Glucosinolate Biosynthesis Genes Research Articles

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.

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.

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.

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.

Melatonin regulated glucosinolate profile via modulation of genes related with sulfur and nitrogen metabolism in Brassica rapa ssp. pekinensis

Melatonin (MT) is a multi–regulatory molecule eliciting pleiotropic effects on plant growth and development. Glucosinolates (GS) are a group of sulfur (S)-rich and nitrogen (N)-containing secondary metabolites that play an important role in plant defense. Although MT treatment is known to strongly induce expression of GS metabolism related genes, it still remains unclear how MT modulates GS profile. In order to better understand the molecular mechanisms of MT-regulated GS profile in Brassica rapa, comparative transcriptomics and HPLC analyses were conducted. Upon MT treatment, overall GS accumulation increased 3.2-fold, along with emergence of three new GS of glucocochlearin (GCR), glucoerucin (GEC) and 4-hydroxyglucobrassicin (4-OHGBC), and aliphatic GS was the most abundant form constituting 63% of the total GS content. Besides, MT treatment generated eleven differentially expressed genes involved in GS biosynthesis, among which eight encode GS pathway enzymes, and three encode MYB transcription factors modulating expression of GS biosynthesis genes. This was accompanied by significantly elevated expression of three S assimilation-related genes and glutamine synthetase gene involved in N assimilation. In vitro disk-diffusion bioassay demonstrated that lyophilized powder of MT-treated rosette leaf, as well as hydrolyzed products of GCR and GEC significantly inhibited the growth of Sclerotinia sclerotiorum. There was a statistically significant negative correlation between 4-OHGBC and colony growth rate, and between 4-methoxyglucobrassicin (4MeGBC) and sclerotia number. Taken together, the results suggested that exogenous MT treatment altered GS profiles and contents by activating the expression of the primary metabolism genes for S and N assimilation, as well as the secondary metabolism genes for GS biosynthesis, thereby playing a prominent role in S. sclerotiorum resistance in B. rapa. MT as an amphiphilic bio-substance, which can easily pass though the cell membrane, may offer a new integrated strategy for ecofriendly and sustainable management to control plant diseases.

Light irradiation maintains the sensory quality, health-promoting phytochemicals, and antioxidant capacity of post-harvest baby mustard.

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

Revisiting a GWAS peak in Arabidopsis thaliana reveals possible confounding by genetic heterogeneity.

Genome-wide association studies (GWAS) have become a standard approach for exploring the genetic basis of phenotypic variation. However, correlation is not causation, and only a tiny fraction of all associations have been experimentally confirmed. One practical problem is that a peak of association does not always pinpoint a causal gene, but may instead be tagging multiple causal variants. In this study, we reanalyze a previously reported peak associated with flowering time traits in Swedish Arabidopsis thaliana population. The peak appeared to pinpoint the AOP2/AOP3 cluster of glucosinolate biosynthesis genes, which is known to be responsible for natural variation in herbivore resistance. Here we propose an alternative hypothesis, by demonstrating that the AOP2/AOP3 flowering association can be wholly accounted for by allelic variation in two flanking genes with clear roles in regulating flowering: NDX1, a regulator of the main flowering time controller FLC, and GA1, which plays a central role in gibberellin synthesis and is required for flowering under some conditions. In other words, we propose that the AOP2/AOP3 flowering-time association may be yet another example of a spurious, "synthetic" association, arising from trying to fit a single-locus model in the presence of two statistically associated causative loci. We conclude that caution is needed when using GWAS for fine-mapping.

Activating the MYB51 and MYB122 to upregulate the transcription of glucosinolates biosynthesis genes by copper ions in Arabidopsis

Copper ions (Cu2+) are key constituents of copper-based antimicrobial compounds (CBACs), which are extensively used in agriculture. Previously, we demonstrated that a low concentration of Cu2+ induced plant defenses associated with callose deposition in Arabidopsis as well as flg22, a microbe-associated molecular pattern (MAMP) peptide. However, the details and differences of the mechanisms between Cu2+- and flg22-mediated callose deposition remain unclear. Here, we reported that Cu2+- and flg22-induced defense responses and callose deposition are dependent on AtACS8 and AtACS2/AtACS6, respectively. After the RNA sequencing data were mined, the expression of MYB51, MYB122, CYP79B2/B3 and CYP83B1 implied that a conserved downstream indole glucosinolate (IGS) metabolic pathway is regulated by Cu2+. In the Cu2+-induced response, the ethylene biosynthesis rate-limiting gene AtACS8 and the signal transduction pathway were found to be required for Cu2+-activated MYB51 and MYB122 transcription. Functional redundancy of MYB51 and MYB122, the key regulators of the IGS metabolic pathway, was identified in the Cu2+-mediated regulation of IGS gene transcription, promotion of callose deposition, and increase in Arabidopsis resistance to bacterial pathogens. Furthermore, IGS genes such as CYP79B2, CYP81F2 and PAD2 were required for Cu2+-induced callose deposition and defense responses. Our results demonstrate that Cu2+ activates MYB51 and MYB122 through distinct ethylene signal transduction to regulate the IGS metabolic pathway, resulting in an enhanced defense response in Arabidopsis.

Sound Vibration-Triggered Epigenetic Modulation Induces Plant Root Immunity Against Ralstonia solanacearum.

Sound vibration (SV) is one of the several environmental stimuli that induce physiological changes in plants including changes in plant immunity. Immune activation is a complicated process involving epigenetic modifications, however, SV-induced epigenetic modifications remain unexplored. Here, we performed an integrative analysis comprising chromatin immunoprecipitation (ChIP) and microRNA sequencing (miRNA-seq) to understand the role of SV-mediated epigenetic modifications in immune activation in Arabidopsis thaliana against the root pathogen Ralstonia solanacearum. Plants exposed to SV (10 kHz) showed abundant H3K27me3 modification in the promoter regions of aliphatic glucosinolate biosynthesis and cytokinin signaling genes, leading to transcriptional changes that promote immunity. Additionally, 10 kHz SV down-regulated miR397b expression, thus activating three target LACCASE transcripts that mediate cell wall reinforcement via lignin accumulation. Taken together, SV triggers epigenetic modification of genes involved in secondary metabolite biosynthesis, defense hormone signaling, and pre-formed defense in A. thaliana, leading to the activation of plant immunity against R. solanacearum.

Leptosphaeria maculans Alters Glucosinolate Accumulation and Expression of Aliphatic and Indolic Glucosinolate Biosynthesis Genes in Blackleg Disease-Resistant and -Susceptible Cabbage Lines at the Seedling Stage.

The fungal pathogen, Leptosphaeria maculans causes a severe and economically important disease to Brassica crops globally, well-known as blackleg. Besides, the anti-oxidative defense response of glucosinolates to fungal pathogens is widely established. Despite notable importance of glucosinolates in blackleg disease resistance the association of glucosinolate pathway genes in glucosinolate mediated defense response after L. maculans infection remains incompletely understood. The current study was designed to identify glucosinolate-biosynthesis specific genes among the eight selected candidates induced by L. maculans and associated alterations in glucosinolate profiles to explore their roles in blackleg resistance at the seedling stage of cabbage plants. The defense responses of four cabbage inbred lines, two resistant and two susceptible, were investigated using two L. maculans isolates, 03-02s and 00-100s. Pathogen-induced glucosinolate accumulation dynamically changed from two days after inoculation to four days after inoculation. In general, glucosinolate biosynthetic genes were induced at 24 h after inoculation and glucosinolate accumulation enhanced at two days after inoculation. An increase in either aliphatic (GIB, GRA) or indolic (GBS and MGBS) glucosinolates was associated with seedling resistance of cabbage. Pearson correlation showed the enhanced accumulation of MGBS, GBS, GIB, GIV and GRA after the inoculation of fungal isolates was associated with expression of specific genes. Principal component analysis separated two resistant cabbage lines—BN4098 and BN4303 from two susceptible cabbage lines—BN4059 and BN4072 for variable coefficients of disease scores, glucosinolate accumulation and expression levels of genes. Enhanced MGBS content against both fungal isolates, contributing to seedling resistance in two interactions—BN4098 × 03-02s and BN4303 × 00-100s and enhanced GBS content only in BN4098 × 03-02s interaction. Aliphatic GRA took part in resistance of BN4098 × 00-100s interaction whereas aliphatic GIB took part is resistance of BN4098 × 03-02s interaction. Aliphatic GIV accumulated upon BN4098 × 03-02s interaction but GSL-OH-Bol033373 and CYP81F2-Bol026044 showed enhanced expression in BN4303 × 03-02s interaction. The association between the selected candidate genes, corresponding glucosinolates, and seedling resistance broaden the horizon of glucosinolate conciliated defense against L. maculans in cabbage seedlings.

FRS7 and FRS12 recruit NINJA to regulate expression of glucosinolate biosynthesis genes.

The sessile lifestyle of plants requires accurate physiology adjustments to be able to thrive in a changing environment. Plants integrate environmental timing signals to control developmental and stress responses. Here, we identified Far1 Related Sequence (FRS) 7 and FRS12, two transcriptional repressors that accumulate in short-day conditions, as regulators of Arabidopsis glucosinolate (GSL) biosynthesis. Loss of function of FRS7 and FRS12 results in plants with increased amplitudes of diurnal expression of GSL pathway genes. Protein interaction analyses revealed that FRS7 and FRS12 recruit the NOVEL INTERACTOR OF JAZ (NINJA) to assemble a transcriptional repressor complex. Genetic and molecular evidence demonstrated that FRS7, FRS12 and NINJA jointly regulate the expression of GSL biosynthetic genes, and thus constitute a molecular mechanism that modulates specialized metabolite accumulation.

Brassinosteroids Antagonize Jasmonate-Activated Plant Defense Responses through BRI1-EMS-SUPPRESSOR1 (BES1).

Brassinosteroids (BRs) and jasmonates (JAs) regulate plant growth, development, and defense responses, but how these phytohormones mediate the growth-defense tradeoff is unclear. Here, we identified the Arabidopsis (Arabidopsis thaliana) dwarf at early stages1 (dwe1) mutant, which exhibits enhanced expression of defensin genes PLANT DEFENSIN1.2a (PDF1.2a) and PDF1.2b The dwe1 mutant showed increased resistance to herbivory by beet armyworms (Spodoptera exigua) and infection by botrytis (Botrytis cinerea). DWE1 encodes ROTUNDIFOLIA3, a cytochrome P450 protein essential for BR biosynthesis. The JA-inducible transcription of PDF1.2a and PDF1.2b was significantly reduced in the BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1) gain-of-function mutant bes1- D, which was highly susceptible to S. exigua and B. cinerea BES1 directly targeted the terminator regions of PDF1.2a/PDF1.2b and suppressed their expression. PDF1.2a overexpression diminished the enhanced susceptibility of bes1- D to B. cinerea but did not improve resistance of bes1- D to S. exigua In response to S. exigua herbivory, BES1 inhibited biosynthesis of the JA-induced insect defense-related metabolite indolic glucosinolate by interacting with transcription factors MYB DOMAIN PROTEIN34 (MYB34), MYB51, and MYB122 and suppressing expression of genes encoding CYTOCHROME P450 FAMILY79 SUBFAMILY B POLYPEPTIDE3 (CYP79B3) and UDP-GLUCOSYL TRANSFERASE 74B1 (UGT74B1). Thus, BR contributes to the growth-defense tradeoff by suppressing expression of defensin and glucosinolate biosynthesis genes.

Comparative transcriptome analysis reveals defense responses against soft rot in Chinese cabbage

Pectobacterium carotovorum ssp. carotovorum (Pcc) is a necrotrophic bacterial species that causes soft rot disease in Chinese cabbage. In this study, plants harboring the resistant mutant sr gene, which confers resistance against Pcc, were screened from an 800 M2 population mutated by ethyl methane sulfonate (EMS) and scored in vitro and in vivo for lesion size. The transcript profiles showed ~512 differentially expressed genes (DEGs) between sr and WT plants occurring between 6 and 12 h postinoculation (hpi), which corresponded to the important defense regulation period (resistance) to Pcc in Chinese cabbage. The downstream defense genes (CPK, CML, RBOH MPK3, and MPK4) of pathogen pattern-triggered immunity (PTI) were strongly activated during infection at 12 hpi in resistant mutant sr; PTI appears to be central to plant defense against Pcc via recognition by three putative pattern recognition receptors (PRRs; BrLYM1-BrCERK1, BrBKK1/SERK4-PEPR1, BrWAKs). Pcc triggered the upregulation of the jasmonic acid (JA) and ethylene (ET) biosynthesis genes in mutant sr, but auxins and other hormones may have affected some negative signals. Endogenous hormones (auxins, JAs, and SA), as well as exogenous auxins (MEJA and BTH), were also verified as functioning in the immune system. Concurrently, the expression of glucosinolate and lignin biosynthesis genes was increased at 12 hpi in resistant mutant sr, and the accumulation of glucosinolate and lignin also indicated that these genes have a functional defensive role against Pcc. Our study provides valuable information and elucidates the resistance mechanism of Chinese cabbage against Pcc infection.

Environmentally friendly fertilizers can enhance yield and bioactive compounds in Chinese cabbage (Brassica rapa ssp. pekinensis)

The objective of this study was to evaluate the performance of 3 environmentally friendly fertilizers and their effect on the growth of Chinese cabbage and its contents of bioactive compounds. The following fertilizers were applied: purslane extract, 5% sulfur, and 15% phosphoric acid with 5% calcium. A total of 9, 9, and 11 components were detected for fatty acids, carotenoids, and glucosinolates, respectively. The contents of cis-7 hexadecenoic acid, linoleic acid (C18:2n6c+t), and linolenic acid (C183n3) were generally increased, and also carotenoid amounts of violaxanthin, antheraxanthin, lutein, 13-cis-ß-carotene, α-carotene, ß-carotene, and 9-cis-ß-carotene were significantly increased by fertilizer treatments. All fertilization treatments decreased the amounts of progoitrin, a strong goitrogenic, by 20.9% (H fertilization) to 53.8% (HJ fertilization). Expression analysis of fatty acid, carotenoid, and glucosinolate biosynthesis genes showed that fertilizer treatments affected the accumulation of bioactive components by changing gene expression levels. These environmentally friendly fertilizers can add value to Chinese cabbage by increasing its yield and nutritional value.

Reconstruction of the Transcriptional Regulatory Network in Arabidopsis thaliana Aliphatic Glucosinolate Biosynthetic Pathway

Aliphatic glucosinolate is an important secondary metabolite responsible in plant defense mechanism and carcinogenic activity. It plays a crucial role in plant adaptation towards changes in the environment such as salinity and drought. However, in many plant genomes, there are thousands of genes encoding proteins still with putative functions and incomplete annotations. Therefore, the genome of Arabidopsis thaliana was selected to be investigated further to identify any putative genes that are potentially involved in the aliphatic glucosinolate biosynthesis pathway, most of its gene are with incomplete annotation. Known genes for aliphatic glucosinolates were retrieved from KEGG and AraCyc databases. Three co-expression databases i.e., ATTED-II, GeneMANIA and STRING were used to perform the co-expression network analysis. The integrated co-expression network was then being clustered, annotated and visualized using Cytoscape plugin, MCODE and ClueGO. Then, the regulatory network of A. thaliana from AtRegNet was mapped onto the co-expression network to build the transcriptional regulatory network. This study showed that a total of 506 genes were co-expressed with the 61 aliphatic glucosinolate biosynthesis genes. Five transcription factors have been predicted to be involved in the biosynthetic pathway of aliphatic glucosinolate, namely SEPALLATA 3 (SEP3), PHYTOCHROME INTERACTING FACTOR 3-like 5 (AtbHLH15/PIL5), ELONGATED HYPOCOTYL 5 (HY5), AGAMOUS-like 15 (AGL15) and GLABRA 3 (GL3). Meanwhile, three other genes with high potential to be involved in the aliphatic glucosinolates biosynthetic pathway were identified, i.e., methylthioalkylmalate-like synthase 4 (MAML-4) and aspartate aminotransferase (ASP1 and ASP4). These findings can be used to complete the aliphatic glucosinolate biosynthetic pathway in A. thaliana and to update the information on the glucosinolate-related pathways in public metabolic databases.

Glucosinolate Profiling and Expression Analysis of Glucosinolate Biosynthesis Genes Differentiate White Mold Resistant and Susceptible Cabbage Lines.

Sclerotinia stem rot (white mold), caused by the fungus Sclerotinia sclerotiorum, is a serious disease of Brassica crops worldwide. Despite considerable progress in investigating plant defense mechanisms against this pathogen, which have revealed the involvement of glucosinolates, the host–pathogen interaction between cabbage (Brassica oleracea) and S. sclerotiorum has not been fully explored. Here, we investigated glucosinolate profiles and the expression of glucosinolate biosynthesis genes in white-mold-resistant (R) and -susceptible (S) lines of cabbage after infection with S. sclerotiorum. The simultaneous rise in the levels of the aliphatic glucosinate glucoiberverin (GIV) and the indolic glucosinate glucobrassicin (GBS) was linked to white mold resistance in cabbage. Principal component analysis showed close association between fungal treatment and cabbage GIV and GBS contents. The correlation analysis showed significant positive associations between GIV content and expression of the glucosinolate biosynthesis genes ST5b-Bol026202 and ST5c-Bol030757, and between GBS content and the expression of the glucosinolate biosynthesis genes ST5a-Bol026200 and ST5a-Bol039395. Our results revealed that S. sclerotiorum infection of cabbage induces the expression of glucosinolate biosynthesis genes, altering the content of individual glucosinolates. This relationship between the expression of glucosinolate biosynthesis genes and accumulation of the corresponding glucosinolates and resistance to white mold extends the molecular understanding of glucosinolate-negotiated defense against S. sclerotiorum in cabbage.

Altered Glucosinolate Profiles and Expression of Glucosinolate Biosynthesis Genes in Ringspot-Resistant and Susceptible Cabbage Lines.

Ringspot, caused by the fungus Mycosphaerella brassicicola, is a serious disease of Brassica crops worldwide. Despite noteworthy progress to reveal the role of glucosinolates in pathogen defense, the host–pathogen interaction between cabbage (Brassica oleracea) and M. brassicicola has not been fully explored. Here, we investigated the glucosinolate profiles and expression of glucosinolate biosynthesis genes in the ringspot-resistant (R) and susceptible (S) lines of cabbage after infection with M. brassicicola. The concomitant rise of aliphatic glucoiberverin (GIV) and indolic glucobrassicin (GBS) and methoxyglucobrassicin (MGBS) was linked with ringspot resistance in cabbage. Pearson’s correlation and principle component analysis showed a significant positive association between GIV contents and the expression of the glucosinolate biosynthesis gene ST5b-Bol026202 and between GBS contents and the expression of the glucosinolate biosynthesis gene MYB34-Bol017062. Our results confirmed that M. brassicicola infection induces the expression of glucosinolate biosynthesis genes in cabbage, which alters the content of individual glucosinolates. This link between the expression of glucosinolate biosynthesis genes and the accumulation of their respective glucosinolates with the resistance to ringspot extends our molecular sense of glucosinolate-negotiated defense against M. brassicicola in cabbage.

Comparative analysis of glucosinolates and metabolite profiling of green and red mustard (brassica juncea) hairy roots.

Here, accumulation of glucosinolates and expression of glucosinolates biosynthesis genes in green and red mustard hairy roots were identified and quantified by HPLC and qRT-PCR analyses. The total glucosinolates content of green mustard hairy root (10.09µg/g dry weight) was 3.88 times higher than that of red mustard hairy root. Indolic glucosinolates (glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin) in green mustard were found at 30.92, 6.95, and 5.29 times higher than in red mustard hairy root, respectively. Conversely, levels of glucotropaeolin (aromatic glucosinolate) was significantly higher in red mustard than in green mustard. Accumulation of glucoraphasatin, an aliphatic glucosinolate, was only observed only in red mustard hairy roots. Quantitative real-time PCR analysis showed that the expression level of genes related to aliphatic and aromatic glucosinolate biosynthesis were higher in red mustard, exception BjCYP83B. The expression of BjCYP79B2, which encodes a key enzyme involved in the indolic glucosinolate biosynthetic pathway, was higher in green mustard than in red mustard. Additionally, to further distinguish between green mustard and red mustard hairy roots, hydrophilic and lipophilic compounds were identified by gas chromatography-mass spectrometry and subjected to principal component analysis. The results indicated that core primary metabolites and glucosinolate levels were higher in the hairy roots of green mustard than in those of red mustard.

Molecular Characterization of MYB28 Involved in Aliphatic Glucosinolate Biosynthesis in Chinese Kale (Brassica oleracea var. alboglabra Bailey).

Glucosinolates are Brassicaceae-specific secondary metabolites that act as crop protectants, flavor precursors, and cancer-prevention agents, which shows strong evidences of anticarcinogentic, antioxidant, and antimicrobial activities. MYB28, the R2R3-MYB28 transcription factor, directly activates genes involved in aliphatic glucosinolate biosynthesis. In this study, the MYB28 homology (BoaMYB28) was identified in Chinese kale (Brassica oleracea var. alboglabra Bailey). Analysis of the nucleotide sequence indicated that the cDNA of BoaMYB28 was 1257 bp with an ORF of 1020 bp. The deduced BoaMYB28 protein was a polypeptide of 339 amino acid with a putative molecular mass of 38 kDa and a pI of 6.87. Sequence homology and phylogenetic analysis showed that BoaMYB28 was most closely related to MYB28 homologs from the Brassicaceae family. The expression levels of BoaMYB28 varies across the tissues and developmental stages. BoaMYB28 transcript levels were higher in leaves and stems compared with those in cotyledons, flowers, and siliques. BoaMYB28 was expressed across all developmental leaf stages, with higher transcript accumulation in mature and inflorescence leaves. Over-expression and RNAi studies showed that BoaMYB28 retains the basic MYB28 gene function as a major transcriptional regulator of aliphatic glucosinolate pathway. The results indicated that over-expression and RNAi lines showed no visible difference on plant morphology. The contents of aliphatic glucosinolates and transcript levels of aliphatic glucosinolate biosynthesis genes increased in over-expression lines and decreased in RNAi lines. In over-expression lines, aliphatic glucosinolate contents were 1.5- to 3-fold higher than those in the wild-type, while expression levels of aliphatic glucosinolate biosynthesis genes were 1.5- to 4-fold higher than those in the wild-type. In contrast, the contents of aliphatic glucosinolates and transcript levels of aliphatic glucosinolate biosynthesis genes in RNAi lines were considerably lower than those in the wild-type. The results suggest that BoaMYB28 has the potential to alter the aliphatic glucosinolates contents in Chinese kale at the genetic level.

A novel Filamentous Flower mutant suppresses brevipedicellus developmental defects and modulates glucosinolate and auxin levels.

BREVIPEDICELLUS (BP) encodes a class-I KNOTTED1-like homeobox (KNOX) transcription factor that plays a critical role in conditioning a replication competent state in the apical meristem, and it also governs growth and cellular differentiation in internodes and pedicels. To search for factors that modify BP signaling, we conducted a suppressor screen on bp er (erecta) plants and identified a mutant that ameliorates many of the pleiotropic defects of the parent line. Map based cloning and complementation studies revealed that the defect lies in the FILAMENTOUS FLOWER (FIL) gene, a member of the YABBY family of transcriptional regulators that contribute to meristem organization and function, phyllotaxy, leaf and floral organ growth and polarity, and are also known to repress KNOX gene expression. Genetic and cytological analyses of the fil-10 suppressor line indicate that the role of FIL in promoting growth is independent of its previously characterized influences on meristem identity and lateral organ polarity, and likely occurs non-cell-autonomously from superior floral organs. Transcription profiling of inflorescences revealed that FIL downregulates numerous transcription factors which in turn may subordinately regulate inflorescence architecture. In addition, FIL, directly or indirectly, activates over a dozen genes involved in glucosinolate production in part by activating MYB28, a known activator of many aliphatic glucosinolate biosynthesis genes. In the bp er fil-10 suppressor mutant background, enhanced expression of CYP71A13, AMIDASE1 (AMI) and NITRILASE genes suggest that auxin levels can be modulated by shunting glucosinolate metabolites into the IAA biosynthetic pathway, and increased IAA levels in the bp er fil-10 suppressor accompany enhanced internode and pedicel elongation. We propose that FIL acts to oppose KNOX1 gene function through a complex regulatory network that involves changes in secondary metabolites and auxin.