Metabolic highways may be orchestrated by the assembly of sequential enzymes into protein complexes, or metabolons, to facilitate efficient channeling of intermediates and to prevent undesired metabolic cross-talk while maintaining metabolic flexibility. Here we report the isolation of the dynamic metabolon that catalyzes the formation of the cyanogenic glucoside dhurrin, a defense compound produced in sorghum plants. The metabolon was reconstituted in liposomes, which demonstrated the importance of membrane surface charge and the presence of the glucosyltransferase for metabolic channeling. We used in planta fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to study functional and structural characteristics of the metabolon. Understanding the regulation of biosynthetic metabolons offers opportunities to optimize synthetic biology approaches for efficient production of high-value products in heterologous hosts.
Metabolic adjustments are a significant, but poorly understood, part of the response of plants to oxidative stress. In a previous study (Baxter et al., 2007), the metabolic response of Arabidopsis cells in culture to induction of oxidative stress by menadione was characterized. An emergency survival strategy was uncovered in which anabolic primary metabolism was largely down-regulated in favour of catabolic and antioxidant metabolism. The response in whole plant tissues may be different and we have therefore investigated the response of Arabidopsis roots to menadione treatment, analyzing the transcriptome, metabolome and key metabolic fluxes with focus on primary as well as secondary metabolism. Using a redox-sensitive GFP, it was also shown that menadione causes redox perturbation, not just in the mitochondrion, but also in the cytosol and plastids of roots. In the first 30 min of treatment, the response was similar to the cell culture: there was a decrease in metabolites of the TCA cycle and amino acid biosynthesis and the transcriptomic response was dominated by up-regulation of DNA regulatory proteins. After 2 and 6 h of treatment, the response of the roots was different to the cell culture. Metabolite levels did not remain depressed, but instead recovered and, in the case of pyruvate, some amino acids and aliphatic glucosinolates showed a steady increase above control levels. However, no major changes in fluxes of central carbon metabolism were observed and metabolic transcripts changed largely independently of the corresponding metabolites. Together, the results suggest that root tissues can recover metabolic activity after oxidative inhibition and highlight potentially important roles for glycolysis and the oxidative pentose phosphate pathway.
Despite vast diversity in metabolites and the matching substrate specificity of their transporters, little is known about how evolution of transporter substrate specificities is linked to emergence of substrates via evolution of biosynthetic pathways. Transporter specificity towards the recently evolved glucosinolates characteristic of Brassicales is shown to evolve prior to emergence of glucosinolate biosynthesis. Furthermore, we show that glucosinolate transporters belonging to the ubiquitous NRT1/PTR FAMILY (NPF) likely evolved from transporters of the ancestral cyanogenic glucosides found across more than 2500 species outside of the Brassicales. Biochemical characterization of orthologs along the phylogenetic lineage from cassava to A. thaliana, suggests that alterations in the electrogenicity of the transporters accompanied changes in substrate specificity. Linking the evolutionary path of transporter substrate specificities to that of the biosynthetic pathways, exemplify how transporter substrate specificities originate and evolve as new biosynthesis pathways emerge.DOI:
http://dx.doi.org/10.7554/eLife.19466.001
Two previously known phenanthroindolizidine alkaloids, (-)-10beta-antofine N-oxide (1) and (-)-10beta, 13aalpha-14beta-hydroxyantofine N-oxide (2), and a novel alkaloid, (-)-10beta,13aalpha-secoantofine N-oxide (3), were isolated from aerial parts of Cynanchum vincetoxicum. Their structures were established by means of NMR methods, including COSY, NOESY, HSQC, and HMBC experiments, as well as from their CD spectra. Cytotoxic activity of the alkaloids was assessed in vitro using both a drug-sensitive KB-3-1 and a multi-drug-resistant KB-V1 cancer cell line. The antofine derivatives (1 and 2) showed pronounced cytotoxicity against the drug-sensitive cell line (IC(50) values about 100 nM), whereas the secoantofine derivative (3) was considerably less active. The KB-V1 cell line showed a marginal resistance against all alkaloids, demonstrating that these compounds are poor substrates for the P-glycoprotein (P-170) efflux pump.
BackgroundThe important cereal crop Sorghum bicolor (L.) Moench biosynthesize and accumulate the defensive compound dhurrin during development. Previous work has suggested multiple roles for the compound including a function as nitrogen storage/buffer. Crucial for this function is the endogenous turnover of dhurrin for which putative pathways have been suggested but not confirmed.ResultsIn this study, the biosynthesis and endogenous turnover of dhurrin in the developing sorghum grain was studied by metabolite profiling and time-resolved transcriptome analyses. Dhurrin was found to accumulate in the early phase of grain development reaching maximum amounts 25 days after pollination. During the subsequent maturation period, the dhurrin content was turned over, resulting in only negligible residual dhurrin amounts in the mature grain. Dhurrin accumulation correlated with the transcript abundance of the three genes involved in biosynthesis. Despite the accumulation of dhurrin, the grains were acyanogenic as demonstrated by the lack of hydrogen cyanide release from macerated grain tissue and by the absence of transcripts encoding dhurrinases. With the missing activity of dhurrinases, the decrease in dhurrin content in the course of grain maturation represents the operation of hitherto uncharacterized endogenous dhurrin turnover pathways. Evidence for the operation of two such pathways was obtained by metabolite profiling and time-resolved transcriptome analysis. By combining cluster- and phylogenetic analyses with the metabolite profiling, potential gene candidates of glutathione S-transferases, nitrilases and glycosyl transferases involved in these pathways were identified. The absence of dhurrin in the mature grain was replaced by a high content of proanthocyanidins. Cluster- and phylogenetic analyses coupled with metabolite profiling, identified gene candidates involved in proanthocyanidin biosynthesis in sorghum.ConclusionsThe results presented in this article reveal the existence of two endogenous dhurrin turnover pathways in sorghum, identify genes putatively involved in these transformations and show that dhurrin in addition to its insect deterrent properties may serve as a storage form of reduced nitrogen. In the course of sorghum grain maturation, proanthocyanidins replace dhurrin as a defense compound. The lack of cyanogenesis in the developing sorghum grain renders this a unique experimental system to study CNglc synthesis as well as endogenous turnover.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3360-4) contains supplementary material, which is available to authorized users.
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). Reduction in seed glucosinolates was stably inherited over multiple generations and maintained in field trials of two mutant populations at three locations. Successful translation of the gtr loss-of-function phenotype from model plant to two Brassica crops suggests that our transport engineering approach could be broadly applied to reduce seed glucosinolate content in other oilseed crops, such as Camelina sativa or Crambe abyssinica.
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