Thylakoid Targeting Improves Stability of a Cytochrome P450 in the Cyanobacterium sp. PCC 6803.

Plants produce a large array of natural products of biotechnological interest. In many cases, these compounds are naturally produced at low titers and involve complex biosynthetic pathways, which often include cytochrome P450 enzymes. P450s are known to be difficult to express in traditional heterotrophic chassis.

Apoplastic barriers are essential for nodule formation and nitrogen fixation in .

Establishment of the apoplastic root barrier known as the Casparian strip occurs early in root development. In legumes, this area overlaps with nitrogen-fixing nodule formation, which raises the possibility that nodulation and barrier formation are connected. Nodules also contain Casparian strips, yet, in this case, their role is unknown.

ZINC FINGER PROTEIN2 suppresses funiculus lignification to ensure seed loading efficiency in Arabidopsis.

The plant funiculus anchors the developing seed to the placenta within the inner dorsal pod strands of the silique wall and directly transports nutrients to the seeds. The lignified vasculature critically supports nutrient transport through the funiculus. However, molecular mechanisms underlying lignified secondary cell wall (SCW) biosynthesis in the funiculus remain elusive.

A quest for the potato of the future: characterization of wild tuber-bearing Solanum species for de novo domestication.

Potato (Solanum tuberosum) is a staple food worldwide, but modern potato cultivation relies heavily on the use of pesticides to control pests and diseases. However, many wild Solanum species are highly resistant to biotic and abiotic stresses relevant to potato production. Several of those species have been used in potato breeding to confer resistance but this has only been moderately successful.

Site-directed genotype screening for elimination of antinutritional saponins in quinoa seeds identifies TSARL1 as a master controller of saponin biosynthesis selectively in seeds.

Climate change may result in a drier climate and increased salinization, threatening agricultural productivity worldwide. Quinoa (Chenopodium quinoa) produces highly nutritious seeds and tolerates abiotic stresses such as drought and high salinity, making it a promising future food source. However, the presence of antinutritional saponins in their seeds is an undesirable trait.

Inverse pH Gradient-Assay for Facile Characterization of Proton-Antiporters in Oocytes.

oocytes represent one of the most versatile model systems for characterizing the properties of membrane transporters. However, for studying proton-coupled antiporters, the use of oocytes has so far been limited to so-called injection-based transport assays. In such assays, where the compound is injected directly into the oocytes' cytosol and transport is detected by monitoring substrate efflux, poor control over internal diffusion and concentration are incompatible with mechanistic characterizations.

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 .

Phytoalexins of the crucifer Barbarea vulgaris: Structural profile and correlation with glucosinolate turnover.

Phytoalexins are antimicrobial plant metabolites elicited by microbial attack or abiotic stress. We investigated phytoalexin profiles after foliar abiotic elicitation in the crucifer Barbarea vulgaris and interactions with the glucosinolate-myrosinase system. The treatment for abiotic elicitation was a foliar spray with CuCl solution, a usual eliciting agent, and three independent experiments were carried out.

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.

Gibberellin and abscisic acid transporters facilitate endodermal suberin formation in Arabidopsis.

The plant hormone gibberellin (GA) regulates multiple developmental processes. It accumulates in the root elongating endodermis, but how it moves into this cell file and the significance of this accumulation are unclear. Here we identify three NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER (NPF) transporters required for GA and abscisic acid (ABA) translocation.

Multi-Knock-a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants.

Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components.

Species-specific dynamics of specialized metabolism in germinating sorghum grain revealed by temporal and tissue-resolved transcriptomics and metabolomics.

Seed germination is crucial for plant productivity, and the biochemical changes during germination affect seedling survival, plant health and yield. While the general metabolism of germination is extensively studied, the role of specialized metabolism is less investigated. We therefore analyzed the metabolism of the defense compound dhurrin during sorghum (Sorghum bicolor) grain germination and early seedling development.

SWEET11b transports both sugar and cytokinin in developing barley grains.

Even though Sugars Will Eventually be Exported Transporters (SWEETs) have been found in every sequenced plant genome, a comprehensive understanding of their functionality is lacking. In this study, we focused on the SWEET family of barley (Hordeum vulgare). A radiotracer assay revealed that expressing HvSWEET11b in African clawed frog (Xenopus laevis) oocytes facilitated the bidirectional transfer of not only just sucrose and glucose, but also cytokinin.

Exploiting photosynthesis-driven P450 activity to produce indican in tobacco chloroplasts.

Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical diversity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules.

Artificial Fluorescent Glucosinolates (F-GSLs) Are Transported by the Glucosinolate Transporters GTR1/2/3.

The glucosinolate transporters 1/2/3 (GTR1/2/3) from the Nitrate and Peptide transporter Family (NPF) play an essential role in the transport, accumulation, and distribution of the specialized plant metabolite glucosinolates. Due to representing both antinutritional and health-promoting compounds, there is increasing interest in characterizing GTRs from various plant species. We generated seven artificial glucosinolates (either aliphatic or benzenic) bearing different fluorophores (Fluorescein, BODIPY, Rhodamine, Dansylamide, and NBD) and investigated the ability of GTR1/2/3 from to import the fluorescent glucosinolates (F-GSLs) into oocytes from .

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.

Structure-guided engineering of key amino acids in UGT85B1 controlling substrate and stereo-specificity in aromatic cyanogenic glucoside biosynthesis.

Cyanogenic glucosides are important defense molecules in plants with useful biological activities in animals. Their last biosynthetic step consists of a glycosylation reaction that confers stability and increases structural diversity and is catalyzed by the UDP-dependent glycosyltransferases (UGTs) of glycosyltransferase family 1. These versatile enzymes have large and varied substrate scopes, and the structure-function relationships controlling scope and specificity remain poorly understood.

Structural Insights into the Substrate Transport Mechanisms in GTR Transporters through Ensemble Docking.

Glucosinolate transporters (GTRs) are part of the nitrate/peptide transporter (NPF) family, members of which also transport specialized secondary metabolites as substrates. Glucosinolates are defense compounds derived from amino acids. We selected 4-methylthiobutyl (4MTB) and indol-3-ylmethyl (I3M) glucosinolates to study how GTR1 from transports these substrates in computational simulation approaches.

Cyanogenesis in the Genus: From Genotype to Phenotype.

Domestication has resulted in a loss of genetic diversity in our major food crops, leading to susceptibility to biotic and abiotic stresses linked with climate change. Crop wild relatives (CWR) may provide a source of novel genes potentially important for re-gaining climate resilience. is an important cereal crop with wild relatives that are endemic to Australia.

The biosynthesis of thymol, carvacrol, and thymohydroquinone in Lamiaceae proceeds via cytochrome P450s and a short-chain dehydrogenase.

Thymol and carvacrol are phenolic monoterpenes found in thyme, oregano, and several other species of the Lamiaceae. Long valued for their smell and taste, these substances also have antibacterial and anti-spasmolytic properties. They are also suggested to be precursors of thymohydroquinone and thymoquinone, monoterpenes with anti-inflammatory, antioxidant, and antitumor activities.

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.

Cytosolic phosphofructokinases are important for sugar homeostasis in leaves of Arabidopsis thaliana.

Background And Aims: ATP-dependent phosphofructokinases (PFKs) catalyse phosphorylation of the carbon-1 position of fructose-6-phosphate, to form fructose-1,6-bisphosphate. In the cytosol, this is considered a key step in channelling carbon into glycolysis. Arabidopsis thaliana has seven genes encoding PFK isoforms, two chloroplastic and five cytosolic.