Terpenoid Biosynthesis Pathway Research Articles

De novo assembly and characterization of transcriptome in the medicinal plant Euphorbia jolkini

Euphorbia jolkini, a medicinal herb that grows on the warm beaches in Japan and South Korea, is known to be used for traditional medicines to treat a variety of ailments, including bruises, stiffness, indigestion, toothache, and diabetes. It is to analyze the whole transcriptome and identify the genes related to the phenylpropanoid biosynthesis in the medicinally important herb E jolkini. Paired-end Illumina HiSeq™ 2500 sequencing technology was employed for cDNA library construction and Illumina sequencing. Public databases like TAIR (The Arabidopsis Information Resource), Swissprot and KEGG (Kyoto Encyclopedia of Genes and Genomes) were used for annotations of unigenes obtained. The transcriptome of E. jolkini generated 139,215 assembled transcripts with an average length of 868bp and an N50 value of 1460bp that were further clustered using CD-HIT into 93,801 unigenes with an average length of 847bp (N50-1410bp). Sixty-three percent of the coding sequences (CDS) were annotated from the longest open reading frame (ORF). A remarkable percentage of unigenes were annotated against various databases. The differentially expressed gene analysis revealed that the expression of genes related to the terpenoid backbone biosynthesis pathway was higher in the flowers, whereas that of genes related to the phenylpropanoid biosynthesis pathway was both up- and downregulated in flowers and leaves. A search of against the transcription factor domain found 1023 transcription factors (TFs) that were from 54 TF families. Assembled sequences of the E. jolkini transcriptome are made available for the first time in this study E. jolkini and lay a foundation for the investigation of secondary metabolite biosynthesis.

Mining of candidate genes involved in the biosynthesis of dextrorotatory borneol in Cinnamomum burmannii by transcriptomic analysis on three chemotypes.

BackgroundDextrorotatory borneol (D-borneol), a cyclic monoterpene, is widely used in traditional Chinese medicine as an efficient topical analgesic drug. Fresh leaves of Cinnamomum trees, e.g., C. burmannii and C. camphor, are the main sources from which D-borneol is extracted by steam distillation, yet with low yields. Insufficient supply of D-borneol has hampered its clinical use and production of patent remedies for a long time. Biological synthesis of D-borneol offers an additional approach; however, mechanisms of D-borneol biosynthesis remain mostly unresolved. Hence, it is important and necessary to elucidate the biosynthetic pathway of D-borneol.ResultsComparative analysis on the gene expression patterns of different D-borneol production C. burmannii samples facilitates elucidation on the underlying biosynthetic pathway of D-borneol. Herein, we collected three different chemotypes of C. burmannii, which harbor different contents of D-borneol.A total of 100,218 unigenes with an N50 of 1,128 bp were assembled de novo using Trinity from a total of 21.21 Gb clean bases. We used BLASTx analysis against several public databases to annotate 45,485 unigenes (45.38%) to at least one database, among which 82 unigenes were assigned to terpenoid biosynthesis pathways by KEGG annotation. In addition, we defined 8,860 unigenes as differentially expressed genes (DEGs), among which 13 DEGs were associated with terpenoid biosynthesis pathways. One 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and two monoterpene synthase, designated as CbDXS9, CbTPS2 and CbTPS3, were up-regulated in the high-borneol group compared to the low-borneol and borneol-free groups, and might be vital to biosynthesis of D-borneol in C. burmannii. In addition, we identified one WRKY, two BHLH, one AP2/ERF and three MYB candidate genes, which exhibited the same expression patterns as CbTPS2 and CbTPS3, suggesting that these transcription factors might potentially regulate D-borneol biosynthesis. Finally, quantitative real-time PCR was conducted to detect the actual expression level of those candidate genes related to the D-borneol biosynthesis pathway, and the result showed that the expression patterns of the candidate genes related to D-borneol biosynthesis were basically consistent with those revealed by transcriptome analysis.ConclusionsWe used transcriptome sequencing to analyze three different chemotypes of C. burmannii, identifying three candidate structural genes (one DXS, two monoterpene synthases) and seven potential transcription factor candidates (one WRKY, two BHLH, one AP2/ERF and three MYB) involved in D-borneol biosynthesis. These results provide new insight into our understanding of the production and accumulation of D-borneol in C. burmannii.

RNA-Seq transcriptomic analyses of Antrodia camphorata to determine antroquinonol and antrodin C biosynthetic mechanisms in the in situ extractive fermentation.

In situ extractive fermentation (ISEF) is an important technique for improving metabolite productivity. The different extractants can induce the synthesis of different bioactive metabolites of Antrodia camphorata during ISEF. However, a lack of research on the molecular genetics of A. camphorata during ISEF currently hinders such studies on metabolite biosynthetic mechanisms. To clarify the differentially expressed genes during ISEF, the gene transcriptional expression features of A. camphorata S-29 were analysed. The addition of n-tetradecane as an extractant during ISEF showed more pronounced up-regulation of ubiquinone and other terpenoid-quinone biosynthesis pathway genes (CoQ2, wrbA and ARO8). When oleic acid was used as an extractant, the terpenoid backbone biosynthesis and ubiquinone and other terpenoid-quinone biosynthesis pathways were significantly enriched, and genes (IDI, E2.3.3.10, HMGCR atoB, and CoQ2) related to these two pathways were also significantly up-regulated. The CoQ2 genes encode puru-hydroxybenzoate:polyprenyltransferase, playing an important role in antroquinonol synthesis. The IDI, E2.3.3.10, HMGCR and atoB genes of the terpenoid backbone biosynthesis pathway might play an important role in the synthesis of the triquine-type sesquiterpene antrodin C. This investigation advances our understanding of how two different extractants of n-tetradecane and oleic acid affect the biosynthesis of metabolites in A. camphorata. It is beneficial to provide potential strategies for improving antrodin C and antroquinonol production by genetic means. © 2020 Society of Chemical Industry.

Comparative transcriptomics characterized the distinct biosynthetic abilities of terpenoid and paeoniflorin biosynthesis in herbaceous peony strains.

The herbaceous peony (Paeonia lactiflora Pall.) is a perennial flowering plant of the Paeoniaceae species that is widely cultivated for medical and ornamental uses. The monoterpene glucoside paeoniflorin and its derivatives are the active compounds of the P. lactiflora roots. However, the gene regulation pathways associated with monoterpene and paeoniflorin biosynthesis in P. lactiflora are still unclear. Here, we selected three genotypes of P. lactiflora with distinct morphologic features and chemical compositions that were a result of long-term reproductive isolation. We performed an RNA-sequencing experiment to profile the transcriptome changes of the shoots and roots. Using de novo assembly analysis, we identified 36,264 unigenes, including 521 genes responsible for encoding transcription factors. We also identified 28,925 unigenes that were differentially expressed in different organs and/or genotypes. Pathway enrichment analysis showed that the P. lactiflora unigenes were significantly overrepresented in several secondary metabolite biosynthesis pathways. We identified and profiled 33 genes responsible for encoding the enzymescontrolling the major catalytic reactions in the terpenoid backbone and in monoterpenoid biosynthesis. Our study identified the candidate genes in the terpenoid biosynthesis pathways, providing useful information for metabolic engineering of P. lactiflora intended for pharmaceutical uses and facilitating the development of strategies to improve marker-assist P. lactiflora in the future.

Phytochemical and comparative transcriptome analyses reveal different regulatory mechanisms in the terpenoid biosynthesis pathways between Matricaria recutita L. and Chamaemelum nobile L.

BackgroundMatricaria recutita (German chamomile) and Chamaemelum nobile (Roman chamomile) belong to the botanical family Asteraceae. These two herbs are not only morphologically distinguishable, but their secondary metabolites – especially the essential oils present in flowers are also different, especially the terpenoids. The aim of this project was to preliminarily identify regulatory mechanisms in the terpenoid biosynthetic pathways that differ between German and Roman chamomile by performing comparative transcriptomic and metabolomic analyses.ResultsWe determined the content of essential oils in disk florets and ray florets in these two chamomile species, and found that the terpenoid content in flowers of German chamomile is greater than that of Roman chamomile. In addition, a comparative RNA-seq analysis of German and Roman chamomile showed that 54% of genes shared > 75% sequence identity between the two species. In particular, more highly expressed DEGs (differentially expressed genes) and TF (transcription factor) genes, different regulation of CYPs (cytochrome P450 enzymes), and rapid evolution of downstream genes in the terpenoid biosynthetic pathway of German chamomile could be the main reasons to explain the differences in the types and levels of terpenoid compounds in these two species. In addition, a phylogenetic tree constructed from single copy genes showed that German chamomile and Roman chamomile are closely related to Chrysanthemum nankingense.ConclusionThis work provides the first insights into terpenoid biosynthesis in two species of chamomile. The candidate unigenes related to terpenoid biosynthesis will be important in molecular breeding approaches to modulate the essential oil composition of Matricaria recutita and Chamaemelum nobile.

Deciphering the high-quality genome sequence of coriander that causes controversial feelings.

SummaryCoriander (Coriandrum sativum L. 2n = 2x = 22), a plant from the Apiaceae family, also called cilantro or Chinese parsley, is a globally important crop used as vegetable, spice, fragrance and traditional medicine. Here, we report a high‐quality assembly and analysis of its genome sequence, anchored to 11 chromosomes, with total length of 2118.68 Mb and N50 scaffold length of 160.99 Mb. We found that two whole‐genome duplication events, respectively, dated to ~45–52 and ~54–61 million years ago, were shared by the Apiaceae family after their split from lettuce. Unbalanced gene loss and expression are observed between duplicated copies produced by these two events. Gene retention, expression, metabolomics and comparative genomic analyses of terpene synthase (TPS) gene family, involved in terpenoid biosynthesis pathway contributing to coriander’s special flavour, revealed that tandem duplication contributed to coriander TPS gene family expansion, especially compared to their carrot counterparts. Notably, a TPS gene highly expressed in all 4 tissues and 3 development stages studied is likely a major‐effect gene encoding linalool synthase and myrcene synthase. The present genome sequencing, transcriptome, metabolome and comparative genomic efforts provide valuable insights into the genome evolution and spice trait biology of Apiaceae and other related plants, and facilitated further research into important gene functions and crop improvement.

Differential Gene Profiling of the Heartwood Formation Process in Taiwania cryptomerioides Hayata Xylem Tissues.

Taiwania (Taiwania cryptomerioides) is an important tree species in Taiwan because of the excellent properties of its wood and fascinating color qualities of its heartwood (HW), as well as the bioactive compounds therein. However, limited information is available as to the HW formation of this species. The objective of this research is to analyze the differentially expressed genes (DEGs) during the HW formation process from specific Taiwania xylem tissues, and to obtain genes that might be closely associated with this process. The results indicated that our analyses have captured DEGs representative to the HW formation process of Taiwania. DEGs related to the terpenoid biosynthesis pathway were all up-regulated in the transition zone (TZ) to support the biosynthesis and accumulation of terpenoids. Many DEGs related to lignin biosynthesis, and two DEGs related to pinoresinol reductase (PrR)/pinoresinol lariciresinol reductase (PLR), were up-regulated in TZ. These DEGs together are likely involved in providing the precursors for the subsequent lignan biosynthesis. Several transcription factor-, nuclease-, and protease-encoding DEGs were also highly expressed in TZ, and these DEGs might be involved in the regulation of secondary metabolite biosynthesis and the autolysis of the cellular components of ray parenchyma cells in TZ. These results provide further insights into the process of HW formation in Taiwania.

Fusarium oxysporum infection activates the plastidial branch of the terpenoid biosynthesis pathway in flax, leading to increased ABA synthesis

Main conclusionUpregulation of the terpenoid pathway and increased ABA content in flax uponFusarium infection leads to activation of the early plant’s response (PR genes, cell wall remodeling, and redox status).Plants have developed a number of defense strategies against the adverse effects of fungi such as Fusarium oxysporum. One such defense is the production of antioxidant secondary metabolites, which fall into two main groups: the phenylpropanoids and the terpenoids. While functions and biosynthesis of phenylpropanoids have been extensively studied, very little is known about the genes controlling the terpenoid synthesis pathway in flax. They can serve as antioxidants, but are also substrates for a plethora of different compounds, including those of regulatory functions, like ABA. ABA’s function during pathogen attack remains obscure and often depends on the specific plant-pathogen interactions. In our study we showed that in flax the non-mevalonate pathway is strongly activated in the early hours of pathogen infection and that there is a redirection of metabolites towards ABA synthesis. The elevated synthesis of ABA correlates with flax resistance to F. oxysporum, thus we suggest ABA to be a positive regulator of the plant’s early response to the infection.

Dissection of Dynamic Transcriptome Landscape of Leaf, Bract, and Lupulin Gland in Hop (Humulus lupulus L.).

The hop plant (Humulus lupulus L.) produces several valuable secondary metabolites, such as prenylflavonoid, bitter acids, and essential oils. These compounds are biosynthesized in glandular trichomes (lupulin glands) endowed with pharmacological properties and widely implicated in the beer brewing industry. The present study is an attempt to generate exhaustive information of transcriptome dynamics and gene regulatory mechanisms involved in biosynthesis and regulation of these compounds, developmental changes including trichome development at three development stages, namely leaf, bract, and mature lupulin glands. Using high-throughput RNA-Seq technology, a total of 61.13, 50.01, and 20.18 Mb clean reads in the leaf, bract, and lupulin gland libraries, respectively, were obtained and assembled into 43,550 unigenes. The putative functions were assigned to 30,996 transcripts (71.17%) based on basic local alignment search tool similarity searches against public sequence databases, including GO, KEGG, NR, and COG families, which indicated that genes are principally involved in fundamental cellular and molecular functions, and biosynthesis of secondary metabolites. The expression levels of all unigenes were analyzed in leaf, bract, and lupulin glands tissues of hop. The expression profile of transcript encoding enzymes of BCAA metabolism, MEP, and shikimate pathway was most up-regulated in lupulin glands compared with leaves and bracts. Similarly, the expression levels of the transcription factors and structural genes that directly encode enzymes involved in xanthohumol, bitter acids, and terpenoids biosynthesis pathway were found to be significantly enhanced in lupulin glands, suggesting that production of these metabolites increases after the leaf development. In addition, numerous genes involved in primary metabolism, lipid metabolism, photosynthesis, generation of precursor metabolites/energy, protein modification, transporter activity, and cell wall component biogenesis were differentially regulated in three developmental stages, suggesting their involvement in the dynamics of the lupulin gland development. The identification of differentially regulated trichome-related genes provided a new foundation for molecular research on trichome development and differentiation in hop. In conclusion, the reported results provide directions for future functional genomics studies for genetic engineering or molecular breeding for augmentation of secondary metabolite content in hop.

Effects of ammonia-N exposure on the growth, metabolizing enzymes, and metabolome of Macrobrachium rosenbergii.

Ammonia nitrogen elevated is one of the commonest problem in the aquatic system, which caused a great threat to the survival and growth of prawn. However, little is know about the ammonia metabolism and detoxification strategy of prawn. In this study, the effects of ammonia-N (0, 0.108, 0.216, 0.324, or 0.54mgL-1) on growth and metabolizing enzymes in hepatopancreas of Macrobrachium rosenbergii, including glutamine synthetase (GS), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutamate dehydrogenase (GDH), were investigated. The metabolome of its muscle was also analyzed after exposure to ammonia-N (0, 0.108, 0.324, or 0.54mgL-1) for 20 days. The survival rate of M. rosenbergii decreased significantly after treatment with 0.54mgL-1 ammonia-N compared with that in the other groups. However, ammonia-N had no significant effect on the growth of the river prawn after exposure for 20 days. GS activity increased significantly after exposure to 0.108mgL-1 ammonia-N compared with the control and other ammonia-N-treated groups. Hepatopancreatic GDH activity was lower in the prawns treated with 0.216, 0.324, or 0.54mgL-1 ammonia-N than in the control by 34.70%, 38.80%, or 41.94%, respectively. Ammonia-N had no significant effect on hepatopancreatic AST or ALT activity. Urea nitrogen was higher in the prawns treated with 0.216mgL-1 ammonia-N than in the control or those treated with 0.54mgL-1 ammonia-N. Ammonia-N had significant effects on the lipid, carbohydrate. and protein metabolism of M. rosenbergii, including purine metabolism, amino sugar and nucleotide sugar metabolism, α-linolenic acid metabolism, arginine and proline metabolism, glutathione metabolism, and phosphonate and phosphate metabolism, and on the terpenoid biosynthesis, lysine degradation, and lysine biosynthesis pathways. High concentrations of ammonia-N stress increased the content of glutamate and arginine, which may participate in the urea cycle, which synthesizes glutamine or urea to eliminate ammonia toxicity.

Analysis of terpenoid biosynthesis pathways in German chamomile (Matricaria recutita) and Roman chamomile (Chamaemelum nobile) based on co-expression networks

German chamomile and Roman chamomile are the two most widely known chamomile species due to the medicinal properties of volatile compounds from their flowers. We determined the volatile compound content of different organs of these two chamomiles, and found that main volatile compounds in German chamomile were terpenoids and those in Roman chamomile were esters. Furthermore, 24 tissues from two chamomiles were sequenced and analyzed by gene co-expression network. The results showed higher terpene synthase expression levels and more modules correlated with sesquiterpenoids in German chamomile, which may explain its high sesquiterpenoid content. In both chamomiles, unigenes in volatile compound-correlated modules were significantly enriched in pathways related to plant-pathogen interactions and circadian rhythm, demonstrating that volatile compounds of chamomiles are influenced by these factors. There were ten times more unigenes related to plant-pathogen interactions in German chamomile than in Roman chamomile, which indicates German chamomile has higher resistance to pathogens.

Transcriptome analysis of the palmarosa Cymbopogon martinii inflorescence with emphasis on genes involved in essential oil biosynthesis

Cymbopogon martinii belongs to the family Poaceae and provides an aromatic essential oil of large applications in fragrance, perfumery, cosmetics and pharmaceuticals. The essential oil from this genus has two dominating monoterpenes, geranyl acetate and geraniol. Owing to the tremendous applications of the essential oil from this genus, knowledge of the biochemical and molecular regulatory processes and expressions of genes involved in terpenoid biosynthesis pathway, is highly desirable to improve the quality and yield of essential oil. We performed high-throughput transcriptome sequencing of C. martinii for identification of the genes related to terpenoids biosynthesis using Illumina HiSeq-2000 platform. In the present study, approximately 286,309,78 reads of high quality were generated. De novo assembly yielded 96,470 unigenes (≥200 bp) and 84,168 (≥500 bp). Functional annotation of total 96,470 (56%) unigenes was done. More than 4500 enriched unigenes were assigned to 367 KEGG pathways among various categories, i.e. protein processing, cellular processes, metabolism, steroid biosynthesis, and others. 913 unigenes in the metabolism category were assigned to 287 metabolic pathways linked with secondary metabolism. To explore the putative functions of six genes encoding alcohol dehydrogenase, geranyldiphosphate synthase, fernasyldiphosphate synthase, aldehyde dehydrogenase, aldo-keto reductase and alcohol acyltransferase expression patterns were studies using qRT-PCR. We have also identified 60 transcription factors in C. martini transcriptome using Plant Transcription factor database. To the best of our knowledge, this is the first-ever report on the C. martinii inflorescence transcriptome which will facilitate the understanding of the molecular mechanisms of essential oil biosynthesis in the genus Cymbopogon and related plant families.

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.

Transcriptomic analysis of postharvest toon buds and key enzymes involved in terpenoid biosynthesis during cold storage

Toon buds have been used in China as an important woody vegetable for its unique flavor. However, the changes of volatile terpenoids related to flavor and their biosynthesis in postharvest toon buds after cold storage remain largely unknown. Therefore, a transcriptomic database must be constructed to analyze the molecular mechanism of terpenoid biosynthesis in toon buds during cold storage. The chemical profiles of volatile terpenoids over postharvest storage periods were comparatively analyzed by gas chromatography–mass spectrometry. Results showed that the total content of volatile terpenoids and their oxidates, except for monoterpenes and a small portion of sesquiterpenes, increased significantly after cold storage. The transcriptome database derived from different cold storage times was established using BGIseq500 technology. Approximately 6.5 g of clean nucleotides were obtained and de novo assembled into 152 127 non-redundant unigenes, approximately 72.36% of which could be aligned to public databases. Many candidate genes involved in terpenoid biosynthesis were identified. Furthermore, 29 513 unigenes were demonstrated to be differentially expressed under different cold storage times. These differentially expressed genes were functionally annotated by gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. The expression of 20 putative unigenes involved in terpenoid pathways was confirmed by qRT-PCR, the results of which were consistent with the RNA-seq data. These genes with different expression patterns correlated well with the changes in the volatile terpenoids in the toon buds during low-temperature storage. In summary, our results showed that the high expression levels of terpenoid backbone biosynthesis pathways contributed to sesquiterpene biosynthesis after cold storage, which led to high sesquiterpene accumulation in toon buds during low-temperature storage.

Agrobacterium rhizogenes-mediated RNAi of Tripterygium wilfordii and application for functional study of terpenoid biosynthesis pathway genes

Tripterygium wilfordii is an important medical plants which is rich in several valuable bioactive terpenoids, such as triptolide and wilforine. Developing a fast and efficient transformation method is imperative for exploring these metabolites biosynthesis pathway by using RNA interference (RNAi) technology. In the present study, an efficient transformation system using Agrobacterium rhizogenes was established by optimizing four parameters including vacuum infection time, pre-culture duration, appropriate concentration of Agrobacterium, and co-cultivation duration. The transformation efficiency could reach to 62.22% by using leaf sections pre-cultured 5 days as explants, vacuum infection with Agrobacterium at optical density (OD600) of 0.6 for 3 min and co-cultivation for 3 d. The T. wilfordii 3-hydroxy-3-methylglutaryl- coenzyme A reductase (TwHMGR) RNAi hairy root was induced to validate the applicability of this system. The identification of transgenic hairy root was monitored by fluorescence microscopy and verified by PCR analysis. Real-time quantitative PCR analysis showed that the expression of TwHMGR was significantly decreased in transgenic hairy roots compared with control lines. The detection of metabolites showed that the silence of TwHMGR could highly decrease the content of triptolide and wilforine. These results indicated that this A. rhizogenes-mediated RNAi system is an effective protocol for characterizing the function of genes involved in the terpenoid biosynthetic pathway in Tripterygium wilfordii.

Expanding the Isoprenoid Building Block Repertoire with an IPP Methyltransferase from Streptomyces monomycini

Many synthetic biology approaches aim at expanding the product diversity of enzymes or whole biosynthetic pathways. However, the chemical structure space of natural product forming routes is often restricted by the limited cellular availability of different starting intermediates. Although the terpene biosynthesis pathways are highly modular, their starting intermediates are almost exclusively the C5 units IPP and DMAPP. To amplify the possibilities of terpene biosynthesis through the modification of its building blocks, we identified and characterized a SAM-dependent methyltransferase converting IPP into a variety of C6 and C7 prenyl pyrophosphates. Heterologous expression in Escherichia coli not only extended the intracellular prenyl pyrophosphate spectrum with mono- or dimethylated IPP and DMAPP, but also enabled the biosynthesis of C11, C12, C16, and C17 prenyl pyrophosphates. We furthermore demonstrated the general high promiscuity of terpenoid biosynthesis pathways toward uncommon building blocks by the E. coli-based production of polymethylated C41, C42, and C43 carotenoids. Integration of the IPP methyltransferase in terpene synthesis pathways enables an expansion of the terpenoid structure space beyond the borders predetermined by the isoprene rule which indicates a restricted synthesis by condensation of C5 units.

Draft genome of Santalum album L. provides genomic resources for accelerated trait improvement

Sandalwood (Santalum album L.) is a valuable commodity in the international trade due to its use in fragrance and essential oil industries. In the present study, a draft genome of S. album representing the natural population from Kerala, India, was sequenced and assembled into 74,900 major scaffolds with N50 of 12,068 bp and the estimated genome size was 286 Mb. A total of 37,500 genes were predicted including 30 genes from terpene synthase gene family. Repetitive sequence was mined and spanned 12.52% of the sandalwood genome. Domain analysis of predicted proteins revealed that transcription factors and kinases were the major category of proteins followed by ARM repeat proteins and WD domain proteins. The draft genome encoded for 574 miRNAs belonging to 23 families including two miRNAs associated with terpenoid biosynthesis pathway. Further, the consensus chloroplast genome of 147.25 kb size was reconstructed and phylogenetic analysis with 40 plastid genes grouped members of Santalales with Asterids in accordance with its taxonomic status recommended in APG III and APG IV. Variant calling was conducted against the published genome and a total of 1,191,838 (0.42%) SNPs and 98,312 (0.034%) InDels were predicted in the present genome. The draft genome presented in this study has provided additional genomic resource for S. album for subsequent research in population diversity estimation and accelerated trait breeding in this species.

Proteomic and metabolomic analyses provide insight into the off-flavour of fruits from citrus trees infected with \u2018Candidatus Liberibacter asiaticus\u2019

Orange fruit from trees infected by ‘Candidatus Liberibacter asiaticus’ (CaLas) often do not look fully mature and exhibit off-flavours described as bitter, harsh, and metallic rather than juicy and fruity. Although previous studies have been carried out to understand the effect of CaLas on the flavour of orange juice using metabolomic methods, the mechanisms leading to the off-flavour that occurs in Huanglongbing (HLB)-symptomatic fruit are not well understood. In this study, fruits were collected from symptomatic and healthy Valencia sweet orange (Citrus sinensis) trees grafted on Swingle (C. paradisi X Poncirus trifoliata) rootstock. Isobaric tags for relative and absolute quantification (iTRAQ) and gas chromatography-mass spectrometry (GC-MS) were used to measure the proteins, sugars, organic acids, amino acids, and volatile terpenoids. The results showed that most of the differentially expressed proteins involved in glycolysis, the tricarboxylic acid (TCA) cycle and amino-acid biosynthesis were degraded, and terpenoid metabolism was significantly downregulated in the symptomatic fruit. Valencene, limonene, 3-carene, linalool, myrcene, and α-terpineol levels were significantly lower in fruit from CaLas-infected trees than from healthy trees. Similar phenomena were observed for sucrose and glucose. Our study indicated that off-flavour of symptomatic fruit was associated with a reduction in the levels of terpenoid products and the downregulation of proteins in glycolysis, the TCA cycle, and the terpenoid biosynthesis pathway.

Chronic exposure of bumblebees to neonicotinoid imidacloprid suppresses the entire mevalonate pathway and fatty acid synthesis

Determining the side effects of pesticides on pollinators is an important topic due to the increasing loss of pollinators. We aimed to determine the effects of chronic sublethal exposure of the neonicotinoid pesticide imidacloprid on the bumblebee Bombus terrestris under laboratory conditions. The analytical standard of imidacloprid in sugar solution was used for the treatment. Verification of pesticides using UHPLC-QqQ-MS/MS in the experimental bumblebees showed the presence of only two compounds, imidacloprid and imidacloprid-olefin, which were found in quantities of 0.57 ± 0.22 and 1.95 ± 0.43 ng/g, respectively. Thus, the level of the dangerous metabolite imidacloprid-olefin was 3.4-fold higher than that of imidacloprid. Label-free nanoLC-MS/MS quantitative proteomics of bumblebee heads enabled quantitative comparison of 2883 proteins, and 206 proteins were significantly influenced by the imidacloprid treatment. The next analysis revealed that the highly downregulated markers are members of the terpenoid backbone biosynthesis pathway (KEGG: bter00900) and that imidacloprid treatment suppressed the entire mevalonate pathway, fatty acid synthesis and associated markers. The proteomics results indicate that the consequences of imidacloprid treatment are complex, and the marker changes are associated with metabolic and neurological diseases and olfaction disruption. This study provides important markers and can help to explain the widely held assumptions from biological observations. SignificanceThe major finding is that all markers of the mevalonate pathway were substantially downregulated due to the chronic imidacloprid exposure. The disbalance of mevalonate pathway has many important consequences. We suggest the mechanism associated with the novel toxicogenic effect of imidacloprid. The results are helpful to explain that imidacloprid impairs the cognitive functions and possesses the delayed and time cumulative effect.

Transcriptome analysis provides insight into prickle development and its link to defense and secondary metabolism in Solanum viarumDunal

Prickles are epidermal outgrowth found on the aerial surface of several terrestrial plants. Microscopic studies on prickles of S. viarum Dunal indicated a crucial role of glandular trichomes (GTs) in their development. A spontaneously obtained prickleless mutant showed normal epidermal GTs, but its downstream developmental process to prickle was perturbed. Thus, prickleless mutant offers an ideal opportunity to unveil molecular regulators working downstream to GTs in the prickle formation. Differential transcriptome analysis of epidermis of prickly and prickleless mutant revealed that expression of several defense regulators like ethylene, salicylic acid, PR-proteins, etc. were significantly down-regulated in prickleless mutant, provide an important link between defense and prickle development. It was also noteworthy that the expression of few essential development related TFs like MADS-box, R2R3-MYB, REM, DRL1, were also down-regulated in the stem, petioles, and leaves of prickleless mutant indicating their potential role in prickle development. Interestingly, the gene expression of terpenoid, steroid, flavonoid, glucosinolate, and lignin biosynthesis pathways were up-regulated in prickleless mutant. The biochemical and qRT-PCR analysis also confirmed metabolite elevation. These results indicated that the loss of prickle was compensated by elevated secondary metabolism in the prickleless mutant which played important role in the biotic and abiotic stress management.