Glucosinolate Biosynthesis Pathway Research Articles

Integrated Analysis of Transcriptome and Metabolome Reveals Differential Responses to Alternaria brassicicola Infection in Cabbage (Brassica oleracea var. capitata).

Black spot, caused by Alternaria brassicicola (Ab), poses a serious threat to crucifer production, and knowledge of how plants respond to Ab infection is essential for black spot management. In the current study, combined transcriptomic and metabolic analysis was employed to investigate the response to Ab infection in two cabbage (Brassica oleracea var. capitata) genotypes, Bo257 (resistant to Ab) and Bo190 (susceptible to Ab). A total of 1100 and 7490 differentially expressed genes were identified in Bo257 (R_mock vs. R_Ab) and Bo190 (S_mock vs. S_Ab), respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that "metabolic pathways", "biosynthesis of secondary metabolites", and "glucosinolate biosynthesis" were the top three enriched KEGG pathways in Bo257, while "metabolic pathways", "biosynthesis of secondary metabolites", and "carbon metabolism" were the top three enriched KEGG pathways in Bo190. Further analysis showed that genes involved in extracellular reactive oxygen species (ROS) production, jasmonic acid signaling pathway, and indolic glucosinolate biosynthesis pathway were differentially expressed in response to Ab infection. Notably, when infected with Ab, genes involved in extracellular ROS production were largely unchanged in Bo257, whereas most of these genes were upregulated in Bo190. Metabolic profiling revealed 24 and 56 differentially accumulated metabolites in Bo257 and Bo190, respectively, with the majority being primary metabolites. Further analysis revealed that dramatic accumulation of succinate was observed in Bo257 and Bo190, which may provide energy for resistance responses against Ab infection via the tricarboxylic acid cycle pathway. Collectively, this study provides comprehensive insights into the Ab-cabbage interactions and helps uncover targets for breeding Ab-resistant varieties in cabbage.

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.

Metabolome and Transcriptome Analyses Provide Insights into Glucosinolate Accumulation in the Novel Vegetable Crop Cardamine violifolia

Cardamine violifolia, a species belonging to the Brassicaceae family, is a novel vegetable crop that is rich in glucosinolates. However, the specific glucosinolate profiles in this species remain unknown. In the present study, four parts of C. violifolia were collected including central leaves (CLs), outer leaves (OLs), petiole (P), and root (R). The highest level of total glucosinolate was observed in the R. A total of 19 glucosinolates were found in C. violifolia. The predominant glucosinolate compounds were 3-methylbutyl glucosinolate, 6-methylsulfinylhexyl glucosinolate, Indol-3-ylmethyl glucosinolate, 4-methoxyglucobrassicin, and neoglucobrassicin. A transcriptome analysis showed that 16 genes, including BCAT1, BCAT3-6, CYP79A2, CYP79B2-3, CYP83A1, CYP83B1, and SOT17-18, and nine metabolites, such as valine, tryptophan, and 1-methylpropyl glucosinolate, were enriched in the glucosinolate biosynthesis pathway. These genes may be involved in the regulation of glucosinolate accumulation among the four parts. A weighted gene co-expression analysis showed that five genes were predicted to regulate glucosinolate accumulation, including ABC transporter G family member 19, 3-ketoacyl-CoA synthase 19, and pyruvate decarboxylase 1. This study deepens our understanding of the nutrient quality of C. violifolia and provides insights into the regulatory mechanism of glucosinolate accumulation in C. violifolia.

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.

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.

Dynamic changes in the transcriptome landscape of Arabidopsis thaliana in response to cold stress.

Plants must reprogram gene expression to adapt constantly changing environmental temperatures. With the increased occurrence of extremely low temperatures, the negative effects on plants, especially on growth and development, from cold stress are becoming more and more serious. In this research, strand-specific RNA sequencing (ssRNA-seq) was used to explore the dynamic changes in the transcriptome landscape of Arabidopsis thaliana exposed to cold temperatures (4°C) at different times. In total, 7,623 differentially expressed genes (DEGs) exhibited dynamic temporal changes during the cold treatments. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the DEGs were enriched in cold response, secondary metabolic processes, photosynthesis, glucosinolate biosynthesis, and plant hormone signal transduction pathways. Meanwhile, long non-coding RNAs (lncRNAs) were identified after the assembly of the transcripts, from which 247 differentially expressed lncRNAs (DElncRNAs) and their potential target genes were predicted. 3,621 differentially alternatively spliced (DAS) genes related to RNA splicing and spliceosome were identified, indicating enhanced transcriptome complexity due to the alternative splicing (AS) in the cold. In addition, 739 cold-regulated transcription factors (TFs) belonging to 52 gene families were identified as well. This research analyzed the dynamic changes of the transcriptome landscape in response to cold stress, which reveals more complete transcriptional patterns during short- and long-term cold treatment and provides new insights into functional studies of that how plants are affected by cold stress.

UVA-Radiation Exposure of Different Durations Promoted the Growth, Phytochemicals and Glucosinolate Biosynthesis of Chinese Kale.

Ultraviolet-A (UVA) (315–400 nm) is an essential environmental signal that regulates plant development and affects phytochemicals biosynthesis, including glucosinolate biosynthesis. The effects of different UVA (380 ± 10 nm, 40 μmol/m2/s) exposure durations, including 0 h/d (UV0), 6 h/d (UV6) and 12 h/d (UV12), on the growth and phytochemicals of Chinese kale (Brassica alboglabra) under white 250 μmol/m2/s LEDs were investigated. UVA exposure of different durations influenced the growth and phytochemicals biosynthesis of Chinese kale. Prolonging UVA irradiation throughout the growth cycle positively affected the growth and the development of Chinese kale, with evident increases in the dry weights of shoots and roots, plant height, stem diameter, specific leaf weight and flower budding rate. The application of UVA increased the soluble sugar content, whereas higher flavonoid content and antioxidant capacity (FRAP) and lower nitrate content were only observed in Chinese kale exposed to UV6 treatment. Besides, the qPCR assay showed that supplemental UVA-radiation exposure up-regulated the gene expressions of UVR8, transcription factors genes and genes related to the glucosinolate biosynthesis pathway, thereby promoting the accumulation of glucosinolates. Therefore, supplemental UVA-radiation exposure for 12 h/d was more conducive to plant growth, while supplemental UVA-radiation exposure for 6 h/d was better for phytochemical biosynthesis in Chinese kale in an artificial-light plant factory.

Issue Information

AbstractFront cover: Barbour et al. created an experimental food web, as depicted in this drawing, consisting of four species, Arabidopsis thaliana, two species of aphids, and a parasitoid wasp parasitizing the aphids, and examined the effects of alleles of three genes in the aliphatic glucosinolate biosynthesis pathway in A. thaliana on the survival of the four species (Barbour et al. (2022) Science 376: 70‐73, DOI: 10.1126/science.abf2232). Their finding was that the null allele of one gene (AOP2) significantly reduced the extinction rate of the four. They call AOP2 a ‘keystone gene,’ in analogy to a keystone species. We would like to thank Dr. Toshiyuki Harumoto (Hakubi Center for Advanced Research, Kyoto University) for his advice on the drawing. Designed by TRAIS Co., Ltd. (Kobe, Japan). image

The beneficial functions of blue light supplementary on the biosynthesis of glucosinolates in pakchoi (Brassica rapa L. ssp. chinensis) under greenhouse conditions

Blue light can improve the nutritional value of vegetables grown in greenhouse, while the mechanisms about the biosynthesis and accumulation of glucosinolates regulated by light signaling still unknown. Here, the soluble amino acid and glucosinolate profiles under supplementary different blue light intensities (T50, T100, T150) for 10 days were investigated in green-leaf and red-leaf pakchoi. The results indicated that blue light could significantly increase the contents of glucoalyssin, glucobrassicin, glucobrassicanapin, gluconapin and total glucosinolates, and shown a dose effect. The soluble amino acid profiles and the contents of glucosinolates precursor amino acid were also enhanced to varying degrees. Besides, in green-leaf and red-leaf pakchoi, the expression levels of photoreceptor genes under T100 were significantly higher compared with the CK. Supplementary blue light also promoted the expression of key transcription factors and key genes of glucosinolate biosynthesis pathway in two cultivars pakchoi. Therefore, the beneficial functions of blue light supplementary on the biosynthesis of glucosinolates in pakchoi under greenhouse conditions are owing to the promoted in the contents of precursor amino acids and the expressions of glucosinolate-related genes mediated by light signal transduction. • Supplementary bule light could increase the glucosinolate profiles of pakchoi leaves. • The increasing of glucosinolate content be linked to glucosinolate biosynthesis and photoreceptor gene-related expressions. • The increasing of light-controlled amino acids profiles provides the material basis for glucosinolate biosynthesis. • The respond sensitivity to bule light was different in green-leaf and red-leaf pakchoi.

Analysis of Glucosinolate Content, Composition and Expression Level of Biosynthesis Pathway Genes in Different Chinese Kale Varieties

The content and component of glucosinolates in edible stems and leaves of eight Chinese kale varieties from Japan and eight varieties from China were determined by HPLC-MS. Simultaneously, the expression levels of glucosinolate biosynthesis pathway genes from four varieties with high and low total glucosinolate contents were analyzed by the qRT-PCR method. Four types of aliphatic glucosinolates (A-GLSs: GRA, SIN, GNA and GER) and indole glucosinolates (I-GLSs: 4-HGBS, GBS, 4-MGBS and NGBS) were detected in the stems and leaves of 16 varieties, and no aromatic glucosinolates (R-GLSs) were detected. A-GLSs account for more than 80.69% of the total content of total glucosinolates (T-GLSs), in which GNA and GRA are the main components of stems and leaves. Among Japanese varieties, QB1 has higher content of A- and T-GLSs, while that of XLB was lower; however, the corresponding varieties were ZH and DSHH in Chinese varieties. Among the above four varieties, the expression levels of SOT16, CYP83B1, SOT17, CYP83A1 and MAM1 genes were significantly higher in the varieties with higher GLSs; the expression levels of SOT16 and CYP83B1 were consistent with the content of I-GLSs; and SOT17, CYP83A1 and MAM1 expression levels were consistent with A-GLSs content. At the same time, the expression levels of SOT16 and CYP83B1 in the leaves were higher than those in the stems. CYP83A1 and MAM1 genes were less expressed in the leaves than in the stems of lower content varieties. It is speculated that these genes may be the key genes regulating GLS biosynthesis in Chinese kale.

Glucosinolate biosynthesis: role of MAM synthase and its perspectives.

Glucosinolates, synthesized by the glucosinolate biosynthesis pathway, are the secondary metabolites used as a defence mechanism in the Brassicaceae plants, including Arabidopsis thaliana. The first committed step in the pathway, catalysed by methylthioalkylmalate (MAM) synthase (EC: 2.3.3.17), is to produce different variants of glucosinolates. Phylogenetic analyses suggest that possibly MAM synthases have been evolved from isopropylmalate synthase (IPMS) by the substitutions of five amino acid residues (L143I, H167L, S216G, N250G and P252G) in the active site of IPMS due to point mutations. Considering the importance of MAM synthase in Brassicaceae plants, Petersen et al. (2019) made an effort to characterise the MAM synthase (15 MAM1 variants) in vitro by single substitution or double substitutions. In their study, the authors have expressed the variants in Escherichia coli and analysed the amino acids in the cultures of E. coli in vivo. Since modifying the MAM synthases by transgenic approaches could increase the resistance of Brassicaceae plants for enhancing the defence effect of glucosinolates and their degraded products; hence, MAM synthases should be characterized in detail in vivo in A. thaliana along with the structural analysis of the enzyme for meaningful impact and for its imminent use in vivo.

Exploration of the Characteristics of Intestinal Microbiota and Metabolomics in Different Rat Models of Mongolian Medicine.

Background Mongolian medicine is a systematic theoretical system, which is based on the balance among Heyi, Xila, and Badagan. However, the underlying mechanisms remain unclear. This study aimed to explore the characteristics of intestinal microbiota and metabolites in different rat models of Mongolian medicine. Methods After establishing rat models of Heyi, Xila, and Badagan, we integrated 16S rRNA gene sequencing and metabolomics. Results Heyi, Xila, and Badagan rats had significantly altered intestinal microbial composition compared with rats in the MCK group. They showed 11, 18, and 8 significantly differential bacterial biomarkers and 22, 11, and 15 differential metabolites, respectively. The glucosinolate biosynthesis pathway was enriched only in Heyi rats; the biosynthesis of phenylpropanoids pathway and phenylpropanoid biosynthesis pathway were enriched only in Xila rats; the isoflavonoid biosynthesis pathway, the glycine, serine, and threonine metabolism pathway, and the arginine and proline metabolism pathway were enriched only in Badagan rats. Conclusions The intestinal microbiota, metabolites, and metabolic pathways significantly differed among Heyi, Xila, and Badagan rats compared with control group rats.

Phytohormonal and Transcriptomic Response of Hulless Barley Leaf in Response to Powdery Mildew Infection

Powdery mildew (PM) caused by Blumeria graminis (DC.) Golovin ex Speer f. sp. hordei Marchal (Bgh) is one of the major yield reducing diseases in hulless barley (Hordeum vulgare L. var. nudum Hook. f.). Genotypes with contrasting resistance to PM offer unique opportunities to explore the transcriptome in order to understand the expression changes in genes and pathways. In this study, we explored the phytohormone levels and transcriptome of a Bgh susceptible (XL19) and resistant (ZYM1288) hulless barley genotypes at 0, 5, 12, 24, and 36 h post infection (hpi) with Bgh. We found relatively higher levels of abscisic acid, jasmonic acid, salicylic acid, and cytokinins in ZYM1288. The transcriptome analyses identified 31,354 genes that were enriched in signaling, energy, and defense related pathways. Higher numbers of differentially expressed genes (DEGs) were found in XL19 as compared to ZYM1288 after 5 (3603 vs. 2341) and 12 hpi (3530 vs. 2416). However, after 24 and 36 hpi, the number of DEGs was higher in ZYM1288 as compared to XL19 i.e., 3625 vs. 3034 and 5855 vs. 2725, respectively. Changes in hormone levels drove downstream expression changes in plant-hormone signaling that helped ZYM1288 to perform better under Bgh infection. The expression of DEGs in MAPK-signaling and Toll-like receptor signaling pathways, glucosinolate biosynthesis, glutathione metabolism, brassinosteroid metabolism, and energy related pathways indicated their common roles in defense against PM. Key genes related to PM-resistance were upregulated in the resistant genotype. These genes provide key information towards differences in both genotypes towards resistance to PM. The transcriptomic signatures explored in this study will broaden our understanding towards molecular regulation of resistance to PM in hulless barley.

Genetic Analysis of a Horizontal Resistance Locus BLMR2 in Brassica napus.

Leptosphaeria maculans causes blackleg disease in Brassica napus. The blackleg disease is mainly controlled by resistance genes in B. napus. Previous studies have shown that the blackleg resistant BLMR2 locus that conferred horizontal resistance under field conditions, is located on chromosome A10 of B. napus. The purpose of this study is to fine map this locus and hence identify a candidate gene underlying horizontal resistance. The spectrum of resistance to L. maculans isolates of the resistance locus BLMR2 was analyzed using near isogenic lines, resistant, and susceptible cultivars. The results showed that this locus was horizontally resistant to all isolates tested. Sequence characterized amplified regions (SCAR), simple sequence repeats (SSR), and single nucleotide polymorphism (SNP) markers were developed in the chromosome region of BLMR2 and a fine genetic map was constructed. Two molecular markers narrowed BLMR2 in a 53.37 kb region where six genes were annotated. Among the six annotated genes, BnaA10g11280D/BnaA10g11290D encoding a cytochrome P450 protein were predicted as the candidate of BLMR2. Based on the profiling of pathogen induced transcriptome, three expressed genes in the six annotated genes were identified while only cytochrome P450 showed upregulation. The candidate corresponds to the gene involved in the indole glucosinolate biosynthesis pathway and plant basal defense in Arabidopsis thaliana. The molecular markers identified in this study will allow the quick incorporation of the BLMR2 allele in rapeseed cultivars to enhance blackleg resistance.

RcTGA1 and glucosinolate biosynthesis pathway involvement in the defence of rose against the necrotrophic fungus Botrytis cinerea

BackgroundRose is an important economic crop in horticulture. However, its field growth and postharvest quality are negatively affected by grey mould disease caused by Botrytis c. However, it is unclear how rose plants defend themselves against this fungal pathogen. Here, we used transcriptomic, metabolomic and VIGS analyses to explore the mechanism of resistance to Botrytis c.ResultIn this study, a protein activity analysis revealed a significant increase in defence enzyme activities in infected plants. RNA-Seq of plants infected for 0 h, 36 h, 60 h and 72 h produced a total of 54 GB of clean reads. Among these reads, 3990, 5995 and 8683 differentially expressed genes (DEGs) were found in CK vs. T36, CK vs. T60 and CK vs. T72, respectively. Gene annotation and cluster analysis of the DEGs revealed a variety of defence responses to Botrytis c. infection, including resistance (R) proteins, MAPK cascade reactions, plant hormone signal transduction pathways, plant-pathogen interaction pathways, Ca2+ and disease resistance-related genes. qPCR verification showed the reliability of the transcriptome data. The PTRV2-RcTGA1-infected plant material showed improved susceptibility of rose to Botrytis c. A total of 635 metabolites were detected in all samples, which could be divided into 29 groups. Metabonomic data showed that a total of 59, 78 and 74 DEMs were obtained for T36, T60 and T72 (T36: Botrytis c. inoculated rose flowers at 36 h; T60: Botrytis c. inoculated rose flowers at 60 h; T72: Botrytis c. inoculated rose flowers at 72 h) compared to CK, respectively. A variety of secondary metabolites are related to biological disease resistance, including tannins, amino acids and derivatives, and alkaloids, among others; they were significantly increased and enriched in phenylpropanoid biosynthesis, glucosinolates and other disease resistance pathways. This study provides a theoretical basis for breeding new cultivars that are resistant to Botrytis c.ConclusionFifty-four GB of clean reads were generated through RNA-Seq. R proteins, ROS signalling, Ca2+ signalling, MAPK signalling, and SA signalling were activated in the Old Blush response to Botrytis c. RcTGA1 positively regulates rose resistance to Botrytis c. A total of 635 metabolites were detected in all samples. DEMs were enriched in phenylpropanoid biosynthesis, glucosinolates and other disease resistance pathways.

Combined transcriptomic, proteomic and biochemical approaches to identify the cadmium hyper-tolerance mechanism of turnip seedling leaves.

Cadmium (Cd) pollution is a prominent environment problem, and great interests have been developed towards the molecular mechanism of Cd accumulation in plants. In this study, we conducted combined transcriptomic, proteomic and biochemical approaches to explore the detoxification of a Cd-hyperaccumulating turnip landrace exposed to 5 μM (T5) and 25 μM (T25) Cd treatments. A total of 1090 and 2111 differentially expressed genes (DEGs) and 161 and 303 differentially expressed proteins (DEPs) were identified in turnips under T5 and T25, respectively. However, poor correlations were observed in expression changes between mRNA and protein levels. The enriched KEGG pathways of DEGs with a high proportion (> 80%) of upregulated genes were focused on the flavonoid biosynthesis, sulphur metabolism and glucosinolate biosynthesis pathways, whereas those of DEPs were enriched on the glutathione metabolism pathway. This result suggests that these pathways contribute to Cd detoxification in turnips. Furthermore, induced antioxidant enzymes, heat stock proteins and stimulated protein acetylation modification seemed to play important roles in Cd tolerance in turnips. In addition, several metal transporters were found responsible for the Cd accumulation capacity of turnips. This study may serve as a basis for breeding low-Cd-accumulating vegetables for foodstuff or high-Cd-abstracting plants for phytoremediation.

Overexpression and Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA Synthase Affect Central Metabolic Pathways in Tobacco.

Little has been established on the relationship between the mevalonate (MVA) pathway and other metabolic pathways except for the sterol and glucosinolate biosynthesis pathways. In the MVA pathway, 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to form 3-hydroxy-3-methylglutaryl-coenzyme A. Our previous studies had shown that, while the recombinant Brassica juncea HMGS1 (BjHMGS1) mutant S359A displayed 10-fold higher enzyme activity than wild-type (wt) BjHMGS1, transgenic tobacco overexpressing S359A (OE-S359A) exhibited higher sterol content, growth rate and seed yield than OE-wtBjHMGS1. Herein, untargeted proteomics and targeted metabolomics were employed to understand the phenotypic effects of HMGS overexpression in tobacco by examining which other metabolic pathways were affected. Sequential window acquisition of all theoretical mass spectra quantitative proteomics analysis on OE-wtBjHMGS1 and OE-S359A identified the misregulation of proteins in primary metabolism and cell wall modification, while some proteins related to photosynthesis and the tricarboxylic acid cycle were upregulated in OE-S359A. Metabolomic analysis indicated corresponding changes in carbohydrate, amino acid and fatty acid contents in HMGS-OEs, and F-244, a specific inhibitor of HMGS, was applied successfully on tobacco to confirm these observations. Finally, the crystal structure of acetyl-CoA-liganded S359A revealed that improved activity of S359A likely resulted from a loss in hydrogen bonding between Ser359 and acyl-CoA, which is evident in wtBjHMGS1. This work suggests that regulation of plant growth by HMGS can influence the central metabolic pathways. Furthermore, this study demonstrates that the application of the HMGS-specific inhibitor (F-244) in tobacco represents an effective approach for studying the HMGS/MVA pathway.

An Integrated Metabolomics Study of Glucosinolate Metabolism in Different Brassicaceae Genera.

Glucosinolates are a group of plant secondary metabolites that can be hydrolyzed into a variety of breakdown products such as isothiocyanates, thiocyanates, and nitriles. These breakdown products can facilitate plant defense and function as attractants to natural enemies of insect pests. As part of the diet, some of these compounds have shown cancer-preventing activities, and the levels of these metabolites in the edible parts of the plants are of interest. In this study, we systematically examined variations in glucosinolates, their precursors, and their breakdown products in 12 commonly consumed vegetables of the Brassicaceae family with gas chromatography—quadrupole time-of-flight mass spectrometer (GC-Q-TOF/MS), liquid chromatography–quadrupole time-of-flight mass spectrometer (LC-Q-TOF/MS), and liquid chromatography—triple quadrupole mass spectrometer (LC-QQQ/MS), using both untargeted and targeted approaches. The findings were integrated with data from literature to provide a comprehensive map of pathways for biosynthesis of glucosinolates and isothiocyanates. The levels of precursor glucosinolates are found to correlate well with their downstream breakdown products. Further, the types and abundances of glucosinolates among different genera are significantly different, and these data allow the classification of plants based on morphological taxonomy. Further validation on three genera, which are grown underground, in damp soil, and above ground, suggests that each genus has its specific biosynthetic pathways and that there are variations in some common glucosinolate biosynthesis pathways. Our methods and results provide a good starting point for further investigations into specific aspects of glucosinolate metabolism in the Brassica vegetables.

Ultraviolet-B radiation exposure lowers the antioxidant capacity in the Arabidopsis thaliana pdx1.3-1 mutant and leads to glucosinolate biosynthesis alteration in both wild type and mutant

Pyridoxine (vitamin B6) and its vitamers are used by living organisms both as enzymatic cofactors and as antioxidants. We used Arabidopsis pyridoxine biosynthesis mutant pdx1.3-1 to study the involvement of the PLP-synthase main polypeptide PDX1 in plant responses to ultraviolet radiation of two different qualities, one containing primarily UV-A (315–400 nm) and the other containing both UV-A and UV-B (280–315 nm). The antioxidant capacity and the flavonoid and glucosinolate (GS) profiles were examined. As an indicator of stress, Fv/Fm of photosystem II reaction centers was used. In pdx1.3-1, UV−A + B exposure led to a significant 5% decrease in Fv/Fm on the last day (day 15), indicating mild stress at this time point. The antioxidant capacity of Col-0 wildtype increased significantly (50–73%) after 1 and 3 days of UV−A + B. Instead, in pdx1.3-1, the antioxidant capacity significantly decreased by 44–52% over the same time period, proving the importance of a full complement of functional PDX1 genes for the detoxification of reactive oxygen species. There were no significant changes in the flavonoid glycoside profile under any light condition. However, the GS profile was significantly altered, both with respect to Arabidopsis accession and exposure to UV. The difference in flavonoid and GS profiles reflects that the GS biosynthesis pathway contains at least one pyridoxine-dependent enzyme, whereas no such enzyme is used in flavonoid biosynthesis. Also, there was strong correlation between the antioxidant capacity and the content of some GS compounds. Our results show that vitamin B6 vitamers, functioning both as antioxidants and co-factors, are of importance for the physiological fitness of plants.

Copper toxicity affects indolic glucosinolates and gene expression of key enzymes for their biosynthesis in Chinese cabbage

ABSTRACT Excessive levels of Cu2+ are phytotoxic and exposure of Chinese cabbage to elevated Cu2+ concentrations led to reduction of the plant biomass. To get more insight into the role of glucosinolates upon copper stress, the impact of elevated Cu2+ levels on glucosinolates biosynthesis were studied in Chinese cabbage. The content of total glucosinolates was only elevated in the roots, mostly due to indolic and aromatic glucosinolates. The results showed a higher contribution of indolic glucosinolates, notably glucobrassicin, a 2- and 4-fold increase in Chinese cabbage exposed to 5 and 10 µM Cu2+, respectively. Furthermore, the increase in the indolic glucosinolates was accompanied by enhanced transcript levels of CYP79B2 and CYP83B1, two genes involved in biosynthesis of indolic glucosinolates, and that of the MYB51, a transcription factor involved in regulation of indolic glucosinolate biosynthesis pathway, at elevated Cu2+ concentrations. In addition, total sulfur and nitrogen remained unaffected in the root, but total glucosinolate was significantly enhanced upon exposure to elevated Cu2+. This result may show that relatively more sulfur and nitrogen was channeled into glucosinolates in the root. In conclusion, accumulation of indolic glucosinolates in the root can be considered as a strategy for Chinese cabbage to combat elevated Cu2+ concentrations.