Integrated transcriptomic and WGCNA analyses reveal candidate genes regulating mainly flavonoid biosynthesis in Litsea coreana var. sinensis.

Litsea coreana Levl. var. sinensis (Allen) Yang et P. H. Huang is a popular ethnic herb and beverage plant known for its high flavonoid content, which has been linked to a variety of pharmacological benefits and crucial health-promoting impacts in humans. The progress in understanding the molecular mechanisms of flavonoid accumulation in this plant has been hindered due to the deficiency of genomic and transcriptomic resources. We utilized a combination of Illumina and Oxford Nanopore Technology (ONT) sequencing to generate a de novo hybrid transcriptome assembly. In total, 126,977 unigenes were characterized, out of which 107,977 were successfully annotated in seven public databases. Within the annotated unigenes, 3,781 were categorized into 58 transcription factor families. Furthermore, we investigated the presence of four valuable flavonoids-quercetin-3-O-ß-D-galactoside, quercetin-3-O-ß-D-glucoside, kaempferol-3-O-ß-D-galactoside, and kaempferol-3-O-ß-D-glucoside in 98 samples, using high-performance liquid chromatography. A weighted gene co-expression network analysis identified two co-expression modules, MEpink and MEturquoise, that showed strong positive correlation with flavonoid content. Within these modules, four transcription factor genes (R2R3-MYB, NAC, WD40, and ARF) and four key enzyme-encoding genes (CHI, F3H, PAL, and C4H) emerged as potential hub genes. Among them, the R2R3-MYB (LcsMYB123) as a homologous gene to AtMYB123/TT2, was speculated to play a significant role in flavonol biosynthesis based on phylogenetic analysis. Our findings provided a theoretical foundation for further research into the molecular mechanisms of flavonoid biosynthesis. Additionally, The hybrid transcriptome sequences will serve as a valuable molecular resource for the transcriptional annotation of L. coreana var. sinensis, which will contribute to the improvement of high-flavonoid materials.

The Association between BZIP Transcription Factors and Flower Development in Litsea cubeba.

The basic leucine zipper (bZIP) family is one of the largest families of transcription factors among eukaryotic organisms. Members of the bZIP family play various roles in regulating the intricate process of flower development in plants. Litsea cubeba (Lour.) (family: Lauraceae) is an aromatic, dioecious plant used in China for a wide range of applications. However, no study to date has undertaken a comprehensive analysis of the bZIP gene family in L. cubeba. In this work, we identified 68 members of the bZIP gene family in L. cubeba and classified them into 12 subfamilies based on previous studies on Arabidopsis thaliana. Transcriptome data analysis revealed that multiple LcbZIP genes exhibit significantly high expression levels in the flowers of L. cubeba, while some also demonstrate distinct temporal specificity during L. cubeba flower development. In particular, some LcbZIP genes displayed specific and high expression levels during the stamen and pistil degradation process. Using differential gene expression analysis, weighted gene co-expression network analysis, and Gene Ontology enrichment analysis, we identified six candidate LcbZIP genes that potentially regulate stamen or pistil degradation during flower development. In summary, our findings provide a framework for future functional analysis of the LcbZIP gene family in L. cubeba and offer novel insights for investigating the mechanism underlying pistil and stamen degeneration in this plant.

LcMYB106 suppresses monoterpene biosynthesis by negatively regulating LcTPS32 expression in Litsea cubeba.

Litsea cubeba, the core species of the Lauraceae family, is valuable for the production of essential oils due to its high concentration of monoterpenes (90%). The key monoterpene synthase and metabolic regulatory network of monoterpene biosynthesis have provided new insights for improving essential oil content. However, there are few studies on the regulation mechanism of monoterpenes in L. cubeba. In this study, we investigated LcTPS32, a member of the TPS-b subfamily, and identified its function as an enzyme for the synthesis of monoterpenes, including geraniol, α-pinene, ß-pinene, ß-myrcene, linalool and eucalyptol. The quantitative real-time PCR analysis showed that LcTPS32 was highly expressed in the fruits of L. cubeba and contributed to the characteristic flavor of its essential oil. Overexpression of LcTPS32 resulted in a significant increase in the production of monoterpenes in L. cubeba by activating both the MVA and MEP pathways. Additionally, the study revealed that LcMYB106 played a negative regulatory role in monoterpenes biosynthesis by directly binding to the promoter of LcTPS32. Our study indicates that LcMYB106 could serve as a crucial target for metabolic engineering endeavors, aiming at enhancing the monoterpene biosynthesis in L. cubeba.

LcWRKY17, a WRKY Transcription Factor from Litsea cubeba, Effectively Promotes Monoterpene Synthesis.

The WRKY gene family is one of the most significant transcription factor (TF) families in higher plants and participates in many secondary metabolic processes in plants. Litsea cubeba (Lour.) Person is an important woody oil plant that is high in terpenoids. However, no studies have been conducted to investigate the WRKY TFs that regulate the synthesis of terpene in L. cubeba. This paper provides a comprehensive genomic analysis of the LcWRKYs. In the L. cubeba genome, 64 LcWRKY genes were discovered. According to a comparative phylogenetic study with Arabidopsis thaliana, these L. cubeba WRKYs were divided into three groups. Some LcWRKY genes may have arisen from gene duplication, but the majority of LcWRKY evolution has been driven by segmental duplication events. Based on transcriptome data, a consistent expression pattern of LcWRKY17 and terpene synthase LcTPS42 was found at different stages of L. cubeba fruit development. Furthermore, the function of LcWRKY17 was verified by subcellular localization and transient overexpression, and overexpression of LcWRKY17 promotes monoterpene synthesis. Meanwhile, dual-Luciferase and yeast one-hybrid (Y1H) experiments showed that the LcWRKY17 transcription factor binds to W-box motifs of LcTPS42 and enhances its transcription. In conclusion, this research provided a fundamental framework for future functional analysis of the WRKY gene families, as well as breeding improvement and the regulation of secondary metabolism in L. cubeba.

LcERF134 increases the production of monoterpenes by activating the terpene biosynthesis pathway in Litsea cubeba.

Litsea cubeba, an aromatic species of the Lauraceae family, produces a diverse array of monoterpenes. The biosynthesis of monoterpenes is regulated by transcriptional factors (TFs), such as APETALA2/ethylene response factor (AP2/ERF). However, the regulatory mechanisms that control the AP2/ERF gene responsible for the biosynthesis of monoterpenes in L. cubeba have yet to be elucidated. Here, we identified an AP2/ERF gene, LcERF134, as an activator for the accumulation of citral and other monoterpenes. The expression level of LcERF134 was consistent with terpene synthase LcTPS42 in the pericarp. The transient overexpression of LcERF134 significantly increased monoterpene production in L. cubeba as well as the expression of rate-limiting genes involved in the monoterpene biosynthesis pathway. Furthermore, yeast one-hybrid, dual-luciferase and electrophoretic mobility shift assays demonstrated that LcERF134 activated the monoterpene biosynthesis pathway by directly binding to the GCC-box elements of the LcTPS42 and LcGPPS.SSU1 promoters. However, the overexpression of LcERF134 in tomatoes had no impact on the synthesis of monoterpenes, thus indicating that LcERF134 is a species-specific TF. Our research demonstrated that LcERF134 significantly increased the biosynthesis of monoterpenes by inducing the expression of LcTPS42 and LcGPPS.SSU1, thus offering insight into how to enhance the flavor of L. cubeba essential oil.

Comparative Analysis of Complete Chloroplast Genomes of Nine Species of Litsea (Lauraceae): Hypervariable Regions, Positive Selection, and Phylogenetic Relationships.

Litsea is a group of evergreen trees or shrubs in the laurel family, Lauraceae. Species of the genus are widely used for a wide range of medicinal and industrial aspects. At present, most studies related to the gene resources of Litsea are restricted to morphological analyses or features of individual genomes, and currently available studies of select molecular markers are insufficient. In this study, we assembled and annotated the complete chloroplast genomes of nine species in Litsea, carried out a series of comparative analyses, and reconstructed phylogenetic relationships within the genus. The genome length ranged from 152,051 to 152,747 bp and a total of 128 genes were identified. High consistency patterns of codon bias, repeats, divergent analysis, single nucleotide polymorphisms (SNP) and insertions and deletions (InDels) were discovered across the genus. Variations in gene length and the presence of the pseudogene ycf1Ψ, resulting from IR contraction and expansion, are reported. The hyper-variable gene rpl16 was identified for its exceptionally high Ka/Ks and Pi values, implying that those frequent mutations occurred as a result of positive selection. Phylogenetic relationships were recovered for the genus based on analyses of full chloroplast genomes and protein-coding genes. Overall, both genome sequences and potential molecular markers provided in this study enrich the available genomic resources for species of Litsea. Valuable genomic resources and divergent analysis are also provided for further research of the evolutionary patterns, molecular markers, and deeper phylogenetic relationships of Litsea.

High-quality chromosome-level genome assembly of Litsea coreana L. provides insights into Magnoliids evolution and flavonoid biosynthesis.

The magnoliid Litsea coreana has been the subject of a substantial amount of research owing to its production of many flavonoid metabolites, high food processing value, and a controversial phylogenetic position. For this study, we assembled a high-grade genome at the chromosome scale and annotation of L. coreana that was anchored to 12 chromosomes. The total genome was 1139.45 Mb, while the N50 scaffold was 97.18 Mb long. The analysis of phylogenetic trees constructed by different methods show that the phylogeny of Magnoliids is inconsistent, indicating that the differentiation process of monocots, eudicots, and Magnoliids still remains in dispute. An ancient whole-genome duplication (WGD) event was shown to have occurred before the Magnoliales and Laurels had differentiated. Subsequently, an independent WGD appeared in the Lauralean lineage. A total of 27 types of flavonoids were detected in all five tissues of L. coreana. Chalcone synthases (CHSs) that are responsible for production of flavonoids have been validated at the bioinformatics level. The retention of comparative genomic analyses of the CHS gene family showed that this family had contracted significantly in L. coreana. Our research further elaborated the evolution of Lauraceae and perfected the genetic basis of flavonoid biosynthesis in L. coreana. SIGNIFICANCE STATEMENT: Provides evidence that determines the evolutionary status of Magnoliids. The chalcone synthase gene family was significantly contracted in Litsea coreana.

The Litsea genome and the evolution of the laurel family.

The laurel family within the Magnoliids has attracted attentions owing to its scents, variable inflorescences, and controversial phylogenetic position. Here, we present a chromosome-level assembly of the Litsea cubeba genome, together with low-coverage genomic and transcriptomic data for many other Lauraceae. Phylogenomic analyses show phylogenetic discordance at the position of Magnoliids, suggesting incomplete lineage sorting during the divergence of monocots, eudicots, and Magnoliids. An ancient whole-genome duplication (WGD) event occurred just before the divergence of Laurales and Magnoliales; subsequently, independent WGDs occurred almost simultaneously in the three Lauralean lineages. The phylogenetic relationships within Lauraceae correspond to the divergence of inflorescences, as evidenced by the phylogeny of FUWA, a conserved gene involved in determining panicle architecture in Lauraceae. Monoterpene synthases responsible for production of specific volatile compounds in Lauraceae are functionally verified. Our work sheds light on the evolution of the Lauraceae, the genetic basis for floral evolution and specific scents.

Ectopic Expression of Litsea cubeba LcMADS20 Modifies Silique Architecture.

Litsea cubeba (Lour.) Pers. (mountain pepper, Lauraceae) is an important woody essential oil crop that produces fragrant oils in its fruits, especially in its peels. Identification of genes involved in the regulation of fruits and peel architecture is of economic significance for L. cubeba industry. It has been well known that the MADS-box genes are essential transcription factors that control flowers and fruits development. Here, we obtained 33 MADS-box genes first from the RNA-seq data in L. cubeba, and 27 of these genes were of the MIKC-type. LcMADS20, an AGAMOUS-like gene, was highly expressed in the developing stages of fruits, particularly at 85 days after full bloom. The ectopic expression of LcMADS20 in Arabidopsis resulted in not only curved leaves, early flowering and early full-opened inflorescences, but also shorter siliques and decreased percentage of peel thickness. Moreover, in the LcMADS20 transgenic Arabidopsis, the expression modes of several intrinsic ABC model class genes were influenced, among which the expression of FUL was significantly reduced and AP3, AG, and STK were significantly increased. This study systematically analyzed the MADS-box genes in L. cubeba at the transcriptional level and showed that LcMADS20 plays important roles in the regulation of fruit architecture.

A Model of Hormonal Regulation of Stamen Abortion during Pre-Meiosis of Litseacubeba.

Abstract:Litseacubeba (Lour.) Pers., a popular essential oil plant, is a dioecious species with degenerative sexual organs in both male and female individuals. Yet, the mechanism of degenerative organs development in male and female flowers is poorly understood. Here, we analyzed the morphological characters of degenerative organ development by morphological and histological observations, and determined the critical stage of abortion that occurs at pre-meiosis in male and female flowers. We also conducted RNA sequencing (RNA-seq) to understand the genetic basis of stamen abortion in female flowers. The differentially expressed genes (DEGs) were identified during the staminode development in female flowers; functional enrichment analysis revealed some important biological pathways involved the regulation of stamen abortion, including plant hormone signal transduction, phenylpropanoid biosynthesis, flavonoid biosynthesis and monoterpenoid biosynthesis. Furthermore, 15 DEGs involved in the hormone pathways were found to regulate stamen development. By HPLC-MS/MS analysis, there were a salicylic acid (SA) content peak and the gibberellin (GA) content lowest point in the abortion processes in female flowers, suggesting a vital function of hormonal processes. Co-expression network analysis further identified several hub genes that potentially played significant roles in the stamen abortion of L. cubeba. Taken together, we proposed a model involved in plant hormones pathways underlying stamen abortion during pre-meiosis in female flowers of L.cubeba.

Transcriptome profile of cup-shaped galls in Litsea acuminata leaves.

BACKGROUND: Insect galls are atypical plant tissues induced by the invasion of insects. Compared to the host leaf, gall tissues lose photosynthetic ability, but have higher soluble sugar content. Although the physiological and biochemical regulation of gall tissues have been demonstrated, the mechanism of genetic regulation has only been analyzed in few studies. RESULTS: In this study, the transcriptome of cup-shaped galls and its host leaf were de novo assembled. Cellular functional enrichment and differentially expressed gene groups in the gall tissues were analyzed. The genes associated with primary metabolism, including photosynthesis, cell wall turnover, and sugar degradation, were expressed differently in galls and leaves. The examination of gene expression demonstrated that the genes involved in brassinosteroid synthesis and responses exhibited a remarkable modulation in cup-shaped galls, suggesting a potential role of steroid hormones in regulating gall development. CONCLUSIONS: This study revealed the genetic responses, including those involved in source-sink reallocation and phytohormone metabolism, of galls induced by a dipteran insect.

Transcriptome Analysis of Litsea cubeba Floral Buds Reveals the Role of Hormones and Transcription Factors in the Differentiation Process.

BACKGROUND: Litsea cubeba (Lour.) Pers. is an important economic plant that is rich in valuable essential oil. The essential oil is often used as a raw material for perfumes, food additives, insecticides and bacteriostats. Most of the essential oil is contained in the fruit, and the quantity and quality of fruit are dependent on the flowers. To explore the molecular mechanism of floral bud differentiation, high-throughput RNA sequencing was used to detect differences in the gene expression of L. cubeba female and male floral buds at three differentiation stages. RESULTS: This study obtained 160.88 Gbp of clean data that were assembled into 100,072 unigenes, and a total of 38,658 unigenes were annotated. A total of 27,521 simple sequence repeats (SSRs) were identified after scanning the assembled transcriptome, and the mono-nucleotide repeats were predominant, followed by di-nucleotide and tri-nucleotide repeats. A total of 12,559 differentially expressed genes (DEGs) were detected from the female (F) and male (M) floral bud comparisons. The gene ontology (GO) databases revealed that these DEGs were primarily contained in "metabolic processes", "cellular processes", and "single-organism processes". The Kyoto Encyclopedia of Genes and Genomes (KEGG) databases suggested that the DEGs belonged to "plant hormone signal transduction" and accounted for a relatively large portion in all of these comparisons. We analyzed the expression level of plant hormone-related genes and detected the contents of several relevant plant hormones in different stages. The results revealed that the dynamic changes in each hormone content were almost consistent with the expression levels of relevant genes. The transcription factors selected from the DEGs were analyzed. Most DEGs of MADS-box were upregulated and most DEGs of bZIP were downregulated. The expression trends of the DEGs were nearly identical in female and male floral buds, and qRT-PCR analysis revealed consistency with the transcriptome data. CONCLUSIONS: We sequenced and assembled a high-quality L. cubeba floral bud transcriptome, and the data appeared to be well replicated (n = 3) over three developmental time points during flower development. Our study explored the changes in the contents of several plant hormones during floral bud differentiation using biochemical and molecular biology techniques, and the changes in expression levels of several flower development related transcription factors. These results revealed the role of these factors (i.e., hormones and transcription factors) and may advance our understanding of their functions in flower development in L. cubeba.

Sex differential marker FD for rapid sex identification of Litsea cubeba.

Litsea cubeba is an important economic tree in China. Sex identification of the species is required to reduce breeding costs. Molecular biology is an ideal method to achieve this aim because of the lack of morphological differences between male and female plants, especially at the seedling stage. Sequence-related amplified polymorphism was used to amplify sex-related bands. Following sequencing, the amplified fragment Dwas used to create a sequence-characterized amplified region (SCAR) marker, FD. The SCAR marker is approximately 750 bp, is female-specific, and is expected to be useful for L. cubeba breeding programs. Furthermore, the amplified fragment L had homology to sex-determining-related genes of other species. Quantitative real-time polymerase chain reaction analysis of this fragment during flower bud development identified expression differences between male and female plants.

Transcriptome sequencing and expression analysis of terpenoid biosynthesis genes in Litsea cubeba.

BACKGROUND: Aromatic essential oils extracted from fresh fruits of Litsea cubeba (Lour.) Pers., have diverse medical and economic values. The dominant components in these essential oils are monoterpenes and sesquiterpenes. Understanding the molecular mechanisms of terpenoid biosynthesis is essential for improving the yield and quality of terpenes. However, the 40 available L. cubeba nucleotide sequences in the public databases are insufficient for studying the molecular mechanisms. Thus, high-throughput transcriptome sequencing of L. cubeba is necessary to generate large quantities of transcript sequences for the purpose of gene discovery, especially terpenoid biosynthesis related genes. RESULTS: Using Illumina paired-end sequencing, approximately 23.5 million high-quality reads were generated. De novo assembly yielded 68,648 unigenes with an average length of 834 bp. A total of 38,439 (56%) unigenes were annotated for their functions, and 35,732 and 25,806 unigenes could be aligned to the GO and COG database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 16,130 unigenes were assigned to 297 KEGG pathways, and 61 unigenes, which contained the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathways, could be related to terpenoid backbone biosynthesis. Of the 12,963 unigenes, 285 were annotated to the terpenoid pathways using the PlantCyc database. Additionally, 14 terpene synthase genes were identified from the transcriptome. The expression patterns of the 16 genes related to terpenoid biosynthesis were analyzed by RT-qPCR to explore their putative functions. CONCLUSION: RNA sequencing was effective in identifying a large quantity of sequence information. To our knowledge, this study is the first exploration of the L. cubeba transcriptome, and the substantial amount of transcripts obtained will accelerate the understanding of the molecular mechanisms of essential oils biosynthesis. The results may help improve future genetic and genomics studies on the molecular mechanisms behind the chemical composition of essential oils in L. cubeba fruits.

Identification of appropriate reference genes for normalizing transcript expression by quantitative real-time PCR in Litsea cubeba.

Quantitative real-time PCR has emerged as a highly sensitive and widely used method for detection of gene expression profiles, via which accurate detection depends on reliable normalization. Since no single control is appropriate for all experimental treatments, it is generally advocated to select suitable internal controls prior to use for normalization. This study reported the evaluation of the expression stability of twelve potential reference genes in different tissue/organs and six fruit developmental stages of Litsea cubeba in order to screen the superior internal reference genes for data normalization. Two softwares-geNorm, and NormFinder-were used to identify stability of these candidate genes. The cycle threshold difference and coefficient of variance were also calculated to evaluate the expression stability of candidate genes. F-BOX, EF1α, UBC, and TUA were selected as the most stable reference genes across 11 sample pools. F-BOX, EF1α, and EIF4α exhibited the highest expression stability in different tissue/organs and different fruit developmental stages. Besides, a combination of two stable reference genes would be sufficient for gene expression normalization in different fruit developmental stages. In addition, the relative expression profiles of DXS and DXR were evaluated by EF1α, UBC, and SAMDC. The results further validated the reliability of stable reference genes and also highlighted the importance of selecting suitable internal controls for L. cubeba. These reference genes will be of great importance for transcript normalization in future gene expression studies on L. cubeba.

Characterization of microsatellite loci from Litsea hypophaea (Lauraceae), a tree endemic to Taiwan.

PREMISE OF THE STUDY: Microsatellite primers were developed for the endemic tree Litsea hypophaea (Lauraceae) in Taiwan to investigate its genetic diversity and population genetic structure and to investigate species delimitation within Litsea. METHODS AND RESULTS: Fifteen new simple sequence repeat markers were developed from L. hypophaea with a magnetic bead enrichment method. Most loci were also amplified from three closely related species, L. coreana, L. lii, and L. acutivena. The number of alleles and observed and expected heterozygosities across loci varied with a range of 1-25, 0.000-1.000, and 0.000-0.956, respectively. CONCLUSIONS: The application of these microsatellite markers of L. hypophaea provides a tool for understanding genetic diversity and population differentiation. In addition, interspecific amplification suggests that these markers will also be useful for species identification of related taxa within Litsea in Taiwan.