Aliphatic Glucosinolates Research Articles

Unravelling Glucoraphanin and Glucoerucin Metabolism across Broccoli Sprout Development: Insights from Metabolite and Transcriptome Analysis.

Variations in the concentration of glucoraphanin (GRA) and glucoerucin (GER), as well as the corresponding breakdown products, isothiocyanates (ITCs) and nitriles, were investigated during the growth of broccoli sprouts. The concentrations of GRA and GER decreased sharply from 33.66 µmol/g to 11.48 µmol/g and 12.98 µmol/g to 8.23 µmol/g, respectively, after seed germination. From the third to the seventh day, both GRA and GER were maintained as relatively stable. The highest concentrations of sulforaphane (17.16 µmol/g) and erucin (12.26 µmol/g) were observed on the first day. Hereafter, the concentrations of nitrile hydrolyzed from GRA or GER were higher than those of the corresponding ITCs. Moreover, the ratio of sulforaphane to sulforaphane nitrile decreased from 1.35 to 0.164 from 1 d to 5 d, with a similar trend exhibited for erucin/erucin nitrile after 2 d. RNA-seq analysis showed that BolMYB28 and BolCYP83A1, involved in aliphatic glucosinolate (GSL) biosynthesis, remained largely unexpressed until the third day. In contrast, the genes operating within the GSL-myrosinase hydrolysis pathway were highly expressed right from the beginning, with their expression levels increasing significantly after the third day. Additionally, we identified two BolESPs and six BolNSPs that might play important roles in promoting the production of nitriles during the development of broccoli sprouts.

Does fungal infection increase the palatability of oilseed rape to insects?

Multiple and simultaneous attacks by pathogens and insect pests frequently occur in nature. Plants respond to biotic stresses by activating distinct defense mechanisms, but little is known about how plants cope with multiple stresses. The focus of this study was the combined interaction of fungal infection caused by Leptosphaeria maculans (synonym Plenodomus lingam) and arthropod infestation by the diamondback moth (Plutella xylostella) in oilseed rape (Brassica napus). We hypothesized that infection by the fungal pathogen L. maculans could alter oilseed rape palatability to P. xylostella-chewing caterpillars. Feeding preference tests were complemented with analyses of defense gene transcription, and levels of glucosinolates (GLSs) and volatile organic compounds (VOCs) in L. maculans-inoculated and non-inoculated (control) leaves to determine possible causes of larval choice. Caterpillars preferred true leaves to cotyledons, hence true leaves were used for further experiments. True leaves inoculated with L. maculans were more palatable to caterpillars over control leaves during the early stage of infection at 3 days post inoculation (dpi), but this preference disappeared in the later stages of infection at 7 dpi. In parallel, genes involved in the salicylic acid and ethylene pathways were up-regulated in L. maculans-inoculated leaves at 3 and 7 dpi; L. maculans increased the level of total aliphatic GLSs, specifically glucobrassicanapin, and decreased the level of glucoiberin at 3 dpi and altered the content of specific VOCs. A group of 55 VOCs with the highest variability between treatments was identified. We suggest that the P. xylostella preference for L. maculans-inoculated leaves in the early stage of disease development could be caused by the underlying mechanisms leading to changes in metabolic composition. Further research should pinpoint the compounds responsible for driving larval preference and evaluate whether the behavior of the adult moths, i.e. the stage that makes the first choice regarding host plant selection in field conditions, correlates with our results on larval host acceptance. © 2024 Society of Chemical Industry.

Plant glucosinolate biosynthesis and breakdown pathways shape the rhizosphere bacterial/archaeal community.

Rhizosphere microbial community assembly results from microbe-microbe-plant interactions mediated by small molecules of plant and microbial origin. Studies with Arabidopsis thaliana have indicated a critical role of glucosinolates in shaping the root and/or rhizosphere microbial community, likely through breakdown products produced by plant or microbial myrosinases inside or outside of the root. Plant nitrile-specifier proteins (NSPs) promote the formation of nitriles at the expense of isothiocyanates upon glucosinolate hydrolysis with unknown consequences for microbial colonisation of roots and rhizosphere. Here, we generated the A. thaliana triple mutant nsp134 devoid of nitrile formation in root homogenates. Using this line and mutants lacking aliphatic or indole glucosinolate biosynthesis pathways or both, we found bacterial/archaeal alpha-diversity of the rhizosphere to be affected only by the ability to produce aliphatic glucosinolates. In contrast, bacterial/archaeal community composition depended on functional root NSPs as well as on pathways of aliphatic and indole glucosinolate biosynthesis. Effects of NSP deficiency were strikingly distinct from those of impaired glucosinolate biosynthesis. Our results demonstrate that rhizosphere microbial community assembly depends on functional pathways of both glucosinolate biosynthesis and breakdown in support of the hypothesis that glucosinolate hydrolysis by myrosinases and NSPs happens before secretion of products to the rhizosphere.

Characterization of Arabidopsis eskimo1 reveals a metabolic link between xylan O-acetylation and aliphatic glucosinolate metabolism.

Glucuronoxylan is present mainly in the dicot of the secondary cell walls, often O-acetylated, which stabilizes cell structure by maintaining interaction with cellulose and other cell wall components. Some members of the Golgi localized Trichome Birefringence-Like (TBL) family function as xylan O-acetyl transferase (XOAT). The primary XOAT in the stem of Arabidopsis is ESKIMO1/TBL29, and its disruption results in decreased xylan acetylation, stunted plant growth, and collapsed xylem vessels. To elucidate the effect on metabolic reprogramming and identify the underlying cause of the stunted growth in eskimo1, we performed transcriptomic, targeted, and untargeted metabolome analysis, mainly in the inflorescence stem tissue. RNA sequencing analysis revealed that the genes involved in the biosynthesis, regulation, and transport of aliphatic glucosinolates (GSLs) were upregulated, whereas those responsible for indolic GSL metabolism were unaffected in the eskimo1 inflorescence stem. Consistently, aliphatic GSLs, such as 4-methylsulfinylbutyl (4MSOB), were increased in stem tissues and seeds. This shift in the profile of aliphatic GSLs in eskimo1 was further supported by the quantification of the soluble acetate, decrease in accumulation of GSL precursor, i.e., methionine, and increase in the level of jasmonic acid.

4-Aldrithiol-based photometric assay for detection of methylthioalkylmalate synthase activity.

In Brassica plants, glucosinolates are a diverse class of natural products, of which aliphatic methionine-derived glucosinolates are the most abundant form. Their structural diversity comes from the elongation of some side-chains by up to 9 carbons, which, after the formation of the core glucosinolate structure, can undergo further chemical modifications. Methylthioalkylmalate synthase (MAMS) catalyzes the iterative elongation process for aliphatic methionine-derived glucosinolates. Most biochemical studies on MAMS have been performed using liquid chromatography/mass spectrometry (LC/MS)-based assays or high-performance liquid chromatography (HPLC)-based assays. The LC/MS- and HPLC-based methods are endpoint assays, which cannot be monitored in real time and require a laborious process for data collection. These analytical methods are inefficient for performing multiple enzymatic assays needed to determine steady-state kinetic parameters or for mechanistic evaluation of pH-dependence and kinetic isotope effect studies. Although the function of MAMS has long been defined, there is a gap in knowledge as it pertains to biochemical characterization of this plant enzyme. Part of this may be due to the lack of efficient methods that can be used for this type of research. This chapter describes a continuous photometric assay to track MAMS activity in real time using the 4-aldrithiol reagent for reaction detection.

Flavonols affect the interrelated glucosinolate and camalexin biosynthetic pathways in Arabidopsis thaliana.

Flavonols are structurally and functionally diverse biomolecules involved in plant biotic and abiotic stress tolerance, pollen development, and inhibition of auxin transport. However, their effects on global gene expression and signaling pathways are unclear. To explore the roles of flavonol metabolites in signaling, we performed comparative transcriptome and targeted metabolite profiling of seedlings from the flavonol-deficient Arabidopsis loss-of-function mutant flavonol synthase1 (fls1) with and without exogenous supplementation of flavonol derivatives (kaempferol, quercetin, and rutin). RNA-seq results indicated that flavonols modulate various biological and metabolic pathways, with significant alterations in camalexin and aliphatic glucosinolate synthesis. Flavonols negatively regulated camalexin biosynthesis but appeared to promote the accumulation of aliphatic glucosinolates via transcription factor-mediated up-regulation of biosynthesis genes. Interestingly, upstream amino acid biosynthesis genes involved in methionine and tryptophan synthesis were altered under flavonol deficiency and exogenous supplementation. Quercetin treatment significantly up-regulated aliphatic glucosinolate biosynthesis genes compared with kaempferol and rutin. In addition, expression and metabolite analysis of the transparent testa7 mutant, which lacks hydroxylated flavonol derivatives, clarified the role of quercetin in the glucosinolate biosynthesis pathway. This study elucidates the molecular mechanisms by which flavonols interfere with signaling pathways, their molecular targets, and the multiple biological activities of flavonols in plants.

Large-scale identification of novel transcriptional regulators of the aliphatic glucosinolate pathway in Arabidopsis.

Aliphatic glucosinolates are a large group of plant secondary metabolites characteristic of Brassicaceae, including the model plant Arabidopsis. The diverse and complex degradation products of aliphatic glucosinolates contribute to plant responses to herbivory, pathogen attack, and environmental stresses. Most of the biosynthesis genes in the aliphatic glucosinolate pathway have been cloned in Arabidopsis, and the research focus has recently shifted to the regulatory mechanisms controlling aliphatic glucosinolate accumulation. Up till now, more than 40 transcriptional regulators have been identified as regulating the aliphatic glucosinolate pathway, but many more novel regulators likely remain to be discovered based on research evidence over the past decade. In the current study, we took a systemic approach to functionally test 155 candidate transcription factors in Arabidopsis identified by yeast one-hybrid assay, and successfully validated at least 30 novel regulators that could significantly influence the accumulation of aliphatic glucosinolates in our experimental set-up. We also showed that the regulators of the aliphatic glucosinolate pathway have balanced positive and negative effects, and glucosinolate metabolism and plant development can be coordinated. Our work is the largest scale effort so far to validate transcriptional regulators of a plant secondary metabolism pathway, and provides new insights into how the highly diverse plant secondary metabolism is regulated at the transcriptional level.

Effect of Exogenous Substance K2SO4 on the Nutritional Quality of Broccoli and Its Metabolic Regulation Mechanism

The impact of exogenous sulfate components on the nutritional quality of vegetables has been well documented. In this study, we examined the effects of adding K2SO4 to broccoli on its nutritional quality, active components, and the genes involved in glucosinolate synthesis. Different concentrations of exogenous K2SO4 of 25, 75, and 150 g·m−2 were applied to the “Naihan Youxiu” broccoli cultivar, while the control treatment received no potassium sulfate. Our primary objective was to gain insights into strategies for enhancing broccoli’s nutritional and active components. The results showed that broccoli’s vitamin C content in each treatment was lower than that in the control treatment. The contents of soluble protein, soluble sugar, polyphenols, and total flavonoids in the treatment of 150 g·m−2 K2SO4 were the highest. They increased by 23.51%, 87.49%, 146.00%, and 22.73% more than the control, respectively. MDA was significantly inhibited after the 75 g·m−2 treatment, whereas SOD had the highest activity under the 75 g·m−2 treatment. Therefore, the 150 g·m−2 treatment was beneficial in improving the nutritional quality and antioxidant capacity of broccoli. The contents of PRO, SIN, NAP, GBC, 4ME, NEO, total indole glucosinolates, and total glucosinolates reached the peak at the 150 g·m−2 K2SO4 treatment, RAA and total aliphatic glucosinolates reached the peak at the 75 g·m−2 K2SO4 treatment, and ERU and 4OH reached the highest at the 25 g·m−2 K2SO4 treatment. The sulforaphane content was the highest in the 150 g·m−2 treatment, and myrosinase activity was the highest in the 75 g·m−2 treatment. It can be seen that the 150 g·m−2 treatment significantly increased the content of glucosinolates, total indole glucosinolates, total glucosinolates, and sulforaphane in broccoli. CYP79B2, CYP83B1, CYP83A1, AOP2, UGT74B1, and MYB34 were significantly up-regulated under 150 g·m−2 K2SO4 treatment and reached the peak value. CYP79F1, CYP81F4, and MAM1 showed significant inhibitory effects when treated with 150 g·m−2 of K2SO4. The expression levels of BCAT4, CYP81F1, ST5a, ST5c, and SUR1 were down-regulated under the 150 g·m−2 K2SO4 treatment, but not significantly. In summary, the K2SO4 150 g·m−2 treatment had the best effect on nutritional quality, antioxidant activity, the content of glucosinolates, total glucosinolates, sulforaphane, and expression of CYP79B2, CYP83B1, CYP83A1, FMO2, UGT74B1, AOP2, and MYB34 genes.

Molecular networking and equivalently quantitative ion strategy for discovery and quantification of glucosinolates in cauliflower and broccoli by liquid chromatography tandem mass spectrometry

Glucosinolates (GSLs) are unique secondary metabolites in cauliflower and broccoli, which have diverse biological activities. In this study, a molecular networking combined with the equivalently quantitative ion strategy was built based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for the discovery and quantification of GSLs in cauliflower and broccoli. Firstly, 19 GSLs were identified including 2 new compounds, 4-hydroxyneoglucobrassicin and gluco-4-hydroxyneoglucobrassicin. Moreover, the shared fragment ion m/z 96.0 was set as the equivalently quantitative ion to quantify GSLs. This method could use only one reference standard (or one calibration curve) to efficiently quantify all aliphatic and phenyl GSLs in cauliflower and broccoli. This method was fully validated with good linearity, recovery and sensitivity, and subsequently applied in GSLs quantification. The developed strategy could also be applied in GSLs discovery and quantification in other Brassicaceae vegetables.

Overexpression of BoLSU1 and BoLSU2 Confers Tolerance to Sulfur Deficiency in Arabidopsis by Manipulating Glucosinolate Metabolism.

Sulfur is an essential element for plant growth, development and resistance to environmental stresses. Glucosinolates (GSLs), a group of sulfur rich secondary metabolites found in Brassicaceae plants, are known for their defensive properties against pathogens and herbivores. Due to their integration of a large proportion of total sulfur, their biosynthesis and degradation are closely linked to sulfur metabolism. It has been demonstrated that GSLs can be broken down to release sulfur and facilitate the production of other thio-metabolites when the plant is under stress. However, the regulation of this process is still not fully understood. In this study, we constructed two broccoli LSU (low sulfur responsive) gene overexpressing lines, 35S::BoLSU1 and 35S::BoLSU2, to detect changes in GSL metabolism after sulfur deficiency treatment. The results showed that BoLSU1 and BoLSU2 inhibit the biosynthesis of aliphatic GSLs, while also promoting their degradation and increasing the content of glutathione (GSH), leading to the reallocation of sulfur from the GSL pool to other thio-metabolites such as GSH. Furthermore, this regulation of GSL metabolism mediated by BoLSU1 and BoLSU2 is found to be dependent on myrosinases BGLU28 and BGLU30. Our study provides insight into the physiological role of LSU proteins and their regulation of sulfur metabolism.

A Cold Case—Glucosinolate Levels in Kale Cultivars Are Differently Influenced by Cold Temperatures

Among the Brassica oleracea L. crops, kale has gained increased global recognition in recent years as a healthy food item due to its high nutritional value and versatility. Additionally, the diversity of different kale varieties has started to be explored across large latitudes from the Mediterranean to north temperate climates. Specifically, glucosinolates are the predominant phytochemicals found in kale leaves, contributing to the specific taste of this vegetable, and they are affected by environmental factors such as temperature. To date, no study has investigated the effect of chilling on glucosinolate diversity and, thus, the taste in genetically different kale cultivars at the same time. Given the variability of glucosinolates observed among cultivars, we evaluated the impact of acclimation to cold temperatures on glucosinolate levels in curly kale, Lacinato kale, and a feral type using high-performance liquid chromatography coupled with time-of-flight mass spectrometry (HPLC-ESI-qTOF-MS). We targeted the short-term impact (after 12 h) on glucosinolates as well as the longer-term effect (after seven days) of cold acclimation. Our results revealed different molecular patterns regarding the change in glucosinolates in the feral type compared to curly kale and Lacinato-type kale. In the latter ones, primary aliphatic glucosinolates were induced (the glucoraphanin in Lacinato kale increased by more than 200%). The indole glucobrassicin was not significantly affected. Conversely, in the feral type the indole glucobrassicin was reduced by 35% after cold acclimation, whereas aliphatic glucosinolates were hardly affected. The results indicate that both genetic and environmental factors are important for the composition of glucosinolate patterns in kale. In conclusion, to obtain plants with an improved nutritional value, considering both temperature and the choice of cultivar is crucial during kale cultivation. Future breeding attempts of kale should also emphasize the cultivar-dependent cold acclimation patterns reported here.

Comparative Transcriptomics Reveal the Mechanisms Underlying the Glucosinolate Metabolic Response in Leaf Brassica juncea L. under Cold Stress

Glucosinolates (GSLs) are not only a unique flavor substance from leaf B. juncea but also a major secondary metabolite produced in response to abiotic stresses. Cold stress is one of the most common abiotic stresses in leaf B. juncea; however, the metabolic response pattern of GSLs in leaf B. juncea under cold stress has not yet been reported. In the present study, we analyzed the GSLs content of leaf B. juncea under cold stress and found that it increased and subsequently decreased. According to RNA-seq data, genes related to the synthesis of aliphatic GSLs were significantly upregulated following 24 h of cold stress; genes related to the synthesis of indole GSLs were significantly upregulated following 48 h of cold stress; and BjBGLU25 and BjBGLU27 were significantly upregulated. Further analysis of the correlation between transcription factors and GSLs content revealed that MYB, ERF, IQD, and bHLH may be involved in regulating the GSLs response pattern in leaf B. juncea under cold stress. In particular, an unreported transcription factor, BjMYBS3 (BjuVA05G33250), was found to play a possible role in the synthesis of aliphatic GSLs. And the external application of GSLs increased the ability of leaf B. juncea to cope with cold stress.

Glucosinolate Diversity Analysis in Choy Sum (Brassica rapa subsp. chinensis var. parachinensis) Germplasms for Functional Food Breeding.

The aim of this study was to analyze glucosinolates (GSLs) in germplasm that are currently conserved at the RDA-Genebank. The analysis focused on the glucosinolate diversity among the analyzed germplasms, with the goal of identifying those that would be most useful for future breeding efforts to produce nutritionally rich Choy sum plants. In total, 23 accessions of Choy sums that possessed ample background passport information were selected. On analyzing the glucosinolate content for 17 different glucosinolates, we observed aliphatic GSLs to be the most common (89.45%) and aromatic GSLs to be the least common (6.94%) of the total glucosinolates detected. Among the highly represented aliphatic GSLs, gluconapin and glucobrassicanapin were found to contribute the most (>20%), and sinalbin, glucoraphanin, glucoraphasatin, and glucoiberin were detected the least (less than 0.05%). We identified one of the accessions, IT228140, to synthesize high quantities of glucobrassicanapin and progoitrin, which have been reported to contain several therapeutic applications. These conserved germplasms are potential bioresources for breeders, and the availability of information, including therapeutically important glucosinolate content, can help produce plant varieties that can naturally impact public health.

Widely targeted metabolomics reveal the glucosinolate profile and odor-active compounds in flowering Chinese cabbage powder

Widely targeted metabolomics were performed to explore the differences in glucosinolate and odor-active compound levels between flowering Chinese cabbage powder (FCCP) under vacuum-drying and oven-drying conditions. Twenty-three aliphatic, five indole, and three aromatic glucosinolates were identified in two pretreated FCCP. Higher aliphatic glucosinolate levels were retained in vacuum-dried cabbage powder compared to oven-dried samples, and they were negatively correlated with treated temperatures. A total of 36 major odor contributing compounds were detected, including 5 sulfur compounds, 10 aldehydes, 9 heterocyclic compounds, 7 nitriles, 3 acids, and 2 others. 5-Hexenenitrile and (methyldisulfanyl)methan, provide typical pungent, sulfous, and vegetable notes in FCCP. Four major GSLs, namely 2(R)-hydroxy-3-butenyl glucosinolate, (2S)-2-hydroxy-4-pentenyl glucosinolate, 5-(methylthio)pentyl glucosinolate and 2-phenylethyl glucosinolate were the key precursors to form odor-active compounds. Higher temperatures in thermal effects promotes the formation of sulfur-containing and nitrile compounds compared to the vacuum-dried ones. This work can provide a guide for flavor and nutrition retention in FCCP process.

Glucosinolates: Structure, classification, biosynthesis and functions in higher plants

ABSTRACT
 Objective: To analyze concepts, structure, classification, biosynthesis and functions of glucosinolates (GSLs) in higher plants.
 Design/methodology/approach: A search was performed into recent high-impact literature related to glucosinolates (GSLs).
 Results: GSLs are secondary metabolites rich in N and S. They are divided into aliphatic, aromatic, and indole GSLs depending on the amino acid from which they arise. The products of their hydrolysis, mediated by thioglucoside glucohydrolase, thioglucosidase or myrosinase enzymes (EC 3.2.1.147), play a role in increasing tolerance to biotic and abiotic stress factors. Furthermore, given their composition, they can serve as a nutrient reservoir under nutrient deficiency conditions.
 Limitations on study/implications: GSLs are synthesized only in species of the Capparidaceae, Brassicaceae, Resedaceae, and Moringaceae families.
 Findings/conclusions: GSLs are sulfur compounds that can serve as defense mechanisms against biotic and abiotic stress factors and as sources of nutrients in plants, and molecules with important nutraceutical properties in food and human health.

Plant Glucosinolate Content and Host-Plant Preference and Suitability in the Small White Butterfly (Lepidoptera: Pieridae) and Comparison with Another Specialist Lepidopteran.

Glucosinolates are used in host-plant recognition by insects specialized on Brassicaceae, such as Pieris rapae L. (Lepidoptera: Pieridae). This research investigated the association between P. rapae oviposition and larval survival and host-plant glucosinolate content using 17 plant species in which glucosinolate content had previously been determined. Two-choice oviposition tests (comparing each plant species to Arabidopsis thaliana L.) and larval survival experiments showed that indolic glucosinolate content had a positive effect on oviposition preference and larval survival in P. rapae. In the host plants tested, the effects of indolic glucosinolates on oviposition preference and of glucosinolate complexity index and aliphatic glucosinolates without sulfur-containing side chains on total oviposition were smaller on P. rapae than on Plutella xylostella L. (Lepidoptera: Plutellidae), another lepidopteran specialized on glucosinolate-containing plants. This study suggests that high indolic glucosinolate content could make crop plants more susceptible to both P. rapae and P. xylostella, but this effect seems to be greater for P. xylostella. Additionally, as some differences in oviposition and larval survival between P. rapae and P. xylostella occurred in some individual plants, it cannot be concluded that bottom-up factors are always similar in these two specialist insects.

Overexpression of LSU1 and LSU2 confers cadmium tolerance by manipulating sulfur metabolism in Arabidopsis

Phytoremediation using plants is an environmentally friendly and cost-effective strategy for removing cadmium (Cd) from soil. Plants used for phytoremediation must have a high Cd accumulation capacity and strong Cd tolerance. Therefore, understanding the molecular mechanism of Cd tolerance and accumulation in plants is of great interest. In response to Cd exposure, plants produce various thio-rich compounds, such as glutathione, phytochelatins, and metallothioneins, which play important roles in Cd immobilization, sequestration, and detoxification. Therefore, sulfur (S) metabolism is crucial for Cd tolerance and accumulation. In this study, we report that the overexpression of low-S responsive genes, LSU1 and LSU2, confers Cd tolerance in Arabidopsis. First, LSU1 and LSU2 promoted S assimilation under Cd stress. Second, LSU1 and LSU2 inhibited the biosynthesis and promoted the degradation of aliphatic glucosinolates, which could limit the consumption and enhance the release of S, thus, facilitating the production of the S-rich metabolites, glutathione, phytochelatins, and metallothioneins. We further demonstrated that the Cd tolerance mediated by LSU1 and LSU2 was dependent on the myrosinases BGLU28 and BGLU30, which catalyze the degradation of aliphatic glucosinolates. In addition, the overexpression of LSU1 and LSU2 improved Cd accumulation, which has great potential for the phytoremediation of Cd-contaminated soil.

CRISPR/Cas9-mediated BoaAOP2s editing alters aliphatic glucosinolate side-chain metabolic flux and increases the glucoraphanin content in Chinese kale

Glucoraphanin (GRA) is an aliphatic glucosinolate (GSL), and its hydrolysis product has powerful anticancer activity. ALKENYL HYDROXALKYL PRODUCING 2 (AOP2) gene, encodes a 2-oxoglutarate-dependent dioxygenase, which can catalyze GRA to form gluconapin (GNA). However, GRA only present in trace amounts in Chinese kale. To increase the content of GRA in Chinese kale, three copies of BoaAOP2 were isolated and edited using CRISPR/Cas9 system. The content of GRA was 11.71- to 41.29-fold (0.082–0.289 μmol g−1 FW) higher in T1 generation of boaaop2 mutants than in wild-type plants, and this was accompanied by an increase in the GRA/GNA ratio and reductions in the content of GNA and total aliphatic GSLs. BoaAOP2.1 is an effective gene for the alkenylation of aliphatic GSLs in Chinese kale. Overall, targeted editing of CRISPR/Cas9-mediated BoaAOP2s altered aliphatic GSL side-chain metabolic flux and enhanced the GRA content in Chinese kale, suggesting that metabolic engineering of BoaAOP2s has huge potential in improving nutritional quality of Chinese kale.

Daytime of leaf wounding determines plant biomass and affect the interplay between growth and defense in Brassica crops.

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 aim to study the connection between daytime, growth and defense to understand how plant fitness is affected by diurnal stress inputs. To accomplish that, we examined if simulated herbivory (leaf wounding) in the morning, mid-day or evening differentially influenced plant defensive states vs growth in three different crop species of the Brassica genus, broccoli (Brassica oleraceae), turnip greens (B. rapa) and rapeseed (B. napus). The data presented here revealed that plant's ability to tolerate wounding stress is diurnally regulated in Brassica crops. Trade-offs between plant biomass and investment in glucosinolates (GSL) and phenolics were affected by daytime of leaf stress. Negative correlations between plant biomass and induction of defensive compounds were found for morning and evening treated plants. However, these correlations were positives for mid-day treatment. Interestingly, we revealed new connection between plant growth and changes of aliphatic GSLs and flavonoids in response to wounding. These data suggest that metabolic stress-dependent circadian oscillations of leaves defenses could be one mechanism conferring competitive advantage to plants able to anticipate daily environmental variations by synchronize growth with them. Moreover, this work provides first insights of how secondary metabolites are linked to growth response in a timely manner.

Identification of glucosinolates and volatile odor compounds in microwaved radish (Raphanus sativus L.) seeds and the corresponding oils by UPLC-IMS-QTOF-MS and GC × GC-qMS analysis

The effect of microwave treatment on the content of glucosinolates (GSL) in radish seeds and volatile odor compounds in the microwaved radish seed oils (MRSO) is still unclear. In this study, a total of 13 GSL were identified and quantified in five radish seed varieties by UPLC-IMS-QTOF-MS, among which glucoraphenin, glucoraphasatin, glucoerucin accounting for up to 90 %. Total GSL decreased by 47.39–67.88% after microwave processing. Moreover, 58 odor compounds were identified in MRSO, including 6 sulfides, 12 nitriles, 2 isothiocyanates, 10 alcohols, 12 aldehydes, 5 ketones, 6 acids, and 5 others. The major odor compounds were (methyldisulfanyl)methane, dimethyltrisulfane, (methylsulfinyl)methane, 3-(methylsulfanyl)-1-propanol, methyl thiocyanate, hexanenitrile, 5-(methylsulfanyl)pentanenitrile, and 4-isothiocyanato-1-butene with odor activity value (OAV) higher than 1. The principal components analysis (PCA) results can distinguish MRSO from five different radish seed varieties, three of which (H20-18, H20-19 and H20-28) were in one group and other two (H20-23 and H20-26) were in another group. In addition, aliphatic GSL showed positive correlations with sulfides, isothiocyanates, and nitriles, while negative correlations with alcohols. This work provides a new insight into the odor contribution of GSL degradation products.