OXIDOSQUALENE CYCLASE 1 and 2 influence triterpene biosynthesis and defense in Nicotiana attenuata.

Triterpenes are a class of bioactive compounds with diverse biological functions, playing pivotal roles in plant defense against biotic stressors. Oxidosqualene cyclases (OSCs) serve as gatekeepers in the biosynthesis of triterpenes. In this study, we utilized a Nicotiana benthamiana heterologous expression system to characterize NaOSC1 from Nicotiana attenuata as a multifunctional enzyme capable of synthesizing lupeol, dammarenediol II, 3-alpha,20-lupanediol, and 7 other triterpene scaffolds. We also demonstrated that NaOSC2 is, in contrast, a selective enzyme, producing only the ß-amyrin scaffold. Through virus-induced gene silencing and in vitro toxicity assays, we elucidated the roles of NaOSC1 and NaOSC2 in the defense of N. attenuata against Manduca sexta larvae. Metabolomic and feature-based molecular network analyses of leaves with silenced NaOSC1 and NaOSC2 unveiled 3 potential triterpene glycoside metabolite clusters. Interestingly, features identified as triterpenes within these clusters displayed a significant negative correlation with larval mass. Our study highlights the pivotal roles of NaOSC1 and NaOSC2 from N. attenuata in the initial steps of triterpene biosynthesis, subsequently influencing defense against M. sexta through the modulation of downstream triterpene glycoside compounds.

Field-work reveals a novel function for MAX2 in a native tobacco's high-light adaptions.

Laboratory studies have revealed that strigolatone (SL) and karrikin (KAR) signalling mediate responses to abiotic and biotic stresses, and reshape branching architecture that could increase reproductive performance and crop yields. To understand the ecological function of SL and KAR signalling, transgenic lines of wild tobacco Nicotiana attenuata, silenced in SL/KAR biosynthesis/signalling were grown in the glasshouse and in two field plots in the Great Basin Desert in Utah over four field seasons. Of the lines silenced in SL and KAR signalling components (irMAX2, irD14, irKAI2 and irD14 × irKAI2 plants), which exhibited the expected increases in shoot branching, only irMAX2 plants showed a strong leaf-bleaching phenotype when grown in the field. In the field, irMAX2 plants had lower sugar and higher leaf amino acid contents, lower lifetime fitness and were more susceptible to herbivore attack compared to wild-type plants. These irMAX2 phenotypes were not observed in glasshouse-grown plants. Transcriptomic analysis revealed dramatic responses to high-light intensity in irMAX2 leaves in the field: lutein contents decreased, and transcriptional responses to high-intensity light, singlet oxygen and hydrogen peroxide increased. PAR and UV-B manipulations in the field revealed that the irMAX2 bleaching phenotype is reversed by decreasing PAR, but not UV-B fluence. We propose that NaMAX2 functions in high-light adaptation and fitness optimisation by regulating high-light responses independently of its roles in the SL and KAR signalling pathways. The work provides another example of the value of studying the function of genes in the complex environments in which plants evolved, namely nature.

Exploring the metabolic basis of growth/defense trade-offs in complex environments with Nicotiana attenuata plants cosilenced in NaMYC2a/b expression.

In response to challenges from herbivores and competitors, plants use fitness-limiting resources to produce (auto)toxic defenses. Jasmonate signaling, mediated by MYC2 transcription factors (TF), is thought to reconfigure metabolism to minimize these formal costs of defense and optimize fitness in complex environments. To study the context-dependence of this metabolic reconfiguration, we cosilenced NaMYC2a/b by RNAi in Nicotiana attenuata and phenotyped plants in the field and increasingly realistic glasshouse setups with competitors and mobile herbivores. NaMYC2a/b had normal phytohormonal responses, and higher growth and fitness in herbivore-reduced environments, but were devastated in high herbivore-load environments in the field due to diminished accumulations of specialized metabolites. In setups with competitors and mobile herbivores, irMYC2a/b plants had lower fitness than empty vector (EV) in single-genotype setups but increased fitness in mixed-genotype setups. Correlational analyses of metabolic, resistance, and growth traits revealed the expected defense/growth associations for most sectors of primary and specialized metabolism. Notable exceptions were some HGL-DTGs and phenolamides that differed between single-genotype and mixed-genotype setups, consistent with expectations of a blurred functional trichotomy of metabolites. MYC2 TFs mediate the reconfiguration of primary and specialized metabolic sectors to allow plants to optimize their fitness in complex environments.

Natural history-guided omics reveals plant defensive chemistry against leafhopper pests.

Although much is known about plant traits that function in nonhost resistance against pathogens, little is known about nonhost resistance against herbivores, despite its agricultural importance. Empoasca leafhoppers, serious agricultural pests, identify host plants by eavesdropping on unknown outputs of jasmonate (JA)-mediated signaling. Forward- and reverse-genetics lines of a native tobacco plant were screened in native habitats with native herbivores using high-throughput genomic, transcriptomic, and metabolomic tools to reveal an Empoasca-elicited JA-JAZi module. This module induces an uncharacterized caffeoylputrescine-green leaf volatile compound, catalyzed by a polyphenol oxidase in a Michael addition reaction, which we reconstitute in vitro; engineer in crop plants, where it requires a berberine bridge enzyme-like 2 (BBL2) for its synthesis; and show that it confers resistance to leafhoppers. Natural history-guided forward genetics reveals a conserved nonhost resistance mechanism useful for crop protection.

Changing light promotes isoflavone biosynthesis in soybean pods and enhances their resistance to mildew infection.

Mildew severely reduces soybean yield and quality, and pods are the first line of defence against pathogens. Maize-soybean intercropping (MSI) reduces mildew incidence on soybean pods; however, the mechanism remains unclear. Changing light (CL) from maize shading is the most important environmental feature in MSI. We hypothesized that CL affects isoflavone accumulation in soybean pods, affecting their disease resistance. In the present study, shading treatments were applied to soybean plants during different developmental stages according to various CL environments under MSI. Chlorophyll fluorescence imaging (CFI) and classical evaluation methods confirmed that CL, especially vegetative stage shading (VS), enhanced pod resistance to mildew. Further metabolomic analyses and exogenous jasmonic acid (JA) and biosynthesis inhibitor experiments revealed the important relationship between JA and isoflavone biosynthesis, which had a synergistic effect on the enhanced resistance of CL-treated pods to mildew. VS promoted the biosynthesis and accumulation of constitutive isoflavones upstream of the isoflavone pathway, such as aglycones and glycosides, in soybean pods. When mildew infects pods, endogenous JA signalling stimulated the biosynthesis of downstream inducible malonyl isoflavone (MIF) and glyceollin to improve pod resistance.

Drought Tolerance of Soybean (Glycine max L. Merr.) by Improved Photosynthetic Characteristics and an Efficient Antioxidant Enzyme Activities Under a Split-Root System.

Water deficiency significantly affects photosynthetic characteristics. However, there is little information about variations in antioxidant enzyme activities and photosynthetic characteristics of soybean under imbalanced water deficit conditions (WDC). We therefore investigated the changes in photosynthetic and chlorophyll fluorescence characteristics, total soluble protein, Rubisco activity (RA), and enzymatic activities of two soybean varieties subjected to four different types of imbalanced WDC under a split-root system. The results indicated that the response of both cultivars was significant for all the measured parameters and the degree of response differed between cultivars under imbalanced WDC. The maximum values of enzymatic activities (SOD, CAT, GR, APX, and POD), chlorophyll fluorescence (Fv/Fm, qP, ɸPSII, and ETR), proline, RA, and total soluble protein were obtained with a drought-tolerant cultivar (ND-12). Among imbalanced WDC, the enhanced net photosynthesis, transpiration, and stomatal conductance rates in T2 allowed the production of higher total soluble protein after 5 days of stress, which compensated for the negative effects of imbalanced WDC. Treatment T4 exhibited greater potential for proline accumulation than treatment T1 at 0, 1, 3, and 5 days after treatment, thus showing the severity of the water stress conditions. In addition, the chlorophyll fluorescence values of FvFm, ɸPSII, qP, and ETR decreased as the imbalanced WDC increased, with lower values noted under treatment T4. Soybean plants grown in imbalanced WDC (T2, T3, and T4) exhibited signs of oxidative stress such as decreased chlorophyll content. Nevertheless, soybean plants developed their antioxidative defense-mechanisms, including the accelerated activities of these enzymes. Comparatively, the leaves of soybean plants in T2 displayed lower antioxidative enzymes activities than the leaves of T4 plants showing that soybean plants experienced less WDC in T2 compared to in T4. We therefore suggest that appropriate soybean cultivars and T2 treatments could mitigate abiotic stresses under imbalanced WDC, especially in intercropping.

[A clinical follow-up study of children with well-controlled asthma after withdrawal of low-dose inhaled corticosteroids].

OBJECTIVE: To study the incidence of acute attacks of asthma and dynamic changes in laboratory markers in children with well-controlled asthma after the withdrawal of low-dose inhaled corticosteroids (ICS), and to provide a basis for optimal long-term control regimens for children with asthma. METHODS: A total of 63 children with well-controlled asthma were enrolled as subjects. According to their parents' wishes, they were continuously administered with ICS (ICS treatment group; n=35) and without ICS (ICS withdrawal group; n=28). They were followed up for 18 months. The incidence of acute attacks of asthma was evaluated, dynamic monitoring was performed for pulmonary function and fractional exhaled nitric oxide (FeNO), and childhood asthma control test (C-ACT) was performed every three months. RESULTS: At 3, 6, 9, and 12 months of follow-up, there was no significant difference in FeNO between the ICS treatment and withdrawal groups (P>0.05). However, at 15 and 18 months of follow-up, the withdrawal group had a significantly higher level of FeNO than the ICS treatment group (P<0.05). There was no significant difference in the C-ACT score between the two groups at all time points of follow-up (P>0.05). At 3, 6, 9, and 12 months of follow-up, there were no significant differences between the two groups in the percentage of forced expiratory volume in 1 second, the ratio of forced expiratory volume in 1 second to forced vital capacity, percentage of predicted maximum mid-expiratory flow (MMEF%), and maximal expiratory flow at 50% of vital capacity (MEF50) (P>0.05), while at 15 and 18 months of follow-up, the ICS treatment group had significantly higher MMEF% and MEF50 than the withdrawal group (P<0.05). During follow-up, 3 children (9%) in the ICS treatment group and 8 (29%) in the withdrawal group experienced acute attacks of asthma (P=0.0495). CONCLUSIONS: Continuous inhalation of low-dose ICS can maintain the stability of pulmonary function and reduce acute attacks of asthma in children with well-controlled asthma.

DA-6 promotes germination and seedling establishment from aged soybean seeds by mediating fatty acid metabolism and glycometabolism.

Soybean seeds contain higher concentrations of oil (triacylglycerol) and fatty acids than do cereal crop seeds, and the oxidation of these biomolecules during seed storage significantly shortens seed longevity and decreases germination ability. Here, we report that diethyl aminoethyl hexanoate (DA-6), a plant growth regulator, increases germination and seedling establishment from aged soybean seeds by increasing fatty acid metabolism and glycometabolism. Phenotypic analysis showed that DA-6 treatment markedly promoted germination and seedling establishment from naturally and artificially aged soybean seeds. Further analysis revealed that DA-6 increased the concentrations of soluble sugars during imbibition of aged soybean seeds. Consistently, the concentrations of several different fatty acids in DA-6-treated aged seeds were higher than those in untreated aged seeds. Subsequently, quantitative PCR analysis indicated that DA-6 induced the transcription of several key genes involved in the hydrolysis of triacylglycerol to sugars in aged soybean seeds. Furthermore, the activity of invertase in aged seeds, which catalyzes the hydrolysis of sucrose to form fructose and glucose, increased following DA-6 treatment. Taken together, DA-6 promotes germination and seedling establishment from aged soybean seeds by enhancing the hydrolysis of triacylglycerol and the conversion of fatty acids to sugars.

Extraction optimization, purification and characterization of polysaccharides from the seed coat of black soybean.

In this study, the extraction of water-soluble polysaccharides from the seed coat of black soybean (BSCP) was investigated and optimized. A response surface methodology based on a Box-Behnken design (BBD) was used to optimize the extraction conditions as follows: extraction temperature, 100°C; ratio of water to material, 22.3 mL/g; and extraction time, 133.2 min. Under these conditions, the experimental yield of polysaccharides was 10.56%, which was consistent with the predictive yield. A novel galactomannan, BSCP-1, with a molecular weight of 7.55 × 105 Da determined by high-performance gel permeation chromatography, was isolated from the black soybean seed coat. Through gas chromatography-mass spectrometry analysis, BSCP-1 was identified as a galactomannan consisting of galactose, mannose and rhamnose in a molar ratio of 6.01:3.56:1.00. Cytotoxicity against the human gastric carcinoma cancer cell line was also determined.

Fungal Diversity in Field Mold-Damaged Soybean Fruits and Pathogenicity Identification Based on High-Throughput rDNA Sequencing.

Continuous rain and an abnormally wet climate during harvest can easily lead to soybean plants being damaged by field mold (FM), which can reduce seed yield and quality. However, to date, the underlying pathogen and its resistance mechanism have remained unclear. The objective of the present study was to investigate the fungal diversity of various soybean varieties and to identify and confirm the FM pathogenic fungi. A total of 62,382 fungal ITS1 sequences clustered into 164 operational taxonomic units (OTUs) with 97% sequence similarity; 69 taxa were recovered from the samples by internal transcribed spacer (ITS) region sequencing. The fungal community compositions differed among the tested soybeans, with 42 OTUs being amplified from all varieties. The quadratic relationships between fungal diversity and organ-specific mildew indexes were analyzed, confirming that mildew on soybean pods can mitigate FM damage to the seeds. In addition, four potentially pathogenic fungi were isolated from FM-damaged soybean fruits; morphological and molecular identification confirmed these fungi as Aspergillus flavus, A. niger, Fusarium moniliforme, and Penicillium chrysogenum. Further re-inoculation experiments demonstrated that F. moniliforme is dominant among these FM pathogenic fungi. These results lay the foundation for future studies on mitigating or preventing FM damage to soybean.

Organ-Specific Differential NMR-Based Metabonomic Analysis of Soybean [Glycine max (L.) Merr.] Fruit Reveals the Metabolic Shifts and Potential Protection Mechanisms Involved in Field Mold Infection.

Prolonged, continuous rainfall is the main climatic characteristic of autumn in Southwest China, and it has been found to cause mildew outbreaks in pre-harvest soybean fields. Low temperature and humidity (LTH) stress during soybean maturation in the field promotes pre-harvest mildew, resulting in damage to different organs of soybean fruits to different extents, but relatively little information on the resistance mechanisms in these fruits is available. Therefore, to understand the metabolic responses of soybean fruits to field mold (FM), the metabonomic variations induced by LTH were characterized using proton nuclear magnetic resonance spectroscopy (1H-NMR), and the primary metabolites from the pod, seed coat and cotyledon of pre-harvest soybean were quantified. Analysis of FM-damaged soybean germplasms with different degrees of resistance to FM showed that extracts were dominated by 66 primary metabolites, including amino acids, organic acids and sugars. Each tissue had a characteristic metabolic profile, indicating that the metabolism of proline in the cotyledon, lysine in the seed coat, and sulfur in the pod play important roles in FM resistance. The primary-secondary metabolism interface and its potential contribution to FM resistance was investigated by targeted analyses of secondary metabolites. Both the seed coat and the pod have distinct but nonexclusive metabolic responses to FM, and these are functionally integrated into FM resistance mechanisms.

Metabolomic tool to identify soybean [Glycine max (L.) Merrill] germplasms with a high level of shade tolerance at the seedling stage.

The isoflavone profiles of seeds of various soybean genotypes with different levels of shade tolerance at the seedling stage were investigated. High-performance liquid chromatography (HPLC) was used to quantify 12 isoflavones, and the data were analyzed using a multivariate statistical analysis. Combined with field experimental data and an orthogonal partial least-squares discriminant analysis (OPLS-DA), several aglycones (genistein (GE), daidzein (DE), and glycitein (GLE)) were selected and identified as key compounds involved in the shade tolerance of soybean seedlings. Additional correlation analysis and laboratory shading stress experiments with soybean seedlings also confirmed the function of these selected isoflavones, especially GE, in the discrimination of soybean seedlings with different levels of shade tolerance. Furthermore, the structure-antioxidant activity relationships between a range of isoflavones and the plant shade-tolerance mechanism are discussed. Targeted metabolomic analyses of isoflavones could reveal the diversity of shade tolerance in soybean seedlings, thus contributing to the breeding of excellent soybean varieties.

Metabolism variation and better storability of dark- versus light-coloured soybean (Glycine max L. Merr.) seeds.

The effects of storage duration on the seed germination and metabolite profiling of soybean seeds with five different coloured coats were studied. Their germination, constituents and transcript expressions of isoflavones and free fatty acids (FFAs) were compared using chromatographic metabolomic profiling and transcriptome sequencing. The seed water content was characterized using nuclear magnetic resonance (NMR) relaxometry. Results showed that dark-coloured seeds were less inactivated than light-coloured seeds. The aglycone and ß-glucoside concentrations of upstream constituents increased significantly, whereas the acetylglucosides and malonylglucosides of downstream constituents decreased with an increase in the storage period. FFAs increased considerably in the soybean seeds as a result of storage. These results indicate that dark-coloured soybean seeds have better storability than light-coloured seeds, and seed water content plays a role in seed inactivation. It was concluded that there are certain metabolic regularities that are associated with different coloured seed coats of soybeans under storage conditions.

A New 5(6→7)abeo-sterol from the twigs of Podocarpus fleuryi.

A new 5(6→7)abeo-sterol with rarely reported [6-5-6-5]-fused rings, 3ß,5ß,6-trihydroxyl-B-norsitostane (1), along with 10 known compounds were isolated from the twigs of Podocarpus fleuryi. Their structures were elucidated by means of extensive spectroscopic analysis. Compound 1 was assessed for their cytotoxicity against five human tumour cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW-480), and the result showed that it had no activity.

Pod Mildew on Soybeans Can Mitigate the Damage to the Seed Arising from Field Mold at Harvest Time.

Seedpods are the outermost barrier of legume plants encountered by pests and pathogens, but research on this tissue, especially regarding their chemical constituents, is limited. In the present study, a mildew-index-model-based cluster analysis was used to evaluate and identify groups of soybean genotypes with different organ-specific resistance against field mold. The constituents of soybean pods, including proteins, carbohydrates, fatty acids, and isoflavones, were analyzed. Linear regression and correlation analyses were also conducted between these main pod constituents and the organ-specific mildew indexes of seed (MIS) and pod (MIP). With increases in the contents of infection constituents, such as proteins, carbohydrates, and fatty acids, the MIP increased and the MIS decreased. The MIS decreased with increases in the contents of glycitein (GLE)-type isoflavonoids, which act as antibiotic constituents. Although the infection constituents in the soybean pods caused pod mildew, they also helped mitigate the corresponding seed mildew to a certain extent.

Two new flavans from the trunk and leaves of Horsfieldia glabra.

Two new flavans, (2R, 4R)-4'-hydroxy-3',5'-methyl-6,7-methylenedioxy-4-O-2'-cycloflavan (1), and (2R, 4R)-4'-hydroxy-3'-methyl-6,7-methylenedioxy-4-O-2'-cycloflavan (2), along with six known compounds were isolated from the trunk and leaves of Horsfieldia glabra. Their structures were elucidated by means of extensive spectroscopic analysis. Compounds 1 and 2, together with 4-7 were assessed for their cytotoxicity against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW-480), and the result showed that they had no activity.

Chemical Constituents of Plants from the Genus Psychotria.

Psychotria is a genus of ca. 1500 species in the family Rubiaceae. Up to now, 41 species of the Psychotria genus have been chemically investigated, and 159 compounds, including alkaloids of indole, quinoline and benzoquinolizidine type, terpenoids, steroids, phenolics and aliphatic compounds have been isolated. These compounds show potent bioactivities, such as antimicrobial, antiviral, and antiparasitic activities.

Partial improvements in the flavor quality of soybean seeds using intercropping systems with appropriate shading.

The profiles of isoflavone and fatty acids constitute important quality traits in soybean seeds, for making soy-based functional food products, due to their important contributions to the flavor and nutritional value of these products. In general, the composition of these constituents in raw soybeans is affected by cultivation factors, such as sunlight; however, the relationship of the isoflavone and fatty acid profiles with cultivation factors is not well understood. This study evaluated the isoflavone and fatty acid profiles in soybeans grown under a maize-soybean relay strip intercropping system with different row spacings, and with changes in the photosynthetic active radiation (PAR) transmittance. The effects of PAR on the isoflavone and fatty acid contents were found to be quadratic. Appropriate intercropping shading may reduce the bitterness of soybeans caused by soy aglycone and could improve their fatty acid composition.

A new sesquiterpenoid from Polyalthia petelotii.

A new sesquiterpenoid (‒)-ethyl dihydrophaseate (1) was isolated from the stems and leaves of Polyalthia petelotii, together with two clerodane diterpenoids, 16α-hydroxycleroda-3,13(14)Z-dien-15,16-olide (2), 15-hydroxy-cis-ent-cleroda-3,13(E)-diene (3), a eudesmane sesquiterpenoid, eudesm-4(15)-ene-7α,11-diol (4), an aromatic aldehyde, vanillin (5), a bisisoquinolines alkaloid, spinosine (6) and an aporphine alkaloid, (‒)-oliveroline-ß-N-oxide (7). Their structures were established by extensive spectroscopic analysis, including 2D-NMR techniques. Compounds 3, 4 and 6 were isolated from the genus Polyalthia for the first time and the others obtained originally from P. petelotii. The isolates were assessed for their cytotoxicity against five human tumour lines (HL-60, SMMC-7721, A-549, MCF-7 and SW-480), and the result showed that only 2 displayed weak inhibitory activity.