The long noncoding RNAs lnc10 and lnc11 regulating flavonoid biosynthesis in Ginkgo biloba.

Although long non-coding RNAs have been recognized to play important roles in plant, their possible functions and potential mechanism in Ginkgo biloba flavonoid biosynthesis are poorly understood. Flavonoids are important secondary metabolites and healthy components of Ginkgo biloba. They have been widely used in food, medicine, and natural health products. Most previous studies have focused on the molecular mechanisms of structural genes and transcription factors that regulate flavonoid biosynthesis. Few reports have examined the biological functions of flavonoid biosynthesis by long non-coding RNAs in G. biloba. Long noncoding RNAs associated with flavonoid biosynthesis in G. biloba have been identified through RNA sequencing, but the function of lncRNAs has not been reported. In this study, the expression levels of lnc10 and lnc11 were identified. Quantitative real-time polymerase chain reaction analysis revealed that lnc10 and lnc11 were expressed in all detected organs, and they showed significantly higher levels in immature and mature leaves than in other organs. In addition, to fully identify the function of lnc10 and lnc11 in flavonoid biosynthesis in G. biloba, lnc10 and lnc11 were cloned from G. biloba, and were transformed into Arabidopsis and overexpressed. Compared with the wild type, the flavonoid content was increased in transgenic plants. Moreover, the RNA-sequencing analysis of wild-type, lnc10-overexpression, and lnc11-overexpression plants screened out 2019 and 2552 differentially expressed genes, and the transcript levels of structural genes and transcription factors associated with flavonoid biosynthesis were higher in transgenic Arabidopsis than in the wild type, indicating that lnc10 and lnc11 activated flavonoid biosynthesis in the transgenic lines. Overall, these results suggest that lnc10 and lnc11 positively regulate flavonoid biosynthesis in G. biloba.

Contemporary understanding of transcription factor regulation of terpenoid biosynthesis in plants.

KEY MESSAGE: This review summarized how TFs function independently or in response to environmental factors to regulate terpenoid biosynthesis via fine-tuning the expression of rate-limiting enzymes. Terpenoids are derived from various species and sources. They are essential for interacting with the environment and defense mechanisms, such as antimicrobial, antifungal, antiviral, and antiparasitic properties. Almost all terpenoids have high medicinal value and economic performance. Recently, the control of enzyme genes on terpenoid biosynthesis has received a great deal of attention, but transcriptional factors regulatory network on terpenoid biosynthesis and accumulation has yet to get a thorough review. Transcription factors function as activators or suppressors independently or in response to environmental stimuli, fine-tuning terpenoid accumulation through regulating rate-limiting enzyme expression. This study investigates the advancements in transcription factors related to terpenoid biosynthesis and systematically summarizes previous works on the specific mechanisms of transcription factors that regulate terpenoid biosynthesis via hormone signal-transcription regulatory networks in plants. This will help us to better comprehend the regulatory network of terpenoid biosynthesis and build the groundwork for terpenoid development and effective utilization.

Role of bZIP transcription factors in the regulation of plant secondary metabolism.

MAIN CONCLUSION: This study provides an overview of the structure, classification, regulatory mechanisms, and biological functions of the basic (region) leucine zipper transcription factors and their molecular mechanisms in flavonoid, terpenoid, alkaloid, phenolic acid, and lignin biosynthesis. Basic (region) leucine zippers (bZIPs) are evolutionarily conserved transcription factors (TFs) in eukaryotic organisms. The bZIP TFs are widely distributed in plants and play important roles in plant growth and development, photomorphogenesis, signal transduction, resistance to pathogenic microbes, biotic and abiotic stress, and secondary metabolism. Moreover, the expression of bZIP TFs not only promotes or inhibits the accumulation of secondary metabolites in medicinal plants, but also affects the stress response of plants to the external adverse environment. This paper describes the structure, classification, biological function, and regulatory mechanisms of bZIP TFs. In addition, the molecular mechanism of bZIP TFs regulating the biosynthesis of flavonoids, terpenoids, alkaloids, phenolic acids, and lignin are also elaborated. This review provides a summary for in-depth study of the molecular mechanism of bZIP TFs regulating the synthesis pathway of secondary metabolites and plant molecular breeding, which is of significance for the generation of beneficial secondary metabolites and the improvement of plant varieties.

Advances in Research on the Involvement of Selenium in Regulating Plant Ecosystems.

Selenium is an essential trace element which plays an important role in human immune regulation and disease prevention. Plants absorb inorganic selenium (selenite or selenate) from the soil and convert it into various organic selenides (such as seleno amino acids, selenoproteins, and volatile selenides) via the sulfur metabolic pathway. These organic selenides are important sources of dietary selenium supplementation for humans. Organoselenides can promote plant growth, improve nutritional quality, and play an important regulatory function in plant ecosystems. The release of selenium-containing compounds into the soil by Se hyperaccumulators can promote the growth of Se accumulators but inhibit the growth and distribution of non-Se accumulators. Volatile selenides with specific odors have a deterrent effect on herbivores, reducing their feeding on plants. Soil microorganisms can effectively promote the uptake and transformation of selenium in plants, and organic selenides in plants can improve the tolerance of plants to pathogenic bacteria. Although selenium is not an essential trace element for plants, the right amount of selenium has important physiological and ecological benefits for them. This review summarizes recent research related to the functions of selenium in plant ecosystems to provide a deeper understanding of the significance of this element in plant physiology and ecosystems and to serve as a theoretical basis and technical support for the full exploitation and rational application of the ecological functions of selenium-accumulating plants.

SMRT and Illumina RNA sequencing reveal the complexity of terpenoid biosynthesis in Zanthoxylum armatum.

Sichuan pepper (Zanthoxylum armatum DC) is a popular spice and is often prescribed in traditional Chinese medicine to treat vomiting, diarrhea, ascariasis and eczema, among other conditions. Volatile oils from Z. armatum leaves contain active ingredients, with terpenoids being one of the main components. In the present study, the combination of sequencing data of Z. armatum from PacBio single molecule real time (SMRT) and Illumina RNA sequencing (RNA-Seq) platforms facilitated an understanding of the gene regulatory network of terpenoid biosynthesis in pepper leaves. The leaves of three developmental stages from two Z. armatum cultivars, 'Rongchangwuci' (WC) and 'Zhuye' (ZY), were selected as test materials to construct sequencing libraries. A total of 143,122 predictions of unique coding sequences, 105,465 simple sequence repeats, 20,145 transcription factors and 4719 long non-coding RNAs (lncRNAs) were identified, and 142,829 transcripts were successfully annotated. The occurrence of alternative splicing events was verified by reverse transcription PCR, and quantitative real-time PCR was used to confirm the expression pattern of six randomly selected lncRNAs. A total of 96,931 differentially expressed genes were filtered from different samples. According to functional annotation, a total of 560 candidate genes were involved in terpenoid synthesis, of which 526 were differentially expressed genes (DEGs). To identify the key genes involved in terpenoid biosynthesis, the module genes in different samples, including structural and transcription factors genes, were analyzed using the weighted gene co-expression network method, and the co-expression network of genes was constructed. Thirty-one terpenoids were identified by gas chromatography-mass spectrometry. The correlation between 18 compounds with significantly different contents and genes with high connectivity in the module was jointly analyzed in both cultivars, yielding 12 candidate DEGs presumably involved in the regulation of terpenoid biosynthesis. Our findings showed that full-length transcriptome SMRT and Illumina RNA-Seq can play an important role in studying organisms without reference genomes and elucidating the gene regulation of a biosynthetic pathway.

Effects of selenate on Se, flavonoid, and glucosinolate in broccoli florets by combined transcriptome and metabolome analyses.

Broccoli is a nutritious vegetable popular all over the world. This study investigated the effects of different concentrations of selenate (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mmol/L) on the selenium (Se), glucosinolate, and flavonoid contents of broccoli florets. Results showed that the total Se, selenomethionine, and methyl selenocysteine contents increased following selenate dosage. Interestingly, selenate treatment of 0.4 mmol/L decreased the flavonoid but increased the glucosinolate content. Metabolome analysis revealed changes in the individual contents of glucosinolates and flavonoids. Conjoint analysis of transcriptome and metabolome showed that the glucosinolate and flavonoid compounds were potentially regulated by two sulfate transporter genes (Sultr3;1 and Sultr4;2) and several cytochrome P450 genes (e.g., CYP71B21, CYP72C1, and CYP81F1). These new findings indicated that Se treatment may influence glucosinolate and flavonoid accumulation by regulating the expression of these genes. The results of this study provide some novel insights into the effects of Se on glucosinolates and flavonoids in broccoli florets and deepen our understanding of the regulatory network between some specific genes and these compounds.

Integration analysis of PacBio SMRT- and Illumina RNA-seq reveals candidate genes and pathway involved in selenium metabolism in hyperaccumulator Cardamine violifolia.

BACKGROUND: Cardamine violifolia, native to China, is one of the selenium (Se) hyperaccumulators. The mechanism of Se metabolism and tolerance remains unclear, and only limited genetic information is currently available. Therefore, we combined a PacBio single-molecule real-time (SMRT) transcriptome library and the Illumina RNA-seq data of sodium selenate (Na2SeO4)-treated C. violifolia to further reveal the molecular mechanism of Se metabolism. RESULTS: The concentrations of the total, inorganic, and organic Se in C. violifolia seedlings significantly increased as the Na2SeO4 treatment concentration increased. From SMRT full-length transcriptome of C. violifolia, we obtained 26,745 annotated nonredundant transcripts, 14,269 simple sequence repeats, 283 alternative splices, and 3407 transcription factors. Fifty-one genes from 134 transcripts were identified to be involved in Se metabolism, including transporter, assimilatory enzyme, and several specific genes. Analysis of Illumina RNA-Seq data showed that a total of 948 differentially expressed genes (DEGs) were filtered from the four groups with Na2SeO4 treatment, among which 11 DEGs were related to Se metabolism. The enrichment analysis of KEGG pathways of all the DEGs showed that they were significantly enriched in five pathways, such as hormone signal transduction and plant-pathogen interaction pathways. Four genes related to Se metabolism, adenosine triphosphate sulfurase 1, adenosine 5'-phosphosulfate reductase 3, cysteine (Cys) desulfurase 1, and serine acetyltransferase 2, were regulated by lncRNAs. Twenty potential hub genes (e.g., sulfate transporter 1;1, Cys synthase, methionine gamma-lyase, and Se-binding protein 1) were screened and identified to play important roles in Se accumulation and tolerance in C. violifolia as concluded by weighted gene correlation network analysis. Based on combinative analysis of expression profiling and annotation of genes as well as Se speciation and concentration in C. violifolia under the treatments with different Na2SeO4 concentrations, a putative Se metabolism and assimilation pathway in C. violifolia was proposed. CONCLUSION: Our data provide abundant information on putative gene transcriptions and pathway involved in Se metabolism of C. violifolia. The findings present a genetic resource and provide novel insights into the mechanism of Se hyperaccumulation in C. violifolia.

Screening and identification of miRNAs related to sexual differentiation of strobili in Ginkgo biloba by integration analysis of small RNA, RNA, and degradome sequencing.

BACKGROUND: Ginkgo biloba, a typical dioecious plant, is a traditional medicinal plant widely planted. However, it has a long juvenile period, which severely affected the breeding and cultivation of superior ginkgo varieties. RESULTS: In order to clarify the complex mechanism of sexual differentiation in G. biloba strobili. Here, a total of 3293 miRNAs were identified in buds and strobili of G. biloba, including 1085 known miRNAs and 2208 novel miRNAs using the three sequencing approaches of transcriptome, small RNA, and degradome. Comparative transcriptome analysis screened 4346 and 7087 differentially expressed genes (DEGs) in male buds (MB) _vs_ female buds (FB) and microstrobilus (MS) _vs_ ovulate strobilus (OS), respectively. A total of 6032 target genes were predicted for differentially expressed miRNA. The combined analysis of both small RNA and transcriptome datasets identified 51 miRNA-mRNA interaction pairs that may be involved in the process of G. biloba strobili sexual differentiation, of which 15 pairs were verified in the analysis of degradome sequencing. CONCLUSIONS: The comprehensive analysis of the small RNA, RNA and degradome sequencing data in this study provided candidate genes and clarified the regulatory mechanism of sexual differentiation of G. biloba strobili from multiple perspectives.

Characterization and functional analysis of two novel 3-hydroxy-3-methylglutaryl-coenzyme A reductase genes (GbHMGR2 and GbHMGR3) from Ginkgo biloba.

Terpene trilactones (TTLs) are the main secondary metabolites of Ginkgo biloba. As one of the rate-limiting enzymes in the mevalonic acid (MVA) pathway of TTL biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the 3-hydroxy-3-methylglutaryl coenzyme A to form MVA. In this study, two cDNA sequences of HMGR genes, namely, GbHMGR2 and GbHMGR3, were cloned from G. biloba. The protein sequences of GbHMGR2 and GbHMGR3, which contain several functional domains, were analyzed. Regulatory elements related to light, hormone, and stress response were detected in the promoter regions of GbHMGR2 and GbHMGR3. The catalytic activity of these genes was verified by a functional complement experiment in yeast. Quantitative real-time PCR (qRT-PCR) showed the distinct expression patterns of the two genes in different organs. The TTL contents in the organs were detected by high-performance liquid chromatography- evaporative light scattering detector. GbHMGR2 and GbHMGR3 were responded to cold, dark, methyl jasmonate (MJ), abscisic acid (ABA), salicylic acid (SA), and ethephon (Eth) treatments. The TTL contents were also regulated by cold, dark, MJ, ABA, SA, and Eth treatment. In conclusion, GbHMGR2 and GbHMGR3 may participate in the MVA pathway of TTL biosynthesis.

De novo assembly and comparative transcriptome analysis: novel insights into terpenoid biosynthesis in Chamaemelum nobile L.

KEY MESSAGE: Analysis of terpenoids content, transcriptome from Chamaemelum nobile showed that the content of the terpenoids in the roots was the highest and key genes involved in the terpenoids synthesis pathway were identified. Chamaemelum nobile is a widely used herbaceous medicinal plant rich in volatile oils, mainly composed of terpenoids. It is widely used in food, cosmetics, medicine, and other fields. In this study, we analyzed the transcriptome and the content and chemical composition of the terpenoids in different organs of C. nobile. Gas chromatography-mass spectrometry analysis showed that the total content of the terpenoids among C. nobile organs was highest in the roots, followed by the flowers. Illumina HiSeq 2500 high-throughput sequencing technology was used to sequence the transcripts of roots, stems, leaves, and flowers of C. nobile. We obtained 139,757 unigenes using the Trinity software assembly. A total of 887 unigenes were annotated to secondary metabolism. In total, 55,711 differentially expressed genes were screened among different organs of C. nobile. We identified 16 candidate genes that may be involved in the terpenoid biosynthesis from C. nobile and analyzed their expression patterns using real-time PCR. Results showed that the expression pattern of these genes was tissue-specific and had significant differential expression levels in different organs of C. nobile. Among these genes, 13 were expressed in roots with the highest levels. Furthermore, the transcript levels of these 13 genes were positively correlated with the content of α-pinene, ß-phellandrene, 1,8-cineole, camphor, α-terpineol, carvacrol, (E,E)-farnesol and chamazulene, suggesting that these 13 genes may be involved in the regulation of the synthesis of the volatile terpenoids. These results laid the foundation for the subsequent improvement of C. nobile quality through genetic engineering.

Cloning, Expression Profiling and Functional Analysis of CnHMGS, a Gene Encoding 3-hydroxy-3-Methylglutaryl Coenzyme A Synthase from Chamaemelum nobile.

Roman chamomile (Chamaemelum nobile L.) is renowned for its production of essential oils, which major components are sesquiterpenoids. As the important enzyme in the sesquiterpenoid biosynthesis pathway, 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) catalyze the crucial step in the mevalonate pathway in plants. To isolate and identify the functional genes involved in the sesquiterpene biosynthesis of C. nobile L., a HMGS gene designated as CnHMGS (GenBank Accession No. KU529969) was cloned from C. nobile. The cDNA sequence of CnHMGS contained a 1377 bp open reading frame encoding a 458-amino-acid protein. The sequence of the CnHMGS protein was highly homologous to those of HMGS proteins from other plant species. Phylogenetic tree analysis revealed that CnHMGS clustered with the HMGS of Asteraceae in the dicotyledon clade. Further functional complementation of CnHMGS in the mutant yeast strain YSC6274 lacking HMGS activity demonstrated that the cloned CnHMGS cDNA encodes a functional HMGS. Transcript profile analysis indicated that CnHMGS was preferentially expressed in flowers and roots of C. nobile. The expression of CnHMGS could be upregulated by exogenous elicitors, including methyl jasmonate and salicylic acid, suggesting that CnHMGS was elicitor-responsive. The characterization and expression analysis of CnHMGS is helpful to understand the biosynthesis of sesquiterpenoid in C. nobile at the molecular level and also provides molecular wealth for the biotechnological improvement of this important medicinal plant.