Infection by Fusarium verticillioides causes field mold in soybean and reduces seed quality, yet the defensive role of pods as the first protective barrier remains poorly understood. Here, we compared a resistant cultivar (D49) and a susceptible cultivar (ND12). Phenotypic analysis showed that D49 exhibits stronger resistance to pathogen infection. Integrated transcriptomic and metabolomic analyses revealed that isoflavonoid and lipid biosynthetic pathways are specifically activated in the resistant genotype upon infection. Metabolite quantification demonstrated that isoflavonoids accumulate earlier and to higher levels in D49, whereas ND12 shows delayed and weaker induction. In addition, D49 undergoes dynamic remodeling of the cuticle wax barrier, characterized by reduced cutin monomers and increased wax accumulation, while ND12 displays a weaker structural response and a decline in wax content after infection. These results suggest that resistance is achieved through the coordinated action of early isoflavonoid accumulation and adaptive restructuring of the cuticle-wax barrier. Using WGCNA we found three candidate transcription factor genes (GmEGL3, BHLH149, and GmERF9) as potential regulators linking isoflavonoid metabolism and lipid barrier formation, providing molecular insights for soybean resistance breeding.

Pentacyclic triterpenoids, recognized for their natural bioactivity, display complex spatiotemporal accumulation patterns within the ecological model plant, Nicotiana attenuata . Despite their ecological significance, the underlying biosynthetic enzymes and functional attributes of triterpenoid synthesis in N. attenuata remain unexplored. Three multifunctional cytochrome P450 monooxygenases (NaCYP716A419, NaCYP716C87, NaCYP716E107) from N. attenuata were shown to oxidize the pentacyclic triterpene skeleton as evidenced by heterologous expression in Nicotiana benthamiana . NaCYP716A419 catalyzed a consecutive three-step oxidation reaction at the C28 position of β-amyrin/lupeol/lupanediol, yielding the corresponding alcohol, aldehyde, and carboxylic acid. NaCYP716C87 hydroxylated the C2α position of β-amyrin/lupeol/lupanediol/erythrodiol/oleanolic acid/betulinic acid, while NaCYP716E107 hydroxylated the C6β position of β-amyrin/oleanolic acid. Three CYP716 enzymes are highly expressed in flowers and respond to induction by ABA, MeJA, SA, GA3, and abiotic stress treatments. Using VIGS technology, we revealed that silencing of NaCYP716A419 affects the growth and reproduction of N. attenuata , suggesting the ecological significance of these specialized metabolite biosynthetic steps.One-sentence summary Three CYP716 enzymes diversify N. attenuata’s triterpenoid sector with potential roles in growth and development.

Pentacyclic triterpenoids, recognized for their natural bioactivity, display complex spatiotemporal accumulation patterns within the ecological model plant Nicotiana attenuata. Despite their ecological importance, the underlying biosynthetic enzymes and functional attributes of triterpenoid synthesis in N. attenuata remain unexplored. Here, we show that 3 cytochrome P450 monooxygenases (NaCYP716A419, NaCYP716C87, and NaCYP716E107) from N. attenuata oxidize the pentacyclic triterpene skeleton, as evidenced by heterologous expression in Nicotiana benthamiana. NaCYP716A419 catalyzed a consecutive 3-step oxidation reaction at the C28 position of beta-amyrin/lupeol/lupanediol, yielding the corresponding alcohol, aldehyde, and carboxylic acid. NaCYP716C87 hydroxylated the C2 alpha position of beta-amyrin/lupeol/lupanediol/erythrodiol/oleanolic acid/betulinic acid, while NaCYP716E107 hydroxylated the C6 beta position of beta-amyrin/oleanolic acid. The genes encoding these 3 CYP716 enzymes are highly expressed in flowers and respond to induction by ABA, MeJA, SA, GA3, and abiotic stress treatments. Using VIGS technology, we revealed that silencing of NaCYP716A419 affects the growth and reproduction of N. attenuata, suggesting the ecological significance of these specialized metabolite biosynthetic steps. Three cytochrome P450 monooxygenases from Nicotiana attenuata participate in triterpenoid biosynthesis and may influence its growth and development.

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 β-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.

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.

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.

In response to challenges from herbivores and competitors, plants use fitness-limiting resources to produce (auto)toxic defenses. Jasmonate (JA) 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 co-silenced 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 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.

Excessive rainfall provides a favorable condition for field mold infection of plants, which triggers field mold (FM) stress. If FM stress occurs during the late maturation stage of soybean seed, it negatively affects seed yield and quality. To investigate the responses of soybean seed against FM stress and identify the underlying biochemical pathways involved, a greenhouse was equipped with an artificial rain producing system to allow the induction of mold growth on soybean seed. The induced quality changes and stress responses were revealed on the levels of both transcriptome and metabolome. The results showed that soybean seeds produced under FM stress conditions had an abnormal and inferior appearance, and also contained less storage reserves, such as protein and polysaccharide. Transcriptional analysis demonstrated that genes involved in amino acid metabolism, glycolysis, tricarboxylic acid, β-oxidation of fatty acids, and isoflavone biosynthesis were induced by FM stress. These results were supported by a multiple metabolic analysis which exhibited increases in the concentrations of a variety of amino acids, sugars, organic acids, and isoflavones, as well as reductions of several fatty acids. Reprogramming of these metabolic pathways mobilized and consumed stored protein, sugar and fatty acid reserves in the soybean seed in order to meet the energy and substrate demand on the defense system, but led to deterioration of seed quality. In general, FM stress induced catabolism of storage reserves and diminished the quality of soybean seed in the field. This study provides a more profound insight into seed deterioration caused by FM stress.

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.

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.

Black soybean seed (BSS) is a traditional edible and medical food. The cotyledons of BSS are either yellow or green, but that difference's effect on functional and bioactivities are not clear. In the present study, the concentrations of the main functional components, including anthocyanins, isoflavones, fatty acids, protein, and polysaccharides, and in vitro antioxidant activities of yellow-cotyledon and green-cotyledon BSS (YBS and GBS, respectively) were compared. The anti-fatigue activities of the ethanol extract of BSS (EEB) were evaluated using mouse swimming endurance tests. Additionally, a metabolomics comparison between YBS and GBS was done using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) on high doses of EEB-treated mouse plasma. The results indicated that YBS showed significantly higher contents of anthocyanin and protein, and stronger antioxidant activity and anti-fatigue activity than GBS; while GBS showed higher levels of isoflavone, stearic acid, and polysaccharide. By infusing the feed with EEB, the swimming time of mice increased in a dose-dependent manner. The metabolomics results suggested that the anti-fatigue effect of EEB correlated well with serotonin and melatonin biosynthesis in vivo. All of the above results showed that BSS has anti-fatigue potential and might be used as a potential source of functional food ingredients.

As a type of secondary metabolites produced by plants during long-term ecological adaptation, cutin and wax are widely involved in many resistance physiological processes including stress defense and resistance to pests and diseases, playing critical roles in the plant-pathogen interaction, thus becoming an important part of plant disease resistance mechanism. With the development of molecular biology, there is an increasing understanding on the cutin and wax metabolism and their mechanisms against fungal disease in plant. With prior researches mainly focused on the constitutive resistance and inducible resistance of plant cutin and wax, the present study, with a review of the research progress achieved on the plant cutin and wax biosynthesis and its disease resistance mechanism, is aimed to put forward prospects for future research. It was concluded that (1) as the main components of the cuticle, the first line of defense for plants against pathogen infection, cutin and wax play a critical role in physical resistance (physical barrier) and chemical resistance (bacteriostasis) as constitutive resistance components, (2) They can also play the role of inducible resistance components and (3) in addition to being the main component of the cuticle to exert physical resistance, the inducible cutin and wax component can also act as a signal molecule or inducer to activate downstream resistance reactions and exert its chemical resistance function. In the future, the research concerning cutin and wax can be focused on an in-depth explanation of the mechanism of cutin and wax inducible resistance, so as to further enrich the theoretical system of plant chemical ecology. In addition, cutin and wax biopesticides (plant immunity inducers) can be developed based on the inducible resistance of cutin and wax to provide new insight for the plant diseases control. [Ch, 1 fig. 71 ref.]

Continuous rainfall during the harvest period of soybean leads to the delayed harvest of mature seeds and favors the infection of field mold that eventually causes seed mildew in the field under high humid conditions. Soybean isoflavones are a subgroup of flavonoids which possess crucial roles in the responses of soybean to multiple abiotic and/or biotic stresses. To fully reveal the role of isoflavones in the responses of soybean seeds to filed mildew (FM) stress, two soybean genotypes with different resistance to FM stress were treated in a controlled greenhouse which provided high humid conditions in this current research. The dynamic changes of isoflavones contents and the expression levels of four major genes involved in isoflavones biosynthesis pathway were measured by liquid chromatography–mass spectrometry (LC–MS) and quantitative real-time PCR (qRT-PCR), respectively. The current results showed that the contents of major isoflavones components in resistant genotype C103 were higher than the susceptive genotype ND12 before FM treatment. Whereas after FM treatment, the dynamic changing patterns of isoflavones contents and some major genes expression were opposite between these two genotypes, and the biosynthesis of isoflavones was more rapid in resistant genotype compared to susceptible one. Noticeably, the biosynthesis of isoflavones aglycones which exhibit better bioactivities was remarkably promoted after FM treatment in resistant genotype C103, further revealing the important role of isoflavones in the resistance against FM stress. In sum, this research investigated the role of isoflavones against FM stress, the results demonstrated higher inherent contents and more rapid biosynthesis of isoflavones after FM treatment could establish stronger resistant foundation in soybean seeds against the FM stress. These results would provide new insights into the mechanism research of soybean against FM stress, but how the stress systematically regulates isoflavones and other resistant network still needs further research.

[目的]探讨半根交替干旱胁迫对大豆农艺性状及籽粒代谢物的影响,为研究大豆品质提升及抗旱代谢调控机理奠定基础.[方法]以两个耐旱性差异显著的大豆品种(耐旱型ND12、敏感型C103)为研究材料,开展室内盆栽试验,考察了不同水分胁迫下大豆的株高、单株荚数、百粒重等农艺性状,并采用高效液相色谱法分析籽粒代谢群体的变化规律.[结果]在半根交替干旱胁迫下,两个大豆品种的外观性状变化存在差异,但并不显著(P>0.05),抗性品种ND12的株高、茎粗、分枝数、主茎节数等指标呈下降趋势,敏感型品种C103却出现了小幅增长;胁迫对大豆产量性状的影响较大,两个品种的百粒重均极显著降低,单株产量显著降低,ND12的降幅低于C103;干旱敏感型大豆籽粒化学成分对胁迫的响应更剧烈,皂苷合成通路及丙酮醛降解通路上调,大豆皂苷和谷胱甘肽含量显著上升;木质素糖苷合成通路及苯丙氨酸合成通路显著上调,松柏醇、芥子醛等含量显著上升.[结论]有望通过适度的半根干旱栽培,在维持大豆正常生长的前提下,提升皂苷、谷胱甘肽等品质性状;通过籽粒代谢群体的变化情况,能够为抗旱性大豆种质资源的筛选提供参考;胁迫环境下大豆籽粒木质化程度的加剧,可能对大豆代际间应对非生物胁迫有重要作用.

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.

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.

Soybean seeds are vulnerable to aging and deterioration during storage.Deep investigation of molecular mechanisms of soybean seed aging will be helpful for the genetic improvement in the storage stability of soybean cultivars.In this study,two soybean cultivars with different storage-tolerance,"JP6" and "JP16",were used as the experimental materials to investigate the transcriptome changes in response to storage by RNA-Seq technology.The results showed that there were 1683 differentially expressed genes in "JP16" and 832 differentially expressed genes in "JP6" in the aging process of soybean seed,most of which were up regulated.Gene ontology analyses showed that differentially expressed genes were enriched in carbohydrate biosynthetic process,response to water deprivation,mitochondrial electron transport,protein phosphorylation and so on.Moreover,differentially expressed genes in response to protein monoubiquitination and fatty acid omega-oxidation were up-regulated,but genes in response to regulation of anthocyanin and lignin metabolic process were down-regulated in "JP16".Similarly,genes in response to proanthocyanidin and isoflavonoid biosynthetic process,and decreased oxygen levels were up regulated,but genes in response to wax metabolic process were down regulated.The accumulation of carbohydrate decreased in soybean seeds during storage,meanwhile,the albuminous degeneration and intracellular water balance disturbed,and the accumulation of reactive oxygen species increased.The above factors made an aggravated seed aging level.During storage,there were significant differences exist in genes between "JP6" and "JP16",of which "JP6" had more wax,proanthocyanidin,isoflavonoid and other protective components to increase antioxidant and call wall strength via regulation of reactive oxygen species level,resulting in low sensitivity to aging.

Maize-soybean strip intercropping system is an important ecological planting system; the wide development of intercropped soybean has contributed to the soybean industry in China. Soybean growth and seed production were affected by the shading from maize. Previous studies confirmed that relay strip intercropping shading facilitates the accumulation of isoflavone glucosides but does not take advantage of the accumulation of isoflavone aglycone. However, the regulation of isoflavones accumulation under strip intercropping system was unknown; in which soybean and maize were sown at the same time, and soybean was mainly shaded in the reproductive stage. In order to reveal the effects of intercropping shading on the isoflavones accumulation in soybean seed, two independent field experiments were conducted in eastern China. We compared the isoflavones profiles of soybean seeds, which were grown under various maize-soybean intercropping systems with different shade levels. The results showed that the intercropping shading was not benefited to the accumulation of soy-isoflavones, including aglycones and isoflavones glycosides and various forms of isoflavones were decreased with the decline of photosynthetic active radiation. Multivariate statistical analysis and significance analysis showed that acylated isoflavones may be more susceptible to intercropping shading.

类黄酮在植物中广泛分布,具有重要的化学生态学功能,包括微生物信息交流、病虫害防御、化感作用等.本文综述了近年来植物类黄酮与生物环境互作机理方面的研究进展,以期为类黄酮化学生态学功能的开发利用提供参考.