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Numerous herbivores orally secrete defense compounds to detoxify plant toxins. However, little is known about the role of orally secreted enzymes by a specialized pest

Glutathione (GSH) functions as a major sulfur repository and hence occupies an important position in primary sulfur metabolism. GSH degradation results in sulfur reallocation and is believed to be carried out mainly by γ-glutamyl cyclotransferases (GGCT2;1, GGCT2;2, and GGCT2;3), which, however, do not fully explain the rapid GSH turnover. Here, we discovered that γ-glutamyl peptidase 1 (GGP1) contributes to GSH degradation through a yeast complementation assay. Recombinant proteins of GGP1, as well as GGP3, showed high degradation activity of GSH, but not of oxidized glutathione (GSSG), in vitro. Notably, the GGP1 transcripts were highly abundant in rosette leaves, in agreement with the ggp1 mutants constantly accumulating more GSH regardless of nutritional conditions. Given the lower energy requirements of the GGP- than the GGCT-mediated pathway, the GGP-mediated pathway could be a more efficient route for GSH degradation than the GGCT-mediated pathway. Therefore, we propose a model wherein cytosolic GSH is degraded chiefly by GGP1 and likely also by GGP3. Another noteworthy fact is that GGPs are known to process GSH conjugates in glucosinolate and camalexin synthesis; indeed, we confirmed that the ggp1 mutant contained higher levels of O-acetyl-l-Ser, a signaling molecule for sulfur starvation, and lower levels of glucosinolates and their degradation products. The predicted structure of GGP1 further provided a rationale for this hypothesis. In conclusion, we suggest that GGP1 and possibly GGP3 play vital roles in both primary and secondary sulfur metabolism.

Brassicaceae products have been extensively studied for their biofumigant activity; despite this, few investigate their effect on soil proprieties. This paper aims to describe the effect on soil fertility by adding the seed meal of Brassica carinata at three different doses corresponding to field dose (3 tons/ha), 10 and 100 fold this dose in organic soil. The organic carbon balance was evaluated by analysing the oxidisable, humified and mineralised carbon fractions. Microbial activity was measured by enzymes linked to the carbon, phosphorous and sulphur cycles—β-glucosidase, phosphatase and arylsulphatase, respectively. The amount corresponding to 30 t/ha was the best solution for the mineralised carbon and humic carbon ratio. In contrast, there was a substantial increase in the organic substance’s mineralisation level at the maximum dose, not offset by a relative rise in humification. No repression was observed in the metabolic activity of the microorganisms, their abundance or the main enzymatic activities and, in the conditions tested, a release of only a volatile isothiocyanate occurred, limiting the biofumigant effect. Therefore, these combined factors allow us to assert that the amendment with this type of material at the intermediate dose could positively affect the phosphorus cycle, the soil structure, and fertility.

Plum (Prunus spp.) is an economically and nutritionally important stone fruit that is grown worldwide. Gummosis disease (GD) is one of the most common limiting factors that adversely affects the yield and quality of stone fruits such as plum. Elucidating plum fruit metabolomics responses is essential to develop sustainable agricultural practices to combat GD in the future. Herein, an ultra-high-performance liquid chromatography coupled to mass-spectrometry (UHPLC-MS) pseudo-targeted metabolomic profiling was first performed to elucidate the overall metabolic alterations in Asian plum (Prunus salicina Lindl.) fruit in response to GD. The most pivotal differential metabolites, including certain amino acids and proanthocyanidins, in GD and control groups were identified by combining multivariate data analysis with strict statistical criteria. Metabolic pathway enrichment analysis showed that GD induced a series of coordinated defence responses and reprogramming of various metabolic pathways, including glucosinolate biosynthesis, 2-oxocarboxylic acid metabolism, valine, leucine and isoleucine degradation, and isoquinoline alkaloid biosynthesis pathways. Using UHPLC-MS-based pseudo-targeted metabolomic profiling, we systematically evaluated overall metabolic modifications in Asian plum fruits in response to GD for the first time. The identified metabolic pathway alterations helped to better understand the internal relationships and related metabolic networks.

Robust plant immunity negatively affects other fitness traits, including growth and seed production. Jasmonate (JA) confers broad-spectrum protection against plant consumers by stimulating the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins, which in turn relieves repression on transcription factors (TFs) coincident with reduced growth and fecundity. The molecular mechanisms underlying JA-mediated decreases in fitness remain largely unknown. To assess the contribution of MYC TFs to growth and reproductive fitness at high levels of defence, we mutated three MYC genes in a JAZ-deficient mutant (jazD) of Arabidopsis thaliana that exhibits strong defence and low seed yield. Genetic epistasis analysis showed that de-repression of MYC TFs in jazD not only conferred strong resistance to insect herbivory but also reduced shoot and root growth, fruit size and seed yield. We also provided evidence that the JAZ-MYC module coordinates the supply of tryptophan with the production of indole glucosinolates and the proliferation of endoplasmic reticulum bodies that metabolise glucosinolates through the action of β-glucosidases. Our results establish MYCs as major regulators of growth- and reproductive-defence trade-offs and further indicate that these factors coordinate tryptophan availability with the production of amino acid-derived defence compounds.

The tryptophan (Trp)-derived plant secondary metabolites, including camalexin, 4-hydroxy-indole-3-carbonylnitrile, and indolic glucosinolate (IGS), show broad-spectrum antifungal activity. However, the distinct regulations of these metabolic pathways among different plant species in response to fungus infection are rarely studied. In this study, our results revealed that WRKY33 directly regulates IGS biosynthesis, notably the production of 4-methoxyindole-3-ylmethyl glucosinolate (4MI3G), conferring resistance to Alternaria brassicicola, an important pathogen which causes black spot in Brassica crops. WRKY33 directly activates the expression of CYP81F2, IGMT1, and IGMT2 to drive side-chain modification of indole-3-ylmethyl glucosinolate (I3G) to 4MI3G, in both Arabidopsis and Chinese kale (Brassica oleracea var. alboglabra Bailey). However, Chinese kale showed a more severe symptom than Arabidopsis when infected by Alternaria brassicicola. Comparative analyses of the origin and evolution of Trp metabolism indicate that the loss of camalexin biosynthesis in Brassica crops during evolution might attenuate the resistance of crops to Alternaria brassicicola. As a result, the IGS metabolic pathway mediated by WRKY33 becomes essential for Chinese kale to deter Alternaria brassicicola. Our results highlight the differential regulation of Trp-derived camalexin and IGS biosynthetic pathways in plant immunity between Arabidopsis and Brassica crops.

Climate changes such as temperature shifts are leading to several consequences, including intense drought, storms, and melting glaciers. These last phenomena can have an impact on food production and consequently a potential effect on our health. In this context, the aim of this study is to investigate the knowledge regarding the role of climate changes on food composition in terms of glucosinolates content.

Climate changes such as temperature shifts are leading to several consequences, including intense drought, storms, and melting glaciers. These last phenomena can have an impact on food production and consequently a potential effect on our health. In this context, the aim of this study is to investigate the knowledge regarding the role of climate changes on food composition in terms of glucosinolates content.

PremiseCapparis spinosa is a widespread charismatic plant, in which the nocturnal floral habit contrasts with the high visitation by diurnal bees and the pronounced scarcity of hawkmoths. To resolve this discrepancy and elucidate floral evolution of C. spinosa, we analyzed the intrafloral patterns of visual and olfactory cues in relation to the known sensory biases of the different visitor guilds (bees, butterflies, and hawkmoths). MethodsWe measured the intrafloral variation of scent, reflectance spectra, and colorimetric properties according to three guilds of known visitors of C. spinosa. Additionally, we sampled visitation rates using a motion-activated camera. ResultsCarpenter bees visited the flowers eight times more frequently than nocturnal hawkmoths, at dusk and in the following morning. Yet, the floral headspace of C. spinosa contained a typical sphingophilous scent with high emission rates of certain monoterpenes and amino-acid derived compounds. Visual cues included a special case of multisensory nectar guide and color patterns conspicuous to the visual systems of both hawkmoths and bees. ConclusionsThe intrafloral patterns of sensory stimuli suggest that hawkmoths have exerted strong historical selection on C. spinosa. Our study revealed two interesting paradoxes: (a) the flowers phenotypically biased towards the more inconsistent pollinator; and (b) floral display demands an abundance of resources that seems maladaptive in the habitats of C. spinosa. The transition to a binary pollination system accommodating large bees has not required phenotypic changes, owing to specific eco-physiological adaptations, unrelated to pollination, which make this plant an unusual case in pollination ecology., American Journal of Botany, 110 (1), ISSN:1914-2016, ISSN:0002-9122

Coevolutionary interactions are responsible for much of the Earth’s biodiversity, with key innovations driving speciation bursts on both sides of the interaction. One persistent question is whether macroevolutionary traits identified as key innovations accurately predict functional performance and selection dynamics within species, as this necessitates characterizing their function, investigating their fitness consequences, and exploring the selection dynamics acting upon them. Here, we used CRISPR-Cas9 mediating nonhomologous end joining (NHEJ) in the butterfly species Pieris brassicae to knock out and directly assess the function and fitness impacts of nitrile specifier protein ( NSP ) and major allergen ( MA ). These are two closely related genes that facilitate glucosinolate (GSL) detoxification capacity, which is a key innovation in mustard feeding Pierinae butterflies. We find NSP and MA are both required for survival on plants containing GSLs, with expression differences arising in response to variable GSL profiles, concordant with detoxification performance. Importantly, this concordance was only observed when using natural host plants, likely reflecting the complexity of how these enzymes interact with natural plant variation in GSLs and myrosinases. Finally, signatures of positive selection for NSP and MA were detected across Pieris species, consistent with these genes’ importance in recent coevolutionary interactions. Thus, the war between these butterflies and their host plants involves more than the mere presence of chemical defenses and detoxification mechanisms, as their regulation and activation represent key components of complex interactions. We find that inclusion of these dynamics, in ecologically relevant assays, is necessary for coevolutionary insights in this system and likely others.

Introduction The high mortality rate in severe cases of COVID-19 is mainly due to the strong upregulation of cytokines, called a cytokine storm. Hyperinflammation and multiple organ failure comprise the main clinical features of a cytokine storm. Nrf2 is a transcription factor which regulates the expression of genes involved in immune and inflammatory processes. Furthermore, Nrf2, as a master regulator, controls the activity of NF-κB which binds to the promoter of many pro-inflammatory genes inducible of various inflammatory factors. Inhibition of Nrf2 response was recently demonstrated in biopsies from patients with COVID-19, and Nrf2 agonists inhibited SARS-CoV-2 replication across cell lines in vitro. Glucosinolates and their hydrolysis products have excellent anti-inflammatory and antioxidant effects via the Nrf2 activation pathway, reduction in the NF-κB activation, and subsequent reduced cytokines levels. Conclusion Accordingly, these compounds can be helpful in combating the cytokine storm associated with COVID-19. Graphical abstract

Broccoli, kale, and cauliflower contain phenolic compounds and glucosinolates, which have several biological effects on the body. However, because they are thermolabile, many of these substances are lost in the cooking process. Electrospinning encapsulation, using zein as a preservative wall material, can expand the applications of the compounds in the food and pharmaceutical industries. The objective of this research was to characterize broccoli, kale, and cauliflower extracts and encapsulate them with the electrospinning technique using zein.Broccoli, kale, and cauliflower extracts contain five phenolic compounds and three glucosinolates. Fibers from broccoli, kale, and cauliflower showed high encapsulation efficiency, good thermal stability, and nanometric size, especially those containing extract and zein in proportions of up to 35:65.Fibers from broccoli, kale, and cauliflower containing extract and zein in proportions of up to 35:65 have the potential for effective nutraceutical application for the control of non-communicable chronic diseases or application in food packaging. © 2022 Society of Chemical Industry.

A low seed glucosinolate resource was developed in polyploid B. napus using a method that identifies the functions of genes with rare or no genetic variation.

The phenotypic plasticity of flowering plants in response to herbivore damage to vegetative tissues can affect plant interactions with flower-feeding organisms. Such induced systemic responses are probably regulated by defence-related phytohormones that signal flowers to alter secondary chemistry that affects resistance to florivores. Current knowledge on the effects of damage to vegetative tissues on plant interactions with florivores and the underlying mechanisms is limited. We compared the preference and performance of two florivores on flowering Brassica nigra plants damaged by one of three herbivores feeding from roots or leaves. To investigate the underlying mechanisms, we quantified expression patterns of marker genes for defence-related phytohormonal pathways, and concentrations of phytohormones and glucosinolates in buds and flowers. Florivores displayed contrasting preferences for plants damaged by herbivores feeding on roots and leaves. Chewing florivores performed better on plants damaged by folivores, but worse on plants damaged by the root herbivore. Chewing root and foliar herbivory led to specific induced changes in the phytohormone profile of buds and flowers. This resulted in increased glucosinolate concentrations for leaf-damaged plants, and decreased glucosinolate concentrations for root-damaged plants. The outcome of herbivore–herbivore interactions spanning from vegetative tissues to floral tissues is unique for the inducing root/leaf herbivore and receiving florivore combination.

As important flavor precursors, glucosinolates are ubiquitous in the plant family of Brassicaceae. Glucosinolate degradation products are the major volatile flavor compounds of rapeseed oil, accounting for up to 80% of the total volatiles. However, up to now, little attention has been paid to the volatile flavor products of the nonenzymatic thermal degradation of glucosinolates. One of the most important factors that determine the flavor of hot-pressed rapeseed oil is the roasting process, where the thermal degradation of glucosinolates mainly occurs. The thermal degradation behavior and volatile products of progoitrin (the main glucosinolate of rapeseed) in different matrices (phosphate buffer at a pH value of 5.0, 7.0, or 9.0, sea sand, and rapeseed powder) at different temperatures (150-200 °C) and times (0-60 min) were studied using HPLC and GC-TOF-MS. Thereby, the degradation rate of progoitrin decreased in the following order: pH 9.0sea sandrapeseed powderpH 7.0pH 5.0. Further, a higher degradation was observed with increasing temperature and time. Under the applied conditions in this study, 2,4-pentadienenitrile was the major nitrile and thiophenes were the major sulfur-containing volatile compounds formed. Possible formation pathways of main sulfur-containing and nitrogen-containing volatiles were proposed.

Glucosinolates are plant natural products which on degradation by myrosinases give rise to the beneficial bioactive isothiocyanates. Recently, a myrosinase activity was detected in a Citrobacter strain isolated from soil. This enzyme was purified enabling its amino acid sequence and gene sequence (cmyr) to be determined. In order to study this myrosinase it was necessary to establish an expression system that would enable future work such as a structural determination of the protein to be carried out. The myrosinase gene was amplified, cloned and expressed in Escherichia coli with a 6XHis-tag. The heterologous expression of cmyr enabled relatively large amounts of myrosinase to be produced (3.4 mg cmyr/100 ml culture). Myrosinase activity was determined by mixing substrate and enzyme and determining glucose release. Optimum pH and temperature were determined to be pH 6.0 and 25 °C for the Ni-NTA purified protein. The kinetic parameters of the purified myrosinase were determined using sinigrin as a substrate. K

We wished to prepare detoxified rapeseed protein isolate (RPI) by phytase/ethanol treatment based on alkaline extraction and acidic precipitation. Contents of protein, fat, ash, moisture, crude fiber, glucosinolates, phytic acid, phenolics and color were determined. To evaluate the safety of detoxified RPI, five groups of C57 mice (detoxified RPI [10 and 20 g/kg]; commercial soybean protein isolate (SPI) [10 g/kg]; non-detoxified RPI [10 g/kg]; control) were used in the acute-toxicity test. Bodyweight and pathology parameters were recorded at different time points, followed by macroscopic examination, organ-weight measurement and microstructure examination. After pretreatment of rapeseed meals with phytase (enzyme:substrate ratio, 1:5 mg/g) for 1.5 h and two-time ethanol extraction for precipitated protein, the chemical characteristics in RPI were protein (88.26%), fat (0.57%), ash (2.72%), moisture (1.90%), crude fiber (0.77%), glucosinolates (0 μmol/g), phytic acid (0.17%), phenolics (0.36%) and whiteness (73.38). Treatment resulted in significant removal of anti-nutritional factors (ANFs) and increased whiteness from detoxified RPI compared with that from non-detoxified RPI, and lower than that of cruciferin-rich canola protein isolate (Puratein®). Experimental-related effects on bodyweight, clinical observations, or clinicopathology, in mice treated with detoxified RPI were not observed except for a decreased Thyroid Gland/Parathyroid Gland Index in mice treated with non-detoxified RPI. Furthermore, the no-observed-effect level (NOEL) was 10g/kg of detoxicating RPI, whereas the no-observed-adverse-effect-level was (NOAEL) the highest fed level of 20g/kg of detoxicating RPI. Overall, the detoxified RPI prepared by combined treatment of phytase and ethanol was considered safe under the conditions tested, in which the contents of the main ANFs were reduced significantly.

Abstract The study has proved the stimulating effects of different strategies of treatments with elicitors on the production of glucosinolates (GSLs), flavonoids, polyphenols, saccharides, and photosynthetic pigments in watercress (Nasturtium officinale) microshoot cultures. The study also assessed antioxidant and anti-melanin activities. The following elicitors were tested: ethephon (ETH), methyl jasmonate (MeJA), sodium salicylate (NaSA), and yeast extract (YeE) and were added on day 10 of the growth period. Cultures not treated with the elicitor were used as control. The total GSL content estimations and UHPLC-DAD-MS/MS analyses showed that elicitation influenced the qualitative and quantitative profiles of GSLs. MeJA stimulated the production of gluconasturtiin (68.34 mg/100 g dried weight (DW)) and glucobrassicin (65.95 mg/100 g DW). The elicitation also increased flavonoid accumulation (max. 1131.33 mg/100 g DW, for 100 μM NaSA, collection after 24 h). The elicitors did not boost the total polyphenol content. NaSA at 100 μM increased the production of total chlorophyll a and b (5.7 times after 24 h of treatment), and 50 μM NaSA caused a 6.5 times higher production of carotenoids after 8 days of treatment. The antioxidant potential (assessed with the CUPRAC FRAP and DPPH assays) increased most after 24 h of treatment with 100 μM MeJA. The assessment of anti-melanin activities showed that the microshoot extracts were able to cause inhibition of tyrosinase (max. 27.84% for 1250 µg/mL). Key points • Elicitation stimulated of the metabolite production in N. officinale microshoots. • High production of pro-health glucosinolates and polyphenols was obtained. • N. officinale microshoots have got tyrosinase inhibition potential. • The antioxidant potential of N. officinale microshoots was evaluated. Graphical abstract

Coronavirus disease 2019 (COVID-19) is a rapidly growing pandemic that requires urgent therapeutic intervention. Finding potential anti COVID-19 drugs aside from approved vaccines is progressively going on. The chemically diverse natural products represent valuable sources for drug leads. In this study, we aimed to find out safe and effective COVID-19 protease inhibitors from a library of natural products which share the main nucleus/skeleton of FDA-approved drugs that were employed in COVID-19 treatment guidelines or repurposed by previous studies. Our library was subjected to virtual screening against SARS-CoV Main protease (Mpro) using Molecular Operating Environment (MOE) software. Twenty-two out of those natural candidates showed higher binding scores compared to their analogues. We repurpose these natural products including alkaloids, glucosinolates, and phenolics as potential platforms for the development of anti-SARS-CoV-2 therapeutics. This study paves the way towards discovering a lead used in the treatment of COVID-19 from natural sources and introduces phytomedicines with dual therapeutic effects against COVID-19 besides their original pharmacological effects. We recommend further in vitro evaluation of their anti-COVID-19 activity and future clinical studies. Supplementary Information The online version contains supplementary material available at 10.1007/s11356-021-17642-9.

Intake of brassicaceous vegetables such as cabbage is associated with numerous health benefits. The major defense compounds in the Brassicales order are the amino acid-derived glucosinolates that have been associated with the health-promoting effects. This has primed a desire to build glucosinolate-producing microbial cell factories as a stable and reliable source. Here, we established-for the first time-production of the phenylalanine-derived benzylglucosinolate (BGLS) in Saccharomyces cerevisiae using two different engineering strategies: stable genome integration versus plasmid-based introduction of the biosynthetic genes. Although the plasmid-engineered strain showed a tendency to generate higher expression level of each gene (except CYP83B1) in the biosynthetic pathway, the genome-engineered strain produced 8.4-fold higher BGLS yield compared to the plasmid-engineered strain. Additionally, we optimized the genome-engineered strain by overexpressing the entry point genes CYP79A2 and CYP83B1, resulting in a 2-fold increase in BGLS production but also a 4.8-fold increase in the level of the last intermediate desulfo-benzylglucosinolate (dsBGLS). We applied several approaches to alleviate the metabolic bottleneck in the step where dsBGLS is converted to BGLS by sulfotransferase, SOT16 dependent on 39-phosphoadenosine-59-phosphosulfate (PAPS). BGLS production increased 1.7-fold by overexpressing SOT16 and 1.7-fold by introducing APS kinase, APK1, from Arabidopsis thaliana involved in the PAPS regeneration cycle. Modulating the endogenous sulfur assimilatory pathway through overexpression of MET3 and MET14 resulted in 2.4-fold to 12.81 mmol/L (=5.2 mg/L) for BGLS production. IMPORTANCE Intake of brassicaceous vegetables such as cabbage is associated with numerous health benefits. The major defense compounds in the Brassicales order are the amino acid-derived glucosinolates that have been associated with the health-promoting effects. This has primed a desire to build glucosinolate-producing microbial cell factories as a stable and reliable source. In this study, we engineered for the first time the production of phenylalanine-derived benzylglucosinolate in Saccharomyces cerevisiae with two engineering strategies: stable genome integration versus plasmid-based introduction of the biosynthetic genes. Although the plasmid-engineered strain generally showed higher expression level of each gene (except CYP83B1) in the biosynthetic pathway, the genome-engineered strain produced higher production level of benzylglucosinolate. Based on the genome-engineered strain, the benzylglucosinolate level was improved by optimization. Our study compared different approaches to engineer a multigene pathway for production of the plant natural product benzylglucosinolate. This may provide potential application in industrial biotechnology.

Camelina sativa (camelina) seed, oil, and defatted meal are widely used for food, animal feed, and other purposes. The accurate quantification of camelina glucosinolates is critical as their functionalities are highly dose-dependent. The classic quantification of glucosinolates in camelina products involves tedious desulfation steps, toxic reagents, and a lengthy instrument time because glucosinolates are easy to degrade and subject to interference in the liquid chromatography. Thus, we developed and validated an eco-efficient UPLC-DAD method for determining glucoarabin (GS9), glucocamelinin (GS10), and homoglucocamelinin (GS11) in camelina seed, oil, and defatted meal. Glucosinolates were extracted using 80% cold methanol to denature myrosinase, and were separated by an HSS T3 column without desulfation. Glucotropaeolin was used as an internal standard to track analyte degradation and loss during sample preparation. The method has shown high precision (relative standard deviations ranging from 4.12% to 6.54%) and accuracy (>94.4% spike recovery) for GS9-11, and all validation parameters passed the industry-consensus AOAC Appendix F criteria. To our best knowledge, this is the first eco-efficient and low-cost analytical method that is validated against strict AOAC criteria for the quantification of intact camelina glucosinolates. The method is suitable to be adopted as a new industrial testing standard to assist in the quality control of camelina products.

Air pollutants—such as nitrogen oxides, emitted in diesel exhaust, and ozone (O 3 )—disrupt interactions between plants, the insect herbivore pests that feed upon them and natural enemies of those herbivores (e.g. parasitoids). Using eight field-based rings that emit regulated quantities of diesel exhaust and O 3 , we investigated how both pollutants, individually and in combination, altered the attraction and parasitism rate of a specialist parasitoid ( Diaeretiella rapae ) on aphid-infested and un-infested Brassica napus plants. Individual effects of O 3 decreased D. rapae abundance and emergence by 37% and 55%, respectively, compared with ambient (control) conditions. When O 3 and diesel exhaust were emitted concomitantly, D. rapae abundance and emergence increased by 79% and 181%, respectively, relative to control conditions. This attraction response occurred regardless of whether plants were infested with aphids and was associated with an increase in the concentration of aliphatic glucosinolates, especially gluconapin (3-butenyl-glucosinolate), within B. napus leaves. Plant defensive responses and their ability to attract natural aphid enemies may be beneficially impacted by pollution exposure. These results demonstrate the importance of incorporating multiple air pollutants when considering the effects of air pollution on plant–insect interactions.

The glucosinolates (GLs) and myrosinase defensive systems in cruciferous plants were circumvented by Plutella xylostella using glucosinolate sulfatases (PxGSSs) during pest-plant interaction. Despite identifying three duplicated GSS-encoding genes in P. xylostella, limited information regarding their spatiotemporal and induced expression is available. Here, we investigated the tissue- and stage-specific expression and induction in response to GLs of PxGSS1 and PxGSS2 (PxGSS1/2) at the protein level, which shares a high degree of similarity in protein sequences. Western blotting (WB) analysis showed that PxGSS1/2 exhibited a higher protein level in mature larvae, their guts, and gut content. A significantly high protein and transcript levels of PxGSS1/2 were also detected in the salivary glands using WB and qRT-PCR. The immunofluorescence (IF) and immunohistochemistry (IHC) results confirmed that PxGSS1/2 is widely expressed in the larval body. The IHC was more appropriate than IF when autofluorescence interference was present in collected samples. Furthermore, the content of PxGSS1/2 did not change significantly under treatments of GL mixture from Arabidopsis thaliana ecotype Col-0, or commercial ally (sinigrin), 4-(methylsulfinyl)butyl, 3-(methylsulfinyl)propyl, and indol-3-ylmethyl GLs indicating that the major GLs from leaves of A. thaliana Col-0 failed to induce the expression of proteins for both PxGSS1 and PxGSS2. Our study systemically characterized the expression properties of PxGSS1/2 at the protein level, which improves our understanding of PxGSS1/2-center adaptation in P. xylostella during long-term insect-plant interaction.

Glucosinolates (GSLs) from

Brassicaceae are blessed with specialized metabolites called glucosinolates (GSLs), which along with their degradation products, are beneficial in agriculture and human health. To date, more than 130 GSL structures have been identified, mostly derived from the amino acid methionine. The biosynthesis of methionine-derived aliphatic GSLs starts with a side-chain elongation step involving a recursive three-step cyclic process that incorporates a new methylene group into the 2-oxo acid to form a series of elongated 2-oxo acids. Methylthioalkylmalate synthase (MAMS) catalyzes the first committed step in the side-chain elongation of methionine-derived GSLs. The substrate specificity of MAMS with different 2-oxo acids determines whether reaction products of a given cycle enter for an additional round of chain elongation or enter into core GSLs structure formation. Multiple MAMS encoding genes are present in the Brassicaceae species and are known to play a central role in shaping the diverse profile of aliphatic GSLs. We recently established a highly sensitive LC-MS/MS-based methodology that quantifies the MAMS activity by estimating the amount of the next intermediate of the pathway, the 2-malate derivatives. Overall, this chapter describes the protocol for the expression, purification, and steady-state kinetic analysis of the recombinant MAMS protein.

An analytical approach based on reversed-phase liquid chromatography coupled to electrospray ionization Fourier-transform mass spectrometry in negative ion mode (RPLC-ESI-(-)-FTMS) was developed for the untargeted characterization of glucosinolates (GSL) in the polar extracts of four

The glucosinolate-myrosinase system is a two-component defense system characteristic of cruciferous plants. To evade the glucosinolate-myrosinase system, the crucifer specialist insect

Broccoli heads are typically packaged and marketed in petroleum-based heat-shrinkable or stretch-cling films that are not biodegradables. These types of films are not compatible with global strategies to reduce plastics waste. One of these strategies is development of biodegradable packaging materials that can be used as an alternative food package. Therefore, a biodegradable stretch-cling film, based on poly(butylene-adipate-co-terephthalate) and poly(lactide) blend (Nature Fresh®), for its application in broccoli head packaging has been studied. To determine suitability of Nature Fresh® film for broccoli packaging, broccoli heads were wrapped in this film and stored at 2°C for 35 d. Broccolis without packaging and wrapped in linear low density polyethylene (LLDPE) were used as controls. Physicochemical and sensory evaluation were determined during storage to study the effect of films on quality and shelf life of broccoli. On the one hand, broccolis packaged in Nature Fresh® and LLDPE presented similar evolution in internal atmosphere composition, color, hydroxycinnamic acids and carotenoids content and overall appearance. On the other hand, weight loss and decrease in firmness were significantly higher in broccoli wrapped in Nature Fresh® compared to LLDPE, but this had no impact on overall appearance. Nature Fresh® extended postharvest storage of broccoli heads at least 21 d at 2ºC, which is an adequate time to market. In conclusion, Nature Fresh® film was a viable packaging alternative to LLDPE to preserve broccoli quality during postharvest storage, while fulfilling global strategies to reduce plastics waste., Authors are grateful to ANII (Agencia Nacional de Innovación e Investigación) for conceding Erika Paulsen an internship at Phytochemistry and Healthy Foods Laboratory of the Department of Food Science and Technology at CEBAS-CSIC (scholarship reference: POS_EMHE_2018_1_1007740). This work has been funded by the University Miguel Hernández (UMH) through Proyect PAR3265/19.

Glucosinolates, specialized metabolites of the Brassicales including

Rising temperatures and pronounced drought are significantly affecting biodiversity worldwide and reducing yields and quality of Brassica crops. To elucidate the mechanisms of tolerance, 33 kale accessions (

Myrosinase can hydrolyze glucosinolates to generate isothiocyanates, which have cancer prevention and anti-cancer properties. The main sources of myrosinase are cruciferous plants. To further improve the efficiency of isothiocyanates preparation, it is necessary to explore novel sources of myrosinases. In this study, we described a bacterium

Aphids that attack canola (Brassica napus L.) exhibit feeding preferences for different parts of canola plants, which may be associated with brassica-specific glucosinolates. However, this idea remains untested. Furthermore, canola aphid species employ different strategies for tolerating glucosinolates. While the green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae), excretes glucosinolates, the cabbage aphid Brevicoryne brassicae (L.) (Hemiptera: Aphididae) sequesters them. Given the different detoxification mechanisms, we predicted that both aphid species and aphid feeding location would affect prey suitability for larvae of the predator, Hippodamia convergens (Guérin-Méneville) (Coleoptera: Coccinellidae). We hypothesized that aphids, specifically glucosinolate-sequestering cabbage aphid, reared on reproductive structures that harbor higher glucosinolates concentrations would have greater negative effects on predators than those reared on vegetative structures which have lower levels of glucosinolates, and that the impact of aphid feeding location would vary depending on the prey detoxification mechanism. To test these predictions, we conducted experiments to compare 1) glucosinolates profiles between B. brassicae and M. persicae reared on reproductive and vegetative canola structures, 2) aphid population growth on each structure, and 3) their subsequent impact on fitness traits of H. convergens. Results indicate that the population growth of both aphids was greater on reproductive structures, with B. brassicae having the highest population growth. B. brassicae reared on reproductive structures had the highest concentrations of glucosinolates, and the greatest adverse effects on H. convergens. These findings suggest that both aphid-prey species and feeding location on canola could influence populations of this predator and, thus, its potential for biological control of canola aphids.

Diabetes mellitus (DM) is a common metabolic disorder which arises due to the improper carbohydrate metabolism, decreased secretion/activity of insulin, and genetic abnormalities, which result in the increased blood glucose level generally known as hyperglycemia. Diabetes holds an increased global prevalence in each year and is responsible for increased morbidity and mortality rates. Hence, the current investigation focusses to assess the antidiabetic potential of sinigrin on diabetic animal model through the suppression of inflammation. Diabetes was initiated to the animals via administering streptozotocin (STZ) and supplemented with the sinigrin at 25- and 50-mg/kg dose via oral route. The diabetic rats demonstrated the elevated glucose, food and water intake, kidney and liver weights, and reduced bodyweight and depleted insulin status. The sinigrin treatment remarkably improved and modulated these changes in diabetic animals. Additionally, the sinigrin supplementation also modulated the changes in glucose-6-phosphatase; fructose 1,6-bisphosphatase; AST; ALT; creatinine; and inflammatory mediators in the STZ-provoked diabetic animals. The levels of hexokinase, protein, and antioxidants also improved by the sinigrin treatment. The histological investigations of pancreas also witnessed the therapeutic actions of sinigrin, which is supported by the findings of biochemical examinations. Therefore, it was clear that the sinigrin supplementation displayed remarkable antidiabetic effect on STZ-initiated diabetic animals via modulating inflammation and other biochemical changes, which recommends that sinigrin could be a talented candidate for diabetes management in the future.

Summary A high content of seed glucosinolates and their degradation products imposes anti‐nutritional effects on livestock; therefore, persistent efforts are made to reduce the seed GSL content to increase the commercial value of rapeseed meal. Here, we dissected the genetic structure of SGC by genome‐wide association studies (GWAS) combined with transcriptome‐wide association studies (TWAS). Fifteen reliable quantitative trait loci (QTLs) were identified to be associated with the reduced SGC in modern B. napus cultivars by GWAS. Analysis of the selection strength and haplotypes at these QTLs revealed that low SGC was predominantly generated by the co‐selection of qGSL.A02.2, qGSL.C02.1, qGSL.A09.2, and qGSL.C09.1. Integration of the results from TWAS, comprehensive bioinformatics, and POCKET algorithm analyses indicated that BnaC02.GTR2 (BnaC02g42260D) is a candidate gene underlying qGSL.C02.1. Using CRISPR/Cas9‐derived Bna.gtr2s knockout mutants, we experimentally verified that both BnaC02.GTR2 and its three paralogs positively regulate seed GSL accumulation but negatively regulated vegetative tissue GSL contents. In addition, we observed smaller seeds with higher seed oil content in these Bna.gtr2 mutants. Furthermore, both RNA‐seq and correlation analyses suggested that Bna.GTR2s might play a comprehensive role in seed development, such as amino acid accumulation, GSL synthesis, sugar assimilation, and oil accumulation. This study unravels the breeding selection history of low‐SGC improvement and provides new insights into the molecular function of Bna.GTR2s in both seed GSL accumulation and seed development in B. napus.

Mainly found in brussels sprouts, broccoli, and black mustard seeds, sinigrin (2-propenyl glucosinolate) has enjoyed some attention currently for its effects on health and disease prevention. The present research design is aimed at investigating the effects of sinigrin on inflammation, oxidative stress (OS) and memory. Randomly, six groups of male Wistar rats were categorized into the control and experimental groups. The experimental groups were treated with sinigrin (10 and 20 mg/kg, orally). The control positive group was given the pentylenetetrazole (PTZ) treatment and the control negative one was given normal saline. All groups were kindled by the sub-threshold dose (35 mg/kg, i.p.) of PTZ for 12 times in one month. When the kindling procedure was done, the seizure behaviors and the behavioral function were evaluated. For cognitive parameters, the shuttle box test was employed. When the experiment was terminated, the rats were euthanized and their blood serum as well as brain samples were isolated for respective measuring of OS and gene expression parameters. The treatment with sinigrin significantly delayed the appearance of the seizure symptoms in comparison to that of the PTZ group. It also significantly increased the memory parameters like retention latency and the total time having been spent in the light compartment in the epileptic rats. In addition, sinigrin increased the superoxide dismutase and catalase levels. Treatment with sinigrin suppressed the Il1b and Nlrp3 gene expression at hippocampal level. In sum, sinigrin prevents inflammation, OS and memory impairment against the PTZ-kindling epilepsy in rats.

Biostimulants are receiving increasing attention for their beneficial effects on crops, driving interest in identifying new plant extracts that could exert such stimulatory effects. This work aimed to evaluate the potential of an aqueous extract obtained from duckweed (Lemna minor L.), a freshwater species, to act as a biostimulant in maize. For this purpose, duckweed plants were collected from a natural basin and then transferred, stabilized, and grown under controlled conditions. The duckweed extract was first characterized through untargeted profiling, which revealed an abundance of bioactive phytochemicals. A relatively high amount of low-molecular-weight secondary metabolites such as phenolics (6714.99 mg kg−1) and glucosinolates (4563.74 mg kg−1) were present in the plant extract. Maize seeds were primed with different concentrations of this extract (0.01%, 0.05%, 0.50%, and 1.00%, dry weight/water volume), and some physiological and biochemical traits of the crop were recorded. The duckweed extract improved maize germination, biomass, leaf area, pigment content, and vigor index. The most effective treatment was the 0.50% concentration, which improved the majority of the measured growth traits. The extract at concentrations of 0.05%, 0.50%, and 1.00% stimulated the assimilation of nitrogen (N), phosphorous (P), potassium (K), calcium (Ca), magnesium (Mg), sodium (Na), iron (Fe), and copper (Cu). In summary, this study revealed that duckweed is a promising species that can be cultured and grown under controlled conditions for obtaining extracts with biostimulant properties.

Rapeseed contains high levels of glucosinolates (GSLs), playing pivotal roles in defense against herbivores and pests. As their presence in rapeseed reduces the value of the meal for animal feeding, intensive efforts to reduce them produced low-seed GSL cultivars. However, there is no such variety suitable for the south part of Japan. Here, we tested the effects of cold oxygen plasma (oxygen CP) on seed germination and GSL and lipid content, in 3 rapeseed cultivars. According to the cultivars, oxygen CP slightly stimulated seed germination and modified the GSL levels, and decreased GSL levels in Kizakinonatane but increased those in Nanashikibu. In contrast, it negligibly affected the lipid content and composition in the 3 cultivars. Thus, oxygen CP modulated seed GSL levels without affecting seed viability and lipid content. Future optimization of this technique may help optimize rapeseed GSL content without plant breeding.

We investigated the influence of cryogenic grinding on the quality of rapeseed cake. Rapeseed cake is a good source of valuable proteins (30%) and oil (14%), with a balanced fatty acid composition and a fair amount of sterols, which may reduce the risk of cardiovascular diseases. However, the presence of antinutritive compounds prevents its use as a food source. Grinding under cryogenic conditions is much more efficient than grinding at room temperature in terms of particle size reduction. The additional cryogenic grinding of the cake had little effect on the nutritional components, as phytosterols and soluble dietary fiber increased slightly. It had no effect on insoluble dietary fiber, polyphenols, and tannins. Prolonged grinding time, both at room and subzero temperatures, reduced the total amount of glucosinolates by 34 and 43%, respectively. However, the reduction in undesirable components is not sufficient to use cryogenic grinding as the sole treatment for the cake, but it could be a good pretreatment for chemical or biological treatments.

Broccoli is a vegetable that offers valuable components, such as glucosinolates (GLS), flavonoids, and hydroxycinnamic acids (HCA), for our daily food intake. These substances have been associated with reducing the risk of cancer and cardiovascular diseases (CVD). Broccoli florets are also highly perishable, given their elevated respiration rates and their sensitivity to ethylene. Experiments have been carried out on broccoli to investigate the consequences of abiotic stress post-harvest treatments in retarding the senescence process. Nevertheless, the influence of these treatments on the phytochemicals of broccoli has not been extensively examined. Florets of broccoli (Brassica oleracea) were exposed to an atmosphere consisting of 10,000 µL.L−1 ethanol at room temperature for 30 min and 120 min. The exposure to methyl jasmonate (MeJA) treatments was carried out at room temperature using 1 µL.L−1 for 45 min and 180 min. The yellowing of florets was delayed using 10,000 µL.L−1 of ethanol at both exposure times as compared to untreated florets, and the chlorophyll titers were also superior with both doses over the control. The total phenols of the florets increased by 15% and 18% with the application of the hormetic and high doses, respectively, throughout the storage period compared to unexposed broccoli. The GLS and HCA yields were also increased by both ethanol doses. The exposure of florets to 1 µL.L1 MeJA for 45 min resulted in delayed yellowing of florets; however, longer exposures resulted in yellowing after 21 d and significantly (p < 0.05) increased respiration rates relative to untreated florets. Overall antioxidant capacity of the florets was significantly reduced with both doses of methyl jasmonate; however, HCA titers were increased at both doses. The amount of total glucobrassicins within broccoli was increased following exposure of florets to both doses, but no significant differences in glucoraphanin content were observed. As a conclusion, the ethanol treatment could indeed delay senescence and lead to the induction of phytochemicals. In contrast, MeJA’s effect on quality is not quite substantial; it can, however, be used to improve the phytochemical content of florets, particularly indole-type GLS.

Soil salinity can have various negative consequences on agricultural products, from their quality and production to their aesthetic traits. In this work, the possibility to use salt-affected vegetables, that otherwise would be discarded, as a source of nutraceuticals was explored. To this aim, rocket plants, a vegetable featuring bioactive compounds such as glucosinolates, were exposed to increasing NaCl concentrations in hydroponics and analysed for their content in bioactive compounds. Salt levels higher than 68 mM produced rocket plants that did not comply with European Union regulations and would therefore be considered a waste product. Anyway, our findings, obtained by Liquid Chromatography-High Resolution Mass Spectrometry, demonstrated a significant increase in glucosinolates levels in such salt-affected plants. opening the opportunity for a second life of these market discarded products to be recycled as glucosinolates source. Furthermore, an optimal situation was found at NaCl 34 mM in which not only were the aesthetic traits of rocket plants not affected, but also the plants revealed a significant enrichment in glucosinolates. This can be considered an advantageous situation in which the resulting vegetables still appealed to the market and showed improved nutraceutical aspects.

Light-emitting diode (LED) technology is one of the most important light sources in the plant industry for enhancing growth and specific metabolites in plants. In this study, we analyzed the growth, primary and secondary metabolites of 10 days old kohlrabi (Brassica oleracea var. gongylodes) sprouts exposed to different LED light conditions. The results showed that the highest fresh weight was achieved under red LED light, whereas the highest shoot and root lengths were recorded below the blue LED light. Furthermore, high-performance liquid chromatography (HPLC) analysis revealed the presence of 13 phenylpropanoid compounds, 8 glucosinolates (GSLs), and 5 different carotenoids. The phenylpropanoid and GSL contents were highest under blue LED light. In contrast, the carotenoid content was found to be maximum beneath white LED light. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) of the 71 identified metabolites using HPLC and gas chromatography–time-of-flight mass spectrometry (GC-TOF-MS) showed a clear separation, indicating that different LEDs exhibited variation in the accumulation of primary and secondary metabolites. A heat map and hierarchical clustering analysis revealed that blue LED light accumulated the highest amount of primary and secondary metabolites. Overall, our results demonstrate that exposure of kohlrabi sprouts to blue LED light is the most suitable condition for the highest growth and is effective in increasing the phenylpropanoid and GSL content, whereas white light might be used to enhance carotenoid compounds in kohlrabi sprouts.

Glucosinolates are precursors of bioactive and health-promoting isothiocyanates (ITCs). Upon enzymatic hydrolysis, Brassica vegetables, such as cabbage, also often yield nitriles and epithionitriles as main products next to ITCs. Here, we show that amines can be additional main enzymatic hydrolysis products of glucosinolates in Brassica vegetables. We propose that a plant endogenous ITC hydrolase (ITCase) is responsible for the enzymatic-like conversion of ITCs to amines in cabbage samples. This ITCase seems to have high activity towards alkenyl ITCs like allyl ITC and lower activity towards methylthioalkyl ITCs, and not to converting methylsulfinylalkyl ITCs like sulforaphane. In contrast, during heat treatment of homogenized cabbage material, methylsulfinylalkylamine levels increased by 400 % after 2 h of heating, which is likely due to thermal decomposition of ITCs, whereas alkenyl amine levels did not change due to heat treatment. The results show that amines from glucosinolates are part of the human diet.

The flavor of Guizhou suancai fermented by the emerging varieties Zhuchang-2 (ZC-2) and Zhuchang Red (ZC Red) is superior to that of ZC-1, a typical local variety of leaf mustard in Guizhou Province, China. Here, changes in phytochemical qualities during the fermentation of three Guizhou suancai were characterized to identify the causes underlying differences in flavor. The content of pigments, antioxidants, gluconapin, gluconasturtiin, and total isothiocyanates, and antioxidant capacity were highest in fresh ZC Red. The content of sinigrin, indolic glucosinolates, and total glucosinolate breakdown products was highest in fresh ZC-2. The content of phytochemicals, with the exception of carotenoids, was significantly decreased after fermentation. Odor was correlated with gluconapin, while taste was correlated with sinigrin. These findings provide detailed insights into the sensory and phytochemical properties of three Guizhou suancai that could facilitate the selection of raw material varieties.

Biofortification is the process that aims to enrich crops in micronutrients and valuable compounds. Selenium (Se) biofortification has particularly attracted increasing interest in recent times due to the growing number of individuals suffering from Se deficiency. Selenate and selenite are the Se forms most frequently administered to crops. In this study, Se was applied foliarly as selenate at 2.5, 5, or 10 mg per plant to two rocket species, Diplotaxis tenuifolia and Eruca sativa, grown in soil and the effects in terms of Se enrichment and content of primary and secondary metabolites were comparatively analyzed. We also compared our results with those obtained previously when selenate was supplied to the same species in hydroponics by addition to the nutrient solution. In most cases, the results were the opposite. In E. sativa, foliar Se treatment was more effective in promoting Se accumulation, sulfur (S), cysteine, and glucosinolates. No significant effect of Se was evident on total phenolic content, but there were individual phenols. Among amino acids, the content of proline was increased by Se, perhaps to counteract osmotic stress due to high Se accumulation. In D. tenuifolia, the content of S and cysteine decreased under Se treatment, but the amount of glutathione was steady, suggesting a preferred assimilation of cysteine toward the synthesis of this antioxidant. Consistent, the content of methionine and glucosinolates was reduced. The content of total phenolics was enhanced only by the low Se dosage. In both species, selenocysteine (SeCys) was identified, the content of which was higher compared to plants grown hydroponically. Concluding, most metabolic differences between rocket species were observed at high Se supplementation. Low Se foliar fertilization was effective in an enriching rocket in Se without affecting other phytochemicals. However, the Se dosages sufficient for biofortification could be even lower, as the Se concentration in rocket treated with 2.5 mg Se per plant was still very high and the edible part should not be eaten undiluted. Also, a single method of Se supplementation does not appear to be optimal for all plant species or the same species, as the metabolic responses could be very different.

Glucoraphanin (GRA), a glucosinolate particularly abundant in broccoli (