Folate metabolic profiling and expression of folate metabolism-related genes during panicle development in foxtail millet (Setaria italica (L.) P. Beauv).

BACKGROUND: Foxtail millet grain has higher folate content than other cereal crops. However, the folate metabolite content and the expression patterns of folate metabolite-related genes are unknown. RESULTS: Liquid chromatography-mass spectrometry was used to investigate 12 folate metabolites in a foxtail millet panicle. The content of total folate and derivatives gradually decreased during panicle development. Polyglutamate 5-formyl-tetrahydrofolate was the major form. Twenty-eight genes involved in the folate metabolic pathway were identified through bioinformatic analysis. These genes in Setaria italica, S. viridis and Zea mays showed genomic collinearity. Phylogenetic analysis revealed that the folate-related genes were closely related among the C4 plants compared to C3 plants. The gene expressions were then studied at three panicle development stages. The gene expression patterns were classified into two groups, namely SiADCL1 and SiGGH as two key enzymes, which are responsible for folate synthesis and degradation; their expression levels were highest at the early panicle development stage, up to 179.11- and 163.88-fold, respectively. Their expression levels had a similar downward trend during panicle development and were significantly positively correlated with the concentration of total folate and folate derivatives. However, SiSHMT3 expression levels were significantly negatively correlated with total folate concentration. CONCLUSION: Besides being the major determinants of folate and folate derivatives accumulation, SiADCL1 and SiGGH expression levels are key limiting factors in the foxtail millet panicle. Therefore, SiADCL1 and SiGGH expression levels can be targeted in genetic modification studies to improve folate content in foxtail millet seeds in the future. © 2021 Society of Chemical Industry.

Genome-wide identification and functional characterization of LEA genes during seed development process in linseed flax (Linum usitatissimum L.).

BACKGROUND: LEA proteins are widely distributed in the plant and animal kingdoms, as well as in micro-organisms. LEA genes make up a large family and function in plant protection against a variety of adverse conditions. RESULTS: Bioinformatics approaches were adopted to identify LEA genes in the flax genome. In total, we found 50 LEA genes in the genome. We also conducted analyses of the physicochemical parameters and subcellular location of the genes and generated a phylogenetic tree. LuLEA genes were unevenly mapped among 15 flax chromosomes and 90% of the genes had less than two introns. Expression profiles of LuLEA showed that most LuLEA genes were expressed at a late stage of seed development. Functionally, the LuLEA1 gene reduced seed size and fatty acid contents in LuLEA1-overexpressed transgenic Arabidopsis lines. CONCLUSION: Our study adds valuable knowledge about LEA genes in flax which can be used to improve related genes of seed development.

Methylglucosylation of aromatic amino and phenolic moieties of drug-like biosynthons by combinatorial biosynthesis.

Glycosylation is a prominent strategy to optimize the pharmacokinetic and pharmacodynamic properties of drug-like small-molecule scaffolds by modulating their solubility, stability, bioavailability, and bioactivity. Glycosyltransferases applicable for "sugarcoating" various small-molecule acceptors have been isolated and characterized from plants and bacteria, but remained cryptic from filamentous fungi until recently, despite the frequent use of some fungi for whole-cell biocatalytic glycosylations. Here, we use bioinformatic and genomic tools combined with heterologous expression to identify a glycosyltransferase-methyltransferase (GT-MT) gene pair that encodes a methylglucosylation functional module in the ascomycetous fungus Beauveria bassiana The GT is the founding member of a family nonorthologous to characterized fungal enzymes. Using combinatorial biosynthetic and biocatalytic platforms, we reveal that this GT is a promiscuous enzyme that efficiently modifies a broad range of drug-like substrates, including polyketides, anthraquinones, flavonoids, and naphthalenes. It yields both O- and N-glucosides with remarkable regio- and stereospecificity, a spectrum not demonstrated for other characterized fungal enzymes. These glucosides are faithfully processed by the dedicated MT to afford 4-O-methylglucosides. The resulting "unnatural products" show increased solubility, while representative polyketide methylglucosides also display increased stability against glycoside hydrolysis. Upon methylglucosidation, specific polyketides were found to attain cancer cell line-specific antiproliferative or matrix attachment inhibitory activities. These findings will guide genome mining for fungal GTs with novel substrate and product specificities, and empower the efficient combinatorial biosynthesis of a broad range of natural and unnatural glycosides in total biosynthetic or biocatalytic formats.

Identification and Characterization of microRNAs in the Developing Seed of Linseed Flax (Linum usitatissimum L.).

Seed development plays an important role during the life cycle of plants. Linseed flax is an oil crop and the seed is a key organ for fatty acids synthesis and storage. So it is important to understand the molecular mechanism of fatty acid biosynthesis during seed development. In this study, four small RNA libraries from early seeds at 5, 10, 20 and 30 days after flowering (DAF) were constructed and used for high-throughput sequencing to identify microRNAs (miRNAs). A total of 235 miRNAs including 114 known conserved miRNAs and 121 novel miRNAs were identified. The expression patterns of these miRNAs in the four libraries were investigated by bioinformatics and quantitative real-time polymerase chain reaction (qPCR) analysis. It was found that several miRNAs, including Lus-miRNA156a was significantly correlated with seed development process. In order to confirm the actual biological function of Lus-miRNA156a, over-expression vector was constructed and transformed to Arabidopsis. The phenotypes of homozygous transgenic lines showed decreasing of oil content and most of the fatty acid content in seeds as well as late flowering time. The results provided a clue that miRNA156a participating the fatty acid biosynthesis pathway and the detailed molecular mechanism of how it regulates the pathway needs to be further investigated.

Simultaneous extraction and determination of mono-/polyglutamyl folates using high-performance liquid chromatography-tandem mass spectrometry and its applications in starchy crops.

Folates are typically present in polyglutamyl form in organisms. In traditional extraction methods, polyglutamyl folates are hydrolyzed to monoglutamates, sacrificing valuable information. To advance folate metabolism research, we developed an accurate, sensitive, and reproducible extraction method for polyglutamyl folate species in maize, the main crop in most parts of the world. Twelve folates, including six polyglutamyl folates, were simultaneously determined in maize for the first time using high-performance liquid chromatography-tandem mass spectrometry. The glutamation states of the folates were protected by boiling, which inactivated the native conjugases. α-Amylase and protease were added to obtain better recoveries and decrease difficulties in centrifugation and filtration. The recoveries (n = 5) of six polyglutamyl folates were between 80.5 and 101%. All calibration curves showed good linear regression (r2 ≥ 0.994) within the working range. The instrumental limits of detection and quantitation ranged from 0.070 to 2.4 ng/mL and 0.22 to 8.0 ng/mL, respectively. Intra- and inter-day precision was below 7.81% and 11.9%, respectively (n = 5). Using this method, changes in poly- and monoglutamyl folates during maize germination were determined for the first time. The results suggest that folates were largely synthesized as germination initiated, and 5-methyltetrahydrofolate was the most abundant species. Tetraglutamyl 5-methyltetrahydrofolate contributed more than 50% of the 5-methyltetrahydrofolate species. Inverse changes in contents of 5,10-methenyltetrahydrofolate, and 10-formyl folic acid, monoglutamate, and diglutamate of 5-formyltetrahydrofolate were also observed, indicating potential regulation. Additionally, polyglutamyl folates in sweet potatoes were determined using this method, indicating its applications in starchy crops.

Tetrahydrofolate Modulates Floral Transition through Epigenetic Silencing.

Folates, termed from tetrahydrofolate (THF) and its derivatives, function as coenzymes in one-carbon transfer reactions and play a central role in synthesis of nucleotides and amino acids. Dysfunction of cellular folate metabolism leads to serious defects in plant development; however, the molecular mechanisms of folate-mediated cellular modifications and physiological responses in plants are still largely unclear. Here, we reported that THF controls flowering time by adjusting DNA methylation-regulated gene expression in Arabidopsis (Arabidopsis thaliana). Wild-type seedlings supplied with THF as well as the high endogenous THF content mutant dihydrofolate synthetase folypoly-Glu synthetase homolog B exhibited significant up-regulation of the flowering repressor of Flowering Wageningen and thereby delaying floral transition in a dose-dependent manner. Genome-wide transcripts and DNA methylation profiling revealed that THF reduces DNA methylation so as to manipulate gene expression activity. Moreover, in accompaniment with elevated cellular ratios between monoglutamylated and polyglutamylated folates under increased THF levels, the content of S-adenosylhomo-Cys, a competitive inhibitor of methyltransferases, was obviously higher, indicating that enhanced THF accumulation may disturb cellular homeostasis of the concerted reactions between folate polyglutamylation and folate-dependent DNA methylation. In addition, we found that the loss-of-function mutant of CG DNA methyltransferase MET1 displayed much less responsiveness to THF-associated flowering time alteration. Taken together, our studies revealed a novel regulatory role of THF on epigenetic silencing, which will shed lights on the understanding of interrelations in folate homeostasis, epigenetic variation, and flowering control in plants.

Imaging mass spectrometry-guided fast identification of antifungal secondary metabolites from Penicillium polonicum.

The discovery of antibiotics from microorganisms using classic bioactivity screens suffers from heavy labor and high re-discovery rate. Recently, largely uncovered biosynthetic potentials were unveiled by new approaches, such as genetic manipulation of "silent" biosynthetic gene clusters, innovative data acquisition, and processing methods. In this work, a fast and efficient antibiotic identification pipeline based on the MALDI-TOF imaging mass spectrometry was applied to study the antifungal metabolites during the confrontation of two fungal species, Penicillium polonicum and wilt-inducing fungus Fusarium oxysporum. By visualizing the spatial distribution of metabolites directly on the microbial colony and surrounding media, we predicted the antifungal candidates before isolating pure compounds and individually testing their bioactivity, which subsequently guided the identification of target molecules using classic chromatographic methods. Via this procedure, we successfully identified two antifungal metabolites, fructigenine A and B, which belong to indole alkaloid class and were not reported for antifungal activity. Our work assigned new bioactivity to previously reported compounds and more importantly showed the efficiency of this approach towards quick discovery of bioactive compounds, which can help study the vast unexploited synthetic potential of microbial secondary metabolites.

GLUCAN SYNTHASE-LIKE 5 (GSL5) plays an essential role in male fertility by regulating callose metabolism during microsporogenesis in rice.

Callose plays an important role in pollen development in flowering plants. In rice, 10 genes encoding putative callose synthases have been identified; however, none of them has been functionally characterized. In this study, a rice Glucan Synthase-Like 5 (GSL5) knock-out mutant was isolated that exhibited a severe reduction in fertility. Pollen viability tests indicated that the pollen of the mutant was abnormal while the embryo sac was normal. Further, GSL5-RNA interference transgenic plants phenocopied the gsl5 mutant. The RNA expression of GSL5 was found to be knocked out in the gsl5 mutant and knocked down in GSL5-RNA interference transgenic plants by real-time reverse transcripion-PCR (RT-PCR) analysis. The male sterility of the mutant was due to abnormal microspore development; an analysis of paraffin sections of the mutant anthers at various developmental stages revealed that abnormal microspore development began in late meiosis. Both the knock-out and knock-down of GSL5 caused a lack of callose in the primary cell wall of meiocytes and in the cell plate of tetrads. As a result, the callose wall of the microspores was defective. This was demonstrated by aniline blue staining and an immunogold labeling assay; the microspores could not maintain their shape, leading to premature swelling and even collapsed microspores. These data suggest that the callose synthase encoded by GSL5 plays a vital role in microspore development during late meiosis and is essential for male fertility in rice.

The non-metabolizable glucose analog D-glucal inhibits aflatoxin biosynthesis and promotes kojic acid production in Aspergillus flavus.

BACKGROUND: Aflatoxins (AFs) are potent carcinogenic compounds produced by several Aspergillus species, which pose serious threats to human health. As sugar is a preferred carbohydrate source for AF production, we examined the possibility of using sugar analogs to inhibit AF biosynthesis. RESULTS: We showed that although D-glucal cannot be utilized by A. flavus as the sole carbohydrate source, it inhibited AF biosynthesis and promoted kojic acid production without affecting mycelial growth when applied to a glucose-containing medium. The inhibition occurred before the production of the first stable intermediate, norsolorinic acid, suggesting a complete inhibition of the AF biosynthetic pathway. Further studies showed that exogenous D-glucal in culture led to reduced accumulation of tricarboxylic acid (TCA) cycle intermediates and reduced glucose consumption, indicating that glycolysis is inhibited. Expression analyses revealed that D-glucal suppressed the expression of AF biosynthetic genes but promoted the expression of kojic acid biosynthetic genes. CONCLUSIONS: D-glucal as a non-metabolizable glucose analog inhibits the AF biosynthesis pathway by suppressing the expression of AF biosynthetic genes. The inhibition may occur either directly through interfering with glycolysis, or indirectly through reduced oxidative stresses from kojic acid biosynthesis.

Identification of LncRNAs and Functional Analysis of ceRNA Related to Fatty Acid Synthesis during Flax Seed Development.

Flax is a flowering plant cultivated for its oil and contains various unsaturated fatty acids. Linseed oil is known as the "deep-sea fish oil" of plants, and is beneficial to brain and blood lipids, among other positive effects. Long non-coding RNAs (lncRNAs) play an important role in plant growth and development. There are not many studies assessing how lncRNAs are related to the fatty acid synthesis of flax. The relative oil contents of the seeds of the variety Heiya NO.14 (for fiber) and the variety Macbeth (for oil) were determined at 5 day, 10 day, 20 day, and 30 day after flowering. We found that 10-20 day is an important period for ALA accumulation in the Macbeth variety. The strand-specific transcriptome data were analyzed at these four time points, and a series of lncRNAs related to flax seed development were screened. A competing endogenous RNA (ceRNA) network was constructed and the accuracy of the network was verified using qRT-PCR. MSTRG.20631.1 could act with miR156 on the same target, squamosa promoter-binding-like protein (SPL), to influence fatty acid biosynthesis through a gluconeogenesis-related pathway during flax seed development. This study provides a theoretical basis for future studies assessing the potential functions of lncRNAs during seed development.

The domestication-associated L1 gene encodes a eucomic acid synthase pleiotropically modulating pod pigmentation and shattering in soybean.

Pod coloration is a domestication-related trait in soybean, with modern cultivars typically displaying brown or tan pods, while their wild relative, Glycine soja, possesses black pods. However, the factors regulating this color variation remain unknown. In this study, we cloned and characterized L1, the classical locus responsible for black pods in soybean. By using map-based cloning and genetic analyses, we identified the causal gene of L1 and revealed that it encodes a hydroxymethylglutaryl-coenzyme A (CoA) lyase-like (HMGL-like) domain protein. Biochemical assays showed that L1 functions as a eucomic acid synthase and facilitates the synthesis of eucomic acid and piscidic acid, both of which contribute to coloration of pods and seed coats in soybean. Interestingly, we found that L1 plants are more prone to pod shattering under light exposure than l1 null mutants because dark pigmentation increases photothermal efficiency. Hence, pleiotropic effects of L1 on pod color and shattering, as well as seed pigmentation, likely contributed to the preference for l1 alleles during soybean domestication and improvement. Collectively, our study provides new insights into the mechanism of pod coloration and identifies a new target for future de novo domestication of legume crops.

Identification of seven undescribed cucurbitacins in Cucumis sativus (cucumber) and their cytotoxic activity.

Cucurbitacin C-type cucurbitacins that are only identified in Cucumis sativus (cucumber) are, in part, responsible for the health benefits and bitter flavor. Nevertheless, detailed information about those functional ingredients in cucumber is scarce. In this study, ten cucurbitacin C analogues including seven undescribed ones have been isolated from the bitter leaves of cucumber, in which six compounds showed growth inhibition capabilities against tumor cell lines HepG2, A549, DU145 and HCT116. Intriguingly, cucurbitacin C6 and C7 exhibited a significant inhibition effect compared to the positive control taxol (IC50 = 1.86 ± 0.17 µM) on HepG2 cell line with IC50 values of 10.06 ± 0.34 µM and 4.16 ± 0.42 µM, respectively. The mechanism of cucurbitacin-induced apoptosis is likely down-regulating the expression of caspase-related proteins. This work enlarges the knowledge of the cucurbitacins in cucumber and highlights the importance of cucumber as a source of specialized metabolites in the food and medicinal industries.

Enhanced chemoselectivity of a plant cytochrome P450 through protein engineering of surface and catalytic residues.

Cytochrome P450s (P450s) are the most versatile catalysts utilized by plants to produce structurally and functionally diverse metabolites. Given the high degree of gene redundancy and challenge to functionally characterize plant P450s, protein engineering is used as a complementary strategy to study the mechanisms of P450-mediated reactions, or to alter their functions. We previously proposed an approach of engineering plant P450s based on combining high-accuracy homology models generated by Rosetta combined with data-driven design using evolutionary information of these enzymes. With this strategy, we repurposed a multi-functional P450 (CYP87D20) into a monooxygenase after redesigning its active site. Since most plant P450s are membrane-anchored proteins that are adapted to the micro-environments of plant cells, expressing them in heterologous hosts usually results in problems of expression or activity. Here, we applied computational design to tackle these issues by simultaneous optimization of the protein surface and active site. After screening 17 variants, effective substitutions of surface residues were observed to improve both expression and activity of CYP87D20. In addition, the identified substitutions were additive and by combining them a highly efficient C11 hydroxylase of cucurbitadienol was created to participate in the mogrol biosynthesis. This study shows the importance of considering the interplay between surface and active site residues for P450 engineering. Our integrated strategy also opens an avenue to create more tailoring enzymes with desired functions for the metabolic engineering of high-valued compounds like mogrol, the precursor of natural sweetener mogrosides. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-021-00056-z.

Overexpression of the caryophyllene synthase gene GhTPS1 in cotton negatively affects multiple pests while attracting parasitoids.

BACKGROUD: Volatile terpenes can act as ecological signals to affect insect behavior. It has been proposed that the manipulation of terpenes in plants can help to control herbivore pests. In order to investigate the potential pest management function of (E)-ß-caryophyllene in cotton plants, the (E)-ß-caryophyllene synthase gene (GhTPS1) was inserted into Gossypium hirsutum variety R15 to generate overexpression lines. RESULTS: Four GhTPS1-transgenic lines were generated, and GhTPS1 expression in transgenic L18 and L46 lines was 3-5-fold higher than in R15 plants. The transgenic L18 and L46 lines also emitted significantly more (E)-ß-caryophyllene than R15. In laboratory bioassays, L18 and L46 plants reduced pests Apolygus lucorum, Aphis gossypii and Helicoverpa armigera, and attracted parasitoids Peristenus spretus and Aphidius gifuensis, but not Microplitis mediator. In open-field trials, L18 and L46 plants reduced A. lucorum, Adelphocoris suturalis and H. armigera, but had no significant effects on predators. CONCLUSION: Our findings suggest that L18 and L46 plants reduce several major hemipteran and lepidopteran cotton pests, whereas, two parasitoids P. spretus and A. gifuensis, were attracted by L18 and L46 plants. This study shows that overexpressing GhTPS1 in cotton may help to improve pest management in cotton fields. © 2019 Society of Chemical Industry.

Rapid identification of "mad honey" from Tripterygium wilfordii Hook. f. and Macleaya cordata (Willd) R. Br using UHPLC/Q-TOF-MS.

Cases of honey poisoning have been reported widely, meaning there is a need for methods that detect "mad honey" or honey contaminated with plant-derived toxins to protect human health. In this study, we compared whole flower extracts and honey from Tripterygium wilfordii Hook. f. (TwHf) and Macleaya cordata (Willd) R. Br (McRB) using QuEChERS (quick, easy, cheap, effective, rugged, and safe) and ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS). The results revealed several compounds common to whole flowers and honey samples. Triptolide and protopine were selected as potential markers for identifying "mad honeys" from these plants. The developed method can easily detect different honey varieties that were spiked with 5% TwHf and McRB honey samples. Additionally, 90 commercial honey samples were analyzed and determined as free from contamination. The method described in this report could be useful for studies on honey from other poisonous nectar and pollen plants.

A structural and data-driven approach to engineering a plant cytochrome P450 enzyme.

Functional manipulation of biosynthetic enzymes such as cytochrome P450s (or P450s) has attracted great interest in metabolic engineering of plant natural products. Cucurbitacins and mogrosides are plant triterpenoids that share the same backbone but display contrasting bioactivities. This structural and functional diversity of the two metabolites can be manipulated by engineering P450s. However, the functional redesign of P450s through directed evolution (DE) or structure-guided protein engineering is time consuming and challenging, often because of a lack of high-throughput screening methods and crystal structures of P450s. In this study, we used an integrated approach combining computational protein design, evolutionary information, and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450 (CYP87D20) from cucumber. After three rounds of iterative design and evaluation of 96 protein variants, CYP87D20, which is involved in the cucurbitacin C biosynthetic pathway, was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of cucurbitadienol. This integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol, the precursor of the natural sweetener mogroside, or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450s.

Biomarkers for early diagnosis of type 2 diabetic nephropathy: a study based on an integrated biomarker system.

Diabetic nephropathy is a devastating disease that affects a growing number of diabetic patients. A complete cure is very hard to achieve once the disease has been diagnosed, therefore the diagnosis of early stages in diabetic nephropathy has become a hot area. Numbers of molecules have been proposed to be potential biomarkers for this purpose. However, some problems still remain, such as discovering effective biomarkers to diagnose the disease before obvious clinical evidence appears. Thus, the main purpose of this study was to find plasma biomarkers for early diagnosis of type 2 diabetic nephropathy stage 1 and stage 2, as well as separating them from diabetes. 182 subjects (Chinese) were recruited for this study, including 50 healthy controls, 33 type 2 diabetic patients and 99 type 2 diabetic nephropathy patients (33 of these were stage 3). Important clinical indicators including proteinuria, serum creatinine, and urea nitrogen were measured and the glomerular filtration rate was estimated to assess kidney function; fasting blood glucose, postprandial blood glucose and glycated hemoglobin were measured to assess the blood glucose control. Key metabolites and genes in plasma samples were identified and determined using -omic and quantitative techniques. The potential biomarkers were then combined and carefully screened to determine the most informative ones for early diagnosis of type 2 diabetic nephropathy. An integrated biomarker system (IBS) incorporating 6 clinical indicators, 40 metabolites and 5 genes was established. Correlation analysis results revealed that most of the potential biomarkers significantly correlated with the 6 clinical indicators. Discriminant analysis results showed that the developed IBS gave the highest total predictive accuracy (98.9%). Significant test and receiver operating characteristic analysis results indicated that inosine had the highest sensitivity (0.889), specificity (1.000), positive predictive rate (1.000) and negative predictive rate (0.900) amongst the 48 potential biomarkers when separating patients with diabetes from patients with diabetic nephropathy stage 3. Finally, inosine with a cutoff of 0.086 mg L(-1) was combined with estimated GFR to differentiate between diabetic nephropathy stages 1 and 2 from diabetes. The results demonstrate that IBS combined with a proper statistical analysis technique is a powerful tool for biomarker screening.

Plasma esterified and non-esterified fatty acids metabolic profiling using gas chromatography-mass spectrometry and its application in the study of diabetic mellitus and diabetic nephropathy.

Using gas chromatography-mass spectrometry (GC-MS), a new metabolic profiling method was established to assess the levels of non-esterified fatty acids (NEFAs) and esterified fatty acids (EFAs) in plasma. The extraction method was simple and robust without removing protein process. With this method 25 fatty acids (FAs), both EFAs and NEFAs, can be recognized simultaneously with only 10 µL plasma. 15 of the 25 can be precisely quantified. The method was validated and then applied into clinical metabonomics research. Five clinical groups including 150 cases were involved. The relationship between FA levels and diabetic mellitus (DM) as well as diabetic nephropathy (DN) pathology was speculated. Furthermore, the possible pathological causes and effects were discussed in detail. Potential biomarkers (p value <0.01) were screened with Student's t-test. With the application of partial least squares-discriminant analysis (PLS-DA), different stages were distinguished. The result may be useful for the pathology study of metabolic syndromes, and may also be helpful for monitoring the progression of DM and DN.