Natural H2S-donors: A new pharmacological opportunity for the management of overweight and obesity.

The prevalence of overweight and obesity has progressively increased in the last few years, becoming a real threat to healthcare systems. To date, the clinical management of body weight gain is an unmet medical need, as there are few approved anti-obesity drugs and most require an extensive monitoring and vigilance due to risk of adverse effects and poor patient adherence/persistence. Growing evidence has shown that the gasotransmitter hydrogen sulfide (H2S) and, therefore, H2S-donors could have a central role in the prevention and treatment of overweight/obesity. The main natural sources of H2S-donors are plants from the Alliaceae (garlic and onion), Brassicaceae (e.g., broccoli, cabbage, and wasabi), and Moringaceae botanical families. In particular, polysulfides and isothiocyanates, which slowly release H2S, derive from the hydrolysis of alliin from Alliaceae and glucosinolates from Brassicaceae/Moringaceae, respectively. In this review, we describe the emerging role of endogenous H2S in regulating adipose tissue function and the potential efficacy of natural H2S-donors in animal models of overweight/obesity, with a final focus on the preliminary results from clinical trials. We conclude that organosulfur-containing plants and their extracts could be used before or in combination with conventional anti-obesity agents to improve treatment efficacy and reduce inflammation in obesogenic conditions. However, further high-quality studies are needed to firmly establish their clinical efficacy.

Stability and bioaccessibility of micronutrients and phytochemicals present in processed leek and Brussels sprouts during static in vitro digestion.

Vegetables are frequently processed before consumption. However, vegetable functionalization continues beyond ingestion as the human digestive tract exposes vegetable products to various conditions (e.g. elevated temperature, pH alterations, enzymes, electrolytes, mechanical disintegration) which can affect the stability of micronutrients and phytochemicals. Besides the extent to which these compounds withstand the challenges posed by digestive conditions, it is equally important to consider their accessibility for potential absorption by the body. Therefore, this study investigated the impact of static in vitro digestion on the stability (i.e. concentration) and bioaccessibility of vitamin C, vitamin K1, glucosinolates, S-alk(en)yl-l-cysteine sulfoxides (ACSOs) and carotenoids in Brussels sprouts (Brassica oleracea var. gemmifera) and leek (Allium ampeloprasum var. porrum). Water-soluble compounds, glucosinolates and ACSOs, remained stable during digestion while vitamin C decreased by >48%. However, all water-soluble compounds were completely bioaccessible. Lipid-soluble compounds were also stable during digestion but were only bioaccessible for 26-81%.

n-Butanol fraction of moringa seed attenuates arsenic intoxication by regulating the uterine inflammatory and apoptotic pathways.

The adverse effects of arsenic-chelating drugs make it essential to replace invasive chelating therapy with non-invasive oral therapy for arsenic poisoning. The goal of the current investigation was to determine whether the uterine damage caused by arsenization could be repaired by the n-butanol fraction of Moringa oleifera seed (NB). The rats were orally administered with arsenic (10 mg/kg BW) for the initial 8 days, followed by NB (50 mg/kg) for the next 8 days without arsenic. The probable existence of different components in NB was evaluated by HPLC-MS. Pro and anti-inflammatory indicators were assessed by RT-PCR and western blot. ESR-α was detected via immunostaining. Arsenic-exposed rats had significantly increased lipid peroxidation and decreased antioxidant enzyme activity, which were markedly reduced after NB treatment. Weaker ESR-α expression and distorted uterine histomorphology following arsenication were retrieved significantly by NB. Meaningful restoration by NB was also achieved for altered mRNA and protein expression of various inflammatory and apoptotic indicators. Molecular interaction predicted that glucomoringin and methyl glucosinolate of moringa interact with the catalytic site of caspase-3 in a way that limits its activity. However, NB was successful in restoring the arsenic-mediated uterine hypofunction. The glucomoringin and methyl glucosinolate present in n-butanol fraction may play a critical role in limiting apoptotic event in the arsenicated uterus.

Comparative Studies of Extracts Obtained from Brassica oleracea L. Plants at Different Stages of Growth by Isolation and Determination of Isothiocyanates: An Assessment of Chemopreventive Properties of Broccoli.

The main goal of this work was to develop analytical procedures for the isolation and determination of selected isothiocyanates. As an example, particularly sulforaphane from plants of the Brassicaceae Burnett or Cruciferae Juss family. The applied methodology was mainly based on classical extraction methods and high-performance liquid chromatography coupled with tandem mass spectrometry. Moreover, the effect of temperature on the release of isothiocyanates from plant cells was considered. The cytotoxic activity of the obtained plant extracts against a selected cancer cell line has also been included. The results allow evaluating the usefulness of obtained plant extracts and raw sprouts regarding their content of isothiocyanates-bioactive compounds with chemopreventive properties.

Impact of refrigerated storage on (bio)chemical conversions of health-related compounds in pretreated, pasteurized Brussels sprouts and leek.

Vegetable processing often consists of multiple processing steps. Research mostly focused on the impact of individual processing steps on individual health-related compounds. However, there is a need for more holistic approaches to understand the overall impact of the processing chain on the health potential of vegetables. Therefore, this work studied the impact of pretreatment (relatively intact versus pureed vegetable systems), pasteurization and subsequent refrigerated storage (kinetic evaluation) on multiple health-related compounds (vitamin C, vitamin K1, carotenoids, glucosinolates and S-alk(en)yl-L-cysteine sulfoxides (ACSOs)) in Brussels sprouts and leek. It could be shown that differences introduced by different types of pretreatment were not nullified during pasteurization and refrigerated storage. Clearly, enzymatic conversions controlled during pretreatment resulted in different health-related compound profiles still observable after pasteurization. Moreover, about -42% and -100% relative concentration differences of ACSOs and dehydroascorbic acid, respectively, were detected immediately after pasteurization, while glucosinolates concentrations decreased by about 47% during refrigerated storage. All other compounds were stable during pasteurization and refrigerated storage.

Transcriptome Dynamics of Brassica juncea Leaves in Response to Omnivorous Beet Armyworm (Spodoptera exigua, Hübner).

Cruciferous plants manufacture glucosinolates (GSLs) as special and important defense compounds against insects. However, how insect feeding induces glucosinolates in Brassica to mediate insect resistance, and how plants regulate the strength of anti-insect defense response during insect feeding, remains unclear. Here, mustard (Brassica juncea), a widely cultivated Brassica plant, and beet armyworm (Spodoptera exigua), an economically important polyphagous pest of many crops, were used to analyze the changes in GSLs and transcriptome of Brassica during insect feeding, thereby revealing the plant-insect interaction in Brassica plants. The results showed that the content of GSLs began to significantly increase after 48 h of herbivory by S. exigua, with sinigrin as the main component. Transcriptome analysis showed that a total of 8940 DEGs were identified in mustard challenged with beet armyworm larvae. The functional enrichment results revealed that the pathways related to the biosynthesis of glucosinolate and jasmonic acid were significantly enriched by upregulated DEGs, suggesting that mustard might provide a defense against herbivory by inducing JA biosynthesis and then promoting GSL accumulation. Surprisingly, genes regulating JA catabolism and inactivation were also activated, and both JA signaling repressors (JAZs and JAMs) and activators (MYCs and NACs) were upregulated during herbivory. Taken together, our results indicate that the accumulation of GSLs regulated by JA signaling, and the regulation of active and inactive JA compound conversion, as well as the activation of JA signaling repressors and activators, collectively control the anti-insect defense response and avoid over-stunted growth in mustard during insect feeding.

Selenium Biofortification Effect on Glucosinolate Content of Brassica oleracea var. italic and Eruca vesicaria.

Glucosinolates (GSLs) in different plant parts of broccoli (Brassica oleracea var. italic) and rocket (Eruca vesicaria) were analyzed qualitatively and quantitatively before and after treatment with sodium selenate (2 and 5 mM), by their desulfo-counterparts using the UHPLC-DAD-MS/MS technique. Twelve GSLs were detected in broccoli (five aliphatic, one arylaliphatic, and six indolic), where 4-(methylsulfanyl)butyl GSL (glucoerucin) was the main one in the roots (4.88-9.89 µmol/g DW), 4-(methylsulfinyl)butyl GSL (glucoraphanin) in stems (0.44-1.11 µmol/g DW), and 4-hydroxyindol-3-ylmethyl GSL (4-hydroxyglucobrassicin) in leaves (0.51-0.60 µmol/g DW). No GSL containing selenium was detected in the treated broccoli. Ten GSLs were detected in rocket (seven aliphatic and three indolic), where 4-(methylsulfanyl)butyl GSL (glucoerucin) was the main one in the roots (4.50-20.59 µmol/g DW) and 4-methoxyindol-3-ylmethyl GSL (4-methoxyglucobrassicin) in the aerial part (0.57-5.69 µmol/g DW). As a result of induced stress by selenium fertilization, the total GSL content generally increased in both plants. In contrast to broccoli, the roots and the aerial part of the rocket treated with a high concentration of sodium selenate contained 4-(methylseleno)butyl GSL (glucoselenoerucin) (0.36-4.48 µmol/g DW). Although methionine-derived GSLs are the most abundant in both plants, the plants' ability to tolerate selenate and its regulation by selenoglucosinolate production is species- and growth-stage-dependent.

Short-term effects and safety of a natural oral supplement containing glucosinolates, phytosterols, and citrus flavonoids compared with hormone treatment for the management of postmenopausal symptomatic women: a pilot single-center randomized phase 2 clinical trial.

OBJECTIVE: This study aimed to evaluate the short-term efficacy and safety of an oral herbal supplement containing glucosinolates, phytosterols, and citrus flavonoids for menopausal symptoms in comparison with estrogen plus progestogen therapy (EPT) among postmenopausal women. METHODS: This was a pilot single-blinded, three-armed phase II randomized clinical trial, controlled with EPT. Sixty participants were randomly assigned to receive treatment for 3 months: (1) an oral herbal supplement of 1,500 mg/d (G1, n = 20), (2) an oral herbal supplement of 3,000 mg/d (G2, n = 20), or (3) conjugated equine estrogens 0.625 mg/d plus medroxyprogesterone acetate of 5 mg/d (EPT group, n = 20). The primary endpoint was the intensity of menopausal symptoms as measured using the Menopause-Specific Quality of Life Questionnaire (global and domain scores). The Menopause-Specific Quality of Life Questionnaire uses a 7-point scale to rate the symptom intensity, with higher scores indicating severity. The secondary endpoints were hormonal, lipid, and safety profiles. RESULTS: Fifty-four participants (n = 54) completed the study. The mean, model-estimated, and global menopausal symptom scores at 3 months were 85.8 in the EPT group, 61.3 in G1, and 62.5 in G2. Participants treated with the herbal compound had lower global (13.7 [6.9-20.4], P < 0.001) and physical symptom scores (6.6 [1.6-11.5], P = 0.002) on the second month and lower psychosocial symptom scores (3.8 [1.3 to 6.3], P < 0.001) on the third month of follow-up, compared with EPT. Conversely, participants receiving EPT showed better outcomes on vasomotor symptoms since the first month of treatment (-6.1 [-8.3 to -4.0], P < 0.001). The EPT group exhibited higher values of estradiol and lower follicle-stimulating hormone and luteinizing hormone since the first month of follow-up. Also, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were significantly higher in this group than in G2. CONCLUSIONS: In this small single-blind exploratory trial, the oral herbal supplement was more efficacious in reducing global, physical, and psychosocial menopausal symptoms in the short term than EPT. However, further studies are needed to adequately assess the efficacy and safety of this herbal supplement in the treatment of menopausal symptoms.

Glucosinolates Extracts from Brassica juncea Ameliorate HFD-Induced Non-Alcoholic Steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) is mainly characterized by excessive fat accumulation in the liver. It spans a spectrum of diseases from hepatic steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Brassica juncea is rich in glucosinolates and has been proven to possess many potential pharmacological properties, including hypoglycemic, anti-oxidation, anti-inflammatory, and anti-carcinogenic activities. This study aims to investigate whether whole-plant Brassica juncea (WBJ) and its glucosinolates extracts (BGE) have hepatoprotective effects against a high-fat diet (HFD)-induced NAFLD and further explore the mechanism underlying this process in vivo and in vitro. WBJ treatment significantly reduced body fat, dyslipidemia, hepatic steatosis, liver injury, and inflammation; WBJ treatment also reversed the antioxidant enzyme activity to attenuate oxidative stress in HFD-fed rat liver. Moreover, WBJ and BGE enhanced the activation of AMPK to reduce SREBPs, fatty acid synthase, and HMG-CoA reductase but increased the expression of CPT-I and PPARα to improve hepatic steatosis. In addition, WBJ and BGE could ameliorate NAFLD by inhibiting TNF-α and NF-κB. Based on the above results, this study demonstrates that WBJ and BGE ameliorate HFD-induced hepatic steatosis and liver injury. Therefore, these treatments could represent an unprecedented hope toward improved strategies for NAFLD.

Micro-grinding-based production for sulforaphene-enriched radish seeds extract via facilitating glucosinolates-myrosinase reaction, and evaluation of its anti-adipogenic effects.

Sulforaphene (SFEN), an isothiocyanate (ITC) abundant in radish (Raphanus sativus) seeds (RS), has many health benefits, including anti-obesity effects. SFEN content is affected by multiple factors during processing, such as glucoraphenin (GLE) (the precursor of SFEN) availability, myrosinase (essential for conversion from GLE to SFEN) activity, and SFEN stability. We examined the physiochemical-properties and anti-adipogenic effects of SFEN-enriched RSE produced by two processes, roasting and micro-grinding. The roasting process lowered SFEN content and myrosinase activity over 50 °C. However, among micro-grinding conditions, smaller particle size (#2 grind, ≈11.31 µm) more effectively increased SFEN content in RS compared to larger particles (#1 grind, ≈ 179.50 µm) by accelerating available GLE and myrosinase release from RS. Grind #2 also effectively inhibited the adipogenesis of 3T3-L1 pre-adipocytes compared to #1. Thus, micro-grinding can be suggested for producing SFEN-enriched RSE with anti-adipogenic activity as a functional material for obesity prevention or treatment.

Proteome reveals the mechanism of selenium-sulfur interaction in regulating isothiocyanate biosynthesis and the physiological metabolism of broccoli sprouts.

Broccoli sprouts have a strong ability to accumulate isothiocyanate and selenium. In this study, the isothiocyanate content increased significantly as a result of ZnSO4 stress. Particularly, based on the isothiocyanate content is not affected, the combined ZnSO4 and Na2SeO3 treatment alleviated the inhibition of ZnSO4 and induced selenium content. Gene transcription and protein expression analyses revealed the changes in isothiocyanate and selenium metabolite levels in broccoli sprouts. ZnSO4 combined with Na2SeO3 was proven to activate a series of isothiocyanate metabolite genes (UGT74B1, OX1, and ST5b) and selenium metabolite genes (BoSultr1;1, BoCOQ5-2, and BoHMT1). The relative abundance of the total 317 and 203 proteins, respectively, in 4-day-old broccoli sprouts varied, and the metabolic and biosynthetic pathways for secondary metabolites were significantly enriched in ZnSO4/control and ZnSO4 combined Na2SeO3/ZnSO4 comparisons. The findings demonstrated how ZnSO4 combined with Na2SeO3 treatment reduced stress inhibition and the accumulation of encouraged selenium and isothiocyanates during the growth of broccoli sprouts.

Unveiling the functional components and antivirulence activity of mustard leaves using an LC-MS/MS, molecular networking, and multivariate data analysis integrated approach.

Plant extracts have recently received increased attention as alternative sources of antimicrobial agents in the fight against multidrug-resistant bacteria. Non-targeted metabolomics liquid chromatography-quadrupole time-of-flight tandem mass spectrometry, molecular networking, and chemometrics were used to evaluate the metabolic profiles of red and green leaves of two Brassica juncea (L.) varieties, var. integrifolia (IR and IG) and var. rugosa (RR and RG), as well as to establish a relationship between the elucidated chemical profiles and antivirulence activity. In total, 171 metabolites from different classes were annotated and principal component analysis revealed higher levels of phenolics and glucosinolates in var. integrifolia leaves and color discrimination, whereas fatty acids were enriched in var. rugosa, particularly trihydroxy octadecadienoic acid. All extracts demonstrated significant antibacterial activity against Staphylococcus aureus and Enterococcus faecalis, presenting the IR leaves the highest antihemolytic activity against S. aureus (99 % inhibition), followed by RR (84 %), IG (82 %), and RG (37 %) leaves. Antivirulence of IR leaves was further validated by reduction in alpha-hemolysin gene transcription (∼4-fold). Using various multivariate data analyses, compounds positively correlated to bioactivity, primarily phenolic compounds, glucosinolates, and isothiocyanates, were also identified.

Metabolic profiling of Ochradenus baccatus Delile. utilizing UHPLC-HRESIMS in relation to the in vitro biological investigations.

Ochradenus baccatus Delile (Resedaceae) is a desert plant with edible fruits native to the Middle East. Few investigators have reported antibacterial, antiparasitic and anti-cancer activities of the plant. Herein we evaluated the cytotoxic activity of O. baccatus using four cell lines and a zebrafish embryo model. Additionally, liquid chromatography coupled with mass spectroscopy was performed to characterize the extract's main constituents. The highest cytotoxicity was observed against human cervical adenocarcinoma (HeLa), with CC50 of 39.1 µg/mL and a selectivity index (SI) of 7.23 (p < 0.01). Metabolic analysis of the extract resulted in the annotation of 57 metabolites, including fatty acids, flavonoids, glucosinolates, nitrile glycosides, in addition to organic acids. The extract showed an abundance of hydroxylated fatty acids (16 peaks). Further, 3 nitrile glycosides have been identified for the first time in Ochradenus sp., in addition to 2 glucosinolates. These identified phytochemicals may partially explain the cytotoxic activity of the extract. We propose O. baccatus as a possible safe food source for further utilization to partially contribute to the increasing food demand specially in Saharan countries.

Glucosinolates or erucic acid, which one contributes more to volatile flavor of fragrant rapeseed oil?

This study aims to investigate the effect of three rapeseed varieties with different erucic acid (EA) and glucosinolates (GLSs) content, and different degumming methods on the volatile flavor profiles of fragrant rapeseed oil (FRO). A total of 171 volatile compounds were identified by headspace solid-phase microextraction combine with gas chromatography-mass spectrometry (HS-SPME/GC-MS), and 87 compounds were identified as key odorants owing to their relative odor activity values (ROAV) ≥ 1. Methyl furfuryl disulfide was identified in rapeseed oil for the first time, with highest ROAVs (up to 26805.46). The volatile flavor profile of rapeseed oil was affected by GLSs content to a certain extent rather than EA content. Rapeseed varieties with low-EA and high-GLSs are suitable to produce FRO. Silicon dioxide adsorbing was an effective alternative method to water degumming in FRO. This work provided a new idea for selection of raw materials and degumming methods in FRO production.

Glucosinolates and Cytotoxic Activity of Collard Volatiles Obtained Using Microwave-Assisted Extraction.

Glucosinolates (GSLs) in Brassica oleracea L. convar. acephala var. viridis (collard) flower, leaf, stem, and root were analyzed qualitatively and quantitatively via their desulfo-counterparts using UHPLC-DAD-MS/MS. Twelve GSLs were identified, including Met-derived GSLs (sinigrin, glucoibervirin, glucoerucin, glucoiberin, glucoraphanin, progoitrin), Trp-derived GSLs (4-hydroxyglucobrassicin, glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin), and Phe-derived GSLs (glucotropaeolin and gluconasturtiin). Total GSL content was highest in the root, having 63.40 µmol/g dried weight (DW), with gluconasturtiin (34.02 µmol/g DW) as the major GSL, followed by sinigrin and glucoibervirin (12.43 and 7.65 µmol/g DW, respectively). Total GSL contents in the flower, leaf, and stem were lower than in root, having 6.27, 2.64, and 1.84 µmol/g DW, respectively, with Trp and/or Met-derived GSLs as the predominant ones. GSL breakdown products were obtained via microwave hydrodiffusion and gravity (MHG) and volatile breakdown products were analyzed using GC-MS techniques. Volatile isolates were tested for their cytotoxic activity using MTT assay. MHG volatile extract from the root demonstrated the best cytotoxic activity against human bladder cancer cell line T24 and breast cancer cell line MDA-MB-231 during an incubation time of 72 h (IC50 21.58, and 11.62 µg/mL, respectively). The activity of the root extract can be attributed to its major volatile, 2-phenylethyl isothiocyanate (gluconasturtiin breakdown product).

Byproducts of Globe Artichoke and Cauliflower Production as a New Source of Bioactive Compounds in the Green Economy Perspective: An NMR Study.

The recovery of bioactive compounds from crop byproducts leads to a new perspective way of waste reutilization as a part of the circular economy. The present study aimed at an exhaustive metabolite profile characterization of globe artichoke and cauliflower byproducts (leaves, stalks, and florets for cauliflower only) as a prerequisite for their valorization and future implementations. The metabolite profile of aqueous and organic extracts of byproducts was analyzed using the NMR-based metabolomics approach. Free amino acids, organic acids, sugars, polyols, polyphenols, amines, glucosinolates, fatty acids, phospho- and galactolipids, sterols, and sesquiterpene lactones were identified and quantified. In particular, globe artichoke byproducts are a source of health-beneficial compounds including chiro-inositol (up to 10.1 mg/g), scyllo-inositol (up to 1.8 mg/g), sesquiterpene lactones (cynaropicrin, grosheimin, dehydrocynaropicrin, up to 45.5 mg/g in total), inulins, and chlorogenic acid (up to 7.5 mg/g), whereas cauliflower byproducts enclose bioactive sulfur-containing compounds S-methyl-L-cysteine S-oxide (methiin, up to 20.7 mg/g) and glucosinolates. A variable content of all metabolites was observed depending on the crop type (globe artichoke vs. cauliflower) and the plant part (leaves vs. stalks). The results here reported can be potentially used in different ways, including the formulation of new plant biostimulants and food supplements.

In vitro and in vivo biotransformation of glucosinolates from mashua (Tropaeolum tuberosum) by lactic acid bacteria.

Mashua (Tropaeolum tuberosum) is an Andean tuber with a high content of glucosinolates (GLSs). GLSs subjected to biotransformation by plant enzymes or enzymes of the gastrointestinal microbiota give rise to biologically active compounds, to which chemo preventive properties are attributed. In this work, the biotransformation of mashua GLSs was evaluated in vitro by six strains of lactic acid bacteria (LAB) and in vivo using rats with and without previous LAB dosing. The results showed that L. rhamnosus GG utilized the totality of glucosinalbin and glucotropaeolin, and 46.7 % of glucoaubrietin. Four GLSs derivatives were detected. The GLSs were absorbed and metabolized by the rats with low contents in feces (0.02 %) and urine (0.59 %) and were detected up to 3 h after consumption in plasma. The results showed that probiotic bacteria play an important role in transforming GLSs into beneficial compounds for the health of consumers.

Comprehensive two-dimensional gas chromatography-time of flight mass spectrometry as a tool for tracking roasting-induced changes in the volatilome of cold-pressed rapeseed oil.

The aim of this study was to track changes in the volatilome of cold-pressed oil and press cakes obtained from roasted seeds and to combine it with the profile of non-volatile metabolites in a single study, in order to understand pathways of volatile organic compound (VOC) formation caused by thermal processing. Comprehensive two-dimensional gas chromatography-time of flight mass spectrometry was used for the analysis of VOCs in cold-pressed oils and corresponding press cakes obtained after roasting of seeds at 140 and 180 °C prior to pressing. Contents of primary metabolites (amino acids, saccharides, fatty acids) as well as selected secondary metabolites (glucosinolates, polyphenols) were determined, as many of them serve as precursors to volatile compounds formed especially in thermal reactions. After roasting, the formation of Maillard reaction products increased, which corresponded to the reduction of free amino acids and monosaccharides. Moreover, levels of the products of thermal oxidation of fatty acids, such as aldehydes and ketones, increased with the increasing temperature of roasting, although no significant changes were noted for fatty acids. Among sulphur-containing compounds, contents of the products and intermediates of methionine Strecker degradation increased significantly with the increasing temperature of roasting. Degradation of glucosinolates to nitriles occurred after thermal treatment. The results of this study confirmed that seed roasting before cold pressing has a significant effect on the volatiles, but also indicated roasting-induced changes in non-volatile metabolites of oil and press cake. Such an approach helps to understand metabolic changes occurring during rapeseed processing in cold-pressed oil production.

Sulfate supplementation affects nutrient and photosynthetic status of Arabidopsis thaliana and Nicotiana tabacum differently under prolonged exposure to cadmium.

Hydroponic experiments were performed to examine the effect of prolonged sulfate limitation combined with cadmium (Cd) exposure in Arabidopsis thaliana and a potential Cd hyperaccumulator, Nicotiana tabacum. Low sulfate treatments (20 and 40 µM MgSO4) and Cd stress (4 µM CdCl2) showed adverse effects on morphology, photosynthetic and biochemical parameters and the nutritional status of both species. For example, Cd stress decreased NO3- root content under 20 µM MgSO4 to approximately 50% compared with respective controls. Interestingly, changes in many measured parameters, such as chlorophyll and carotenoid contents, the concentrations of anions, nutrients and Cd, induced by low sulfate supply, Cd exposure or a combination of both factors, were species-specific. Our data showed opposing effects of Cd exposure on Ca, Fe, Mn, Cu and Zn levels in roots of the studied plants. In A. thaliana, levels of glutathione, phytochelatins and glucosinolates demonstrated their distinct involvement in response to sub-optimal growth conditions and Cd stress. In shoot, the levels of phytochelatins and glucosinolates in the organic sulfur fraction were not dependent on sulfate supply under Cd stress. Altogether, our data showed both common and species-specific features of the complex plant response to prolonged sulfate deprivation and/or Cd exposure.

Testing hypotheses of a coevolutionary key innovation reveals a complex suite of traits involved in defusing the mustard oil bomb.

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