This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ http://www.plantmethods.commostviewed/ Most viewed articles in last 30 days from Plant Methods (ISSN 1746-4811) published by BioMed Central We describe an efficient method for the rapid quantitative determination of the abundance of three acidic plant hormones from a single crude extract directly by LC/MS/MS. The method exploits the sensitivity of MS and uses multiple reaction monitoring and isotopically labelled samples to quantify the phytohormones abscisic acid, jasmonic acid and salicylic acid in Arabidopsis leaf tissue. http://www.plantmethods.com/content/4/1/16 Silvia Forcat, Mark H Bennett, John W Mansfield and Murray R Grant Plant Methods 2008, 4:16 Number of accesses: 1204 2008-06-30 doi:10.1186/1746-4811-4-16 Plant Methods 1746-4811 4 16 2008-06-30 Background: Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. Results: Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy. The dyes were selective for non-viable cells and showed very little background staining in living cells. Simultaneous detection of SYTOX dye and fluorescent protein (e.g. GFP) fluorescence was possible. Conclusion: The fluorescent SYTOX dyes are useful for an easy and quick first assay of plant cell viability in living plant samples using fluorescence and confocal laser-scanning microscopy. http://www.plantmethods.com/content/4/1/15 Elisabeth Truernit and Jim Haseloff Plant Methods 2008, 4:15 Number of accesses: 746 2008-06-23 doi:10.1186/1746-4811-4-15 Plant Methods 1746-4811 4 15 2008-06-23 Background: Kinome profiling aims at the parallel analysis of kinase activities in a cell. Novel developed arrays containing consensus substrates for kinases are used to assess those kinase activities. The arrays described in this paper were already used to determine kinase activities in mammalian systems, but since substrates from many organisms are present we decided to test these arrays for the determination of kinase activities in the model plant species Arabidopsis thaliana. Results: Kinome profiling using Arabidopsis cell extracts resulted in the labelling of many consensus peptides by kinases from the plant, indicating the usefulness of this kinome profiling tool for plants. Method development showed that fresh and frozen plant material could be used to make cell lysates containing active kinases. Dilution of the plant extract increased the signal to noise ratio and non-radioactive ATP enhances full development of spot intensities.Upon infection of Arabidopsis with an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. tomato, we could detect differential kinase activities by measuring phosphorylation of consensus peptides. Conclusion: We show that kinome profiling on arrays with consensus substrates can be used to monitor kinase activities in plants. In a case study we show that upon infection with avirulent P. syringae differential kinase activities can be found. The PepChip can for example be used to purify (unknown) kinases that play a role in P. syringae infection.This paper shows that kinome profiling using arrays of consensus peptides is a valuable new tool to study signal-transduction in plants. It complements the available methods for genomics and proteomics research. http://www.plantmethods.com/content/3/1/3 Tita Ritsema, Jos Joore, Wilbert van Workum and Corné MJ Pieterse Plant Methods 2007, 3:3 Number of accesses: 725 2007-02-12 doi:10.1186/1746-4811-3-3 Plant Methods 1746-4811 3 3 2007-02-12 Background: Transcription factors (TFs) co-ordinately regulate target genes that are dispersed throughout the genome. This co-ordinate regulation is achieved, in part, through the interaction of transcription factors with conserved cis-regulatory motifs that are in close proximity to the target genes. While much is known about the families of transcription factors that regulate gene expression in plants, there are few well characterised cis-regulatory motifs. In Arabidopsis, over-expression of the MYB transcription factor PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) leads to transgenic plants with elevated anthocyanin levels due to the co-ordinated up-regulation of genes in the anthocyanin biosynthetic pathway. In addition to the anthocyanin biosynthetic genes, there are a number of un-associated genes that also change in expression level. This may be a direct or indirect consequence of the over-expression of PAP1. Results: Oligo array analysis of PAP1 over-expression Arabidopsis plants identified genes co-ordinately up-regulated in response to the elevated expression of this transcription factor. Transient assays on the promoter regions of 33 of these up-regulated genes identified eight promoter fragments that were transactivated by PAP1. Bioinformatic analysis on these promoters revealed a common cis-regulatory motif that we showed is required for PAP1 dependent transactivation. Conclusion: Co-ordinated gene regulation by individual transcription factors is a complex collection of both direct and indirect effects. Transient transactivation assays provide a rapid method to identify direct target genes from indirect target genes. Bioinformatic analysis of the promoters of these direct target genes is able to locate motifs that are common to this sub-set of promoters, which is impossible to identify with the larger set of direct and indirect target genes. While this type of analysis does not prove a direct interaction between protein and DNA, it does provide a tool to characterise cis-regulatory sequences that are necessary for transcription activation in a complex list of co-ordinately regulated genes. http://www.plantmethods.com/content/4/1/17 Andrew P Dare, Robert J Schaffer, Kui Lin-Wang, Andrew C Allan and Roger P Hellens Plant Methods 2008, 4:17 Number of accesses: 590 2008-07-07 doi:10.1186/1746-4811-4-17 Plant Methods 1746-4811 4 17 2008-07-07 Background: The diploid, Solanum caripense, a wild relative of potato and tomato, possesses valuable resistance to potato late blight and we are interested in the genetic base of this resistance. Due to extremely low levels of genetic variation within the S. caripense genome it proved impossible to generate a dense genetic map and to assign individual Solanum chromosomes through the use of conventional chromosome-specific SSR, RFLP, AFLP, as well as gene- or locus-specific markers. The ease of detection of DNA polymorphisms depends on both frequency and form of sequence variation. The narrow genetic background of close relatives and inbreds complicates the detection of persisting, reduced polymorphism and is a challenge to the development of reliable molecular markers. Nonetheless, monomorphic DNA fragments representing not directly usable conventional markers can contain considerable variation at the level of single nucleotide polymorphisms (SNPs). This can be used for the design of allele-specific molecular markers. The reproducible detection of allele-specific markers based on SNPs has been a technical challenge. Results: We present a fast and cost-effective protocol for the detection of allele-specific SNPs by applying Sequence Polymorphism-Derived (SPD) markers. These markers proved highly efficient for fingerprinting of individuals possessing a homogeneous genetic background. SPD markers are obtained from within non-informative, conventional molecular marker fragments that are screened for SNPs to design allele-specific PCR primers. The method makes use of primers containing a single, 3'-terminal Locked Nucleic Acid (LNA) base. We demonstrate the applicability of the technique by successful genetic mapping of allele-specific SNP markers derived from monomorphic Conserved Ortholog Set II (COSII) markers mapped to Solanum chromosomes, in S. caripense. By using SPD markers it was possible for the first time to map the S. caripense alleles of 16 chromosome-specific COSII markers and to assign eight of the twelve linkage groups to consensus Solanum chromosomes. Conclusion: The method based on individual allelic variants allows for a level-of-magnitude higher resolution of genetic variation than conventional marker techniques. We show that the majority of monomorphic molecular marker fragments from organisms with reduced heterozygosity levels still contain SNPs that are sufficient to trace individual alleles. http://www.plantmethods.com/content/3/1/2 Joy Nakitandwe, Friederike Trognitz and Bodo Trognitz Plant Methods 2007, 3:2 Number of accesses: 496 2007-02-07 doi:10.1186/1746-4811-3-2 Plant Methods 1746-4811 3 2 2007-02-07 Background: Medicago truncatula is a model legume species that is currently the focus of an international genome sequencing effort. Although several different oligonucleotide and cDNA arrays have been produced for genome-wide transcript analysis of this species, intrinsic limitations in the sensitivity of hybridization-based technologies mean that transcripts of genes expressed at low-levels cannot be measured accurately with these tools. Amongst such genes are many encoding transcription factors (TFs), which are arguably the most important class of regulatory proteins. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the most sensitive method currently available for transcript quantification, and one that can be scaled up to analyze transcripts of thousands of genes in parallel. Thus, qRT-PCR is an ideal method to tackle the problem of TF transcript quantification in Medicago and other plants. Results: We established a bioinformatics pipeline to identify putative TF genes in Medicago truncatula and to design gene-specific oligonucleotide primers for qRT-PCR analysis of TF transcripts. We validated the efficacy and gene-specificity of over 1000 TF primer pairs and utilized these to identify sets of organ-enhanced TF genes that may play important roles in organ development or differentiation in this species. This community resource will be developed further as more genome sequence becomes available, with the ultimate goal of producing validated, gene-specific primers for all Medicago TF genes. Conclusion: High-throughput qRT-PCR using a 384-well plate format enables rapid, flexible, and sensitive quantification of all predicted Medicago transcription factor mRNAs. This resource has been utilized recently by several groups in Europe, Australia, and the USA, and we expect that it will become the 'gold-standard' for TF transcript profiling in Medicago truncatula. http://www.plantmethods.com/content/4/1/18 Klementina Kakar, Maren Wandrey, Tomasz Czechowski, Tanja Gaertner, Wolf-Rudiger Scheible, Mark Stitt, Ivone Torres-Jerez, Yongli Xiao, Julia Redman, Hank Wu, Foo Cheung, Christopher Town and Michael Udvardi Plant Methods 2008, 4:18 Number of accesses: 479 2008-07-08 doi:10.1186/1746-4811-4-18 Plant Methods 1746-4811 4 18 2008-07-08 Background: In plant functional genomic studies, gene cloning into binary vectors for plant transformation is a routine procedure. Traditionally, gene cloning has relied on restriction enzyme digestion and ligation. In recent years, however, Gateway® cloning technology (Invitrogen Co.) has developed a fast and reliable alternative cloning methodology which uses a phage recombination strategy. While many Gateway- compatible vectors are available, we frequently encounter problems in which antibiotic resistance genes for bacterial selection are the same between recombinant vectors. Under these conditions, it is difficult, if not sometimes impossible, to use antibiotic resistance in selecting the desired transformants. We have, therefore, developed a practical procedure to solve this problem. Results: An integrated protocol for cloning genes of interest from PCR to Agrobacterium transformants via the Gateway® System was developed. The protocol takes advantage of unique characteristics of the replication origins of plasmids used and eliminates the necessity for restriction enzyme digestion in plasmid selections. Conclusion: The protocol presented here is a streamlined procedure for fast and reliable cloning of genes of interest from PCR to Agrobacterium via the Gateway® System. This protocol overcomes a key problem in which two recombinant vectors carry the same antibiotic selection marker. In addition, the protocol could be adapted for high-throughput applications. http://www.plantmethods.com/content/4/1/4 Ruqiang Xu and Qingshun Quinn Li Plant Methods 2008, 4:4 Number of accesses: 413 2008-01-22 doi:10.1186/1746-4811-4-4 Plant Methods 1746-4811 4 4 2008-01-22 Intact protein complexes can be separated by apparent molecular mass using a standard polyacrylamide gel electrophoresis system combining mild detergents and the dye Coomassie Blue. Referring to the blue coloured gel and the gentle method of solubilization yielding native and enzymatically active protein complexes, this technique has been named Blue-Native Polyacrylamide Gel-Electrophoresis (BN-PAGE). BN-PAGE has become the method of choice for the investigation of the respiratory protein complexes of the electron transfer chains of a range of organisms, including bacteria, yeasts, animals and plants. It allows the separation in two dimensions of extremely hydrophobic protein sets for analysis and also provides information on their native interactions. In this review we discuss the capabilities of BN-PAGE in proteomics and the wider investigation of protein:protein interactions with a focus on its use and potential in plant science. http://www.plantmethods.com/content/1/1/11 Holger Eubel, Hans-Peter Braun and A Harvey Millar Plant Methods 2005, 1:11 Number of accesses: 406 2005-11-16 doi:10.1186/1746-4811-1-11 Plant Methods 1746-4811 1 11 2005-11-16 Background: Chloroplast genomes evolve slowly and many primers for PCR amplification and analysis of chloroplast sequences can be used across a wide array of genera. In some cases 'universal' primers have been designed for the purpose of working across species boundaries. However, the essential information on these primer sequences is scattered throughout the literature. Results: A database is presented here which assembles published primer information for chloroplast DNA. Additional primers were designed to fill gaps where little or no primer information could be found. Amplicons are either the genes themselves (typically useful in studies of sequence variation in higher-order phylogeny) or they are spacers, introns, and intergenic regions (for studies of phylogeographic patterns within and among species). The current list of 'generic' primers consists of more than 700 sequences. Wherever possible, we give the locations of the primers in the thirteen fully sequenced chloroplast genomes (Nicotiana tabacum, Atropa belladonna, Spinacia oleracea, Arabidopsis thaliana, Populus trichocarpa, Oryza sativa, Pinus thunbergii, Marchantia polymorpha, Zea mays, Oenothera elata, Acorus calamus, Eucalyptus globulus, Medicago trunculata). Conclusion: The database described here is designed to serve as a resource for researchers who are venturing into the study of poorly described chloroplast genomes, whether for large- or small-scale DNA sequencing projects, to study molecular variation or to investigate chloroplast evolution. http://www.plantmethods.com/content/3/1/4 Berthold Heinze Plant Methods 2007, 3:4 Number of accesses: 371 2007-02-27 doi:10.1186/1746-4811-3-4 Plant Methods 1746-4811 3 4 2007-02-27 Background: Chromatin remodeling, histone modifications and other chromatin-related processes play a crucial role in gene regulation. A very useful technique to study these processes is chromatin immunoprecipitation (ChIP). ChIP is widely used for a few model systems, including Arabidopsis, but establishment of the technique for other organisms is still remarkably challenging. Furthermore, quantitative analysis of the precipitated material and normalization of the data is often underestimated, negatively affecting data quality. Results: We developed a robust ChIP protocol, using maize (Zea mays) as a model system, and present a general strategy to systematically optimize this protocol for any type of tissue. We propose endogenous controls for active and for repressed chromatin, and discuss various other controls that are essential for successful ChIP experiments. We experienced that the use of quantitative PCR (QPCR) is crucial for obtaining high quality ChIP data and we explain why. The method of data normalization has a major impact on the quality of ChIP analyses. Therefore, we analyzed different normalization strategies, resulting in a thorough discussion of the advantages and drawbacks of the various approaches. Conclusion: Here we provide a robust ChIP protocol and strategy to optimize the protocol for any type of tissue; we argue that quantitative real-time PCR (QPCR) is the best method to analyze the precipitates, and present comprehensive insights into data normalization. http://www.plantmethods.com/content/3/1/11 Max Haring, Sascha Offermann, Tanja Danker, Ina Horst, Christoph Peterhansel and Maike Stam Plant Methods 2007, 3:11 Number of accesses: 369 2007-09-24 doi:10.1186/1746-4811-3-11 Plant Methods 1746-4811 3 11 2007-09-24