A telomere-to-telomere reference genome provides genetic insight into the pentacyclic triterpenoid biosynthesis in Chaenomeles speciosa.

Chaenomeles speciosa (2n = 34), a medicinal and edible plant in the Rosaceae, is commonly used in traditional Chinese medicine. To date, the lack of genomic sequence and genetic studies has impeded efforts to improve its medicinal value. Herein, we report the use of an integrative approach involving PacBio HiFi (third-generation) sequencing and Hi-C scaffolding to assemble a high-quality telomere-to-telomere genome of C. speciosa. The genome comprised 650.4 Mb with a contig N50 of 35.5 Mb. Of these, 632.3 Mb were anchored to 17 pseudo-chromosomes, in which 12, 4, and 1 pseudo-chromosomes were represented by a single contig, two contigs, and four contigs, respectively. Eleven pseudo-chromosomes had telomere repeats at both ends, and four had telomere repeats at a single end. Repetitive sequences accounted for 49.5% of the genome, while a total of 45 515 protein-coding genes have been annotated. The genome size of C. speciosa was relatively similar to that of Malus domestica. Expanded or contracted gene families were identified and investigated for their association with different plant metabolisms or biological processes. In particular, functional annotation characterized gene families that were associated with the biosynthetic pathway of oleanolic and ursolic acids, two abundant pentacyclic triterpenoids in the fruits of C. speciosa. Taken together, this telomere-to-telomere and chromosome-level genome of C. speciosa not only provides a valuable resource to enhance understanding of the biosynthesis of medicinal compounds in tissues, but also promotes understanding of the evolution of the Rosaceae.

Molecular understanding of anthocyanin biosynthesis activated by PAP1 and regulated by 2, 4-dichlorophenoxyacetic acid in engineered red Artemisia annua cells.

MAIN CONCLUSION: Eight promoters were cloned, from which AC and G-box cis-elements were identified. PAP1 enhanced the promoter activity. 2,4-D reduced the anthocyanin biosynthesis via downregulating the expression of the PAP1 transgene. Artemisia annua is an effective antimalarial medicinal crop. We have established anthocyanin-producing red cell cultures from this plant with the overexpression of Production of Anthocyanin Pigment 1 (PAP1) encoding a R2R3MYB transcription factor. To understand the molecular mechanism by which PAP1 activated the entire anthocyanin pathway, we mined the genomic sequences of A. annua and obtained eight promoters of the anthocyanin pathway genes. Sequence analysis identified four types of AC cis-elements from six promoters, the MYB response elements (MRE) bound by PAP1. In addition, six promoters were determined to have at least one G-box cis-element. Eight promoters were cloned for activity analysis. Dual luciferase assays showed that PAP1 significantly enhanced the promoting activity of seven promoters, indicating that PAP1 turned on the biosynthesis of anthocyanins via the activation of these pathway gene expression. To understand how 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin, regulates the PAP1-activated anthocyanin biosynthesis, five different concentrations (0, 0.05, 0.5, 2.5, and 5 µM) were tested to characterize anthocyanin production and profiles. The resulting data showed that the concentrations tested decreased the fresh weight of callus growth, anthocyanin levels, and the production of anthocyanins per Petri dish. HPLC-qTOF-MS/MS-based profiling showed that these concentrations did not alter anthocyanin profiles. Real-time RT-PCR was completed to characterize the expression PAP1 and four representative pathway genes. The results showed that the five concentrations reduced the expression levels of the constitutive PAP1 transgene and three pathway genes significantly and eliminated the expression of the chalcone synthase gene either significantly or slightly. These data indicate that the constitutive PAP1 expression depends on gradients added in the medium. Based on these findings, the regulation of 2,4-D is discussed for anthocyanin engineering in red cells of A. annua.

Identification and biosynthesis of plant papanridins, a group of novel oligomeric flavonoids.

The discovery of novel flavonoids and elucidation of their biosynthesis are fundamental to understanding their roles in plants and their benefits for human and animal health. Here, we report a new pathway for polymerization of a group of novel oligomeric flavonoids in plants. We engineered red cells for discovering genes of interest involved in the flavonoid pathway and identified a gene encoding a novel flavanol polymerase (FP) localized in the central vacuole. FP catalyzes the polymerization of flavanols, such as epicatechin and catechin, to produce yellowish dimers or oligomers. Structural elucidation shows that these compounds feature a novel oligomeric flaven-flavan (FF) skeleton linked by interflavan-flaven and interflaven bonds, distinguishing them from proanthocyanidins and dehydrodicatechins. Detailed chemical and physical characterizations further confirmed the novel FFs as flavonoids. Mechanistic investigations demonstrated that FP polymerizes flavan-3-ols and flav-2-en-3-ol carbocation, forming dimeric or oligomeric flaven-4→8-flavans, which we term "papanridins." Data from transgenic experiments, mutant analysis, metabolic profiling, and phylogenetic analyses show that the biosynthesis of papanridins is prevalent in cacao, grape, blueberry, corn, rice, Arabidopsis, and other species in the plant kingdom. In summary, our study discoveries a group of novel oligomeric flavonoids, namely papanridins, and reveals that a novel FP-mediated polymerization mechanism for the biosynthesis of papanridins in plants.

Metabolic engineering of the anthocyanin biosynthetic pathway in Artemisia annua and relation to the expression of the artemisinin biosynthetic pathway.

MAIN CONCLUSION: Four types of cells were engineered from Artemisia annua to produce approximately 17 anthocyanins, four of which were elucidated structurally. All of them expressed the artemisinin pathway. Artemisia annua is the only medicinal crop to produce artemisinin for the treatment of malignant malaria. Unfortunately, hundreds of thousands of people still lose their life every year due to the lack of sufficient artemisinin. Artemisinin is considered to result from the spontaneous autoxidation of dihydroartemisinic acid in the presence of reactive oxygen species (ROS) in an oxidative condition of glandular trichomes (GTs); however, whether increasing antioxidative compounds can inhibit artemisinin biosynthesis in plant cells is unknown. Anthocyanins are potent antioxidants that can remove ROS in plant cells. To date, no anthocyanins have been structurally elucidated from A. annua. In this study, we had two goals: (1) to engineer anthocyanins in A. annua cells and (2) to understand the artemisinin biosynthesis in anthocyanin-producing cells. Arabidopsis Production of Anthocyanin Pigment 1 was used to engineer four types of transgenic anthocyanin-producing A. annua (TAPA1-4) cells. Three wild-type cell types were developed as controls. TAPA1 cells produced the highest contents of total anthocyanins. LC-MS analysis detected 17 anthocyanin or anthocyanidin compounds. Crystallization, LC/MS/MS, and NMR analyses identified cyanidin, pelargonidin, one cyanin, and one pelargonin. An integrative analysis characterized that four types of TAPA cells expressed the artemisinin pathway and TAPA1 cells produced the highest artemisinin and artemisinic acid. The contents of arteannuin B were similar in seven cell types. These data showed that the engineering of anthocyanins does not eliminate the biosynthesis of artemisinin in cells. These data allow us to propose a new hypothesis that enzymes catalyze the formation of artemisinin from dihydroartemisinic acid in non-GT cells. These findings show a new platform to increase artemisinin production via non-GT cells of A. annua.

Transcriptional and Metabolic Characterization of Feeding Ramie Growth Enhanced by a Combined Application of Gibberellin and Ethrel.

Feeding ramie cultivars (Boehmaria nivea L.) are an important feedstock for livestock. Increasing their biomass and improving their nutritional values are essential for animal feeding. Gibberellin (GA3) and ethylene (ETH) are two plant hormones that regulate the growth, development, and metabolism of plants. Herein, we report effects of the GA3 and ETH application on the growth and plant metabolism of feeding ramie in the field. A combination of GA3 and ETH was designed to spray new plants. The two hormones enhanced the growth of plants to produce more biomass. Meanwhile, the two hormones reduced the contents of lignin in leaves and stems, while increased the content of flavonoids in leaves. To understand the potential mechanisms behind these results, we used RNA-seq-based transcriptomics and UPLC-MS/MS-based metabolomics to characterize gene expression and metabolite profiles associated with the treatment of GA3 and ETH. 1562 and 2364 differentially expressed genes (DEGs) were obtained from leaves and stems (treated versus control), respectively. Meanwhile, 99 and 88 differentially accumulated metabolites (DAMs) were annotated from treated versus control leaves and treated versus control stems, respectively. Data mining revealed that both DEGs and DAMs were associated with multiple plant metabolisms, especially plant secondary metabolism. A specific focus on the plant phenylpropanoid pathway identified candidates of DEGs and DEMs that were associated with lignin and flavonoid biosynthesis. Shikimate hydroxycinnamoyl transferase (HCT) is a key enzyme that is involved in the lignin biosynthesis. The gene encoding B. nivea HCT was downregulated in the treated leaves and stems. In addition, genes encoding 4-coumaryl CoA ligase (4CL) and trans-cinnamate 4-monooxygenase (CYP73A), two lignin pathway enzymes, were downregulated in the treated stems. Meanwhile, the reduction in lignin in the treated leaves led to an increase in cinnamic acid and p-coumaryl CoA, two shared substrates of flavonoids that are enhanced in contents. Taken together, these findings indicated that an appropriate combination of GA3 and ETH is an effective strategy to enhance plant growth via altering gene expression and plant secondary metabolism for biomass-enhanced and value-improved feeding ramie.

A de novo regulation design shows an effectiveness in altering plant secondary metabolism.

Introduction: Transcription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants. Objectives: We selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering. Methods: G-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism. Results: PAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes. Conclusion: G-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

Molecular and biochemical characterization of two 4-coumarate: CoA ligase genes in tea plant (Camellia sinensis).

KEY MESSAGE: Two 4-coumarate: CoA ligase genes in tea plant involved in phenylpropanoids biosynthesis and response to environmental stresses. Tea plant is rich in flavonoids benefiting human health. Lignin is essential for tea plant growth. Both flavonoids and lignin defend plants from stresses. The biosynthesis of lignin and flavonoids shares a key intermediate, 4-coumaroyl-CoA, which is formed from 4-coumaric acid catalyzed by 4-coumaric acid: CoA ligase (4CL). Herein, we report two 4CL paralogs from tea plant, Cs4CL1 and Cs4CL2, which are a member of class I and II of this gene family, respectively. Cs4CL1 was mainly expressed in roots and stems, while Cs4CL2 was mainly expressed in leaves. The promoter of Cs4CL1 had AC, nine types of light sensitive (LSE), four types of stress-inducible (SIE), and two types of meristem-specific elements (MSE). The promoter of Cs4CL2 also had AC and nine types of LSEs, but only had two types of SIEs and did not have MSEs. In addition, the LSEs varied in the two promoters. Based on the different features of regulatory elements, three stress treatments were tested to understand their expression responses to different conditions. The resulting data indicated that the expression of Cs4CL1 was sensitive to mechanical wounding, while the expression of Cs4CL2 was UV-B-inducible. Enzymatic assays showed that both recombinant Cs4CL1 and Cs4CL2 transformed 4-coumaric acid (CM), ferulic acid (FR), and caffeic acid (CF) to their corresponding CoA ethers. Kinetic analysis indicated that the recombinant Cs4CL1 preferred to catalyze CF, while the recombinant Cs4CL2 favored to catalyze CM. The overexpression of both Cs4CL1 and Cs4CL2 increased the levels of chlorogenic acid and total lignin in transgenic tobacco seedlings. In addition, the overexpression of Cs4CL2 consistently increased the levels of three flavonoid compounds. These findings indicate the differences of Cs4CL1 and Cs4CL2 in the phenylpropanoid metabolism.

Flavonols and dihydroflavonols inhibit the main protease activity of SARS-CoV-2 and the replication of human coronavirus 229E.

Since December 2019, the deadly novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the current COVID-19 pandemic. To date, vaccines are available in the developed countries to prevent the infection of this virus; however, medicines are necessary to help control COVID-19. Human coronavirus 229E (HCoV-229E) causes the common cold. The main protease (Mpro) is an essential enzyme required for the multiplication of these two viruses in the host cells, and thus is an appropriate candidate to screen potential medicinal compounds. Flavonols and dihydroflavonols are two groups of plant flavonoids. In this study, we report docking simulation with two Mpro enzymes and five flavonols and three dihydroflavonols, in vitro inhibition of the SARS-CoV-2 Mpro, and in vitro inhibition of the HCoV 229E replication. The docking simulation results predicted that (+)-dihydrokaempferol, (+)- dihydroquercetin, (+)-dihydromyricetin, kaempferol, quercetin, myricentin, isoquercitrin, and rutin could bind to at least two subsites (S1, S1', S2, and S4) in the binding pocket and inhibit the activity of SARS-CoV-2 Mpro. Their affinity scores ranged from -8.8 to -7.4 (kcal/mol). Likewise, these compounds were predicted to bind and inhibit the HCoV-229E Mpro activity with affinity scores ranging from -7.1 to -7.8 (kcal/mol). In vitro inhibition assays showed that seven available compounds effectively inhibited the SARS-CoV-2 Mpro activity and their IC50 values ranged from 0.125 to 12.9 µM. Five compounds inhibited the replication of HCoV-229E in Huh-7 cells. These findings indicate that these antioxidative flavonols and dihydroflavonols are promising candidates for curbing the two viruses.

Insights into acylation mechanisms: co-expression of serine carboxypeptidase-like acyltransferases and their non-catalytic companion paralogs.

Serine carboxypeptidase-like acyltransferases (SCPL-ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan-3-ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL-AT paralogs are involved in galloylation of flavan-3-ols: CsSCPL4, which contains the conserved catalytic triad S-D-H, and CsSCPL5, which has the alternative triad T-D-Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non-catalytic companion paralog (NCCP). Co-expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull-down and co-immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post-translational processing. Moreover, phylogenetic analyses revealed that their homologs co-exist in galloylated flavan-3-ol- or hydrolyzable tannin-rich plant species. Enzymatic assays further revealed the necessity of co-expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co-expression of SCPL-ATs and their NCCPs contributes to the acylation of flavan-3-ols in the plant kingdom.

Alternative splicing of CsJAZ1 negatively regulates flavan-3-ol biosynthesis in tea plants.

Flavan-3-ols are abundant in the tea plant (Camellia sinensis) and confer tea with flavor and health benefits. We recently found that alternative splicing of genes is likely involved in the regulation of flavan-3-ol biosynthesis; however, the underlying regulatory mechanisms remain unknown. Here, we integrated metabolomics and transcriptomics to construct metabolite-gene networks in tea leaves, collected over five different months and from five spatial positions, and found positive correlations between endogenous jasmonic acid (JA), flavan-3-ols, and numerous transcripts. Transcriptome mining further identified CsJAZ1, which is negatively associated with flavan-3-ols formation and has three CsJAZ1 transcripts, one full-length (CsJAZ1-1), and two splice variants (CsJAZ1-2 and -3) that lacked 3' coding sequences, with CsJAZ1-3 also lacking the coding region for the Jas domain. Confocal microscopy showed that CsJAZ1-1 was localized to the nucleus, while CsJAZ1-2 and CsJAZ1-3 were present in both the nucleus and the cytosol. In the absence of JA, CsJAZ1-1 was bound to CsMYC2, a positive regulator of flavan-3-ol biosynthesis; CsJAZ1-2 functioned as an alternative enhancer of CsJAZ1-1 and an antagonist of CsJAZ1-1 in binding to CsMYC2; and CsJAZ1-3 did not interact with CsMYC2. In the presence of JA, CsJAZ1-3 interacted with CsJAZ1-1 and CsJAZ1-2 to form heterodimers that stabilized the CsJAZ1-1-CsMYC2 and CsJAZ1-2-CsMYC2 complexes, thereby repressing the transcription of four genes that act late in the flavan-3-ol biosynthetic pathway. These data indicate that the alternative splicing variants of CsJAZ1 coordinately regulate flavan-3-ol biosynthesis in the tea plant and improve our understanding of JA-mediated flavan-3-ol biosynthesis.

Docking Characterization and in vitro Inhibitory Activity of Flavan-3-ols and Dimeric Proanthocyanidins Against the Main Protease Activity of SARS-Cov-2.

We report to use the main protease (Mpro) of SARS-Cov-2 to screen plant flavan-3-ols and proanthocyanidins. Twelve compounds, (-)-afzelechin (AF), (-)-epiafzelechin (EAF), (+)-catechin (CA), (-)-epicatechin (EC), (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (+)-catechin-3-O-gallate (CAG), (-)-epicatechin-3-O-gallate (ECG), (-)-gallocatechin-3-O-gallate (GCG), (-)-epigallocatechin-3-O-gallate (EGCG), procyanidin A2 (PA2), and procyanidin B2 (PB2), were selected for docking simulation. The resulting data predicted that all 12 metabolites could bind to Mpro. The affinity scores of PA2 and PB2 were predicted to be -9.2, followed by ECG, GCG, EGCG, and CAG, -8.3 to -8.7, and then six flavan-3-ol aglycones, -7.0 to -7.7. Docking characterization predicted that these compounds bound to three or four subsites (S1, S1', S2, and S4) in the binding pocket of Mpro via different spatial ways and various formation of one to four hydrogen bonds. In vitro analysis with 10 available compounds showed that CAG, ECG, GCG, EGCG, and PB2 inhibited the Mpro activity with an IC50 value, 2.98 ± 0.21, 5.21 ± 0.5, 6.38 ± 0.5, 7.51 ± 0.21, and 75.3 ± 1.29 µM, respectively, while CA, EC, EGC, GC, and PA2 did not have inhibitory activities. To further substantiate the inhibitory activities, extracts prepared from green tea (GT), two muscadine grapes (MG), cacao, and dark chocolate (DC), which are rich in CAG, ECG, GAG, EGCG, or/and PB2, were used for inhibitory assay. The resulting data showed that GT, two MG, cacao, and DC extracts inhibited the Mpro activity with an IC50 value, 2.84 ± 0.25, 29.54 ± 0.41, 29.93 ± 0.83, 153.3 ± 47.3, and 256.39 ± 66.3 µg/ml, respectively. These findings indicate that on the one hand, the structural features of flavan-3-ols are closely associated with the affinity scores; on the other hand, the galloylation and oligomeric types of flavan-3-ols are critical in creating the inhibitory activity against the Mpro activity.

Functional demonstration of plant flavonoid carbocations proposed to be involved in the biosynthesis of proanthocyanidins.

The plant flavonoid dogma proposes that labile plant flavonoid carbocations (PFCs) play vital roles in the biosynthesis of proanthocyanidins (PAs). However, whether PFCs exist in plants and how PFCs function remain unclear. Here, we report the use of an integrative strategy including enzymatic assays, mutant analysis, metabolic engineering, isotope labeling and metabolic profiling to capture PFCs and demonstrate their functions. In anthocyanidin reductase (ANR) assays, an (-)-epicatechin conjugate was captured in protic polar nucleophilic methanol alone or methanol-HCl extracts. Tandem mass spectrum (MS/MS) analysis characterized this compound as an (-)-epicatechin-4-O-methyl (EOM) ether, which resulted from (-)-epicatechin carbocation and the methyl group of methanol. Acid-based catalysis of procyanidin B2 and B3 produced four compounds, which were annotated as two EOM and two (+)-catechin-4-O-methyl (COM) ethers. Metabolic profiling of seven PA pathway mutants showed an absence or reduction of two EOM ether isomers in seeds. Camellia sinensis ANRa (CsANRa), leucoanthocyanidin reductase c (CsLARc), and CsMYB5b (a transcription factor) were independently overexpressed for successful PA engineering in tobacco. The EOM ether was remarkably increased in CsANRa and CsMYB5b transgenic flowers. Further metabolic profiling for eight green tea tissues revealed two EOM and two COM ethers associated with PA biosynthesis. Moreover, an incubation of (-)-epicatechin or (+)-catechin with epicatechin carbocation in CsANRa transgenic flower extracts formed dimeric procyanidin B1 or B2, demonstrating the role of flavan-3-ol carbocation in the formation of PAs. Taken together, these findings indicated that flavan-3-ol carbocations exist in extracts and are involved in the biosynthesis of PAs of plants.

Untargeted Metabolomics of Nicotiana tabacum Grown in United States and India Characterizes the Association of Plant Metabolomes With Natural Climate and Geography.

Climate change and geography affect all the living organisms. To date, the effects of climate and geographical factors on plant metabolome largely remain open for worldwide and local investigations. In this study, we designed field experiments with tobacco (Nicotiana tabacum) in India versus USA and used untargeted metabolomics to understand the association of two weather factors and two different continental locations with respect to tobacco metabolism. Field research stations in Oxford, North Carolina, USA, and Rajahmundry, Andhra Pradesh India were selected to grow a commercial tobacco genotype (K326) for 2 years. Plant growth, field management, and leaf curing followed protocols standardized for tobacco cultivation. Gas chromatography-mass spectrometry based unbiased profiling annotated 171 non-polar and 225 polar metabolites from cured tobacco leaves. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed that two growing years and two field locations played primary and secondary roles affecting metabolite profiles, respectively. PCA and Pearson analysis, which used nicotine, 11 other groups of metabolites, two locations, temperatures, and precipitation, revealed that in North Carolina, temperature changes were positively associated with the profiles of sesquiterpenes, diterpenes, and triterpenes, but negatively associated with the profiles of nicotine, organic acids of tricarboxylic acid, and sugars; in addition, precipitation was positively associated with the profiles of triterpenes. In India, temperature was positively associated with the profiles of benzenes and polycyclic aromatic hydrocarbons, but negatively associated with the profiles of amino acids and sugar. Further comparative analysis revealed that nicotine levels were affected by weather conditions, nevertheless, its trend in leaves was independent of two geographical locations and weather changes. All these findings suggested that climate and geographical variation significantly differentiated the tobacco metabolism.

A polyketide synthase gene cluster associated with the sexual reproductive cycle of the banana pathogen, Pseudocercospora fijiensis.

Disease spread of Pseudocercospora fijiensis, causal agent of the black Sigatoka disease of banana, depends on ascospores produced through the sexual reproductive cycle. We used phylogenetic analysis to identify P. fijiensis homologs (PKS8-4 and Hybrid8-3) to the PKS4 polyketide synthases (PKS) from Neurospora crassa and Sordaria macrospora involved in sexual reproduction. These sequences also formed a clade with lovastatin, compactin, and betaenone-producing PKS sequences. Transcriptome analysis showed that both the P. fijiensis Hybrid8-3 and PKS8-4 genes have higher expression in infected leaf tissue compared to in culture. Domain analysis showed that PKS8-4 is more similar than Hybrid8-3 to PKS4. pPKS8-4:GFP transcriptional fusion transformants showed expression of GFP in flask-shaped structures in mycelial cultures as well as in crosses between compatible and incompatible mating types. Confocal microscopy confirmed expression in spermagonia in leaf substomatal cavities, consistent with a role in sexual reproduction. A disruption mutant of pks8-4 retained normal pathogenicity on banana, and no differences were observed in growth, conidial production, and spermagonia production. GC-MS profiling of the mutant and wild type did not identify differences in polyketide metabolites, but did identify changes in saturated fatty acid methyl esters and alkene and alkane derivatives. To our knowledge, this is the first report of a polyketide synthase pathway associated with spermagonia.

Artemisinin Biosynthesis in Non-glandular Trichome Cells of Artemisia annua.

Artemisinin-based combination therapy (ACT) forms the first line of malaria treatment. However, the yield fluctuation of artemisinin has remained an unsolved problem in meeting the global demand for ACT. This problem is mainly caused by the glandular trichome (GT)-specific biosynthesis of artemisinin in all currently used Artemisia annua cultivars. Here, we report that non-GT cells of self-pollinated inbred A. annua plants can express the artemisinin biosynthetic pathway. Gene expression analysis demonstrated the transcription of six known pathway genes in GT-free leaves and calli of inbred A. annua plants. LC-qTOF-MS/MS analysis showed that these two types of GT-free materials produce artemisinin, artemisinic acid, and arteannuin B. Detailed IR-MALDESI image profiling revealed that these three metabolites and dihydroartemisinin are localized in non-GT cells of leaves of inbred A. annua plants. Moreover, we employed all the above approaches to examine artemisinin biosynthesis in the reported A. annua glandless (gl) mutant. The resulting data demonstrated that leaves of regenerated gl plantlets biosynthesize artemisinin. Collectively, these findings not only add new knowledge leading to a revision of the current dogma of artemisinin biosynthesis in A. annua but also may expedite innovation of novel metabolic engineering approaches for high and stable production of artemisinin in the future.

Evaluation of Digital Image Recognition Methods for Mass Spectrometry Imaging Data Analysis.

Analyzing mass spectrometry imaging data can be laborious and time consuming, and as the size and complexity of datasets grow, so does the need for robust automated processing methods. We here present a method for comprehensive, semi-targeted discovery of molecular distributions of interest from mass spectrometry imaging data, using widely available image similarity scoring algorithms to rank images by spatial correlation. A fast and powerful batch search method using a MATLAB implementation of structural similarity (SSIM) index scoring with a pre-selected reference distribution is demonstrated for two sample imaging datasets, a plant metabolite study using Artemisia annua leaf, and a drug distribution study using maraviroc-dosed macaque tissue. Graphical Abstract ᅟ.

HPLC-qTOF-MS/MS-Based Profiling of Flavan-3-ols and Dimeric Proanthocyanidins in Berries of Two Muscadine Grape Hybrids FLH 13-11 and FLH 17-66.

FLH 13-11 FL and FLH 17-66 FL are two interspecific hybrid varieties of muscadine grape resulting from the cross of Vitis munsoniana (Simpson) ex Munson and V. rotundifolia. However, profiles of flavan-3-ols and proanthocyanidins in these two hybrids have not been characterized. Herein, we report the use of high-performance liquid chromatography-quadrupole, time-of-flight, tandem mass spectrometry (HPLC-qTOF-MS/MS) to characterize these two groups of metabolites in berries. Ripe berries collected from two consecutive cropping years were used to extract metabolites. Metabolites were ionized using the negative mode. Collision-induced dissociation was performed to fragmentize ions to obtain feature fragment profiles. Based on standards, MS features, and fragments resulted from MS/MS, four flavan-3-ol aglycones, 18 gallated or glycosylated conjugates, and eight dimeric procyanidins, were annotated from berry extracts. Of these 30 metabolites, six are new methylated flavan-3-ol gallates. Furthermore, comparative profiling analysis showed obvious effects of each cultivar on the composition these 30 metabolites, indicating that genotypes control biosynthesis. In addition, cropping seasons altered profiles of these metabolites, showing effects of growing years on metabolic composition. These data are informative to enhance the application of the two cultivars in grape and wine industries in the future.

Overexpression of Artemisia annua Cinnamyl Alcohol Dehydrogenase Increases Lignin and Coumarin and Reduces Artemisinin and Other Sesquiterpenes.

Artemisia annua is the only medicinal crop that produces artemisinin for malarial treatment. Herein, we describe the cloning of a cinnamyl alcohol dehydrogenase (AaCAD) from an inbred self-pollinating (SP) A. annua cultivar and its effects on lignin and artemisinin production. A recombinant AaCAD was purified via heterogeneous expression. Enzyme assays showed that the recombinant AaCAD converted p-coumaryl, coniferyl, and sinapyl aldehydes to their corresponding alcohols, which are key intermediates involved in the biosynthesis of lignin. Km, Vmax, and Vmax/Km values were calculated for all three substrates. To characterize its function in planta, AaCAD was overexpressed in SP plants. Quantification using acetyl bromide (AcBr) showed significantly higher lignin contents in transgenics compared with wild-type (WT) plants. Moreover, GC-MS-based profiling revealed a significant increase in coumarin contents in transgenic plants. By contrast, HPLC-MS analysis showed significantly reduced artemisinin contents in transgenics compared with WT plants. Furthermore, GC-MS analysis revealed a decrease in the contents of arteannuin B and six other sesquiterpenes in transgenic plants. Confocal microscopy analysis showed the cytosolic localization of AaCAD. These data demonstrate that AaCAD plays a dual pathway function in the cytosol, in which it positively enhances lignin formation but negatively controls artemisinin formation. Based on these data, crosstalk between these two pathways mediated by AaCAD catalysis is discussed to understand the metabolic control of artemisinin biosynthesis in plants for high production.

Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula.

Vitis bellula is a new grape crop in southern China. Berries of this species are rich in antioxidative anthocyanins and proanthocyanidins. This study reports cloning and functional characterization of a cDNA encoding a V. bellula dihydroflavonol reductase (VbDFR) involved in the biosynthesis of anthocyanins and proanthocyanidins. A cDNA including 1014 bp was cloned from young leaves and its open reading frame (ORF) was deduced encoding 337 amino acids, highly similar to V. vinifera DFR (VvDFR). Green florescence protein fusion and confocal microscopy analysis determined the cytosolic localization of VbDFR in plant cells. A soluble recombinant VbDFR was induced and purified from E. coli for enzyme assay. In the presence of NADPH, the recombinant enzyme catalyzed dihydrokaempferol (DHK) and dihydroquercetin (DHQ) to their corresponding leucoanthocyanidins. The VbDFR cDNA was introduced into tobacco plants via Agrobacterium-mediated transformation. The overexpression of VbDFR increased anthocyanin production in flowers. Anthocyanin hydrolysis and chromatographic analysis revealed that transgenic flowers produced pelargonidin and delphinidin, which were not detected in control flowers. These data demonstrated that the overexpression of VbDFR produced new tobacco anthocyanidins. In summary, all data demonstrate that VbDFR is a useful gene to provide three types of substrates for metabolic engineering of anthocyanins and proanthocyanidins in grape crops and other crops.