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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.

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.

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.

Two consecutive trials were conducted to investigate the effects of glucosinolates (GLS) in rapeseed cake (RSC) on nitrogen (N) metabolism and urine nitrous oxide (N2O) emissions in steers. In trial 1, 8 steers and 4 levels of RSC, i.e. 0, 2.7%, 5.4% and 8.0% dry matter (DM) (0, 6.0, 12.1, 18.1 μmol GLS/g DM) were allocated in a replicated 4 × 4 Latin square. In trial 2, the static incubation technique was used for measuring the N2O emissions of the urine samples collected from trial 1. The results of trial 1 indicated that dietary inclusion of RSC decreased the digested N and increased the fecal N excretion (P 0.10). Dietary inclusion of RSC decreased the urinary excretion of urea while it increased allantoin, total purine derivatives, the predicted rumen microbial N flow and thiocyanate (SCN) (P

Key message Phosphoglycerate Dehydrogenase 1 of the phosphorylated pathway of serine biosynthesis, active in heterotrophic plastids, is required for the synthesis of serine to enable plant growth at high rates of indolic glucosinolate biosynthesis. Abstract Plants have evolved effective strategies to defend against various types of pathogens. The synthesis of a multitude of specialized metabolites represents one effective approach to keep plant attackers in check. The synthesis of those defense compounds is cost intensive and requires extensive interaction with primary metabolism. However, how primary metabolism is adjusted to fulfill the requirements of specialized metabolism is still not completely resolved. Here, we studied the role of the phosphorylated pathway of serine biosynthesis (PPSB) for the synthesis of glucosinolates, the main class of defensive compounds in the model plant Arabidopsis thaliana. We show that major genes of the PPSB are co-expressed with genes required for the synthesis of tryptophan, the unique precursor for the formation of indolic glucosinolates (IG). Transcriptional and metabolic characterization of loss-of-function and dominant mutants of ALTERED TRYPTOPHAN1-like transcription factors revealed demand driven activation of PPSB genes by major regulators of IG biosynthesis. Trans-activation of PPSB promoters by ATR1/MYB34 transcription factor in cultured root cells confirmed this finding. The content of IGs were significantly reduced in plants compromised in the PPSB and these plants showed higher sensitivity against treatment with 5-methyl-tryptophan, a characteristic behavior of mutants impaired in IG biosynthesis. We further found that serine produced by the PPSB is required to enable plant growth under conditions of high demand for IG. In addition, PPSB-deficient plants lack the growth promoting effect resulting from interaction with the beneficial root-colonizing fungus Colletotrichum tofieldiae.

In natural and agricultural ecosystems, plants are often simultaneously or sequentially exposed to combinations of stressors. Here we tested whether limited water availability (LWA) affects plant response to insect herbivory using two populations of Eruca sativa from desert and Mediterranean habitats that differ in their induced defenses. Considering that such differences evolved as responses to biotic and possibly abiotic stress factors, the two populations offered an opportunity to study ecological aspects in plant response to combined stresses. Analysis of chemical defense mechanisms showed that LWA significantly induced total glucosinolate concentrations in the Mediterranean plants, but their concentrations were reduced in the desert plants. However, LWA, with and without subsequent jasmonate elicitation, significantly induced the expression of proteinase inhibitor in the desert plants. Results of a no-choice feeding experiment showed that LWA significantly increased desert plant resistance to Spodoptera littoralis larvae, whereas it did not affect the relatively strong basal resistance of the Mediterranean plants. LWA and subsequent jasmonate elicitation increased resistance against the generalist insect in Mediterranean plants, possibly due to both increased proteinase inhibitor expression and glucosinolate accumulation. The effect of LWA on the expression of genes involved in phytohormone signaling, abscisic acid (ABA-1) and jasmonic acid (AOC1), and the jasmonate responsive PDF1.2, suggested the involvement of abscisic acid in the regulation of defense mechanisms in the two populations. Our results indicate that specific genotypic responses should be considered when estimating general patterns in plant response to herbivory under water deficiency conditions.

Background Rose is an important economic crop in horticulture. However, its field growth and postharvest quality are negatively affected by grey mould disease caused by Botrytis c. However, it is unclear how rose plants defend themselves against this fungal pathogen. Here, we used transcriptomic, metabolomic and VIGS analyses to explore the mechanism of resistance to Botrytis c. Result In this study, a protein activity analysis revealed a significant increase in defence enzyme activities in infected plants. RNA-Seq of plants infected for 0 h, 36 h, 60 h and 72 h produced a total of 54 GB of clean reads. Among these reads, 3990, 5995 and 8683 differentially expressed genes (DEGs) were found in CK vs. T36, CK vs. T60 and CK vs. T72, respectively. Gene annotation and cluster analysis of the DEGs revealed a variety of defence responses to Botrytis c. infection, including resistance (R) proteins, MAPK cascade reactions, plant hormone signal transduction pathways, plant-pathogen interaction pathways, Ca2+ and disease resistance-related genes. qPCR verification showed the reliability of the transcriptome data. The PTRV2-RcTGA1-infected plant material showed improved susceptibility of rose to Botrytis c. A total of 635 metabolites were detected in all samples, which could be divided into 29 groups. Metabonomic data showed that a total of 59, 78 and 74 DEMs were obtained for T36, T60 and T72 (T36: Botrytis c. inoculated rose flowers at 36 h; T60: Botrytis c. inoculated rose flowers at 60 h; T72: Botrytis c. inoculated rose flowers at 72 h) compared to CK, respectively. A variety of secondary metabolites are related to biological disease resistance, including tannins, amino acids and derivatives, and alkaloids, among others; they were significantly increased and enriched in phenylpropanoid biosynthesis, glucosinolates and other disease resistance pathways. This study provides a theoretical basis for breeding new cultivars that are resistant to Botrytis c. Conclusion Fifty-four GB of clean reads were generated through RNA-Seq. R proteins, ROS signalling, Ca2+ signalling, MAPK signalling, and SA signalling were activated in the Old Blush response to Botrytis c. RcTGA1 positively regulates rose resistance to Botrytis c. A total of 635 metabolites were detected in all samples. DEMs were enriched in phenylpropanoid biosynthesis, glucosinolates and other disease resistance pathways.

Many herbivorous insects selectively accumulate plant toxins for defense against predators; however, little is known about the transport processes that enable insects to absorb and store defense compounds in the body. Here, we investigate how a specialist herbivore, the horseradish flea beetle, accumulates glucosinolate defense compounds from Brassicaceae in the hemolymph. Using phylogenetic analyses of coleopteran major facilitator superfamily transporters, we identify a clade of glucosinolate-specific transporters (PaGTRs) belonging to the sugar porter family. PaGTRs are predominantly expressed in the excretory system, the Malpighian tubules. Silencing of PaGTRs leads to elevated glucosinolate excretion, significantly reducing the levels of sequestered glucosinolates in beetles. This suggests that PaGTRs reabsorb glucosinolates from the Malpighian tubule lumen to prevent their loss by excretion. Ramsay assays corroborated the selective retention of glucosinolates by Malpighian tubules of P. armoraciae in situ. Thus, the selective accumulation of plant defense compounds in herbivorous insects can depend on the ability to prevent excretion.

The invasive pest whitefly (Bemisia tabaci) is a complex species, of which Middle East-Minor Asia 1 (MEAM1) and Mediterranean (MED) are the two most damaging members. Previous research showed that cabbage is frequently infested with MEAM1 but seldomly with MED, and this difference in performance is associated with glucosinolate (GS) content. Some insects can modify GS using glucosinolate sulfatase (SULF), the activity of which is regulated by sulfatase modifying factor 1 (SUMF1); therefore, to increase our understanding of different performances of MEAM1 and MED on cabbage plants, we identified and compared nine putative SULFs and one SUMF in MEAM1 and MED. We found that the lengths of two genes, BtSulf2 and BtSulf4, differed between MEAM1 and MED. The messenger RNA levels of BtSulf4 increased more than 20-fold after MEAM1 and MED adults were exposed to GS, but BtSulf2 expression was only induced by GS in MEAM1. Knockdown of BtSulf2 and BtSulf4 in MEAM1 resulted in a substantial increase in the mortality of GS-treated adults but not in MED. These results indicate that differences in BtSulf2 and BtSulf4 sequences and/or expression may explain why MEAM1 performs better than MED on cabbage. Our results provide a basis for future functional research on SULF and SUMF in B. tabaci.

Glucosinolates are secondary defense metabolites produced by plants of the order Brassicales, which includes the model species Arabidopsis and many crop species. In the past 13 years, the regulation of glucosinolate synthesis in plants has been intensively studied, with recent research revealing complex molecular mechanisms that connect glucosinolate production with responses to other central pathways. In this review, we discuss how the regulation of glucosinolate biosynthesis is ecologically relevant for plants, how it is controlled by transcription factors, and how this transcriptional machinery interacts with hormonal, environmental, and epigenetic mechanisms. We present the central players in glucosinolate regulation, MYB and basic helix–loop–helix transcription factors, as well as the plant hormone jasmonate, which together with other hormones and environmental signals allow the coordinated and rapid regulation of glucosinolate genes. Furthermore, we highlight the regulatory connections between glucosinolates, auxin, and sulfur metabolism and discuss emerging insights and open questions on the regulation of glucosinolate biosynthesis.

Wasabi, horseradish and mustard are popular pungent crops in which the characteristic bioactive hydrolysis of specialized glucosinolates (GSLs) occurs. Although the metabolic pathways of GSLs are well elucidated, how plants have evolved convergent mechanisms to accumulate identical GSL components remains largely unknown. In this study, we discovered that sinigrin is predominantly synthesized in wasabi, horseradish and mustard in Brassicaceae. We de novo assembled the transcriptomes of the three species, revealing the expression patterns of gene clusters associated with chain elongation, side chain modification and transport. Our analysis further revealed that several gene clusters were convergently selected during evolution, exhibiting convergent shifts in amino acid preferences in mustard, wasabi and horseradish. Collectively, our findings provide insights into how unrelated crop species evolve the capacity for sinigrin super-accumulation and thus promise a potent strategy for engineering metabolic pathways at multiple checkpoints to fortify bioactive compounds for condiment or pharmaceutical purposes., Jinghua Yang et al. provide the de novo assemblies of the transcriptomes of wasabi, horseradish and mustard to examine the evolution of specialized glucosinolates. This study demonstrates convergent evolution of super-accumulation of sinigrin in these taxa and may allow for potential strategies to engineer metabolic pathways of bioactive compounds for human food sources or pharmaceutical purposes.

Purpose Brassica are an important food source worldwide and are characterised by the presence of compounds called glucosinolates. Studies indicate that the glucosinolate derived bioactive metabolite sulphoraphane can elicit chemoprotective benefits on human cells. Glucosinolates can be metabolised in vivo by members of the human gut microbiome, although the prevalence of this activity is unclear. Brassica and Allium plants also contain S-methylcysteine sulphoxide (SMCSO), that may provide additional health benefits but its metabolism by gut bacteria is not fully understood. Methods We examined the effects of a broccoli leachate (BL) on the composition and function of human faecal microbiomes of five different participants under in vitro conditions. Bacterial isolates from these communities were then tested for their ability to metabolise glucosinolates and SMCSO. Results Microbial communities cultured in vitro in BL media were observed to have enhanced growth of lactic acid bacteria, such as lactobacilli, with a corresponding increase in the levels of lactate and short-chain fatty acids. Members of Escherichia isolated from these faecal communities were found to bioconvert glucosinolates and SMCSO to their reduced analogues. Conclusion This study uses a broccoli leachate to investigate the bacterial-mediated bioconversion of glucosinolates and SMCSO, which may lead to further products with additional health benefits to the host. We believe that this is the first study that shows the reduction of the dietary compound S-methylcysteine sulphoxide by bacteria isolated from human faeces.

Background Recently, it become an important strategy using light to regulate plant growth and quality, especially on daily edible leafy vegetable. Pak-choi is rich in healthy functional compounds, e.g. flavonoid and glucosinolate. Many studies have focused on the plant response to increased radiation and transformed visible light quality, however, we know less about different blue and UV-A light wavelengths. Therefore, the goal of this study was to identify whether different blue and UV-A light wavelengths could improve quality in two cultivars of pak-choi and further cultivate potentially healthy functional plants. Results The different blue and UV-A light wavelength treatments significantly increased the fresh and dry weight in two cultivars of pak-choi. Compared with control, the content of soluble protein was higher after the different blue and UV-A light treatments. Similarly, the contents of total phenolics and total flavonoids increased significantly under the light treatments, and the highest content presented under T430 (supplemental blue light at 430 nm) in red-leaf pak-choi and under T400 (supplemental UV-A light at 400 nm) in green-leaf pak-choi. The total anthocyanins content and 2,2-diphenyl-1-picrylhydrazyl (DPPH) of two pak-choi cultivars improved positively with decreasing treatment wavelength, and other healthy compounds were affected to varying degrees under supplemental light treatments. Conclusion The growth and healthy compound contents of pak-choi were significantly improved by supplemental blue and UV-A light, and there were wavelength- and cultivar-dependent effects. Compared with control, T430 presented the higher biomass and the contents of total phenolics, flavonoids and pigment in two pak-choi cultivars, and T380 was an efficient strategy to increase antioxidants and health-promoting compounds of red-leaf pak-choi. © 2020 Society of Chemical Industry.

Rutabaga (Brassica napus L.) belonging to Brassicaceae family, is a rich source of polyphenols and glucosinolates. Its consumption in human diet is highly appreciated for its nutritional contribution and health benefits. Brassica napus L. is recognized as the world's most widely grown temperate oilseed crop containing erucic acid for industrial applications, plants germination, animal feed and fuel. In this work we prepared two different extracts of Rutabaga root’s pulp and peel, e.g. ultrasound assisted extract (UAE) and homogenizer assisted extract (HAE). The four extracts have been analyzed by HPLC-MS to assess the phytochemical characterization and tested by antioxidant and enzyme inhibitor assays. Rutabaga pulp and peel extracts possess tyrosinase and glucosidase inhibitory activities together with a moderate antioxidant ability. Our results show a high level of glucosinolates, in particular neoglucobrassicin in the peel extract, which let us suppose a potential application as crop in industry and as supplement in human diet.

Purpose Studies on broccoli (Brassica oleracea var. italica) indicate beneficial effects against a range of chronic diseases, commonly attributed to their bioactive phytochemicals. Sulforaphane, the bioactive form of glucoraphanin, is formed by the action of the indigenous enzyme myrosinase. This study explored the role that digestion and cooking practices play in bioactivity and bioavailability, especially the rarely considered dose delivered to the colon. Methods The antimicrobial activity of sulforaphane extracts from raw, cooked broccoli and cooked broccoli plus mustard seeds (as a source myrosinase) was assessed. The persistence of broccoli phytochemicals in the upper gastrointestinal tract was analysed in the ileal fluid of 11 ileostomates fed, in a cross-over design, broccoli soup prepared with and without mustard seeds. Results The raw broccoli had no antimicrobial activity, except against Bacillus cereus, but cooked broccoli (with and without mustard seeds) showed considerable antimicrobial activity against various tested pathogens. The recovery of sulforaphane in ileal fluids post soup consumption was Escherichia coli K12, no inhibitory effects were observed. Analysis of glucosinolates composition in ileal fluids revealed noticeable inter-individual differences, with six “responding” participants showing increases in glucosinolates after broccoli soup consumption. Conclusions Sulforaphane-rich broccoli extracts caused potent antimicrobial effects in vitro, and the consumption of sulforaphane-enriched broccoli soup may inhibit bacterial growth in the stomach and upper small intestine, but not in the terminal ileum or the colon.

Meristem function is underpinned by numerous genes that affect hormone levels, ultimately controlling phyllotaxy, the transition to flowering and general growth properties. Class I KNOX genes are major contributors to this process, promoting cytokinin biosynthesis but repressing gibberellin production to condition a replication competent state. We identified a suppressor mutant of the KNOX1 mutant brevipedicellus (bp) that we termed flasher (fsh), which promotes stem and pedicel elongation, suppresses early senescence, and negatively affects reproductive development. Map-based cloning and complementation tests revealed that fsh is due to an E40K change in the flavin monooxygenase GS-OX5, a gene encoding a glucosinolate (GSL) modifying enzyme. In vitro enzymatic assays revealed that fsh poorly converts substrate to product, yet the levels of several GSLs are higher in the suppressor line, implicating FSH in feedback control of GSL flux. FSH is expressed predominantly in the vasculature in patterns that do not significantly overlap those of BP, implying a non-cell autonomous mode of meristem control via one or more GSL metabolites. Hormone analyses revealed that cytokinin levels are low in bp, but fsh restores cytokinin levels to near normal by activating cytokinin biosynthesis genes. In addition, jasmonate levels in the fsh suppressor are significantly lower than in bp, which is likely due to elevated expression of JA inactivating genes. These observations suggest the involvement of the GSL pathway in generating one or more negative effectors of growth that influence inflorescence architecture and fecundity by altering the balance of hormonal regulators.

Secondary plant metabolites, represented by indole glucosinolates (IGS) and camalexin, play important roles in Arabidopsis immunity. Previously, we demonstrated the importance of MPK3 and MPK6, two closely related MAPKs, in regulating Botrytis cinerea (Bc)‐induced IGS and camalexin biosynthesis. Here we report that CPK5 and CPK6, two redundant calcium‐dependent protein kinases (CPKs), are also involved in regulating the biosynthesis of these secondary metabolites. The loss‐of‐function of both CPK5 and CPK6 compromises plant resistance to Bc. Expression profiling of CPK5‐VK transgenic plants, in which a truncated constitutively active CPK5 is driven by a steroid‐inducible promoter, revealed that biosynthetic genes of both IGS and camalexin pathways are coordinately upregulated after the induction of CPK5‐VK, leading to high‐level accumulation of camalexin and 4‐methoxyindole‐3‐yl‐methylglucosinolate (4MI3G). Induction of camalexin and 4MI3G, as well as the genes in their biosynthesis pathways, is greatly compromised in cpk5 cpk6 mutant in response to Bc. In a conditional cpk5 cpk6 mpk3 mpk6 quadruple mutant, Bc resistance and induction of IGS and camalexin are further reduced in comparison to either cpk5 cpk6 or conditional mpk3 mpk6 double mutant, suggesting that both CPK5/CPK6 and MPK3/MPK6 signaling pathways contribute to promote the biosynthesis of 4MI3G and camalexin in defense against Bc., Plants produce secondary metabolites as defense chemicals in responses to biotic stimuli, including pathogens and herbivores. In this report, CPK5 and CPK6, two redundant calcium‐dependent protein kinases, are identified to play an important role in regulating the biosynthesis of indole glucosinolates and camalexin, represented antimicrobial metabolites in Arabidopsis immunity.

Specifier proteins (SPs) are components of the glucosinolate-myrosinase defense system found in plants of the order Brassicales (brassicas). Glucosinolates (GLSs) comprise at least 150 known S-(β-d...

Brassicales plants produce glucosinolates and myrosinases that generate toxic isothiocyanates conferring broad resistance against pathogens and herbivorous insects. Nevertheless, some cosmopolitan fungal pathogens, such as the necrotrophic white mold Sclerotinia sclerotiorum, are able to infect many plant hosts including glucosinolate producers. Here, we show that S. sclerotiorum infection activates the glucosinolate-myrosinase system, and isothiocyanates contribute to resistance against this fungus. S. sclerotiorum metabolizes isothiocyanates via two independent pathways: conjugation to glutathione and, more effectively, hydrolysis to amines. The latter pathway features an isothiocyanate hydrolase that is homologous to a previously characterized bacterial enzyme, and converts isothiocyanate into products that are not toxic to the fungus. The isothiocyanate hydrolase promotes fungal growth in the presence of the toxins, and contributes to the virulence of S. sclerotiorum on glucosinolate-producing plants., Some plants produce toxic isothiocyanates that protect them against pathogens. Here, Chen et al. show that the plant pathogenic fungus Sclerotinia sclerotiorum converts isothiocyanates into non-toxic compounds via glutathione conjugation and, more effectively, via hydrolysis to amines using an isothiocyanate hydrolase.

Glucosinolates are secondary plant metabolites present in Brassica vegetables. The endogenous enzyme myrosinase is responsible for the hydrolysis of glucosinolates, yielding a variety of compounds, including health-promoting isothiocyanates. The influence of cabbage accession and growing conditions on myrosinase activity, glucosinolates (GSL) and their hydrolysis products (GHPs) of 18 gene-bank cabbage accessions was studied. Growing conditions, cabbage morphotype and accession all significantly affected myrosinase activity and concentration of glucosinolates and their hydrolysis products. In general, cabbages grown in the field with lower growth temperatures had significantly higher myrosinase activity than glasshouse samples. Profile and concentration of glucosinolates and their hydrolysis products differed across the accessions studied. Aliphatic glucosinolates accounted for more than 60 % of total glucosinolates in most of the samples assessed. Nitriles and epithionitriles were the most abundant hydrolysis products formed. The results obtained showed that consumption of raw cabbages might reduce the amount of beneficial hydrolysis products available to the consumer, as more nitriles were produced from hydrolysis compared to beneficial isothiocyanates. However, red and white cabbages contained high concentrations of glucoraphanin and its isothiocyanate, sulforaphane. This implies that careful selection of accessions with ample concentrations of certain glucosinolates can improve the health benefits derived from raw cabbage consumption.

Glucosinolates are well known as natural antimicrobials and anticarcinogenic agents. However, these compounds can lose their properties and transform into antinutrients, depending on processing conditions. In addition, the bitterness of some glucosinolate in rapeseed meal can affect the likability of the final product. Therefore, it is important to identify and determine each glucosinolate and its derived form, not just the total glucosinolate content, in order to evaluate the potential of the final rapeseed protein product. This study provides a comprehensive report of the types and quantities of glucosinolates and their derived forms (isothiocyanates) associated with different rapeseed processing conditions. Glucosinolates and isothiocyanates were determined by HPLC-DAD-qTOF. In our study, the enzymatic degradation of glucosinolates by myrosinase was the main factor affecting either glucosinolate or isothiocyanate content. Other factors such as pH seemed to influence the concentration and the presence of glucosinolates. In addition, process parameters, such as extraction time and separation technology, seemed to affect the amount and type of isothiocyanates in the final protein extracts. Overall, both determined intact glucosinolates and their derived forms of isothiocyanates can give different types of biological effects. More studies should be performed to evaluate the impact of glucosinolates and isothiocyanates on human health.

Numerous plants protect themselves from attackers by using specialized metabolites. The biosynthesis of these deterrent, often toxic metabolites is costly, as their synthesis diverts energy and resources on account of growth and development. How plants diversify investments into growth and defense is explained by the optimal defense theory. The central prediction of the optimal defense theory is that plants maximize growth and defense by concentrating specialized metabolites in tissues that are decisive for fitness. To date, supporting physiological evidence relies on the correlation between plant metabolite presence and animal feeding preference. Here, we use glucosinolates as a model to examine the effect of changes in chemical defense distribution on feeding preference. Taking advantage of the uniform glucosinolate distribution in transporter mutants, we show that high glucosinolate accumulation in tissues important to fitness protects them by guiding larvae of a generalist herbivore to feed on other tissues. Moreover, we show that the mature leaves of Arabidopsis thaliana supply young leaves with glucosinolates to optimize defense against herbivores. Our study provides physiological evidence for the central hypothesis of the optimal defense theory and sheds light on the importance of integrating glucosinolate biosynthesis and transport for optimizing plant defense.

The traditional Japanese fermented pickle, takuan-zuke, is produced via dehydration and salt-aging of the Japanese winter radish root (Raphanus sativus L.). It has been reported that γ-aminobutyric acid (GABA; antihypertensive factor) accumulates during this production. Herein, the results of the comprehensive study (metabolites, enzyme activity, and gene expression levels were investigated) on the mechanism of branched-chain amino acid (BCAA) synthesis and glucosinolate (GLS)-myrosinase system of daikon dehydration have been reported. BCAAs, GLS, and their precursors were temporarily upregulated, and the genes linked to BCAA, GLS, and isothiocyanate synthesis (BCAT4, MAM3, IPMDH1, RMB1, RMB2, and GRS1) were upregulated during daikon dehydration. BCAAs and GLS accumulated in daikon during dehydration owing to the upregulation of genes, encoding these synthases as a stress response. These results suggest that the biological response of daikon adds characteristic flavor and health functionality to takuan-zuke and helps optimize the processing parameters to produce pickles with improved health benefits. PRACTICAL APPLICATIONS: Takuan-zuke is a popular pickle in Japan, which is produced from a Japanese winter radish root (Raphanus sativus L.), commonly referred to as daikon in Japan. Takuan-zuke is produced by dehydrating daikon either by the process of salt-pressing (shio-oshi) or sun-drying (hoshi). The processes influence the variations in the nutritional value of the final product. The results reveal that both the daikon dehydration processes can be followed to generate increased amounts of health-promoting components (e.g., branched-chain amino acid and glucosinolate) in takuan-zuke.

As of recent, microgreen vegetable production in controlled environments are being investigated for their bioactive properties. Phytochemicals like glucosinolates (GLS) are highly sensitive to varying spectral qualities of light, especially in leafy greens of Brassica where the responses are highly species-dependent. The accumulation of bioactive GLS were studied under 8 different treatments of combined amber (590 nm), blue (455 nm), and red (655 nm) light-emitting diodes (rbaLED). A semi-targeted metabolomics approach was carried out to profile common intact-GLS in microgreen extracts of Brassica by means of LC-HRMS/MS. Thirteen GLS were identified, among them were 8 aliphatic, 4 indolic and 1 aromatic GLS. Mass spectrometry data showed sinigrin had the highest average concentration and was highest in B. juncea, progoitrin was highest in B. rapa and glucobrassicin in R. sativus. The individual and total GLS in the microgreens of the present study were largely different under rbaLED; B. rapa microgreens contained the highest profile of total GLS, followed by R. sativus and B. juncea. Sinigrin was increased and gluconasturtiin was decreased under rbaLED lighting in most microgreens, glucoalyssin uniquely increased in R. sativus and decreased in B. rapa and glucobrassicin uniquely decreased in both B. rapa and B. juncea. The present study showed that rbaLED contributed to the altered profiles of GLS resulting in their significant modulation. Optimizing the light spectrum for improved GLS biosynthesis could lead to production of microgreens with targeted health-promoting properties. Graphical Abstract

Poultry is the major sources of foodborne salmonellosis. Antibiotic resistance and a surge in zoonotic diseases warrant the use of natural alternatives. Glucosinolates (GLs) are naturally occurring antimicrobial compounds in rapeseed and canola. This study investigated the effect of feeding rapeseed, canola meal, and allyl isothiocyanate (AITC; Brassica secondary metabolites) on growth performance (GP), gut health, and the potential antimicrobial activity against nalidixic acid-resistant Salmonella Typhimurium (STNR) in chickens. A total of 640 one-day-old male Cobb 500 broilers were randomly allocated to 8 treatments with 8 replicated cages and 10 birds per cage. Dietary treatments were nonchallenge control (NC, corn-SBM based), challenge (Salmonella) control (CC), 10% rapeseed (10RS), 30% rapeseed (30RS), 20% canola meal (20CLM), 40% canola meal (40CLM), 500 ppm AITC (500AITC), and 1,500 ppm AITC (1500AITC). On d 1, all the birds except NC were orally challenged with STNR (7 log CFU/bird). The chickens were reared for 21 d, and their FI and BW were recorded weekly. Salmonella cecal colonization and fecal shedding were quantified, whereas organ translocation (OT) of STNR to the spleen, liver, and kidney was tested on 0, 3, 6, 13, and 20-d postchallenge (dpc). Data were subjected to one-way ANOVA, and the means were separated by Duncan's test, except mortality and OT data analyzed after transformation by square root of (n +1) (P < 0.05). Overall, feeding 30RS resulted in reduced BW (P = 0.003), BWG (P = 0.003), and FI (P = 0.001) compared to CC, 500AITC, and 1500AITC. Similarly, feeding 20CLM resulted in lower BW and BWG compared to CC (P < 0.05) and increased FCR compared to 1500AITC (P = 0.03). Feeding CC resulted in higher mortality compared to NC and 30RS (P = 0.03). Cecal colonization of STNR was reduced (P < 0.0001) for 30RS on 6 dpc and 500AITC on 6 and 13 dpc (P < 0.0001). Although no difference in gut permeability was observed 6 dpc (P > 0.05), OT of STNR population was the highest for CC in the spleen (P = 0.05). In the liver, 10RS showed reduced OT compared to 20CLM on 13 dpc (P = 0.03), whereas 30RS showed the lowest OT on 6 dpc in the kidney. Fecal shedding was lowest for 30RS on 6 dpc (P = 0.004). Histomorphology showed 30RS had the highest duodenum (P = 0.01) and jejunum (P = 0.02) villus height (VH) and VH to crypt depth (CD) ratio compared to the other treatments, whereas 1500AITC showed similar results to 30RS. Both 30RS and 1500AITC contained comparatively higher functional GL metabolites and were able to maintain gut health. Including higher levels of rapeseed or AITC in poultry feed can reduce Salmonella colonization in the feces and their translocation to other organs.

The ability of white, red, and blue irradiation to maintain sensory quality, health-promoting compounds, and antioxidant capacity, and regulate glucosinolate metabolism-related gene expression in post-harvest baby mustard was studied. Irradiation with 80 µmol m-2 s-1 extended the shelf life of post-harvest baby mustard. Irradiation delayed the increase in weight loss and the decrease in sensory parameter scores and the levels of ascorbic acid, total phenolics, glucosinolate, and antioxidant capacity during storage of baby mustard. Irradiation induced the expression of glucosinolate biosynthesis genes and inhibited glucosinolate degradation gene expression. The glucosinolate content and glucosinolate metabolism-related gene expression in post-harvest baby mustard were higher under white and red light irradiation compared with blue light irradiation. These findings indicate that irradiation (80 µmol m-2 s-1 ), especially of white and red light, is an effective technique for maintaining the sensory and nutritional qualities in post-harvest baby mustard stored at 20°C. PRACTICAL APPLICATION: This study was to evaluate the effect of white, red, and blue irradiation on the sensory quality, health-promoting compounds, antioxidant capacity, and glucosinolate metabolism-related gene expression of baby mustard during post-harvest storage, providing an effective and sustainable post-harvest method to extend shelf life and maintain the post-harvest quality of baby mustard under ambient temperature storage. Irradiation (80 µmol m-2 s-1 ), especially of white and red light, is an effective technique for maintaining the sensory and nutritional qualities in post-harvest baby mustard stored at 20°C.

Glucoraphanin (GRA) is found in the seeds and vegetative organs of broccoli (Brassica oleracea L. var. italica Planch) as the precursor of anti-carcinogen sulforaphane (SF). The yield of GRA obtained from these materials is weak and the cost is high. In recent years, the production of plant secondary metabolites by large-scale hairy roots culture in vitro has succeeded in some species. Melatonin (MT) is a natural hormone which existed in numerous organisms. Studies have demonstrated that MT can improve the synthesis of secondary metabolites in plants. At present, it has not been reported that MT regulates the biosynthesis of glucoraphanin in broccoli hairy roots. In this study, the broccoli hairy roots that grew for 20 d were respectively treated by 500 µM MT for 0, 6, 12, 20 and 32. To explore the reason of changes in secondary metabolites and reveal the biosynthetic pathway of glucoraphanin at transcriptional level. Compared with 0 h, the yield of GRA under other treatments was increased, and the overall trend was firstly increased and then decreased. The total yield of GRA reached the highest at 12 h, which was 1.22-fold of 0 h. Then, the genome of broccoli as the reference, a total of 13234 differentially expressed genes (DEGs) were identified in broccoli hairy roots under treatment with 500 µM MT for 0, 6, 12, 20 and 32 h, respectively. Among these DEGs, 6266 (47.35%) were upregulated and 6968 (52.65%) were downregulated. It was found that the pathway of ‘Glucosinolates biosynthesis (ko00966)’ was enriched in the 16th place by Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the upregulated DEGs. The expression of key genes in the GRA biosynthesis pathway was upregulated at all time points, and a deduced GRA biosynthesis pathway map was constructed for reference.

To investigate the effects of supplementary UV-A intensity on growth and antioxidant compounds in Chinese kale (Brassica alboglabra Bailey) baby-leaves, three different UV-A intensity treatments (5, 10, 15 W·m−2, respectively) were applied 10 days before harvest in artificial light plant factory. In Chinese kale baby-leaves, supplemental 5 and 10 W·m−2 UV-A (UVA-5 and UVA-10) were beneficial for inter-node length, stem diameter, canopy diameter, fresh weight and dry weight, particularly in UVA-10 treatment, while these above-mentioned growth parameters all significantly decreased in UVA-15 treatment. The soluble sugar content decreased under UVA-5, but there was no significant difference under UVA-10 and UVA-15. Soluble protein contents decreased under UVA-5 and UVA-10, but significantly increased under UVA-15. UVA-10 played a predominant role in increasing FRAP and contents of total phenolics and total flavonoids compared to other treatments. Contents of total glucosinolates (GLs), aliphatic GLs and indolic GLs in Chinese kale baby-leaves significantly increased with UV-A intensity increasing, and the highest contents were found under UVA-15. The percentage of total aliphatic GLs (about 80%) was significantly higher than those of total indolic GLs. Glucobrassicanapin and sinigrin were two major individual GLs in Chinese kale baby-leaves, variation trends of which were consistent with the contents of total GLs and aliphatic GLs. From the heatmap analysis, and taking economic benefits into account, UVA-10 might be optimal for the production of high-quality Chinese kale baby-leaves in an artificial light plant factory.

Background Microalgae and nanoparticles are currently considered promising tools for numerous agricultural and biotechnological applications. The green microalga Chlorella sp. MF1 and its biosynthesized silver nanoparticles (AgNPs) were used in this study as biofortification agents to enhance glucosinolate and kaempferol levels in Eruca sativa. UV–visible spectroscopy, XRD, FTIR and TEM were comprehensively used for characterizing Chlorella-based AgNPs. Results The biosynthesized AgNPs were found to be spherical in shape, with size ranging from 1.45 to 5.08 nm. According to FTIR measurements, silver ions were reduced to AgNPs by functional groups such as amide, hydroxyl and carboxylate. Different experimental treatments were conducted, including either soaking seeds of E. sativa or foliar spray with various concentrations of Chlorella suspension (1, 2, 3 and 4 g L−1) and AgNPs (5, 10, 20 and 40 mg L−1). Expression levels of five key genes in the biosynthetic pathway of glucosinolates (MAM1, SUR1, MYB34 and MYB51) and kaempferol (CHS) were assessed using qRT-PCR. The results indicated an upregulation in the gene expression levels in all treatments compared to control, recording the highest level at 40 mg L−1 AgNPs and 4 g L−1Chlorella suspension. In addition, high glucosinolates and kaempferol content was detected in plants whose leaves were sprayed with AgNPs and Chlorella suspension (40 mg L−1 and 4 g L−1) based on HPLC analysis. Sequence analysis of amplified CHS fragments from E. sativa plants treated with AgNPs (40 mg L−1) showed high sequence similarity to A. thaliana CHS gene. However, there were several CHS regions with sequence polymorphism (SNPs and Indels) in foliar sprayed plants. Conclusions Results of this study evidenced that the application of AgNPs and Chlorella suspension increased glucosinolates and kaempferol content in E. sativa through upregulation of key genes in their biosynthetic pathway.

Plant specialized metabolites are often subject to within-plant transport and have tissue-specific distribution patterns. Among plants in the Brassicaceae, the genus Erysimum is unique in producing not only glucosinolates but also cardenolides as defense against insect herbivory. Ten cardenolides were detected with varying abundance in different tissues of Erysimum cheiranthoides (wormseed wallflower). As is predicted by the optimal defense theory, cardenolides were most abundant in young leaves and reproductive tissues. The lowest concentrations were observed in senescing leaves and roots. Crosses between wildtype E. cheiranthoides and a mutant line with an altered cardenolide profile showed that the seed cardenolide phenotype is determined entirely by the maternal genotype. Prior to the development of the first true leaves, seedling cotyledons also had the maternal cardenolide profile. Hypocotyl grafting experiments showed that the root cardenolide profile is determined entirely by the aboveground plant genotype. In further grafting experiments, there was no evidence of cardenolide transport into the leaves, but a mixed cardenolide profile was observed in the stems and inflorescences of plants that had been grafted at vegetative and flowering growth stages, respectively. Together, these results indicate that E. cheiranthoides leaves are a site of cardenolide biosynthesis and therefore also the plant tissue that is most likely to be expressing the relevant biosynthetic genes.

An increasing body of evidence highlights the strong potential for a diet rich in fruit and vegetables to delay, and often prevent, the onset of chronic diseases, including cardiometabolic, neurological, and musculoskeletal conditions, and certain cancers. A possible protective component, glucosinolates, which are phytochemicals found almost exclusively in cruciferous vegetables, have been identified from preclinical and clinical studies. Current research suggests that glucosinolates (and isothiocyanates) act via several mechanisms, ultimately exhibiting anti-inflammatory, antioxidant, and chemo-protective effects. This review summarizes the current knowledge surrounding cruciferous vegetables and their glucosinolates in relation to the specified health conditions. Although there is evidence that consumption of a high glucosinolate diet is linked with reduced incidence of chronic diseases, future large-scale placebo-controlled human trials including standardized glucosinolate supplements are needed.

Kohlrabi is considered an important dietary vegetable worldwide. In this study, we investigated the growth and accumulation of phenolic compounds (PCs) and glucosinolates in sprouts of pale green and purple kohlrabi (Brassica oleracea var. gongylodes) in response to light and dark conditions. Pale green kohlrabi presented high fresh weight and root length irrespective of light treatment, whereas under dark conditions, it presented higher fresh weight and shoot length than purple kohlrabi. In contrast, the root length of both kohlrabies increased markedly under light conditions compared to that under dark conditions. Thirteen PCs and eight glucosinolates were detected and quantified in 10-day-old pale green and purple kohlrabies. In both kohlrabies, the individual and total phenolic levels were much higher under the light treatment than under the dark treatment. Under light and dark conditions, the total phenolic content was 6362.13 and 5475.04 µg/g dry weight in the pale green kohlrabi, respectively, whereas in the purple kohlrabi, it was 10,115.76 and 9361.74 µg/g dry weight, respectively. Dark conditions favored higher accumulation of glucosinolates than light conditions. Progoitrin, neoglucobrassicin, glucoerucin, and 4-methoxyglucobrassicin were the predominant glucosinolates in both kohlrabies and were present in much higher amounts in the pale green kohlrabi. In pale green kohlrabi under dark conditions, the total glucosinolates content was 4.75 and 2.62 times higher than that of the purple kohlrabi under light and dark conditions, respectively. Among individual glucosinolates, in the pale green kohlrabi under the dark condition, progoitrin was found to have the highest content, which was 90.28 and 54.51 times higher than that in the purple kohlrabi under light and dark conditions, respectively. These results show that the phenolic and glucosinolates levels varied widely, and these variations between the two types of kohlrabi under both light and dark conditions were significant. Our findings suggest that light and dark conditions enhance the accumulation of PCs and glucosinolates, respectively, during the development of kohlrabi seedlings.

Aim Epidemiological data suggest that maternal immune activation (MIA) plays a role in the etiology of neuropsychiatric disorders including autism spectrum disorder (ASD) and schizophrenia. However, there is no prophylactic nutrition that can prevent the onset of neurodevelopmental disorders in offspring after MIA. The aim of this study was undertaken to examine whether dietary intake of glucoraphanin (GF: the precursor of a natural anti‐inflammatory compound sulforaphane) can prevent the onset of behavioral abnormalities in offspring after MIA. Methods One percent of GF food pellet or normal food pellet was given into female mice during pregnancy and lactation (from E5 to P21). Saline (5 mL/kg/d) or poly(I:C) (5 mg/kg/d) was injected into pregnant mice from E12 to E17. Behavioral tests and immunohistochemistry of parvalbumin (PV) were performed in male offspring. Results Dietary intake of GF during pregnancy and lactation prevented cognitive deficits and social interaction deficits in the juvenile offspring after MIA. Furthermore, dietary intake of GF during pregnancy and lactation prevented cognitive deficits in the adult offspring after MIA. Moreover, dietary intake of GF prevented the reduction of PV immunoreactivity in the medial prefrontal cortex of adult offspring after MIA. Conclusion These data suggest that dietary intake of GF during pregnancy and lactation could prevent behavioral abnormalities in offspring after MIA.

Background Glucosinolates are an important class of secondary metabolites characteristic to the order Brassicales. They are known to play a major role in plant defense and from the human perspective, can be anticarcinogenic or antinutritive. GTRs are plasma-membrane localized high affinity glucosinolate transporters, which are important components of the source (leaf) to sink (seed) translocation of intact glucosinolates in members of Brassicaceae family. GTRs are identified as major candidates for Brassica crop improvement, thus dictating a need for their functional characterization. However, currently there are limitations in availability of heterologous assay systems for functional characterization of plant secondary metabolite transporters. To date, the animal-based Xenopus oocyte system is the best established heterologous system for functional characterization of these transporters. Inherent biochemical and physiological attributes unique to the plant membranes necessitate the need for developing plant-based transporters assay systems as well. Methods In this study, Agrobacterium mediated transformation was used to develop GTR expressing cotton cell lines (CCL-1) for functional characterization of the Arabidopsis high affinity glucosinolate transporters, AtGTR1 and AtGTR2. Following sub-cellular localization of AtGTRs, we standardized the glucosinolate uptake assays using cell suspension cultures of AtGTR expressing CCL-1 its requirement of pH, salt, and time based glucosinolate uptake. Using the GTR expressing CCL-1, we subsequently performed kinetic analysis of AtGTR1 and AtGTR2 for different glucosinolate substrates, sinigrin, gluconapin and sinalbin. Results Several clones expressing each of AtGTR1 and AtGTR2 were obtained showing high level of GTR expression and were maintained through regular sub-culturing. Both AtGTR1 and AtGTR2 are predominantly plasma-localized proteins when overexpressed in CCL-1 cells. Uptake assays were standardized, suggesting that glucosinolate uptake of GTR expressing CCL-1 is robust within the physiological pH range 5–6, and at lower concentration of nitrate salts. GTR expressing CCL-1 cells show increasing glucosinolate accumulation in time course experiment. Kinetic studies over a wide glucosinolate concentrations (10–800 µM) revealed that our novel assay system displayed robust GTR-mediated uptake of different glucosinolates and unambiguously helps elucidate the saturable kinetics of GTRs. Our system confirms the high affinity of AtGTRs for both aliphatic and aromatic glucosinolates. Conclusion The transporter assay system described in this study holds potential for studying sub-functionalization amongst GTR homologs present across Brassicaceae family. The fast growing CCL-1 cells, confer the benefits of an in vitro system for quick assays and is plant based thus enabling optimal expression without sequence modifications. The efficient functioning of the GTR transporters in the heterologous CCL-1 opens the possibility of using this plant cell suspension system for functional characterization of other metabolite transporters.

Glucosinolates (GSLs) are sulfur-containing defense metabolites produced in the Brassicales, including the model plant Arabidopsis (Arabidopsis thaliana). Previous work suggests that specific GSLs may function as signals to provide direct feedback regulation within the plant to calibrate defense and growth. These GSLs include allyl-GSL, a defense metabolite that is one of the most widespread GSLs in Brassicaceae and has also been associated with growth inhibition. Here we show that at least three separate potential catabolic products of allyl-GSL or closely related compounds affect growth and development by altering different mechanisms influencing plant development. Two of the catabolites, raphanusamic acid and 3-butenoic acid, differentially affect processes downstream of the auxin signaling cascade. Another catabolite, acrylic acid, affects meristem development by influencing the progression of the cell cycle. These independent signaling events propagated by the different catabolites enable the plant to execute a specific response that is optimal to any given environment.Allyl-glucosinolate and its catabolites use multiple mechanisms to affect plant growth and development through specific responses that are optimal to any given environment.

Gln is a key player in plant metabolism. It is one of the major free amino acids that is transported into the developing seed and is central for nitrogen metabolism. However, Gln natural variation and its regulation and interaction with other metabolic processes in seeds remain poorly understood. To investigate the latter, we performed a metabolic genome-wide association study (mGWAS) of Gln-related traits measured from the dry seeds of the Arabidopsis (Arabidopsis thaliana) diversity panel using all potential ratios between Gln and the other members of the Glu family as traits. This semicombinatorial approach yielded multiple candidate genes that, upon further analysis, revealed an unexpected association between the aliphatic glucosinolates (GLS) and the Gln-related traits. This finding was confirmed by an independent quantitative trait loci mapping and statistical analysis of the relationships between the Gln-related traits and the presence of specific GLS in seeds. Moreover, an analysis of Arabidopsis mutants lacking GLS showed an extensive seed-specific impact on Gln levels and composition that manifested early in seed development. The elimination of GLS in seeds was associated with a large effect on seed nitrogen and sulfur homeostasis, which conceivably led to the Gln response. This finding indicates that both Gln and GLS play key roles in shaping the seed metabolic homeostasis. It also implies that select secondary metabolites might have key functions in primary seed metabolism. Finally, our study shows that an mGWAS performed on dry seeds can uncover key metabolic interactions that occur early in seed development.

MEAM1 (Middle East-Asia Minor 1, "B" biotype) and MED (Mediterranean, "Q" biotype) are the two most destructive cryptic species of the Bemisia tabaci complex on the planet. Our previous studies have shown that MEAM1 outcompetes MED on cabbage; the underlying mechanism is unknown. In the Brassicaceae family, the glucosinolate-myrosinase defense system plays a crucial role in deterring feeding, inhibiting growth, and causing acute toxicity against a wide range of generalist herbivores. In the present study, we first compared the survival of MEAM1 and MED exposed to sinigrin (a glucosinolate) and myrosinase (an enzyme that degrades glucosinolates); we found that survival of both species was high in response to sinigrin alone but was near zero in response to sinigrin + myrosinase. We then used electropenetrography (electrical penetration graphs, EPG) to assess the feeding behaviors of MEAM1 and MED whiteflies on cabbage. The EPG results revealed that the mean duration of each potential drop (pd, indicating an intracellular puncture) was substantially longer for MED than MEAM1 on cabbage, indicating that the exposure to the toxic hydrolysates of glucosinolate and myrosinase is greater for MED than for MEAM1. We therefore conclude that differences in penetrating behaviors may help explain the different effects of cabbage on MEAM1 and MED whitefly species.

Amines synthesized by plants may be considered a dietary source of bioactive compounds, which are of interest due to possible health promoting effects. Developing Sinapis alba sprouts are known to produce 4-hydroxybenzylamine, but the reaction mechanism has not yet been established. We propose here a suggested metabolic pathway for the formation of 4-hydroxybenzylamine in S. alba plants. The catabolic sequence starts with a reaction between l-glutamine (Gln) as ammonia donor and 4-hydroxybenzyl carbocation, the enzymatic catalyzed hydrolysis product from sinalbin (4-hydroxybenzylglucosinolate). The suggested reactions are compared with alternative plant metabolic reactions used in the biosynthesis of biogenic amines.

Sulfur (S) is an essential element for plants, and S deficiency causes severe growth retardation. Although the catabolic process of glucosinolates (GSLs), the major S-containing metabolites specific to Brassicales including Arabidopsis, has been recognized as one of the S deficiency (−S) responses in plants, the physiological function of this metabolic process is not clear. Two β-glucosidases (BGLUs), BGLU28 and BGLU30, are assumed to be responsible for this catabolic process as their transcript levels were highly upregulated by −S. To clarify the physiological function of BGLU28 and BGLU30 and their roles in GSL catabolism, we analyzed the accumulation of GSLs and other S-containing compounds in the single and double mutant lines of BGLU28 and BGLU30 and in wild-type plants under different S conditions. GSL levels were highly increased, while the levels of sulfate, cysteine, glutathione and protein were decreased in the double mutant line of BGLU28 and BGLU30 (bglu28/30) under −S. Furthermore, transcript level of Sulfate Transporter1;2, the main contributor of sulfate uptake from the environment, was increased in bglu28/30 mutants under −S. With these metabolic and transcriptional changes, bglu28/30 mutants displayed obvious growth retardation under −S. Overall, our results indicate that BGLU28 and BGLU30 are required for −S-induced GSL catabolism and contribute to sustained plant growth under −S by recycling sulfate to primary S metabolism.

This study was conducted to determine the nutritional composition [acid detergent fibre (ADF), neutral detergent fibre (NDF), ether extract (EE) and crude protein (CP)], digestible energy (DE) and metabolizable energy (ME) of rapeseed meals (RSM) differing in glucosinolates (GLS) contents fed to growing pigs. One hundred and thirty-eight crossbred barrows with initial body weight (BW) of 39.0 ± 1.9 kg were allotted to 1 of 23 diets. Results showed that the content of GLS in RSM ranged from 1.72 to 62.94 mol/g DM with a coefficient of variation (CV) of 89%. The concentration of ADF ranged from 22.78 to 35.71% with a mean of 28.79% [DM (CV = 16.22%)]. The concentration of ether extract (EE) ranged from 1.42% to 14.55% with a mean of 7.78% [DM (CV = 45.16%)]. DE and ME values of the 22 RSM ranged from 10.10 to 15.42 MJ/kg DM and 9.09 to 14.94 MJ/kg DM (P

While the effects of phytohormones on plant gene expression have been well characterized, comparatively little is known about how hormones influence metabolite profiles. This study examined the effects of elevated auxin and ethylene on the metabolome of Arabidopsis roots using a high-resolution 24 h time course, conducted in parallel to time-matched transcriptomic analyses. Mass spectrometry using orthogonal UPLC separation strategies (reversed phase and HILIC) in both positive and negative ionization modes was used to maximize identification of metabolites with altered levels. The findings show that the root metabolome responds rapidly to hormone stimulus and that compounds belonging to the same class of metabolites exhibit similar changes. The responses were dominated by changes in phenylpropanoid, glucosinolate, and fatty acid metabolism, although the nature and timing of the response was unique for each hormone. These alterations in the metabolome were not directly predicted by the corresponding transcriptome data, suggesting that post-transcriptional events such as changes in enzyme activity and/or transport processes drove the observed changes in the metabolome. These findings underscore the need to better understand the biochemical mechanisms underlying the temporal reconfiguration of plant metabolism, especially in relation to the hormone-metabolome interface and its subsequent physiological and morphological effects.

The oilseed crops Crambe abyssinica and Camelina sativa produce oils rich in erucic acid and n-3 polyunsaturated fatty acids (PUFA), respectively. After pressing the oil, a seed cake remains as a protein-rich by-product. Edible insects may convert this seed press cake and the defatted seed meal produced from it into insect biomass suitable for animal feed. Black soldier fly larvae (BSF, Hermetia illucens) can grow on a wide range of organic waste types, but may be hindered by excess protein or the plant toxins characteristic for these two oilseed crops, i.e. glucosinolates and their breakdown products. We tested the effects of 25, 50 and 100% oilseed by-product inclusion in the diet on survival, development, biomass production and fatty acid composition of BSF larvae. Larval performance on diets with up to 50% camelina by-product or 25% crambe by-product was similar to performance on control diet (chicken feed), and decreased with higher inclusion percentages. Larval fatty acid profiles differed significantly among diets, with larvae fed press cake more distinct from control than larvae fed seed meal. Larvae fed camelina press cake had more α-linolenic acid, whereas larvae fed crambe contained most oleic acid. The n-6:n-3 PUFA ratio decreased with increasing proportion of by-product, especially on camelina diets. Lauric acid content was highest in larvae fed 100% camelina meal or 50% crambe meal. These results indicate that BSF larvae can be successfully grown on diets with camelina or crambe oilseed by-products, and that the resulting larval n-6:n-3 PUFA ratio is favourable for animal feed. However, the fate of glucosinolates and their derivatives remains to be determined, to guarantee chemical safety of camelina- or crambe-fed BSF larvae for animal feed.

Evolution of important glucosinolates (GLSs), namely, sinigrin, glucoraphanin, glucoerucin and glucobrassicin, in three commonly consumed Brassica vegetables viz. white cabbage, Chinese cabbage and bok choy during their processing into vegetable powder was investigated. Drying was noted to be a major processing step causing significant losses of GLSs. Interestingly, different GLSs and even the same GLSs in different vegetables showed different thermal stabilities during drying. The stability of GLSs in vegetable powder during in vitro gastric digestion was also studied. Glucoraphanin exhibited the highest stability while glucobrassicin was the most vulnerable GLS under in vitro gastric conditions. White cabbage is found to be a promising material for the production of vegetable powder as it contains high contents of GLSs, especially glucoraphanin and glucoerucin, which are important precursors of anticarcinogenic compounds, namely sulforaphane and erucin. These two GLSs were also noted to be stable during in vitro gastric digestion.

Current research was conducted to explore the effects of liquiritoside on the growth and physiochemical features of Chinese flowering cabbage (Brassica rapa subsp. parachinensis) under lead (Pb) stress. Lead stressed B. rapa plants exhibited decreased growth parameters, chlorophyll, and carotenoid contents. Moreover, Pb toxicity escalated the synthesis of malondialdehyde (MDA), hydrogen peroxide (H2O2), flavonoids, phenolics, and proline in treated plants. Nevertheless, foliar application of liquiritoside mitigated Pb toxicity by decreasing oxidative stress by reducing cysteine, H2O2, and MDA contents in applied plants. Liquiritoside significantly increased plant height, shoot fresh weight and dry weight, number of leaves, and marketable value of Chinese flowering cabbage plants exposed to Pb toxicity. This biotic elicitor also enhanced the proline, glutathione, total phenolics, and flavonoid contents in Chinese flowering cabbage plants exposed to Pb stress compared with the control. Additionally, total glucosinolate content, phytochelatins (PCs), and non-protein thiols were effectively increased in plants grown under Pb regimes compared with the control plants. Overall, foliar application of liquiritoside can markedly alleviate Pb stress by restricting Pb translocation in Chinese flowering cabbage.

Indole-3-acetaldoxime (IAOx) and phenylacetaldoxime (PAOx) are precursors for the growth hormones indole-3-acetic acid (IAA) and phenylacetic acid (PAA) and the defense compounds glucosinolates in Brassicales. Our recent work has shown that Arabidopsis transgenic lines overexpressing AtCYP79A2, a PAOx-production enzyme, accumulate the PAOx-derived compounds benzyl glucosinolate and PAA. Here we report that they also accumulate the benzyl glucosinolate hydrolysis products benzyl isothiocyanate and benzyl cyanide, which indicates that the turnover of benzyl glucosinolate can occur in intact tissues. Myrosinases or β-glucosidases are known to catalyze glucosinolate breakdown. However, transcriptomics analysis detected no substantial increase in expression of known myrosinases or putative β-glucosidases in AtCYP79A2 overexpressing lines. It was previously shown that accumulation of aldoximes or their derivatives represses the phenylpropanoid pathway. For instance, ref2 mutant having a defect in one of the aldoxime catabolic enzymes decreases phenylpropanoid production. Considering that AtCYP79A2 is not expressed in most organs under optimal growth condition, ref2 accumulates aliphatic aldoximes but not PAOx. Interestingly, overexpression of AtCYP79A2 in ref2 resulted in a further decrease in sinapoylmalate content compared to ref2. This indicates that accumulation of PAOx has an additive effect on phenylpropanoid pathway suppression mediated by other aldoximes.

Background To understand the mechanism of glucosinolates (GSs) accumulation in the specific organs, combined analysis of physiological change and transcriptome sequencing were applied in the current study. Taking Chinese kale as material, seeds and silique walls were divided into different stages based on the development of the embryo in seeds and then subjected to GS analysis and transcriptome sequencing. Results The main GS in seeds of Chinese kale were glucoiberin and gluconapin and their content changed with the development of the seed. During the transition of the embryo from torpedo- to the early cotyledonary-embryo stage, the accumulation of GS in the seed was accompanied by the salient decline of GS in the corresponding silique wall. Thus, the seed and corresponding silique wall at these two stages were subjected to transcriptomic sequencing analysis. 135 genes related to GS metabolism were identified, of which 24 genes were transcription factors, 81 genes were related to biosynthetic pathway, 25 genes encoded catabolic enzymes, and 5 genes matched with transporters. The expression of GS biosynthetic genes was detected both in seeds and silique walls. The high expression of FMOGS-OX and AOP2, which is related to the production of gluconapin by side modification, was noted in seeds at both stages. Interestingly, the expression of GS biosynthetic genes was higher in the silique wall compared with that in the seed albeit lower content of GS existed in the silique wall than in the seed. Combined with the higher expression of transporter genes GTRs in silique walls than in seeds, it was proposed that the transportation of GS from the silique wall to the seed is an important source for seed GS accumulation. In addition, genes related to GS degradation expressed abundantly in the seed at the early cotyledonary-embryo stage indicating its potential role in balancing seed GS content. Conclusions Two stages including the torpedo-embryo and the early cotyledonary-embryo stage were identified as crucial in GS accumulation during seed development. Moreover, we confirmed the transportation of GS from the silique wall to the seed and proposed possible sidechain modification of GS biosynthesis may exist during seed formation.

Mustard plants have been widely cultivated and used as spice, medicine and as source of edible oils. Currently, the use of the seeds of the mustard species Sinapis alba (white mustard or yellow mustard), Brassica juncea (brown mustard) and Brassica nigra (black mustard) in the food and beverage industry is immensely growing due to their nutritional and functional properties. The seeds serve as a source for a wide range of biologically active components including isothiocyanates that are responsible for the specific flavor of mustard, and tend to reveal conflicting results regarding possible health effects. Other potentially undesirable or toxic compounds, such as bisphenol F, erucic acid or allergens, may also occur in the seeds and in mustard products intended for human consumption. The aim of this article is to provide comprehensive information about potentially harmful compounds in mustard seeds and to evaluate potential health risks as an increasing use of mustard seeds is expected in the upcoming years.

Glucosinolates (GSLs) from Lepidium graminifolium L. were analyzed qualitatively and quantitatively by their desulfo-counterparts using UHPLC-DAD-MS/MS technique and by their volatile breakdown products-isothiocyanates (ITCs) using GC-MS analysis. Thirteen GSLs were identified with arylaliphatic as the major ones in the following order: 3-hydroxybenzyl GSL (glucolepigramin, 7), benzyl GSL (glucotropaeolin, 9), 3,4,5-trimethoxybenzyl GSL (11), 3-methoxybenzyl GSL (glucolimnanthin, 12), 4-hydroxy-3,5-dimethoxybenzyl GSL (3,5-dimethoxysinalbin, 8), 4-hydroxybenzyl GSL (glucosinalbin, 6), 3,4-dimethoxybenzyl GSL (10) and 2-phenylethyl GSL (gluconasturtiin, 13). GSL breakdown products obtained by hydrodistillation (HD) and CH2Cl2 extraction after hydrolysis by myrosinase for 24 h (EXT) as well as benzyl ITC were tested for their cytotoxic activity using MTT assay. Generally, EXT showed noticeable antiproliferative activity against human bladder cancer cell line UM-UC-3 and human glioblastoma cell line LN229, and can be considered as moderately active, while IC50 of benzyl ITC was 12.3 μg/mL, which can be considered as highly active.