http://www.plantmethods.com The most accessed research articles published by Plant Methods 2012-05-04T00:00:00Z Background: Transient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to only few plant species and often requires substantial equipment and facilities. The high content of chloroplasts and chlorophyll can impede downstream applications of transformed cells from green plant tissue. Results: We describe a fast and simple technique for the high-yield isolation and efficient transformation (>70%) of mesophyll-derived protoplasts from red leaves of the perennial plant Poinsettia (Euphorbia pulccherrima) for which no particular growth facilities or expensive equipment are needed. Poinsettia protoplasts display an astonishing robustness and can be employed in all commonly-used downstream applications, such as subcellular localisation (multi-colour fluorescence) or promoter activity studies. Due to a low abundance of chloroplasts or chromoplasts, problems encountered in other mesophyll-derived protoplast systems (particularly autofluorescence) are alleviated. Transgene expression is detectable within 90 min post-transformation and lasts over several days. Conclusions: The simplicity of isolation and transformation renders Poinsettia protoplasts an attractive system for transient gene expression experiments, including multicolour fluorescence, subcellular localisation and promoter activity studies. In addition, they offer hitherto unknown possibilities for anthocyan research and industrial applications. http://www.plantmethods.com/content/8/1/14 Andrea Pitzschke Helene Persak Plant Methods 2012, null:14 2012-05-04T00:00:00Z doi:10.1186/1746-4811-8-14 Transient expression is an extremely valuable component of the molecular biologist's toolbox. The authors describe a new version of this tool that promises to make the procedure more reliable - as well as being accessible to more laboratories. /content/figures/1746-4811-8-14-toc.gif Plant Methods 1746-4811 ${item.volume} 14 2012-05-04T00:00:00Z PDF Large-scale genetic screens in Arabidopsis are a powerful approach for molecular dissection of complex signaling networks. However, map-based cloning can be time-consuming or even hampered due to low chromosomal recombination. Current strategies using next generation sequencing for molecular identification of mutations require whole genome sequencing and advanced computational devises and skills, which are not readily accessible or affordable to every laboratory. We have developed a streamlined method using parallel massive sequencing for mutant identification in which only targeted regions are sequenced. This targeted parallel sequencing (TPSeq) method is more cost-effective, straightforward enough to be easily done without specialized bioinformatics expertise, and reliable for identifying multiple mutations simultaneously. Here, we demonstrate its use by identifying three novel nitrate-signaling mutants in Arabidopsis. http://www.plantmethods.com/content/8/1/12 Kun-hsiang Liu Matthew McCormack Jen Sheen Plant Methods 2012, null:12 2012-03-30T00:00:00Z doi:10.1186/1746-4811-8-12 Making mutant identification by next-generation sequencing more accessible Next generation sequencing (NGS) is becoming a powerful tool for identifying mutations at the molecular level. This paper describes an innovation that has the potential to make the NGS approach accessible to many more laboratories. In the targeted parallel sequencing (TPSeq) method only genetically defined genomic regions are sequenced, making the process less expensive and the downstream bioinformatics more straightforward. /content/figures/1746-4811-8-12-toc.gif Plant Methods 1746-4811 ${item.volume} 12 2012-03-30T00:00:00Z 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, null:11 2007-09-24T00:00:00Z doi:10.1186/1746-4811-3-11 /content/figures/1746-4811-3-11-toc.gif Plant Methods 1746-4811 ${item.volume} 11 2007-09-24T00:00:00Z 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, null:13 2005-12-18T00:00:00Z doi:10.1186/1746-4811-1-13 /content/figures/1746-4811-1-13-toc.gif Plant Methods 1746-4811 ${item.volume} 13 2005-12-18T00:00:00Z XML Generating and identifying transformants is essential for many studies of gene function. In Arabidopsis thaliana, a revolutionary protocol termed floral dip is now the most widely used transformation method. Although robust, it involves a number of relatively time-consuming and laborious steps, including manipulating an Agrobacterium tumefaciens culture and aseptic procedures for the selection of plant lines harboring antibiotic-selection markers. Furthermore, where multiple transgenes are to be introduced, achieving this by sequential transformations over multiple generations adds significantly to the time required. To circumvent these bottlenecks, we have developed three streamlined sub-protocols. First, we find that A. thaliana can be transformed by dipping directly into an A. tumefaciens culture supplemented with surfactant, eliminating the need for media exchange to a buffered solution. Next, we illustrate that A. thaliana lines possessing a double-transformation event can be readily generated by simply by floral-dipping into a mixture of two A. tumefaciens cultures harboring distinct transformation vectors. Finally, we report an alternative method of transformant selection on chromatography sand that does not require surface sterilization of seeds. These sub-protocols, which can be used separately or in combination, save time and money, and reduce the possibility of contamination. http://www.plantmethods.com/content/5/1/3 Amanda Davis Anthony Hall Andrew Millar Chiarina Darrah Seth Davis Plant Methods 2009, null:3 2009-02-27T00:00:00Z doi:10.1186/1746-4811-5-3 /content/figures/1746-4811-5-3-toc.gif Plant Methods 1746-4811 ${item.volume} 3 2009-02-27T00:00:00Z 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, null:6 2009-05-20T00:00:00Z doi:10.1186/1746-4811-5-6 /content/figures/1746-4811-5-6-toc.gif Plant Methods 1746-4811 ${item.volume} 6 2009-05-20T00:00:00Z 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, null:3 2008-01-22T00:00:00Z doi:10.1186/1746-4811-4-3 /content/figures/1746-4811-4-3-toc.gif Plant Methods 1746-4811 ${item.volume} 3 2008-01-22T00:00:00Z XML Background: Protoplasts isolated from leaves are useful materials in plant research. One application, the transient expression of recombinant genes using Arabidopsis mesophyll protoplasts (TEAMP), is currently commonly used for studies of subcellular protein localization, promoter activity, and in vivo protein-protein interactions. This method requires cutting leaves into very thin slivers to collect mesophyll cell protoplasts, a procedure that often causes cell damage, may yield only a few good protoplasts, and is time consuming. In addition, this protoplast isolation method normally requires a large number of leaves derived from plants grown specifically under low-light conditions, which may be a concern when material availability is limited such as with mutant plants, or in large scale experiments. Results: In this report, we present a new procedure that we call the Tape-Arabidopsis Sandwich. This is a simple and fast mesophyll protoplast isolation method. Two kinds of tape (Time tape adhered to the upper epidermis and 3 M Magic tape to the lower epidermis) are used to make a "Tape-Arabidopsis Sandwich". The Time tape supports the top side of the leaf during manipulation, while tearing off the 3 M Magic tape allows easy removal of the lower epidermal layer and exposes mesophyll cells to cell wall digesting enzymes when the leaf is later incubated in an enzyme solution. The protoplasts released into solution are collected and washed for further use. For TEAMP, plasmids carrying a gene expression cassette for a fluorescent protein can be successfully delivered into protoplasts isolated from mature leaves grown under optimal conditions. Alternatively, these protoplasts may be used for bimolecular fluorescence complementation (BiFC) to investigate protein-protein interactions in vivo, or for Western blot analysis. A significant advantage of this protocol over the current method is that it allows the generation of protoplasts in less than 1 hr, and allows TEAMP transfection to be carried out within 2 hr. Conclusion: The protoplasts generated by this new Tape-Arabidopsis Sandwich method are suitable for the same range of research applications as those that use the current method, but require less operator skill, equipment and time. http://www.plantmethods.com/content/5/1/16 Fu-Hui Wu Shu-Chen Shen Lan-Ying Lee Shu-Hong Lee Ming-Tsar Chan Choun-Sea Lin Plant Methods 2009, null:16 2009-11-24T00:00:00Z doi:10.1186/1746-4811-5-16 /content/figures/1746-4811-5-16-toc.gif Plant Methods 1746-4811 ${item.volume} 16 2009-11-24T00:00:00Z XML Background: We have developed a novel assay based on the ability of type I sucrose uptake transporters (SUTs) to transport the fluorescent coumarin β-glucoside, esculin. Budding yeast (Saccharomyces cerevisiae) is routinely used for the heterologous expression of SUTs and does not take up esculin. Results: When type I sucrose transporters StSUT1 from potato or AtSUC2 from Arabidopsis were expressed in yeast, the cells were able to take up esculin and became brightly fluorescent. We tested a variety of incubation times, esculin concentrations, and buffer pH values and found that for these transporters, a 1 hr incubation at 0.1 to 1 mM esculin at pH 4.0 produced fluorescent cells that were easily distinguished from vector controls. Esculin uptake was assayed by several methods including fluorescence microscopy, spectrofluorometry and fluorescence-activiated cell sorting (FACS). Expression of the type II sucrose transporter OsSUT1 from rice did not result in increased esculin uptake under any conditions tested. Results were reproduced successfully in two distinct yeast strains, SEY6210 (an invertase mutant) and BY4742. Conclusions: The esculin uptake assay is rapid and sensitive and should be generally useful for preliminary tests of sucrose transporter function by heterologous expression in yeast. This assay is also suitable for selection of yeast showing esculin uptake activity using FACS. http://www.plantmethods.com/content/8/1/13 Peter Gora Anke Reinders John Ward Plant Methods 2012, null:13 2012-04-04T00:00:00Z doi:10.1186/1746-4811-8-13 Esculin simplifies functional analysis of sucrose transporters Budding yeast is an important model system for the heterologous expression of plant sucrose transporters. The authors have exploited the ability of type I sucrose uptake transporters to transport the fluorescent β-glucoside, esculin, to develop a rapid and sensitive assay system that should be a valuable tool for the functional analysis of members of this sucrose transporter family. /content/figures/1746-4811-8-13-toc.gif Plant Methods 1746-4811 ${item.volume} 13 2012-04-04T00:00:00Z XML MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression. http://www.plantmethods.com/content/3/1/12 Erika Varkonyi-Gasic Rongmei Wu Marion Wood Eric Walton Roger Hellens Plant Methods 2007, null:12 2007-10-12T00:00:00Z doi:10.1186/1746-4811-3-12 /content/figures/1746-4811-3-12-toc.gif Plant Methods 1746-4811 ${item.volume} 12 2007-10-12T00:00:00Z XML