Shikimate hydroxycinnamoyl transferase (HCT) activity assays in Populus nigra

Lignin is a complex phenolic polymer deposited in secondarily-thickened plant cell walls. The polymer is mainly derived from the three primary monolignols: p-coumaryl, coniferyl and sinapyl alcohol which give rise to p-hydroxyphenyl, guaiacyl and syringyl units (H, G and S units, respectively) when coupled into the polymer. The building blocks differ in their degree of methoxylation and their biosynthetic pathway is catalyzed by more than 10 enzymes. HCT plays a crucial role by channeling the phenylpropanoids towards the production of coniferyl and sinapyl alcohols. Interestingly, HCT has been reported to be implicated in the pathway both upstream and downstream of the 3-hydroxylation of the aromatic ring of p-coumaroyl shikimate (Figure 1) (Hoffmann et al., 2003; Hoffmann et al., 2004; Vanholme et al., 2013b). These features highlight the importance of developing an assay to reliably measure HCT activity in planta. Here, we describe a UPLC-MS-based method for the analysis of HCT activity in xylem total protein extracts of Populus nigra, which can be adapted to other woody and herbaceous plant species. The protocol was initially described in Vanholme et al. (2013a)

Chemical genetics uncovers novel inhibitors of lignification, including p-iodobenzoic acid targeting CINNAMATE-4-HYDROXYLASE

Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization

A phytochemical perspective on plant defense against nematodes

Given the large yield losses attributed to plant-parasitic nematodes and the limited availability of sustainable control strategies, new plant-parasitic nematode control strategies are urgently needed. To defend themselves against nematode attack, plants possess sophisticated multi-layered immune systems. One element of plant immunity against nematodes is the production of small molecules with anti-nematode activity, either constitutively or after nematode infection. This review provides an overview of such metabolites that have been identified to date and groups them by chemical class (e.g., terpenoids, flavonoids, glucosinolates, etc.). Furthermore, this review discusses strategies that have been used to identify such metabolites and highlights the ways in which studying anti-nematode metabolites might be of use to agriculture and crop protection. Particular attention is given to emerging, high-throughput approaches for the identification of anti-nematode metabolites, in particular the use of untargeted metabolomics techniques based on nuclear magnetic resonance (NMR) and mass spectrometry (MS)

The complete mitochondrial genome of the house dust mite Dermatophagoides pteronyssinus (Trouessart): a novel gene arrangement among arthropods

<p>Abstract</p> <p>Background</p> <p>The apparent scarcity of available sequence data has greatly impeded evolutionary studies in Acari (mites and ticks). This subclass encompasses over 48,000 species and forms the largest group within the Arachnida. Although mitochondrial genomes are widely utilised for phylogenetic and population genetic studies, only 20 mitochondrial genomes of Acari have been determined, of which only one belongs to the diverse order of the Sarcoptiformes. In this study, we describe the mitochondrial genome of the European house dust mite <it>Dermatophagoides pteronyssinus</it>, the most important member of this largely neglected group.</p> <p>Results</p> <p>The mitochondrial genome of <it>D. pteronyssinus </it>is a circular DNA molecule of 14,203 bp. It contains the complete set of 37 genes (13 protein coding genes, 2 rRNA genes and 22 tRNA genes), usually present in metazoan mitochondrial genomes. The mitochondrial gene order differs considerably from that of other Acari mitochondrial genomes. Compared to the mitochondrial genome of <it>Limulus polyphemus</it>, considered as the ancestral arthropod pattern, only 11 of the 38 gene boundaries are conserved. The majority strand has a 72.6% AT-content but a GC-skew of 0.194. This skew is the reverse of that normally observed for typical animal mitochondrial genomes. A microsatellite was detected in a large non-coding region (286 bp), which probably functions as the control region. Almost all tRNA genes lack a T-arm, provoking the formation of canonical cloverleaf tRNA-structures, and both rRNA genes are considerably reduced in size. Finally, the genomic sequence was used to perform a phylogenetic study. Both maximum likelihood and Bayesian inference analysis clustered <it>D. pteronyssinus </it>with <it>Steganacarus magnus</it>, forming a sistergroup of the Trombidiformes.</p> <p>Conclusion</p> <p>Although the mitochondrial genome of <it>D. pteronyssinus </it>shares different features with previously characterised Acari mitochondrial genomes, it is unique in many ways. Gene order is extremely rearranged and represents a new pattern within the Acari. Both tRNAs and rRNAs are truncated, corroborating the theory of the functional co-evolution of these molecules. Furthermore, the strong and reversed GC- and AT-skews suggest the inversion of the control region as an evolutionary event. Finally, phylogenetic analysis using concatenated mt gene sequences succeeded in recovering Acari relationships concordant with traditional views of phylogeny of Acari.</p

Chimeric LysR-type transcriptional biosensors for customizing ligand specificity profiles toward flavonoids

Transcriptional biosensors enable key applications in both metabolic engineering and synthetic biology. Due to nature's immense variety of metabolites, these applications require biosensors with a ligand specificity profile customized to the researcher's needs. In this work, chimeric biosensors were created by introducing parts of a donor regulatory circuit from Sinorhizobium meliloti, delivering the desired luteolin-specific response, into a nonspecific biosensor chassis from Herbaspirillum seropedicae. Two strategies were evaluated for the development of chimeric LysR-type biosensors with customized ligand specificity profiles toward three closely related flavonoids, naringenin, apigenin, and luteolin. In the first strategy, chimeric promoter regions were constructed at the biosensor effector module, while in the second strategy, chimeric transcription factors were created at the biosensor detector module. Via both strategies, the biosensor repertoire was expanded with luteolin-specific chimeric biosensors demonstrating a variety of response curves and ligand specificity profiles. Starting from the nonspecific biosensor chassis, a shift from 27.5% to 95.3% luteolin specificity was achieved with the created chimeric biosensors. Both strategies provide a compelling, faster, and more accessible route for the customization of biosensor ligand specificity, compared to de novo design and construction of each biosensor circuit for every desired ligand specificity

A unique genetic code change in the mitochondrial genome of the parasitic nematode Radopholus similis

<p>Abstract</p> <p>Background</p> <p>Mitochondria (mt) contain their own autonomously replicating DNA, constituted as a small circular genome encoding essential subunits of the respiratory chain. Mt DNA is characterized by a genetic code which differs from the standard one. Interestingly, the mt genome of nematodes share some peculiar features, such as small transfer RNAs, truncated ribosomal RNAs and - in the class of Chromadorean nematodes - unidirectional transcription.</p> <p>Findings</p> <p>We present the complete mt genomic sequence (16,791 bp) of the plant-parasitic nematode <it>Radopholus similis </it>(class Chromadorea). Although it has a gene content similar to most other nematodes, many idiosyncrasies characterize the extremely AT-rich mt genome of <it>R. similis </it>(85.4% AT). The secondary structure of the large (16S) rRNA is further reduced, the gene order is unique, the large non-coding region contains two large repeats, and most interestingly, the UAA codon is reassigned from translation termination to tyrosine. In addition, 7 out of 12 protein-coding genes lack a canonical stop codon and analysis of transcriptional data showed the absence of polyadenylation. Northern blot analysis confirmed that only one strand is transcribed and processed. Furthermore, using nucleotide content bias methods, regions for the origin of replication are suggested.</p> <p>Conclusion</p> <p>The extraordinary mt genome of <it>R. similis </it>with its unique genetic code appears to contain exceptional features correlated to DNA decoding. Therefore the genome may provide an incentive to further elucidate these barely understood processes in nematodes. This comprehension may eventually lead to parasitic nematode-specific control targets as healthy mitochondria are imperative for organism survival. In addition, the presented genome is an interesting exceptional event in genetic code evolution.</p