An UMAMIT-GTR transporter cascade controls glucosinolate seed loading in Arabidopsis.

Many plant species translocate maternally synthesized specialized metabolites to the seed to protect the developing embryo and later the germinating seedling before it initiates its own de novo synthesis. While the transport route into the seed is well established for primary metabolites, no model exists for any class of specialized metabolites that move from maternal source tissue(s) to embryo. Glucosinolate seed loading in Arabidopsis depends on plasma membrane localized exporters (USUALLY MULTIPLE AMINO ACIDS MOVE IN AND OUT TRANSPORTERs, UMAMITs) and importers (GLUCOSINOLATE TRANSPORTERs, GTRs), but the critical barriers in the seed loading process remain unknown.

Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories.

Production of plant secondary metabolites in engineered microorganisms provides a scalable and sustainable alternative to their sourcing from nature or through chemical synthesis. However, the biosynthesis of many valuable plant-derived products relies on cytochromes P450 - enzymes notoriously difficult to express in microbes. To improve their expression in Escherichia coli, an arsenal of engineering strategies was developed, often paired with an extensive screening of enzyme variants.

Identification of key amino acid residues in AtUMAMIT29 for transport of glucosinolates.

Glucosinolates are key defense compounds of plants in Brassicales order, and their accumulation in seeds is essential for the protection of the next generation. Recently, members of the Usually Multiple Amino acids Move In and Out Transporter (UMAMIT) family were shown to be essential for facilitating transport of seed-bound glucosinolates from site of synthesis within the reproductive organ to seeds. Here, we set out to identify amino acid residues responsible for glucosinolate transport activity of the main seed glucosinolate exporter UMAMIT29 in .

Export of defensive glucosinolates is key for their accumulation in seeds.

Plant membrane transporters controlling metabolite distribution contribute key agronomic traits. To eliminate anti-nutritional factors in edible parts of crops, the mutation of importers can block the accumulation of these factors in sink tissues. However, this often results in a substantially altered distribution pattern within the plant, whereas engineering of exporters may prevent such changes in distribution.

Comparison of Genome and Plasmid-Based Engineering of Multigene Benzylglucosinolate Pathway in Saccharomyces cerevisiae.

Intake of brassicaceous vegetables such as cabbage is associated with numerous health benefits. The major defense compounds in the Brassicales order are the amino acid-derived glucosinolates that have been associated with the health-promoting effects. This has primed a desire to build glucosinolate-producing microbial cell factories as a stable and reliable source.

Correction: The ins and outs of transporters at plasma membrane and tonoplast in plant specialized metabolism.

Correction for 'The ins and outs of transporters at plasma membrane and tonoplast in plant specialized metabolism' by Deyang Xu and Barbara Ann Halkier, , 2022, https://doi.org/10.1039/d2np00016d.

The ins and outs of transporters at plasma membrane and tonoplast in plant specialized metabolism.

Covering: up to 2022Plants are organic chemists par excellence and produce an amazing array of diverse chemical structures. Whereas primary metabolites are essential for all living organisms and highly conserved, the specialized metabolites constitute the taxonomy-specific chemical languages that are key for fitness and survival. Allocation of plants' wide array of specialized metabolites in patterns that are fine-tuned spatiotemporally is essential for adaptation to the ever-changing environment and requires transport processes.

Herbivore feeding preference corroborates optimal defense theory for specialized metabolites within plants.

Numerous plants protect themselves from attackers by 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.

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

2-Phenylethylglucosinolate (2PE) derived from homophenylalanine is present in plants of the Brassicales order as a defense compound. It is associated with multiple biological properties, including deterrent effects on pests and antimicrobial and health-promoting functions, due to its hydrolysis product 2-phenylethyl isothiocyanate, which confers 2PE as a potential application in agriculture and industry. In this study, we characterized the putative key genes for 2PE biosynthesis from Barbarea vulgaris W.

In Substrate Recognition and Tissue- as Well as Plastid Type-Specific Expression Define the Roles of Distinct Small Subunits of Isopropylmalate Isomerase.

In , the heterodimeric isopropylmalate isomerase (IPMI) is composed of a single large (IPMI LSU1) and one of three different small subunits (IPMI SSU1 to 3). The function of IPMI is defined by the small subunits. IPMI SSU1 is required for Leu biosynthesis and has previously also been proposed to be involved in the first cycle of Met chain elongation, the first phase of the synthesis of Met-derived glucosinolates.

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.

Characterization of Arabidopsis CYP79C1 and CYP79C2 by Glucosinolate Pathway Engineering in Shows Substrate Specificity Toward a Range of Aliphatic and Aromatic Amino Acids.

Glucosinolates (GLSs) are amino acid-derived defense compounds characteristic of the Brassicales order. Cytochromes P450s of the CYP79 family are the entry point into the biosynthetic pathway of the GLS core structure and catalyze the conversion of amino acids to oximes. In , CYP79A2, CYP79B2, CYP79B3, CYP79F1, and CYP79F2 have been functionally characterized and are responsible for the biosynthesis of phenylalanine-, tryptophan-, and methionine-derived GLSs, respectively.

GTR-Mediated Radial Import Directs Accumulation of Defensive Glucosinolates to Sulfur-Rich Cells in the Phloem Cap of Arabidopsis Inflorescence Stem.

In the phloem cap region of Arabidopsis plants, sulfur-rich cells (S-cells) accumulate >100 mM glucosinolates (GLS), but are biosynthetically inactive. The source and route of S-cell-bound GLS remain elusive. In this study, using single-cell sampling and scanning electron microscopy with energy-dispersive X-ray analysis we show that two GLS importers, NPF2.

Changing substrate specificity and iteration of amino acid chain elongation in glucosinolate biosynthesis through targeted mutagenesis of methylthioalkylmalate synthase 1.

Methylthioalkylmalate synthases catalyse the committing step of amino acid chain elongation in glucosinolate biosynthesis. As such, this group of enzymes plays an important role in determining the glucosinolate composition of Brassicaceae species, including Based on protein structure modelling of MAM1 from and analysis of 57 MAM sequences from Brassicaceae species, we identified four polymorphic residues likely to interact with the 2-oxo acid substrate. Through site-directed mutagenesis, the natural variation in these residues and the effect on product composition were investigated.

Arabidopsis glucosinolate storage cells transform into phloem fibres at late stages of development.

The phloem cap of Arabidopsis thaliana accumulates glucosinolates that yield toxic catabolites upon damage-induced hydrolysis. These defence compounds are stored in high concentrations in millimetre long S-cells. At early stages of development, S-cells initiate a process indicative of programmed cell death.

Identification of genes involved in shea butter biosynthesis from Vitellaria paradoxa fruits through transcriptomics and functional heterologous expression.

Shea tree (Vitellaria paradoxa) is one economically important plant species that mainly distributes in West Africa. Shea butter extracted from shea fruit kernels can be used as valuable products in the food and cosmetic industries. The most valuable composition in shea butter was one kind of triacylglycerol (TAG), 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0-C18:1-C18:0).

De novo production of benzyl glucosinolate in Escherichia coli.

Microbial production of plant specialised metabolites is challenging as the biosynthetic pathways are often complex and can contain enzymes, which function is not supported in traditional production hosts. Glucosinolates are specialised metabolites of strong commercial interest due to their health-promoting effects. In this work, we engineered the production of benzyl glucosinolate in Escherichia coli.

Biotechnological approaches in glucosinolate production.

Glucosinolates (GLSs) are sulfur-rich, amino acid-derived defense compounds characteristic of the Brassicales order. In the past, GLSs were mostly known as anti-nutritional factors in fodder, biopesticides in agriculture, and flavors in condiments such as mustard. However, in recent times, GLSs have received increased attention as promoters of human health.

How to prove the existence of metabolons?

Sequential enzymes in biosynthetic pathways are organized in metabolons. It is challenging to provide experimental evidence for the existence of metabolons as biosynthetic pathways are composed of highly dynamic protein-protein interactions. Many different methods are being applied, each with strengths and weaknesses.

Uptake Assays in Oocytes Using Liquid Chromatography-mass Spectrometry to Detect Transport Activity.

oocytes are a widely used model system for characterization of heterologously expressed secondary active transporters. Historically, researchers have relied on detecting transport activity by measuring accumulation of radiolabeled substrates by scintillation counting or of fluorescently labelled substrates by spectrofluorometric quantification. These techniques are, however, limited to substrates that are available as radiolabeled isotopes or to when the substrate is fluorescent.

How does a plant orchestrate defense in time and space? Using glucosinolates in Arabidopsis as case study.

The sessile nature of plants has caused plants to develop means to defend themselves against attacking organisms. Multiple strategies range from physical barriers to chemical warfare including pre-formed anticipins as well as phytoalexins produced only upon attack. While phytoalexins require rapid induction, constitutive defenses can impose ecological costs if they deter pollinators or attract specialized herbivores.

Advances in methods for identification and characterization of plant transporter function.

Transport proteins are crucial for cellular function at all levels. Numerous importers and exporters facilitate transport of a diverse array of metabolites and ions intra- and intercellularly. Identification of transporter function is essential for understanding biological processes at both the cellular and organismal level.

Reduction of antinutritional glucosinolates in Brassica oilseeds by mutation of genes encoding transporters.

The nutritional value of Brassica seed meals is reduced by the presence of glucosinolates, which are toxic compounds involved in plant defense. Mutation of the genes encoding two glucosinolate transporters (GTRs) eliminated glucosinolates from Arabidopsis thaliana seeds, but translation of loss-of-function phenotypes into Brassica crops is challenging because Brassica is polyploid. We mutated one of seven and four of 12 GTR orthologs and reduced glucosinolate levels in seeds by 60-70% in two different Brassica species (Brassica rapa and Brassica juncea).