Detection of induced mutations in CaFAD2 genes by next generation sequencing leading to the production of improved oil composition in Crambe abyssinica
Cheng, J. ; Salentijn, E.M.J. ; Huang Bangquan, ; Denneboom, C. ; Dechesne, A.C. ; Krens, F.A. ; Visser, R.G.F. ; Loo, E.N. van - \ 2015
Plant Biotechnology Journal 13 (2015)4. - ISSN 1467-7644 - p. 471 - 481.
induced point mutations - crop improvement - reverse genetics - oleic-acid - functional genomics - fad2 gene - discovery - arabidopsis - populations - cloning
Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down-regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS-mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one-dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non-sense mutations and mis-sense mutations in CaFAD2-C1, -C2 and -C3. The homozygous non-sense mutants for CaFAD2-C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis-sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non-sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents.
Pseudomonas corrugata crpCDE is part of the cyclic lipopeptide corpeptin biosynthetic gene cluster and is involved in bacterial virulence in tomato and in hypersensitive response in Nicotiana benthamiana
Strano, C.P. ; Bella, P. ; Licciardello, G. ; Fiore, A. ; Piero, A.R. Lo; Fogliano, V. ; Fogliano, V. ; Catara, V. - \ 2015
Molecular Plant Pathology 16 (2015)5. - ISSN 1464-6722 - p. 495 - 506.
syringae pv.-syringae - gram-negative bacteria - quorum-sensing system - chromobacterium-violaceum - putisolvin-ii - syringomycin - lipodepsipeptides - syringopeptin - peptide - cloning
Pseudomonas corrugata CFBP 5454 produces two kinds of cyclic lipopeptides (CLPs), cormycin A and corpeptins, both of which possess surfactant, antimicrobial and phytotoxic activities. In this study, we identified genes coding for a putative non-ribosomal peptide synthetase and an ABC-type transport system involved in corpeptin production. These genes belong to the same transcriptional unit, designated crpCDE. The genetic organization of this locus is highly similar to other Pseudomonas CLP biosynthetic clusters. Matrix-assisted laser desorption ionization-time of flightmass spectrometry (MALDI-TOF-MS) analysis revealed that transporter and synthetase genomic knock-out mutants were unable to produce corpeptins, but continued to produce cormycin A. This suggests that CrpCDE is the only system involved in corpeptin production in P. corrugata CFBP 5454. In addition, phylogenetic analysis revealed that the CrpE ABC transporter clustered with the transporters of CLPs with a long peptide chain. Strains depleted in corpeptin production were significantly less virulent than the wildtype strain when inoculated in tomato plants and induced only chlorosis when infiltrated into Nicotiana benthamiana leaves. Thus, corpeptins are important effectors of P. corrugata interaction with plants. Expression analysis revealed that crpC transcription occurs at high cell density. Two LuxR transcriptional regulators, PcoR and RfiA, have a pivotal role in crpC expression and thus in corpeptin production.
Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus
Mohedano, M.L. ; Garcia-Cayuela, T. ; Perez-Ramos, A. ; Gaiser, R.A. ; Requena, T. ; Lopez, P. - \ 2015
Journal of Industrial Microbiology and Biotechnology 42 (2015)2. - ISSN 1367-5435 - p. 247 - 253.
lactic-acid bacteria - controlled gene-expression - streptococcus-pneumoniae - lactococcus-lactis - plasmid - cloning
Lactobacilli are widespread in natural environments and are increasingly being investigated as potential health modulators. In this study, we have adapted the broad-host-range vector pNZ8048 to express the mCherry protein (pRCR) to expand the usage of the mCherry protein for analysis of gene expression in Lactobacillus. This vector is also able to replicate in Streptococcus pneumoniae and Escherichia coli. The usage of pRCR as a promoter probe was validated in Lactobacillus acidophilus by characterizing the regulation of lactacin B expression. The results show that the regulation is exerted at the transcriptional level, with lbaB gene expression being specifically induced by co-culture of the L. acidophilus bacteriocin producer and the S. thermophilus STY-31 inducer bacterium.
Natural loss-of-function mutation of EDR1 conferring resistance to tomato powdery mildew in Arabidopsis thaliana accession C24
Gao, D. ; Appiano, M. ; Huibers, R.P. ; Loonen, A.E.H.M. ; Visser, R.G.F. ; Wolters, A.M.A. ; Bai, Y. - \ 2015
Molecular Plant Pathology 16 (2015)1. - ISSN 1464-6722 - p. 71 - 82.
salicylic-acid - downy mildew - gene - defense - plants - microsatellites - mechanism - evolution - cloning - kinase
To screen for potentially novel types of resistance to tomato powdery mildew Oidium neolycopersici, a disease assay was performed on 123 Arabidopsis thaliana accessions. Forty accessions were fully resistant, and one, C24, was analysed in detail. By quantitative trait locus (QTL) analysis of an F2 population derived from C24 × Sha (susceptible accession), two QTLs associated with resistance were identified in C24. Fine mapping of QTL-1 on chromosome 1 delimited the region to an interval of 58¿kb encompassing 15 candidate genes. One of these was Enhanced Disease Resistance 1 (EDR1). Evaluation of the previously obtained edr1 mutant of Arabidopsis accession Col-0, which was identified because of its resistance to powdery mildew Golovinomyces cichoracearum, showed that it also displayed resistance to O.¿neolycopersici. Sequencing of EDR1 in our C24 germplasm (referred to as C24-W) revealed two missing nucleotides in the second exon of EDR1 resulting in a premature stop codon. Remarkably, C24 obtained from other laboratories does not contain the EDR1 mutation. To verify the identity of C24-W, a DNA region containing a single nucleotide polymorphism (SNP) unique to C24 was sequenced showing that C24-W contains the C24-specific nucleotide. C24-W showed enhanced resistance to O.¿neolycopersici compared with C24 not containing the edr1 mutation. Furthermore, C24-W displayed a dwarf phenotype, which was not associated with the mutation in EDR1 and was not caused by the differential accumulation of pathogenesis-related genes. In conclusion, we identified a natural edr1 mutant in the background of C24.
Expression studies in the embryo and in the micropylar endosperm of germinating coffee (Coffea arabica cv. Rubi) seeds
Farias, E.T. de; Amaral da Silva, E.A. ; Toorop, P.E. ; Bewley, J.D. ; Hilhorst, H.W.M. - \ 2015
Plant Growth Regulation 75 (2015)2. - ISSN 0167-6903 - p. 575 - 581.
beta-mannanase - growth - purification - cloning
Germination of coffee (Coffea arabica L.) seed is slow and uneven. Its germination is the net result of events that occur simultaneously in the embryo and endosperm and which are controlled by abscisic acid (ABA). The aim of the study was to monitor the expression of genes related to the cell cycle and to cell wall modifications, including an actin (ACT), a cyclin-dependent kinase (CDK2a) and a-expansin (a-EXP) in the embryo, and a-galactosidase (a-GAL), ß-mannosidase (LeMSIDE2), endo-ß-mannanase (MANA) in the micropylar endosperm. The first seed germinated after 5 days of imbibition and 50 % germination was reached after 10 days. The embryo grew inside the seed prior to radicle protrusion and ABA inhibited both embryo growth and radicle protrusion. The expression of the genes associated with the embryo growth increased during germination and ABA partially inhibited expression. The expression of ß-mannosidase and endo-ß-mannanase increased during imbibition and ABA completely inhibited expression of these genes. However, a-galactosidase displayed a more constitutive expression and was less affected by ABA. ABA plays a dual role in the regulation of coffee seed germination; it concomitantly controls both endosperm weakening and embryo growth.
Characterisation of a novel endo-xyloglucanase (XcXGHA) from Xanthomonas that accommodates a xylosyl-substituted glucose at subsite -1
Feng, T. ; Yan, K.P. ; Mikkelsen, M.D. ; Meyer, A.S. ; Schols, H.A. ; Westereng, B. ; Mikkelsen, J.D. - \ 2014
Applied Microbiology and Biotechnology 98 (2014)23. - ISSN 0175-7598 - p. 9667 - 9679.
polysaccharide-degrading enzymes - geotrichum sp m128 - plant-cell walls - substrate-specificity - glycoside hydrolase - structural basis - oligosaccharides - expression - cloning - endo-beta-1,4-glucanase
A xyloglucan-specific endo-1,4ß-glucanase (XcXGHA) from Xanthomonas citri pv. mangiferaeindicae has been cloned, expressed in Escherichia coli, purified and characterised. The XcXGHA enzyme belongs to CAZy family GH74 and has catalytic site residues conserved with other xyloglucanases in this family. At its optimal reaction conditions, pH 7.0 and 40 °C, the enzyme has a k cat/K M value of 2.2¿×¿107 min-1 M-1 on a tamarind seed xyloglucan substrate. XcXGHA is relatively stable within a broad pH range (pH 4–9) and up to 50 °C (t 1/2, 50 °C of 74 min). XcXGHA is proven to be xyloglucan-specific, and a glycan microarray study verifies that XcXGHA catalyses cleavage of xyloglucan extracted from both monocot and dicot plant species. The enzyme catalyses hydrolysis of tamarind xyloglucan in a unique way by cleaving XXXG into XX and XG (X is xylosyl-substituted glucose; G is unsubstituted glucose), is able to degrade more complex xyloglucans and notably is able to cleave near more substituted xyloglucan motifs such as L [i.e. a-l-Fucp-(1¿¿¿2)-ß-d-Galp-(1¿¿¿2)-a-d-Xylp-(1¿¿¿6)-ß-d-Glcp]. LC-MS/MS analysis of product profiles of tamarind xyloglucan which had been catalytically degraded by XcXGHA revealed that XcXGHA has specificity for X in subsite -1. The 3D model suggests that XcXGHA consists of two seven-bladed ß-propeller domains with the catalytic center formed by the interface of these two domains, which is conserved in xyloglucanases in the GH74 family. However, the XcXGHA has two amino acids (D264 and R472) that differ from the conserved residues of other GH74 xyloglucanases. These two amino acids were predicted to be located on the opposite side of the active site pocket, facing each other and forming a closing surface above the active site pocket. These two amino acids may contribute to the unique substrate specificity of the XcXGHA enzyme.
Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemolsynthase activity
Yang, T. ; Gao, L. ; Hu, H. ; Stoopen, G.M. ; Wang, C. ; Jongsma, M.A. - \ 2014
Journal of Biological Chemistry 289 (2014). - ISSN 0021-9258 - p. 36325 - 36335.
dimethylallyl diphosphate - squalene synthase - monoterpene synthase - terpene synthases - biosynthesis - expression - arabidopsis - metabolism - cloning - leaves
Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme inthe biosynthesis of pyrethrins, the most widely used plant-derivedpesticide.CDScatalyzes c1’-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteinsare known to catalyzethis cyclopropanation reactionof terpene precursors.Two of them, phytoene and squalenesynthase, arebifunctional enzymes with both prenyltransferase and terpenesynthase activity. CDS, the other member,was reported to perform only the prenyltransferase step.Here, we show thatthe NDXXD catalytic motif of CDS,under lowersubstrate conditions prevalent inplants,also catalyzesthe next step converting CPP into chrysanthemolby hydrolyzing the diphosphatemoiety.The enzymatic hydrolysis reactionfollowed conventional Michaelis-Menten kinetics, withaKM value for CPP of 196 µM.For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemolwas ~100 µM, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemolat arate of 0.12 –0.16 µg•h-1•g-1FW. We propose that CDS should be renamed a chrysanthemol synthase(CHS)utilizingDMAPP as substrate.
Identification of reference genes for gene expression studies during seed germination and seedling establishment Ricinus communis L.
Ribeiro de Jesus, P.R. ; Dekkers, S.J.W. ; Fernandez, L.G. ; Castro, R.D. De; Ligterink, W. ; Hilhorst, H.W.M. - \ 2014
Seed Science Research 24 (2014)4. - ISSN 0960-2585 - p. 341 - 352.
time rt-pcr - fatty-acid - jatropha-curcas - castor-oil - phosphoenolpyruvate carboxylase - arabidopsis - normalization - plant - cloning - family
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is an important technology to analyse gene expression levels during plant development or in response to different treatments. An important requirement to measure gene expression levels accurately is a properly validated set of reference genes. In this context, we analysed the potential use of 17 candidate reference genes across a diverse set of samples, including several tissues, different stages and environmental conditions, encompassing seed germination and seedling growth in Ricinus communis L. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. GeNorm and Normfinder indicated that ACT, POB and PP2AA1 comprise the optimal combination for normalization of gene expression data in inter-tissue (heterogeneous sample panel) studies. We also describe the optimal combination of reference genes for a subset of root, endosperm and cotyledon samples. In general, the most stable genes suggested by GeNorm are very consistent with those indicated by NormFinder, which highlights the strength of the selection of reference genes in our study. We also validated the selected reference genes by normalizing the expression levels of three target genes involved in energy metabolism with the reference genes suggested by GeNorm and NormFinder. The approach used in this study to identify stably expressed genes, and thus potential reference genes, was applied successfully for R. communis and it provides important guidelines for RT-qPCR studies in seeds and seedlings for other species (especially in those cases where extensive microarray data are not available)
Bacillus subtilis spore protein SpoVAC functions as a mechanosensitive channel
Velasquez, J. ; Schuurman-Wolters, G. ; Birkner, J.P. ; Abee, T. ; Poolman, B. - \ 2014
Molecular Microbiology 92 (2014)4. - ISSN 0950-382X - p. 813 - 823.
dipicolinic acid uptake - escherichia-coli - inner membrane - germination proteins - high-pressure - mscl - sporulation - expression - release - cloning
A critical event during spore germination is the release of Ca-DPA (calcium in complex with dipicolinic acid). The mechanism of release of Ca-DPA through the inner membrane of the spore is not clear, but proteins encoded by the Bacillus subtilis spoVA operon are involved in the process. We cloned and expressed the spoVAC gene in Escherichia coli and characterized the SpoVAC protein. We show that SpoVAC protects E.¿coli against osmotic downshift, suggesting that it might act as a mechanosensitive channel. Purified SpoVAC was reconstituted in unilamellar lipid vesicles to determine the gating mechanism and pore properties of the protein. By means of a fluorescence-dequenching assay, we show that SpoVAC is activated upon insertion into the membrane of the amphiphiles lysoPC and dodecylamine. Patch clamp experiments on E.¿coli giant spheroplast as well as giant unilamellar vesicles (GUVs) containing SpoVAC show that the protein forms transient pores with main conductance values of about 0.15 and 0.1 nS respectively. Overall, our data indicate that SpoVAC acts as a mechanosensitive channel and has properties that would allow the release of Ca-DPA and amino acids during germination of the spore.
A cautionary note on the use of split-YFP/BiFC in plant protein-protein interaction studies
Horstman, A. ; Nougalli Tonaco, I.A. ; Boutilier, K.A. ; Immink, R.G.H. - \ 2014
International Journal of Molecular Sciences 15 (2014). - ISSN 1661-6596 - p. 9628 - 9643.
bimolecular fluorescence complementation - bifc-based fret - living cells - ternary complexes - visualization - arabidopsis - assay - expression - fragments - cloning
Since its introduction in plants 10 years ago, the bimolecular fluorescence complementation (BiFC) method, or split-YFP (yellow fluorescent protein), has gained popularity within the plant biology field as a method to study protein-protein interactions. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. The major drawback of BiFC is that the fluorescent protein halves are prone to self-assembly independent of a protein-protein interaction event. To circumvent this problem, several modifications of the technique have been suggested, but these modifications have not lead to improvements in plant BiFC protocols. Therefore, it remains crucial to include appropriate internal controls. Our literature survey of recent BiFC studies in plants shows that most studies use inappropriate controls, and a qualitative rather than quantitative read-out of fluorescence. Therefore, we provide a cautionary note and beginner’s guideline for the setup of BiFC experiments, discussing each step of the protocol, including vector choice, plant expression systems, negative controls, and signal detection. In addition, we present our experience with BiFC with respect to self-assembly, peptide linkers, and incubation temperature. With this note, we aim to provide a guideline that will improve the quality of plant BiFC experiments.
A novel class of fungal lipoxygenases
Heshof, R. ; Jylhä, S. ; Haarmann, T. ; Jørgensen, A.L.W. ; Dalsgaard, T.K. ; Graaff, L.H. de - \ 2014
Applied Microbiology and Biotechnology 98 (2014)3. - ISSN 0175-7598 - p. 1261 - 1270.
manganese lipoxygenase - cloning - hydroperoxide - stereocontrol - oxygenation - oxylipins - pathway
Lipoxygenases (LOXs) are well-studied enzymes in plants and mammals. However, fungal LOXs are less studied. In this study, we have compared fungal LOX protein sequences to all known characterized LOXs. For this, a script was written using Shell commands to extract sequences from the NCBI database and to align the sequences obtained using Multiple Sequence Comparison by Log-Expectation. We constructed a phylogenetic tree with the use of Quicktree to visualize the relation of fungal LOXs towards other LOXs. These sequences were analyzed with respect to the signal sequence, C-terminal amino acid, the stereochemistry of the formed oxylipin, and the metal ion cofactor usage. This study shows fungal LOXs are divided into two groups, the Ile- and the Val-groups. The Ile-group has a conserved WRYAK sequence that appears to be characteristic for fungal LOXs and has as a C-terminal amino acid Ile. The Val-group has a highly conserved WL-L/F-AK sequence that is also found in LOXs of plant and animal origin. We found that fungal LOXs with this conserved sequence have a Val at the C-terminus in contrast to other LOXs of fungal origin. Also, these LOXs have signal sequences implying these LOXs will be expressed extracellularly. Our results show that in this group, in addition to the Gaeumannomyces graminis and the Magnaporthe salvinii LOXs, the Aspergillus fumigatus LOX uses manganese as a cofactor
Fine mapping of the Ph-3 gene conferring resistance to late blight (Phytophthora infestans) in tomato
Zhang, C.Z. ; Liu, L. ; Zheng, Z. ; Sun, Y.Y. ; Zhou, L.X. ; Yang, Y.H. ; Cheng, F. ; Zhang, Z.H. ; Wang, X.W. ; Huang, S.W. ; Xie, B.Y. ; Du, Y.C. ; Bai, Y. ; Li, J.M. - \ 2013
Theoretical and Applied Genetics 126 (2013)10. - ISSN 0040-5752 - p. 2643 - 2653.
species solanum-pimpinellifolium - disease resistance - united-states - potato - cloning - virus - accession - effectors - genomics - venturii
Late blight, caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary, is a devastating disease for tomato and potato crops. In the past decades, many late blight resistance (R) genes have been characterized in potato. In contrast, less work has been conducted on tomato. The Ph-3 gene from Solanum pimpinellifolium was introgressed into cultivated tomatoes and conferred broad-spectrum resistance to P. infestans. It was previously assigned to the long arm of chromosome 9. In this study, a high-resolution genetic map covering the Ph-3 locus was constructed using an F-2 population of a cross between Solanum lycopersicum CLN2037B (containing Ph-3) and S. lycopersicum LA4084. Ph-3 was mapped in a 0.5 cM interval between two markers, Indel_3 and P55. Eight putative genes were found in the corresponding 74 kb region of the tomato Heinz1706 reference genome. Four of these genes are resistance gene analogs (RGAs) with a typical nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 domain. Each RGA showed high homology to the late blight R gene Rpi-vnt1.1 from Solanum venturii. Transient gene silencing indicated that a member of this RGA family is required for Ph-3-mediated resistance to late blight in tomato. Furthermore, this RGA family was also found in the potato genome, but the number of the RGAs was higher than in tomato.
Detection of novel strains of cyprinid herpesvirus closely related to koi herpesvirus
Engelsma, M.Y. ; Way, K. ; Dodge, M.J. ; Voorbergen-Laarman, H.A. ; Panzarin, V.M. ; Abbadi, M. ; El-Matbouli, M. ; Skall, H.F. ; Kahns, S. ; Stone, D.M. - \ 2013
Diseases of Aquatic Organisms 107 (2013)2. - ISSN 0177-5103 - p. 113 - 120.
common carp - thymidine kinase - 1st detection - genome - aquaculture - cloning - disease - khv
Cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus (KHV) is a devastating virus of carp. Using generic primers for the DNA polymerase and the major capsid protein genes of cyprinid herpesviruses, nucleotide sequences divergent from previously described CyHV-3 were obtained. At least 3 novel groups of putative CyHV-3-like viruses were identified, sharing 95 to 98% nucleotide identity with CyHV-3 strains. Carp carrying the CyHV-3 variants did not show clinical signs consistent with CyHV-3 infection and originated from locations with no actual CyHV-3 outbreaks. These strains might represent low- or non-pathogenic variants of CyHV-3.
Geraniol hydroxylase and hydroxygeraniol oxidase activities of the CYP76 family of cytochrome P450 enzymes and potential for engineering the early steps of the (seco)iridoid pathway
Hofer, R. ; Dong, L. ; Andre, F. ; Ginglinger, J.F. ; Lugan, R. ; Gavira, C. ; Grec, S. ; Lang, G. ; Memelink, J. ; Krol, A.R. van der; Bouwmeester, H.J. ; Werck-Reichhart, D. - \ 2013
Metabolic Engineering 20 (2013). - ISSN 1096-7176 - p. 221 - 232.
indole alkaloid biosynthesis - catharanthus-roseus - madagascar periwinkle - expression - 10-hydroxylase - metabolomics - organization - arabidopsis - iridoids - cloning
The geraniol-derived (seco)iridoid skeleton is a precursor for a large group of bioactive compounds with diverse therapeutic applications, including the widely used anticancer molecule vinblastine. Despite of this economic prospect, the pathway leading to iridoid biosynthesis from geraniol is still unclear. The first geraniol hydroxylation step has been reported to be catalyzed by cytochrome P450 enzymes such as CYP76B6 from Catharanthus roseus and CYP76C1 from Arabidopsis thaliana. In the present study, an extended functional analysis of CYP76 family members was carried-out to identify the most effective enzyme to be used for pathway reconstruction. This disproved CYP76C1 activity and led to the characterization of CYP76C4 from A. thaliana as a geraniol 9- or 8-hydroxylase. CYP76B6 emerged as a highly specialized multifunctional enzyme catalyzing two sequential oxidation steps leading to the formation of 8-oxogeraniol from geraniol. This dual function was confirmed in planta using a leaf-disc assay. The first step, geraniol hydroxylation, was very efficient and fast enough to outcompete geraniol conjugation in plant tissues. When the enzyme was expressed in leaf tissues, 8-oxogeraniol was converted into further oxidized and/or reduced compounds in the absence of the next enzyme of the iridoid pathway
Bioluminescent and spectroscopic properties of His-Trp-Tyr triad mutants of obelin and aequorin.
Eremeeva, E. ; Markova, S.V. ; Frank, L.A. ; Visser, A.J.W.G. ; Berkel, W.J.H. van; Vysotski, E.S. - \ 2013
Photochemistry and Photobiological Sciences 12 (2013). - ISSN 1474-905X - p. 1016 - 1024.
ca2+-regulated photoproteins - ca2+-binding photoprotein - sequence-analysis - crystal-structure - violet bioluminescence - angstrom resolution - mnemiopsis-leidyi - light-emission - w92f obelin - cloning
Ca2+-regulated photoproteins are responsible for the bioluminescence of a variety of marine organisms, mostly coelenterates. The photoproteins consist of a single polypeptide chain to which an imidazopyrazinone derivative (2-hydroperoxycoelenterazine) is tightly bound. According to photoprotein spatial structures the side chains of His175, Trp179, and Tyr190 in obelin and His169, Trp173, Tyr184 in aequorin are at distances that allow hydrogen bonding with the peroxide and carbonyl groups of the 2-hydroperoxycoelenterazine ligand. We replaced these amino acids in both photoproteins by residues with different hydrogen bond donor–acceptor capacity. All mutants exhibited luciferase-like bioluminescence activity, hardly present in the wild-type photoproteins, and showed low or no photoprotein activity, except for aeqH169Q (24% of wild-type activity), obeW179Y (23%), obeW179F (67%), obeY190F (14%), and aeqY184F (22%). The results clearly support the supposition made from photoprotein spatial structures that the hydrogen bond network formed by His–Trp–Tyr triad participates in stabilizing the 2-hydroperoxy adduct of coelenterazine. These residues are also essential for the positioning of the 2-hydroperoxycoelenterazine intermediate, light emitting reaction, and for the formation of active photoprotein. In addition, we demonstrate that although the positions of His–Trp–Tyr residues in aequorin and obelin spatial structures are almost identical the substitution effects might be noticeably different.
Characterization of low-molecular-weight-glutenin subunit genes from the D-genome of Triticum aestivum, Aegilops crassa, Ae. cylindrica and Ae. tauschii
Naghavi, M.R. ; Ahmadi, S. ; Shanejat-Boushehri, A.A. ; Komaei, G. ; Struik, P.C. - \ 2013
Biochemical Systematics and Ecology 50 (2013). - ISSN 0305-1978 - p. 23 - 29.
endosperm storage proteins - group-1 chromosomes - wheat endosperm - hexaploid wheat - common wheat - cloning - pcr - electrophoresis - identification - polymorphism
Twenty low-molecular-weight-glutenin subunit (LMW-GS) gene sequences from the D-genome from Aegilops crassa (2n ¼ 4x ¼ 28), Ae. cylindrica (2n ¼ 4x ¼ 28), Ae. tauschii (2n ¼ 2x ¼ 14) and Triticum aestivum (2n ¼ 6x ¼ 42) were obtained using five sets of specific allele primer pairs. Only the sequences of the first primer pair were complete coding sequences (cds) of LMW-GS, and had 305, 304, 306 and 305 LMW-m amino acid residues in Ae. crassa, Ae. cylindrica, Ae. tauschii and T. aestivum, respectively. The repetitive domain and repeat motif numbers of all LMW glutenin subunits showed eight conserved cysteine residues that lead to the same functional activity in different genome. Based on DNA and predicted protein sequences, phylogenetic trees for all sets of sequences were drawn. At the DNA level, the species closest to T. aestivum for the second, third, fourth and fifth set of sequences were Ae. cylindrica, Ae. tauschii and Ae. crassa, respectively. At the protein level, the species closest to T. aestivum based on the first, second and fifth set of sequences were Ae. cylindrica, Ae. crassa and Ae. crassa, respectively. For other sets of sequences, bread wheat proved to be a distinct species. The LMW-GS gene sequences have been recorded in the GenBank with accession numbers JQ726549–JQ726568.
Biochemical and functional characterization of recombinant fungal immunomodulatory proteins (rFIPs)
Bastiaan-Net, S. ; Chanput, W. ; Hertz, A. ; Zwittink, R.D. ; Mes, J.J. ; Wichers, H.J. - \ 2013
International Immunopharmacology 15 (2013)1. - ISSN 1567-5769 - p. 167 - 175.
ganoderma-lucidium - murine macrophages - fip-fve - expression - lectins - cloning - gene - lz-8 - mushroom - sinense
In this study two novel FIPs have been identified and characterized. The first is FIP-nha, identified in the ascomycete Nectria haematococca, and as such, FIP-nha would be the first FIP to be identified outside the order of Basidiomycota. The second is LZ-9, an LZ-8 like protein identified in Ganoderma lucidum. Recombinant FIP proteins were produced in Pichia pastoris and purified using His-affinity magnetic beads. The bioactive characteristics of FIP-nha and LZ-9 were compared to two other well-known FIP proteins, LZ-8 from G. lucidum and FIP-fve from Flammulina velutipes, which were produced and purified using the same method. The produced recombinant FIPs (rFIPs): rLZ-8, rLZ-9, rFIP-fve and rFIP-nha were investigated for their hemagglutinating activity which revealed that rLZ-8, rLZ-9 and rFIP-nha were able to agglutinate rabbit, mouse and sheep red blood cells while rFIP-fve only agglutinated rabbit red blood cells. None of the rFIPs were able to agglutinate human red blood cells unless the cells were trypsinized. In addition, all rFIPs were studied and compared to several lectins for their effect on Caco-2 intestinal cell layer integrity using transepithelial electrical resistance (TEER) measurement. rLZ-9 appeared to have the highest effect in lowering TEER, similar to one of the tested lectins. Testing of rFIPs for their activation of inflammation-related genes of THP-1 macrophages showed rFIP-fve to be the strongest inducer of pro-inflammatory cytokine transcription. These results indicate that each rFIP has a unique bioactive profile as well as each lectin, creating the basis for further studies to relate structure to biological activity.
Melanin biosynthesis pathway in Agaricus bisporus mushrooms
Weijn, A. ; Bastiaan-Net, S. ; Wichers, H.J. ; Mes, J.J. - \ 2013
Fungal Genetics and Biology 55 (2013). - ISSN 1087-1845 - p. 42 - 53.
quantitative trait locus - lecanicillium-fungicola - polyphenol oxidase - tyrosinase - expression - resistance - cloning - genes - metabolism
With the full genome sequence of Agaricus bisporus available, it was possible to investigate the genes involved in the melanin biosynthesis pathway of button mushrooms. Based on different BLAST and alignments, genes were identified in the genome which are postulated to be involved in this pathway. Seven housekeeping genes were tested of which 18S rRNA was the only housekeeping gene that was stably expressed in various tissues of different developmental stages. Gene expression was determined for most gene homologs (26 genes) involved in the melanin pathway. Of the analysed genes, those encoding polyphenol oxidase (PPO), the PPO co-factor L-chain (unique for Agaricus bisporus), and a putative transcription factor (photoregulator B) were among the highest expressed in skin tissue. An in depth look was taken at the clustering of several PPO genes and the PPO co-factor gene on chromosome 5, which showed that almost 25% of the protein encoding genes in this cluster have a conserved NACHT and WD40 domain or a P-loop nucleoside triphosphate hydrolase. This article will be the start for an in depth study of the melanin pathway and the role in quality losses of this economically important product.
Enhanced production of Aspergillus niger laccase-like multicopper oxidases through mRNA optimization of the glucoamylase expression system
Tamayo Ramos, J.A. ; Barends, S. ; Lange, D. ; Jel, A. de; Verhaert, R.M. ; Graaff, L.H. de - \ 2013
Biotechnology and Bioengineering 110 (2013)2. - ISSN 0006-3592 - p. 543 - 551.
5'-untranslated region - protein expression - gene - translation - transformation - promoter - nidulans - cloning - oryzae
In filamentous fungi, most of the strategies used for the improvement of protein yields have been based on an increase in the transcript levels of a target gene. Strategies focusing at the translational level have been also described, but are far less explored. Here the 5' untranslated sequence of the glaA mRNA, a widely used expression system for the expression of recombinant proteins, was modified by the introduction of different nucleotide elements that have positive role in the translation process. Five Aspergillus niger laccase-like multicopper oxidases (MCOs) coding genes were fused to the native glaA 5'UTR and the three synthetic versions (sUTR1, sUTR2, and sUTR3) as well, and placed under the control of the glucoamylase gene promoter. Afterwards, a total of 20 fungal transformations were done using A. niger N593 as a recipient strain and 50 transformants per transformation were isolated and analyzed. The result of the incorporation of the synthetic 5'UTRs on the overall productivity of the transformants was assessed, on one hand by monitoring the laccase activity of all the isolated transformants, and on the other hand by quantifying and comparing the activity of those secreting the highest level of each MCO. For this purpose, a high-throughput method for the screening and selection of the best producers was developed. Once the best transformants producing the highest yield of McoA, McoB, McoC, McoD, and McoJ laccases were selected, their production level was quantified in supernatants of liquid cultures. The results obtained in this work indicate that modifications in the native glaA 5'UTR can lead to improvements in protein yields. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc
The Tomato FRUITFULL Homologs TDR4/FUL1 and MBP7/FUL2 Regulate Ethylene-Independent Aspects of Fruit Ripening
Bemer, M. ; Karlova, R.B. ; Ballester, A.R. ; Tikunov, Y.M. ; Bovy, A.G. ; Wolters-Arts, M. ; Barros Rossetto, P. de; Angenent, G.C. ; Maagd, R.A. de - \ 2012
The Plant Cell 24 (2012)11. - ISSN 1040-4651 - p. 4437 - 4451.
mads-box gene - d-glucuronate 4-epimerase - differential expression - 1-aminocyclopropane-1-carboxylate oxidase - agrobacterium-tumefaciens - carotenoid biosynthesis - systems biology - arabidopsis - protein - cloning
Tomato (Solanum lycopersicum) contains two close homologs of the Arabidopsis thaliana MADS domain transcription factor FRUITFULL (FUL), FUL1 (previously called TDR4) and FUL2 (previously MBP7). Both proteins interact with the ripening regulator RIPENING INHIBITOR (RIN) and are expressed during fruit ripening. To elucidate their function in tomato, we characterized single and double FUL1 and FUL2 knockdown lines. Whereas the single lines only showed very mild alterations in fruit pigmentation, the double silenced lines exhibited an orange-ripe fruit phenotype due to highly reduced lycopene levels, suggesting that FUL1 and FUL2 have a redundant function in fruit ripening. More detailed analyses of the phenotype, transcriptome, and metabolome of the fruits silenced for both FUL1 and FUL2 suggest that the genes are involved in cell wall modification, the production of cuticle components and volatiles, and glutamic acid (Glu) accumulation. Glu is responsible for the characteristic umami taste of the present-day cultivated tomato fruit. In contrast with previously identified ripening regulators, FUL1 and FUL2 do not regulate ethylene biosynthesis but influence ripening in an ethylene-independent manner. Our data combined with those of others suggest that FUL1/2 and TOMATO AGAMOUS-LIKE1 regulate different subsets of the known RIN targets, probably in a protein complex with the latter.