RESUMO
The biochemical profile of coffee beans translates directly into quality traits, nutraceutical and health promoting properties of the coffee beverage. Ent-kaurene is the ubiquitous precursor for gibberellin biosynthesis in plants, but it also serves as an intermediate in specialized (i.e., secondary) diterpenoid metabolism that leads to a diversity of more than 1,000 different metabolites. Nutraceutical effects on human health attributed to diterpenes include antioxidant, anticarcinogenic, and anti-inflammatory properties. Cafestol (CAF) and kahweol (KAH) are two diterpenes found exclusively in the Coffea genus. Our objective was to identify and functionally characterize genes involved in the central step of ent-kaurene production. We identified 17 putative terpene synthase genes in the transcriptome of Coffea arabica. Two ent-copalyl diphosphate synthase (CaCPS) and three kaurene synthase (CaKS) were selected and manually annotated. Transcript expression profiles of CaCPS1 and CaKS3 best matched the CAF and KAH metabolite profiles in different tissues. CaCPS1 and CaKS3 proteins were heterologously expressed and functionally characterized. CaCPS1 catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by CaKS3. Knowledge about the central steps of diterpene formation in coffee provides a foundation for future characterization of the subsequent enzymes involved in CAF and KAH biosynthesis.
Assuntos
Alquil e Aril Transferases , Coffea , Diterpenos do Tipo Caurano , Diterpenos , Humanos , Coffea/genética , Coffea/metabolismo , Diterpenos/química , Diterpenos do Tipo Caurano/metabolismo , Alquil e Aril Transferases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Stone cells are a specialized, highly lignified cell type found in both angiosperms and gymnosperms. In conifers, abundance of stone cells in the cortex provides a robust constitutive physical defense against stem feeding insects. Stone cells are a major insect-resistance trait in Sitka spruce (Picea sitchensis), occurring in dense clusters in apical shoots of trees resistant (R) to spruce weevil (Pissodes strobi) but being rare in susceptible (S) trees. To learn more about molecular mechanisms of stone cell formation in conifers, we used laser microdissection and RNA sequencing to develop cell-type-specific transcriptomes of developing stone cells from R and S trees. Using light, immunohistochemical, and fluorescence microscopy, we also visualized the deposition of cellulose, xylan, and lignin associated with stone cell development. A total of 1293 genes were differentially expressed at higher levels in developing stone cells relative to cortical parenchyma. Genes with potential roles in stone cell secondary cell wall formation (SCW) were identified and their expression evaluated over a time course of stone cell formation in R and S trees. The expression of several transcriptional regulators was associated with stone cell formation, including a NAC family transcription factor and several genes annotated as MYB transcription factors with known roles in SCW formation.
Assuntos
Picea , Gorgulhos , Animais , Transcriptoma/genética , Picea/genética , Fenótipo , Insetos , Regulação da Expressão Gênica de PlantasRESUMO
The highly diverse insect family of true weevils, Curculionidae, includes many agricultural and forest pests. Pissodes strobi, commonly known as the spruce weevil or white pine weevil, is a major pest of spruce and pine forests in North America. Pissodes strobi larvae feed on the apical shoots of young trees, causing stunted growth and can destroy regenerating spruce or pine forests. Here, we describe the nuclear and mitochondrial Pissodes strobi genomes and their annotations, as well as the genome of an apparent Wolbachia endosymbiont. We report a substantial expansion of the weevil nuclear genome, relative to other Curculionidae species, possibly driven by an abundance of class II DNA transposons. The endosymbiont observed belongs to a group (supergroup A) of Wolbachia species that generally form parasitic relationships with their arthropod host.
Assuntos
Picea , Gorgulhos , Wolbachia , Animais , Florestas , Insetos , Picea/genética , Gorgulhos/genética , Wolbachia/genéticaRESUMO
Spruce weevil (Pissodes strobi) is a significant pest of regenerating spruce (Picea) and pine (Pinus) forests in North America. Weevil larvae feed in the bark, phloem, cambium, and outer xylem of apical shoots, causing stunted growth or mortality of young trees. We identified and characterized constitutive and weevil-induced patterns of Sitka spruce (Picea sitchensis) transcriptomes in weevil-resistant (R) and susceptible (S) trees using RNA sequencing (RNA-seq) and differential expression (DE) analyses. We developed a statistical model for the analysis of RNA-seq data from treatment experiments with a 2 × 3 factorial design to differentiate insect-induced responses from the effects of mechanical damage. Across the different comparisons, we identified two major transcriptome contrasts: A large set of genes that was constitutively DE between R and S trees, and another set of genes that was DE in weevil-induced S-trees. The constitutive transcriptome unique to R trees appeared to be attuned to defense, while the constitutive transcriptome unique to S trees was enriched for growth-related transcripts. Notably, a set of transcripts annotated as "fungal" was detected consistently in the transcriptomes. Fungal transcripts were identified as DE in the comparison of R and S trees and in the weevil-affected DE transcriptome of S trees, suggesting a potential microbiome role in this conifer-insect interaction.
RESUMO
Specialized metabolites constitute key layers of immunity that underlie disease resistance in crops; however, challenges in resolving pathways limit our understanding of the functions and applications of these metabolites. In maize (Zea mays), the inducible accumulation of acidic terpenoids is increasingly considered to be a defence mechanism that contributes to disease resistance. Here, to understand maize antibiotic biosynthesis, we integrated association mapping, pan-genome multi-omic correlations, enzyme structure-function studies and targeted mutagenesis. We define ten genes in three zealexin (Zx) gene clusters that encode four sesquiterpene synthases and six cytochrome P450 proteins that collectively drive the production of diverse antibiotic cocktails. Quadruple mutants in which the ability to produce zealexins (ZXs) is blocked exhibit a broad-spectrum loss of disease resistance. Genetic redundancies ensuring pathway resiliency to single null mutations are combined with enzyme substrate promiscuity, creating a biosynthetic hourglass pathway that uses diverse substrates and in vivo combinatorial chemistry to yield complex antibiotic blends. The elucidated genetic basis of biochemical phenotypes that underlie disease resistance demonstrates a predominant maize defence pathway and informs innovative strategies for transferring chemical immunity between crops.
Assuntos
Antibacterianos/biossíntese , Resistência à Doença/genética , Imunidade Inata/genética , Redes e Vias Metabólicas/genética , Zea mays/genética , Resistência à Doença/fisiologia , Perfilação da Expressão Gênica , Genes de Plantas/genética , Genes de Plantas/fisiologia , Metabolômica , Família Multigênica/genética , Família Multigênica/fisiologia , Proteômica , Zea mays/imunologia , Zea mays/metabolismo , Zea mays/microbiologiaRESUMO
Surveys of microbial systems indicate that in many situations taxonomy and function may constitute largely independent ('decoupled') axes of variation. However, this decoupling is rarely explicitly tested experimentally, partly because it is hard to directly induce taxonomic variation without affecting functional composition. Here we experimentally evaluate this paradigm using microcosms resembling lake sediments and subjected to two different levels of salinity (0 and 19) and otherwise similar environmental conditions. We used DNA sequencing for taxonomic and functional profiling of bacteria and archaea and physicochemical measurements to monitor metabolic function, over 13 months. We found that the taxonomic composition of the saline systems gradually but strongly diverged from the fresh systems. In contrast, the metabolic composition (in terms of proportions of various genes) remained nearly identical across treatments and over time. Oxygen consumption rates and methane concentrations were substantially lower in the saline treatment, however, their similarity either increased (for oxygen) or did not change significantly (for methane) between the first and last sampling time, indicating that the lower metabolic activity in the saline treatments was directly and immediately caused by salinity rather than the gradual taxonomic divergence. Our experiment demonstrates that strong taxonomic shifts need not directly affect metabolic rates.
Assuntos
Archaea/classificação , Archaea/metabolismo , Bactérias/classificação , Bactérias/metabolismo , Sedimentos Geológicos/microbiologia , Archaea/genética , Archaea/isolamento & purificação , Bactérias/genética , Bactérias/isolamento & purificação , Sedimentos Geológicos/química , Lagos/química , Lagos/microbiologia , Metano/metabolismo , Microbiota , Oxigênio/metabolismo , Filogenia , SalinidadeRESUMO
Cannabis (Cannabis sativa) resin is the foundation of a multibillion dollar medicinal and recreational plant bioproducts industry. Major components of the cannabis resin are the cannabinoids and terpenes. Variations of cannabis terpene profiles contribute much to the different flavor and fragrance phenotypes that affect consumer preferences. A major problem in the cannabis industry is the lack of proper metabolic characterization of many of the existing cultivars, combined with sometimes incorrect cultivar labeling. We characterized foliar terpene profiles of plants grown from 32 seed sources and found large variation both within and between sets of plants labeled as the same cultivar. We selected five plants representing different cultivars with contrasting terpene profiles for clonal propagation, floral metabolite profiling, and trichome-specific transcriptome sequencing. Sequence analysis of these five cultivars and the reference genome of cv Purple Kush revealed a total of 33 different cannabis terpene synthase (CsTPS) genes, as well as variations of the CsTPS gene family and differential expression of terpenoid and cannabinoid pathway genes between cultivars. Our annotation of the cv Purple Kush reference genome identified 19 complete CsTPS gene models, and tandem arrays of isoprenoid and cannabinoid biosynthetic genes. An updated phylogeny of the CsTPS gene family showed three cannabis-specific clades, including a clade of sesquiterpene synthases within the TPS-b subfamily that typically contains mostly monoterpene synthases. The CsTPSs described and functionally characterized here include 13 that had not been previously characterized and that collectively explain a diverse range of cannabis terpenes.
Assuntos
Alquil e Aril Transferases/metabolismo , Cannabis/enzimologia , Cannabis/metabolismo , Terpenos/metabolismo , Alquil e Aril Transferases/classificação , Alquil e Aril Transferases/genética , Cannabis/genética , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Duplication and divergence of primary pathway genes underlie the evolution of plant specialized metabolism; however, mechanisms partitioning parallel hormone and defence pathways are often speculative. For example, the primary pathway intermediate ent-kaurene is essential for gibberellin biosynthesis and is also a proposed precursor for maize antibiotics. By integrating transcriptional coregulation patterns, genome-wide association studies, combinatorial enzyme assays, proteomics and targeted mutant analyses, we show that maize kauralexin biosynthesis proceeds via the positional isomer ent-isokaurene formed by a diterpene synthase pair recruited from gibberellin metabolism. The oxygenation and subsequent desaturation of ent-isokaurene by three promiscuous cytochrome P450s and a new steroid 5α reductase indirectly yields predominant ent-kaurene-associated antibiotics required for Fusarium stalk rot resistance. The divergence and differential expression of pathway branches derived from multiple duplicated hormone-metabolic genes minimizes dysregulation of primary metabolism via the circuitous biosynthesis of ent-kaurene-related antibiotics without the production of growth hormone precursors during defence.
Assuntos
Diterpenos do Tipo Caurano/metabolismo , Genes de Plantas , Reguladores de Crescimento de Plantas/genética , Zea mays/genética , Ascomicetos , Sistema Enzimático do Citocromo P-450/metabolismo , Resistência à Doença/genética , Estudo de Associação Genômica Ampla , Giberelinas/metabolismo , Redes e Vias Metabólicas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Zea mays/imunologia , Zea mays/metabolismo , Zea mays/microbiologiaRESUMO
Type 2 diabetes (T2D) affects over 320 million people worldwide. Healthy lifestyles, improved drugs and effective nutraceuticals are different components of a response against the growing T2D epidemic. The specialized metabolite montbretin A (MbA) is being developed for treatment of T2D and obesity due to its unique pharmacological activity as a highly effective and selective inhibitor of the human pancreatic α-amylase. MbA is an acylated flavonol glycoside found in small amounts in montbretia (Crocosmia × crocosmiiflora) corms. MbA cannot be obtained in sufficient quantities for drug development from its natural source or by chemical synthesis. To overcome these limitations through metabolic engineering, we are investigating the genes and enzymes of MbA biosynthesis. We previously reported the first three steps of MbA biosynthesis from myricetin to myricetin 3-O-(6'-O-caffeoyl)-glucosyl rhamnoside (mini-MbA). Here, we describe the sequence of reactions from mini-MbA to MbA, and the discovery and characterization of the gene and enzyme responsible for the glucosylation of mini-MbA. The UDP-dependent glucosyltransferase CcUGT3 (UGT703E1) catalyzes the 1,2-glucosylation of mini-MbA to produce myricetin 3-O-(glucosyl-6'-O-caffeoyl)-glucosyl rhamnoside. Co-expression of CcUGT3 with genes for myricetin and mini-MbA biosynthesis in Nicotiana benthamiana validated its biological function and expanded the set of genes available for metabolic engineering of MbA.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Flavonas/biossíntese , Glucosiltransferases/metabolismo , Hipoglicemiantes/metabolismo , Engenharia Metabólica/métodos , Trissacarídeos/biossíntese , Ácidos Cafeicos/química , Ácidos Cafeicos/metabolismo , Flavonas/química , Flavonas/farmacologia , Flavonas/uso terapêutico , Flavonoides/química , Flavonoides/metabolismo , Flavonóis/química , Flavonóis/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Glucose/química , Glucose/metabolismo , Glicosídeos/química , Glicosídeos/metabolismo , Glicosilação , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Iridaceae/química , Iridaceae/enzimologia , Filogenia , Proteínas de Plantas/metabolismo , Caules de Planta/química , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , Ramnose/química , Ramnose/metabolismo , Metabolismo Secundário , Biologia Sintética/métodos , Nicotiana/metabolismo , Transcriptoma/genética , Trissacarídeos/química , Trissacarídeos/farmacologia , Trissacarídeos/uso terapêutico , Xilose/química , Xilose/metabolismoRESUMO
Conifers have evolved complex oleoresin terpene defenses against herbivores and pathogens. In co-evolved bark beetles, conifer terpenes also serve chemo-ecological functions as pheromone precursors, chemical barcodes for host identification, or nutrients for insect-associated microbiomes. We highlight the genomic, molecular and biochemical underpinnings of the large chemical space of conifer oleoresin terpenes and volatiles. Conifer terpenes are predominantly the products of the conifer terpene synthase (TPS) gene family. Terpene diversity is increased by cytochromes P450 of the CYP720B class. Many conifer TPS are multiproduct enzymes. Multisubstrate CYP720B enzymes catalyse multistep oxidations. We summarise known terpenoid gene functions in various different conifer species with reference to the annotated terpenoid gene space in a spruce genome. Overall, biosynthesis of terpene diversity in conifers is achieved through a system of biochemical radiation and metabolic grids. Expression of TPS and CYP720B genes can be specific to individual cell types of constitutive or traumatic resin duct systems. Induced terpenoid transcriptomes in resin duct cells lead to dynamic changes of terpene composition and quantity to fend off herbivores and pathogens. While terpenoid defenses have contributed much to the evolutionary success of conifers, under new conditions of climate change, these defences may become inconsequential against range-expanding forest pests.
Assuntos
Alquil e Aril Transferases/metabolismo , Herbivoria , Extratos Vegetais/metabolismo , Traqueófitas/química , Traqueófitas/fisiologia , Alquil e Aril Transferases/genética , Animais , Mudança Climática , Besouros , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Filogenia , Extratos Vegetais/química , Terpenos/metabolismoRESUMO
Insect pests are part of natural forest ecosystems contributing to forest rejuvenation but can also cause ecological disturbance and economic losses that are expected to increase with climate change. The white pine or spruce weevil (Pissodes strobi) is a pest of conifer forests in North America. Weevil-host interactions with various spruce (Picea) species have been explored as a genomic and molecular reference system for conifer defence against insects. Interactions occur in two major phases of the insect life cycle. In the exophase, adult weevils are free-moving and display behaviour of host selection for oviposition that is affected by host traits. In the endophase, insects live within the host where mobility and development from eggs to young adults are affected by a complex system of host defences. Genetic resistance exists in several spruce species and involves synergism of constitutive and induced chemical and physical defences that comprise the conifer defence syndrome. Here, we review conifer defences that disrupt the weevil life cycle and mechanisms by which trees resist weevil attack. We highlight molecular and genomic aspects and a possible role for the weevil microbiome. Knowledge of this conifer defence system is supporting forest health strategies and tree breeding for insect resistance.
Assuntos
Genômica , Interações Hospedeiro-Parasita/imunologia , Insetos , Doenças das Plantas/imunologia , Traqueófitas/imunologia , Animais , Sistema Enzimático do Citocromo P-450 , Microbiota , Oviposição , Fenótipo , Pinus , Doenças das Plantas/parasitologia , Proteoma , Terpenos/farmacologia , Transcriptoma , GorgulhosRESUMO
Cannabis sativa (cannabis) produces a resin that is valued for its psychoactive and medicinal properties. Despite being the foundation of a multi-billion dollar global industry, scientific knowledge and research on cannabis is lagging behind compared to other high-value crops. This is largely due to legal restrictions that have prevented many researchers from studying cannabis, its products, and their effects in humans. Cannabis resin contains hundreds of different terpene and cannabinoid metabolites. Many of these metabolites have not been conclusively identified. Our understanding of the genomic and biosynthetic systems of these metabolites in cannabis, and the factors that affect their variability, is rudimentary. As a consequence, there is concern about lack of consistency with regard to the terpene and cannabinoid composition of different cannabis 'strains'. Likewise, claims of some of the medicinal properties attributed to cannabis metabolites would benefit from thorough scientific validation.
Assuntos
Cannabis/metabolismo , Terpenos/metabolismo , Canabinoides/biossíntese , Cannabis/genética , Variação Genética , Genoma de Planta , Genótipo , Técnicas de Genotipagem , Redes e Vias MetabólicasRESUMO
Conifers depend on complex defense systems against herbivores. Stone cells (SC) and oleoresin are physical and chemical defenses of Sitka spruce that have been separately studied in previous work. Weevil oviposit at the tip of the previous year's apical shoot (PYAS). We investigated interactions between weevil larvae and trees in controlled oviposition experiments with resistant (R) and susceptible (S) Sitka spruce. R trees have an abundance of SC in the PYAS cortex. SC are mostly absent in S trees. R trees and S trees also differ in the composition of oleoresin terpenes. Transcriptomes of R and S trees revealed differences in long-term weevil-induced responses. Performance of larvae was significantly reduced on R trees compared with S trees under experimental conditions that mimicked natural oviposition behavior at apical shoot tips and may be attributed to the effects of SC. In oviposition experiments designed for larvae to feed below the area of highest SC abundance, larvae showed an unusual feeding behavior and oleoresin appeared to function as the major defense. The results support a role for both SC and oleoresin terpenes and possible synergies between these traits in the defense syndrome of weevil-resistant Sitka spruce.
Assuntos
Extratos Vegetais/metabolismo , Terpenos/metabolismo , Traqueófitas/citologia , Traqueófitas/metabolismo , Animais , Comportamento Alimentar , Trato Gastrointestinal/fisiologia , Regulação da Expressão Gênica de Plantas , Larva/fisiologia , Oviposição , Traqueófitas/genética , Transcriptoma/genética , Árvores/genética , Árvores/fisiologia , Gorgulhos/fisiologiaRESUMO
Plant genomes encode isopentenyl phosphate kinases (IPKs) that reactivate isopentenyl phosphate (IP) via ATP-dependent phosphorylation, forming the primary metabolite isopentenyl diphosphate (IPP) used generally for isoprenoid/terpenoid biosynthesis. Therefore, the existence of IPKs in plants raises unanswered questions concerning the origin and regulatory roles of IP in plant terpenoid metabolism. Here, we provide genetic and biochemical evidence showing that IP forms during specific dephosphorylation of IPP catalysed by a subset of Nudix superfamily hydrolases. Increasing metabolically available IP by overexpression of a bacterial phosphomevalonate decarboxylase (MPD) in Nicotiana tabacum resulted in significant enhancement in both monoterpene and sesquiterpene production. These results indicate that perturbing IP metabolism results in measurable changes in terpene products derived from both the methylerythritol phosphate (MEP) and mevalonate (MVA) pathways. Moreover, the unpredicted peroxisomal localization of bacterial MPD led us to discover that the step catalysed by phosphomevalonate kinase (PMK) imposes a hidden constraint on flux through the classical MVA pathway. These complementary findings fundamentally alter conventional views of metabolic regulation of terpenoid metabolism in plants and provide new metabolic engineering targets for the production of high-value terpenes in plants.
Assuntos
Hemiterpenos/metabolismo , Compostos Organofosforados/metabolismo , Terpenos/metabolismo , Arabidopsis/metabolismo , Redes e Vias Metabólicas , Fosfotransferases/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Nicotiana/metabolismoRESUMO
Terpenoids are a major component of maize (Zea mays) chemical defenses that mediate responses to herbivores, pathogens, and other environmental challenges. Here, we describe the biosynthesis and elicited production of a class of maize diterpenoids, named dolabralexins. Dolabralexin biosynthesis involves the sequential activity of two diterpene synthases, ENT-COPALYL DIPHOSPHATE SYNTHASE (ZmAN2) and KAURENE SYNTHASE-LIKE4 (ZmKSL4). Together, ZmAN2 and ZmKSL4 form the diterpene hydrocarbon dolabradiene. In addition, we biochemically characterized a cytochrome P450 monooxygenase, ZmCYP71Z16, which catalyzes the oxygenation of dolabradiene to yield the epoxides 15,16-epoxydolabrene (epoxydolabrene) and 3ß-hydroxy-15,16-epoxydolabrene (epoxydolabranol). The absence of dolabradiene and epoxydolabranol in Zman2 mutants under elicited conditions confirmed the in vivo biosynthetic requirement of ZmAN2. Combined mass spectrometry and NMR experiments demonstrated that much of the epoxydolabranol is further converted into 3ß,15,16-trihydroxydolabrene (trihydroxydolabrene). Metabolite profiling of field-grown maize root tissues indicated that dolabralexin biosynthesis is widespread across common maize cultivars, with trihydroxydolabrene as the predominant diterpenoid. Oxidative stress induced dolabralexin accumulation and transcript expression of ZmAN2 and ZmKSL4 in root tissues, and metabolite and transcript accumulation were up-regulated in response to elicitation with the fungal pathogens Fusarium verticillioides and Fusarium graminearum Consistently, epoxydolabranol significantly inhibited the growth of both pathogens in vitro at 10 µg mL-1, while trihydroxydolabrene-mediated inhibition was specific to Fverticillioides These findings suggest that dolabralexins have defense-related roles in maize stress interactions and expand the known chemical space of diterpenoid defenses as genetic targets for understanding and ultimately improving maize resilience.
Assuntos
Vias Biossintéticas , Diterpenos/metabolismo , Estresse Fisiológico , Zea mays/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Resistência à Doença/genética , Diterpenos/química , Fusarium/classificação , Fusarium/fisiologia , Regulação da Expressão Gênica de Plantas , Estrutura Molecular , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especificidade da Espécie , Zea mays/genética , Zea mays/microbiologiaRESUMO
The development of heartwood (HW) and the associated accumulation of secondary metabolites, which are also known as 'specialized metabolites' or 'extractives', is an important feature of tree biology. Heartwood development can affect tree health with broader implications for forest health. Heartwood development also defines a variety of wood quality traits that are important in the forest industry such as durability and colour of wood products. In the bioproducts industry, HW provides a source of high-value small molecules such as fragrances and antimicrobials. The HW properties of decay resistance in living trees, durability and colour of wood products, and small molecule bioproducts are largely defined by secondary metabolites, the biosynthesis of which appears to be activated during the onset of HW formation. Traditionally, it is thought that HW formation involves a spike in the activity of secondary metabolism in parenchyma cells in a transition zone between sapwood and HW, followed by programmed cell-death. The resulting HW tissue is thought to consist entirely of dead cells. Here, we discuss a variation of existing models of HW formation, based on the recent discovery of HW-specific transcriptome signatures of terpenoid biosynthesis in sandalwood (Santalum album L.) that invokes the activity of living cells in HW.
Assuntos
Genômica , Santalum/genética , Santalum/metabolismo , Metabolismo Secundário , Terpenos/metabolismo , Transcriptoma , Madeira/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Plant defenses often involve specialized cells and tissues. In conifers, specialized cells of the bark are important for defense against insects and pathogens. Using laser microdissection, we characterized the transcriptomes of cortical resin duct cells, phenolic cells and phloem of white spruce (Picea glauca) bark under constitutive and methyl jasmonate (MeJa)-induced conditions, and we compared these transcriptomes with the transcriptome of the bark tissue complex. Overall, ~3700 bark transcripts were differentially expressed in response to MeJa. Approximately 25% of transcripts were expressed in only one cell type, revealing cell specialization at the transcriptome level. MeJa caused cell-type-specific transcriptome responses and changed the overall patterns of cell-type-specific transcript accumulation. Comparison of transcriptomes of the conifer bark tissue complex and specialized cells resolved a masking effect inherent to transcriptome analysis of complex tissues, and showed the actual cell-type-specific transcriptome signatures. Characterization of cell-type-specific transcriptomes is critical to reveal the dynamic patterns of spatial and temporal display of constitutive and induced defense systems in a complex plant tissue or organ. This was demonstrated with the improved resolution of spatially restricted expression of sets of genes of secondary metabolism in the specialized cell types.
Assuntos
Resistência à Doença/genética , Picea/genética , Doenças das Plantas/imunologia , Transcriptoma , Acetatos/farmacologia , Animais , Análise por Conglomerados , Ciclopentanos/farmacologia , Regulação da Expressão Gênica de Plantas , Insetos/fisiologia , Microdissecção e Captura a Laser , Especificidade de Órgãos , Oxilipinas/farmacologia , Floema/anatomia & histologia , Floema/genética , Floema/imunologia , Picea/anatomia & histologia , Picea/imunologia , Casca de Planta/anatomia & histologia , Casca de Planta/genética , Casca de Planta/imunologia , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Análise de Sequência de RNA , Terpenos/metabolismoRESUMO
Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as ß-myrcene, (E)-ß-ocimene, (-)-limonene, (+)-α-pinene, ß-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.
Assuntos
Alquil e Aril Transferases/genética , Cannabis/genética , Regulação da Expressão Gênica de Plantas , Inflorescência/genética , RNA Mensageiro/genética , Monoterpenos Acíclicos , Alcenos/metabolismo , Alquil e Aril Transferases/metabolismo , Monoterpenos Bicíclicos , Cannabis/classificação , Cannabis/enzimologia , Cicloexenos/metabolismo , Inflorescência/enzimologia , Isoenzimas/genética , Isoenzimas/metabolismo , Limoneno , Redes e Vias Metabólicas/genética , Sesquiterpenos Monocíclicos , Monoterpenos/metabolismo , Família Multigênica , Filogenia , Sesquiterpenos Policíclicos , RNA Mensageiro/metabolismo , Sesquiterpenos/metabolismo , Terpenos/metabolismoRESUMO
The diversity of small molecules formed via plant diterpene metabolism offers a rich source of known and potentially new biopharmaceuticals. Among these, the microtubule-destabilizing activity of pseudolaric acid B (PAB) holds promise for new anticancer agents. PAB is found, perhaps uniquely, in the coniferous tree golden larch (Pseudolarix amabilis, Pxa). Here we describe the discovery and mechanistic analysis of golden larch terpene synthase 8 (PxaTPS8), an unusual diterpene synthase (diTPS) that catalyzes the first committed step in PAB biosynthesis. Mining of the golden larch root transcriptome revealed a large TPS family, including the monofunctional class I diTPS PxaTPS8, which converts geranylgeranyl diphosphate into a previously unknown 5,7-fused bicyclic diterpene, coined "pseudolaratriene." Combined NMR and quantum chemical analysis verified the structure of pseudolaratriene, and co-occurrence with PxaTPS8 and PAB in P amabilis tissues supports the intermediacy of pseudolaratriene in PAB metabolism. Although PxaTPS8 adopts the typical three-domain structure of diTPSs, sequence phylogeny places the enzyme with two-domain TPSs of mono- and sesqui-terpene biosynthesis. Site-directed mutagenesis of PxaTPS8 revealed several catalytic residues that, together with quantum chemical calculations, suggested a substantial divergence of PxaTPS8 from other TPSs leading to a distinct carbocation-driven reaction mechanism en route to the 5,7-trans-fused bicyclic pseudolaratriene scaffold. PxaTPS8 expression in microbial and plant hosts provided proof of concept for metabolic engineering of pseudolaratriene.
Assuntos
Alquil e Aril Transferases/metabolismo , Diterpenos/metabolismo , Larix/metabolismo , Proteínas de Plantas/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Alquil e Aril Transferases/genética , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , DNA Complementar/genética , Larix/enzimologia , Larix/genética , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Raízes de Plantas/enzimologia , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Alinhamento de SequênciaRESUMO
Salvia divinorum commonly known as diviner's sage, is an ethnomedicinal plant of the mint family (Lamiaceae). Salvia divinorum is rich in clerodane-type diterpenoids, which accumulate predominantly in leaf glandular trichomes. The main bioactive metabolite, salvinorin A, is the first non-nitrogenous natural compound known to function as an opioid-receptor agonist, and is undergoing clinical trials for potential use in treating neuropsychiatric diseases and drug addictions. We report here the discovery and functional characterization of two S. divinorum diterpene synthases (diTPSs), the ent-copalyl diphosphate (ent-CPP) synthase SdCPS1, and the clerodienyl diphosphate (CLPP) synthase SdCPS2. Mining of leaf- and trichome-specific transcriptomes revealed five diTPSs, two of which are class II diTPSs (SdCPS1-2) and three are class I enzymes (SdKSL1-3). Of the class II diTPSs, transient expression in Nicotiana benthamiana identified SdCPS1 as an ent-CPP synthase, which is prevalent in roots and, together with SdKSL1, exhibits a possible dual role in general and specialized metabolism. In vivo co-expression and in vitro assays combined with nuclear magnetic resonance (NMR) analysis identified SdCPS2 as a CLPP synthase. A role of SdCPS2 in catalyzing the committed step in salvinorin A biosynthesis is supported by its biochemical function, trichome-specific expression and absence of additional class II diTPSs in S. divinorum. Structure-guided mutagenesis revealed four catalytic residues that enabled the re-programming of SdCPS2 activity to afford four distinct products, thus advancing our understanding of how neo-functionalization events have shaped the array of different class II diTPS functions in plants, and may promote synthetic biology platforms for a broader spectrum of diterpenoid bioproducts.