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Rapeseed cake is a protein-rich by-product from the rapeseed oil extraction process. It contains high levels of glucosinolates (GSLs), which possess anti-nutritional functions after being hydrolyzed. Moreover, the hydrolyzed products of GSLs produce an extremely bitter taste. These adverse properties hinder the use of seedcake for human consumption and restrict its use in livestock feed manufacturing. The GSLs, especially aliphatic GSLs, significantly contribute to importing adverse properties compared to aromatic and indole GSLs. Thus, this study focused on partially down-regulating the aliphatic GSL biosynthesis by inducing targeted mutagenesis in the MYB28 transcription factor genes using CRISPR/Cas9. Six out of seven MYB28 paralogs were identified in the cv. Kumily genome of rapeseed and two sgRNAs were designed at the transcriptional regions for both MYB-type HTH domains of MYB28. The ribonucleoprotein (RNP), in which sgRNAs and Cas9 are mixed as a complex, was introduced into the rapeseed protoplasts. The transfected protoplasts were maintained for callus and shoot formation under in vitro conditions. For screening of mutation lines, genomic DNA was extracted from the true leaves of three regenerated shoots and seven randomly selected callus samples. The sgRNA target sites of five paralogs were amplified using gene-specific primers and screened for the presence of mutations. Screening results showed a single nucleotide insertion in one MYB28 paralog, LOC106428039, in one callus. This confirms that at least one selected sgRNA was successful inducing mutation in MYB28 using the RNA-based CRISPR-edited method.

Greenhouse gases are a driving force of climate change and the annual greenhouse gas emissions were higher between 2010-2019 than any other time in human history (Skea et. al. 2022). Cover crops are used to mitigate the effect of climate change, but recent studies indicate that the use of the cover crop oilseed radish results in significantly larger emissions of nitrous oxide (N2O) compared to other cover crops (Dörsch et. al. 2022; Müller Júnior et. al. 2019; Olofsson and Ernfors 2022; Thomas et. al. 2017). Results from Olofsson and Ernfors (2022) study showed that Raphanus sativus var oleiformis (OR) causes significantly higher emission of N2O compared to Phacelia tanacetifolia (PH) even though the quality and quantity were similar. The hypothesis in this study was that the glucosinolates in OR provides a carbon source for denitrifying bacteria, thus causing a significantly higher N2O emission compared to PH. The hypothesis was tested in a laboratory setting during 32 days of gas measurement with plant material of OR and PH incubated with and without added glucose. Surprisingly, aboveground plant material of PH showed highest emissions throughout the study. The result of this study could not confirm the hypothesis since added glucose did not affect the N2O emission. In further studies, it is recommended to increase the number of replicates and optimize the methodology to be able to draw any conclusions.

Neonicotinoids, systemic insecticides that are distributed into all plant tissues and protect against pests, have become a common part of crop production, but can unintentionally also affect non-target organisms, including pollinators. Such effects can be direct effects from insecticide exposure, but neonicotinoids can affect plant physiology, and effects could therefore also be indirectly mediated by changes in plant phenology, attractiveness and nutritional value. Under controlled greenhouse conditions, we tested if seed treatment with the neonicotinoid clothianidin affected oilseed rape’s production of flower resources for bees and the content of the secondary plant products glucosinolates that provide defense against herbivores. Additionally, we tested if seed treatment affected the attractiveness of oilseed rape to flower visiting bumblebees, using outdoor mesocosms. Flowers and leaves of clothianidin-treated plants had different profiles of glucosinolates compared with untreated plants. Bumblebees in mesocosms foraged slightly more on untreated plants. Neither flower timing, flower size nor the production of pollen and nectar differed between treatments, and therefore cannot explain any preference for untreated oilseed rape. We instead propose that this small but significant preference for untreated plants was related to the altered glucosinolate profile caused by clothianidin. Thereby, this study contributes to the understanding of the complex relationships between neonicotinoid-treated crops and pollinator foraging choices, by suggesting a potential mechanistic link by which insecticide treatment can affect insect behavior., Funding was provided by the Royal Physiographic Society in Lund and The Swedish Research Council for Sustainable Development FORMAS (grant 2016-00632). Annemarie Wurz was supported by the German Academic Exchange Service (DAAD) within the PROMOS initiative. BdLV was funded by the Science without Borders program at Lund University, sponsored by the Capes Foundation within the Ministry of Education, Brazil. Open access funding provided by Lund University., Evaluation and balance of combined effects from insecticides and climate change on bees and pollination

Neonicotinoids, systemic insecticides that are distributed into all plant tissues and protect against pests, have become a common part of crop production, but can unintentionally also affect non-target organisms, including pollinators. Such effects can be direct effects from insecticide exposure, but neonicotinoids can affect plant physiology, and effects could therefore also be indirectly mediated by changes in plant phenology, attractiveness and nutritional value. Under controlled greenhouse conditions, we tested if seed treatment with the neonicotinoid clothianidin affected oilseed rape’s production of flower resources for bees and the content of the secondary plant products glucosinolates that provide defense against herbivores. Additionally, we tested if seed treatment affected the attractiveness of oilseed rape to flower visiting bumblebees, using outdoor mesocosms. Flowers and leaves of clothianidin-treated plants had different profiles of glucosinolates compared with untreated plants. Bumblebees in mesocosms foraged slightly more on untreated plants. Neither flower timing, flower size nor the production of pollen and nectar differed between treatments, and therefore cannot explain any preference for untreated oilseed rape. We instead propose that this small but significant preference for untreated plants was related to the altered glucosinolate profile caused by clothianidin. Thereby, this study contributes to the understanding of the complex relationships between neonicotinoid-treated crops and pollinator foraging choices, by suggesting a potential mechanistic link by which insecticide treatment can affect insect behavior., Funding was provided by the Royal Physiographic Society in Lund and The Swedish Research Council for Sustainable Development FORMAS (grant 2016-00632). Annemarie Wurz was supported by the German Academic Exchange Service (DAAD) within the PROMOS initiative. BdLV was funded by the Science without Borders program at Lund University, sponsored by the Capes Foundation within the Ministry of Education, Brazil. Open access funding provided by Lund University., Evaluation and balance of combined effects from insecticides and climate change on bees and pollination

Raphanus sativus L. (Brassicaceae), commonly known as radish, is consumed worldwide as a vegetable. However, its benefits on mental health are unknown. The aim of this study was to evaluate its anxiolytic-like effects and safety using different experimental models. An aqueous extract of R. sativus sprouts (AERSS) was pharmacologically evaluated by intraperitoneal route (i.p.) at 10, 30, and 100 mg/kg and orally (p.o.) at 500 mg/kg on behavior by using open-field and plus-maze tests. In addition, its acute toxicity (LD50) was determined by the Lorke's method. Diazepam (1 mg/kg, i.p.) and buspirone (4 mg/kg, i.p.) were the reference drugs. A significant and anxiolytic-like dosage of AERSS (30 mg/kg, i.p.) resembling the effects of reference drugs was chosen to explore the involvement of GABAA/BDZs site (flumazenil, 5 mg/kg, i.p.) and serotonin 5-HT1A receptors (WAY100635, 1 mg/kg, i.p.) as a possible mechanism of action. A 500 mg/kg, p.o. dosage of AERSS produced an anxiolytic-like response equivalent to 100 mg/kg, i.p. No acute toxicity was observed since a LD50 > 2000 mg/kg, i.p. The phytochemical analysis allowed the identification and quantification of major presence of sulforaphene (2500 µM), sulforaphane (15 µM), iberin (0.75 µM), and indol-3-carbinol (0.75 µM), as major constituents. Both the GABAA/BDZs site and serotonin 5-HT1A receptors were involved in the anxiolytic-like activity of AERSS, depending on the pharmacological parameter or the experimental assay tested. Our results demonstrate that the anxiolytic activity of R. sativus sprouts involves GABAA/BDZs site and serotonin 5-HT1A receptors supporting its health benefits in the treatment of anxiety beyond the satisfaction of basic nutritional needs

In the search for new alternatives to avoid the problems associated with the use of synthetic chemical fungicides in agriculture, the use of green manure (GrM) could help combat fungal diseases of crops, such as those produced by the necrotrophic pathogen Rhizoctonia solani. In the case of the use of Brassica tissues as GrM, it could have an elicitor capacity for systemic plant resistance.

This data set underlies the study that aimed to describe bacterial, fungal and nematode diversity in soil associated with wild cabbage plants (Brassica oleracea) from five different population growing naturally along the Dorset coast in the UK. Samples were taken from bulk soil, rhizosphere soil and the roots of cabbage plants in 2011 and 2012. Nematode composition and abundance was determined in bulk soil, rhizosphere and roots. Presence/absence of fungal and bacterial OTU's was determined only in rhizosphere soil in 2011., This data set underlies the study that aimed to describe bacterial, fungal and nematode diversity in soil associated with wild cabbage plants (Brassica oleracea) from five different population growing naturally along the Dorset coast in the UK. Samples were taken from bulk soil, rhizosphere soil and the roots of cabbage plants in 2011 and 2012. Nematode composition and abundance was determined in bulk soil, rhizosphere and roots. Presence/absence of fungal and bacterial OTU's was determined only in rhizosphere soil in 2011.

Neonicotinoids, systemic insecticides that are distributed into all plant tissues and protect against pests, have become a common part of crop production, but can unintentionally also affect non-target organisms, including pollinators. Such effects can be direct effects from insecticide exposure, but neonicotinoids can affect plant physiology, and effects could therefore also be indirectly mediated by changes in plant phenology, attractiveness and nutritional value. Under controlled greenhouse conditions, we tested if seed treatment with the neonicotinoid clothianidin affected oilseed rape’s production of flower resources for bees and the content of the secondary plant products glucosinolates that provide defense against herbivores. Additionally, we tested if seed treatment affected the attractiveness of oilseed rape to flower visiting bumblebees, using outdoor mesocosms. Flowers and leaves of clothianidin-treated plants had different profiles of glucosinolates compared with untreated plants. Bumblebees in mesocosms foraged slightly more on untreated plants. Neither flower timing, flower size nor the production of pollen and nectar differed between treatments, and therefore cannot explain any preference for untreated oilseed rape. We instead propose that this small but significant preference for untreated plants was related to the altered glucosinolate profile caused by clothianidin. Thereby, this study contributes to the understanding of the complex relationships between neonicotinoid-treated crops and pollinator foraging choices, by suggesting a potential mechanistic link by which insecticide treatment can affect insect behavior., Funding was provided by the Royal Physiographic Society in Lund and The Swedish Research Council for Sustainable Development FORMAS (grant 2016-00632). Annemarie Wurz was supported by the German Academic Exchange Service (DAAD) within the PROMOS initiative. BdLV was funded by the Science without Borders program at Lund University, sponsored by the Capes Foundation within the Ministry of Education, Brazil. Open access funding provided by Lund University., Evaluation and balance of combined effects from insecticides and climate change on bees and pollination

Determining plant responses to hazardous air pollutants is critical in predicting food security programs and challenges in the future. This study aimed to determine the effects of various ozone levels on plant growth responses (leaf area, dry matter, and number of leaves) and biochemical quality (photopigments and glucosinolates) on Brassica campestris L. ssp. chinensis (Pak-Choi). The experiment was conducted within test chambers under different ozone concentrations (60, 150, and 240 ppb for 2 h/day). Leaf area and dry matter were negatively correlated with increasing ozone concentrations, but the number of leaves was not affected by ozone treatment. Lycopene and chlorophylls also showed the same tendency. Even if the ambient ozone concentration was only elevated for a short time, various glucosnilates (GLS) have been diversely affected. The total aliphatic GLS content was reduced. In contrast, the total indole GLS increased at the highest ozone concentration, and the aromatic GLS significantly increased and then decreased as the ozone concentration level increased. These results provide evidence of the strong effect of ozone stress on the plant quality of Pak-Choi with respect to certain secondary plant metabolites. These findings provide an understanding of elevated ozone effects in urban horticulture sites on the growth and metabolite profiling of Brassica plants., Peer Reviewed

Plants exhibit significant variation in morphological and chemical traits of shoots and roots in response to an array of biotic and abiotic selection pressures, and this variation in turn affects their interactions with the biotic and abiotic environment. Thus far, most studies examining these interactions have focused on the aboveground domain, which is easier to study than the belowground domain. However, soil organisms significantly affect plant fitness directly through mutualisms e.g. growth promotion, or antagonisms e.g. herbivory and disease. Natural populations of wild Brassica oleracea L. growing along the south coastline of Great Britain exhibit significant differences in growth form and secondary chemistry. Studies in the field have shown that these differences affect aboveground plant-insect interactions, whereas soil communities have not been explored. We sampled belowground communities of nematodes, bacteria and fungi associated with roots, rhizosphere and bulk soil in five coastal wild cabbage populations in Dorset, England, and found significant differences among these communities. Site-related differences in nematode community composition were primarily found for nematodes in bulk soil and were consistent over two years of sampling. Nematode communities in roots of wild cabbage did not significantly differ across the cabbage populations but did differ between the two years. Results for communities in rhizosphere soil were spatially and temporally variable. The composition of nematode communities in cabbage roots differed strongly from those in the rhizosphere and bulk soil, showing that plants attract a subset of nematodes from the bulk soil community. For microbes, we analysed only rhizosphere samples, and found that fungal communities differed more strongly among plant populations than bacterial communities. Thus, while there is spatio-temporal variation in belowground communities, soil and/or plant properties differentially affect the assembly of

Recent discoveries pointed out the importance of the mutual correlation between timing of environmental stress and plant fitness. However, the internal reshaping of plant growth under daily stress sensing, and their metabolic coordination remain to be investigated. Thus, we studied the connection between time of day, growth and defence to understand how plant fitness is affected by diurnal stress inputs. We examined if simulated herbivory (leaf wounding) in the morning, at midday or the evening differentially influence plant defence state vs growth in three crop species of Brassica: broccoli (Brassica oleraceae), turnip greens (B. rapa) and rapeseed (B. napus). The data revealed that plant's tolerance of wounding stress is diurnally regulated in Brassica crops. Trade-offs between biomass and investment in glucosinolates (GSL) and phenolics were affected by timing of leaf stress. Negative correlations between biomass and induction of defence compounds were found for plants treeated in the morning and evening. However, the correlations were positive for midday treatment. Interestingly, we revealed a new connection between plant growth and changes in aliphatic GSLs and flavonoids in response to wounding. These data suggest that metabolic stress-dependent circadian oscillations in leaf defences could be one mechanism conferring a competitive advantage to plants to anticipate daily environmental variations by synchronizing them with growth. Moreover, this work provides first insights into how secondary metabolites are linked to growth response in a timing-related manner.

While the "farm to fork" strategy ticks many boxes in the sustainability agenda, it does not go far enough in addressing how we can improve crop nutraceutical quality. Here, we explored whether supplementary ultraviolet (UV) radiation exposure during growth of broccoli and Chinese cabbage can induce bioactive tryptophan- and glucosinolate-specific metabolite accumulation thereby enhancing Aryl hydrocarbon receptor (AhR) activation in human intestinal cells. By combining metabolomics analysis of both plant extracts and in vitro human colonic fermentation extracts with AhR reporter cell assay, we reveal that human colonic fermentation of UVB-exposed Chinese cabbage led to enhanced AhR activation in human intestinal cells by 23% compared to plants grown without supplementary UV. Thus, by exploring aspects beyond "from farm to fork", our study highlights a new strategy to enhance nutraceutical quality of Brassicaceae, while also providing new insights into the effects of cruciferous vegetables on human intestinal health., Funding agencies:German Academic Exchange Service Erasmus+programs

The present study was designed to evaluate the functional potential of common duckweed (Lemna minor L.) as a source of bioactive compounds of nutraceutical interest. The untargeted profiling of the bioactive components of common duckweed was carried out through ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS), in parallel with assessing in vitro antioxidant and enzymatic inhibition properties. The optimization of extraction parameters was determined using the response surface methodology (RSM) through a 3-factor central composite design. The process parameters included extraction temperature, % of ethanol, and ultrasound power, while the response variables were the phenolic content (considering each main phenolic class), total glucosinolates, total carotenoids, the antioxidant potential, and enzyme inhibition activities. The results revealed that common duckweed was a rich source of carotenoids and total flavonoids (mainly flavones and flavonols), followed by phenolic acids, low-molecular-weight phenolics, and glucosinolates. Interestingly, the total flavones, total flavonols and total carotenoid equivalents showed the highest and most positive correlation values with the bioactive properties measured. Finally, the combined RSM approach and unsupervised statistics allowed us to point out the pivotal impact of ethanol percentage in the extraction solvent to recover the highest amounts of bioactive compounds efficiently.

Producción Científica, Bee pollen is currently one of the most commonly consumed food supplements, as it is considered to be a good source of bioactive substances and energy. It contains various health-promoting compounds, such as proteins, amino acids, lipids, as well as glucosinolates. In the present study, the glucosinolate content was determined, by means of ultra-performance liquid chromatography coupled to a quadrupole time-of-flight mass detector, in 72 bee pollen samples from four different apiaries in Guadalajara (Spain), harvested in three different periods. In addition, 11 commercial multifloral samples from different Spanish regions were also analyzed. The aim was to verify the suitability of these compounds as biomarkers of their geographical origin, and to test their potential for distinguishing the harvesting period. By means of a canonical discriminant analysis, it was possible to differentiate the apiary of origin of most of the samples, and these could also be clearly differentiated from the commercial ones, simply as a result of the glucosinolate content. In addition, it was also demonstrated for the first time that bee pollen samples were capable of being differentiated according to the time of harvesting and their glucosinolate content., Ministerio de Economía y Competitividad e Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria - (projects RTA 2015-00013-C03-01 and 03)

Watercress (Nasturtium officinale R. Br.) is a highly nutritious leafy crop with a rich health-related phytonutrient profile including secondary plant metabolites such as glucosinolates. This semi-aquatic crop is well-suited for indoor hydroponic cultivation and has a growing market for its distinctive peppery taste and health benefits. We describe crop trials in three contrasting environments and report the changes to plant morphology, yield, and nutritional profile under varying blue light treatments. Yield and glucosinolate quantity were significantly increased in a vertical farm system enriched in blue light, relative to field-grown crops. Additionally, PBITC, a glucosinolate not previously identified in field-grown watercress, was found in plants grown in a vertical farm. This work demonstrates the great potential of watercress in a new era of controlled environment agriculture to deliver improved health benefits to customers.

The receptor kinase FERONIA (FER) is a versatile regulator of plant growth and development, biotic and abiotic stress responses, and reproduction. To gain new insights into the molecular interplay of these processes and to identify new FER functions, we carried out quantitative transcriptome, proteome, and phosphoproteome profiling of Arabidopsis (Arabidopsis thaliana) wild-type and fer-4 loss-of-function mutant plants. Gene ontology terms for phytohormone signaling, abiotic stress, and biotic stress were significantly enriched among differentially expressed transcripts, differentially abundant proteins, and/or misphosphorylated proteins, in agreement with the known roles for FER in these processes. Analysis of multiomics data and subsequent experimental evidence revealed previously unknown functions for FER in endoplasmic reticulum (ER) body formation and glucosinolate biosynthesis. FER functions through the transcription factor NAI1 to mediate ER body formation. FER also negatively regulates indole glucosinolate biosynthesis, partially through NAI1. Furthermore, we found that a group of abscisic acid (ABA)-induced transcription factors is hypophosphorylated in the fer-4 mutant and demonstrated that FER acts through the transcription factor ABA INSENSITIVE5 (ABI5) to negatively regulate the ABA response during cotyledon greening. Our integrated omics study, therefore, reveals novel functions for FER and provides new insights into the underlying mechanisms of FER function.

The synthesis of indole-3-acetonitrile (IAN) from the indolic glucosinolate (iGSL) glucobrassicin (GB) is a unique trait of members of the Brassicales. To assess the contribution of this pathway to indole-3-acetic acid (IAA) synthesis under stress conditions, drought stress (DS) experiments with Arabidopsis thaliana were performed in vitro. Analysis of GSLs in DS plants revealed higher contents of GB in shoots and roots compared to control plants. Deuterium incorporation experiments showed the highest turnover of GB compared to all other GSLs during drought conditions. Evidence suggests the involvement of the thioglucosidase BGLU18 in the degradation of GB. The nitrile specifier proteins NSP1 and NSP5 are known to direct the GSL hydrolysis towards formation of IAN. Nitrilases like NIT2 are able to subsequently synthesize IAA from IAN. Expression of BGLU18, NSP1, NSP5 and NIT2 and contents of GB, IAN and IAA were significantly elevated in DS plants compared to control plants suggesting the increased use of GB as IAA source. Significantly higher contents of reactive oxygen species in DS bglu18 and epithionitrile specifier protein (esp) mutants compared to Col-0 indicate higher stress levels in these mutants highlighting the need for both proteins in DS plants. Furthermore, GB accumulation in leaves was higher in both mutants during DS when compared to Col-0 indicating enhanced synthesis of GB due to a lack of breakdown products. This work provides the first evidence for the breakdown of iGSLs to IAN which seems to be used for synthesis of IAA in DS A. thaliana plants.

The use of plant biostimulants such as seaweed extracts (SWE) and protein hydrolysates (PH) has grown in the recent years due to their beneficial effects on yield under both optimal and sub-optimal conditions such as salt stress. The comprehension of the mode of action of these two important categories of biostimulants on plant performance will allow to use them more efficiently under different growth conditions. This study aimed to examine the efficacy of a seaweed and plant-based biostimulants on greenhouse lettuce (Lactuca sativa L.) grown under non-saline (0 mM NaCl) and saline conditions (40 mM NaCl) in terms of growth, yield, SPAD index, leaf mineral composition and metabolomic profiling. Shoot fresh weight of lettuce was reduced by 15.3% under 40 mM of NaCl. Nonetheless, it was boosted by both used biostimulants by 9 and 18%, on average, under 0 and 40 mM NaCl salinity, respectively. Na content under saline conditions was reduced in the presence of the biostimulants treatment, where PH reduced it significantly by 15.6% and SWE by 9.4%. On the other hand, Cl content was significantly reduced only under PH treatment. Both biostimulants elicited a broad metabolic reprogramming, involving the accumulation of stress-related compounds such as glucosinolates, terpenoid phytoalexins, and jasmonates. Interestingly, distinctive metabolomic signatures could be observed following the application of the different biostimulants under salinity conditions. In more detail, PH promoted the accumulation of glucosinolates and phytoalexins precursors, while SWE induced a down accumulation of secondary metabolites. Our findings indicate different processes being modulated by PH and SWE, with possible synergistic effects, thus paving the way towards integrated strategies to alleviate the detrimental effects of salinity in lettuce.

Spices, widely used to improve the sensory characteristics of food, contain several bioactive compounds as well, including polyphenols, carotenoids, and glucosynolates. Acting through multiple pathways, these bioactive molecules affect a wide variety of cellular processes involved in molecular mechanisms important in the onset and progress of human diseases. Capparis spinosa L. is an aromatic plant characteristic of the Mediterranean diet. Previous studies have reported that different parts (aerial parts, roots, and seeds) of C. spinosa exert various pharmacological activities. Flower buds of C. spinosa contain several bioactive compounds, including polyphenols and glucosinolates. Two different subspecies of C. spinosa L., namely, C. spinosa L. subsp. spinosa, and C. spinosa L. subsp. rupestris, have been reported. Few studies have been carried out in C. spinosa L. subsp. rupestris. The aim of our study was to investigate the phytochemical profile of floral buds of the less investigated species C. spinosa subsp. rupestris. Moreover, we investigated the effect of the extract from buds of C. spinosa subsp. rupestris (CSE) on cell proliferation, intracellular ROS levels, and expression of the antioxidant and anti-apoptotic enzyme paraoxonase-2 (PON2) in normal and cancer cells. T24 cells and Caco-2 cells were selected as models of advanced-stage human bladder cancer and human colorectal adenocarcinoma, respectively. The immortalized human urothelial cell line (UROtsa) and human dermal fibroblast (HuDe) were chosen as normal cell models. Through an untargeted metabolomic approach based on ultra-high-performance liquid chromatography quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS), our results demonstrate that C. spinosa subsp. rupestris flower buds contain polyphenols and glucosinolates able to exert a higher cytotoxic effect and higher intracellular reactive oxygen species (ROS) production in cancer cells compared to normal cells. Moreover, upregulation of

Glucoraphanin is a plant specialized metabolite found in cruciferous vegetables that has long been a target for production in a heterologous host because it can subsequently be hydrolyzed to form the chemopreventive compound sulforaphane before and during consumption. However, previous studies have only been able to produce small amounts of glucoraphanin in heterologous plant and microbial systems compared to the levels found in glucoraphanin-producing plants, suggesting that there may be missing auxiliary genes that play a role in improving production in planta. In an effort to identify auxiliary genes required for high glucoraphanin production, we leveraged transient expression in Nicotiana benthamiana to screen a combination of previously uncharacterized coexpressed genes and rationally selected genes alongside the glucoraphanin biosynthetic pathway. This strategy alleviated metabolic bottlenecks, which improved glucoraphanin production by 4.74-fold. Our optimized glucoraphanin biosynthetic pathway provides a pathway amenable for high glucoraphanin production.

Brassicaceae family includes an important group of plants of great scientific interest, e.g., the model plant Arabidopsis thaliana, and of economic interest, such as crops of the genus Brassica (Brassica oleracea, Brassica napus, Brassica rapa, etc.). This group of plants is characterized by the synthesis and accumulation in their tissues of secondary metabolites called glucosinolates (GSLs), sulfur-containing compounds mainly involved in plant defense against pathogens and pests. Brassicaceae plants are among the 30% of plant species that cannot establish optimal associations with mycorrhizal hosts (together with other plant families such as Proteaceae, Chenopodiaceae, and Caryophyllaceae), and GSLs could be involved in this evolutionary process of non-interaction. However, this group of plants can establish beneficial interactions with endophytic fungi, which requires a reduction of defensive responses by the host plant and/or an evasion, tolerance, or suppression of plant defenses by the fungus. Although much remains to be known about the mechanisms involved in the Brassicaceae-endophyte fungal interaction, several cases have been described, in which the fungi need to interfere with the GSL synthesis and hydrolysis in the host plant, or even directly degrade GSLs before they are hydrolyzed to antifungal isothiocyanates. Once the Brassicaceae-endophyte fungus symbiosis is formed, the host plant can obtain important benefits from an agricultural point of view, such as plant growth promotion and increase in yield and quality, increased tolerance to abiotic stresses, and direct and indirect control of plant pests and diseases. This review compiles the studies on the interaction between endophytic fungi and Brassicaceae plants, discussing the mechanisms involved in the success of the symbiosis, together with the benefits obtained by these plants. Due to their unique characteristics, the family Brassicaceae can be seen as a fruitful source of novel beneficial endophyte

Introductions of novel plant species can disturb the historical resource environment of herbivorous insects, resulting in strong selection to either adopt or exclude the novel host. However, an adaptive response depends on heritable genetic variation for preference or performance within the targeted herbivore population, and it is unclear how heritability of host-use preference may differ between novel and historical hosts. Pieris macdunnoughii butterflies in the Rocky Mountains lay eggs on the nonnative mustard Thlaspi arvense, which is lethal to their offspring. Heritability analyses revealed considerable sex-linked additive genetic variation in host preference within a population of this butterfly. This was contrary to general predictions about the genetic basis of preference variation, which are hypothesized to be sex linked between populations but autosomal within populations. Evidence of sex linkage disappeared when butterflies were tested on methanol-based chemical extracts, suggesting these chemicals in isolation may not be the primary driver of female choice among available host plants. Although unexpected, evidence for within-population sex-linked genetic variation in preference for T. arvense over native hosts indicates that persistent maladaptive oviposition on this lethal plant must be maintained by alternative evolutionary dynamics such as migration- or drift-selection balance or pleiotropic constraints.

Plant metabolic pathways and gene networks involved in the response to herbivory are well-established, but the impact of epigenetic factors as modulators of those responses is less understood. Here, we studied the role of DNA cytosine methylation on phenotypic responses after short-term herbivory in Thlaspi arvense plants with two contrasting flowering phenotypes. We investigated the effect of experimental demethylation and herbivory treatments following a 2x3 factorial design. First, half the seeds were submerged in a water solution of the demethylating agent 5-azacytidine and the other half only in water, as controls. Then, we assigned control and demethylated plants to three herbivory categories (i) insect herbivory, (ii) artificial herbivory, and (iii) undamaged plants. The effects of the demethylation and herbivory treatments were assessed by quantifying genome-wide global DNA cytosine methylation, concentration of leaf glucosinolates, final stem biomass, fruit and seed production, and seed size. For most of the plant traits analysed, individuals from the two plant-types responded differently. In late-flowering plants, global DNA methylation did not differ between control and demethylated plants but it was significantly reduced by herbivory. Conversely, in early-flowering plants, demethylation at seed stage was still evident in leaf genomes of reproductive individuals whereas herbivory did not affect their global DNA methylation. In late-flowering plants, artificial herbivory imposed a stronger reduction than insect herbivory in global DNA methylation and final stem biomass, and induced higher concentration of aliphatic glucosinolates. In early-flowering plants, the effects of herbivory were non-significant for the same traits. Finally, the effect of herbivory on reproductive parameters varied with the level of demethylation and the plant flowering-type. The complexity of our results suggests that the genetic background of experimental plants can affect the res

Plant metabolic pathways and gene networks involved in the response to herbivory are well-established, but the impact of epigenetic factors as modulators of those responses is less understood. Here, we use the demethylating agent 5-azacytidine to uncover the role of DNA cytosine methylation on phenotypic responses after short-term herbivory in Thlaspi arvense plants that came from two European populations with contrasting flowering phenotypes expected to differ in the response to experimental demethylation. The experimental design followed a 2 × 3 factorial design, that was replicated for each flowering-type. First, half the seeds were submerged in a water solution of 5-azacytidine and the other half only in water, as controls. Then, we assigned control and demethylated plants to three herbivory categories (i) insect herbivory, (ii) artificial herbivory, and (iii) undamaged plants. The effects of the demethylation and herbivory treatments were assessed by quantifying genome-wide global DNA cytosine methylation, concentration of leaf glucosinolates, final stem biomass, fruit and seed production, and seed size. For most of the plant traits analysed, individuals from the two flowering-types responded differently. In late-flowering plants, global DNA methylation did not differ between control and demethylated plants but it was significantly reduced by herbivory. Conversely, in early-flowering plants, demethylation at seed stage was still evident in leaf DNA of reproductive individuals whereas herbivory did not affect their global DNA methylation. In late-flowering plants, artificial herbivory imposed a stronger reduction than insect herbivory in global DNA methylation and final stem biomass, and induced higher concentration of aliphatic glucosinolates. In early-flowering plants, the effects of herbivory were non-significant for the same traits. Finally, the effect of herbivory on reproductive parameters varied with the level of demethylation and the plant flowering-type

Plant metabolic pathways and gene networks involved in the response to herbivory are well-established, but the impact of epigenetic factors as modulators of those responses is less understood. Here, we studied the role of DNA cytosine methylation on phenotypic responses after short-term herbivory in Thlaspi arvense plants with two contrasting flowering phenotypes. We investigated the effect of experimental demethylation and herbivory treatments following a 2x3 factorial design. First, half the seeds were submerged in a water solution of the demethylating agent 5-azacytidine and the other half only in water, as controls. Then, we assigned control and demethylated plants to three herbivory categories (i) insect herbivory, (ii) artificial herbivory, and (iii) undamaged plants. The effects of the demethylation and herbivory treatments were assessed by quantifying genome-wide global DNA cytosine methylation, concentration of leaf glucosinolates, final stem biomass, fruit and seed production, and seed size. For most of the plant traits analysed, individuals from the two plant-types responded differently. In late-flowering plants, global DNA methylation did not differ between control and demethylated plants but it was significantly reduced by herbivory. Conversely, in early-flowering plants, demethylation at seed stage was still evident in leaf genomes of reproductive individuals whereas herbivory did not affect their global DNA methylation.

Endophytic fungi of crops can promote plant growth through various mechanisms of action (i.e., improve nutrient uptake and nutrient use efficiency, and produce and modulate plant hormones). The genus Brassica includes important horticultural crops, which have been little studied in their interaction with endophytic fungi. Previously, four endophytic fungi were isolated from kale roots (Brassica oleracea var. acephala), with different benefits for their host, including plant growth promotion, cold tolerance, and induction of resistance to pathogens (Xanthomonas campestris) and pests (Mamestra brassicae). In the present work, the molecular and morphological identification of the four different isolates were carried out, describing them as the species Acrocalymma vagum, Setophoma terrestris, Fusarium oxysporum, and the new species Pyrenophora gallaeciana. In addition, using a representative crop of each Brassica U's triangle species and various in vitro biochemical tests, the ability of these fungi to promote plant growth was described. In this sense, the four fungi used promoted the growth of B. rapa, B. napus, B. nigra, B. juncea, and B. carinata, possibly due to the production of auxins, siderophores, P solubilization or cellulase, xylanase or amylase activity. Finally, the differences in root colonization between the four endophytic fungi and two pathogens (Leptosphaeria maculans and Sclerotinia sclerotiorum) and the root glucosinolate profile were studied, at different times. In this way, how the presence of progoitrin in the roots reduces their colonization by endophytic and pathogenic fungi was determined, while the possible hydrolysis of sinigrin to fungicidal products controls the colonization of endophytic fungi, but not of pathogens.

Thermal stress causes the reduction in productivity and harvest quality. To adapt to different temperature ranges, plants activate protecting metabolic pathways. Previous studies have reported that stressful environments due to abiotic stresses have an impact on the accumulation of glucosinolates (GSLs) in Brassicaceae plants. In order to determine the role of GSLs in the plant response to thermal stress, we conducted a study comparing four populations with a high and low GSL content. The GSL levels were analysed at different temperatures [control (20), 12 and 32 °C], detecting that populations with a higher GSL content increased their resistance to the cold. In addition, populations subjected to the cold increased the content of indolic GSLs. Populations with high levels of GSLs show higher levels of glucobrassicin (GBS) and sinigrin (SIN) under cold temperatures than plants grown under control conditions. High temperatures have a lower impact on GSLs accumulation. To elucidate the induced metabolic changes due to the accumulation of GSLs under cold conditions, we performed an untargeted metabolomic analysis and identified 25 compounds differentially expressed under cold conditions in the populations with a high GSL content. Almost 50% of these compounds are classified as lipids (fatty amides, monoradylglycerols, diterpenes, glycosylglycerols, linoleic acids and derivatives). Organoheterocyclic and nitrogenous organic compounds are also over-represented. Therefore, the current results suggest that GSLs play a key role in cold tolerance. Although the associated molecular mechanisms have not been elucidated, the non-targeted metabolomics assay shows a significant change in the lipid profile, with compounds that need to be studied further.

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.

Microplastics (MPs) can affect plant biomass, tissue composition, and root traits. However, the effects of MPs on the synthesis of secondary metabolites and on the accumulation of bioactive compounds remain poorly studied. The objective of this work was to analyze accumulation of bioactive compounds in broccoli and radish sprouts grown hydroponically in a substrate containing seven different toxic amounts (from very low to extremely high) of low-density polyethylene (PE). Radish was more severely affected by microplastic pollution than broccoli. The effect on the phytochemical composition was statistically significant in both species compared to control. In this aspect, glucosinolate (GSL) content was negatively affected by MPs decreasing from 182 to 124 mg 100 g at medium doses of MPs in broccoli, whereas these compounds drastically decreased from 253 to 151 mg 100 g at the same doses in radish. Anthocyanin content significantly increased until medium doses of MPs ranging from 6.28 to 11.44 mg 100 g in broccoli whereas in radish was from 2.44 to approximately 4 mg 100 g. In addition, other morphological and physiological parameter were considered. The analysis of malondialdehyde (MDA) showed significant effects on broccoli and radish in all the MP treatments. The results revealed that high loads of MPs in the substrate affect growth parameters, lipid peroxidation rate estimated by MDA, and phytochemicals of broccoli and radish sprouts, with differences in response to MPs pollution and intensity between species.

In northwest Spain and Portugal, there is a long tradition of cultivating B. rapa subsp. rapa to obtain turnip greens and turnip tops. Brassica rapa L. subsp. rapa (turnip greens and turnip tops) were grown under conventional and organic conditions in two Farms in southern Spain. Glucosinolatescontents were higher in Brassicas grown under conventional conditions than those grown under organic ones. Average Ca total and bioaccessible contents ranged between 14.6-23.4 mg/g; 8.9-12.0 mg/g for turnip greens and 6.4-8.9 mg/g; 4.3-4.8 mg/g for turnip tops. According to these concentrations, an intake of 100-200 g (fresh weight) of the studied Brassica rapa fulfills Ca dietary reference intakes (DRI) (considering the total content data) and complies with 72-100% Ca DRI percentage (considering the bioaccessible data). Se concentrations ranged between 0.061-0.073 µg/g and 0.039-0.053 µg/g for turnip greens and turnip tops respectively. Se bioaccessibility values were high, with percentages of around 90%. Finally, the total glucosinolate content ranged between 13.23-21.28 µmol/g for turnip greens and 13.36-20.20 µmol/g for turnip tops. In general, the bioaccessibility of the total glucosinolates analyzed in this study was high, with mean values of around 73% and 66% for turnip greens and turnip tops, respectively. Brassica rapa vegetables grown under both organic and conventional conditions in southern Spain are an excellent dietary source of Ca, Se, and glucosinolates with a high bioaccessibility.

The diamondback moth, "Plutella xylostella" L. (Lepidoptera: Plutellidae), is an important insect pest of cruciferous crops. Understanding its preference patterns can lead to more efficient management methods, such as trap crops. Several strains of "P. xylostella" were used to test whether there were differences in oviposition preference in a four-choice setting, on abaxial versus adaxial leaf surfaces in 28 different plant species, and on substrates with different concentrations of sinigrin (allylglucosinolate). Additionally, the larval preference of "P. xylostella" was studied with 17 plant species of known glucosinolate content that were compared to "Arabidopsis thaliana" L. in two-choice tests. Our research shows that the diet on which "P. xylostella" has fed hardly affects multiple-choice host-plant preference, abaxial and adaxial oviposition preference, or oviposition response to pure glucosinolates. Our study also shows that glucosinolate content affects larval preference, which together with the known correlation between glucosinolate content and "P. xylostella" oviposition, indicates that crops with high glucosinolate content could be more susceptible to damage by "P. xylostella" than crops with low glucosinolate content. These findings are discussed in regards to their significance in the management of "P. xylostella".

Sulfur is essential for the health of plants and is an indispensable dietary component for human health and disease prevention. Its incorporation into our food supply is heavily reliant upon the uptake of sulfur into plant tissue and our subsequent intake. Dietary requirements for sulfur are largely calculated based upon requirements for the sulfur-containing amino acids (SAA), cysteine and methionine, to meet the demands for synthesis of proteins, enzymes, co-enzymes, vitamins, and hormones. SAA are found in abundance in animal sources and are relatively low in plants. However, some plants, particularly cruciferous and allium vegetables, produce many protective sulfur-containing secondary metabolites, such as glucosinolates and cysteine sulfoxides. The variety and quantity of these sulfur-containing metabolites are extensive and their effects on human health are wide-reaching. Many benefits appear to be related to sulfur’s role in redox biochemistry, protecting against uncontrolled oxidative stress and inflammation; features consistent within cardiometabolic dysfunction and many chronic metabolic diseases of aging. This narrative explores the origins and importance of sulfur, its incorporation into our food supply and dietary sources. It also explores the overarching potential of sulfur for human health, particularly around the amelioration of oxidative stress and chronic inflammation, and subsequent chronic disease prevention.

Plants face attackers aboveground and belowground. Insect root herbivores can lead to severe crop losses, yet the underlying transcriptomic responses have rarely been studied. We studied the dynamics of the transcriptomic response of Brussels sprouts (Brassica oleracea var. gemmifera) primary roots to feeding damage by cabbage root fly larvae (Delia radicum), alone or in combination with aboveground herbivory by cabbage aphids (Brevicoryne brassicae) or diamondback moth caterpillars (Plutella xylostella). This was supplemented with analyses of phytohormones and the main classes of secondary metabolites; aromatic, indole and aliphatic glucosinolates. Root herbivory leads to major transcriptomic rearrangement that is modulated by aboveground feeding caterpillars, but not aphids, through priming soon after root feeding starts. The root herbivore downregulates aliphatic glucosinolates. Knocking out aliphatic glucosinolate biosynthesis with CRISPR-Cas9 results in enhanced performance of the specialist root herbivore, indicating that the herbivore downregulates an effective defence. This study advances our understanding of how plants cope with root herbivory and highlights several novel aspects of insect–plant interactions for future research. Further, our findings may help breeders develop a sustainable solution to a devastating root pest.

[ES] Para hacer frente a las condiciones ambientales cambiantes, las plantas han sido capaces de desarrollar una inmensa variedad de mecanismos de defensa y adaptación. Uno de ellos es el llamado metabolismo secundario, cuya complejidad le otorga la capacidad de producir alrededor de 200.000 compuestos, gracias a una gran variedad de rutas metabólicas diferentes. En el presente trabajo nos centraremos en aquellos metabolitos encargados de la defensa que, aunque su principal función no sea el crecimiento o el desarrollo, son necesarios para la supervivencia. Para ello, algunos de los objetivos planteados en este trabajo son: conocer las respuesta hormonal y de otros metabolitos frente a un elicitor altamente corrosivo, el nitrato de plata (AgNO3), averiguando a través de qué mecanismos funciona; evaluar el papel de las hormonas vegetales de defensa, ácido salicílico (SA) y ácido jasmónico (JA), frente a diferentes elicitores, en presencia de otros metabolitos cuya función está relacionada con la de dichas hormonas; profundizar en el rol defensivo de los glucosinolatos, en ausencia de otros compuestos encargados de esta función. En cuanto al proceso metodológico, con el fin de conseguir estos objetivos se buscará la integración de datos metabolómicos y datos genéticos. Para lograr la primera aproximación, se analizará el perfil cromatográfico de una selección de metabolitos secundarios de Arabidopsis thaliana en el ecotipo Columbia 0 (Col0), y en los mutantes atr1D, cyp79b2/b3, qKO (myb28 myb29 cyp79b2 cyp79b3), jai1, npr1, ics2 y jar1 tratados con agentes de estrés abiótico y hormonas como el nitrato de plata (AgNO3), el metil jasmonato (MeJA) y el ácido salicílico (SA) a una concentración de 5mM, 500µM y 100µM respectivamente. Siguiendo un análisis metabolómico dirigido se buscará poder identificar metabolitos relacionados con los mecanismos de defensa de Arabidopsis, priorizando algunos como glucosinolatos (indólicos, y alifáticos), hormonas y precursores, y otros

[ES] Para hacer frente a las condiciones ambientales cambiantes, las plantas han sido capaces de desarrollar una inmensa variedad de mecanismos de defensa y adaptación. Uno de ellos es el llamado metabolismo secundario, cuya complejidad le otorga la capacidad de producir alrededor de 200.000 compuestos, gracias a una gran variedad de rutas metabólicas diferentes. En el presente trabajo nos centraremos en aquellos metabolitos encargados de la defensa que, aunque su principal función no sea el crecimiento o el desarrollo, son necesarios para la supervivencia. Para ello, algunos de los objetivos planteados en este trabajo son: conocer las respuesta hormonal y de otros metabolitos frente a un elicitor altamente corrosivo, el nitrato de plata (AgNO3), averiguando a través de qué mecanismos funciona; evaluar el papel de las hormonas vegetales de defensa, ácido salicílico (SA) y ácido jasmónico (JA), frente a diferentes elicitores, en presencia de otros metabolitos cuya función está relacionada con la de dichas hormonas; profundizar en el rol defensivo de los glucosinolatos, en ausencia de otros compuestos encargados de esta función. En cuanto al proceso metodológico, con el fin de conseguir estos objetivos se buscará la integración de datos metabolómicos y datos genéticos. Para lograr la primera aproximación, se analizará el perfil cromatográfico de una selección de metabolitos secundarios de Arabidopsis thaliana en el ecotipo Columbia 0 (Col0), y en los mutantes atr1D, cyp79b2/b3, qKO (myb28 myb29 cyp79b2 cyp79b3), jai1, npr1, ics2 y jar1 tratados con agentes de estrés abiótico y hormonas como el nitrato de plata (AgNO3), el metil jasmonato (MeJA) y el ácido salicílico (SA) a una concentración de 5mM, 500µM y 100µM respectivamente. Siguiendo un análisis metabolómico dirigido se buscará poder identificar metabolitos relacionados con los mecanismos de defensa de Arabidopsis, priorizando algunos como glucosinolatos (indólicos, y alifáticos), hormonas y precursores, y otros

[ES] Para hacer frente a las condiciones ambientales cambiantes, las plantas han sido capaces de desarrollar una inmensa variedad de mecanismos de defensa y adaptación. Uno de ellos es el llamado metabolismo secundario, cuya complejidad le otorga la capacidad de producir alrededor de 200.000 compuestos, gracias a una gran variedad de rutas metabólicas diferentes. En el presente trabajo nos centraremos en aquellos metabolitos encargados de la defensa que, aunque su principal función no sea el crecimiento o el desarrollo, son necesarios para la supervivencia. Para ello, algunos de los objetivos planteados en este trabajo son: conocer las respuesta hormonal y de otros metabolitos frente a un elicitor altamente corrosivo, el nitrato de plata (AgNO3), averiguando a través de qué mecanismos funciona; evaluar el papel de las hormonas vegetales de defensa, ácido salicílico (SA) y ácido jasmónico (JA), frente a diferentes elicitores, en presencia de otros metabolitos cuya función está relacionada con la de dichas hormonas; profundizar en el rol defensivo de los glucosinolatos, en ausencia de otros compuestos encargados de esta función. En cuanto al proceso metodológico, con el fin de conseguir estos objetivos se buscará la integración de datos metabolómicos y datos genéticos. Para lograr la primera aproximación, se analizará el perfil cromatográfico de una selección de metabolitos secundarios de Arabidopsis thaliana en el ecotipo Columbia 0 (Col0), y en los mutantes atr1D, cyp79b2/b3, qKO (myb28 myb29 cyp79b2 cyp79b3), jai1, npr1, ics2 y jar1 tratados con agentes de estrés abiótico y hormonas como el nitrato de plata (AgNO3), el metil jasmonato (MeJA) y el ácido salicílico (SA) a una concentración de 5mM, 500µM y 100µM respectivamente. Siguiendo un análisis metabolómico dirigido se buscará poder identificar metabolitos relacionados con los mecanismos de defensa de Arabidopsis, priorizando algunos como glucosinolatos (indólicos, y alifáticos), hormonas y precursores, y otros

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.

Glucosinolate transporters (GTRs) are part of the nitrate/peptide transporter (NPF) family, members of which also transport specialized secondary metabolites as substrates. Glucosinolates are defense compounds derived from amino acids. We selected 4‐methylthiobutyl (4MTB) and indol‐3‐ylmethyl (I3M) glucosinolates to study how GTR1 from Arabidopsis thaliana transports these substrates in computational simulation approaches. The designed pipeline reported here includes massive docking of 4MTB and I3M in an ensemble of GTR1 conformations (in both inward and outward conformations) extracted from molecular dynamics simulations, followed by clustered and substrate–protein interactions profiling. The identified key residues were mutated, and their role in substrate transport was tested. We were able to identify key residues that integrate a major binding site of these substrates, which is critical for transport activity. In silico approaches employed here represent a breakthrough in the plant transportomics field, as the identification of key residues usually takes a long time if performed from a purely wet‐lab experimental perspective. The inclusion of structural bioinformatics in the analyses of plant transporters significantly speeds up the knowledge‐gaining process and optimizes valuable time and resources.

Sulfur is essential for the health of plants and is an indispensable dietary component for human health and disease prevention. Its incorporation into our food supply is heavily reliant upon the uptake of sulfur into plant tissue and our subsequent intake. Dietary requirements for sulfur are largely calculated based upon requirements for the sulfur-containing amino acids (SAA), cysteine and methionine, to meet the demands for synthesis of proteins, enzymes, co-enzymes, vitamins, and hormones. SAA are found in abundance in animal sources and are relatively low in plants. However, some plants, particularly cruciferous and allium vegetables, produce many protective sulfur-containing secondary metabolites, such as glucosinolates and cysteine sulfoxides. The variety and quantity of these sulfur-containing metabolites are extensive and their effects on human health are wide-reaching. Many benefits appear to be related to sulfur’s role in redox biochemistry, protecting against uncontrolled oxidative stress and inflammation; features consistent within cardiometabolic dysfunction and many chronic metabolic diseases of aging. This narrative explores the origins and importance of sulfur, its incorporation into our food supply and dietary sources. It also explores the overarching potential of sulfur for human health, particularly around the amelioration of oxidative stress and chronic inflammation, and subsequent chronic disease prevention.

Glucosinolate transporters (GTRs) are part of the nitrate/peptide transporter (NPF) family, members of which also transport specialized secondary metabolites as substrates. Glucosinolates are defense compounds derived from amino acids. We selected 4‐methylthiobutyl (4MTB) and indol‐3‐ylmethyl (I3M) glucosinolates to study how GTR1 from Arabidopsis thaliana transports these substrates in computational simulation approaches. The designed pipeline reported here includes massive docking of 4MTB and I3M in an ensemble of GTR1 conformations (in both inward and outward conformations) extracted from molecular dynamics simulations, followed by clustered and substrate–protein interactions profiling. The identified key residues were mutated, and their role in substrate transport was tested. We were able to identify key residues that integrate a major binding site of these substrates, which is critical for transport activity. In silico approaches employed here represent a breakthrough in the plant transportomics field, as the identification of key residues usually takes a long time if performed from a purely wet‐lab experimental perspective. The inclusion of structural bioinformatics in the analyses of plant transporters significantly speeds up the knowledge‐gaining process and optimizes valuable time and resources.

Although ammonium (NH4+ ) is a key intermediate of plant nitrogen metabolism, high concentrations of NH4+ in the soil provoke physiological disorders that lead to the development of stress symptoms. Ammonium nutrition was shown to induce the accumulation of glucosinolates (GSLs) in leaves of different Brassicaceae species. To further understand the link between ammonium nutrition and GSLs, we analysed the ammonium stress response of Arabidopsis mutants impaired in GSL metabolic pathway. We showed that the MYB28 and MYB29 double mutant (myb28myb29), which is almost deprived of aliphatic GSLs, is highly hypersensitive to ammonium nutrition. Moreover, we evidenced that the stress symptoms developed were not a consequence of the lack of aliphatic GSLs. Transcriptomic analysis highlighted the induction of an iron (Fe) deficiency response in myb28myb29 under ammonium nutrition. Consistently, ammonium-grown myb28myb29 plants showed altered Fe accumulation and homeostasis. Interestingly, we showed overall that growing Arabidopsis with increased Fe availability relieved ammonium stress symptoms and that this was associated with MYB28 and MYB29 expression. Taken together, our data indicated that the control of Fe homeostasis was crucial for the Arabidopsis response to ammonium nutrition and evidenced that MYB28 and MYB29 play a role in this control.

Background and aimsWe investigate patterns of evolution of genome size across a morphologically and ecologically diverse clade of Brassicaceae, in relation to ecological and life history traits. While numerous hypotheses have been put forward regarding autecological and environmental factors that could favour small vs. large genomes, a challenge in understanding genome size evolution in plants is that many hypothesized selective agents are intercorrelated.MethodsWe contribute genome size estimates for 47 species of Streptanthus Nutt. and close relatives, and take advantage of many data collections for this group to assemble data on climate, life history, soil affinity and composition, geographic range and plant secondary chemistry to identify simultaneous correlates of variation in genome size in an evolutionary framework. We assess models of evolution across clades and use phylogenetically informed analyses as well as model selection and information criteria approaches to identify variables that can best explain genome size variation in this clade.Key resultsWe find differences in genome size and heterogeneity in its rate of evolution across subclades of Streptanthus and close relatives. We show that clade-wide genome size is positively associated with climate seasonality and glucosinolate compounds. Model selection and information criteria approaches identify a best model that includes temperature seasonality and fraction of aliphatic glucosinolates, suggesting a possible role for genome size in climatic adaptation or a role for biotic interactions in shaping the evolution of genome size. We find no evidence supporting hypotheses of life history, range size or soil nutrients as forces shaping genome size in this system.ConclusionsOur findings suggest climate seasonality and biotic interactions as potential forces shaping the evolution of genome size and highlight the importance of evaluating multiple factors in the context of phylogeny to understand the effect of

type:Article, Plants take up sulfur (S), an essential element for all organisms, as sulfate, which is mainly attributed to the function of SULTR1;2 in Arabidopsis. A disruption mutant of SULTR1;2, sel1-10, has been characterized with phenotypes similar to plants grown under sulfur deficiency (-S). Although the effects of -S on S metabolism were well investigated in seedlings, no studies have been performed on mature Arabidopsis plants. To study further the effects of -S on S metabolism, we analyzed the accumulation and distribution of S-containing compounds in different parts of mature sel1-10 and of the wild-type (WT) plants grown under long-day conditions. While the levels of sulfate, cysteine, and glutathione were almost similar between sel1-10 and WT, levels of glucosinolates (GSLs) differed between them depending on the parts of the plant. GSLs levels in the leaves and stems were generally lower in sel1-10 than those in WT. However, sel1-10 seeds maintained similar levels of aliphatic GSLs to those in WT plants. GSL accumulation in reproductive tissues is likely to be prioritized even when sulfate supply is limited in sel1-10 for its role in S storage and plant defense.

Plants produce diverse metabolites to cope with the challenges presented by complex and ever-changing environments. These challenges drive the diversification of specialized metabolites within and between plant species. However, we are just beginning to understand how frequently new alleles arise controlling specialized metabolite diversity and how the geographic distribution of these alleles may be structured by ecological and demographic pressures. Here, we measure the variation in specialized metabolites across a population of 797 natural Arabidopsis thaliana accessions. We show that a combination of geography, environmental parameters, demography and different genetic processes all combine to influence the specific chemotypes and their distribution. This showed that causal loci in specialized metabolism contain frequent independently generated alleles with patterns suggesting potential within-species convergence. This provides a new perspective about the complexity of the selective forces and mechanisms that shape the generation and distribution of allelic variation that may influence local adaptation.

Plant metabolism is modulated by a complex interplay between internal signals and external cues. A major goal of all quantitative metabolomic studies is to clone the underlying genes to understand the mechanistic basis of this variation. Using fine-scale genetic mapping, in this work we report the identification and initial characterization of NAD-DEPENDENT MALIC ENZYME 1 (NAD-ME1) as the candidate gene underlying the pleiotropic network Met.II.15 quantitative trait locus controlling variation in plant metabolism and circadian clock outputs in the Bay × Sha Arabidopsis population. Transcript abundance and promoter analysis in NAD-ME1Bay-0 and NAD-ME1Sha alleles confirmed allele-specific expression that appears to be due a polymorphism disrupting a putative circadian cis-element binding site. Analysis of transfer DNA insertion lines and heterogeneous inbred families showed that transcript variation of the NAD-ME1 gene led to temporal shifts of tricarboxylic acid cycle intermediates, glucosinolate (GSL) accumulation, and altered regulation of several GSL biosynthesis pathway genes. Untargeted metabolomic analyses revealed complex regulatory networks of NAD-ME1 dependent upon the daytime. The mutant led to shifts in plant primary metabolites, cell wall components, isoprenoids, fatty acids, and plant immunity phytochemicals, among others. Our findings suggest that NAD-ME1 may act as a key gene to coordinate plant primary and secondary metabolism in a time-dependent manner.

Cooking vegetables in microwave bags is becoming a popular domestic cooking method, being relevant to know how this cooking method affects health-promoting phytochemicals of staples such as broccoli. The aim of this work was to study the effect of microwave bag cooking versus conventional microwaving on bioactive compound content (glucosinolates and hydroxycinnamic acid derivatives) and other quality parameters (such as antioxidant capacity, mineral content and microbial load) of broccoli florets. The influence of cooking time on bioactive compounds content was also evaluated. The study was carried out in two independent experiments; using intact broccoli and broccoli preprocessed in industry. Microwave bag cooked broccoli for 5 min (following label recommendation) showed higher glucosinolate content retention compared to conventional microwaving. Results suggest that volatilization could be an important phenomenon in reduction of glucosinolates during microwave cooking of broccoli florets. Glucosinolate profile did not change after cooking, regardless of cooking method applied. Furthermore, microwave bag cooked broccoli presented higher antioxidant capacity (by DPPH assay) than conventional microwaved broccoli. Hydroxycinnamic acid derivatives content was reduced in microwave cooking, regardless of method applied. Altogether, the use of microwave bags for microwaving is a novel method that retains main bioactive components of broccoli. This option is a fast, easy and considerably clean cooking option to fulfill modern consumer needs.

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.

Background and Aims: We investigate patterns of evolution of genome size across a morphologically and ecologically diverse clade of Brassicaceae, in relation to ecological and life history traits. While numerous hypotheses have been put forward regarding autecological and environmental factors that could favour small vs. large genomes, a challenge in understanding genome size evolution in plants is that many hypothesized selective agents are intercorrelated. Methods: We contribute genome size estimates for 47 species of Streptanthus Nutt. and close relatives, and take advantage of many data collections for this group to assemble data on climate, life history, soil affinity and composition, geographic range and plant secondary chemistry to identify simultaneous correlates of variation in genome size in an evolutionary framework. We assess models of evolution across clades and use phylogenetically informed analyses as well as model selection and information criteria approaches to identify variables that can best explain genome size variation in this clade. Key Results: We find differences in genome size and heterogeneity in its rate of evolution across subclades of Streptanthus and close relatives. We show that clade-wide genome size is positively associated with climate seasonality and glucosinolate compounds. Model selection and information criteria approaches identify a best model that includes temperature seasonality and fraction of aliphatic glucosinolates, suggesting a possible role for genome size in climatic adaptation or a role for biotic interactions in shaping the evolution of genome size. We find no evidence supporting hypotheses of life history, range size or soil nutrients as forces shaping genome size in this system. Conclusions: Our findings suggest climate seasonality and biotic interactions as potential forces shaping the evolution of genome size and highlight the importance of evaluating multiple factors in the context of phylogeny to understand

Plants produce diverse metabolites to cope with the challenges presented by complex and ever-changing environments. These challenges drive the diversification of specialized metabolites within and between plant species. However, we are just beginning to understand how frequently new alleles arise controlling specialized metabolite diversity and how the geographic distribution of these alleles may be structured by ecological and demographic pressures. Here, we measure the variation in specialized metabolites across a population of 797 natural Arabidopsis thaliana accessions. We show that a combination of geography, environmental parameters, demography and different genetic processes all combine to influence the specific chemotypes and their distribution. This showed that causal loci in specialized metabolism contain frequent independently generated alleles with patterns suggesting potential within-species convergence. This provides a new perspective about the complexity of the selective forces and mechanisms that shape the generation and distribution of allelic variation that may influence local adaptation.

In traditional, small-scale agriculture in the Andes, potatoes are frequently co-cultivated with the Andean edible tuber Tropaeolum tuberosum, commonly known as mashua, which is believed to exert a pest and disease protective role due to its content of the phenylalanine-derived benzylglucosinolate (BGLS). We bioengineered the production of BGLS in potato by consecutive generation of stable transgenic events with two polycistronic constructs encoding for expression of six BGLS biosynthetic genes from Arabidopsis thaliana. First, we integrated a polycistronic construct coding for the last three genes of the pathway (SUR1, UGT74B1 and SOT16) into potato driven by the cauliflower mosaic virus 35S promoter. After identifying the single-insertion transgenic event with the highest transgene expression, we stacked a second polycistronic construct coding for the first three genes in the pathway (CYP79A2, CYP83B1 and GGP1) driven by the leaf-specific promoter of the rubisco small subunit from chrysanthemum. We obtained transgenic events producing as high as 5.18 pmol BGLS/mg fresh weight compared to the non-transgenic potato plant producing undetectable levels of BGLS. Preliminary bioassays suggest a possible activity against Phytophthora infestans, causing the late blight disease and Premnotrypes suturicallus, referred to as the Andean potato weevil. However, we observed altered leaf morphology, abnormally thick and curlier leaves, reduced growth and tuber production in five out of ten selected transgenic events, which indicates that the expression of BGLS biosynthetic genes has an undesirable impact on the potato. Optimization of the expression of the BGLS biosynthetic pathway in potato is required to avoid alterations of plant development. Graphical abstract: [Figure not available: see fulltext.]