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In the last decades, growing evidence showed the therapeutic capabilities of Cannabis plants. These capabilities were attributed to the specialized secondary metabolites stored in the glandular trichomes of female inflorescences, mainly phytocannabinoids and terpenoids. The accumulation of the metabolites in the flower is versatile and influenced by a largely unknown regulation system, attributed to genetic, developmental and environmental factors. As Cannabis is a dioecious plant, one main factor is fertilization after successful pollination. Fertilized flowers are considerably less potent, likely due to changes in the contents of phytocannabinoids and terpenoids; therefore, this study examined the effect of fertilization on metabolite composition by crossbreeding (-)-Δ9-trans-tetrahydrocannabinol (THC)- or cannabidiol (CBD)-rich female plants with different male plants: THC-rich, CBD-rich, or the original female plant induced to develop male pollen sacs. We used advanced analytical methods to assess the phytocannabinoids and terpenoids content, including a newly developed semi-quantitative analysis for terpenoids without analytical standards. We found that fertilization significantly decreased phytocannabinoids content. For terpenoids, the subgroup of monoterpenoids had similar trends to the phytocannabinoids, proposing both are commonly regulated in the plant. The sesquiterpenoids remained unchanged in the THC-rich female and had a trend of decrease in the CBD-rich female. Additionally, specific phytocannabinoids and terpenoids showed an uncommon increase in concentration followed by fertilization with particular male plants. Our results demonstrate that although the profile of phytocannabinoids and their relative ratios were kept, fertilization substantially decreased the concentration of nearly all phytocannabinoids in the plant regardless of the type of fertilizing male. Our findings may point to the functional roles of secondary metabolites in Cannabis.

Background: Farmers harvest two batches fruits of Lemons (Citrus limon L. Burm. f.) i.e., spring flowering fruit and autumn flowering fruit in dry-hot valley in Yunnan, China. Regular lemons harvested in autumn have smooth skin. However, lemons harvested in spring have rough skin, which makes them less attractive to customers. Furthermore, the rough skin causes a reduction in commodity value and economical losses to farmers. This is a preliminary study that investigates the key transcriptomic and metabolomic differences in peels of lemon fruits (variety Yuning no. 1) harvested 30, 60, 90, 120, and 150 days after flowering from the same trees in different seasons.Results: We identified 5,792, 4,001, 3,148, and 5,287 differentially expressed genes (DEGs) between smooth peel (C) and rough peel (D) 60, 90, 120, and 150 days after flowering, respectively. A total of 1,193 metabolites differentially accumulated (DAM) between D and C. The DEGs and DAMs were enriched in the mitogen-activated protein kinase (MAPK) and plant hormone signaling, terpenoid biosynthesis, flavonoid, and phenylalanine biosynthesis, and ribosome pathways. Predominantly, in the early stages, phytohormonal regulation and signaling were the main driving force for changes in peel surface. Changes in the expression of genes associated with asymmetric cell division were also an important observation. The biosynthesis of terpenoids was possibly reduced in rough peels, while the exclusive expression of cell wall synthesis-related genes could be a possible reason for the thick peel of the rough-skinned lemons. Additionally, cell division, cell number, hypocotyl growth, accumulation of fatty acids, lignans and coumarins- related gene expression, and metabolite accumulation changes were major observations.Conclusion: The rough peels fruit (autumn flowering fruit) and smooth peels fruit (spring flowering fruit) matured on the same trees are possibly due to the differential regulation of asymmetric cell division, cell number regulation, and randomization of hypocotyl growth related genes and the accumulation of terpenoids, flavonoids, fatty acids, lignans, and coumarins. The preliminary results of this study are important for increasing the understanding of peel roughness in lemon and other citrus species.

Diosgenin serves as an important precursor of most steroidal drugs in market. Cholesterol was previously deemed as a sterol origin leading to diosgenin biosynthesis. This study reports that cholesterol is not in parallel with diosgenin biosynthesis inTrigonella foenum-graecum. We first perturbed its sterol composition using inhibitors specific for the upstream isoprenoid pathway enzymes, HMGR (3-hydroxy-3-methylgutaryl-CoA reductase) on the mevalonate (MVA) and DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) on the 2-C-methyl-D-erythritol-4-phophate (MEP) pathways, and have revealed that diosgenin and cholesterol reversely or differently accumulated in either the MVA or the MEP pathway-suppressed plants, challenging the previously proposed role of cholesterol in diosgenin biosynthesis. To further investigate this, we altered the sterol composition by suppressing and overexpressing the 24-sterol methyltransferase type 1 (SMT1) gene inT. foenum-graecum, as SMT1 acts in the first committed step of diverting the carbon flux of cholesterol toward biosynthesis of 24-alkyl sterols. Knockdown ofTfSMT1expression led to increased cholesterol level but caused a large reduction of diosgenin. Diosgenin was increased upon theTfSMT1-overexpressing, which, however, did not significantly affect cholesterol biosynthesis. These data consistently supported that diosgenin biosynthesis inT. foenum-graecumis not associated with cholesterol. Rather, campesterol, a 24-alkyl sterol, was indicative of being correlative to diosgenin biosynthesis inT. foenum-graecum.

Host-plant-associated bacteria affect the growth, vigor, and nutrient availability of the host plant. However, phyllosphere bacteria have received less research attention and their functions remain elusive, especially in forest ecosystems. In this study, we collected newly developed needles from sapling (age 5 years), juvenile (15 years), mature (25 years), and overmature (35 years) stands of Chinese fir [Cunninghamia lanceolata (Lamb.) Hook]. We analyzed changes in phyllosphere bacterial communities, their functional genes, and metabolic activity among different stand ages. The results showed that phyllosphere bacterial communities changed, both in relative abundance and in composition, with an increase in stand age. Community abundance predominantly changed in the orders Campylobacterales, Pseudonocardiales, Deinococcales, Gemmatimonadales, Betaproteobacteriales, Chthoniobacterales, and Propionibacteriales. Functional predictions indicated the genes of microbial communities for carbon metabolism, nitrogen metabolism, antibiotic biosynthesis, flavonoids biosynthesis, and steroid hormone biosynthesis varied; some bacteria were strongly correlated with some metabolites. A total of 112 differential metabolites, including lipids, benzenoids, and flavonoids, were identified. Trigonelline, proline, leucine, and phenylalanine concentrations increased with stand age. Flavonoids concentrations were higher in sapling stands than in other stands, but the transcript levels of genes associated with flavonoids biosynthesis in the newly developed needles of saplings were lower than those of other stands. The nutritional requirements and competition between individual trees at different growth stages shaped the phyllosphere bacterial community and host–bacteria interaction. Gene expression related to the secondary metabolism of shikimate, mevalonate, terpenoids, tocopherol, phenylpropanoids, phenols, alkaloids, carotenoids, betains, wax, and flavonoids pathways were clearly different in Chinese fir at different ages. This study provides an overview of phyllosphere bacteria, metabolism, and transcriptome in Chinese fir of different stand ages and highlights the value of an integrated approach to understand the molecular mechanisms associated with biosynthesis.

Trans-isopentenyl diphosphate synthases (TIDSs) genes are known to be important determinants for terpene diversity and the accumulation of terpenoids. The essential oil of Cinnamomum camphora, which is rich in monoterpenes, sesquiterpenes, and other aromatic compounds, has a wide range of pharmacological activities and has therefore attracted considerable interest. However, the TIDS gene family, and its relationship to the camphor tree (C. camphora L. Presl.), has not yet been characterized. In this study, we identified 10 TIDS genes in the genome of the C. camphora borneol chemotype that were unevenly distributed on chromosomes. Synteny analysis revealed that the TIDS gene family in this species likely expanded through segmental duplication events. Furthermore, cis-element analyses demonstrated that C. camphora TIDS (CcTIDS) genes can respond to multiple abiotic stresses. Finally, functional characterization of eight putative short-chain TIDS proteins revealed that CcTIDS3 and CcTIDS9 exhibit farnesyl diphosphate synthase (FPPS) activity, while CcTIDS1 and CcTIDS2 encode geranylgeranyl diphosphate synthases (GGPPS). Although, CcTIDS8 and CcTIDS10 were found to be catalytically inactive alone, they were able to bind to each other to form a heterodimeric functional geranyl diphosphate synthase (GPPS) in vitro, and this interaction was confirmed using a yeast two-hybrid assay. Furthermore, transcriptome analysis revealed that the CcTIDS3, CcTIDS8, CcTIDS9, and CcTIDS10 genes were found to be more active in C. camphora roots as compared to stems and leaves, which were verified by quantitative real-time PCR (qRT-PCR). These novel results provide a foundation for further exploration of the role of the TIDS gene family in camphor trees, and also provide a potential mechanism by which the production of camphor tree essential oil could be increased for pharmacological purposes through metabolic engineering.

Chinese cedar (Cryptomeria fortunei) is a tree species with important ornamental, medicinal, and economic value. Terpenoids extracted from the essential oil of C. fortunei needles have been considered valuable ingredients in the pharmaceutical and cosmetic industries. However, the possible gene regulation mechanisms that limit terpenoid biosynthesis in this genus are poorly understood. Here, we adopted integrated metabolome analysis, transcriptome, small-RNA (sRNA), and degradome sequencing to analyze the differences in terpenoid regulatory mechanisms in two different overwintering C. fortunei phenotypes (wild-type and an evergreen mutant). A total of 1447/6219 differentially synthesized metabolites (DSMs)/unigenes (DEGs) were detected through metabolome/transcriptome analyses, and these DSMs/DEGs were significantly enriched in flavonoid and diterpenoid biosynthesis pathways. In C. fortunei needles, 587 microRNAs (miRNAs), including 67 differentially expressed miRNAs (DERs), were detected. Among them, 8346 targets of 571 miRNAs were predicted using degradome data, and a 72-miRNA-target regulatory network involved in the metabolism of terpenoids and polyketides was constructed. Forty-one targets were further confirmed to be involved in terpenoid backbone and diterpenoid biosynthesis, and target analyses revealed that two miRNAs (i.e., aly-miR168a-5p and aof-miR396a) may be related to the different phenotypes and to differential regulation of diterpenoid biosynthesis. Overall, these results reveal that C. fortunei plants with the evergreen mutation maintain high terpenoid levels in winter through miRNA-target regulation, which provides a valuable resource for essential oil-related bioengineering research.

Prohydrojasmon (PDJ), an analog of jasmonic acid (JA), was found to induce direct and indirect defenses against herbivores in non-infested plants. To test whether PDJ can be used for pest control in crop production, we conducted experiments in pesticide-free Japanese radish fields from October 4 to December 12 in 2015. Twenty-four Japanese radish plants in three plots were treated with a 100 times-diluted commercial formulation (5%) of PDJ (treated plants), and 24 plants in three different plots were treated with water (control plants) until November 29 every week. Throughout the observation period, the number of aphids, leaf-mining fly larvae, vegetable weevils, and thrips was significantly lower on the treated plants than on the control plants. In contrast, the number of lepidopteran larvae was not significantly different between the treated and control plants throughout the study period. Parasitized aphids (mummies) were also observed in both plots. Poisson regression analyses showed that a significantly higher number of mummies was recorded on the treated plants as compared to that on the control plants when the number of aphids increased. This suggested that PDJ application to Japanese radish plants attracted more parasitoid wasps on the treated plants than on the control plants. We also identified eight terpenoids and methyl salicylate as the PDJ-induced plant volatiles in the headspace of the treated plants. Some of these volatiles might be responsible for attracting aphid-parasitoid wasps in the field. However, for other insect pests, we did not find any natural enemies. Interestingly, the genes of the JA and salicylic acid signaling pathways were differentially upregulated in the treated plants. We also observed that the PDJ treatments induced the expression of the genes related to glucosinolate biosynthesis and the subsequent isothiocyanate formation. Additionally, the weights of both the aboveground and belowground parts of the treated plants were significantly lower than those of the respective parts of the control plants. These results indicated that the treatment of Japanese radish plants with a 100 times-diluted commercial formulation of PDJ induced their direct and indirect defenses against several insect pest species to reduce their numbers, and negatively affected their biomass.

Rehmannia glutinosa has important medicinal value; terpenoid is one of the main active components in R. glutinosa. In this study, iTRAQ technique was used to analyze the relative abundance of proteins in roots of R. glutinosa, and 6,752 reliable proteins were quantified. GO enrichment results indicated that most proteins were involved in metabolic process or cellular process, 57.63% proteins had catalytic activity, and 65.80% proteins were enriched in membrane-bounded organelle. In roots of R. glutinosa, there were 38 KEGG enrichments with significance, more DEPs were found in some pathways, especially the proteasome pathway and TCA cycle with 15.0% DEPs between elongation stage and expansion stage of roots. Furthermore, five KEGG pathways of terpenoid synthesis were found. Most prenyltransferases belong to FPP/GGPP synthase family, involved in terpenoid backbone biosynthesis, and all interacted with biotin carboxylase CAC2. Compared with that at the elongation stage, many prenyltransferases exhibited higher expression at the expansion stage or maturation stage of roots. In addition, eight FPP/GGPP synthase encoding genes were cloned from R. glutinosa, namely FPPS, FPPS1, GGPS, GGPS3, GGPS4, GGPS5, GPPS and GPPS2, introns were also found in FPPS, FPPS1, GGPS5 and GGPS2, and FPP/GPP synthases were more conservative in organisms, especially in viridiplantae, in which the co-occurrence of GPPS or GPPS2 was significantly higher in plants. Further analysis found that FPP/GGPP synthases of R. glutinosa were divided into three kinds, GGPS, GPPS and FPPS, and their gene expression was significantly diverse in different varieties, growth periods, or tissues of R. glutinosa. Compared with that of GGPS, the expression of GPPS and FPPS was much higher in R. glutinosa, especially at the expansion stage and maturation stage. Thus, the synthesis of terpenoids in roots of R. glutinosa is intricately regulated and needs to be further studied.

It is known that undamaged plants that have been exposed to volatiles from damaged con- or heterospecific plants become more resistant against herbivores. This is one of the plants’ induced resistant responses against herbivores. To test whether this response can be used for rice production, we conducted the following experiments over 2 years (2012 and 2013). Rice seedlings were first planted in the rice seedling bed for 2 weeks in early May. There, half of the rice seedlings were exposed to artificially damaged weed volatiles three times for 12 days (treated plants). Weeds were randomly collected from the areas that were >100 m away from the seedling bed and the rice paddy fields. The remaining seedlings were not exposed (control plants). In the middle of May, bunches (ca. three seedlings per bunch) were transplanted to the rice paddy field. In July, leaf damage was observed. The total number of leaves in the treated and control plants was not significantly different. In contrast, the total number of damaged leaves in the treated plants was significantly lower than that in the control plants. In September, rice grains were harvested. The average weight of a rice grain from the treated and control plants was not significantly different. However, the weight of grains per bunch of treated plants was significantly higher than that of control plants; this indicated a significant increase of the number of grains by 23% in 2012 and by 18% in 2013 in the treated plants compared to that in the control plants. The volatiles emitted from the weeds included monoterpenoids (40.4% in total), green leaf volatiles (46.5%), short-chain alcohols (5.3%), short-chain ketone (5.4%), short-chain acetate (0.5%), short-chain aldehyde (1.1%), and hydrocarbon (0.7%). These results suggest that exposure of volatiles from artificially damaged weeds to rice seedlings has the potential to increase rice production.

The isoprenoids play important ecological and physiological roles in plants. They also have a tremendous impact on human lives as food additives, medicines, and industrial raw materials, among others. Though some isoprenoids are highly abundant in nature, plants produce many at extremely low levels. Glandular trichomes (GT), which cover the aerial parts of more than 25% of vascular plants, have been considered as natural biofactories for the mass production of rare industrially important isoprenoids. In several plant genera (e.g., Lavandula and Mentha), GTs produce and store large quantities of the low molecular weight isoprenoids, in particular mono- and sesquiterpenes, as essential oil constituents. Within each trichome, a group of secretory cells is specialized to strongly and specifically express isoprenoid biosynthetic genes, and to synthesize and deposit copious amounts of terpenoids into the trichome’s storage reservoir. Despite the abundance of certain metabolites in essential oils and defensive resins, plants, particularly those lacking glandular trichomes, accumulate small quantities of many of the biologically active and industrially important isoprenoids. Therefore, there is a pressing need for technologies to enable the mass production of such metabolites, and to help meet the ever-increasing demand for plant-based bioproducts, including medicines and renewable materials. Considerable contemporary research has focused on engineering isoprenoid metabolism in GTs, with the goal of utilizing them as natural biofactories for the production of valuable phytochemicals. In this review, we summarize recent advances related to the engineering of isoprenoid biosynthetic pathways in glandular trichomes.

Previous chemotaxonomic studies of cannabis only focused on tetrahydrocannabinol (THC) dominant strains while excluded the cannabidiol (CBD) dominant strains and intermediate strains (THC ≈ CBD). This study investigated the utility of the full spectrum of secondary metabolites in different plant parts in three cannabis chemotypes (THC dominant, intermediate, and CBD dominant) for chemotaxonomic discrimination. Hierarchical clustering, principal component analysis (PCA), and canonical correlation analysis assigned 21 cannabis varieties into three chemotypes using the content and ratio of cannabinoids, terpenoids, flavonoids, sterols, and triterpenoids across inflorescences, leaves, stem bark, and roots. The same clustering results were obtained using secondary metabolites, omitting THC and CBD. Significant chemical differences were identified in these three chemotypes. Cannabinoids, terpenoids, flavonoids had differentiation power while sterols and triterpenoids had none. CBD dominant strains had higher amounts of total CBD, cannabidivarin (CBDV), cannabichromene (CBC), α-pinene, β-myrcene, (−)-guaiol, β-eudesmol, α-eudesmol, α-bisabolol, orientin, vitexin, and isovitexin, while THC dominant strains had higher total THC, total tetrahydrocannabivarin (THCV), total cannabigerol (CBG), camphene, limonene, ocimene, sabinene hydrate, terpinolene, linalool, fenchol, α-terpineol, β-caryophyllene, trans-β-farnesene, α-humulene, trans-nerolidol, quercetin, and kaempferol. Compound levels in intermediate strains were generally equal to or in between those in CBD dominant and THC dominant strains. Overall, with higher amounts of β-myrcene, (−)-guaiol, β-eudesmol, α-eudesmol, and α-bisabolol, intermediate strains more resemble CBD dominant strains than THC dominant strains. The results of this study provide a comprehensive profile of bioactive compounds in three chemotypes for medical purposes. The simultaneous presence of a predominant number of identified chemotype markers (with or without THC and CBD) could be used as chemical fingerprints for quality standardization or strain identification for research, clinical studies, and cannabis product manufacturing.

Orchids are the most species-rich plants and highly interactive with pollinators via visual or olfactory cues. Biosynthesis and emission of volatile organic compounds (VOCs) to the atmosphere facilitate the olfactory cues and ensure successful pollination. Phalaenopsis bellina is a scented orchid with monoterpenes as major VOCs, comprising linalool, geraniol, and their derivatives. Comparative transcriptomics analysis identified four terpene synthase-b (TPS-b) genes and two TPS-e/f genes with differential gene expression between scented and scentless Phalaenopsis species. Here, we confirmed their differential expression between scented and scentless Phalaenopsis orchids and excluded one TPS-b candidate. We analyzed the temporal and spatial expression and functionally characterized these TPSs. Both TPS-b and TPS-e/f genes showed an increased expression on blooming day or 3 days post-anthesis (D + 3) before the optimal emission of floral scent on D + 5, with especially high expression of PbTPS5 and PbTPS10. The TPS-b genes are expressed exclusively in reproductive organs, whereas the TPS-e/f genes are expressed in both reproductive and vegetative organs. In planta functional characterization of both PbTPS5 and PbTPS10 in tobacco and scentless Phalaenopsis plants did not produce terpenoids. Further ectopic expression in scented Phalaenopsis cultivar P. I-Hsin Venus showed that linalool was the main product, with PbTPS10 displaying 3-fold higher activity than PbTPS5. On in vitro enzyme assay with purified recombinant TPS-b proteins ectopically expressed in Escherichia coli, geraniol was the product catalyzed by PbTPS5 and PbTPS9. PbTPS3 was a linalool/(β)-cis-ocimene synthase and PbTPS4 a linalool synthase. In conclusion, both TPS-b and TPS-e/f enzymes orchestrated floral monoterpene biosynthesis in P. bellina.

Disease lesion mimic (Les/les) mutants display disease-like spontaneous lesions in the absence of pathogen infection, implying the constitutive activation of defense responses. However, the genetic and biochemical bases underlying the activated defense responses in those mutants remain largely unknown. Here, we performed integrated transcriptomics and metabolomics analysis on three typical maize Les mutants Les4, Les10, and Les17 with large, medium, and small lesion size, respectively, thereby dissecting the activated defense responses at the transcriptional and metabolomic level. A total of 1,714, 4,887, and 1,625 differentially expressed genes (DEGs) were identified in Les4, Les10, and Les17, respectively. Among them, 570, 3,299, and 447 specific differentially expressed genes (SGs) were identified, implying a specific function of each LES gene. In addition, 480 common differentially expressed genes (CGs) and 42 common differentially accumulated metabolites (CMs) were identified in all Les mutants, suggesting the robust activation of shared signaling pathways. Intriguingly, substantial analysis of the CGs indicated that genes involved in the programmed cell death, defense responses, and phenylpropanoid and terpenoid biosynthesis were most commonly activated. Genes involved in photosynthetic biosynthesis, however, were generally repressed. Consistently, the dominant CMs identified were phenylpropanoids and flavonoids. In particular, lignin, the phenylpropanoid-based polymer, was significantly increased in all three mutants. These data collectively imply that transcriptional activation of defense-related gene expression; increase of phenylpropanoid, lignin, flavonoid, and terpenoid biosynthesis; and inhibition of photosynthesis are generalnatures associated with the lesion formation and constitutively activated defense responses in those mutants. Further studies on the identified SGs and CGs will shed new light on the function of each LES gene as well as the regulatory network of defense responses in maize.

This study aimed to evaluate the changes in aromatic components and other chemical properties of Tainong mango during fruit development, ripening, and storage. As the volatiles of Tainong mango and their related molecular mechanisms remain unclear, volatile profile, metabonomics, and transcriptome analyses were applied to investigate the molecular determinants of the synthesis of aroma components in mango during fruit development and storage. Total acids, total sugar, total carotenoids, enzyme activities of the mango pulp samples were also determined. Volatile components of the mango pulp samples were identified using a gas chromatography-mass spectrometric method. Ribonucleic acid (RNA) sequences of the samples were analyzed by real-time polymerase chain reaction. The results showed that 181 volatiles were isolated and identified in the fruit at seven stages. Compared to the other stages, mango collected on day 8 and day 12 had higher concentrations of 17 volatile components, especially (E,Z)-2,6-nonadienal, 53384 transcripts were also detected through RNA sequencing. The differentially expressed genes analyses included catalytic activity, transferase activity, adenosine diphosphate binding, transcription factor activity, and oxidoreductase activity. α-Pinene content and expression of the differentially expressed genes involved in terpenoid metabolism and enzyme activities in the terpenoid metabolic pathways gradually increased during the maturity of the fruit, and had maximum values at day 8 of storage. Moreover, the integrative analyses revealed potential molecular insights of mango development and aroma formation in the fruit.

Amygdalus pedunculataPall [Rosaceae, Prunus,Prunus pedunculata(Pall.) Maxim.] belongs to the Rosaceae family and is resistant to cold and drought. Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry and metabolomics were used to track the changes in bioactive metabolites during several stages ofAmygdalus pedunculata Pallgrowth. A total of 827 different metabolites were detected, including 169 flavonoids, 68 organic acids, 35 terpenoids and 2 tannins. Flavonoid biosynthesis and flavone and flavonol biosynthesis were the main synthetic sources of flavonoids. Quercetin, isoquercitrin, and epicatechin as biomarkers related to growth and development were found. Quercetin connects the biosynthesis of flavonoids and the biosynthesis of flavones and flavonols. The contents of isoquercitrin and epicatechin increased uniformly during the whole growth process from the flowering stage to the fruit ripening stage, indicating that play key roles in the fruit growth and ripening stages of this plant. The tissue location and quantitative analysis of flavonoids in leaves at different stages were performed by confocal laser scanning microscopy. The flavonoids were mainly distributed in the palisade tissue and spongy tissue, indicating the need for protection of these sensitive tissues in particular. Through comprehensive and systematic analysis, the temporal distribution of flavonoids in the process of their leaves growth was determined. These results clarify the important role of flavonoids in the developmental process ofAmygdalus pedunculataPall.

The fruits of Amomum villosum and Amomum longiligulare are both used medicinally as Fructus Amomi the famous traditional Chinese medicine, however, the medicinal quality of A. villosum is better than that of A. longiligulare. Volatile terpenoids in the seeds, especially bornyl acetate and borneol, are the medicinal components of Fructus Amomi. The volatile terpenoids and transcriptome of developing seeds of A. villosum and A. longiligulare were compared in this study. The result revealed that the bornyl acetate and borneol contents were higher in A. villosum than in A. longiligulare. Additionally, six terpenoid synthase genes (AlTPS1–AlTPS6) were screened from the transcriptome of A. longiligulare, and AlTPS2 and AlTPS3 were found to share 98 and 83% identity with AvTPS2 and AvBPPS (bornyl diphosphate synthase) from A. villosum, respectively. BPPS is the key enzyme for the biosynthesis of borneol and bornyl acetate. Biochemical assays improved that AlTPS2 had an identical function to AvTPS2 as linalool synthase; however, AlTPS3 produced camphene as the major product and bornyl diphosphate (BPP) as the secondary product, whereas AvBPPS produced BPP as its major product. There was only one different amino acid between AlTPS3 (A496) and AvBPPS (G495) in their conserved motifs, and the site-directed mutation of A496G in DTE motif of AlTPS3 changed the major product from camphene to BPP. Molecular docking suggests that A496G mutation narrows the camphene-binding pocket and decreases the BPP-binding energy, thus increases the product BPP selectivity of enzyme. In addition, the expression level of AvBPPS was significantly higher than that of AlTPS3 in seeds, which was consistent with the related-metabolites contents. This study provides insight into the TPS-related molecular bases for the biosynthesis and accumulation differences of the bioactive terpenoids between A. villosum and A. longiligulare. BPPS, the key gene involved in the biosynthesis of the active compound, was identified as a target gene that could be applied for the quality-related identification and breeding of Fructus Amomi.

Floral volatiles and reward traits are major drivers for the behavior of mutualistic as well as antagonistic flower visitors, i.e., pollinators and florivores. These floral traits differ tremendously between species, but intraspecific differences and their consequences on organism interactions remain largely unknown. Floral volatile compounds, such as terpenoids, function as cues to advertise rewards to pollinators, but should at the same time also repel florivores. The reward composition, e.g., protein and lipid contents in pollen, differs between individuals of distinct plant families. Whether the nutritional value of rewards within the same plant species is linked to their chemotypes, which differ in their pattern of specialized metabolites, has yet not been investigated. In the present study, we compared Tanacetum vulgare plants of five terpenoid chemotypes with regard to flower production, floral headspace volatiles, pollen macronutrient and terpenoid content, and floral attractiveness to florivorous beetles. Our analyses revealed remarkable differences between the chemotypes in the amount and diameter of flower heads, duration of bloom period, and pollen nutritional quality. The floral headspace composition of pollen-producing mature flowers, but not of premature flowers, was correlated to that of pollen and leaves in the same plant individual. For two chemotypes, florivorous beetles discriminated between the scent of mature and premature flower heads and preferred the latter. In semi-field experiments, the abundance of florivorous beetles and flower tissue miners differed between T. vulgare chemotypes. Moreover, the scent environment affected the choice and beetles were more abundant in homogenous plots composed of one single chemotype than in plots with different neighboring chemotypes. In conclusion, flower production, floral metabolic composition and pollen quality varied to a remarkable extend within the species T. vulgare, and the attractiveness of floral scent differed also intra-individually with floral ontogeny. We found evidence for a trade-off between pollen lipid content and pollen amount on a per-plant-level. Our study highlights that chemotypes which are more susceptible to florivory are less attacked when they grow in the neighborhood of other chemotypes and thus gain a benefit from high overall chemodiversity.

Nematicidal potential of essential oils (EOs) has been widely reported. Terpenoids present in most of the essential oils have been reported responsible for their bioactivity though very less is known about their modes of action. In the present study, anin vitroscreening of nine Eos, namely,Citrus sinensis(OEO),Myrtus communis(MTEO),Eucalyptus citriodora(CEO),Melaleuca alternifolia(TEO),Acorus calamus(AEO),Commiphora myrrha(MREO),Cymbopogon nardus(CNEO),Artemisia absinthium(WEO), andPogostemon cablin(PEO) againstMeloidogyne incognitarevealed OEO, CNEO, and TEO as most effective with LC5039.37, 43.22, and 76.28 μg ml–1respectively. EOs had varying compositions of mono- and sesquiterpenes determined by gas chromatography-mass spectrometry (GC-MS) analysis. Thein silicomolecular interactions screening of major EO constituents and the seven selected target proteins of the nematode indicated highest binding affinity of geraniol-ODR1 (odorant response gene 1) complex (ΔG = -36.9 kcal mol–1), due to extensive H-bonding, hydrophobic and π-alkyl interactions. The relative binding affinity followed the order: geraniol-ODR1 > β-terpineol-ODR1 > citronellal-ODR1 >l-limonene-ODR1 > γ-terpinene-ODR1. Taken together, the cumulativein vitroand computational bioefficacy analysis related to the chemoprofiles of EOs provides useful leads on harnessing the potential of EOs as bionematicides. The insight on biochemical ligand–target protein interactions described in the present work will be helpful in logical selection of biomolecules and essential oils for development of practically viable bionematicidal products.

The therapeutic use of medical Cannabis is growing, and so is the need for standardized and therapeutically stable Cannabis products for patients. The therapeutic effects of Cannabis largely depend on the content of its pharmacologically active secondary metabolites and their interactions, mainly terpenoids and phytocannabinoids. Once harvested and during storage, these natural compounds may decarboxylate, oxidize, isomerize, react photochemically, evaporate and more. Despite its widespread and increasing use, however, data on the stability of most of the plant's terpenoids and phytocannabinoids during storage is scarce. In this study, we therefore aimed to determine postharvest optimal storage conditions for preserving the composition of naturally biosynthesized secondary metabolites in Cannabis inflorescences and Cannabis extracts. To this end, Cannabis inflorescences (whole versus ground samples) and Cannabis extracts (dissolved in different solvents) from (-)-Δ9-trans-tetrahydrocannabinol- or cannabidiol-rich chemovars, were stored in the dark at various temperatures (25, 4, -30 and -80°C), and their phytocannabinoid and terpenoid profiles were analyzed over the course of 1 year. We found that in both Cannabis inflorescences and extracts, a storage temperature of 25°C led to the largest changes in the concentrations of the natural phytocannabinoids over time, making this the most unfavorable temperature compared with all others examined here. Olive oil was found to be the best vehicle for preserving the natural phytocannabinoid composition of the extracts. Terpenoid concentrations were found to decrease rapidly under all storage conditions, but temperatures lower than -20°C and grinding of the inflorescences were the least favorable conditions. Overall, our conclusions point that storage of whole inflorescences and extracts dissolved in olive oil, at 4°C, were the optimal postharvest conditions for Cannabis.

Phytochemicals or secondary metabolites present in fruit are key components contributing to sensory attributes like aroma, taste, and color. In addition, these compounds improve human nutrition and health. Stone fruits are an important source of an array of secondary metabolites that may reduce the risk of different diseases. The first part of this review is dedicated to the description of the main secondary organic compounds found in plants which include (a) phenolic compounds, (b) terpenoids/isoprenoids, and (c) nitrogen or sulfur containing compounds, and their principal biosynthetic pathways and their regulation in stone fruit. Then, the type and levels of bioactive compounds in different stone fruits of the Rosaceae family such as peach (Prunus persica), plum (P. domestica, P. salicina and P. cerasifera), sweet cherries (P. avium), almond kernels (P. dulcis, syn. P. amygdalus), and apricot (P. armeniaca) are presented. The last part of this review encompasses pre- and postharvest treatments affecting the phytochemical composition in stone fruit. Appropriate management of these factors during pre- and postharvest handling, along with further characterization of phytochemicals and the regulation of their synthesis in different cultivars, could help to increase the levels of these compounds, leading to the future improvement of stone fruit not only to enhance organoleptic characteristics but also to benefit human health. Fil: Lara, Maria Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Bonghi, Claudio. Università di Padova; Italia Fil: Famiani, Franco. Università di Perugia; Italia Fil: Vizzotto, Giannina. Università di Udine; Italia Fil: Walker, Robert P.. Università di Perugia; Italia Fil: Drincovich, Maria Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina

The recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) started in December 2019, resulting in the coronavirus disease-19 (COVID-19) pandemic. Coronaviruses are solely accountable for rising mortality and socioeconomic saddles. Presently, there are few repurposed drugs such as remdesivir or favipiravir approved for the treatment of COVID-19, although vaccines and plasma therapy is also subject to emergency approval. However, some potential natural treatments and cures have also been proposed. Molecules of natural origin showed therapeutic importance such as antiviral, anti-inflammatory, and antioxidant activity, and could be useful drug candidates for treating COVID-19. In recent years, essential oils have shown promising therapeutic effects against many viral diseases. Carvacrol is one of the monoterpene phenol with abundant presence in essential oils of many aromatic plants, including thyme and oregano. It is being used as food flavoring, additive, and preservatives. Carvacrol is also used as a fragrance in cosmetic products. A number of research studies have shown biological actions of carvacrol with its therapeutic potential is of clinical significance. The in vitro and in vivo studies have shown multiple pharmacological properties such as anticancer, anti-fungal, anti-bacterial, anti-oxidant, anti-inflammatory, vasorelaxant, hepatoprotective, and spasmolytic. This review highlights the various biological and pharmacological properties of carvacrol within the scope of COVID-19.

Terpenoids are considered to be the largest group of secondary metabolites and natural products. Studies have revealed 1-deoxy-D-xylulose 5-phosphate synthase (DXS) is the first and rate-limiting enzyme in the plastidial methylerythritol phosphate pathway, which produces isopentenyl diphosphate and its isoform dimethylallyl diphosphate as terpenoid biosynthesis precursors. Mulberry (Morus L.) is an economically and ecologically important perennial tree with diverse secondary metabolites, including terpenoids that protect plants against bacteria and insects and may be useful for treating human diseases. However, there has been relatively little research regarding DXS genes in mulberry and other woody plant species. In this study, we cloned and functionally characterized three Morus notabilis DXS genes (MnDXS1, MnDXS2A, and MnDXS2B). Bioinformatics analyses indicated MnDXS1 belongs to clade 1, whereas MnDXS2A and MnDXS2B are in clade 2. The three encoded MnDXS proteins are localized to chloroplasts. Additionally, substantial differences in MnDXS expression patterns were observed in diverse tissues and in response to insect feeding and methyl jasmonate treatment. Moreover, overexpression of MnDXS1 in Arabidopsis thaliana increased the gibberellic acid content and resulted in early flowering, whereas overexpression of MnDXS2A enhanced root growth and increased the chlorophyll and carotenoid content. Our findings indicate that MnDXS functions vary among the clades, which may be useful for further elucidation of the functions of the DXS genes in mulberry.

Cyanobacteria are photosynthetic prokaryotes that perform oxygenic photosynthesis. Due to their ability to use the photon energy of sunlight to fix carbon dioxide into biomass, cyanobacteria are promising hosts for the sustainable production of terpenoids, also known as isoprenoids, a diverse class of natural products with potential as advanced biofuels and high-value chemicals. However, the cyanobacterial enzymes involved in the biosynthesis of the terpene precursors needed to make more complicated terpenoids are poorly characterized. Here we show that the predicted type II prenyltransferase CrtE encoded by the model cyanobacterium Synechococcus sp. PCC 7002 is homodimeric and able to synthesize C20-geranylgeranyl pyrophosphate (GGPP) from C5-isopentenyl pyrophosphate (IPP) and C5-dimethylallyl pyrophosphate (DMAPP). The crystal structure of CrtE solved to a resolution of 2.7 Å revealed a strong structural similarity to the large subunit of the heterodimeric geranylgeranyl pyrophosphate synthase 1 from Arabidopsis thaliana with each subunit containing 14 helices. Using mutagenesis, we confirmed that the fourth and fifth amino acids (Met-87 and Ser-88) before the first conserved aspartate-rich motif (FARM) play important roles in controlling chain elongation. While the WT enzyme specifically produced GGPP, variants M87F and S88Y could only generate C15-farnesyl pyrophosphate (FPP), indicating that residues with large side chains obstruct product elongation. In contrast, replacement of M87 with the smaller Ala residue allowed the formation of the longer C25-geranylfarnesyl pyrophosphate (GFPP) product. Overall, our results provide new structural and functional information on the cyanobacterial CrtE enzyme that could lead to the development of improved cyanobacterial platforms for terpenoid production.

Plants are known to respond to Ultraviolet-B radiation (UV-B: 280–320 nm) by generating phenolic metabolites which absorbs UV-B light. Phenolics are extraordinarily abundant in Camellia sinensis leaves and are considered, together with pleasant volatile terpenoids, as primary flavor determinants in tea beverages. In this study, we focused on the effects of UV-B exposure (at 35 μW cm–2 for 0, 0.5, 2, and 8 h) on tea transcriptional and metabolic alterations, specifically related to tea flavor metabolite production. Out of 34,737 unigenes, a total of 18,081 differentially expressed genes (DEGs) due to UV-B treatments were identified. Additionally, the phenylpropanoid pathway was found as one of the most significantly UV-B affected top 20 KEGG pathways while flavonoid and monoterpenoid pathway-related genes were enhanced at 0.5 h. In the UVR8-signal transduction pathway, UVR8 was suppressed at both short and long exposure of UV-B with genes downstream differentially expressed. Divergent expression of MYB4 at different treatments could have differentially altered structural and regulatory genes upstream of flavonoid biosynthesis pathways. Suppression of MYB4-1&3 at 0.5 h could have led to the up-regulation of structural CCOAOMT-1&2, HST-1&2, DFR-4, ANR-2, and LAR-1&3 genes resulting in accumulation of specialized metabolites at a shorter duration of UV-B exposure. Specialized metabolite profiling revealed the correlated alterations in the abundances of catechins and some volatile terpenoids in all the treatments with significant accumulation of specialized metabolites at 0.5 h treatment. A significant increase in specialized metabolites at 0.5 h treatment and no significant alteration observed at longer UVB treatment suggested that shorter exposure to UV-B led to different display in gene expression and accumulation of specialized metabolites in tea shoots in response to UV-B stress. Taken together, our results indicated that the UV-B treatment applied in this study differentially altered the UVR8-signal transduction, flavonoid and terpenoid pathways at transcriptional and metabolic levels in tea plants. Our results show strong potential for UV-B application in flavor improvement in tea at the industrial level.

Oolong tea is a popular and semi-fermented beverage. During the processing of tea leaves, withering is the first indispensable process for improving flavor. However, the roles of long non-coding RNAs (lncRNAs) and the characteristic secondary metabolites during the withering of oolong tea leaves remain unknown. In this study, phytochemical analyses indicated that total polyphenols, flavonoids, catechins, epigallocatechin (EGC), catechin gallate (CG), gallocatechin gallate (GCG), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG) were all less abundant in the solar-withered leaves (SW) than in the fresh leaves (FL) and indoor-withered leaves (IW). In contrast, terpenoid, jasmonic acid (JA), and methyl jasmonate (MeJA) contents were higher in the SW than in the FL and IW. By analyzing the transcriptome data, we detected 32,036 lncRNAs. On the basis of the Kyoto Encyclopedia of Genes and Genomes analysis, the flavonoid metabolic pathway, the terpenoid metabolic pathway, and the JA/MeJA biosynthesis and signal transduction pathway were enriched pathways. Additionally, 63 differentially expressed lncRNAs (DE-lncRNAs) and 23 target genes were identified related to the three pathways. A comparison of the expression profiles of the DE-lncRNAs and their target genes between the SW and IW revealed four up-regulated genes (FLS, CCR, CAD, and HCT), seven up-regulated lncRNAs, four down-regulated genes (4CL, CHI, F3H, and F3’H), and three down-regulated lncRNAs related to flavonoid metabolism; nine up-regulated genes (DXS, CMK, HDS, HDR, AACT, MVK, PMK, GGPPS, and TPS), three up-regulated lncRNAs, and six down-regulated lncRNAs related to terpenoid metabolism; as well as six up-regulated genes (LOX, AOS, AOC, OPR, ACX, and MFP2), four up-regulated lncRNAs, and three down-regulated lncRNAs related to JA/MeJA biosynthesis and signal transduction. These results suggested that the expression of DE-lncRNAs and their targets involved in the three pathways may be related to the low abundance of the total polyphenols, flavonoids, and catechins (EGC, CG, GCG, ECG, and EGCG) and the high abundance of terpenoids in the SW. Moreover, solar irradiation, high JA and MeJA contents, and the endogenous target mimic (eTM)-related regulatory mechanism in the SW were also crucial for increasing the terpenoid levels. These findings provide new insights into the greater contribution of solar-withering to the high-quality flavor of oolong tea compared with the effects of indoor-withering.

The monoterpenes linalool and its oxides are the key aroma-active compounds in Osmanthus fragrans Lour. flowers. The glycosides of these monoterpenes accumulate throughout flowering, leading to considerable storage of potential aroma constituents that account for the majority of non-volatile aroma compounds. However, the UDP-glycosyltransferase (UGT) responsible for the glycosylation of linalool and its oxides has not been clarified. Four candidate OfUGTs (UGT85A82, UGT85A83, UGT85AF3, and UGT85A84) with high homology to the known terpenoid UGTs were screened by transcriptome sequencing. Over-expression of the candidate OfUGTs in tobacco showed that UGT85A84 glycosylated linalool oxides in planta. Since the transcript levels of UGT85A84 were positively correlated with glycoside accumulation, the recombinant UGT85A84 protein was subjected to reactions with aglycones and sugar donors. Two formate adducts were exclusively detected in UDP-Glc with linalool and linalool oxide reactions by liquid chromatography-mass spectrometry (LC-MS), indicating that UDP-Glc was the specific sugar donor. The kinetic parameters demonstrated that UGT85A84 glycosylated both linalool and lianlool oxides in vitro. Further analysis demonstrated that the transcription levels of MEP pathway genes might play an important role in mediating terpenoid glycosylation. Our findings unraveled the mechanism underlying the glycosylation of essential aroma compounds in flowers. This study will facilitate the application of potential aroma contributors in future industries.

Terpenoids comprise tens of thousands of small molecule natural products that are widely distributed across all domains of life. Plants produce by far the largest array of terpenoids with various roles in development and chemical ecology. Driven by selective pressure to adapt to their specific ecological niche, individual species form only a fraction of the myriad plant terpenoids, typically representing unique metabolite blends. Terpene synthase (TPS) enzymes are the gatekeepers in generating terpenoid diversity by catalyzing complex carbocation-driven cyclization, rearrangement, and elimination reactions that enable the transformation of a few acyclic prenyl diphosphate substrates into a vast chemical library of hydrocarbon and, for a few enzymes, oxygenated terpene scaffolds. The seven currently defined clades (a-h) forming the plant TPS family evolved from ancestral triterpene synthase- and prenyl transferase–type enzymes through repeated events of gene duplication and subsequent loss, gain, or fusion of protein domains and further functional diversification. Lineage-specific expansion of these TPS clades led to variable family sizes that may range from a single TPS gene to families of more than 100 members that may further function as part of modular metabolic networks to maximize the number of possible products. Accompanying gene family expansion, the TPS family shows a profound functional plasticity, where minor active site alterations can dramatically impact product outcome, thus enabling the emergence of new functions with minimal investment in evolving new enzymes. This article reviews current knowledge on the functional diversity and molecular evolution of the plant TPS family that underlies the chemical diversity of bioactive terpenoids across the plant kingdom.

Switchgrass (Panicum virgatum L.), a perennial C4 grass, represents an important species in natural and anthropogenic grasslands of North America. Its resilience to abiotic and biotic stress has made switchgrass a preferred bioenergy crop. However, little is known about the mechanisms of resistance of switchgrass against pathogens and herbivores. Volatile compounds such as terpenes have important activities in plant direct and indirect defense. Here, we show that switchgrass leaves emit blends of monoterpenes and sesquiterpenes upon feeding by the generalist insect herbivore Spodoptera frugiperda (fall armyworm) and in a systemic response to the treatment of roots with defense hormones. Belowground application of methyl jasmonate also induced the release of volatile terpenes from roots. To correlate the emission of terpenes with the expression and activity of their corresponding biosynthetic genes, we identified a gene family of 44 monoterpene and sesquiterpene synthases (mono-and sesqui-TPSs) of the type-a, type-b, type-g, and type-e subfamilies, of which 32 TPSs were found to be functionally active in vitro. The TPS genes are distributed over the K and N subgenomes with clusters occurring on several chromosomes. Synteny analysis revealed syntenic networks for approximately 30-40% of the switchgrass TPS genes in the genomes of Panicum hallii, Setaria italica, and Sorghum bicolor, suggesting shared TPS ancestry in the common progenitor of these grass lineages. Eighteen switchgrass TPS genes were substantially induced upon insect and hormone treatment and the enzymatic products of nine of these genes correlated with compounds of the induced volatile blends. In accordance with the emission of volatiles, TPS gene expression was induced systemically in response to belowground treatment, whereas this response was not observed upon aboveground feeding of S. frugiperda. Our results demonstrate complex above and belowground responses of induced volatile terpene metabolism in switchgrass and provide a framework for more detailed investigations of the function of terpenes in stress resistance in this monocot crop. Community Science Program grant of Department of Energy Joint Genome Institute [WIP 2568]; Translational Plant Sciences Program at Virginia Tech; Office of Science of the US Department of EnergyUnited States Department of Energy (DOE) [DE-AC02-05CH11231] This work was supported by Community Science Program grant (WIP 2568) of the Department of Energy Joint Genome Institute and funds by the Translational Plant Sciences Program at Virginia Tech. The work conducted by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy under contract no. DE-AC02-05CH11231.