NAD+ deficiency primes defense metabolism via 1O2-escalated jasmonate biosynthesis in plants.

Nicotinamide adenine dinucleotide (NAD+) is a redox cofactor and signal central to cell metabolisms. Disrupting NAD homeostasis in plant alters growth and stress resistance, yet the underlying mechanisms remain largely unknown. Here, by combining genetics with multi-omics, we discover that NAD+ deficiency in qs-2 caused by mutation in NAD+ biosynthesis gene-Quinolinate Synthase retards growth but induces biosynthesis of defense compounds, notably aliphatic glucosinolates that confer insect resistance. The elevated defense in qs-2 is resulted from activated jasmonate biosynthesis, critically hydroperoxidation of α-linolenic acid by the 13-lipoxygenase (namely LOX2), which is escalated via the burst of chloroplastic ROS-singlet oxygen (1O2). The NAD+ deficiency-mediated JA induction and defense priming sequence in plants is recapitulated upon insect infestation, suggesting such defense mechanism operates in plant stress response. Hence, NAD homeostasis is a pivotal metabolic checkpoint that may be manipulated to navigate plant growth and defense metabolism for stress acclimation.

ELONGATED HYPOCOTYL 5 interacts with HISTONE DEACETYLASE 9 to suppress glucosinolate biosynthesis in Arabidopsis.

Glucosinolates (GSLs) are defensive secondary metabolites produced by Brassicaceae species in response to abiotic and biotic stresses. The biosynthesis of GSL compounds and the expression of GSL-related genes are highly modulated by endogenous signals (i.e. circadian clocks) and environmental cues, such as temperature, light, and pathogens. However, the detailed mechanism by which light signaling influences GSL metabolism remains poorly understood. In this study, we found that a light-signaling factor, ELONGATED HYPOCOTYL 5 (HY5), was involved in the regulation of GSL content under light conditions in Arabidopsis (Arabidopsis thaliana). In hy5-215 mutants, the transcript levels of GSL pathway genes were substantially upregulated compared with those in wild-type (WT) plants. The content of GSL compounds was also substantially increased in hy5-215 mutants, whereas 35S::HY5-GFP/hy5-215 transgenic lines exhibited comparable levels of GSL-related transcripts and GSL content to those in WT plants. HY5 physically interacts with HISTONE DEACETYLASE9 and binds to the proximal promoter region of MYB29 and IMD1 to suppress aliphatic GSL biosynthetic processes. These results demonstrate that HY5 suppresses GSL accumulation during the daytime, thus properly modulating GSL content daily in Arabidopsis plants.

Characterization of unique EDTA-insensitive methylthioalkylmalate synthase from Eutrema japonicum and its potential application in synthetic biology.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), a derivative of glucosinolate with a six-carbon chain, is a compound found in wasabi and has diverse health-promoting properties. The biosynthesis of glucosinolates from methionine depends on a crucial step catalyzed methylthioalkylmalate synthases (MAMs), which are responsible for the generation of glucosinolates with varying chain lengths. In this study, our primary focus was the characterization of two methylthioalkyl malate synthases, MAM1-1 and MAM1-2, derived from Eutrema japonicum, commonly referred to as Japanese wasabi. Eutremajaponicum MAMs (EjMAMs) were expressed in an Escherichiacoli expression system, subsequently purified, and in vitro enzymatic activity was assayed. We explored the kinetic properties, optimal pH conditions, and cofactor preferences of EjMAMs and compared them with those of previously documented MAMs. Surprisingly, EjMAM1-2, categorized as a metallolyase family enzyme, displayed 20% of its maximum activity even in the absence of divalent metal cofactors or under high concentrations of EDTA. Additionally, we utilized AlphaFold2 to generate structural homology models of EjMAMs, and used in silico analysis and mutagenesis studies to investigate the key residues participating in catalytic activity. Moreover, we examined in vivo biosynthesis in E. coli containing Arabidopsis thaliana branched-chain amino acid transferase 3 (AtBCAT3) along with AtMAMs or EjMAMs and demonstrated that EjMAM1-2 exhibited the highest conversion rate among those MAMs, converting l-methionine to 2-(2-methylthio) ethyl malate (2-(2-MT)EM). EjMAM1-2 shows a unique property in vitro and highest activity on converting l-methionine to 2-(2-MT)EM in vivo which displays high potential for isothiocyanate biosynthesis in E. coli platform.

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.

Transcriptomics analysis of genes induced by melatonin related to glucosinolates synthesis in broccoli hairy roots.

Glucoraphanin (GRA) is found in the seeds and vegetative organs of broccoli (Brassica oleracea L. var. italica Planch) as the precursor of anti-carcinogen sulforaphane (SF). The yield of GRA obtained from these materials is weak and the cost is high. In recent years, the production of plant secondary metabolites by large-scale hairy roots culture in vitro has succeeded in some species. Melatonin (MT) is a natural hormone which existed in numerous organisms. Studies have demonstrated that MT can improve the synthesis of secondary metabolites in plants. At present, it has not been reported that MT regulates the biosynthesis of glucoraphanin in broccoli hairy roots. In this study, the broccoli hairy roots that grew for 20 d were respectively treated by 500 µM MT for 0, 6, 12, 20 and 32. To explore the reason of changes in secondary metabolites and reveal the biosynthetic pathway of glucoraphanin at transcriptional level. Compared with 0 h, the yield of GRA under other treatments was increased, and the overall trend was firstly increased and then decreased. The total yield of GRA reached the highest at 12 h, which was 1.22-fold of 0 h. Then, the genome of broccoli as the reference, a total of 13234 differentially expressed genes (DEGs) were identified in broccoli hairy roots under treatment with 500 µM MT for 0, 6, 12, 20 and 32 h, respectively. Among these DEGs, 6266 (47.35%) were upregulated and 6968 (52.65%) were downregulated. It was found that the pathway of 'Glucosinolates biosynthesis (ko00966)' was enriched in the 16th place by Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the upregulated DEGs. The expression of key genes in the GRA biosynthesis pathway was upregulated at all time points, and a deduced GRA biosynthesis pathway map was constructed for reference.

The phosphorylated pathway of serine biosynthesis is crucial for indolic glucosinolate biosynthesis and plant growth promotion conferred by the root endophyte Colletotrichum tofieldiae.

KEY MESSAGE: Phosphoglycerate Dehydrogenase 1 of the phosphorylated pathway of serine biosynthesis, active in heterotrophic plastids, is required for the synthesis of serine to enable plant growth at high rates of indolic glucosinolate biosynthesis. Plants have evolved effective strategies to defend against various types of pathogens. The synthesis of a multitude of specialized metabolites represents one effective approach to keep plant attackers in check. The synthesis of those defense compounds is cost intensive and requires extensive interaction with primary metabolism. However, how primary metabolism is adjusted to fulfill the requirements of specialized metabolism is still not completely resolved. Here, we studied the role of the phosphorylated pathway of serine biosynthesis (PPSB) for the synthesis of glucosinolates, the main class of defensive compounds in the model plant Arabidopsis thaliana. We show that major genes of the PPSB are co-expressed with genes required for the synthesis of tryptophan, the unique precursor for the formation of indolic glucosinolates (IG). Transcriptional and metabolic characterization of loss-of-function and dominant mutants of ALTERED TRYPTOPHAN1-like transcription factors revealed demand driven activation of PPSB genes by major regulators of IG biosynthesis. Trans-activation of PPSB promoters by ATR1/MYB34 transcription factor in cultured root cells confirmed this finding. The content of IGs were significantly reduced in plants compromised in the PPSB and these plants showed higher sensitivity against treatment with 5-methyl-tryptophan, a characteristic behavior of mutants impaired in IG biosynthesis. We further found that serine produced by the PPSB is required to enable plant growth under conditions of high demand for IG. In addition, PPSB-deficient plants lack the growth promoting effect resulting from interaction with the beneficial root-colonizing fungus Colletotrichum tofieldiae.

BrPP5.2 Overexpression Confers Heat Shock Tolerance in Transgenic Brassica rapa through Inherent Chaperone Activity, Induced Glucosinolate Biosynthesis, and Differential Regulation of Abiotic Stress Response Genes.

Plant phosphoprotein phosphatases are ubiquitous and multifarious enzymes that respond to developmental requirements and stress signals through reversible dephosphorylation of target proteins. In this study, we investigated the hitherto unknown functions of Brassica rapa protein phosphatase 5.2 (BrPP5.2) by transgenic overexpression of B. rapa lines. The overexpression of BrPP5.2 in transgenic lines conferred heat shock tolerance in 65-89% of the young transgenic seedlings exposed to 46 °C for 25 min. The examination of purified recombinant BrPP5.2 at different molar ratios efficiently prevented the thermal aggregation of malate dehydrogenase at 42 °C, thus suggesting that BrPP5.2 has inherent chaperone activities. The transcriptomic dynamics of transgenic lines, as determined using RNA-seq, revealed that 997 and 1206 (FDR < 0.05, logFC ≥ 2) genes were up- and down-regulated, as compared to non-transgenic controls. Statistical enrichment analyses revealed abiotic stress response genes, including heat stress response (HSR), showed reduced expression in transgenic lines under optimal growth conditions. However, most of the HSR DEGs were upregulated under high temperature stress (37 °C/1 h) conditions. In addition, the glucosinolate biosynthesis gene expression and total glucosinolate content increased in the transgenic lines. These findings provide a new avenue related to BrPP5.2 downstream genes and their crucial metabolic and heat stress responses in plants.

CRISPR-Cas9-Mediated Gene Editing of MYB28 Genes Impair Glucoraphanin Accumulation of Brassica oleracea in the Field.

Discoveries in model plants grown under optimal conditions can provide important directions for crop improvement. However, it is important to verify whether results can be translated to crop plants grown in the field. In this study, we sought to study the role of MYB28 in the regulation of aliphatic glucosinolate (A-GSL) biosynthesis and associated sulfur metabolism in field-grown Brassica oleracea with the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene-editing technology. We describe the first myb28 knockout mutant in B. oleracea, and the first CRISPR field trial in the United Kingdom approved and regulated by the UK Department for Environment, Food & Rural Affairs after the reclassification of gene-edited crops as genetically modified organisms by the European Court of Justice on July 25, 2018. We report that knocking out myb28 results in downregulation of A-GSL biosynthesis genes and reduction in accumulation of the methionine-derived glucosinolate, glucoraphanin, in leaves and florets of field-grown myb28 mutant broccoli plants, whereas accumulation of sulfate, S-methyl cysteine sulfoxide, and indole glucosinolate in leaf and floret tissues remained unchanged. These results demonstrate the potential of gene-editing approaches to translate discoveries in fundamental biological processes for improved crop performance.

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

BACKGROUND: Rose 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. RESULT: In 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. CONCLUSION: Fifty-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.

LATE ELONGATED HYPOCOTYL potentiates resistance conferred by CIRCADIAN CLOCK ASSOCIATED1 to aphid by co-regulating the expression of indole glucosinolate biosynthetic genes.

CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are core components of the circadian clock in Arabidopsis thaliana that impacts plant response to biotic stresses. Their clock-regulating functions are believed to be partially redundant, and mutation of either gene leads to shortened periods of the circadian cycle. Our recent study has demonstrated that CCA1 promotes plant resistance to the green peach aphid (Myzus persicae) through modulation of indole glucosinolate biosynthesis, but the role of LHY remains to be elucidated. Here we showed that, similar to cca1-11, single mutant lhy-21 became more susceptible to aphid infestation. Damage to the cca1-11 lhy-21 double mutant by aphids was most pronounced, indicating that the defensive roles of CCA1 and LHY were not entirely redundant. Also, the cyclic expression pattern of key indole glucosinolate biosynthetic genes was considerably disturbed in both single mutants and this was more severe in the double mutant. Apparently, both CCA1 and LHY were necessary for circadian-regulated indole glucosinolate biosynthesis. Taken together, LHY-CCA1 coordination in transcriptional regulation of indole glucosinolate biosynthetic genes most likely contributed to plant defensive capacity against aphids.

Overexpression of the ribosomal S30 subunit leads to indole-3-carbinol tolerance in Arabidopsis thaliana.

Indole-3-carbinol (I3C), a hydrolysis product of indole-3-methylglucosinolate, is toxic to herbivorous insects and pathogens. In mammals, I3C is extensively studied for its properties in cancer prevention and treatment. Produced in Brassicaceae, I3C reversibly inhibits root elongation in a concentration-dependent manner. This inhibition is partially explained by the antagonistic action of I3C on auxin signaling through TIR1. To further elucidate the mode of action of I3C in plants, we have identified and characterized a novel Arabidopsis mutant tolerant to I3C, ICT1. This mutant was identified following screening of the Full-length cDNA Over-eXpression library (FOX) seed collection for root growth in the presence of exogenous I3C. ICT1 carries the AT2G19750 gene, which encodes an S30 ribosomal protein. Overexpression, but not knockout, of the S30 gene causes tolerance to I3C. The tolerance is specific to I3C, since ICT1 did not exhibit pronounced tolerance to other indole or benzoxazinoid molecules tested. ICT1 maintains I3C-induced antagonism of auxin signaling, indicating that the tolerance is due to an auxin-independent mechanism. Transcript profiling experiments revealed that ICT1 is transcriptionally primed to respond to I3C treatment.

Effects of long light exposure and drought stress on plant growth and glucosinolate production in pak choi (Brassica rapa subsp. chinensis).

Glucosinolates (GLs), found in Brassicaceae family, are precursor metabolites with anti-cancer properties. Increased GLs have been studied under various environmental growth conditions. Pak choi (Brassica rapa subsp. chinensis) is a GL-rich vegetable. We hypothesize that long exposure to light and drought will increase the biomass of, and GL production in, pak choi. The experiment was conducted for 6 weeks. Long light exposure (20 h/day) increased, whilst drought exposure (12 h/week) decreased the plant growth. The plants exposed to a combination of drought and long light conditions showed similar growth pattern as control plants. GL production increased at week 6 in plants exposed to long light, while drought exposure had no impact on GL production, with the exception of glucoraphanin. Significant positive correlations were observed between plant growth and GL yield with accumulated light exposure time. Our findings suggest that long exposure to light can be used to increase both the biomass and GL production in pak choi.

Glucosinolates: Natural Occurrence, Biosynthesis, Accessibility, Isolation, Structures, and Biological Activities.

Glucosinolates (GSLs) are secondary plant metabolites abundantly found in plant order Brassicales. GSLs are constituted by an S-ß-d-glucopyrano unit anomerically connected to O-sulfated (Z)-thiohydroximate moiety. The side-chain of the O-sulfate thiohydroximate moiety, which is derived from a different amino acid, contributes to the diversity of natural GSL, with more than 130 structures identified and validated to this day. Both the structural diversity of GSL and their biological implication in plants have been biochemically studied. Although chemical syntheses of GSL have been devised to give access to these secondary metabolites, direct extraction from biomass remains the conventional method to isolate natural GSL. While intact GSLs are biologically inactive, various products, including isothiocyanates, nitriles, epithionitriles, and cyanides obtained through their hydrolysis of GSLs, exhibit many different biological activities, among which several therapeutic benefits have been suggested. This article reviews natural occurrence, accessibility via chemical, synthetic biochemical pathways of GSL, and the current methodology of extraction, purification, and characterization. Structural information, including the most recent classification of GSL, and their stability and storage conditions will also be discussed. The biological perspective will also be explored to demonstrate the importance of these prominent metabolites.

The Combination of Selenium and LED Light Quality Affects Growth and Nutritional Properties of Broccoli Sprouts.

Selenium (Se) supplement was combined with different LED light qualities to investigate mutual effects on the growth, nutritional quality, contents of glucosinolates and mineral elements in broccoli sprouts. There were five treatments: CK:1R1B1G, 1R1B1G+Se (100 µmol L-1 Na2SeO3), 1R1B+Se, 1R2B+Se, 2R1B+Se, 60 µmol m-2 s-1 PPFD, 12 h/12 h (light/dark). Sprouts under a combination of selenium and LED light quality treatment exhibited no remarkable change fresh weight, but had a shorter hypocotyl length, lower moisture content and heavier dry weight, especially with 1R2B+Se treatment. The contents of carotenoid, soluble protein, soluble sugar, vitamin C, total flavonoids, total polyphenol and contents of total glucosinolates and organic Se were dramatically improved through the combination of Se and LED light quality. Moreover, heat map and principal component analysis showed that broccoli sprouts under 1R2B+Se treatment had higher nutritional quality and health-promoting compound contents than other treatments. This suggests that the Se supplement under suitable LED lights might be beneficial to selenium-biofortified broccoli sprout production.

Induction of Glucoraphasatin Biosynthesis Genes by MYB29 in Radish (Raphanus sativus L.) Roots.

Glucoraphasatin (GRH) is a specific aliphatic glucosinolate (GSL) that is only abundant in radish (Raphanus sativus L.). The gene expression regulating GRH biosynthesis in radish is still poorly understood. We employed a total of 59 radish accessions to analyze GSL profiles and showed that GRH was specific and predominant among the aliphatic GSLs in radish roots. We selected five accessions roots with high, moderate and low GSL biosynthesis, respectively, to conduct a comparative transcriptome analysis and the qRT-PCR of the biosynthesis genes for aliphatic GSLs. In this study, among all the accessions tested, roots with the accession RA157-74 had a high GRH content and showed a significant expression of the aliphatic GSL biosynthesis genes. We defined the genes involved in the GRH biosynthesis process and found that they were regulated by a transcription factor (RSG00789) at the MYB29 locus in radish roots. We found 13 aliphatic GSL biosynthesis genes regulated by the RSG00789 gene in the GRH biosynthesis pathway.

The Influence of Nasturtium officinale R. Br. Agar and Agitated Microshoot Culture Media on Glucosinolate and Phenolic Acid Production, and Antioxidant Activity.

This paper presents an optimization of conditions for microshoot cultures of Nasturtium officinale R. Br. (watercress). Variants of the Murashige and Skoog (MS) medium containing different plant growth regulators (PGRs): cytokinins-BA (6-benzyladenine), 2iP (6-γ,γ-dimethylallylaminopurine), KIN (kinetin), Zea (zeatin), and auxins-IAA (3-indoleacetic acid), IBA (indole-3-butyric acid), 2,4-d (2,4-dichlorophenoxyacetic acid), IPA (indole-3-pyruvic acid), NAA (naphthalene-1-acetic acid), total 27 MS variants, were tested in agar and agitated cultures. Growth cycles were tested for 10, 20, or 30 days in the agar cultures, and 10 or 20 days in the agitated cultures. Glucosinolate and phenolic acid production, total phenolic content and antioxidant potential were evaluated. The total amounts of glucosinolates ranged from 100.23 to 194.77 mg/100 g dry weight of biomass (DW) in agar cultures, and from 78.09 to 182.80 mg/100 g DW in agitated cultures. The total phenolic acid content varied from 15.89 to 237.52 mg/100 g DW for the agar cultures, and from 70.80 to 236.74 mg/100 g DW for the agitated cultures. Extracts of the cultured biomass contained higher total amounts of phenolic acids, lower total amounts of glucosinolates, a higher total phenolic content and similar antioxidant potentials compared to plant material. The analyses performed confirmed for the first time the explicit influence on secondary metabolite production and on the antioxidant potential. The significance was statistically estimated in a complex manner.

A Comprehensive Gene Inventory for Glucosinolate Biosynthetic Pathway in Arabidopsis thaliana.

Glucosinolates (GSLs) are plant secondary metabolites comprising sulfur and nitrogen mainly found in plants from the order of Brassicales, such as broccoli, cabbage, and Arabidopsis thaliana. The activated forms of GSL play important roles in fighting against pathogens and have health benefits to humans. The increasing amount of data on A. thaliana generated from various omics technologies can be investigated more deeply in search of new genes or compounds involved in GSL biosynthesis and metabolism. This review describes a comprehensive inventory of A. thaliana GSLs identified from published literature and databases such as KNApSAcK, KEGG, and AraCyc. A total of 113 GSL genes encoding for 23 transcription components, 85 enzymes, and five protein transporters were experimentally characterized in the past two decades. Continuous efforts are still on going to identify all molecules related to the production of GSLs. A manually curated database known as SuCCombase (http://plant-scc.org) was developed to serve as a comprehensive GSL inventory. Realizing lack of information on the regulation of GSL biosynthesis and degradation mechanisms, this review also includes relevant information and their connections with crosstalk among various factors, such as light, sulfur metabolism, and nitrogen metabolism, not only in A. thaliana but also in other crucifers.

Production of plant-derived anticancer precursor glucoraphanin in chromosomally engineered Escherichia coli.

Glucoraphanin is a methionine-derived glucosinolate that imparts numerous health-benefits with broad bioactivity. Low amounts in plant tissues and high cost of extraction have limited the production of glucoraphanin. Metabolic engineering in heterologous microorganisms is an attractive approach to achieve efficient production of valuable natural products. In this study, a microbial fermentation process for glucoraphanin production was demonstrated. The engineered bacterial strain stably expressed 10 allogeneic enzymes in E. coli chromosome, including nine heterologous genes from Arabidopsis and Brassica and one from fungus Neurospora crassa, which could produce the specialized glucosinolate compound glucoraphanin with a titer of 0.675 µg/L by fermentation from glucose. The cofactor supplements and individual gene overexpression for glucoraphanin production were also investigated. This work highlights the possibility of supplying specialized plant glucosinolates by microbial fermentation process, instead of chemical extraction. Additionally, the limiting step enzyme, UDP-glucose-thiohydroximate glucosyltransferase, identified in this study also laid a foundation for further optimizing the glucoraphanin-producing cell factory.

De novo indol-3-ylmethyl glucosinolate biosynthesis, and not long-distance transport, contributes to defence of Arabidopsis against powdery mildew.

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolate is constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolate is activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis demonstrate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.