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Cannabinoids are a therapeutically valuable class of secondary metabolites with a vast number of substituents. Currently over 100 cannabinoids have been isolated form the plant species Cannabis sativa, with many more non-natural analogues being chemically synthesised. Cannabinoids are primarily sourced from the cannabis plant, which has drawbacks including the resource costs of crop cultivation, inconsistent yields, and extraction and purification difficulties. In the native cannabinoid biosynthetic pathway of C. sativa, cannabigerolic acid is the gateway to the synthesis of many of these compounds, being the common substrate to multiple cannabinoid synthases. The bioactive decarboxylated analogue of this compound, cannabigerol, represents an alternate gateway into the cannabinoid space either as a substrate to non-canonical cannabinoid synthase homologues or to chemical reactions. The construction of a synthetic biology production platform to these compounds would open opportunities for sustainable production of well researched and rarer cannabinoids. Herein, we describe the identification and repurposing of the aromatic prenyl transferase, AtaPT, which when coupled with native enzymes of C. sativa can form an E. coli production system for cannabigerolic acid in cell lysates, and cannabigerol in whole cells. Engineering of AtaPT, guided by structural analysis, was performed to enhance its kinetics towards cannabigerolic acid production for subsequent use in a proof-of-concept lysate system. For the first time, we show cannabigerol production in E. coli cells by employing AtaPT under an optimised microbial system. Our results have therefore set the foundation for a cannabinoid production platform in an E. coli chassis.

Metabolic engineering technologies have been employed with increasing success over the last three decades for the engineering and optimization of industrial host strains to competitively produce high-value chemical targets. To this end, continued reductions in the time taken from concept, to development, to scale-up are essential. Design–Build–Test–Learn pipelines that are able to rapidly deliver diverse chemical targets through iterative optimization of microbial production strains have been established. Biofoundries are employing in silico tools for the design of genetic parts, alongside combinatorial design of experiments approaches to optimize selection from within the potential design space of biological circuits based on multi-criteria objectives. These genetic constructs can then be built and tested through automated laboratory workflows, with performance data analysed in the learn phase to inform further design. Successful examples of rapid prototyping processes for microbially produced compounds reveal the potential role of biofoundries in leading the sustainable production of next-generation bio-based chemicals.

Bio-based production of industrial chemicals using synthetic biology can provide alternative green routes from renewable resources, allowing for cleaner production processes. To efficiently produce chemicals on-demand through microbial strain engineering, biomanufacturing foundries have developed automated pipelines that are largely compound agnostic in their time to delivery. Here we benchmark the capabilities of a biomanufacturing pipeline to enable rapid prototyping of microbial cell factories for the production of chemically diverse industrially relevant material building blocks. Over 85 days the pipeline was able to produce 17 potential material monomers and key intermediates by combining 160 genetic parts into 115 unique biosynthetic pathways. To explore the scale-up potential of our prototype production strains, we optimized the enantioselective production of mandelic acid and hydroxymandelic acid, achieving gram-scale production in fed-batch fermenters. The high success rate in the rapid design and prototyping of microbially-produced material building blocks reveals the potential role of biofoundries in leading the transition to sustainable materials production., Graphical abstract Image 1, Highlights • An automated biomanufacturing pipeline is benchmarked for the production of material monomers. • Over 85 days, 160 genes were screened for activity, assembled into 115 unique pathways and tested for in vivo production. • E. coli production strains were successfully constructed to produce 17 target compounds at competitive titers. • Scale-up potential is demonstrated through enantioselective production of mandelic acid targets at gram-scale in bioreactors. • The high success rate demonstrates the capabilities of biofoundries to rapidly prototype microbial production strains.

Natural plant-based flavonoids have drawn significant attention as dietary supplements due to their potential health benefits, including anti-cancer, anti-oxidant and anti-asthmatic activities. Naringenin, pinocembrin, eriodictyol and homoeriodictyol are classified as (2S)-flavanones, an important sub-group of naturally occurring flavonoids, with wide-reaching applications in human health and nutrition. These four compounds occupy a central position as branch point intermediates towards a broad spectrum of naturally occurring flavonoids. Here, we report the development of Escherichia coli production chassis for each of these key gatekeeper flavonoids. Selection of key enzymes, genetic construct design and the optimization of process conditions resulted in the highest reported titers for naringenin (484 mg/l), improved production of pinocembrin (198 mg/l) and eriodictyol (55 mg/l from caffeic acid), and provided the first example of in vivo production of homoeriodictyol directly from glycerol (17 mg/l). This work provides a springboard for future production of diverse downstream natural and non-natural flavonoid targets.

Chromatography-based mass spectrometry approaches (xC-MS) are commonly used in untargeted metabolomics, providing retention time, m/z values and metabolite-specific fragments, all of which are used to identify and validate an unknown analyte. Ion mobility-mass spectrometry (IM-MS) is emerging as an enhancement to classic xC-MS strategies, by offering additional ion separation as well as collision cross section (CCS) determination. In order to apply such an approach to a metabolomics workflow, verified data from metabolite standards is necessary. In this work we present experimental DTCCSN2 values for a range of metabolites in positive and negative ionisation modes using drift tube-ion mobility-mass spectrometry (DT-IM-MS) with nitrogen as the buffer gas. The value of DTCCSN2 measurements for application in metabolite identification relies on a robust technique that acquires measurements of high reproducibility. We report that the CCS values found for 86% of metabolites measured in replicate have a relative standard deviation lower than 0.2%. Examples of metabolites with near identical mass are demonstrated to be separated by ion mobility with over 4% difference in DTCCSN2 values. We conclude that the integration of ion mobility into current LC-MS workflows can aid in small molecule identification for both targeted and untargeted metabolite screening.

Natural plant-based flavonoids have drawn significant attention as dietary supplements due to their potential health benefits, including anti-cancer, anti-oxidant and anti-asthmatic activities. Naringenin, pinocembrin, eriodictyol and homoeriodictyol are classified as (2S)-flavanones, an important sub-group of naturally occurring flavonoids, with wide-reaching applications in human health and nutrition. These four compounds occupy a central position as branch point intermediates towards a broad spectrum of naturally occurring flavonoids. Here, we report the development of Escherichia coli production chassis for each of these key gatekeeper flavonoids. Selection of key enzymes, genetic construct design and the optimization of process conditions resulted in the highest reported titers for naringenin (484 mg/l), improved production of pinocembrin (198 mg/l) and eriodictyol (55 mg/l from caffeic acid), and provided the first example of in vivo production of homoeriodictyol directly from glycerol (17 mg/l). This work provides a springboard for future production of diverse downstream natural and non-natural flavonoid targets.

Synthetic biology utilizes the Design–Build–Test–Learn pipeline for the engineering of biological systems. Typically, this requires the construction of specifically designed, large and complex DNA assemblies. The availability of cheap DNA synthesis and automation enables high-throughput assembly approaches, which generates a heavy demand for DNA sequencing to verify correctly assembled constructs. Next-generation sequencing is ideally positioned to perform this task, however with expensive hardware costs and bespoke data analysis requirements few laboratories utilize this technology in-house. Here a workflow for highly multiplexed sequencing is presented, capable of fast and accurate sequence verification of DNA assemblies using nanopore technology. A novel sample barcoding system using polymerase chain reaction is introduced, and sequencing data are analyzed through a bespoke analysis algorithm. Crucially, this algorithm overcomes the problem of high-error rate nanopore data (which typically prevents identification of single nucleotide variants) through statistical analysis of strand bias, permitting accurate sequence analysis with single-base resolution. As an example, 576 constructs (6 × 96 well plates) were processed in a single workflow in 72 h (from Escherichia coli colonies to analyzed data). Given our procedure’s low hardware costs and highly multiplexed capability, this provides cost-effective access to powerful DNA sequencing for any laboratory, with applications beyond synthetic biology including directed evolution, single nucleotide polymorphism analysis and gene synthesis.

In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three‐step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 → C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 → C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an ‘aldol switch’ is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable ‘aldol switch’ consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue‐specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high‐resolution structural analysis of multiple enzyme homologues with classical site‐directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes. Enzymes TKS, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/206.html; OAC, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC4/4/1/26.html; chalcone synthase, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/74.html; stilbene synthase, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/95.html; 2‐PS, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/-.html. Accession numbers The atomic coordinates and structure factors for the crystal structure of TKS have been deposited in the Protein Data Bank with accession number http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6GW3., Tetraketide synthase from Cannabis sativa is a type III polyketide synthase involved in cannabinoid production. Unlike chalcone and stilbene synthases, it cannot catalyse classical cyclization reactions to generate chalcone or stilbene acid products. Instead, it releases a linear tetraketide product that undergoes a non‐enzymatic C2→C7 decarboxylative aldol condensation to form a stilbene. In this study by Nigel Scrutton and co‐authors, structure determination and mutagenesis studies are performed to investigate mechanistic details of this Cannabis sativa tetraketide synthase.

The UK Synthetic Biology Research Centre, SYNBIOCHEM, hosted by the Manchester Institute of Biotechnology at the University of Manchester is delivering innovative technology platforms to facilitate the predictable engineering of microbial bio-factories for fine and speciality chemicals production. We provide an overview of our foundry activities that are being applied to grand challenge projects to deliver innovation in bio-based chemicals production for industrial biotechnology.

Chromatography based mass spectrometry approaches (xC-MS) are commonly used in untargeted metabolomics, providing retention time, m/z values and metabolite specific-fragments all of which are used to identify and validate an unknown analyte. Ion mobility-mass spectrometry (IM-MS) is emerging as an enhancement to classic xC-MS strategies, by offering additional separation as well as collision cross section (CCS) determination. In order to apply such an approach to a synthetic biology workflow, verified data from metabolite standards is necessary. In this work we present experimental DTCCSN2 values for a range of metabolites in positive and negative ionisation modes using drift time-ion mobility-mass spectrometry (DT-IM-MS) with nitrogen as the buffer gas. Creating a useful database containing DTCCSN2 measurements for application in metabolite identification relies on a robust technique that acquires measurements of high reproducibility. We report that 86% of the metabolites measured in replicate have a relative standard deviation lower than 0.2 %. Examples of metabolites with near identical mass are demonstrated to be separated by ion mobility with over 4% difference in DTCCSN2 values. We conclude that the integration of ion mobility into current LC-MS workflows can aid in small molecule identification for both targeted and untargeted metabolite screening which is commonly performed in synthetic biology.

Cryptochrome 1 (CRY1) is a blue light receptor that mediates primarily blue-light inhibition of hypocotyl elongation. Very little is known of the mechanisms by which CRY1 affects growth. Blue light and temperature are two key environmental signals that profoundly affect plant growth and development, but how these two abiotic factors integrate remains largely unknown. Here, we show that blue light represses high temperature-mediated hypocotyl elongation via CRY1. Furthermore, CRY1 interacts directly with PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) in a blue light-dependent manner to repress the transcription activity of PIF4. CRY1 represses auxin biosynthesis in response to elevated temperature through PIF4. Our results indicate that CRY1 signal by modulating PIF4 activity, and that multiple plant photoreceptors [CRY1 and PHYTOCHROME B (PHYB)] and ambient temperature can mediate morphological responses through the same signaling component-PIF4.

Highlight Two ubiquitin ligases control fumonisin B1-elicited programmed cell death by modulating jasmonate signalling transduction in Arabidopsis., The mycotoxin fumonisin B1 (FB1) is a strong inducer of programmed cell death (PCD) in plants, but its underlying mechanism remains unclear. Here, we describe two ubiquitin ligases, RING DOMAIN LIGASE3 (RGLG3) and RGLG4, which control FB1-triggered PCD by modulating the jasmonate (JA) signalling pathway in Arabidopsis thaliana. RGLG3 and RGLG4 transcription was sensitive to FB1. Arabidopsis FB1 sensitivity was suppressed by loss of function of RGLG3 and RGLG4 and was increased by their overexpression. Thus RGLG3 and RGLG4 have coordinated and positive roles in FB1-elicited PCD. Mutated JA perception by coi1 disrupted the RGLG3- and RGLG4-related response to FB1 and interfered with their roles in cell death. Although FB1 induced JA-responsive defence genes, it repressed growth-related, as well as JA biosynthesis-related, genes. Consistently, FB1 application reduced JA content in wild-type plants. Furthermore, exogenously applied salicylic acid additively suppressed JA signalling with FB1 treatment, suggesting that FB1-induced salicylic acid inhibits the JA pathway during this process. All of these effects were attenuated in rglg3 rglg4 plants. Altogether, these data suggest that the JA pathway is hijacked by the toxin FB1 to elicit PCD, which is coordinated by Arabidopsis RGLG3 and RGLG4.

The direct separation of five acidic phytohormones including auxin (indole-3-acetic acid, indole-3-propionic acid, and indole-3-butyric acid), jasmonic acid, and abscisic acid by differential mobility spectrometry–mass spectrometry (DMS–MS) with the use of the polar gas-phase chemical modifier 2-propanol was demonstrated. The method was rapid and simple to operate with selectivity and repeatability. The influence of experimental conditions on the separation, such as modifier types and concentration, separation voltage, and temperature, was systematically investigated. The field mobilities dependence was measured as the normalized α function using the developed DMS–MS method. All the analytes behaved as type A ions and the clustering separation mechanism dominated. These results not only gave insights of ion properties but also contributed to the prediction of DMS separation by choosing targeted and appropriate conditions for the study of acidic phytohormones.

In this work, the glucose-containing disaccharide isomers were studied using negative electrospray ionization tandem mass spectrometry (ESI-MS/MS). Interestingly, the full-scan mass spectra of the disaccharides revealed that the deprotonated dimers were the predominant gas phase ions during ionization process. Importantly, several diagnostic fragment ions relative to linkage positions and anomeric configurations, arising from the covalent bond dissociation of dimers without breakdown of the non-covalent complexes, can be detected in the tandem mass spectra. Based on the scarce fragmentation characteristic, an original and simple approach for structural discrimination of disaccharide isomers was put forward. In addition, density functional theory (DFT) was employed to find out the reason why several fragmentations of intramolecular sugar bonds had preceded breakdown of the non-covalent complexes.

Increasing grain yields is a major focus of crop breeders around the world. Here we report that overexpression of the rice microRNA (miRNA) OsmiR397, which is naturally highly expressed in young panicles and grains, enlarges grain size and promotes panicle branching, leading to an increase in overall grain yield of up to 25% in a field trial. To our knowledge, no previous report has shown a positive regulatory role of miRNA in the control of plant seed size and grain yield. We determined that OsmiR397 increases grain yield by downregulating its target, OsLAC, whose product is a laccase-like protein that we found to be involved in the sensitivity of plants to brassinosteroids. As miR397 is highly conserved across different species, our results suggest that manipulating miR397 may be useful for increasing grain yield not only in rice but also in other cereal crops.

Screening of bacterial colonies to identify new biocatalytic activities is a widely adopted tool in biotechnology, but is constrained by the requirements for colorimetric or tag-based detection methods. Herein we report a label-free screening platform for biotransformations in live colonies using desorption electrospray ionization coupled with ion mobility mass spectrometry imaging (DiBT-IMMS). The screening method is demonstrated for both ammonia lyases and P450 monooxygenases expressed within live bacterial colonies and is shown to enable multiplexing of enzyme variants and substrate libraries simultaneously.

The exploration and identification of new brassinosteroid (BR) compounds is critical to improve the biosynthetic research of BRs and expand the chemodiversity of active BRs. However, traditional methods are labor-intensive, time-consuming, and less sensitive. Here, we present a facile screening strategy for discovering and identifying novel BRs from plant tissues based on ultra performance liquid chromatography-mass spectrometry (UPLC-MS). A total of 14 potential BRs were discovered from only 1 g of rice tissues and structurally elucidated by following a MS-based clue, acquired through multiple reaction monitoring (MRM) data-dependent enhanced product ion (EPI) scan, high resolution MS, and MS survey-dependent MS/MS. One of the 14 candidates was identified as 6-deoxo-28-homotyphasterol, a brand new BR compound that is reported for the first time in the BRs biosynthesis pathway. Detailed comparison with reference standards and quantitative level analysis in rice BRs mutants confirmed the availability of the other candidates. This effective, yet simple method provides an efficient way to find more and more chemically new BR biosynthetic intermediates in plants, which is significant for complementing the biosynthesis and metabolism network of BRs. This strategy may also be used to discover unknown compounds of other plant hormone species as well as their key metabolites.

The combination of stable isotope labelling with direct infusion ion mobility mass spectrometry (IM-MS) enabled qualitative and quantitative monitoring of biocatalytic reactions with reduced analysis times, enhanced sensitivity and μL-level assay volumes. The new approach was demonstrated by applying to both lipase and monooxygenase enzymes, including multi-substrate screening.

Although the role of auxin in biotrophic pathogenesis has been extensively studied, relatively little is known about its role in plant resistance to necrotrophs. Arabidopsis thaliana mutants defective in different aspects of the auxin pathway are generally more susceptible than wild-type plants to the necrotrophic pathogen Alternaria brassicicola. We show that A. brassicicola infection up-regulates auxin biosynthesis and down-regulates the auxin transport capacities of infected plants, these effects being partially dependent on JA signaling. We also show that these effects of A. brassicicola infection together lead to an enhanced auxin response in host plants. Application of IAA and MeJA together synergistically induces the expression of defense marker genes PDF1.2 (PLANT DEFENSIN 1.2) and HEL (HEVEIN-LIKE), suggesting that enhancement of JA-dependent defense signaling may be part of the auxin-mediated defense mechanism involved in resistance to necrotrophic pathogens. Our results provide molecular evidence supporting the hypothesis that JA and auxin interact positively in regulating plant resistance to necrotrophic pathogens and that activation of auxin signaling by JA may contribute to plant resistance to necrotrophic pathogens.

Chrysanthemum (Dendranthema grandiflorum cv. Jinba) shoot branching is determined by bud outgrowth during the vegetative growth stage. The degree of axillary bud outgrowth is highly influenced by environmental conditions, such as nutrient availability. Here, we demonstrated that phosphorus (Pi) starvation significantly reduces axillary bud outgrowth in chrysanthemum. A strigolactone (SL) biosynthesis gene, DgCCD7, was isolated and characterized as an ortholog of MAX3/DAD3/RMS5/D17. By using ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS), three putative SLs were identified and levels of all three SLs showed strong increase under Pi starvation conditions. Determinations of the distribution of SLs and regulation of DgCCD7/8 in response to Pi changes in root indicate that SL acts systemically. However, temporal expression patterns of biosynthesis and signaling genes in nodes revealed that Pi starvation causes a local response of SL pathway. Treatment of node segments with or without auxin and Pi revealed that in the absence of exogenous auxin, Pi delayed axillary buds outgrowth and up-regulated local SL pathway genes. These data indicated that an auxin-SL regulatory loop responded to Pi starvation for delaying bud outgrowth locally, root biosynthesized SLs were transported acropetally and functioned in shoot branching inhibition under Pi starvation. We proposed that SLs contributed to chrysanthemum shoot branching control in response to Pi-limiting conditions in a systemic way.

The effect of Liuwei Dihuang decoction (LW), a traditional Chinese medicine (TCM) prescription, on voltage-dependent currents and synaptic transmission were investigated in cultured hippocampal neurons of rat by whole-cell patch clamp recording technique. After application with serum from LW-treated rats, termed LW-containing serum (LWCS) for 48 h, the amplitude of delay rectifying K+ current (IK) and voltage-gated Ca2+ current (ICa) decreased. While the frequency of spontaneous excitatory post-synaptic current (sEPSC) and miniature excitatory post-synaptic current (mEPSC) increased significantly. Yet the amplitude of voltage-depended Na+ current (INa) and transient outward K+ current (IA), membrane capacitance and resistance remained unchanged. The results indicated that LWCS possessed the effect of modulating or improving neuronal and synaptic function, which possibly contribute to the cognition enhancing effect of LW.

Electrospray ionization mass spectrometry(ESI-MS) was applied simultaneously in determining norditerpenoid alkaloids from the roots of Aconitum sinomantanum Nakai (RAS) based on molecular mass information. The tandem mass spectra(ESI-MSn) provided the alkaloidal structural information, through which the existence of these alkaloids was further confirmed. Accordingly, six known norditerpenoid alkaloids were simultaneously determined on the basis of their ESI-MSn spectra. Furthermore, based on the diagnostic fragmentation pathways of alkaloidal MSn, a rapid method for direct detection and characterization of alkaloids from an ethanolic extract of RAS was described.

Seed dormancy is an important agronomic trait in cereals. Using deep dormant (N22), medium dormant (ZH11), and non-dormant (G46B) rice cultivars, we correlated seed dormancy phenotypes with abscisic acid (ABA) and gibberellin (GA) metabolism gene expression profiles and phytohormone levels during seed development and imbibition. A time course analysis of ABA and GA content during seed development showed that N22 had a high ABA level at early and middle seed developmental stages, while at late developmental stage it declined to the level of ZH11; however, its ABA/GA ratio maintained at a high level throughout seed development. By contrast, G46B had the lowest ABA content during seed development though at early developmental stage its ABA level was close to that of ZH11, and its ABA/GA ratio peaked at late developmental stage that was at the same level of ZH11. Compared with N22 and G46B, ZH11 had an even and medium ABA level during seed development and its ABA/GA ratio peaked at the middle developmental stage. Moreover, the seed development time-point having high ABA/GA ratio also had relatively high transcript levels for key genes in ABA and GA metabolism pathways across three cultivars. These indicated that the embryo-imposed dormancy has been induced before the late developmental stage and is determined by ABA/GA ratio. A similar analysis during seed imbibition showed that ABA was synthesized in different degrees for the three cultivars. In addition, water uptake assay for intact mature seeds suggested that water could permeate through husk barrier into seed embryo for all three cultivars; however, all three cultivars showed distinct colors by vanillin-staining indicative of the existence of flavans in their husks, which are dormancy inhibition compounds responsible for the husk-imposed dormancy.

A sensitive and reliable in-advance stable isotope labeling strategy was developed for simultaneous relative quantification of 8 acidic plant hormones in sub-milligram amount of plant materials. Bromocholine bromide (BETA) and its deuterated counterpart D9-BETA were used to in-advance derivatize control and sample extracts individually, which were then combined and subjected to solid-phase extraction (SPE) purification followed by UPLC-MS/MS analysis. Relative quantification of target compounds was obtained by calculation of the peak area ratios of BETA/D9-BETA labeled plant hormones. The in-advance stable isotope labeling strategy realized internal standard-based relative quantification of multiple kinds of plant hormones independent of availability of internal standard of every analyte with enhanced sensitivity of 1-3 orders of magnitude. Meanwhile, the in-advance labeling contributes to higher sample throughput and more reliability. The method was successfully applied to determine 8 plant hormones in 0.8mg DW (dry weight) of seedlings and 4 plant hormones from single seed of Arabidopsis thaliana. The results show the potential of the method in relative quantification of multiple plant hormones in tiny plant tissues or organs, which will advance the knowledge of the crosstalk mechanism of plant hormones.

Strigolactones (SLs), a newly discovered class of carotenoid-derived phytohormones, are essential for developmental processes that shape plant architecture and interactions with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Despite the rapid progress in elucidating the SL biosynthetic pathway, the perception and signalling mechanisms of SL remain poorly understood. Here we show that DWARF 53 (D53) acts as a repressor of SL signalling and that SLs induce its degradation. We find that the rice (Oryza sativa) d53 mutant, which produces an exaggerated number of tillers compared to wild-type plants, is caused by a gain-of-function mutation and is insensitive to exogenous SL treatment. The D53 gene product shares predicted features with the class I Clp ATPase proteins and can form a complex with the α/β hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL perception. We demonstrate that, in a D14- and D3-dependent manner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth. Our combined genetic and biochemical data reveal that D53 acts as a repressor of the SL signalling pathway, whose hormone-induced degradation represents a key molecular link between SL perception and responses.

An efficient simplified isotope dilution method was developed to determine four carboxyl containing phytohormones simultaneously in 200 mg of fresh tomato tissues using ultra high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS) with negative electrospray ionization. The four phytohormones are indole-3-acetic acid (IAA), abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA). Only one purification step of Oasis MAX solid phase extraction (SPE) was employed to enrich target phytohormones after crude extraction. In addition, two endogenous isomers of JA, (-)-JA and (+)-7-iso-JA, were separated directly. The validated method has been applied to monitor changes of JA, SA, IAA, and ABA in both local and systemic leaves of wild-type and transgenic 35S::prosystemin (35S::PS) tomato lines. Meanwhile, the JA burst amplified by the overexpressed prosystemin in 35S::PS was verified. Furthermore, the spatial and temporal changes of JA, SA, ABA, and IAA were analyzed.

The allene oxide synthase (AOS) and hydroperoxide lyase (HPL) branches of the oxylipin pathway, which underlie the production of jasmonates and aldehydes, respectively, function in plant responses to a range of stresses. Regulatory crosstalk has been proposed to exist between these two signaling branches; however, there is no direct evidence of this. Here, we identified and characterized a jasmonic acid (JA) overproduction mutant, cea62, by screening a rice T-DNA insertion mutant library for lineages that constitutively express the AOS gene. Map-based cloning was used to identify the underlying gene as hydroperoxide lyase OsHPL3. HPL3 expression and the enzyme activity of its product, (E)-2-hexenal, were depleted in the cea62 mutant, which resulted in the dramatic overproduction of JA, the activation of JA signaling, and the emergence of the lesion mimic phenotype. A time-course analysis of lesion formation and of the induction of defense responsive genes in the cea62 mutant revealed that the activation of JA biosynthesis and signaling in cea62 was regulated in a developmental manner, as was OsHPL3 activity in the wild-type plant. Microarray analysis showed that the JA-governed defense response was greatly activated in cea62 and this plant exhibited enhanced resistance to the T1 strain of the bacterial blight pathogen Xanthomonasoryzaepvoryzae (Xoo). The wounding response was attenuated in cea62 plants during the early stages of development, but partially recovered when JA levels were elevated during the later stages. In contrast, the wounding response was not altered during the different developmental stages of wild-type plants. These findings suggest that these two branches of the oxylipin pathway exhibit crosstalk with regards to biosynthesis and signaling and cooperate with each other to function in diverse stress responses.

Thellungiella salsuginea, a close relative of Arabidopsis , represents an extremophile model for abiotic stress tolerance studies. We present the draft sequence of the T. salsuginea genome, assembled based on ∼134-fold coverage to seven chromosomes with a coding capacity of at least 28,457 genes. This genome provides resources and evidence about the nature of defense mechanisms constituting the genetic basis underlying plant abiotic stress tolerance. Comparative genomics and experimental analyses identified genes related to cation transport, abscisic acid signaling, and wax production prominent in T. salsuginea as possible contributors to its success in stressful environments.

The interactions between phytohormones are crucial for plants to adapt to complex environmental changes. One example is the ethylene-regulated local auxin biosynthesis in roots, which partly contributes to ethylene-directed root development and gravitropism. Using a chemical biology approach, we identified a small molecule, l-kynurenine (Kyn), which effectively inhibited ethylene responses in Arabidopsis thaliana root tissues. Kyn application repressed nuclear accumulation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Moreover, Kyn application decreased ethylene-induced auxin biosynthesis in roots, and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATEDs (TAA1/TARs), the key enzymes in the indole-3-pyruvic acid pathway of auxin biosynthesis, were identified as the molecular targets of Kyn. Further biochemical and phenotypic analyses revealed that Kyn, being an alternate substrate, competitively inhibits TAA1/TAR activity, and Kyn treatment mimicked the loss of TAA1/TAR functions. Molecular modeling and sequence alignments suggested that Kyn effectively and selectively binds to the substrate pocket of TAA1/TAR proteins but not those of other families of aminotransferases. To elucidate the destabilizing effect of Kyn on EIN3, we further found that auxin enhanced EIN3 nuclear accumulation in an EIN3 BINDING F-BOX PROTEIN1 (EBF1)/EBF2-dependent manner, suggesting the existence of a positive feedback loop between auxin biosynthesis and ethylene signaling. Thus, our study not only reveals a new level of interactions between ethylene and auxin pathways but also offers an efficient method to explore and exploit TAA1/TAR-dependent auxin biosynthesis.

Oleanolic acid (OA) and ursolic acid (UA) are isomeric triterpenoid compounds with similar pharmaceutical properties. Usually, modern chromatographic and electrophoretic methods are widely utilized to differentiate these two compounds. Compared with mass spectrometric (MS) methods, these modern separation methods are both time- and sample-consuming. Herein, we present a new method for structural differentiation of OA and UA by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) with the association of heptakis-(2,6-di-O-methyl)beta-cyclodextrin (DM-beta-CD). Exact MS and tandem MS (MS/MS) data showed that there is no perceptible difference between OA and UA, as well as their beta-cyclodextrin and gamma-cyclodextrin complexes. However, there is a remarkable difference in MS/MS spectra of DM-beta-CD complexes of OA and UA. The peak corresponding to the neutral loss of a formic acid and a water molecule could only be observed in the MS/MS spectrum of the complex of DM-beta-CD: OA. Molecular modeling calculations were also employed to further investigate the structural differences of DM-beta-CD: OA and DM-beta-CD : UA complexes. Therefore, by employing DM-beta-CD as a reference reagent, OA and UA could be differentiated with purely MS method. Copyright (C) 2010 John Wiley & Sons, Ltd.

Tillering in rice (Oryza sativa) is one of the most important agronomic traits that determine grain yields. Previous studies on rice tillering mutants have shown that the outgrowth of tiller buds in rice is regulated by a carotenoid-derived MAX/RMS/D (more axillary branching) pathway, which may be conserved in higher plants. Strigolactones, a group of terpenoid lactones, have been recently identified as products of the MAX/RMS/D pathway that inhibits axillary bud outgrowth. We report here the molecular genetic characterization of d27, a classic rice mutant exhibiting increased tillers and reduced plant height. D27 encodes a novel iron-containing protein that localizes in chloroplasts and is expressed mainly in vascular cells of shoots and roots. The phenotype of d27 is correlated with enhanced polar auxin transport. The phenotypes of the d27 d10 double mutant are similar to those of d10, a mutant defective in the ortholog of MAX4/RMS1 in rice. In addition, 2′-epi-5-deoxystrigol, an identified strigolactone in root exudates of rice seedlings, was undetectable in d27, and the phenotypes of d27 could be rescued by supplementation with GR24, a synthetic strigolactone analog. Our results demonstrate that D27 is involved in the MAX/RMS/D pathway, in which D27 acts as a new member participating in the biosynthesis of strigolactones.

Nature 504, 406–410 (2013); doi: 10.1038/nature12878 In this Article, the bottom panel of Fig. 3c was assembled from processed digital images of separate western blot autoradiographs to show approximately equal amounts of MBP–D3 protein input in the presence or absence of GR24 treatment. During figure preparation, a single MBP–D3 lane was duplicated in error to create the third and fourth lanes.

High-resolution Sustained off resonance irradiation (SORI) CID was employed to distinguish four pairs of isomeric diglycosyl flavonoids in the negative mode using the electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR MS). All of these isomers can be distinguished via MS/MS data. For these diglycosyl flavones and flavanones, the deprotonated alpha1--6 linkage diglycosyl flavonoids produce fewer fragments than the alpha1--2 linkage type compounds and the Retro-Diels-Alder (RDA) reaction in MS/MS only takes place when the aglycone is a flavanone and glycosylated with an alpha1--2 intersaccharide linkage disaccharide. The deprotonation sites after collisional activation are discussed according to the high mass accuracy and high-resolution data of tandem spectrometry. Some of these high-resolution SORI CID product ions from alpha1--2 linkage diglycosyl flavonoids involve multibond cleavages; the possible mechanism is discussed based on the computer modeling using Gaussian 03 program package at the B3LYP/6-31G level of theory. Unambiguous elementary composition data provides fragmentation information that has not been reported previously.

The non-covalent complexes between three flavonoid glycosides (quercitrin, hyperoside and rutin) and heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD) were investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). The 1:1 complexation of each flavonoid glycoside (guest) to the DM-beta-CD (host) was monitored in the negative ion mode by mixing each guest with an up to 30-fold molar excess of the host. The binding constants for all complexes were calculated by a linear equation in the order: DM-beta-CD:quercitrin > DM-beta-CD:rutin > DM-beta-CD:hyperoside. A binding model for the complexes has also been proposed based on the binding constants and tandem mass spectrometric data of these complexes.

The fragmentations of four strychnos alkaloids have been investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the positive ion mode. Experiments using multi-stage tandem mass spectrometry (ESI-FT-ICR-MSn) allowed us to obtain precise elemental compositions of product ions at high mass resolution. The experimental data demonstrated that the nitrogen bridge and the coordinated oxygen atom on the nitrogen bridge in the alkaloid compounds were the active sites in the MS2 fragmentations. The loss of CH3 or the OCH3 group in those alkaloids, which have an OCH3 substituent, was the dominant fragmentation mode in the MS3 fragmentations. Logical fragmentation schemes for strychnos alkaloids have been proposed and these should be useful for the identification of these compounds.

The Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI–FT-ICR-MS) using sustained off-resonance irradiation (SORI)/collision-induced dissociation (CID) method at high mass resolution has been first applied to investigate the characteristic fragment ions of four protoberberine alkaloids in medicine herbs. The ESI–FT–ICR SORI-CID experiment results demonstrate that the unambiguous elemental composition of fragment ions can be obtained at high mass resolution, then the logical fragmentation pathways of the protoberberine alkaloids has been proposed. The characteristic fragment ions of CID and MS n of protoberberine alkaloids have been discussed, which are specific and useful for the identification of some protoberberine alkaloid compounds. Then, the extracts of four kinds of medicine herbs have been analyzed by HPLC–ESI–MS n . According to these characteristic fragmentation pathways, the retention time ( t R ) of HPLC and mass spectra of product ion, the structures of six kinds of protoberberine alkaloids have been identified. And, in the present paper, the selected ion monitoring (SIM) method has been used to separate and identify the alkaloid isomers.

Based on the dual role of specific boronate affinity, making use of both novel self-synthesized boronate affinity-functionalized magnetic nanoparticles and a high-efficiency organic boronic acid-type derivatization reagent, we report a simple, convenient and highly-sensitive method for detection of endogenous brassinosteroids from real plant materials.