adaptive trajectory tracking controller based on asymptotic prescribed performance is proposed. First, a velocity planner based on kinematic is constructed to control the velocity signal to improve the motion stability of SBV. Second, the prescribed performance function (PPF) is designed to prescribe transient-state and steady-state performances (TSP). Afterwards, an optimization-based predictive control (OPC) is proposed for accurate trajectory tracking of SBV. Furthermore, a modified radial basis function neural network (RBFNN) approximator is developed to compensate the unmodeled dynamics and the nonlinear external disturbances of the SBV. The overall system stability is proved with the help of Lyapunov theorem. Finally, the tracking performance and anti-interference robustness of the proposed control method are verified by comparative numerical simulations.

Nanoplastics increase the adverse impacts of lead on the growth, morphological structure and photosynthesis of marine microalga Platymonashelgolandica.

Nanoplastics and heavy metals are common pollutants in coastal environments with high concerns, but their joint ecological risk to marine primary productivity remains unclear. In this study, the effects of 7, 70, 700 µg/L lead (Pb) single exposure and in combination with 200 µg/L polystyrene nanoplastics (NPs, 70 nm) on marine microalga Platymonas helgolandica were investigated. Pb single exposure induced a dose-dependent inhibition on the growth of P. helgolandica, which was associated with the reduced photosynthetic efficiency and nutrient accumulation. Compared to Pb single exposure, the addition of NPs significantly reduced the photosynthetic efficiency and aggravated the damage to cell structure. Reduced esterase activity and increased membrane permeability also indicated that NPs exacerbated the adverse effects of Pb on P. helgolandica. Thus, co-exposure to NPs and Pb induced more severe impacts on marine microalgae, suggesting that the joint ecological risk of NPs and heavy metals to marine primary productivity merits more attention.

Comparative Metabolomics and Transcriptome Analysis Reveal the Fragrance-Related Metabolite Formation in Phoebe zhennan Wood.

Nanmu (Phoebe zhennan) has a unique fragrance and is a high-quality tree species for forest conservation. The types and contents of volatile compounds in different tissues of nanmu wood are different, and the study of its volatile metabolites can help us to understand the source of its fragrance and functions. In order to explore the metabolites related to the wood fragrance of nanmu and to find out the unique volatile substances in the heartwood, gas chromatography-mass spectrometry (GC-MS) was performed to analyze the non-targeted metabolomics in five radial tissues from the sapwood to the heartwood of nanmu. A total of 53 volatile metabolites belonging to 11 classes were detected in all tissues, including terpenes, aromatic hydrocarbons, organoheterocyclics, phenols, esters, organic acids, alcohols, alkaloids, alkane, indoles derivatives, and others. And most of the volatile metabolites were identified for the first time in nanmu wood. Among them, terpenes and aromatic hydrocarbons were the main volatile components. In addition, 22 differential metabolites were screened from HW and SW, HW, and TZ via metabolomic analysis. Among these DAMs, three volatile metabolites (cadinene, a sesquiterpenoid; p-cymene, a monoterpenoid; 1,3,5-triisopropylbenzene, an aromatic hydrocarbon) contributed heavily to the characteristic fragrance of the heartwood. Additionally, the expression of transcripts showed that the unigenes in the terpenoid biosynthesis pathway were especially up-regulated in the SW. Therefore, we speculated that fragrance-related metabolites were synthesized in SW and then deposited in heartwood during sapwood transformed to heartwood. The expression levels of transcription factors (e.g., WRKY, C2H2, NAC) acted as the major regulatory factors in the synthesis of terpenoid. The results lay the foundations for further studies on the formation mechanism of fragrance components in nanmu wood and also provide a reference for the further development and utilization of nanmu wood.

Highly sensitive lateral flow immunoassays based on Ag@Au nanoflowers with marine medaka (Oryzias melastigm) vitellogenin as a target analyte.

In order to improve the sensitivity of lateral flow immunoassays (LFIAs) for the detection of piscine vitellogenin (Vtg), a well-established biomarker for environmental estrogens, Au coated Ag nanoflowers (Ag@Au NFs) were used as labeling probes to develop a LFIA for marine medaka Vtg. The synthesized Ag@Au NFs with good monodispersity had an average diameter of 44.1 nm and absorbance peak of 524 nm. When the concentration of goat anti-mouse IgG and anti-Vtg polyclonal antibody (anti-Vtg PAbs) were 1.3 and 0.4 mg/mL, respectively, the detection range of the LFIA was 0.19-25 ng/mL, and the visual detection limit was 0.1 ng/mL, which was approximately 80 times lower than that of LFIAs based on other nanoparticles (Au NPs, Ag NPs, Au NFs, and FM). After evaluation of its specificity and robustness, the usefulness of Ag@Au NFs labeled LFIA was validated by measuring Vtg induction in the plasma of marine medaka exposed to bisphenol A, a weak estrogenic chemical. This highly sensitive lateral flow immunoassay could detect Vtg biomarker within 15 min without the need of expensive and complicated instruments, and thus offered an ultrasensitive and robust on-site detection method for estrogenic activity in field environment.

A novel electrochemical aptasensor for ultrasensitive detection of herbicide prometryn based on its highly specific aptamer and Ag@Au nanoflowers.

Herbicide prometryn has become a common pollutant in aquatic environments and caused adverse impacts on ecosystems. This study developed an ultrasensitive electrochemical aptasensor for prometryn based on its highly affinitive and specific aptamer and Ag@Au nanoflowers (Ag@AuNFs) for signal amplification. Firstly, this study improved the Capture-SELEX strategy to screen aptamers and obtained aptamer P60-1, which had a high affinity (Kd: 23 nM) and could distinguish prometryn from its structural analogues. Moreover, the typical stem-loop structure in aptamer P60-1 was found to be the binding pocket for prometryn. Subsequently, an electrochemical aptasensor for prometryn was established using multiwalled carbon nanotubes and reduced graphene oxide as electrode substrate, Ag@Au NFs as signal amplification element, and aptamer P60-1 as recognition element. The aptasensor had a detection range of 0.16-500 ng/mL and a detection limit of 60 pg/mL, which was much lower than those of existing detection methods. The aptasensor had high stability and good repeatability, and could specifically detecting prometryn. Furthermore, the utility of the aptasensor was validated by measuring prometryn in environmental and biological components. Therefore, this study provides a robust and ultrasensitive aptasensor for accurate detection for prometryn pollution.

Integrated Transcriptomic, Metabolomic, and Physiological Analyses Reveal New Insights into Fragrance Formation in the Heartwood of Phoebe hui.

Phoebe hui is an extremely valuable tree that is the main source of the fragrant golden-thread nanmu wood. Although the fragrance of wood has been investigated in several trees, the potential substances and gene regulation mechanisms that are involved in fragrance formation are poorly understood. Here, three radial tissues, sapwood (SW), heartwood (HW), and the transition zone (TZ) in between them, were compared via integrative physiological, volatile-metabolomic, and transcriptomic analyses to identify the key metabolites and regulatory mechanisms involved in fragrance formation. During heartwood formation, gradual starch grain loss was accompanied by the deposition of lipids and extractives in the cell lumen. Extracts of terpenoids were synthesized and accumulated in the heartwood, including monoterpenoids (limonene and p-cymene) and sesquiterpenes (cubebene and guaiadiene); these were identified as being closely related to the special fragrance of the wood. Additionally, the expression of transcripts showed that the genes related to primary metabolism were specifically upregulated in the SW, whereas genes annotated in terpenoid biosynthesis were specifically upregulated in the HW. Therefore, we speculated that terpenoid biosynthesis occurs in situ in the HW via the HW formation model of Type-III (Santalum) using the precursors that were produced by primary metabolism in the SW. The expression levels of transcription factors (e.g., MYB, WRKY, and C2H2) acted as the major regulatory factors in the synthesis of terpenoids. Our results explain the special fragrance in P. hui and broaden the current knowledge of the regulatory mechanisms of fragrance formation. This work provides a framework for future research that is focused on improving wood quality and value.

Integrative Metabolomic and Transcriptomic Analysis Reveals the Mechanism of Specific Color Formation in Phoebe zhennan Heartwood.

Nanmu (Phoebe zhennan) is an extremely valuable tree plant that is the main source of famous "golden-thread nanmu" wood. The potential metabolites and gene regulation mechanisms involved in golden thread formation are poorly understood, even though the color change from sapwood to heartwood has been investigated in several tree plants. Here, five radial tissues from sapwood to heartwood were compared via integrative metabolomic and transcriptomic analysis to reveal the secondary metabolites and molecular mechanisms involved in golden thread formation. During heartwood formation, gradual starch grain loss is accompanied by the cell lumen deposition of lipids and color-related extractives. Extractives of 20 phenylpropanoids accumulated in heartwood, including cinnamic acids and derivatives, coumarin acid derivatives, and flavonoids, which were identified as being closely related to the golden thread. Phenylpropanoids co-occurring with abundant accumulated metabolites of prenol lipids, fatty acyls, steroids, and steroid derivatives may greatly contribute to the characteristics of golden thread formation. Additionally, the expression of nine genes whose products catalyze phenylpropanoid and flavonoids biosynthesis was upregulated in the transition zone, then accumulated and used to color the heartwood. The expression levels of transcription factors (e.g., MYB, bHLH, and WRKY) that act as the major regulatory factors in the synthesis and deposition of phenylpropanoid and flavonoids responsible for golden thread formation were also higher than in sapwood. Our results not only explain golden thread formation in nanmu, but also broaden current knowledge of special wood color formation mechanisms. This work provides a framework for future research focused on improving wood color.

Structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex.

INTRODUCTION The nuclear pore complex (NPC) resides on the nuclear envelope (NE) and mediates nucleocytoplasmic cargo transport. As one of the largest cellular machineries, a vertebrate NPC consists of cytoplasmic filaments, a cytoplasmic ring (CR), an inner ring, a nuclear ring, a nuclear basket, and a luminal ring. Each NPC has eight repeating subunits. Structure determination of NPC is a prerequisite for understanding its functional mechanism. In the past two decades, integrative modeling, which combines x-ray structures of individual nucleoporins and subcomplexes with cryo-electron tomography reconstructions, has played a crucial role in advancing our knowledge about the NPC. The CR has been a major focus of structural investigation. The CR subunit of human NPC was reconstructed by cryo-electron tomography through subtomogram averaging to an overall resolution of ~20 Å, with local resolution up to ~15 Å. Each CR subunit comprises two Y-shaped multicomponent complexes known as the inner and outer Y complexes. Eight inner and eight outer Y complexes assemble in a head-to-tail fashion to form the proximal and distal rings, respectively, constituting the CR scaffold. To achieve higher resolution of the CR, we used single-particle cryo-electron microscopy (cryo-EM) to image the intact NPC from the NE of Xenopus laevis oocytes. Reconstructions of the core region and the Nup358 region of the X. laevis CR subunit had been achieved at average resolutions of 5 to 8 Å, allowing identification of secondary structural elements. RATIONALE Packing interactions among the components of the CR subunit were poorly defined by all previous EM maps. Additional components of the CR subunit are strongly suggested by the EM maps of 5- to 8-Å resolution but remain to be identified. Addressing these issues requires improved resolution of the cryo-EM reconstruction. Therefore, we may need to enhance sample preparation, optimize image acquisition, and develop an effective data-processing strategy. RESULTS To reduce conformational heterogeneity of the sample, we spread the opened NE onto the grids with minimal force and used the chemical cross-linker glutaraldehyde to stabilize the NPC. To alleviate orientation bias of the NPC, we tilted sample grids and imaged the sample with higher electron dose at higher angles. We improved the image-processing protocol. With these efforts, the average resolutions for the core and the Nup358 regions have been improved to 3.7 and 4.7 Å, respectively. The highest local resolution of the core region reaches 3.3 Å. In addition, a cryo-EM structure of the N-terminal α-helical domain of Nup358 has been resolved at 3.0-Å resolution. These EM maps allow the identification of five copies of Nup358, two copies of Nup93, two copies of Nup205, and two copies of Y complexes in each CR subunit. Relying on the EM maps and facilitated by AlphaFold prediction, we have generated a final model for the CR of the X. laevis NPC. Our model of the CR subunit includes 19,037 amino acids in 30 nucleoporins. A previously unknown C-terminal fragment of Nup160 was found to constitute a key part of the vertex, in which the short arm, long arm, and stem of the Y complex meet. The Nup160 C-terminal fragment directly binds the ß-propeller proteins Seh1 and Sec13. Two Nup205 molecules, which do not contact each other, bind the inner and outer Y complexes through distinct interfaces. Conformational elasticity of the two Nup205 molecules may underlie their versatility in binding to different nucleoporins in the proximal and distal CR rings. Two Nup93 molecules, each comprising an N-terminal extended helix and an ACE1 domain, bridge the Y complexes and Nup205. Nup93 and Nup205 together play a critical role in mediating the contacts between neighboring CR subunits. Five Nup358 molecules, each in the shape of a shrimp tail and named "the clamp," hold the stems of both Y complexes. The innate conformational elasticity allows each Nup358 clamp to adapt to a distinct local environment for optimal interactions with neighboring nucleoporins. In each CR subunit, the α-helical nucleoporins appear to provide the conformational elasticity; the 12 ß-propellers may strengthen the scaffold. CONCLUSION Our EM map-based model of the X. laevis CR subunit substantially expands the molecular mass over the reported composite models of vertebrate CR subunit. In addition to the Y complexes, five Nup358, two Nup205, and two Nup93 molecules constitute the key components of the CR. The improved EM maps reveal insights into the interfaces among the nucleoporins of the CR. [Figure: see text].

Cryo-EM structure of the inner ring from the Xenopus laevis nuclear pore complex.

Nuclear pore complex (NPC) mediates nucleocytoplasmic shuttling. Here we present single-particle cryo-electron microscopy structure of the inner ring (IR) subunit from the Xenopus laevis NPC at an average resolution of 4.2 Å. A homo-dimer of Nup205 resides at the center of the IR subunit, flanked by two molecules of Nup188. Four molecules of Nup93 each places an extended helix into the axial groove of Nup205 or Nup188, together constituting the central scaffold. The channel nucleoporin hetero-trimer of Nup62/58/54 is anchored on the central scaffold. Six Nup155 molecules interact with the central scaffold and together with the NDC1-ALADIN hetero-dimers anchor the IR subunit to the nuclear envelope and to outer rings. The scarce inter-subunit contacts may allow sufficient latitude in conformation and diameter of the IR. Our structure reveals the molecular basis for the IR subunit assembly of a vertebrate NPC.

Cryo-EM structure of the nuclear ring from Xenopus laevis nuclear pore complex.

Nuclear pore complex (NPC) shuttles cargo across the nuclear envelope. Here we present single-particle cryo-EM structure of the nuclear ring (NR) subunit from Xenopus laevis NPC at an average resolution of 5.6 Å. The NR subunit comprises two 10-membered Y complexes, each with the nucleoporin ELYS closely associating with Nup160 and Nup37 of the long arm. Unlike the cytoplasmic ring (CR) or inner ring (IR), the NR subunit contains only one molecule each of Nup205 and Nup93. Nup205 binds both arms of the Y complexes and interacts with the stem of inner Y complex from the neighboring subunit. Nup93 connects the stems of inner and outer Y complexes within the same NR subunit, and places its N-terminal extended helix into the axial groove of Nup205 from the neighboring subunit. Together with other structural information, we have generated a composite atomic model of the central ring scaffold that includes the NR, IR, and CR. The IR is connected to the two outer rings mainly through Nup155. This model facilitates functional understanding of vertebrate NPC.

Polystyrene nanoplastics cause growth inhibition, morphological damage and physiological disturbance in the marine microalga Platymonas helgolandica.

Effects of nanoplastics at low level on the marine primary producer are largely unclear. To assess the potential risk of nanoplastic pollution, this study exposed marine green microalgae Platymonas helgolandica to 20, 200, and 2000 µg/L 70-nm polystyrene nanoplastics for 6 days. Nanoplastics significantly inhibited the growth of P. helgolandica during the first 4 days of exposure, and elevated heterocyst frequency was observed in 200 and 2000 µg/L exposure groups in the early exposure stage. Exposure to 200 and 2000 µg/L nanoplastics for 4 days increased the membrane permeability and mitochondrial membrane potential, and decreased light energy used in photochemical processes of microalgae. Moreover, clear morphological changes, including surface folds, fragmentation, aggregation cluster, and rupture, in the microalgae exposed to nanoplastics were observed under scanning electron microscope and transmission electron microscope. These results demonstrate that nanoplastics could reduce the microalgal vitality by the damage on cell morphology and organelle function.

An HMGA2-p62-ERα axis regulates uterine leiomyomas proliferation.

Uterine leiomyomas (ULM) are a major public health issue contributing to high morbidity and poor pregnancy outcomes. However, its molecular pathogenesis is poorly understood. HMGA2-ULM is the second major subtype of human ULM and associates with large sizes, fast-growth, and high percentages of estrogen receptor α (ERα). As altered ERα expression plays a distinct role in ULM growth, here, we investigate a regulatory mechanism driving ULM growth via HMGA2 and ERα. We reveal a positive correlation of HMGA2 with ERα protein and demonstrate that HMGA2 promotes ULM cells proliferation via ERα. In addition, autophagy pathway and p62/SQSTM1 (a selective autophagy receptor) are found to participate in the regulation of HMGA2 and ERα. Moreover, HMGA2 suppresses the transcription of p62 by binding to its promoter, meanwhile, p62 interacts with ERα, and inhibition of p62 increases ERα expression and enhances cell viability in ULM, suggesting a novel mechanism of the HMGA2-p62-ERα axis in ULM proliferation. Notably, rapamycin, a familiar autophagy agonist, reduces ERα levels and the proliferation ability of ULM cells. This study demonstrates a causal role of the HMGA2-p62-ERα axis in preventing autophagy and increasing ERα expression in HMGA2-ULM. Therefore, blocking HMGA2-p62-ERα axis and targeting autophagy pathway establish a roadmap toward HMGA2-ULM medical treatment.

Evidence for an edge supercurrent in the Weyl superconductor MoTe2.

Edge supercurrents in superconductors have long been an elusive target. Interest in them has reappeared in the context of topological superconductivity. We report evidence for the existence of a robust edge supercurrent in the Weyl superconductor molybdenum ditelluride (MoTe2). In a magnetic field B, fluxoid quantization generates a periodic modulation of the edge condensate observable as a "fast-mode" oscillation of the critical current I c versus B The fast-mode frequency is distinct from the conventional Fraunhofer oscillation displayed by the bulk supercurrent. We confirm that the fast-mode frequency increases with crystal area as expected for an edge supercurrent. In addition, weak excitation branches are resolved that display an unusual broken symmetry.

Structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex by cryo-electron microscopy single particle analysis.

The nuclear pore complex (NPC) exhibits structural plasticity and has only been characterized at local resolutions of up to 15 Å for the cytoplasmic ring (CR). Here we present a single-particle cryo-electron microscopy (cryo-EM) structure of the CR from Xenopus laevis NPC at average resolutions of 5.5-7.9 Å, with local resolutions reaching 4.5 Å. Improved resolutions allow identification and placement of secondary structural elements in the majority of the CR components. The two Y complexes in each CR subunit interact with each other and associate with those from flanking subunits, forming a circular scaffold. Within each CR subunit, the Nup358-containing region wraps around the stems of both Y complexes, likely stabilizing the scaffold. Nup205 connects the short arms of the two Y complexes and associates with the stem of a neighboring Y complex. The Nup214-containing region uses an extended coiled-coil to link Nup85 of the two Y complexes and protrudes into the axial pore of the NPC. These previously uncharacterized structural features reveal insights into NPC assembly.

Molecular architecture of the luminal ring of the Xenopus laevis nuclear pore complex.

The nuclear pore complex (NPC) mediates the flow of substances between the nucleus and cytoplasm in eukaryotic cells. Here we report the cryo-electron tomography (cryo-ET) structure of the luminal ring (LR) of the NPC from Xenopus laevis oocyte. The observed key structural features of the LR are independently confirmed by single-particle cryo-electron microscopy (cryo-EM) analysis. The LR comprises eight butterfly-shaped subunits, each containing two symmetric wings. Each wing consists of four elongated, tubular protomers. Within the LR subunit, the eight protomers form a Finger domain, which directly contacts the fusion between the inner and outer nuclear membranes and a Grid domain, which serves as a rigid base for the Finger domain. Two neighboring LR subunits interact with each other through the lateral edges of their wings to constitute a Bumper domain, which displays two major conformations and appears to cushion neighboring NPCs. Our study reveals previously unknown features of the LR and potentially explains the elastic property of the NPC.

Transcriptome analysis of terpenoid biosynthetic genes and simple sequence repeat marker screening in Eucommia ulmoides.

Trans-polyisoprene rubber is produced in the tissues of leaves, bark, and fruit of Eucommia ulmoides and is considered an important energy source. Transcript profiles of two tissues from E. ulmoides cv. Qinzhong No. 3, leaf and fruit, were analysed using the Illumina HiSeq 2000 system. In total, 104 million clean reads were obtained and assembled into 58,863 unigenes. Through gene functional classification, 28,091 unigenes (47.72%) were annotated and 65 unigenes have been hypothesized to encode proteins involved in terpenoid biosynthesis. In addition, 10,041 unigenes were detected as differentially expressed unigenes, and 29 of them were putatively related to terpenoid biosynthesis. The synthesis of trans-polyisoprene rubbers in E. ulmoides was hypothesised to be dominated by the mevalonate pathway. Farnesyl diphosphate synthase 2 (FPPS2) was considered a key component in the biosynthesis of trans-polyprenyl diphosphate. Rubber elongation factor 3 (REF3) might be involved in stabilising the membrane of rubber particles in E. ulmoides. To date, 351 simple sequence repeats (SSRs) were validated as polymorphisms from eight E. ulmoides plants (two parent plants and six F1 individuals), and these could act as molecular markers for genetic map density increase and breeding improvement of E. ulmoides.

Molecular cloning and function analysis of FAD2 gene in Idesia polycarpa.

Idesia polycarpa is a valuable oil-producing tree and can potentially be used for edible oil and biofuel production. The fruits of I. polycarpa are unique in that they contain both saturated and unsaturated lipids. Fatty acid desaturase 2 (FAD2), also as known as omega-6 fatty acid desaturase in endoplasmic, is a key enzyme for linoleic acid and α-linolenic acid biosynthesis. However, bioinformatics and expression of FAD2 in I. polycarpa are still absent. Here, to gain insight into the lipid and linoleic synthesis of I. polycarpa, we compared the fruits from different growth stages. Lipid accumulation rates, final lipid content, linoleic accumulation rates and final linoleic content were significantly different among the different stages. In a further step, the FAD2 gene from fruits of I. polycarpa, named IpFAD2, was cloned and characterized. A partial fragment of 169 bp of IpFAD2 was amplified by degenerate PCR. Full cDNA of IpFAD2 was obtained by the RACE technique. The open-reading frame of IpFAD2 was 1149 bp in length, encoding 382 amino acids. A comparison of the deduced amino acids sequence of IpFAD2 with FAD2 from other species showed high similarities, ranging from 78.8 to 92.6%. The IpFAD2-predicted protein has a theoretical molecular mass of 44.03 kDa and an isoelectric point (pI) of 8.04. It has five transmembrane helices located on the endoplasmic reticulum. The IpFAD2-predicted protein was classified as belonging to the Membrane-FADS-like superfamily based on its conserved domain analysis. Expression analysis based on qRT-PCR indicated that IpFAD2 was expressed in different fruit growth stages, with the highest expression level at 80 DAP and the lowest at 130 DAP. The expression of IpFAD2 was positively correlated with the linoleic accumulation rates in I. polycarpa fruits. Prokaryotic expression in Escherichia. Coli BL21(DE3) indicated that IpFAD2 gene could encode a bio-functional omega-6 fatty acid desaturase. Heterologous expression in Arabidopsis thaliana confirmed that the isolated IpFAD2 proteins could catalyse linoleic synthesis. This is the first cloning and expression analysis of FAD2 from I. polycarpa, significantly contributing to our understanding of the role of IpFAD2 in linoleic synthesis, esp. in terms of genetic engineering breeding for linoleic production.

Integration of Transcriptomes, Small RNAs, and Degradome Sequencing to Identify Putative miRNAs and Their Targets Related to Eu-Rubber Biosynthesis in Eucommia ulmoides.

Eucommia ulmoides has attracted much attention as a valuable natural rubber (Eu-rubber) production tree. As a strategic material, Eu-rubber plays a vital role in general and defence industries. However, the study of Eu-rubber biosynthesis at a molecular level is scarce, and the regulatory network between microRNAs (miRNAs) and messenger RNAs (mRNAs) in Eu-rubber biosynthesis has not been assessed. In this study, we comprehensively analyzed the transcriptomes, small RNAs (sRNAs) and degradome to reveal the regulatory network of Eu-rubber biosynthesis in E. ulmoides. A total of 82,065 unigenes and 221 miRNAs were identified using high-throughput sequencing; 20,815 targets were predicted using psRNATarget software. Of these targets, 779 miRNA-target pairs were identified via degradome sequencing. Thirty-one miRNAs were differentially expressed; 22 targets of 34 miRNAs were annotated in the terpenoid backbone biosynthesis pathway (ko00900) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG). These miRNAs were putatively related to Eu-rubber biosynthesis. A regulatory network was constructed according to the expression profiles of miRNAs and their targets. These results provide a comprehensive analysis of transcriptomics, sRNAs and degradome to reveal the Eu-rubber accumulation, and provide new insights into genetic engineering techniques which may improve the content of Eu-rubber in E. ulmoides.

Dynamic Changes in Metabolite Accumulation and the Transcriptome during Leaf Growth and Development in Eucommia ulmoides.

Eucommia ulmoides Oliver is widely distributed in China. This species has been used mainly in medicine due to the high concentration of chlorogenic acid (CGA), flavonoids, lignans, and other compounds in the leaves and barks. However, the categories of metabolites, dynamic changes in metabolite accumulation and overall molecular mechanisms involved in metabolite biosynthesis during E. ulmoides leaf growth and development remain unknown. Here, a total of 515 analytes, including 127 flavonoids, 46 organic acids, 44 amino acid derivatives, 9 phenolamides, and 16 vitamins, were identified from four E. ulmoides samples using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) (for widely targeted metabolites). The accumulation of most flavonoids peaked in growing leaves, followed by old leaves. UPLC-MS analysis indicated that CGA accumulation increased steadily to a high concentration during leaf growth and development, and rutin showed a high accumulation level in leaf buds and growing leaves. Based on single-molecule long-read sequencing technology, 69,020 transcripts and 2880 novel loci were identified in E. ulmoides. Expression analysis indicated that isoforms in the flavonoid biosynthetic pathway and flavonoid metabolic pathway were highly expressed in growing leaves and old leaves. Co-expression network analysis suggested a potential direct link between the flavonoid and phenylpropanoid biosynthetic pathways via the regulation of transcription factors, including MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic/helix-loop-helix). Our study predicts dynamic metabolic models during leaf growth and development and will support further molecular biological studies of metabolite biosynthesis in E. ulmoides. In addition, our results significantly improve the annotation of the E. ulmoides genome.