Glucosinolate Biosynthesis Research Articles

Molecular insights: Proteomic and metabolomic dissection of plasma-induced growth and functional compound accumulation in Raphanus sativus

This study investigated the impact of plasma-activated water (PAW) on Raphanus sativus (radish) roots at the level of proteins and metabolites. PAW treatment induced the accumulation of reactive oxygen species (ROS) and nitrogen oxide species (NOx) in radish and enhanced the activities of antioxidant enzymes. Proteomic analysis resulted in the identification of 6054 proteins, including 1845 PAW-modulated proteins that were majorly associated with energy metabolism, ROS-detoxification, phytohormones signaling, and biosynthesis of glucosinolates. Subsequent metabolomics analysis identified 314 metabolites, of which 194 showed significant differences in response to PAW treatment. In particular, PAW treatment triggered the accumulation of functional compounds such as vitamin C, vitamin B5, glutathione, and glucosinolates, the well-known characteristic compounds of the Brassicaceae family. Further, integrating proteomics and metabolomics data provided novel insights into the molecular mechanism governing plasma-induced growth and the accumulation of these functional compounds in radish plants.

Cell-type-specific responses to fungal infection in plants revealed by single-cell transcriptomics

Pathogen infection is a dynamic process. Here, we employ single-cell transcriptomics to investigate plant response heterogeneity. By generating an Arabidopsis thaliana leaf atlas encompassing 95,040 cells during infection by a fungal pathogen, Colletotrichum higginsianum, we unveil cell-type-specific gene expression, notably an enrichment of intracellular immune receptors in vasculature cells. Trajectory inference identifies cells that had different interactions with the invading fungus. This analysis divulges transcriptional reprogramming of abscisic acid signaling specifically occurring in guard cells, which is consistent with a stomatal closure dependent on direct contact with the fungus. Furthermore, we investigate the transcriptional plasticity of genes involved in glucosinolate biosynthesis in cells at the fungal infection sites, emphasizing the contribution of the epidermis-expressed MYB122 to disease resistance. This work underscores spatially dynamic, cell-type-specific plant responses to a fungal pathogen and provides a valuable resource that supports in-depth investigations of plant-pathogen interactions.

Agricultural Jiaosu Enhances the Stress Resistance of Pak Choi (Brassica rapa L. subsp. chinensis) by Recruiting Beneficial Rhizosphere Bacteria and Altering Metabolic Pathways

Agricultural Jiaosu (AJ) is a method of recycling agricultural wastes for improving soil properties, promoting plant growth, and enhancing plant stress resistance. However, the underlying mechanism by which AJ improves plant stress resistance needs to be determined. Therefore, in this study, two treatments of AJ spraying and water spraying were set up to determine the enzyme activities related to the stress resistance of pak choi after 30 days of growth, and the potential mechanism of AJ’s influence on the stress resistance of pak choi was revealed by transcriptome, metabolome, and rhizome microbiome analyses. Microbial community analysis revealed that the application of AJ does not alter microbial abundance in the rhizosphere; however, it can improve microbial diversity and enrich Actinobacteriota, Proteobacteria, and Firmicutes in the pak choi rhizosphere. Metabolomic analysis revealed that these phyla were significantly positively correlated, with highly upregulated metabolites. Our findings suggest that AJ recruits beneficial microorganisms (BMs) in the rhizosphere and stimulates the expression of genes and metabolites involved in phenylpropanoid and glucosinolate biosynthesis, as well as glutathione and alpha-linolenic acid metabolism pathways. The use of AJ could considerably minimise the use of pesticides and fertilisers and improve the quality of the ecological environment.

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.

Transcriptome and metabolome integrated analysis revealed the effects and potential mechanism of hydrogen peroxide on antioxidant system in postharvest broccoli

To investigate the effects and potential mechanism of exogenous H2O2 on antioxidant metabolism and yellowing in postharvest broccoli, the transcriptome and metabolome integrated analysis were performed. A total of 61 differentially expressed genes and 6 differentially accumulated metabolites identified among of H2O2 and diphenylene iodonium (DPI) treatments on 2 and 4 d. Exogenous H2O2 promoted the accumulation of endogenous H2O2 within 1 d by activating RBOHs expressions·H2O2 treatment up-regulated the expressions of SOD, CAT and peroxiredoxin (Prx) genes and APX, DHAR and GPX genes involved in ascorbic acid-glutathione (AsA-GSH) cycle, thereby promoting the scavenging of O2-. and H2O2 and alleviating the oxidative damage. Additionally, H2O2 treatment promoted the biosynthesis of AsA and GSH, and increased the content of AsA, GSH, oxidized glutathione, and γ-glutamylcysteine·H2O2 treatment also promoted glucosinolate biosynthesis, the accumulation of 3-(6′-methylthio)hexylmalic acid, and the production of sulforaphane by up-regulating MYS expression, thereby enhancing the health-care function of postharvest broccoli. This study provided the new insights into regulatory mechanism of H2O2 on antioxidant system.

The effect of glutathione on glucosinolate biosynthesis through the sulfur assimilation pathway in pakchoi associated with the growth conditions

Glucosinolates (GSLs) are a group of nitrogen- and sulfur-containing secondary metabolites, synthesized primarily in members of the Brassicaceae family, that play an important role in food flavor, plant antimicrobial activity, resistance to insect attack, stress tolerance, and human anti-cancer effects. As a sulfur-containing compound, glutathione has a strong connection with GSLs biosynthesis as a sulfur donor or redox system, and exists in reduced (glutathione; GSH) and oxidized (glutathione disulfide; GSSG) forms. However, the mechanism of GSH regulating GSLs biosynthesis remainds unclear. Hence, the exogenous therapy to pakchoi under normal growth condition and sulfur deficiency condition were conducted in this work to explore the relevant mechanism. The results showed that exogenous application of buthionine sulfoximine, an inhibitor of GSH synthesis, decreased the transcript levels of GSLs synthesis-related genes and transcription factors, as well as sulfur assimilation-related genes under the normal growth condition. Application of exogenous GSH inhibited the expression of GSLs synthesis- and sulfur assimilation-related genes under the normal condition, while the GSLs biosynthesis and the sulfur assimilation pathway were activated by exogenous application of GSH when the content of GSH in vivo of plants decreased owing to sulfur deficiency. Moreover, exogenous application of GSSG increased the transcript levels of GSLs synthesis- and sulfur assimilation-related genes under the normal growth condition and under sulfur deficiency. The present work provides new insights into the molecular mechanisms of GSLs biosynthesis underlying glutathione regulation.

Crystal structure of Arabidopsis thaliana sulfotransferase SOT16 involved in glucosinolate biosynthesis

Glucosinolates (GSLs), a class of secondary metabolites found in Brassicaceae plants, play important roles in plant defense and contribute distinct flavors and aromas when used as food ingredients. Following tissue damage, GSLs undergo enzymatic hydrolysis to release bioactive volatile compounds. Understanding GSL biosynthesis and enzyme involvement is crucial for improving crop quality and advancing agriculture. Plant sulfotransferases (SOTs) play a key role in the final step of GSL biosynthesis by transferring sulfate groups to the precursor molecules. In the present study, we investigated the enzymatic reaction mechanism and broad substrate specificity of Arabidopsis thaliana sulfotransferase AtSOT16, which is involved in GSL biosynthesis, using crystal structure analysis. Our analysis revealed the specific catalytic residues involved in the sulfate transfer reaction and supported the hypothesis of a concerted acid-base catalytic mechanism. Furthermore, the docking models showed a strong correlation between the substrates with high predicted binding affinities and those experimentally reported to exhibit high activity. These findings provide valuable insights into the enzymatic reaction mechanisms and substrate specificity of GSL biosynthesis. The information obtained in this study may contribute to the development of novel strategies for manipulating GSL synthesis pathways in Brassica plants and has potential agricultural applications.

The allotetraploid horseradish genome provides insights into subgenome diversification and formation of critical traits

Polyploidization can provide a wealth of genetic variation for adaptive evolution and speciation, but understanding the mechanisms of subgenome evolution as well as its dynamics and ultimate consequences remains elusive. Here, we report the telomere-to-telomere (T2T) gap-free reference genome of allotetraploid horseradish (Armoracia rusticana) sequenced using a comprehensive strategy. The (epi)genomic architecture and 3D chromatin structure of the A and B subgenomes differ significantly, suggesting that both the dynamics of the dominant long terminal repeat retrotransposons and DNA methylation have played critical roles in subgenome diversification. Investigation of the genetic basis of biosynthesis of glucosinolates (GSLs) and horseradish peroxidases reveals both the important role of polyploidization and subgenome differentiation in shaping the key traits. Continuous duplication and divergence of essential genes of GSL biosynthesis (e.g., FMOGS-OX, IGMT, and GH1 gene family) contribute to the broad GSL profile in horseradish. Overall, the T2T assembly of the allotetraploid horseradish genome expands our understanding of polyploid genome evolution and provides a fundamental genetic resource for breeding and genetic improvement of horseradish.

Profiling of Health-Promoting and Taste-Relevant Compounds in Sixteen Radish (Raphanus sativus L.) Genotypes Grown under Controlled Conditions.

It is becoming increasingly challenging to maintain crop yields and quality as the global climate changes. The aim of this study was to determine whether and how the profile of health-promoting and taste-related compounds of radishes changes within a growing season. A total of 16 radish (Raphanus sativus L.) genotypes that are commercially available on the Czech market were assessed by means of chemical analysis. Radishes were cultivated in three independent growing cycles under controlled conditions, and the effects of the genotype and growing cycle, as well as their interactions, on the chemical traits were evaluated. Most of the variability in chemical composition was associated with the growing cycle, which accounted for 51.53% of total variance, followed by the genotype (26% of total variance). The interaction between the growing cycle and genotype explained 22.47% of total variance. The growing cycle had the strongest effect on amino acid profiles. More specifically, the amino acids that are known to contribute to overall taste (glycine, along with glutamic and aspartic acids) showed the highest degree of variation, while the amino acids related to glucosinolate biosynthesis (methionine, isoleucine, tryptophan, and phenylalanine) showed relatively low variability. On the other hand, indole glucosinolates were found to differ the most between genotypes.

Salicylic acid treatment alleviates the loss of glucosinolates accompanying yellowing in harvested broccoli

Commercial value of broccoli is seriously affected by the deterioration of its organoleptical and nutritional quality after harvest. Here, we treated broccoli with salicylic acid (SA) to evaluate its impact on the yellowing process and its protective effect on glucosinolates content during shelf life. SA treatment visibly postponed the yellowing process, as evidenced by higher chlorophyll content and h◦ values as well as lower L* values and yellowing index. Meanwhile, higher levels of glucoraphanin, glucobrassicin, and neoglucobrassicin were accumulated in the treatment group. It was attributed to the activation of glucosinolates biosynthetic capacity by SA treatment, as evidenced by the up-regulated expressions of key regulators BoBCAT3, BoCYP83A1, BoCYP83B1, BoMYB28, BoMYB34, and BoMYB122. In addition, the treatment suppressed the increment of ethylene release while facilitating the biosynthesis of endogenous SA, brassinolide, and jasmonic acid. Statistical analysis provided evidence for SA treatment directly promoting glucosinolate biosynthesis in broccoli and indirectly mitigating glucosinolates loss through hormonal interactions.

Glucosinolates Biosynthesis, Characterization and Chemopreventive Properties

Glucosinolates Biosynthesis, Characterization and Chemopreventive Properties

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.

1-Methylcyclopropene promotes glucosinolate biosynthesis through BrWRKY12 mediated jasmonic acid biosynthesis in postharvest flowering Chinese cabbage

Flowering Chinese cabbage is a Brassica vegetable that is rich in glucosinolates with health-promoting effects. 1-Methylcyclopropene (1-MCP) plays a pivotal role in procrastinating aging and maintaining the quality of postharvest vegetables and fruit products. However, understanding of the underlying molecular mechanisms of 1-MCP involved in regulating glucosinolate biosynthesis is still limited and needs further studies. In present study, a total of eight glucosinolates were detected in flowering Chinese cabbage, containing five aliphatic, two indole, and one aromatic glucosinolates. 1-MCP could delay aging, enhance glucosinolate accumulation, and promote the transcripts of key glucosinolate biosynthesis genes (BrCYP79B2, BrCYP79F1, BrCYP83A2, BrCYP83B1, BrMYB28, and BrCYP83A1). Based on the transcriptome analyses, we noticed that BrWRKY12 was an interesting pivotal WRKY transcription factor and BrLOX4 was a key gene related to the jasmonic acid (JA) biosynthesis pathway involved in improving glucosinolate biosynthesis, which was mediated by 1-MCP. Furthermore, yeast one‐hybrid, dual luciferase, and β‐glucuronidase tissue staining experiments also indicated that BrWRKY12 could directly bind to the BrLOX4 promoter region and activate its transcript. While silencing BrWRKY12 decreased JA content, down-regulated the transcripts of BrLOX4, BrCYP79B2, BrCYP79F1, BrCYP83A2, BrCYP83B1, BrMYB28, and BrCYP83A1, and reduced the glucosinolate accumulation. The results of this work advance our understanding of 1-MCP-enhanced bioactive components and initially confirmed that 1-MCP promoted JA content through BrWRKY12-mediated JA biosynthesis, resulting in improving the glucosinolate accumulation in postharvest flowering Chinese cabbage.

Dynamic Changes in Plant Secondary Metabolites Induced by Botrytis cinerea Infection.

In response to pathogen infection, some plants increase production of secondary metabolites, which not only enhance plant defense but also induce fungicide resistance, especially multidrug resistance (MDR) in the pathogen through preadaptation. To investigate the cause of MDR in Botrytis cinerea, grapes 'Victoria' (susceptible to B. cinerea) and 'Shine Muscat' (resistant to B. cinerea) were inoculated into seedling leaves with B. cinerea, followed by extraction of metabolites from the leaves on days 3, 6, and 9 after inoculation. The extract was analyzed using gas chromatography/quadrupole time-of-flight mass (GC/QTOF) combined with solid-phase microextraction (SPME) for volatile and nonvolatile metabolomic components. Nonvolatile metabolites γ-aminobutyric acid (GABA), resveratrol, piceid, and some carbohydrates or amino acids, coupled with volatile metabolites β-ocimene, α-farnesene, caryophyllene, germacrene D, β-copaene, and alkanes, accumulated at a higher level in grape leaves infected with B. cinerea compared to in noninoculated leaves. Among the established metabolic pathways, seven had greater impacts, including aminoacyl-tRNA biosynthesis, galactose metabolism, valine, leucine, and isoleucine biosynthesis. Furthermore, isoquinoline alkaloid biosynthesis; phenylpropanoid biosynthesis; monobactam biosynthesis; tropane, piperidine, and pyridine alkaloid biosynthesis; phenylalanine metabolism; and glucosinolate biosynthesis were related to antifungal activities. Based on liquid chromatography/quadrupole time-of-flight mass (LC/QTOF) detection and bioassay, B. cinerea infection induced production of plant secondary metabolites (PSMs) including eugenol, flavanone, reserpine, resveratrol, and salicylic acid, which all have inhibitory activity against B. cinerea. These compounds also promoted overexpression of ATP-binding cassette (ABC) transporter genes, which are involved in induction of MDR in B. cinerea.

Effects of light intensity on the biosynthesis of glucosinolate in Chinese cabbage plantlets

Glucosinolates variations in six cultivars of Chinese cabbage plantlets under different light intensities (50, 100, 200, 400, and 600 μmol·m −2·s −1) were analyzed. Nineteen glucosinolates were identified and quantified using UHPLC-QTOF-MS. Different light intensities affected the levels of glucosinolates in Chinese cabbage plantlets. Chinese cabbage plantlets had the highest synthesis capacity of the beneficial glucosinolate glucoraphanin at 200 μmol·m −2·s −1 light intensity while for the anti-nutritional glucosinolate progoitrin at 50 μmol·m −2·s −1 light intensity. BCAT4 was the key gene for the biosynthesis of the aliphatic glucosinolates glucobrassicanapin, glucoerucin, glucoberteroin, and glucoalyssin, whereas the upregulation of SUR1 was attributed to indolic glucosinolate biosynthesis. Chinese cabbage plantlets showed a similar distribution of glucosinolates under 400 μmol·m −2·s −1 with that of 600 μmol·m −2·s −1based on the principal component analysis. Our results indicated that the effects of light intensity on the glucosinolate formations in Chinese cabbage plantlets, which provides new insights.

Identification of new potential downstream transcriptional targets of the strigolactone pathway including glucosinolate biosynthesis.

Strigolactones regulate shoot branching and many aspects of plant growth, development, and allelopathy. Strigolactones are often discussed alongside auxin because they work together to inhibit shoot branching. However, the roles and mechanisms of strigolactones and how they act independently of auxin are still elusive. Additionally, there is still much in general to be discovered about the network of molecular regulators and their interactions in response to strigolactones. Here, we conducted an experiment in Arabidopsis with physiological treatments and strigolactone mutants to determine transcriptional pathways associated with strigolactones. The three physiological treatments included shoot tip removal with and without auxin treatment and treatment of intact plants with the auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA). We identified the glucosinolate biosynthesis pathway as being upregulated across strigolactone mutants indicating strigolactone-glucosinolate crosstalk. Additionally, strigolactone application cannot restore the highly branched phenotype observed in glucosinolate biosynthesis mutants, placing glucosinolate biosynthesis downstream of strigolactone biosynthesis. Oxidative stress genes were enriched across the experiment suggesting that this process is mediated through multiple hormones. Here, we also provide evidence supporting non-auxin-mediated, negative feedback on strigolactone biosynthesis. Increases in strigolactone biosynthesis gene expression seen in strigolactone mutants could not be fully restored by auxin. By contrast, auxin could fully restore auxin-responsive gene expression increases, but not sugar signaling-related gene expression. Our data also point to alternative roles of the strigolactone biosynthesis genes and potential new signaling functions of strigolactone precursors. In this study, we identify a strigolactone-specific regulation of glucosinolate biosynthesis genes indicating that the two are linked and may work together in regulating stress and shoot ranching responses in Arabidopsis. Additionally, we provide evidence for non-auxinmediated feedback on strigolactone biosynthesis and discuss this in the context of sugar signaling.

Identification and in vitro enzymatic activity analysis of the AOP2 gene family associated with glucosinolate biosynthesis in Tumorous stem mustard (Brassica juncea var. tumida).

The major enzyme encoded by the glucosinolate biosynthetic gene AOP2 is involved in catalyzing the conversion of glucoiberin (GIB) into sinigrin (SIN) in Brassicaceae crops. The AOP2 proteins have previously been identified in several Brassicaceae species, but not in Tumorous stem mustard. As per this research, the five identified members of the AOP2 family from the whole genome of Brassica juncea named BjuAOP2.1-BjuAOP2.5 were found to be evenly distributed on five chromosomes. The subcellular localization results implied that BjuAOP2 proteins were mainly concentrated in the cytoplasm. Phylogenetic analysis of the AOP2 proteins from the sequenced Brassicaceae species in BRAD showed that BjuAOP2 genes were more closely linked to Brassica carinata and Brassica rapa than Arabidopsis. In comparison with other Brassicaceae plants, the BjuAOP2 members were conserved in terms of gene structures, protein sequences, and motifs. The light response and hormone response elements were included in the BjuAOP2 genes' cis-regulatory elements. The expression pattern of BjuAOP2 genes was influenced by the different stages of development and the type of tissue being examined. The BjuAOP2 proteins were used to perform the heterologous expression experiment. The results showed that all the five BjuAOP2 proteins can catalyze the conversion of GIB to SIN with different catalytic activity. These results provide the basis for further investigation of the functional study of BjuAOP2 in Tumorous stem mustard glucosinolate biosynthesis.

AtBBX29 integrates photomorphogenesis and defense responses in Arabidopsis

Light is an environmental signal that modulates plant defenses against attackers. Recent research has focused on the effects of light on defense hormone signaling; however, the connections between light signaling pathways and the biosynthesis of specialized metabolites involved in plant defense have been relatively unexplored. Here, we show that Arabidopsis BBX29, a protein that belongs to the B-Box transcription factor (TF) family, integrates photomorphogenic signaling with defense responses by promoting flavonoid, sinapate and glucosinolate accumulation in Arabidopsis leaves. AtBBX29 transcript levels were up regulated by light, through photoreceptor signaling pathways. Genetic evidence indicated that AtBBX29 up-regulates MYB12 gene expression, a TF known to induce genes related to flavonoid biosynthesis in a light-dependent manner, and MYB34 and MYB51, which encode TFs involved in the regulation of glucosinolate biosynthesis. Thus, bbx29 knockout mutants displayed low expression levels of key genes of the flavonoid biosynthetic pathway, and the opposite was true in BBX29 overexpression lines. In agreement with the transcriptomic data, bbx29 mutant plants accumulated lower levels of kaempferol glucosides, sinapoyl malate, indol-3-ylmethyl glucosinolate (I3M), 4-methylsulfinylbutyl glucosinolate (4MSOB) and 3-methylthiopropyl glucosinolate (3MSP) in rosette leaves compared to the wild-type, and showed increased susceptibility to the necrotrophic fungus Botrytis cinerea and to the herbivore Spodoptera frugiperda. In contrast, BBX29 overexpressing plants displayed increased resistance to both attackers. In addition, we found that AtBBX29 plays an important role in mediating the effects of ultraviolet-B (UV-B) radiation on plant defense against B. cinerea. Taken together, these results suggest that AtBBX29 orchestrates the accumulation of specific light-induced metabolites and regulates Arabidopsis resistance against pathogens and herbivores.Graphical

Reduced glucosinolate content in oilseed rape (Brassica napus L.) by random mutagenesis of BnMYB28 and BnCYP79F1 genes

The presence of anti-nutritive compounds like glucosinolates (GSLs) in the rapeseed meal severely restricts its utilization as animal feed. Therefore, reducing the GSL content to < 18 µmol/g dry weight in the seeds is a major breeding target. While candidate genes involved in the biosynthesis of GSLs have been described in rapeseed, comprehensive functional analyses are missing. By knocking out the aliphatic GSL biosynthesis genes BnMYB28 and BnCYP79F1 encoding an R2R3 MYB transcription factor and a cytochrome P450 enzyme, respectively, we aimed to reduce the seed GSL content in rapeseed. After expression analyses on single paralogs, we used an ethyl methanesulfonate (EMS) treated population of the inbred winter rapeseed ‘Express617’ to detect functional mutations in the two gene families. Our results provide the first functional analysis by knock-out for the two GSL biosynthesis genes in winter rapeseed. We demonstrate that independent knock-out mutants of the two genes possessed significantly reduced seed aliphatic GSLs, primarily progoitrin. Compared to the wildtype Express617 control plants (36.3 µmol/g DW), progoitrin levels were decreased by 55.3% and 32.4% in functional mutants of BnMYB28 (16.20 µmol/g DW) and BnCYP79F1 (24.5 µmol/g DW), respectively. Our study provides a strong basis for breeding rapeseed with improved meal quality in the future.

Genome-Wide Association Study of Glucosinolate Metabolites (mGWAS) in Brassica napus L.

Glucosinolates (GSLs) are secondary plant metabolites that are enriched in rapeseed and related Brassica species, and they play important roles in defense due to their anti-nutritive and toxic properties. Here, we conducted a genome-wide association study of six glucosinolate metabolites (mGWAS) in rapeseed, including three aliphatic glucosinolates (m145 gluconapin, m150 glucobrassicanapin and m151 progoitrin), one aromatic glucosinolate (m157 gluconasturtiin) and two indole glucosinolates (m165 indolylmethyl glucosinolate and m172 4-hydroxyglucobrassicin), respectively. We identified 113 candidate intervals significantly associated with these six glucosinolate metabolites. In the genomic regions linked to the mGWAS peaks, 187 candidate genes involved in glucosinolate biosynthesis (e.g., BnaMAM1, BnaGGP1, BnaSUR1 and BnaMYB51) and novel genes (e.g., BnaMYB44, BnaERF025, BnaE2FC, BnaNAC102 and BnaDREB1D) were predicted based on the mGWAS, combined with analysis of differentially expressed genes. Our results provide insight into the genetic basis of glucosinolate biosynthesis in rapeseed and should facilitate marker-based breeding for improved seed quality in Brassica species.