Biology subjects

Halkier, B. A.

Publications and source records attributed to Halkier, B. A..

8 recordsLinked to original sources

bioRxiv2026

Characterisation of gene expression markers and glucosinolates during discrete infection stages of Pyrenopeziza brassicae in Brassica napus

Light leaf spot (LLS), caused by subcuticular hemibiotrophic ascomycete fungus Pyrenopeziza brassicae, is a major constraint on oilseed rape (Brassica napus) production, yet the genetic and biochemical mechanisms of quantitative disease resistance (QDR) remain poorly defined. Here, disease phenotyping, pathogen quantification, microscopy, gene expression profiling and glucosinolate (GSL) analysis were integrated to dissect resistance mechanisms in B. napus. Disease assays of 19 diverse lines revealed clear contrasts between susceptible and resistant genotypes, with the commercial cultivar Ambassador showing a phenotype inconsistent with the UK Recommended List rating. Microscopy demonstrated that resistance within doubled haploid line Cubs Root does not inhibit spore germination or penetration but restricts hyphal branching and subcuticular colonisation from 4 to 8 days post-inoculation. Expression profiling of seven candidate gene expression markers (GEMs) and pathogenesis-related PR1 showed that cinnamate-4-hydroxylase, phospholipase C4, {beta}-adaptin, universal stress protein and the 40S ribosomal subunit protein S24 were strongly pathogen-induced in resistant lines, whereas a BAHD acyltransferase, a putative susceptibility factor, was induced only in susceptible cultivars. GSL profiling identified negative correlations between disease severity and total GSLs, particularly aliphatic and aromatic GSLs, with 2{square}phenylethyl and 7-methylsulfinyl heptyl GSLs showing the strongest associations with resistance. Together, these results highlight coordinated transcriptional and metabolic responses that limit pathogen proliferation and provide targets for breeding durable LLS resistance in B. napus.

plant biology↗

bioRxiv2026

Utilizing raw rapeseed press cake in foods: A case study on sensory quality and profile of selected bitter compounds in snack bars

Todays canola quality rapeseed press cake (RPC) is a protein-rich co-product with potential as human food, but its application is limited due to antinutritional compounds and bitter taste. It remains, however, unknown how introduction of raw RPC to a food matrix affects sensory perception and which metabolites drive the sensation. Here, raw RPC from whole or dehulled seeds was introduced into snack bars at 0%, 7%, 14%, and 21%, and sensory responses were correlated to selected known RPC-derived bitter compounds. A trained panel evaluated 13 RPC-characteristic sensory attributes, and the bitter compounds sinapic acid, kaempferol 3-O-(2'''-O-sinapoyl-{beta}-sophoroside) (KSS), KSS-hexose, selected bitter glucosinolates, and goitrin were quantified using targeted LC-MS/MS. Most dose-dependent sensory responses increased up to 14% RPC and then plateaued, whereas astringent mouthfeel increased almost linearly across the full dose range. Dehulling intensified several odor- and flavor-related attributes but did not increase bitterness or protein content in the final product. Principal component analysis linked bitterness and astringency positively with KSS, KSS-hexose, and goitrin. Dose-over-threshold analysis further showed that goitrin, but not progoitrin, reached concentrations relevant for bitterness perception. Together, the results demonstrate that raw RPC contributes distinct dose-dependent sensory attributes and that metabolite transformations in the food matrix shape final sensory profiles. These findings provide a basis for developing RPC-containing foods and for breeding rapeseed lines with improved sensory characteristics. HIGHLIGHTSO_LIThis study presents the first sensory panel assessment of rapeseed press cake (RPC)-containing in food products (snack bars) made from whole and dehulled seeds. C_LIO_LI13 RPC-characteristic sensory attributes are identified. C_LIO_LISensory profiles of the tasted snack bars differed significantly, influenced by the dosage of RPC and by the dehulling treatment. Bitterness and astringency are positively correlated with the RPC dosage. C_LIO_LIGoitrin, kaempferol 3-O-(2'''-O-sinapoyl-{beta}-sophoroside) (KSS) and sinapic acid are RPC-derived bitter compounds that correlate with bitter taste of RPC-containing snack bars. C_LIO_LIApproximately 90% of glucosinolates introduced with the RPC are not detected in the snack bars, and goitrin levels in snack bars accounts for only [~]10% of introduced progoitrin. C_LIO_LIGoitrin is - for the first time - reported to contribute to the perceived bitterness of an RPC-containing food product. C_LI

plant biology↗

bioRxiv2026

Optimization of the glucosinolate core pathway for production of simple glucosinolates in Escherichia coli

Microbial biosynthesis of plant secondary metabolites aims to provide access to compounds of medicinal or industrial value independently of the native plant. Glucosinolates are plant secondary metabolites characteristic of brassicaceous plants and recognized as promoters of human health. However, plants often contain a complex mixture of glucosinolates with insufficient amounts to elicit a clinical effect through diet. Here, we demonstrate the biosynthesis of defined glucosinolate products in Escherichia coli through combinatorial screening of pathway enzyme homologs, tailoring the optimal biosynthetic route for each individual product. To achieve high product titers, we establish efficient P450 expression by membrane anchor truncation and engineer sulfate assimilation to increase the supply of a sulfate donor 3-phosphoadenosine-5-phosphosulfate. We use benzyl glucosinolate pathway as a model to test the engineering strategies and improve the titer 37-fold over our previous study. Extrapolating the best approaches to other simple glucosinolates, we establish the first microbial synthesis of tyrosine-derived p-glucosinolate. Showing the highest titer overall, we report production of 1250 {+/-} 91 {micro}M indolyl-3-methyl glucosinolate, a 500-fold increase over biosynthesis in yeast.

synthetic biology↗

bioRxiv2025

Isolation of a novel plant growth-promoting Dyella sp. from a Danish natural soil

Environmental soils are natural reservoirs of unexplored microbes, including potentially beneficial microbes to improve plant performance. Here, we isolated 75 bacterial strains from surface-sterilized roots of Arabidopsis thaliana (Arabidopsis) grown in a natural soil derived from an alder swamp. Culture-dependent isolation of individual strains from the roots followed by monoassociation-based screening identified seven bacteria that promoted Arabidopsis seedling weight. Of those, we identified a new species from the Dyella genus which increased biomass of tomato and Arabidopsis seedlings in agar as well as the shoot biomass of Arabidopsis grown in both the alder swamp soil and potting soil. Dyella sp. A4 specifically promoted the elongation of lateral roots without affecting lateral root number and primary root elongation. The new Dyella sp. A4 expands the toolbox of biostimulants for plant growth promotion via modulating root architecture.

plant biology↗

bioRxiv2022

Systematic engineering of plant cytochrome P450 system identifies a comprehensive strategy for expression of highly functional P450 enzymes in Escherichia coli

Cytochrome P450s catalyse diverse and unique chemical reactions, which makes them invaluable enzymes in nature and industry. Metabolic engineers leverage these unique catalytic properties when refactoring plant biosynthetic pathways into microbial cell factories. However, due to their hydrophobic anchor, microbial expression of membrane-bound cytochrome P450s is challenging. An arsenal of protein engineering strategies was developed to improve their expression in Escherichia coli, but extensive screening is often necessary to tailor the engineering approach to an individual enzyme. Here, we propose a universal strategy that allows the expression of highly active cytochrome P450s in E. coli by systematically evaluating six common N-terminal modifications and their effect on in vivo activity of enzymes from the CYP79 and CYP83 families. We identified transmembrane domain truncation as the only strategy that had a significantly positive effect on all seven tested enzymes, increasing product titres between 2- to 170-fold. When comparing the changes in protein titre and product generation, we show that higher expression does not always translate to higher in vivo activity, thus making protein titre an unreliable screening target. Our results demonstrate that transmembrane domain truncation improves in vivo activity across a broad range of cytochrome P450s with diverse N-terminal sequences and could be applied as the modification-of-choice to avoid the time-consuming screening process and accelerate the future design of E. coli cell factories.

bioengineering↗

bioRxiv2021

Herbivore feeding behavior validates optimal defense theory for specialized metabolites within plants

Numerous plants protect themselves from attackers using specialized metabolites. The biosynthesis of these deterrent, often toxic metabolites is costly, as their synthesis diverts energy and resources on account of growth and development. How plants diversify investments into growth and defense is explained by the optimal defense theory. The central prediction of the optimal defense theory is that plants maximize growth and defense by concentrating specialized metabolites in tissues that are decisive for fitness. To date, supporting physiological evidence merely relies on the correlation between plant metabolite distribution and animal feeding preference. Here, we use glucosinolates as a model to examine the effect of changes in chemical defense distribution on actual feeding behavior. Taking advantage of the uniform glucosinolate distribution in transporter mutants, we show that high glucosinolate accumulation in tissues important to fitness protects them by guiding larvae of a generalist herbivore to feed on other tissues. Moreover, we show that mature leaves of Arabidopsis thaliana supply young leaves with glucosinolates to optimize defense against herbivores. Our study provides physiological evidence for the central hypothesis of the optimal defense theory and sheds light on the importance of integrating glucosinolate biosynthesis and transport for optimizing plant defense.

plant biology↗

bioRxiv2020

Engineering and optimization of the 2-phenylethylglucosinolate production in Nicotiana benthamiana by combining biosynthetic genes from Barbarea vulgaris and Arabidopsis thaliana

Among the glucosinolate (GLS) defense compounds characteristic of the Brassicales order, several have been shown to promote human health. This includes 2-phenylethylglucosinolate (2PE) derived from homophenylalanine (HPhe). In this study, we used transient expression in Nicotiana benthamiana to validate and characterize previously predicted key genes in the 2PE biosynthetic pathway from Barbarea vulgaris and demonstrate the feasibility of engineering 2PE production. We used genes from B. vulgaris and Arabidopsis thaliana, in which the biosynthesis of GLSs is predominantly derived from HPhe and dihomomethionine, respectively. The resulting GLS profiles partially mirrored GLS profiles in the gene donor plant, but in both cases the profiles in N. benthamiana were wider than in the native plants. We found that BvBCAT4 is a more efficient entry enzyme for biosynthesis of both HPhe and dihomomethionine and that MAM1 enzymes determine the chain-elongated profile. Co-expression of the chain elongation pathway and CYP79F6 from B. vulgaris with the remaining aliphatic GLS core pathway genes from A. thaliana, demonstrated the feasibility of engineering production of 2PE in N. benthamiana. Noticeably, the HPhe-converting enzyme BvCYP79F6 in the core GLS pathway belongs to the CYP79F subfamily, a family believed to have substrate specificity towards chain-elongated methionine derivatives. Replacing the B. vulgaris chain elongation pathway with a chimeric pathway consisting of BvBCAT4, BvMAM1, AtIPMI and AtIPMDH1 resulted in an additional 2-fold increase in 2PE production, demonstrating that chimeric pathway with genes from different species can increase flux and boost production in an engineered pathway. Our study provides a novel approach to produce the important HPhe and 2PE in a heterologous host. Chimeric engineering of a complex biosynthetic pathway enabled detailed understanding of catalytic properties of individual enzymes - a prerequisite for understanding biochemical evolution - and with biotechnological and plant breeding potentials of new-to-nature gene combinations.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsGLSglucosinolate;2PE2-phenylethylglucosinolate;HPhehomophenylalanine;HMhomomethionine;DHMdihomomethionine;THMtrihomomethionine;HLhomoleucine;DHLdihomoleucine;PEITC2-phenylethyl isothiocyanate;3MTP3-(methylthio)propylglucosinolate;3MSP3-(methylsulfinyl)propylglucosinolate;4MTB4-(methylthio)butylglucosinolate;4MSB4-(methylsulfinyl)butylglucosinolate;BGLSbenzylglucosinolateView Full Text

synthetic biology↗

bioRxiv2019

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 glucosinolates are constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolates are 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 indicate 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.

plant biology↗