A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis.

SignificanceMethanobactins (Mbns), copper-binding peptidic compounds produced by some bacteria, are candidate therapeutics for human diseases of copper overload. The paired oxazolone-thioamide bidentate ligands of methanobactins are generated from cysteine residues in a precursor peptide, MbnA, by the MbnBC enzyme complex. MbnBC activity depends on the presence of iron and oxygen, but the catalytically active form has not been identified. Here, we provide evidence that a dinuclear Fe(II)Fe(III) center in MbnB, which is the only representative of a >13,000-member protein family to be characterized, is responsible for this reaction. These findings expand the known roles of diiron enzymes in biology and set the stage for mechanistic understanding, and ultimately engineering, of the MbnBC biosynthetic complex.

Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC.

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

Myrosinase isogenes in wasabi (Wasabia japonica Matsum) and their putative roles in glucosinolate metabolism.

BACKGROUND: Wasabi, a Brassicaceae member, is well-known for its unique pungent and hot flavor which is produced from glucosinolate (GSL) degradation. Myrosinase (MYR) is a principle enzyme catalyzing the primary conversion of GSLs to GSL hydrolysis products (GHPs) which is responsible for plant defense system and food quality. Due to the limited information in relation to MYRs present in wasabi (Wasabia japonica M.), this study aimed to identify the MYR isogenes in W. japonica and analyze their roles in relation to GSL metabolism. RESULTS: In results, WjMYRI-1 was abundantly expressed in all organs, whereas WjMYRI-2 showed only trace expression levels. WjMYRII was highly expressed in the aboveground tissues. Interestingly, WjMYRII expression was significantly upregulated by certain abiotic factors, such as methyl jasmonate (more than 40-fold in petioles and 15-fold in leaves) and salt (tenfold in leaves). Young leaves and roots contained 97.89 and 91.17 µmol‧g-1 of GSL, whereas less GSL was produced in mature leaves and petioles (38.36 and 44.79 µmol‧g-1, respectively). Similar pattern was observed in the accumulation of GHPs in various plant organs. Notably, despite the non-significant changes in GSL production, abiotic factors treated samples enhanced significantly GHP content. Pearson's correlation analysis revealed that WjMYRI-1 expression significantly correlated with GSL accumulation and GHP formation, suggesting the primary role of WjMYRI-1-encoding putative protein in GSL degradation. In contrast, WjMYRII expression level showed no correlation with GSL or GHP content, suggesting another physiological role of WjMYRII in stress-induced response. CONCLUSIONS: In conclusions, three potential isogenes (WjMYRI-1, WjMYRI-2, and WjMYRII) encoding for different MYR isoforms in W. japonica were identified. Our results provided new insights related to MYR and GSL metabolism which are important for the implications of wasabi in agriculture, food and pharmaceutical industry. Particularly, WjMYRI-1 may be primarily responsible for GSL degradation, whereas WjMYRII (clade II) may be involved in other regulatory pathways induced by abiotic factors.

Understanding the Impact of Nitrogen Availability: A Limiting Factor for Enhancing Fucoxanthin Productivity in Microalgae Cultivation.

This study aimed to investigate the regulation of fucoxanthin (FX) biosynthesis under various nitrogen conditions to optimize FX productivity in Phaeodactylum tricornutum. Apart from light, nitrogen availability significantly affects the FX production of microalgae; however, the underlying mechanism remains unclear. In batch culture, P. tricornutum was cultivated with normal (NN, 0.882 mM sodium nitrate), limited (LN, 0.22 mM), and high (HN, 8.82 mM) initial nitrogen concentrations in f/2 medium. Microalgal growth and photosynthetic pigment production were examined, and day 5 samples were subjected to fucoxanthin-chlorophyll a/c-binding protein (FCP) proteomic and transcriptomic analyses. The result demonstrated that HN promoted FX productivity by extending the exponential growth phase for higher biomass and FX accumulation stage (P1), showing a continuous increase in FX accumulation on day 6. Augmented FX biosynthesis via the upregulation of carotenogenesis could be primarily attributed to enhanced FCP formation in the thylakoid membrane. Key proteins, such as LHC3/4, LHCF8, LHCF5, and LHCF10, and key genes, such as PtPSY, PtPDS, and PtVDE, were upregulated under nitrogen repletion. Finally, the combination of low light and HN prolonged the P1 stage to day 10, resulting in maximal FX productivity to 9.82 ± 0.56 mg/L/day, demonstrating an effective strategy for enhancing FX production in microalgae cultivation.

Secondary Coordination Sphere Influences the Formation of Fe(III)-O or Fe(III)-OH in Nitrite Reduction: A Synthetic and Computational Study.

The reduction of nitrite (NO2-) to generate nitric oxide (NO) is a significant area of research due to their roles in the global nitrogen cycle. Here, we describe various modifications of the tris(5-cyclohexyliminopyrrol-2-ylmethyl)amine H3[N(piR)3] ligand where the steric bulk and acidity of the secondary coordination sphere were explored in the non-heme iron system for nitrite reduction. The cyclohexyl and 2,4,6-trimethylphenyl variants of the ligand were used to probe the mechanism of nitrite reduction. While previously stoichiometric addition of nitrite to the iron(II)-species generated an iron(III)-oxo complex, changing the secondary coordination sphere to mesityl resulted in an iron(III)-hydroxo complex. Subsequent addition of an electron and two protons led to the release of water and regeneration of the starting iron(II) catalyst. This sequence mirrored the proposed mechanism of nitrite reduction in biological systems, where the distal histidine residue shuttles protons to the active site. Computational studies aimed at interrogating the dissimilar behavior of the cyclohexyl and mesityl ligand systems resulting in Fe(III)-oxo and Fe(III)-hydroxo complexes, respectively, shed light on the key role of H-bonds involving the secondary coordination sphere in the relative stability of these species.

Impact of copper treatment on phenylpropanoid biosynthesis in adventitious root culture of Althaea officinalis L.

Althaea officinalis has been widely used in various pharmaceutical applications. The biological effects and significance of phenylpropanoids in numerous industries are well studied. However, fulfilling consumer demand for these commercially important compounds is difficult. The effect of heavy-metal toxic influence on plants is primarily due to a strong and rapid suppression of growth processes, as well as the decline in activity of the photosynthetic apparatus, also associated with progressing senescence processes. Some of the secondary metabolite production was triggered by the application of heavy metals, but there was not a stress response. In the adventitious root culture of A. officinalis, copper-mediated phenylpropanoid biosynthesis has been investigated in both concentration-and duration-dependent manners. High-performance liquid chromatography (HPLC) analysis revealed a total of nine different phenolic compounds in response to different concentrations of copper chloride. In this study, high productivity of phenolic compounds was observed in the copper chloride treated-adventitious root culture of A. officianalis. In particular, a low concentration of copper chloride led to a significant accumulation of phenolic compounds under optimal conditions. Moreover, all genes responsible for phenylpropanoid biosynthesis may be sensitive to phenolic compound production following copper treatment. Especially, the highest change in transcript level was observed from AoANS at 6 h. According to our findings, treatment with copper chloride (0.5 mM) for 48 or 96 h can be an appropriate method to maximize phenylpropanoid levels in A. officinalis adventitious root culture.

Characterization of a Copper-Chelating Natural Product from the Methanotroph Methylosinus sp. LW3.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified peptidic natural products that bind copper with high affinity. Methanotrophic bacteria use Mbns to acquire copper needed for enzymatic methane oxidation. Despite the presence of Mbn operons in a range of methanotroph and other bacterial genomes, few Mbns have been isolated and structurally characterized. Here we report the isolation of a novel Mbn from the methanotroph Methylosinus (Ms.) sp. LW3. Mass spectrometric and nuclear magnetic resonance spectroscopic data indicate that this Mbn, the largest characterized to date, consists of a 13-amino acid backbone modified to include pyrazinedione/oxazolone rings and neighboring thioamide groups derived from cysteine residues. The pyrazinedione ring is more stable to acid hydrolysis than the oxazolone ring and likely protects the Mbn from degradation. The structure corresponds exactly to that predicted on the basis of the Ms. sp. LW3 Mbn operon content, providing support for the proposed role of an uncharacterized biosynthetic enzyme, MbnF, and expanding the diversity of known Mbns.

Yeast extract improved biosynthesis of astragalosides in hairy root cultures of Astragalus membranaceus.

The dried root of Astragalus membranaceus is a well-known herbal medicine, and it is useful in treating chronic diseases and weakness, as well as for improving overall health and vitality. Astragalosides, which are root quality indicators of A. membranaceus, are natural triterpenoid saponins that are used in the treatment of diabetes and cardiovascular diseases. Currently, there is an urgent need to improve their production because of their low quantity in plants and the difficulty of chemical synthesis. In this study, yeast extract was added to facilitate elicitation in Agrobacterium-mediated hairy root cultures, thereby enhancing astragaloside production in A. membranaceus. Results showed that yeast extract could stimulate astragaloside content effectively in the hairy roots of A. membranaceus. Moreover, astragaloside accumulation was positively correlated with the upregulation of mevalonate biosynthetic gene expression in the presence of yeast extract. Our study demonstrated that pretreatment with yeast extract (3.65 mM) for 72 h serves as an effective strategy to enhance astragaloside levels in A. membranaceus hairy root cultures. Thus, these optimal conditions can provide valuable information for the improvement of astragaloside industrial production in A. membranaceus.

Metabolic Profiling of Nine Mentha Species and Prediction of Their Antioxidant Properties Using Chemometrics.

Mentha species are well recognized for their medicinal and aromatic properties. The comprehensive metabolite profiles of nine Mentha species have been determined. The extracts of these Mentha species were also screened for antioxidant and free radical scavenging activities. Forty-seven hydrophilic and seventeen lipophilic compounds were identified and quantified from the selected Mentha species. Also, eleven phenolic compounds, riboflavin and eight carotenoids were present, and their composition and content varied among the various Mentha species. The different Mentha species exhibited a range of antioxidant potencies. Horse mint especially exhibited the strongest antioxidant capacities (1,1-diphenyl-2-picryl-hydrazyl (DPPH), hydrogen peroxide, and reducing power assay) among the nine Mentha species. A difference between different samples from the same species was not observed by multivariate analysis. A high correlation between metabolites involved in closely linked biosynthetic pathways has been indicated. The projection to latent structure method, using the partial least squares (PLS) method, was applied to predict antioxidant capacities based on the metabolite profiles of Mentha leaves. According to the PLS analysis, several carotenoid contents, such as E-ß-carotene, 9Z-ß-carotene, 13Z-ß-carotene and lutein, as well as phenolic compounds, showed a positive relationship in reducing the power of Mentha extracts. Horse mint is a good candidate because of its high antioxidant efficacy among the nine Mentha species included in the study.

Repurposed HisC Aminotransferases Complete the Biosynthesis of Some Methanobactins.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified bacterial natural products with a high affinity for copper. MbnN, a pyridoxal 5'-phosphate-dependent aminotransferase, performs a transamination reaction that is the last step in the biosynthesis of Mbns produced by several Methylosinus species. Our bioinformatic analyses indicate that MbnNs likely derive from histidinol-phosphate aminotransferases (HisCs), which play a key role in histidine biosynthesis. A comparison of the HisC active site with the predicted MbnN structure suggests that MbnN's active site is altered to accommodate the larger and more hydrophobic substrates necessary for Mbn biosynthesis. Moreover, we have confirmed that MbnN is capable of catalyzing the final transamination step in Mbn biosynthesis in vitro and in vivo. We also demonstrate that without this final modification, Mbn exhibits significantly decreased stability under physiological conditions. An examination of other Mbns and Mbn operons suggests that N-terminal protection of this family of natural products is of critical importance and that several different means of N-terminal stabilization have evolved independently in Mbn subfamilies.

Identification and Characterization of Phenylpropanoid Biosynthetic Genes and Their Accumulation in Bitter Melon (Momordica charantia).

Phenylpropanoids and flavonoids belong to a large group of secondary metabolites, and are considered to have antioxidant activity, which protects the cells against biotic and abiotic stresses. However, the accumulation of phenylpropanoids and flavonoids in bitter melon has rarely been studied. Here, we identify ten putative phenylpropanoid and flavonoid biosynthetic genes in bitter melon. Most genes were highly expressed in leaves and/or flowers. HPLC analysis showed that rutin and epicatechin were the most abundant compounds in bitter melon. Rutin content was the highest in leaves, whereas epicatechin was highly accumulated in flowers and fruits. The accumulation patterns of trans-cinnamic acid, p-coumaric acid, ferulic acid, kaempferol, and rutin coincide with the expression patterns of McPAL, McC4H, McCOMT, McFLS, and Mc3GT, respectively, suggesting that these genes play important roles in phenylpropanoid and flavonoid biosynthesis in bitter melon. In addition, we also investigated the optimum light conditions for enhancing phenylpropanoid and flavonoid biosynthesis and found that blue light was the most effective wavelength for enhanced accumulation of phenylpropanoids and flavonoids in bitter melon.

Influence of Indole-3-Acetic Acid and Gibberellic Acid on Phenylpropanoid Accumulation in Common Buckwheat (Fagopyrum esculentum Moench) Sprouts.

We investigated the effects of natural plant hormones, indole-3-acetic (IAA) acid and gibberellic acid (GA), on the growth parameters and production of flavonoids and other phenolic compounds in common buckwheat sprouts. A total of 17 phenolic compounds were identified using liquid chromatography-mass spectrometry (LC-MS) analysis. Among these, seven compounds (4-hydroxybenzoic acid, catechin, chlorogenic acid, caffeic acid, epicatechin, rutin, and quercetin) were quantified by high-performance liquid chromatography (HPLC) after treating the common buckwheat sprouts with different concentrations of the hormones IAA and GA. At a concentration of 0.5 mg/L, both IAA and GA exhibited the highest levels of growth parameters (shoot length, root length, and fresh weight). The HPLC analysis showed that the treatment of sprouts with IAA at concentrations ranging from 0.1 to 1.0 mg/L produced higher or comparable levels of the total phenolic compounds than the control sprout and enhanced the production of rutin. Similarly, the supplementation with 0.1 and 0.5 mg/L GA increased the content of rutin in buckwheat sprouts. Our results suggested that the treatment with optimal concentrations of IAA and GA enhanced the growth parameters and accumulation of flavonoids and other phenolic compounds in buckwheat sprouts.

Accumulation of Carotenoids and Metabolic Profiling in Different Cultivars of Tagetes Flowers.

Species of Tagetes, which belong to the family Asteraceae show different characteristics including, bloom size, shape, and color; plant size; and leaf shape. In this study, we determined the differences in primary metabolites and carotenoid yields among six cultivars from two Tagetes species, T. erecta and T. patula. In total, we detected seven carotenoids in the examined cultivars: violaxanthin, lutein, zeaxanthin, α-carotene, ß-carotene, 9-cis-ß-carotene, and 13-cis-ß-carotene. In all the cultivars, lutein was the most abundant carotenoid. Furthermore, the contents of each carotenoid in flowers varied depending on the cultivar. Principal component analysis (PCA) facilitated metabolic discrimination between Tagetes cultivars, with the exception of Inca Yellow and Discovery Orange. Moreover, PCA and orthogonal projection to latent structure-discriminant analysis (OPLS-DA) results provided a clear discrimination between T. erecta and T. patula. Primary metabolites, including xylose, citric acid, valine, glycine, and galactose were the main components facilitating separation of the species. Positive relationships were apparent between carbon-rich metabolites, including those of the TCA cycle and sugar metabolism, and carotenoids.

Expression of Genes Related to Phenylpropanoid Biosynthesis in Different Organs of Ixeris dentata var. albiflora.

Members of the genus Ixeris have long been used in traditional medicines as stomachics, sedatives, and diuretics. Phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate: coenzyme-A (CoA) ligase (4CL), chalcone synthase (CHS), and dihydroflavonol 4-reductase (DFR) are important enzymes in the phenylpropanoid pathway. In this study, we analyzed seven genes from Ixeris dentata var. albiflora that are involved in phenylpropanoid biosynthesis, using an Illumina/Solexa HiSeq 2000 platform. The amino acid sequence alignments for IdPALs, IdC4H, Id4CLs, IdCHS, and IdDFR showed high identity to sequences from other plants. We also investigated transcript levels using quantitative real-time PCR, and analyzed the accumulation of phenylpropanoids in different organs of I. dentata var. albiflora using high-performance liquid chromatography. The transcript levels of IdC4H, Id4CL1, IdCHS, and IdDFR were highest in the leaf. The catechin, chlorogenic acid, ferulic acid, and quercetin contents were also highest in the leaf. We suggest that expression of IdC4H, Id4CL1, IdCHS, and IdDFR is associated with the accumulation of phenylpropanoids. Our results may provide baseline information for elucidating the mechanism of phenylpropanoid biosynthesis in different organs of I. dentata var. albiflora.

Transcriptome and metabolome analysis in shoot and root of Valeriana fauriei.

BACKGROUND: Valeriana fauriei is commonly used in the treatment of cardiovascular diseases in many countries. Several constituents with various pharmacological properties are present in the roots of Valeriana species. Although many researches on V. fauriei have been done since a long time, further studies in the discipline make a limit due to inadequate genomic information. Hence, Illumina HiSeq 2500 system was conducted to obtain the transcriptome data from shoot and root of V. fauriei. RESULTS: A total of 97,595 unigenes were noticed from 346,771,454 raw reads after preprocessing and assembly. Of these, 47,760 unigens were annotated with Uniprot BLAST hits and mapped to COG, GO and KEGG pathway. Also, 70,013 and 88,827 transcripts were expressed in root and shoot of V. fauriei, respectively. Among the secondary metabolite biosynthesis, terpenoid backbone and phenylpropanoid biosynthesis were large groups, where transcripts was involved. To characterize the molecular basis of terpenoid, carotenoid, and phenylpropanoid biosynthesis, the levels of transcription were determined by qRT-PCR. Also, secondary metabolites content were measured using GC/MS and HPLC analysis for that gene expression correlated with its accumulation respectively between shoot and root of V. fauriei. CONCLUSIONS: We have identified the transcriptome using Illumina HiSeq system in shoot and root of V. fauriei. Also, we have demonstrated gene expressions associated with secondary metabolism such as terpenoid, carotenoid, and phenylpropanoid.

Accessing Pincer Bis(carbene) Ni(IV) Complexes from Ni(II) via Halogen and Halogen Surrogates.

This communication describes the two-electron oxidation of ((DIPP)CCC)NiX ((DIPP)CCC = bis(diisopropylphenyl-benzimidazol-2-ylidene)phenyl); X = Cl or Br) with halogen and halogen surrogates to form ((DIPP)CCC)NiX3. These complexes represent a rare oxidation state of nickel, as well as an unprecedented reaction pathway to access these species through Br2 and halogen surrogate (benzyltrimethylammonium tribromide). The Ni(IV) complexes have been characterized by a suite of spectroscopic techniques and can readily reduce to the Ni(II) counterpart, allowing for cycling between the Ni(II)/Ni(IV) oxidation states.

Expression of Terpenoid Biosynthetic Genes and Accumulation of Chemical Constituents in Valeriana fauriei.

Valeriana fauriei (V. fauriei), which emits a characteristic and unpleasant odor, is important in traditional medicine. In this study, the expression of terpenoid biosynthetic genes was investigated in different organs that were also screened for volatile compounds including valerenic acid and its derivatives. Specific expression patterns from different parts of V. fauriei were observed using quantitative real-time PCR (qRT-PCR). The highest transcript levels of biosynthetic genes involved in mevalonic acid (MVA) and methylerythritol phosphate (MEP) production were found in the stem. Although the amounts of volatile compounds were varied by organ, most of the volatile terpenoids were accumulated in the root. Gas chromatography mass spectrometry (GC-MS) analysis identified 128 volatile compounds, which represented 65.33% to 95.66% of total volatiles. Certain compounds were only found in specific organs. For example, isovalerenic acid and valerenic acid and its derivatives were restricted to the root. Organs with high transcript levels did not necessarily have high levels of the corresponding chemical constituents. According to these results, we hypothesize that translocation may occur between different organs in V. fauriei.

Facile nitrite reduction in a non-heme iron system: formation of an iron(III)-oxo.

Reaction of tetrabutylammonium nitrite with [N(afa(Cy))3Fe(OTf)](OTf) cleanly resulted in the formation of an iron(III)-oxo species, [N(afa(Cy))3Fe(O)](OTf), and NO(g). Formation of NO(g) as a byproduct was confirmed by reaction of the iron(II) starting material with half an equivalent of nitrite, resulting in a mixture of two products, the iron-oxo and an iron-NO species, [N(afa(Cy))3Fe(NO)](OTf)2. Formation of the latter was confirmed through independent synthesis. The results of this study provide insight into the role of hydrogen bonding in the mechanism of nitrite reduction and the binding mode of nitrite in biological heme systems.

Nanoencapsulation enhances the bioavailability of fucoxanthin in microalga Phaeodactylum tricornutum extract.

The marine carotenoid fucoxanthin (FX) has various health benefits but suffers from poor bioavailability. We hypothesize that the bioavailability of FX in microalga Phaeodactylum tricornutum extract (PE) could be improved through nanoencapsulation. Here, we developed two types of nanoparticles: one consisting of alginate and casein (A-C-PE, 246 nm diameter, 79.6% encapsulation efficiency) and the other A-C-PE coated with chitosan (CS-A-C-PE, 258 nm, 78.1%). Both types of nanoparticles incorporating PE showed controlled FX release during simulated gastrointestinal digestion, as well as 1.8-fold improvement of membrane permeability in Caco-2/TC7 cells compared to non-encapsulated PE. Pharmacokinetic behavior of two FX metabolites (fucoxanthinol and amarouciaxanthin A) in mouse plasma was monitored after oral administration. The results showed that 31.8-332.1% more FX metabolites from the nanoparticles were absorbed into plasma than those from PE. In conclusion, encapsulation of PE in both types of nanoparticles significantly promoted the bioavailability of FX.

Identification of drought-responsive phenolic compounds and their biosynthetic regulation under drought stress in Ligularia fischeri.

Ligularia fischeri, a leafy edible plant found in damp shady regions, has been used as an herbal medicine and is also consumed as a horticultural crop. In this study, we investigated the physiological and transcriptomic changes, especially those involved in phenylpropanoid biosynthesis, induced by severe drought stress in L. fischeri plants. A distinguishing characteristic of L. fischeri is a color change from green to purple due to anthocyanin biosynthesis. We chromatographically isolated and identified two anthocyanins and two flavones upregulated by drought stress using liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses in this plant for the first time. In contrast, all types of caffeoylquinic acids (CQAs) and flavonol contents were decreased under drought stress. Further, we performed RNA sequencing to examine the molecular changes in these phenolic compounds at the transcriptome level. In an overview of drought-inducible responses, we identified 2,105 hits for 516 distinct transcripts as drought-responsive genes. Moreover, differentially expressed genes (DEGs) associated with phenylpropanoid biosynthesis accounted for the greatest number of both up- and downregulated DEGs by Kyoto Encyclopedia of Genes and Genomes enrichment analysis. We identified 24 meaningful DEGs based on the regulation of phenylpropanoid biosynthetic genes. Potential drought-responsive genes included upregulated flavone synthase (LfFNS, TRINITY DN31661 c0 g1 i1) and anthocyanin 5-O-glucosyltransferase (LfA5GT1, TRINITY DN782 c0 g1 i1), which could contribute to the high levels of flavones and anthocyanins under drought stress in L. fischeri. In addition, the downregulated shikimate O-hydroxycinnamolytransferase (LfHCT, TRINITY DN31661 c0 g1 i1) and hydroxycinnamoyl-CoA quinate/shikimate transferase (LfHQT4, TRINITY DN15180 c0 g1 i1) genes led to a reduction in CQAs. Only one or two BLASTP hits for LfHCT were obtained for six different Asteraceae species. It is possible that the HCT gene plays a crucial role in CQAs biosynthesis in these species. These findings expand our knowledge of the response mechanisms to drought stress, particularly regarding the regulation of key phenylpropanoid biosynthetic genes in L. fischeri.