http://www.plantmethods.com The most accessed research articles published by Plant Methods 2010-08-20T00:00:00Z 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/ Background: Interphase chromosome organization and dynamics can be studied in living cells using fluorescent tagging techniques that exploit bacterial operator/repressor systems and auto-fluorescent proteins. A nuclear-localized Repressor Protein-Fluorescent Protein (RP-FP) fusion protein binds to operator repeats integrated as transgene arrays at defined locations in the genome. Under a fluorescence microscope, the tagged sites appear as bright fluorescent dots in living cells. This technique has been used successfully in plants, but is often hampered by low expression of genes encoding RP-FP fusion proteins, perhaps owing to one or more gene silencing mechanisms that are prevalent in plant cells. Results: We used two approaches to overcome this problem. First, we tested mutations in four factors involved in different types of gene silencing and/or epigenetic modifications for their effects on nuclear fluorescence. Only mutations in DDM1, a chromatin remodelling ATPase involved in repeat-induced heterochromatin formation and DNA methylation, released silencing of the RP-FP fusion protein. This result suggested that the operator repeats can trigger silencing of the adjacent gene encoding the RP-FP fusion protein. In the second approach, we transformed the tagged lines with a second T-DNA encoding the RP-FP fusion protein but lacking operator repeats. This strategy avoided operator repeat-induced gene silencing and increased the number of interphase nuclei displaying fluorescent dots. In a further extension of the technique, we show that green fluorescent-tagged sites can be visualized on moving mitotic chromosomes stained with red fluorescent-labelled histone H2B. Conclusions: The results illustrate the propensity of operator repeat arrays to form heterochromatin that can silence the neighbouring gene encoding the RP-FP fusion protein. Supplying the RP-FP fusion protein in trans from a second T-DNA largely alleviates this problem. Depending on the promoter used to drive expression of the RP-FP fusion protein gene, the fluorescent tagged sites can be visualized at high frequency in different cell types. The ability to observe fluorescent dots on both interphase and mitotic chromosomes allows tagged sites to be tracked throughout the cell cycle. These improvements enhance the versatility of the fluorescent tagging technique for future studies of chromosome arrangement and dynamics in living plants. http://www.plantmethods.com/content/6/1/2 Antonius Matzke Koichi Watanabe Johannes van der Winden Ulf Naumann Marjori Matzke Plant Methods 2010, 6:2 2010-01-19T00:00:00Z doi:10.1186/1746-4811-6-2 Plant Methods 1746-4811 6 2 2010-01-19T00:00:00Z XML Background: Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression. Results: A high throughput pipeline was developed to generate promoter-reporter (GFP) transgenic lines for Arabidopsis genes expressed at very low levels and to examine their expression patterns in vivo. The promoter region from a total of 627 non- or low-expressed genes in Arabidopsis based on Arabidopsis annotation release 5 were amplified and cloned into a Gateway vector. A total of 353 promoter-reporter (GFP) constructs were successfully transferred into Agrobacterium (GV3101) by triparental mating and subsequently used for Arabidopsis transformation. Kanamycin-resistant transgenic lines were obtained from 266 constructs and among them positive GFP expression was detected from 150 constructs. Of these 150 constructs, multiple transgenic lines exhibiting consistent expression patterns were obtained for 112 constructs. A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes. Conclusions: Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened. More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis. The results of this study are captured in a MySQL database and can be searched at http://www.jcvi.org/arabidopsis/qpcr/index.shtml. Transgenic seeds and constructs are also available for the research community. http://www.plantmethods.com/content/6/1/18 Yong-Li Xiao Julia Redman Erin Monaghan Jun Zhuang Beverly Underwood William Moskal Wei Wang Hank Wu Christopher Town Plant Methods 2010, 6:18 2010-08-06T00:00:00Z doi:10.1186/1746-4811-6-18 Plant Methods 1746-4811 6 18 2010-08-06T00:00:00Z XML Background: The introduction of second generation sequencing technology has enabled the cost effective sequencing of genomes and the identification of large numbers of genes and gene promoters. However, the assembly of DNA sequences to create a representation of the complete genome sequence remains costly, especially for the larger and more complex plant genomes. Results: We have developed an online database, TAGdb, that enables researchers to identify paired read sequences that share identity with a submitted query sequence. These tags can be used to design oligonucleotide primers for the PCR amplification of the region in the target genome. Conclusions: The ability to produce large numbers of paired read genome tags using second generation sequencing provides a cost effective method for the identification of genes and promoters in large, complex or orphan species without the need for whole genome assembly. http://www.plantmethods.com/content/6/1/19 Daniel Marshall Alice Hayward Dominic Eales Michael Imelfort Jiri Stiller Paul Berkman Terry Clark Megan McKenzie Kaitao Lai Chris Duran Jacqueline Batley David Edwards Plant Methods 2010, 6:19 2010-08-20T00:00:00Z doi:10.1186/1746-4811-6-19 Plant Methods 1746-4811 6 19 2010-08-20T00:00:00Z PDF Background: High throughput DNA isolation from plants is a major bottleneck for most studies requiring large sample sizes. A variety of protocols have been developed for DNA isolation from plants. However, many species, including conifers, have high contents of secondary metabolites that interfere with the extraction process or the subsequent analysis steps. Here, we describe a procedure for high-throughput DNA isolation from conifers. Results: We have developed a high-throughput DNA extraction protocol for conifers using an automated liquid handler and modifying the Qiagen MagAttract Plant Kit protocol. The modifications involve change to the buffer system and improving the protocol so that it almost doubles the number of samples processed per kit, which significantly reduces the overall costs. We describe two versions of the protocol: one for medium-throughput (MTP) and another for high-throughput (HTP) DNA isolation. The HTP version works from start to end in the industry-standard 96-well format, while the MTP version provides higher DNA yields per sample processed. We have successfully used the protocol for DNA extraction and genotyping of thousands of individuals of several spruce and a pine species. Conclusion: A high-throughput system for DNA extraction from conifer needles and seeds has been developed and validated. The quality of the isolated DNA was comparable with that obtained from two commonly used methods: the silica-spin column and the classic CTAB protocol. Our protocol provides a fully automatable and cost effective solution for processing large numbers of conifer samples. http://www.plantmethods.com/content/4/1/20 Stanislav Bashalkhanov Om Rajora Plant Methods 2008, 4:20 2008-08-01T00:00:00Z doi:10.1186/1746-4811-4-20 Plant Methods 1746-4811 4 20 2008-08-01T00:00:00Z XML Background: We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. Results: We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. Conclusion: In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays. http://www.plantmethods.com/content/1/1/13 Roger Hellens Andrew Allan Ellen Friel Karen Bolitho Karryn Grafton Matthew Templeton Sakuntala Karunairetnam Andrew Gleave William Laing Plant Methods 2005, 1:13 2005-12-18T00:00:00Z doi:10.1186/1746-4811-1-13 Plant Methods 1746-4811 1 13 2005-12-18T00:00:00Z XML Background: Recent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously. To perform these studies, recombinational cloning methods such as the Gateway system have been adapted to plant transformation vectors to facilitate the creation of overexpression, tagging and silencing vectors on a large scale. Results: Here we present a hybrid cloning strategy which combines advantages of both recombinational and traditional cloning and which is particularly amenable to low-to-medium throughput investigations of protein function using techniques of molecular biochemistry and cell biology. The system consists of a series of twelve Gateway Entry cassettes into which a gene of interest can be inserted using traditional cloning methods to generate either N- or C-terminal fusions to epitope tags and fluorescent proteins. The resulting gene-tag fusions can then be recombined into Gateway-based Destination vectors, thus providing a wide choice of resistance marker, promoter and expression system. The advantage of this modified Gateway cloning strategy is that the entire open reading frame encoding the tagged protein of interest is contained within the Entry vectors so that after recombination no additional linker sequences are added between the tag and the protein that could interfere with protein function and expression. We demonstrate the utility of this system for both transient and stable Agrobacterium-mediated plant transformations. Conclusion: This modified Gateway cloning strategy is complementary to more conventional Gateway-based systems because it expands the choice of tags and higher orders of combinations, and permits more control over the linker sequence lying between a protein of interest and an epitope tag, which can be particularly important for studies of protein function. http://www.plantmethods.com/content/4/1/3 Manu Dubin Chris Bowler Giovanna Benvenuto Plant Methods 2008, 4:3 2008-01-22T00:00:00Z doi:10.1186/1746-4811-4-3 Plant Methods 1746-4811 4 3 2008-01-22T00:00:00Z XML 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 Qingshun Li Plant Methods 2008, 4:4 2008-01-22T00:00:00Z doi:10.1186/1746-4811-4-4 Plant Methods 1746-4811 4 4 2008-01-22T00:00:00Z XML 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 Peterhaensel Maike Stam Plant Methods 2007, 3:11 2007-09-24T00:00:00Z doi:10.1186/1746-4811-3-11 Plant Methods 1746-4811 3 11 2007-09-24T00:00:00Z XML Background: The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7–10 d and high seedling density and fungal contamination may result in failure to recover transformants. Results: A method for identifying transformed seedlings in as little as 3.25 d has been developed. Arabidopsis T1 seeds obtained after floral dip transformation are plated on 1% agar containing MS medium and kanamycin, phosphinothricin or hygromycin B, as appropriate. After a 2-d stratification period, seeds are subjected to a regime of 4–6 h light, 48 h dark and 24 h light (3.25 d). Kanamycin-resistant and phosphinothricin-resistant seedlings are easily distinguished from non-resistant seedlings by green expanded cotyledons whereas non-resistant seedlings have pale unexpanded cotyledons. Seedlings grown on hygromycin B differ from those grown on kanamycin and phosphinothricin as both resistant and non-resistant seedlings are green. However, hygromycin B-resistant seedlings are easily identified as they have long hypocotyls (0.8–1.0 cm) whereas non-resistant seedlings have short hypocotyls (0.2–0.4 cm). Conclusion: The method presented here is an improvement on current selection methods as it allows quicker identification of transformed seedlings: transformed seedlings are easily discernable from non-transformants in as little as 3.25 d in comparison to the 7–10 d required for selection using current protocols. http://www.plantmethods.com/content/2/1/19 Samuel Harrison Ellie Mott Kate Parsley Sue Aspinall John Gray Amanda Cottage Plant Methods 2006, 2:19 2006-11-06T00:00:00Z doi:10.1186/1746-4811-2-19 Plant Methods 1746-4811 2 19 2006-11-06T00:00:00Z XML Background: Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes. Results: We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass. Conclusion: The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis. http://www.plantmethods.com/content/5/1/6 Jian-Feng Li Eunsook Park Albrecht von Arnim Andreas Nebenfuhr Plant Methods 2009, 5:6 2009-05-20T00:00:00Z doi:10.1186/1746-4811-5-6 Plant Methods 1746-4811 5 6 2009-05-20T00:00:00Z XML