Integrative proteome and metabolome unveil the central role of IAA alteration in axillary bud development following topping in tobacco.

Axillary bud is an important aspect of plant morphology, contributing to the final tobacco yield. However, the mechanisms of axillary bud development in tobacco remain largely unknown. To investigate this aspect of tobacco biology, the metabolome and proteome of the axillary buds before and after topping were compared. A total of 569 metabolites were differentially abundant before and 1, 3, and 5 days after topping. KEGG analyses further revealed that the axillary bud was characterized by a striking enrichment of metabolites involved in flavonoid metabolism, suggesting a strong flavonoid biosynthesis activity in the tobacco axillary bud after topping. Additionally, 9035 differentially expressed proteins (DEPs) were identified before and 1, 3, and 5 days after topping. Subsequent GO and KEGG analyses revealed that the DEPs in the axillary bud were enriched in oxidative stress, hormone signal transduction, MAPK signaling pathway, and starch and sucrose metabolism. The integrated proteome and metabolome analysis revealed that the indole-3-acetic acid (IAA) alteration in buds control dormancy release and sustained growth of axillary bud by regulating proteins involved in carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Notably, the proteins related to reactive oxygen species (ROS) scavenging and flavonoid biosynthesis were strongly negatively correlated with IAA content. These findings shed light on a critical role of IAA alteration in regulating axillary bud outgrowth, and implied a potential crosstalk among IAA alteration, ROS homeostasis, and flavonoid biosynthesis in tobacco axillary bud under topping stress, which could improve our understanding of the IAA alteration in axillary bud as an important regulator of axillary bud development.

Metabolomic and transcriptomic analyses of peach leaves and fruits in response to pruning.

BACKGROUND: Pruning is an important cultivation management option that has important effects on peach yield and quality. However, the effects of pruning on the overall genetic and metabolic changes in peach leaves and fruits are poorly understood. RESULTS: The transcriptomic and metabolomic profiles of leaves and fruits from trees subjected to pruning and unpruning treatments were measured. A total of 20,633 genes and 622 metabolites were detected. Compared with those in the control, 1,127 differentially expressed genes (DEGs) and 77 differentially expressed metabolites (DEMs) were identified in leaves from pruned and unpruned trees (pdLvsupdL), whereas 423 DEGs and 29 DEMs were identified in fruits from the pairwise comparison pdFvsupdF. The content of three auxin analogues was upregulated in the leaves of pruned trees, the content of all flavonoids detected in the leaves decreased, and the expression of almost all genes involved in the flavonoid biosynthesis pathway decreased. The phenolic acid and amino acid metabolites detected in fruits from pruned trees were downregulated, and all terpenoids were upregulated. The correlation analysis revealed that DEGs and DEMs in leaves were enriched in tryptophan metabolism, auxin signal transduction, and flavonoid biosynthesis. DEGs and DEMs in fruits were enriched in flavonoid and phenylpropanoid biosynthesis, as well as L-glutamic acid biosynthesis. CONCLUSIONS: Pruning has different effects on the leaves and fruits of peach trees, affecting mainly the secondary metabolism and hormone signalling pathways in leaves and amino acid biosynthesis in fruits.

Spatial distribution of metabolites in processing Ziziphi Spinosae Semen as revealed by matrix-assisted laser desorption/ionization mass spectrometry imaging.

Ziziphi Spinosae Semen (ZSS) is the first choice for the treatment of insomnia. This research aimed to reveal the spatial distribution of identifying quality markers of ZSS and to illustrate the metabolite quality characteristics of this herbal medicine. Here, we performed a matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in situ to detect and image 33 metabolites in ZSS, including three saponins, six flavonoids, four alkaloids, eight fatty acids, and 12 amino acids. The MALDI images of the metabolites clearly showed the heterogeneous spatial distribution in different regions of ZSS tissues, such as the cotyledon, endosperm, and radicle. The distribution area of two saponins, six flavonoids, and three alkaloids increased significantly after the fried processing of ZSS. Based on the ion images, samples with different processing technologies were distinguished unambiguously by the pattern recognition method of orthogonal partial least squares discrimination analysis (OPLS-DA). Simultaneously, 23 major influencing components exerting higher ion intensities were identified as the potential quality markers of ZSS. Results obtained in the current research demonstrate that the processing of ZSS changes its content and distribution of the medicinal components. The analysis of MALDI-MSI provides a novel MS-based molecular imaging approach to investigate and monitor traditional medicinal plants.

The Key Role of Plant Hormone Signaling Transduction and Flavonoid Biosynthesis Pathways in the Response of Chinese Pine (Pinus tabuliformis) to Feeding Stimulation by Pine Caterpillar (Dendrolimus tabulaeformis).

In nature, plants have developed a series of resistance mechanisms to face various external stresses. As understanding of the molecular mechanisms underlying plant resistance continues to deepen, exploring endogenous resistance in plants has become a hot topic in this field. Despite the multitude of studies on plant-induced resistance, how plants respond to stress under natural conditions remains relatively unclear. To address this gap, we investigated Chinese pine (Pinus tabuliformis) using pine caterpillar (Dendrolimus tabulaeformis) under natural conditions. Healthy Chinese pine trees, approximately 10 years old, were selected for studying induced resistance in Huangtuliangzi Forestry, Pingquan City, Chengde City, Hebei Province, China. Pine needles were collected at 2 h and 8 h after feeding stimulation (FS) via 10 pine caterpillars and leaf clipping control (LCC), to simulate mechanical damage caused by insect chewing for the quantification of plant hormones and transcriptome and metabolome assays. The results show that the different modes of treatments significantly influence the contents of JA and SA in time following treatment. Three types of differentially accumulated metabolites (DAMs) were found to be involved in the initial response, namely phenolic acids, lipids, and flavonoids. Weighted gene co-expression network analysis indicated that 722 differentially expressed genes (DEGs) are positively related to feeding stimulation and the specific enriched pathways are plant hormone signal transduction and flavonoid biosynthesis, among others. Two TIFY transcription factors (PtTIFY54 and PtTIFY22) and a MYB transcription factor (PtMYB26) were found to be involved in the interaction between plant hormones, mainly in the context of JA signal transduction and flavonoid biosynthesis. The results of this study provide an insight into how JA activates, serving as a reference for understanding the molecular mechanisms of resistance formation in conifers responding to mandibulate insects.

Application of hydrophobic eutectic solvent in efficient biotransformation of total flavonoids of Herba Epimedii.

Icaritin, a hydrolysate from total flavonoids of Epimedii (TFE), which has better anti-hepatoma activity than its glycosylated form. In this work, immobilized enzymes 4LP-Tpebgl3@Na-Y and DtRha@ES-107 were used to hydrolyze TFE to prepare icaritin. Five different hydrophobic deep eutectic solvents (HDES) were prepared and the most ideal HDES was successfully selected, which was composed of dodecyl alcohol and thymol with the molar ratio of 2:1. The relative enzyme activity of 4LP-Tpebgl3@Na-Y and DtRha@ES-107 was about 102.4 % and 112.5 %, respectively. In addition, the thermal and binding stability of 4LP-Tpebgl3@Na-Y and DtRha@ES-107 in HDES was not affected negatively. In the biphasic system composed of 50 % (v/v) HDES and Na2HPO4-citric acid buffer (50 mM, pH 5.5), 4LP-Tpebgl3@Na-Y (1.0 U/mL) and TFE (1 g/L) were reacted at 80 °C for 1 h, and then reacted with DtRha@ES-107 (20 U/mL) at 80 °C for 2 h. Finally, TFE was completely converted to 301.8 mg/L icaritin (0.82 mM). After 10 cycles, 4LP-Tpebgl3@Na-Y/DtRha@ES-107 still maintained 84.1 % original activity. In this study, we developed an efficient methodology for icaritin preparation through the integration of enzymatic catalysis and adsorption separation, presenting a viable approach for large-scale, cost-effective production of icaritin.

Comprehensive evaluation of drought tolerance of six Chinese chestnut varieties (clones) based on flavonoids and other physiological indexes.

Flavonoids are crucial secondary metabolites that possess the ability to mitigate UV damage and withstand both biotic and abiotic stresses. Therefore, it is of immense significance to investigate the flavonoid content as a pivotal indicator for a comprehensive assessment of chestnut's drought tolerance. This study aimed to determine the flavonoid content and drought tolerance-related physiological and biochemical indices of six chestnut varieties (clones) grafted trees-Qianxi 42 (QX42), Qinglong 45 (QL45), Yanshanzaofeng (YSZF), Yanzi (YZ), Yanqiu (YQ), and Yanlong (YL)-under natural drought stress. The results were used to comprehensively analyze the drought tolerance ability of these varieties. The study revealed that the ranking of drought tolerance indices in terms of their ability to reflect drought tolerance was as follows: superoxide (oxide) dismutase (SOD) activity, ascorbate peroxidase (APX) activity, flavone content, catalase (CAT) activity, proline (PRO) content, soluble sugar content, peroxidase (POD) activity, betaine content, flavonol content, hydrogen peroxide (H2O2) content, soluble protein content, superoxide ion (OFR) content, superoxide (ion OFR) production rate, malondialdehyde (MDA) content, chlorophyll content. Through principal component analysis, the contents of flavonoids and flavonols can be used as indicators for comprehensive evaluation of drought tolerance of chestnut. The comprehensive evaluation order of drought tolerance of grafted trees of 6 chestnut varieties (Clones) was: QL45 > QX42 > YQ > YZ > YSZF > YL.

Transcriptomics and metabolomics reveal the mechanism of metabolites changes in Cymbidium tortisepalum var. longibracteatum colour mutation cultivars.

BACKGROUND: Foliage color is considered an important ornamental character of Cymbidium tortisepalum (C. tortisepalum), which significantly improves its horticultural and economic value. However, little is understood on the formation mechanism underlying foliage-color variations. METHODS: In this study, we applied a multi-omics approach based on transcriptomics and metabolomics, to investigate the biomolecule mechanisms of metabolites changes in C. tortisepalum colour mutation cultivars. RESULTS: A total of 508 genes were identified as differentially expressed genes (DEGs) between wild and foliage colour mutation C. tortisepalum cultivars based on transcriptomic data. KEGG enrichment of DEGs showed that genes involved in phenylalanine metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis and brassinosteroid biosynthesis were most significantly enriched. A total of 420 metabolites were identified in C. tortisepalum using UPLC-MS/MS-based approach and 115 metabolites differentially produced by the mutation cultivars were identified. KEGG enrichment indicated that the most metabolites differentially produced by the mutation cultivars were involved in glycerophospholipid metabolism, tryptophan metabolism, isoflavonoid biosynthesis, flavone and flavonol biosynthesis. Integrated analysis of the metabolomic and transcriptomic data showed that there were four significant enrichment pathways between the two cultivars, including phenylalanine metabolism, phenylpropanoid biosynthesis, flavone and flavonol biosynthesis and flavonoid biosynthesis. CONCLUSION: The results of this study revealed the mechanism of metabolites changes in C. tortisepalum foliage colour mutation cultivars, which provides a new reference for breeders to improve the foliage color of C. tortisepalum.

Multiomics Analyses Reveal MsC3H29 Positively Regulates Flavonoid Biosynthesis to Improve Drought Resistance of Autotetraploid Cultivated Alfalfa (Medicago sativa L.).

Alfalfa (Medicago sativa subsp. sativa), the "queen of forage," is the most important perennial legume, with high productivity and an excellent nutritional profile. Medicago sativa subsp. falcata is a subspecies of the alfalfa complex and exhibits better drought tolerance. However, drought stress significantly hampers their development and yield. The molecular mechanisms underlying the aboveground and underground tissues of sativa and falcata responding to drought stress remain obscure. Here, we performed a comprehensive comparative analysis of the physiological and transcriptomic responses of sativa and falcata under drought stress. The results showed that photosynthesis was inhibited, and antioxidant enzymes were activated under drought stress. MsC3H29, a CCCH-type zinc finger protein, was identified as a hub gene through weighted gene coexpression network analysis (WGCNA) and was significantly induced by drought in underground tissue. The MsC3H29 protein was localized in the nucleus. Overexpression (OE) of MsC3H29 can increase the primary root length and fresh weight of transgenic alfalfa hairy roots, while RNA interference (RNAi) decreases them under drought stress. The 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining revealed that MsC3H29 promoted drought tolerance of alfalfa hairy roots through decreasing ROS accumulation. The targeted metabolome analysis showed that the overexpression of MsC3H29 resulted in higher levels of accumulation for flavonoid monomers, including vicenin, daidzein, apigenin, isorhamnetin, quercetin, and tricin, in transgenic alfalfa hairy roots before and after drought stress, while RNAi led to a reduction. Our study provided a key candidate gene for molecular breeding to improve drought resistance in alfalfa.

The influence of prolonged but low intensity blue light on the physiological properties of root tubers and the accumulation of flavonoids in Tetrastigma hemsleyanum Diels et Gilg.

Tetrastigma hemsleyanum Diel et Gilg is a perennial herbaceous plant native to subtropical China with multiple medicinal applications. Supplementing with low-density blue light (BL) for 45 days (3 h/day) can not only significantly increase the yields of root tubers but also significantly increase the flavonoid content and its antioxidant activity. The chlorophyll content in the leaves of T. hemsleyanum significantly decreased, but the photosynthetic efficiency significantly increased after reaching the light saturation point. The production rate of superoxide anion radical in the leaves reached the highest peak after 1.5 h in BL and decreased at 3 h. The H2O2 content in the leaves decreased significantly, while the H2O2 content in the root tubers increased significantly at 3 h in BL. The objective of this research was to determine how the scavenging system, including antioxidant enzymes, antioxidants, and flavonoids respond to the oxidative stress induced by BL in root tubers. After exposure to BL, significant differences in the activity of APX and SOD were observed in the leaves and tubers within 3 h. By analyzing the upregulated flavonoids metabolites and key genes in metabolic pathways through the combined analysis of the flavonoid metabolic group and transcriptome in the root tubers, the upregulated accumulation of flavanols was found to be the main reason for the improvement in the antioxidant properties of flavonoids.

Efficient and novel biosynthesis of myricetin α-triglucoside with improved solubility using amylosucrase from Deinococcus deserti.

Although myricetin (3,3',4',5,5',7-hexahydroxyflavone, MYR) has a high antioxidant capacity and health functions, its use as a functional food material is limited owing to its low stability and water solubility. Amylosucrase (ASase) is capable of biosynthesizing flavonol α-glycoside using flavonols as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus deserti (DdAS) efficiently biosynthesizes a novel MYR α-triglucoside (MYRαG3) using MYR as the acceptor molecule. Comparative homology analysis and computational simulation revealed that DdAS has a different active pocket for the transglycosylation reaction. DdAS produced MYRαG3 with a conversion efficiency of 67.4 % using 10 mM MYR and 50 mM sucrose as acceptor and donor molecules, respectively. The structure of MYRαG3 was identified as MYR 4'-O-4″,6″-tri-O-α-D-glucopyranoside using NMR and LC-MS. In silico analysis confirmed that DdAS has a distinct active pocket compared to other ASases. In addition, molecular docking simulations predicted the synthetic sequence of MYRαG3. Furthermore, MYRαG3 showed a similar DPPH radical scavenging activity of 49 %, comparable to MYR, but with significantly higher water solubility, which increased from 0.03 µg/mL to 511.5 mg/mL. In conclusion, this study demonstrated the efficient biosynthesis of a novel MYRαG3 using DdAS and highlighted the potential of MYRαG3 as a functional material.

Physiological and molecular mechanism of Populus pseudo-cathayana × Populus deltoides response to Hyphantria cunea.

Populus pseudo-cathayana × Populus deltoides is a crucial artificial forest tree species in Northeast China. The presence of the fall webworm (Hyphantria cunea) poses a significant threat to these poplar trees, causing substantial economic and ecological damage. This study conducted an insect-feeding experiment with fall webworm on P. pseudo-cathayana × P. deltoides, examining poplar's physiological indicators, transcriptome, and metabolome under different lengths of feeding times. Results revealed significant differences in phenylalanine ammonia-lyase activity, total phenolic content, and flavonoids at different feeding durations. Transcriptomic analysis identified numerous differentially expressed genes, including AP2/ERF, MYB, and WRKY transcription factor families exhibiting the highest expression variations. Differential metabolite analysis highlighted flavonoids and phenolic acid compounds of poplar's leaves as the most abundant in our insect-feeding experiment. Enrichment analysis revealed significant enrichment in the plant hormone signal transduction and flavonoid biosynthetic pathways. The contents of jasmonic acid and jasmonoyl-L-isoleucine increased with prolonged fall webworm feeding. Furthermore, the accumulation of dihydrokaempferol, catechin, kaempferol, and naringenin in the flavonoid biosynthesis pathway varied significantly among different samples, suggesting their crucial role in response to pest infestation. These findings provide novel insights into how poplar responds to fall webworm infestation.

Evaluation of Polyphenols Synthesized in Mature Seeds of Common Bean (Phaseolus vulgaris L.) Advanced Mutant Lines.

This study aimed to investigate the availability of flavonoids, anthocyanins, and phenolic acids in mutant bean seeds, focusing on M7 mutant lines, and their corresponding initial and local cultivars. HPLC-DAD-MS/MS and HPLC-MS/MS were used to analyze twenty-eight genotypes of common bean. The obtained results suggest that the mutations resulted in four newly synthesized anthocyanins in the mutant bean seeds, namely, delphinidin 3-O-glucoside, cyanidin 3-O-glucoside, pelargonidin 3-O-glucoside, and petunidin 3-O-glucoside, in 20 accessions with colored seed shapes out of the total of 28. Importantly, the initial cultivar with white seeds, as well as the mutant white seeds, did not contain anthocyanins. The mutant lines were classified into groups based on their colors as novel qualitative characteristics. Five phenolic acids were further quantified: ferulic, p-coumaric, caffeic, sinapic, and traces of chlorogenic acids. Flavonoids were represented by epicatechin, quercetin, and luteolin, and their concentrations in the mutant genotypes were several-fold superior compared to those of the initial cultivar. All mutant lines exhibited higher concentrations of phenolic acids and flavonoids. These findings contribute to the understanding of the genetics and biochemistry of phenolic accumulation and anthocyanin production in common bean seeds, which is relevant to health benefits and might have implications for common bean breeding programs and food security efforts.

Structural and Interactional Analysis of the Flavonoid Pathway Proteins: Chalcone Synthase, Chalcone Isomerase and Chalcone Isomerase-like Protein.

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI's unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.

Comprehensive studies reveal physiological and genetic diversity in traditional rice cultivars for UV-B sensitivity.

Acclimation to crop niches for thousands of years has made indigenous rice cultivars better suited for stress-prone environments. Still, their response to UV-B resiliency is unknown. 38 rice landraces were grown in cemented pots in a randomised block design with three replicates under open field conditions in Sambalpur University in the wet season of 2022. Half of the plants in each of the cultivars were administered UV-B radiation at the panicle emergence stage in an adjustable UV-B chamber permitting sunlight, and the effects of the stress on various morpho-physiological features, such as spikelet sterility, flag leaf photosynthetic and flavonoid pigment contents, and lipid peroxidation activities, were estimated for calibration of stress resistance. The experiment identified Swarnaprabha and Lalkain as the most sensitive and resilient to stress respectively, and the differential response between them was further revealed in the expression of genes related to UV-B sensitivity. Subject to the stress, Swarnaprabha exhibited symptoms of injuries, like leaf burns, and a higher loss of various photosynthetic parameters, such as pigment contents, SPAD and Fv/Fm, ETR and qP values, while NPQ increased only in Lalkain. Exposure to UV-B increased the total phenolic and flavonoid contents in Lalkain while depressing them in Swarnaprabha. Such an effect amounted to a higher release of fluorescent energy in the latter. The levels of expression of gene families controlling flavonoid activation and UV-B signal transduction, such as OsWRKY, OsUGT, OsRLCK, OsBZIP, OsGLP, and CPD photolyase were similar in both the cultivars in the control condition. However, exposure to UV-B stress overexpressed them in resilient cultivars only. The magnitude of expression of the genes and the impact of the stress on photosynthetic parameters, phenolic compounds and pubescent hair structure at the panicle emergence stage could be valid indicators among indigenous rice for UV-B tolerance.

Modifications in gene expression and phenolic compounds content by methyl jasmonate and fungal elicitors in Ficus carica. Cv. Siah hairy root cultures.

BACKGROUND: One of the most effective strategies to increase phytochemicals production in plant cultures is elicitation. In the present study, we studied the effect of abiotic and biotic elicitors on the growth, key biosynthetic genes expression, antioxidant capacity, and phenolic compounds content in Rhizobium (Agrobacterium) rhizogenes-induced hairy roots cultures of Ficus carica cv. Siah. METHODS: The elicitors included methyl jasmonate (MeJA) as abiotic elicitor, culture filtrate and cell extract of fungus Piriformospora indica as biotic elicitors were prepared to use. The cultures of F. carica hairy roots were exposed to elicitores at different time points. After elicitation treatments, hairy roots were collected, and evaluated for growth index, total phenolic (TPC) and flavonoids (TFC) content, antioxidant activity (2,2-diphenyl-1-picrylhydrazyl, DPPH and ferric ion reducing antioxidant power, FRAP assays), expression level of key phenolic/flavonoid biosynthesis genes, and high-performance liquid chromatography (HPLC) analysis of some main phenolic compounds in comparison to control. RESULTS: Elicitation positively or negatively affected the growth, content of phenolic/flavonoid compounds and DPPH and FRAP antioxidant activities of hairy roots cultures in depending of elicitor concentration and exposure time. The maximum expression level of chalcone synthase (CHS: 55.1), flavonoid 3'-hydroxylase (F3'H: 34.33) genes and transcription factors MYB3 (32.22), Basic helix-loop-helix (bHLH: 45.73) was induced by MeJA elicitation, whereas the maximum expression level of phenylalanine ammonia-lyase (PAL: 26.72) and UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT: 27.57) genes was obtained after P. indica culture filtrate elicitation. The P. indica elicitation also caused greatest increase in the content of gallic acid (5848 µg/g), caffeic acid (508.2 µg/g), rutin (43.5 µg/g), quercetin (341 µg/g), and apigenin (1167 µg/g) phenolic compounds. CONCLUSIONS: This study support that elicitation of F. carica cv. Siah hairy roots can be considered as an effective biotechnological method for improved phenolic/flavonoid compounds production, and of course this approach requires further research.

Integrated metabolomic and transcriptomic analysis reveals the regulatory mechanisms of flavonoid and alkaloid biosynthesis in the new and old leaves of Murraya tetramera Huang.

BACKGROUND: Murraya tetramera Huang is a traditional Chinese woody medicine. Its leaves contain flavonoids, alkaloids, and other active compounds, which have anti-inflammatory and analgesic effects, as well as hypoglycemic and lipid-lowering effects, and anti-tumor effects. There are significant differences in the content of flavonoids and alkaloids in leaves during different growth cycles, but the synthesis mechanism is still unclear. RESULTS: In April 2021, new leaves (one month old) and old leaves (one and a half years old) of M. tetramera were used as experimental materials to systematically analyze the changes in differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) with transcriptomics and metabolomics technology. This was done to identify the signaling pathways of flavonoid and alkaloid synthesis. The results showed that the contents of total alkaloids and flavonoids in old leaves were significantly higher than those in new leaves. Thirteen flavonoid compounds, three isoflavone compounds, and nineteen alkaloid compounds were identified, and 125 and 48 DEGs related to flavonoid and alkaloid synthesis were found, respectively. By constructing the KEGG (Kyoto Encyclopedia of Genes and Genomes) network of DEGs and DAMs, it was shown that the molecular mechanism of flavonoid biosynthesis in M. tetramera mainly focuses on the "flavonoid biosynthetic pathway" and the "flavonoid and flavonol biosynthetic pathway". Among them, p-Coumaryl alcohol, Sinapyl alcohol, Phloretin, and Isoquercitrin were significantly accumulated in old leaves, the up-regulated expression of CCR (cinnamoyl-CoA reductase) might promote the accumulation of p-Coumaryl alcohol, upregulation of F5H (ferulate-5-hydroxylase) might promote Sinapyl alcohol accumulation. Alkaloids, including indole alkaloids, pyridine alkaloids, imidazole alkaloids, and quinoline alkaloids, were significantly accumulated in old leaves, and a total of 29 genes were associated with these substances. CONCLUSIONS: These data are helpful to better understand the biosynthesis of flavonoids and alkaloids in M. tetramera and provide a scientific basis for the development of medicinal components in M. tetramera.

Metabolite analysis reveals flavonoids accumulation during flower development in Rhododendron pulchrum sweet (Ericaceae).

The azalea (Rhododendron simsii Planch.) is an important ornamental woody plant with various medicinal properties due to its phytochemical compositions and components. However little information on the metabolite variation during flower development in Rhododendron has been provided. In our study, a comparative analysis of the flavonoid profile was performed in Rhododendron pulchrum sweet at three stages of flower development, bud (stage 1), partially open flower (stage 2), and full bloom (stage 3). A total of 199 flavonoids, including flavone, flavonol, flavone C-glycosides, flavanone, anthocyanin, and isoflavone were identified. In hierarchical clustering analysis (HCA) and principal component analysis (PCA), the accumulation of flavonoids displayed a clear development stage variation. During flower development, 78 differential accumulated metabolites (DAMs) were identified, and most were enriched to higher levels at the full bloom stage. A total of 11 DAMs including flavone (chrysin, chrysoeriol O-glucuronic acid, and chrysoeriol O-hexosyl-O-pentoside), isoflavone (biochanin A), and flavonol (3,7-di-O-methyl quercetin and isorhamnetin) were significantly altered at three stages. In particular, 3,7-di-O-methyl quercetin was the top increased metabolite during flower development. Furthermore, integrative analyses of metabolomic and transcriptomic were conducted, revealing that the contents of isoflavone, biochanin A, glycitin, and prunetin were correlated with the expression of 2-hydroxyisoflavanone dehydratase (HIDH), which provide insight into the regulatory mechanism that controls isoflavone biosynthesis in R. pulchrum. This study will provide a new reference for increasing desired metabolites effectively by more accurate or appropriate genetic engineering strategies.

Increase of secondary metabolites in sweet basil (Ocimum basilicum L.) leaves by exposure to N2O5 with plasma technology.

Exposure to N2O5 generated by plasma technology activates immunity in Arabidopsis through tryptophan metabolites. However, little is known about the effects of N2O5 exposure on other plant species. Sweet basil synthesizes many valuable secondary metabolites in its leaves. Therefore, metabolomic analyses were performed at three different exposure levels [9.7 (Ex1), 19.4 (Ex2) and 29.1 (Ex3) µmol] to assess the effects of N2O5 on basil leaves. As a result, cinnamaldehyde and phenolic acids increased with increasing doses. Certain flavonoids, columbianetin, and caryophyllene oxide increased with lower Ex1 exposure, cineole and methyl eugenol increased with moderate Ex2 exposure and L-glutathione GSH also increased with higher Ex3 exposure. Furthermore, gene expression analysis by quantitative RT-PCR showed that certain genes involved in the syntheses of secondary metabolites and jasmonic acid were significantly up-regulated early after N2O5 exposure. These results suggest that N2O5 exposure increases several valuable secondary metabolites in sweet basil leaves via plant defense responses in a controllable system.

Unveiling phenylpropanoid regulation: the role of DzMYB activator and repressor in durian (Durio zibethinus) fruit.

KEY MESSAGE: DzMYB2 functions as an MYB activator, while DzMYB3 acts as an MYB repressor. They bind to promoters, interact with DzbHLH1, and influence phenolic contents, revealing their roles in phenylpropanoid regulation in durian pulps. Durian fruit has a high nutritional value attributed to its enriched bioactive compounds, including phenolics, carotenoids, and vitamins. While various transcription factors (TFs) regulate phenylpropanoid biosynthesis, MYB (v-myb avian myeloblastosis viral oncogene homolog) TFs have emerged as pivotal players in regulating key genes within this pathway. This study aimed to identify additional candidate MYB TFs from the transcriptome database of the Monthong cultivar at five developmental/postharvest ripening stages. Candidate transcriptional activators were discerned among MYBs upregulated during the ripe stage based on the positive correlation observed between flavonoid biosynthetic genes and flavonoid contents in ripe durian pulps. Conversely, MYBs downregulated during the ripe stage were considered candidate repressors. This study focused on a candidate MYB activator (DzMYB2) and a candidate MYB repressor (DzMYB3) for functional characterization. LC-MS/MS analysis using Nicotiana benthamiana leaves transiently expressing DzMYB2 revealed increased phenolic compound contents compared with those in leaves expressing green fluorescence protein controls, while those transiently expressing DzMYB3 showed decreased phenolic compound contents. Furthermore, it was demonstrated that DzMYB2 controls phenylpropanoid biosynthesis in durian by regulating the promoters of various biosynthetic genes, including phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR). Meanwhile, DzMYB3 regulates the promoters of PAL, 4-coumaroyl-CoA ligase (4CL), CHS, and CHI, resulting in the activation and repression of gene expression. Moreover, it was discovered that DzMYB2 and DzMYB3 could bind to another TF, DzbHLH1, in the regulation of flavonoid biosynthesis. These findings enhance our understanding of the pivotal role of MYB proteins in regulating the phenylpropanoid pathway in durian pulps.

Comparative pharmacognosy and secondary metabolite analysis of Balanophorae herbs from different sources.

The Balanophorae are not only traditional Chinese herbal medicines but also functional foods with diverse sources. This study aimed to distinguish pharmacognostic characteristics and secondary metabolites among different species of Balanophorae. Eight species of Balanophorae herbs were harvested, including 21 batches with 209 samples. Ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used to analyze secondary metabolites of Balanophorae from 21 sources. Targeted metabolomic analysis was performed to compare differences among the groups. Rhopalocnemis phalloide and B. indica can be identified by their pharmacognostic characteristics. Then, 41 secondary metabolites were identified or characterized in the mixed extracts of the 209 samples, mainly phenolic acids, flavonoids, and their derivatives. The distribution of these secondary metabolites revealed apparent differences among different species. In addition, targeted metabolomic analysis suggested that the secondary metabolite profiles of seven species of Balanophorae showed noticeable differences, and differences were also observed among different growing regions. Finally, five important metabolic markers were screened to successfully distinguish B. laxiflora, B. harlandii, and B. polyandra, including three phenolic acids and two flavonoids. This is the first study to systematically compare both the morphology and secondary metabolites among different sources of Balanophorae, which could provide effective information for identifying diverse species.