Correction to: Bioengineering potato plants to produce benzylglucosinolate for improved broad-spectrum pest and disease resistance

Transgenic Research -     

Bioengineering potato plants to produce benzylglucosinolate for improved broad-spectrum pest and disease resistance

Transgenic Research - In traditional, small-scale agriculture in the Andes, potatoes are frequently co-cultivated with the Andean edible tuber Tropaeolum tuberosum, commonly known as mashua, which...     

Cytosolic γ-glutamyl peptidases process glutathione conjugates in the biosynthesis of glucosinolates and camalexin in Arabidopsis

The defense-related plant metabolites known as glucosinolates play important roles in agriculture, ecology, and human health. Despite an advanced biochemical understanding of the glucosinolate pathway, the source of the reduced sulfur atom in the core glucosinolate structure remains unknown. Recent evidence has pointed toward GSH, which would require further involvement of a GSH conjugate processing enzyme. In this article, we show that an Arabidopsis thaliana mutant impaired in the production of the γ-glutamyl peptidases GGP1 and GGP3 has altered glucosinolate levels and accumulates up to 10 related GSH conjugates. We also show that the double mutant is impaired in the production of camalexin and accumulates high amounts of the camalexin intermediate GS-IAN upon induction. In addition, we demonstrate that the cellular and subcellular localization of GGP1 and GGP3 matches that of known glucosinolate and camalexin enzymes. Finally, we show that the purified recombinant GGPs can metabolize at least nine of the 10 glucosinolate-related GSH conjugates as well as GS-IAN. Our results demonstrate that GSH is the sulfur donor in the biosynthesis of glucosinolates and establish an in vivo function for the only known cytosolic plant γ-glutamyl peptidases, namely, the processing of GSH conjugates in the glucosinolate and camalexin pathways.     

USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5′ end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3′ overhangs designed to specifically complement each other. The combination of this principle with the improved USER cloning technique provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest. Around 90% positive clones were obtained when three different PCR products were fused and cloned into a USER-compatible vector in a simple procedure that, apart from the single PCR amplification step and the bacterial transformation, took approximately one hour. We expect this method to replace overlapping PCR and the use of type IIS restriction enzymes in many of their applications.     

Towards engineering glucosinolates into non-cruciferous plants

Glucosinolates are amino acid-derived secondary metabolites present in cruciferous plants. Glucosinolates and their hydrolysis products are involved in defence against insects and pathogens, but are also known for their characteristic flavor and their cancer-preventive and antibacterial properties. This wide range of bioactivities has prompted a desire to engineer glucosinolates into non-cruciferous plants. We report the one-step transfer of the last three steps of the benzylglucosinolate pathway (comprising the C-S lyase, glycosyltransferase and sulfotransferase) from Arabidopsis to tobacco. This was achieved using an expression construct consisting of a single 2A polycistronic open reading frame, which allowed the expression of the three coding-sequences from a single promoter. When compared to wildtype plants, transgenic tobacco lines showed increased ability to convert the intermediate phenylacetothiohydroxamate to benzylglucosinolate upon in vivo feeding. Enzymatic assays using plant extracts demonstrated that the individual activities required for this conversion were enhanced in the transgenic plants. The relatively high conversion by wildtype plants in feeding assays supports the hypothesis that the last part of the glucosinolate pathway was recruited from existing detoxification reactions. Immunoblots confirmed that individual proteins were being successfully produced from the 2A polycistronic open reading frame, albeit fusion proteins could also be detected. In summary, we transferred the last three steps of the benzylglucosinolate pathway to tobacco as a first step towards engineering glucosinolates into non-cruciferous plants.     

Cytochromes P450 in the biosynthesis of glucosinolates and indole alkaloids

Characteristic of cruciferous plants is the synthesis of nitrogen- and sulfur-rich compounds, such as glucosinolates and indole alkaloids. The intact glucosinolates have limited biological activity, but give rise to an array of bio-active breakdown products when hydrolysed by endogenous β-thioglucosidases (myrosinases) upon tissue disruption. Both glucosinolates and indole alkaloids constitute an important part of the defence of plants against herbivores and pathogens, with the difference that a basal level of glucosinolates is ever-present in the plant whereas indole alkaloids are true phytoalexins that are de novo synthesised upon pathogen attack. With the completion of the genome sequence of the model plant, Arabidopsis thaliana, which is a crucifer, many genes involved in the biosynthesis of glucosinolates and indole alkaloids have been identified and cytochromes P450 are key players in these pathways. In the present review, we will focus on the cytochromes P450 in the biosynthesis of both groups of compounds. Their functional roles and regulation will be discussed.     

Modulation of sulfur metabolism enables efficient glucosinolate engineering

Background  

Metabolic engineering in heterologous organisms is an attractive approach to achieve efficient production of valuable natural products. Glucosinolates represent a good example of such compounds as they are thought to be the cancer-preventive agents in cruciferous plants. We have recently demonstrated that it is feasible to engineer benzylglucosinolate (BGLS) in the non-cruciferous plant Nicotiana benthamiana by transient expression of five genes from Arabidopsis thaliana. In the same study, we showed that co-expression of a sixth Arabidopsis gene, γ-glutamyl peptidase 1 (GGP1), resolved a metabolic bottleneck, thereby increasing BGLS accumulation. However, the accumulation did not reach the expected levels, leaving room for further optimization.