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Background Lindera glauca fruit with high quality and quantity of oil has emerged as a novel potential source of biodiesel in China, but the molecular regulatory mechanism of carbon flux and energy source for oil biosynthesis in developing fruits is still unknown. To better develop fruit oils of L. glauca as woody biodiesel, a combination of two different sequencing platforms (454 and Illumina) and qRT-PCR analysis was used to define a minimal reference transcriptome of developing L. glauca fruits, and to construct carbon and energy metabolic model for regulation of carbon partitioning and energy supply for FA biosynthesis and oil accumulation. Results We first analyzed the dynamic patterns of growth tendency, oil content, FA compositions, biodiesel properties, and the contents of ATP and pyridine nucleotide of L. glauca fruits from seven different developing stages. Comprehensive characterization of transcriptome of the developing L. glauca fruit was performed using a combination of two different next-generation sequencing platforms, of which three representative fruit samples (50, 125, and 150 DAF) and one mixed sample from seven developing stages were selected for Illumina and 454 sequencing, respectively. The unigenes separately obtained from long and short reads (201, and 259, respectively, in total) were reconciled using TGICL software, resulting in a total of 60,031 unigenes (mean length = 1061.95 bp) to describe a transcriptome for developing L. glauca fruits. Notably, 198 genes were annotated for photosynthesis, sucrose cleavage, carbon allocation, metabolite transport, acetyl-CoA formation, oil synthesis, and energy metabolism, among which some specific transporters, transcription factors, and enzymes were identified to be implicated in carbon partitioning and energy source for oil synthesis by an integrated analysis of transcriptomic sequencing and qRT-PCR. Importantly, the carbon and energy metabolic model was well established for oil biosynthesis of developing L. glauca fruits, which could help to reveal the molecular regulatory mechanism of the increased oil production in developing fruits. Conclusions This study presents for the first time the application of an integrated two different sequencing analyses (Illumina and 454) and qRT-PCR detection to define a minimal reference transcriptome for developing L. glauca fruits, and to elucidate the molecular regulatory mechanism of carbon flux control and energy provision for oil synthesis. Our results will provide a valuable resource for future fundamental and applied research on the woody biodiesel plants.

Background Lindera glauca with rich resource and fruit oil has emerged as novel source of biodiesel in China, but different germplasms show a variation for fruit oil content and FA profile. To develop L. glauca fruit oils as biodiesel, a concurrent exploration of oil content, FA composition, biodiesel yield, fuel property and prediction model construction was conducted on the fruits from 8 plus germplasms to select superior genotype for ideal biodiesel production. Another vital focus was to highlight mechanism that govern the differences in oil content and FA profile of different germplasms. The cross-accessions comparisons associated with oil-synthesized gene transcriptional level and oil accumulative amount led to the identification of potential determinants (enzymes, transporters or transcription factors) and regulatory mechanisms responsible for high-quality oil accumulation. Results To select superior germplasm and unravel regulatory mechanism of high oil production for developing L. glauca fruit oils as biodiesel, 8 plus trees (accession LG01/02/03/04/05/06/07/08) with high-yield fruits were selected to evaluate the differences in oil content, FA profile, biodiesel yield and fuel property, and to construct fuel property prediction model, revealing a variation in the levels of fruit oil (45.12–60.95%), monounsaturated FA (52.43–78.46%) and polyunsaturated FA (17.69–38.73%), and biodiesel yield (80.12–98.71%) across different accessions. Of note, LG06 had a maximum yield of oil (60.95%) and biodiesel (98.71%), and ideal proportions of C18:1 (77.89%), C18:2 (14.16%) and C18:3 (1.55%), indicating that fruit oils from accession LG06 was the most suitable for high-quality biodiesel production. To highlight molecular mechanism that govern such differences in oil content and FA composition of different accessions, the quantitative relationship between oil-synthesized gene transcription and oil accumulative amount were conducted on different accessions to identify some vital determinants (enzymes, transporters or transcription factors) with a model of carbon metabolic regulatory for high-quality oil accumulation by an integrated analysis of our recent transcriptome data and qRT-PCR detection. Our findings may present strategies for developing L. glauca fruit oils as biodiesel feedstock and engineering its oil accumulation. Conclusions This is the first report on the cross-accessions evaluations of L. glauca fruit oils to determine ideal accession for producing ideal biodiesel, and the associations of oil accumulative amount with oil-synthesized gene transcription was performed to identify some crucial determinants (enzymes, transporters or transcription factors) with metabolic regulation model established for governing high oil production. Our finding may provide molecular basis for new strategies of developing biodiesel resource and engineering oil accumulation.

Research Highlights: genetic diversity in populations were compared among related shrub species with different reproductive systems. Background and Objectives: Lindera species are dioecious trees or shrubs that produce seeds by mating of males and females. To evaluate the importance of genetic diversity for the persistence of natural populations, we compared genetic information among four Lindera species in Japan. Three are dioecious shrubs (Lindera praecox, Lindera umbellata, and Lindera obtusiloba) that produce seeds by sexual reproduction. The remaining species, Lindera glauca, reproduces by apomixis; only female plants are found in Japan. Materials and Methods: all four species were sampled across a wide geographic area, from Tohoku to Kyushu, Japan. Single nucleotide polymorphisms (SNPs) were detected by multiplexed ISSR genotyping by sequencing (MIG-seq) and the resulting genetic diversity parameters were compared among populations. Results: in all sexually reproducing species, the values of observed heterozygosity were close to the expected ones and the inbreeding coefficients were nearly 0. These results were supposed to be caused by their obligate outcrossing. The genetic difference increased, in ascending order, between a mother plant and its seeds, within populations, and across geographic space. We observed a substantial geographic component in the genetic structure of these species. For L. glauca, the genetic difference between a mother and its seeds, within populations, and across space were not significantly different from what would be expected from PCR errors. Genetic diversity within and among populations of L. glauca was extremely low. Conclusions: apomixis has the advantage of being able to found populations from a single individual, without mating, which may outweigh the disadvantages associated with the extremely low genetic diversity of L. glauca. This may explain why this species is so widely distributed in Japan. Provided that the current genotypes remain suited to environmental conditions, L. glauca may not be constrained by its limited genetic diversity.

Diacylglycerol acyltransferase (DGAT) is a vital and sole rate-limiting enzyme involved in triacylglycerol synthesis. Identifying DGAT genes in Lindera glauca is essential for studying lipid metabolism pathways and developing novel oil crops with enhanced value. In the study reported in this paper, 15 LgDGAT family genes were first obtained from the L. glauca genome via bioinformatics analysis. We comprehensively analyzed their chromosome distribution, gene structure, subcellular localization, promoter prediction, phylogenetic relationships, tissue-specific expression, and expression patterns during different stages of fruit development. Our findings revealed that LgDGATs can be classified into DGAT1, DGAT2, DGAT3, and WSD (wax ester synthase/acyl-CoA: diacylglycerol acyltransferase) subfamilies distributed across chromosome 3, 5, 6, 8 and 11. LgDGATs’ promoter region showed abundant elements linked to the light response and plant hormone response. Forms of LgDGAT1, LgDGAT2, and LgDGAT3 were primarily expressed in the early and late phases of fruit development, indicating their potential function in the growth and development of L. glauca, particularly in oil accumulation. Conversely, LgWSDs exhibited predominant expression in stems and leaves. This paper elucidates the gene structure and expression patterns of LgDGATs, providing a theoretical foundation for understanding the functionality of DGAT genes in Lindera species.

Lindera glauca is a shrub or small tree mostly distributed in China, Japan and Korea. However, reports on the biological activities of Lindera glauca fruit essential oil (LGFEO) are limited. The study on its chemical composition, and antioxidant and cholinesterase inhibitory activities were performed, along with molecular docking of six selected compounds. The LGFEO was extracted by hydro distillation and analyzed by GC-MS and GC-FID. Antioxidant activities of LGFEO were evaluated by three methods with different mechanisms. Acetylcholinesterase and butyrylcholinesterase inhibitory activities of LGFEO were tested. A total of 48 components were identified representing 95.74% of the total composition of LGFEO in which the major compounds were (E)-β-ocimene (41.53%), α-copaene (13.17%), δ-cadinene (6.20%), 3-carene (5.89%) and eucalyptol (3.57%). Weak antioxidant activities of LGFEO in three assays (9.52, 11.36 and 38.98 μmol TE/g, respectively) were observed. LGFEO showed obvious cholinesterase inhibitory activities at the final concentrations of 50 and 20 μg/mL. IC50 values for acetylcholinesterase and butyrylcholinesterase were 46.48 and 34.85 μg/mL, respectively. Molecular docking revealed that geranyl acetate, β-caryophyllene and limonene had lower binding affinities in the range of −7.1 to −6.1 kcal/mol through hydrophobic interactions and hydrogen bond. Six compounds including 3-carene, limonene, eucalyptol, (E)-β-ocimene, geranyl acetate and β-caryophyllene could contribute together to cholinesterase inhibitory activities of LGFEO. This essential oil indicated low potential as natural antioxidant, but it could be potentially used as cholinesterase inhibitor with possible application in food, aromatherapy and pharmaceutical industries.

Lindera glauca (Lauraceae) is a tree of economic and ecological significance that reproduces sexually and asexually via apomictic seeds. It is widely distributed in the low-altitude montane forests of East Asia. Despite the potential implications of a mixed reproductive system in terms of genetic diversity, few studies have focused on this aspect. In this study, the genetic structure of wild populations of L. glauca was investigated via genetic analyses. Overall, 13 nuclear microsatellites (nSSRs) and five chloroplast microsatellites (cpSSRs) were used to genotype 300 individual plants, taken from 20 wild populations (a small sample size in some wild populations is due to the limitation of its specific reproduction, leading to certain limitations in the results of this study) and two cultivated populations ranging across nearly the entire natural distribution of mainland China. The populations exhibited low levels of genetic diversity (nSSR: A R = 1.75, Ho = 0.32, He = 0.36; cpSSR: Nb = 2.01, Hrs = 0.40), and no significant effect of isolation by distance between populations existed, regardless of marker type (nSSR: R 2 = 0.0401, P = 0.068; cpSSR: R 2 = 0.033, P = 0.091). Haplotype networks showed complex relationships among populations, and the H12 haplotype was predominant in most populations. Analyses of molecular variance obtained with nuclear markers ( F sc = 0.293, F ST = 0.362) and chloroplast markers ( F sc = 0.299, F ST = 0.312) were similar. The migration ratio of pollen flow versus seed flow in this study was negative ( r = −1.149). Results suggest that weak barriers of dispersal between populations and/or the similarity of founders shared between neighbors and distant populations are indicative of the gene flow between populations more likely involving seeds. Wild L. glauca in mainland China was inferred to have highly skewed sex ratios with predominant females. In addition, some populations experienced a recent bottleneck effect, especially in Gujianshan, Chongqing, and southwest China (population GJS). It is suggested that few wild male individuals should be conserved in order to maintain overall genetic diversity in the wild populations of this species. These findings provide important information for the sustainable utilization and preservation of the overall genetic diversity of L. glauca .

Microplastics enter forest ecosystems in a variety of ways, including through atmospheric deposition, anthropogenic waste, and leaching. There is growing evidence of the ecotoxicity of microplastics to soil decomposers. Soil animals and microorganisms are the main decomposers of plant litter, and their interactions play important roles in determining the terrestrial biochemical cycle. However, how emerging microplastics in forests affect the influence of soil animals on the fungal community in decomposed litter is still unclear. Here, by constructing a rigorous mesocosm experiment, we investigated soil enzyme activities and the variation in fungal community characteristics in the leaf litter of a deciduous tree, Lindera glauca, which was decomposed by contrasting decomposer structures (with or without soil animals) under different contamination conditions (with or without microplastic contamination), aiming to determine the impacts of these factors on litter decomposition. We found that soil animals can significantly depress the litter decomposition rate by reducing fungal diversity and largely changing the community structure in the litter. However, these critical changes caused by soil animals were inhibited in the mesocosms contaminated with high-density polyethylene microplastics (HDPE−MPs), during which soil animal activities were significantly reduced. These findings represent a step forward in illustrating the potential effect of emerging contamination stress on forest litter decomposition and biogeochemical cycles under global environmental change.

Hypertension is one of the most common chronic diseases, and its prevalence is increasing worldwide. Lindera glauca (Siebold & Zucc.) Blume, known as grayblue spicebush (GS), has been used as food and for medicinal purposes; however, studies about its hypotensive or vasorelaxant effects are lacking. Therefore, the hypotensive effect of an ethanolic extract of the GS branch (GSE) was investigated in 15-week-old spontaneously hypertensive rats (SHRs) using the tail cuff method. The GSE administration group (1000 mg/kg SHR body weight) showed a decrease in their systolic and diastolic blood pressure measured 4 h after its administration. In addition, we investigated its vasorelaxant effect using the thoracic aorta dissected from Sprague-Dawley rats. The GSE (0.5, 1, 2, 5, 10, and 20 μg/mL) showed an endothelium-dependent vasorelaxant effect, and its mechanisms were found to be relevant to the inward rectifier, voltage-dependent, and non-selective K+ channels. Moreover, the GSE (20 μg/mL) showed an inhibitory effect on aortic rings constricted with angiotensin II. Considering its hypotensive and vasorelaxant effects, GSE has potential as a functional food to help treat and prevent high blood pressure. However, further studies on the identification of the active components of GSE and safety evaluations of its use are needed.

Lindera glauca (Lauraceae) is an economically important East Asian forest tree characterized by a dioecy in China and apomixis in Japan. However, patterns of population genetic diversity and structure of this species remain unknown for this species due to a lack of efficient molecular markers. In this study, we employed Illumina sequencing to analyze the transcriptomes of the female and male flower buds of L. glauca . We retrieved 59,753 and 75,075 unigenes for the female and male buds, respectively. Based on sequence similarity, 44,379 (74.27 %) unigenes for the female and 45,414 (60.49 %) unigenes for the male were matched to public databases. We identified 11,127 putative differentially expressed genes between the female and male buds and 20,048 expressed sequence tag-simple sequence repeats (EST-SSRs). From 3147 primer pairs designed successfully, 120 were selected for validation of polymorphism, and 13 could reliably amplify polymorphic bands and exhibited moderate levels of genetic diversity (e.g., N A  = 4.42; H E  = 0.56) when surveyed across 96 individuals of altogether six L. glauca populations from China and Japan. One of the three population genetic clusters identified in China was fixed in Japan, suggesting a historical population bottleneck following island immigration. The present study has generated a wealth of transcriptome data for future functional genomic research focused on the variable reproductive system of L. glauca (dioecy, apomixis) as well as EST-SSR markers for population genetics studies and its intriguing evolutionary shift from dioecy to apomixis in the wake of island colonization.

Premise of the study: Microsatellite primers were developed to investigate population genetic structure in Lindera glauca (Lauraceae). Methods and Results: Twenty-five microsatellite primers were developed and optimized for L. glauca using Illumina's Solexa sequencing technology. These novel primers were found to be polymorphic in nine wild L. glauca populations with 81 total alleles confirmed and genotyped via capillary gel electrophoresis. The total number of alleles, observed heterozygosity, and expected heterozygosity for each population ranged from one to four, from 0.00 to 0.90, and from 0.00 to 0.79, respectively. In addition, the 25 primers were tested in 10 additional individuals of the related species L. communis, and all but four markers showed good amplification results. Conclusions: This set of microsatellite primers is the first specifically developed for L. glauca and will facilitate studies of genetic diversity and evolution among populations of this species.